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In this study, an analytical method was established to determine pentachlorophenol (PCP) in milk powder by isotope dilution-liquid chromatography-tandem mass spectrometry (LC-MS/MS). The sample was dissolved in water, extracted by the quick, effective, cheap, easy, rugged and safe (QuEChERS) method using acetonitrile as the extraction solvent and protein precipitant, purified on a Captiva EMR-Lipid column, and diluted with the same volume of water before analysis. The chromatographic separation was achieved on a C18 column (100 mm × 2.1 mm, 1.7 μm) with gradient elution using a mobile phase composed of methanol and 5 mmol/L ammonium acetate solution (containing 0.05% formic acid), and then the analyte was detected with negative electrospray ionization (ESI-) in the multiple reaction monitoring (MRM) mode. Matrix matching-isotope dilution internal standard method was used for identification and quantification of the analyte. The results indicated that the calibration curve of PCP was linear in the concentration range of 0.2‒150 μg/L with correlation coefficients (r) not less than 0.996 0. The limits of detection (LOD) and quantitation (LOQ) of the method were 0.5 and 1.5 μg/kg, respectively. The average recoveries for spiked milk powder ranged from 90.3% to 119.2%, with relative standard deviation (RSD, n = 6) equal to or less than 7.5%. The method has the advantages of simple pretreatment without concentration or dissolution, high sensitivity, accuracy and reliability, and can be used as an effective method for detecting pentachlorophenol in milk powder.

To improve our understanding of farmland surface process and agricultural water management in arid oasis area, we analyzed the characteristics of water and heat fluxes at different time scales, and the influence of arid advection and canopy conductance (Gc) on water and heat fluxes at different growth stages. In addition, we used path analysis to study the influence path of environmental factors on latent heat flux (LE). In this paper, the water and heat fluxes were measured by eddy-covariance in 2017. The results were as follows: (1) LE showed multiple peaks, while the other fluxes were basically unimodal shapes at the daily scale. On the whole, the values of daily net radiation (Rn) were maximum, followed by LE, sensible heat flux (H) and soil heat flux (G) at each growth stage. The variation of diurnal G showed obvious lagged effect compared to that of Rn. (2) LE and H accounted for 86% and 14% (respectively) of daytime energy during the whole growing stages, indicating that LE was the main energy consumption item. The contribution of arid advection to daytime LE ranged from 5% to 59% with an average value of 28% at whole growing season. The influence of Gc on LE was stronger at the new shoot and leaf-fall stages than that in the middle growing season. (3) LE was mainly affected by Rn, followed by vapor pressure deficit (VPD) and air temperature (Ta), and the influence degrees of VPD and Ta on LE were similar. Furthermore, the path analysis showed that Rn directly affected LE; VPD and Ta mainly affected LE indirectly through Rn. As a whole, the water and heat fluxes in vineyards of arid desert oasis in Northwest China display significant diurnal and seasonal variations, and the influences of environmental factors on these exists great discrepancies in magnitude and path.

With the rapid development of the aquaculture industry and the shortage of global fishery resources, the contradiction between supply and demand of fish meal has become increasingly significant. Therefore, finding new substitutes for fish meals and reasonably reducing the amount of fish meal in formula feed has become an important research topic in aquatic feed. Stickwater is a byproduct of fishmeal processing. It contains many water-soluble molecules, such as small molecular peptides, biogenic amines, taurine, and high unsaturated fatty acids. These components are regarded as particular nutrients or bioactive substances in the fish meal. Studies have shown that stickwater can replace part of fish meal in recent years and achieve good results in fish and other aquatic animals. The fermented feed has excellent advantages for fish meal replacement. Studies have shown that after fermentation, the contents of anti-nutritional factors in plant raw materials are decreased.In contrast, the contents of small peptides and free amino acids are increased, the nutrient composition is changed, and the intermediate metabolites of microorganisms are obtained, which can further improve the replacement level of fish meal. There are few studies on the fermentation of feed materials, such as fish meal and stickwater, which do not contain anti-nutritional factors. Our laboratory's preliminary study found that stickwater combined with other animal and plant proteins could replace 40% of fish meal in the feed. This experiment fermented stickwater first, then replace fish meal in high plant protein formula feed with fermented stickwater (FSW), aims to further increase the amount of fish meal replacement, reduce the negative effect of the plant protein source on the turbot, for FSW application in feeds for turbot provide a theoretical reference.The stickwater in the experiment was a brown viscous liquid, with a water content of 48.73%, dry matter crude protein content of 64.08%, and crude fat content of 8.91%. The strains used were Bacillus subtilis and Lactobacillus. The fermentation conditions were as follows: Temperature of 37℃, the addition of 1% sugar as auxiliary material, Bacillus subtilis and Lactobacillus (1:1) added to 1% of the total mass of the stickwater, and the fermentation period was five days. After fermentation, the content of acid-soluble protein decreased significantly. Still, the crude protein, crude fat, and free amino acids showed no significant changes. It was then used for turbot culture.Healthy juvenile turbot with an average bodyweight of (30.00±0.03) g were randomly divided into six groups with three replicates and 30 fish per replicate. The trial lasted for eight weeks. Six diets consisted of a positive control diet with 50% fish meal (positive control group), a harmful control diet with 30% fish meal (negative control group), and experimental diets formulated by FSW were used to replace 2%, 4%, 6%, and 8% of the fish meal with the harmful control diet, respectively (FSW2, FSW4, FSW6, and FSW8). The results showed: There were no significant differences in the survival rate of juvenile turbot among all groups (P > 0.05). There were no significant differences in juvenile turbot's weight gain rate and protein efficiency ratio in the FSW2~FSW8 group compared with that of the positive control group. Still, they were all higher than those in the negative control group. The crude protein content of whole fish and dorsal muscle in the FSW2~FSW8 group was not significantly different from that in the positive control group. Still, it was significantly higher than that in the negative control group. The highest crude lipid content of whole fish and dorsal muscle was found in the negative control group (P < 0.05). Serum ALT, AST, and TG levels in the negative control group were significantly higher than those in the positive control group; the negative control group and FSW2~FSW8 group showed first decreasing and subsequent increasing serum ALT, AST, and T-CHO levels. At the same time, the ALT and AST levels in the liver showed an opposite trend. The serum HDL-C content in the negative control group was significantly lower than that in the positive control group. The activities of PKA, KPC, and LDH in the liver were significantly lower in the negative control group than in the positive control group (P < 0.05). Compared with the positive control group, the expression of b0at1 and pept1 in the intestine was upregulated in the negative control group. In contrast, the expression of cat1 and pat1 was not significantly different. The expression levels of boat1, cat1, pat1, and pept1 in the FSW2~FSW8 group were significantly higher than those in the positive control and negative control groups (P < 0.05).The results showed that FSW was an excellent substitute for fish meals, and the added amount could be up to 8% in the feed. In the feed with high plant protein, the fish meal can be replaced by FSW to reduce the amount of fish meal further, reduce the metabolic abnormality caused by plant proteins, and improve juvenile turbot's antioxidant capacity. Under these experimental conditions, the group supplemented with FSW achieved the same growth effect as the positive control group. In conclusion, the fish meal content of juvenile turbot feed can be reduced to 22% by adding FSW without adverse effects on the growth of juvenile turbot. This provides a theoretical reference for the fermentation process of SW and subsequent application of FSW in seawater fish.

Objective To investigate the gene carrying rate,gene mutation type and hematological characteristics of thalassemia in Dai nationality population of Dehong Prefecture, Yunnan Province. Methods A total of 591 Dai nationality gestational age population in Dehong were tested for thalassemia gene by next-generation sequencing technology. The sensitivity and specificity of the hemoglobin electrophoresis and blood routine tests were performed. Results A total of 289 cases (48.90%) were positive for thalassemia gene. Among them,231 cases were classified as α-thalassemia(39.09%) and 89 cases were β-thalassemia(15.06%). Five α-thalassemia genotypes and seven β-thalassemia genotypes were detected. For α-thalassemia, --SEA showed comparatively lower values for MCV and MCH; αCSα showed comparatively lower values for Hb A2 (P＜0.05). For the β-thalassemia,CD 26 (G＞A)had significantly lower Hb A values,and higher MCH values(P＜0.05). A new cut-off value for the thalassemia with the highest accuracy was proposed(MCV 83.55 fL,MCH 27.95 pg). Conclusions Thalassemia is common in Dai nationality population of Dehong. The most frequently identified types are －α3.7 and CD 26 (G＞A).

【Objective】There are more than 50 kinds of orchid viruses in the world, among which Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are the most common viruses. However, there are no effective measures to control the virus disease. Therefore, the establishment of efficient detection methods for the CymMV and ORSV is of great significance to control infected plants.【Method】CymMV and ORSV were used as experimental materials, and the reverse transcription loop-mediated isothermal amplification method (RT-LAMP) was used to amplify the viral RNA, so as to establish a rapid detection method of CymMV and ORSV. The RT-LAMP primers were designed according to the conserved regions of the cp gene sequences of two viruses. The hydroxylnaphthol blue (HNB, the final concentration of 200 μmol/L)was added to the RT-LAMP reaction system as an indicator of the amplification product. Without opening the tube for gel electrophoresis, the reaction results could be directly interpreted by the color change of the system which could avoid product aerosol pollution.【Result】The detection method could detect the presence of CymMV and ORSV RNA in the samples within 2 h, and its sensitivity was 10 times that of RT-PCR. In order to test the detection effect of RT-LAMP-HNB in actual production, 20 Phalaenopsis samples were detected by one-step RT-LAMP-HNB. According to the results, the detection rates of CymMV and ORSV were 70% and 55%, which were consistent with the results of RT-PCR, indicating that this method was suitable for the detection of field samples.【Conclusion】The one-step RT-LAMP-HNB established in this study is a sensitive, rapid, specific and convenient method for the detection of CymMV and ORSV. Nucleic acid amplification can be carried out only by simple temperature control equipment such as water bath pot, suitable for promotion and use in grass-roots or inconvenient areas.

With the recent exciting achievements of Google’s artificial intelligence system in the game of Go, deep reinforcement learning (DRL) has witnessed considerable development. DRL combines the abilities of sensing and making decisions provided by deep learning and reinforcement learning. Natural language processing (NLP) involves a large number of vocabularies or statements that have to be represented, and its subtasks, such as the dialogue system and machine translation, involve many decision problems that are difficult to model. Because of the aforementioned reasons, DRL can be appropriately applied to various NLP tasks such as named entity recognition, relation extraction, dialogue system, image caption, and machine translation. Further, DRL is helpful in improving the framework or the training pipeline of the aforementioned tasks, and notable achievements have been obtained. DRL is not an algorithm or a method but a paradigm. Many researchers fit plenty of NLP tasks in this paradigm and achieve better performance. Specifically, in text generation based on the reinforcement learning paradigm, the learning process that is used to produce a predicted sequence from the given source sequence can be considered to be the Markov decision process (MDP). In MDP, an agent interacts with the environment by receiving a sequence of observations and scaled rewards and subsequently produces the next action or word. This causes the text generation model to achieve decision-making ability, which can result in future success. Thus, the text generation task integrated with reinforcement learning is an attractive and promising research field. This study presented a comprehensive introduction and a systemic overview. First, we presented the basic methods in DRL and its variations. Then, we showed the main applications of DRL during the text generation task, trace the development of DRL, and summarized the merits and demerits associated with these applications. The final section enumerated some future research directions of DRL combined with NLP.

As the current popular chaos-based image encryption algorithms with the permutation–diffusion structure have security flaws of no immunity to attack.A new image encryption algorithm was proposed based on the analysis of current algorithms,which can well resist the chosen-plaintext and the chosen-ciphertext attacks.The algorithm uses Kent chaotic map to generate key streams,and produces the parameters of the chaotic system and the iteration times according to the characteristics of plaintext pixels and input key.Firstly,the positions of pixels were shuffled totally with the chaotic sequence.Secondly,the 0 and 1 bit positions of image pixels were scrambled by using another chaotic sequence generated by the input key.The experimental results show that the algorithm cannot only resist the chosen plaintext attack and chosen ciphertext attack but also achieve better cryptographic properties,such as key space,statistical analysis.

The misjudgement of spam has always been the difficulty in the anti-spam area.Experiments based on Tsinghua university E-mail systems and anti-spam gateway system(Sep,2011–Oct,2012) were deployed and made,analyzing the history of recalling spam behavior of mail users,and using spam text similarity calculation and intelligent key parameters prediction method to predict the user's interest in the current pending spam,which can help users automatically recall their potential interested spams which were misjudged,based on the users' subjective choices and experience,solving the problem that cannot be eliminated by traditional filtering methods.

A multipurpose image watermarking scheme based on chaotic systems and sub-image lifting wavelet transformation（LWT） was proposed.Firstly, the host image was divided into 8×8 sub-images and each sub-image was performed 2-level LWT.Then, the invisible watermarks for copyright protection and content authentication were respectively embedded into low and intermediate frequency LWT components of some sub-images that were randomly selected by general Arnold map.A new kind of quantization technique was adopted in the embedding process and the extracting process was blind.Secondly, the visible watermarks for copyright notification were embedded into the image spatial domain based on image fusion method, in which the embedding coefficients were generated by the 4D hyperchaotic sequences.Experimental results were presented, showing that the proposed watermarking scheme is effective.

This study aims to explore the influence of Polygonati Rhizoma on the pyroptosis in the rat model of diabetic macroangiopathy via the NOD-like receptor thermal protein domain associated protein 3(NLRP3)/cysteinyl aspartate specific proteinase-1(caspase-1)/gasdermin D(GSDMD) pathway. The rat model of diabetes was established by intraperitoneal injection of streptozotocin(STZ) combined with a high-fat, high-sugar diet. The blood glucose meter, fully automated biochemical analyzer, hematoxylin-eosin(HE) staining, enzyme-linked immunosorbent assay, immunofluorescence, immunohistochemistry, and Western blot were employed to measure blood glucose levels, lipid levels, vascular thickness, inflammatory cytokine levels, and expression levels of pyroptosis-related proteins. The mechanism of pharmacological interventions against the injury in the context of diabetes was thus explored. The results demonstrated the successful establishment of the model of diabetes. Compared with the control group, the model group showed elevated levels of fasting blood glucose, total cholesterol(TC), triglycerides(TG) and low-density lipoprotein cholesterol(LDL-c), lowered level of high-density lipoprotein cholesterol(HDL-c), thickened vascular intima, and elevated serum and aorta levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and interleukin-18(IL-18). Moreover, the model group showed increased NLRP3 inflammasomes and up-regulated levels of caspase-1 and GSDMD in aortic vascular cells. Polygonati Rhizoma intervention reduced blood glucose and lipid levels, inhibited vascular thickening, lowered the levels of TNF-α, IL-1β, IL-18 in the serum and aorta, attenuated NLRP3 inflammasome expression, and down-regulated the expression levels of caspase-1 and GSDMD, compared with the model group. In summary, Polygonati Rhizoma can slow down the progression of diabetic macroangiopathy by inhibiting pyroptosis and alleviating local vascular inflammation.

Abelmoschus manihot (L.) Medic., a folk herbal medicine in China, is a flowering plant belonging to Abelmoschus L. genus and Malvaceae family, which has been reported with an antidepressant activity. The study was designed to isolate flavonoids from Abelmoschus manihot corolla and explore the action mechanism of antidepressant activities. The flavonoids were isolated and purified by D101 macroporous resin column, polyamide column and Sephadex LH-20 sequentially and identified as myricetin-3-O-β-D-glucoside (1), gossypetin-8-O-β-D-glucuronide (2, G-8-G), gossypetin-3'-O-β-D-glucoside (3), quercetin-3'-glucoside (4, Q-3-G), isoquercitrin (5, IQT), hyperoside (6, HY), myricetin (7), quercetin (8, QT). Compounds 2, 4, 5, 6 and 8 (15, 30 and 60 mg·kg−1) were orally administered to mice and the reaction was observed in tail suspension test (TST) and forced swimming test (FST). Western blot analysis was used in determination of the protein expressions of brain-derived neurotrophic factor (BDNF), tyrosine receptor kinase B (TrkB) and phosphorylation eukaryotic elongation factor 2 (p-eEF2). The results revealed that only Q-3-G and G-8-G (15, 30, 60 mg ·kg−1) significantly reduced the immobility time in FST and TST. Furthermore, Q-3-G and G-8-G remarkably increased the expression of BDNF and TrkB, and decreased the expression of p-eEF2. These results suggest that Q-3-G and G-8-G had an obvious antidepressant activity via up-regulation of BDNF expression. The new observation will provide a new direction in the development of antidepressant in the treatment of major depressive disorder (MDD).

Objective: To study the nucleotide and amino acid sequences of norovirus G II.4/Sydney 2012 variants, in China., Methods: Twenty-two stool specimens, confirmed as G II.4/Sydney 2012- positive were collected from Beijing in the winter of 2012-2013. RT-PCR was performed to target the complete capsid gene. G II.4/Sydney 2012 strains from other regions in China were searched and obtained from the GenBank. Nucleotide and amino acid sequences of G II.4/Sydney 2012 strains were analyzed, using the CLUSTAL X(Version 1.83)and followed by phylogenetic analysis using Mega version 5.1., Results: The complete major capsid nucleotide sequences of thirty-eight G II.4/ Sydney 2012 strains from seven regions in China were obtained. The VP1 nucleotide and amino acid sequences diversity were 0.1%-3.3% and 0-3.1%, respectively. Result from phylogenetic analysis demonstrated that the G II.4/Sydney 2012 variant shared a common ancestor with both the dominant norovirus G II.4 variants Apeldoorn 2008 and the New Orleans 2009. G II.4/Sydney 2012 variants appeared to have had two A/D/E site combinations at the amino acid level, TSRN-GTT-SNT and TSRN-STT-SNT., Conclusion: G II.4/Sydney 2012 variant had been circulating in many regions in China. There seemed a high nucleotide and amino acid identity among G II.4/Sydney 2012 strains collected from China. G II.4/Sydney 2012 variants showed different A/D/E site combination from other pandemic G II.4 variants.

Objective: to explore whether there is an interaction between Helicobacter pylori (H. pylori), omeprazole and gastrin on the proliferation and apoptosis of gastric epithelial cells., Methods: With omeprazole, H. pylori and gastrin as treatment factors, the in vitro changes of gastric epithelial cells AGS and GES-1 were detected by MTS, Hoechst33342 and flow cytometry., Results: (1) for a single factor, the gastric mucosal cells (AGS and GES-1) treated by H. pylori or omeprazole show significantly higher apoptosis than that of control group [H. pylori and 104 nmol/L omeprazole group: (17.20 ± 0.90)%, (12.81 ± 0.78)% vs (7.96 ± 1.25)%; (4.60 ± 0.34)%, (6.60 ± 0.76)% vs (3.52 ± 0.16)%; all P < 0.05]. The gastric mucosal cells treated by H. pylori or omeprazole show significantly lower proliferation than that of control group (H. pylori and 104 nmol/L omeprazole group: 13.35 ± 0.55 vs 37.78 ± 1.98, 47.62 ± 2.40 vs 62.44 ± 4.46; 27.15 ± 1.64 vs 32.76 ± 1.57, 29.42 ± 1.44 vs 48.86 ± 4.95; all P < 0.05). The differences were statistically significant and the effects were correlated with the concentration of omeprazole. Gastrin had no direct effect on cellular proliferation and apoptosis (all P > 0.05). (2) For a combinations of factors, the GES-1 cells treated with H. pylori and 500 ng/L gastrin had a significantly higher ratio [(7.25 ± 0.54)% vs (4.60 ± 0.34)%, P < 0.05], while the other groups just showed a rising trend in the proportion of apoptosis cells, but there was no statistical significance. Flow cytometry analysis showed that combinations of H. pylori, omeprazole and gastrin caused lower proliferation than that of H. pylori group, and all changes were statistically significant (AGS: 12.68 ± 0.09, 12.28 ± 0.31 vs 13.35 ± 0.55; GES-1: 22.06 ± 1.61, 18.59 ± 0.09 vs 27.15 ± 1.64; all P < 0.05). And combinations of 104 nmol/L omeprazole and 500 ng/L gastrin caused lower proliferation than that of 104 nmol/L omeprazole group (33.23 ± 5.98 vs 47.62 ± 2.40 and 25.97 ± 0.74 vs 29.42 ± 1.44, both P < 0.05). (3) When comparing the apoptosis and proliferation changes of two cell lines, gastric cancer cells AGS had a more marked changes than immortalized cells GES-1., Conclusion: gastrin significantly enhances the effects of omeprazole and H. pylori on gastric epithelial cells. It is speculated that the factors of omeprazole, H. pylori and gastrin may have a synergistic effect of decreased proliferation and increased apoptosis of gastric mucosal cells. Thus a long-term proton pump inhibitor treatment of H. pylori patients may carry a higher risk of atrophic gastritis. But it needs to be confirmed by further experiments.

Objective: To evaluate the inhibition of CD4+ CD25+ regulatory T cells (Treg) in the chronic hepatitis B patients., Methods: Peripheral blood samples were collected from 22 patients with chronic hepatitis B (CHB) and 18 healthy blood donors to isolate the peripheral blood mononuclear cells (PBMCs). Flow cytometry was used to analyze the proportion of CD4+ CD127(lo)CD25(hi-int) Tregs in the CD4+ T cells so as to calculate the proportion of CD4+ CD25+ Tregs in the CD4+ T cells. BrdU incorporation method was used to evaluate the immune inhibition of the CD4+ CD25+ Tregs. CD4+ CD25- cells were isolated by magnetic bead sorting technique. The CD4- T cells and CD4+ CD25- T cells ere mixed and stimulated by HBVcore 18-27 peptide. The PBMCs of the CHB patients with the Treg depleted and Treg not depleted underwent detection of HBVcore18-27 specific cytotoxic T lymphocytes (CTLs). The IFN-gamma secretion of the CTLs in the PBMCs of CHB patients with Treg depleted and Treg not depleted was detected by HLA-pentamer and enzyme-linked immunospot assay (Elispot)., Results: The proportion of CD4+ CD127(lo)CD25(hi-int) Treg in the CD4+ T cells used to reflect the percentage of CD4+CD25+ Tregs in the CD4+ T cells of the CHB patients was 4.3% +/- 2.4%, significantly higher than that of the healthy controls (2.1% +/- 1.3%, t = 3.74, P <0.01). There was no significant difference in the inhibition of CD4+ CD25- T cells by autogenous CD4+ CD25+ T cells between the CHB patients and healthy controls. The frequency of CTLs induced by HBV core 18-27 of the CHB patients with their CD4+ CD25+ cells in circulation depleted was 0.74% +/- 0.31%, significantly higher than that of the patients whose CD4+ CD25+ cells in circulation were not depleted (0.17% +/- 0.08%, t = 4.75, P <0.01). The frequency of IFN-gamma secreting spots of HBVcore18-27-specific CD8+ T cells of the CHB patients with their CD4+ CD25+ cells depleted was (112 +/- 33), significantly higher than that of the CHB patients whose CD4+ CD25+ cells in circulation were not depleted [(23 +/- 14), t =7.828, P<0.01)]., Conclusion: The proportion of CD4+ CD25+ Treg in CHB patients is increased compared to the healthy blood donor. The proliferative capacity of CD4+ CD25- T cells is inhibited by the presence of CD4+ CD25+ Treg dose-dependently, and the inhibition of CD4+ CD25+ Tregs in the CHB patients is similar to the inhibition of CD4+ CD25+ Tregs in healthy donors. The elimination of Treg cells followed by stimulation with HBVcore18-27 peptide significantly improves the antivirus CTL responses in CHB patients.

Objective: To understand the infection and epidemiology of norovirus and rotavirus and enteral adenovirus among adult with sporadic viral gastroenteritis in Beijing and provide theoretical basis for clinical prevention and control., Methods: Stool specimens were collected from all 312 sporadic outpatient among adult with non-cholera watery diarrhea in Infectious Disease Department of Peking University People's Hospital 2005-2006. PAGE were used for detection of rotavirus RNA in stool specimens; R-Biopharm RIDASCREEN norovirus and RIDASCREEN adenovirus were used for detection of norovirus and adenovirus., Results: Rotavirus RNA was not present in all 312 stool specimens; Norovirus was present in 17.6% (22/125) and in 32.4% (11/34) in October; Adenovirus was present in 3.3% (3/92); Mixed infections of norovirus and adenovirus was present in 1 stool specimen., Conclusions: Norovirus is more common etiologic agents of sporadic acute viral gastroenteritis among adult in Beijing, The infection peak of norovirus is in autumn. Maybe the infection of rotavirus is few.

Objectives: To study the effects of rat endothelial progenitor cell (EPC) transplantation on hepatic fibrosis in carbon tetrachloride (CCl4) induced hepatic fibrosis rats., Methods: Hepatic fibrosis was developed in 24 healthy female SD rats by feeding them 25% CCl4/olive oil for 8 weeks. Eight of them were sacrificed at the end of the 8 weeks. The rats were subdivided into a EPCs transplanting group (n=8) and a saline control group (n=8). After the EPCs were isolated and cultured for 9 days, the cells were injected into the portal veins of the rats in the EPCs transplanting group. Four weeks later all of the rats were sacrificed. The blood biochemical parameters from the serum were examined. The degree of liver fibrosis was evaluated by reading Masson staining liver slides and by detecting the expression of a-SMA and collagen III., Results: Compared with the saline control group, hepatic activity index (HAI), levels of ALT, AST and TBil in the serum were all lower in the EPCs transplanting group, but the level of Alb was higher and the expression of a-SMA and collagen III were lower. Compared with the 8 week hepatic fibrosis group, the levels of ALT, AST and TBil in the serum of the EPCs transplanting group were all lower. In the saline control group, the serum levels of ALT, AST and TBil were higher, the level of Alb was lower, and the expressions of a-SMA and Collagen III were higher., Conclusion: In hepatic fibrosis rats, transplantation of rat EPCs could minimize the hepatic fibrosis process and the injuries.

Objectives: (1) To evaluate the prevalence, phenotypes and suppressive function of CD4+CD25+ regulatory T cells (Tregs) among the in peripheral blood mononuclear cells (PBMCs) and tumor-infiltration lymphocytes (TILs) from hepatocellular carcinoma (HCC) patients and patients with chronic hepatitis B. (2) To investigate the correlation between the frequency of CD4+CD25+ Tregs and clinical characteristics of HCC patients., Methods: PBMCs and TILs in 18 HCC patients, 10 chronic hepatitis B (CHB) patients and 15 healthy donors were evaluated for the phenotypes of CD4+CD25+ Tregs and the proportion of CD4+CD25+ Tregs as a percentage of the total CD4+ cells, by flow cytometric analysis with three or four color staining. The relationship between the frequency of CD4+CD25+ Tregs and tumor TNM stages was analyzed. The CD4+CD25+ Tregs and CD4+CD25- T cells were isolated from PBMC of HCC patients and donors. The suppressive function of CD4+CD25+ Tregs was analyzed., Results: The percentages of CD4+CD25+ Tregs of the HCC patients (6.38% +/- 6.30%) and CHB patients (4.29% +/- 1.82%) were significantly higher than those of the healthy donors (1.58% +/- 0.55%, P less than 0.01). Among the TILs, the percentage of CD4+CD25+ Tregs was higher (t = 4.39, P < 0.01). There were significant differences in the prevalence of CD4+CD25+ Tregs in early and advanced stage HCCs (stage II vs. III, P less than 0.05; stage II vs. IV P < 0.01). The proliferative capacity of CD4+CD25- T cells was inhibited by the presence of CD4+CD25+ T cells in a dose-dependent manner where the level of suppression was correlated to the ratio of the two-cell populations., Conclusion: These results suggest that the increase in frequency of CD4+CD25+ Tregs might play a role in the suppression of the immune response against HCC, which may contribute to the HCC cells that escaped from immunological surveillance.

Objective: To study the effects of interferon-gamma and interferon-gamma combined with interferon-alpha on HCV RNA replication and the possible mediators of interferon-gamma anti-HCV in vitro., Methods: An HCV replicon cell culture system was established and the cells were treated with interferon-gamma or interferon-gamma combined with interferon-alpha. HCV RNA levels in the cells were evaluated by semi-quantitative RT-PCR and real-time PCR, and the levels of NS5A protein were examined by Western blot., Results: Interferon-gamma could inhibit HCV RNA replication and NS5A protein expression effectively; The anti-HCV effects of interferon-gamma were both in time-dependent and does-dependent manners; Pretreating the cells with interferon-gamma could significantly enhance the antiviral effects of interferon-alpha; The expressions of IRF-1, 2'5'-OAS1(p46), 2'5'-OAS2(p69), ISGF3gamma and STAT1 were significantly increased after interferon-gamma treatment., Conclusion: Interferon-gamma has a direct inhibitory effect on HCV replicon RNA replication and NS5A expression, and both are in a dose and time dependent manner; Interferon-gamma has a synergistic anti-HCV effect with interferon-alpha. IRF-1, 2'5'-OAS1(p46), 2'5'-OAS2(p69) and ISGF3gamma may mediate the anti-HCV effects of interferon-gamma.

Objective: To investigate the effects of granulocyte colony-stimulating factor (G-CSF)-mobilized autologous bone marrow stem cells on the liver regeneration of partial liver transplant., Methods: A 50% partial liver transplantation model as established by transplanting parts of the liver of female rats to the male rats with the equal weights that had had parts of their livers resected. Then the transplanted male rats were randomly divided into 3 equal groups: G-CSF + PLTx group that was injected hypodermically with recombinant human G-CSF (rhG-CSF) daily for 5 days and then underwent liver resection and 50% partial liver transplantation (PLTx); PLTx + G-CSF group that underwent PLTx and 3 hours after the transplantation received injection of rhG-CSF daily for 5 days, and PLTx control group that underwent PLTx and 3 hours later received injection of normal saline daily for 5 days. One, 3, 5, 7, and 14 days after the PLTx blood samples and livers were collected from 6 rats from each group, and one hour before the liver was taken bromodeoxyuridine (BrdU) was injected intraperitoneally to be integrated into the synthesis of DNA in the liver cells. Immunohistochemistry was used to detect the CD34 and BrdU-positive cells. In situ hybridization was used to detect the sry (sex-determining region) gene so as to determine the origin of the proliferating cells in the transplanted liver., Results: The 14-day survival rate of the G-CSF + PLTx group was 90%, significantly higher than those of the G-CSF + PLTx (60%) and PLTx group (50%) (both P < 0.05) with a significant difference between the latter 2 groups too (P < 0.05). The mitosis index of liver cells 3 days after the transplantation of the PLTx + G-CSF group was 30% +/- 5%, significantly higher than those of the G-CSF + PLTx group (24% +/- 7%) and PLTx group (24% +/- 6%) (both P < 0.05) without a significant difference between the latter 2 groups. The rate of BrdU-positive cells of the PLTx + G-CSF group was 42% +/- 6%, significantly higher than those of the G-CSF + PLTx group (38% +/- 4%) and PLTx group (34% +/- 8%) (both P < 0.05). The transplantation the mitosis index and rate of BrdU-positive cells decreased since the 7th days after transplantation in all 3 groups, however, with the same relationship among them. The numbers of CD34(+) cell around the portal area of the 2 G-CSF groups increased since the 3rd day after transplantation in comparison with the PLTx group, was the highest on the 3rd day for the G-CSF + PLTx group, and continued to increase in the PLTx + G-CSF group. Since the 3rd day after transplantation, sry-positive cells were seen in the hepatic sinusoid and portal area in the 2 G-CSF groups, and were rarely seen in the PLTx group., Conclusion: G-CSF treatment after 50% PLTx significantly promotes liver regeneration and ameliorates the liver damage, thus raising the survival rate.

Objective: To explore the role of stem cell mobilization on regeneration of partially grafted livers., Methods: Rats models with cross-sex 50% PLTx (partial liver transplantation) were established. The rats were divided into three groups: PLTx, WLTx (whole liver transplantation) and sham operation groups. Bone marrow and liver samples were collected on days 1, 3, 5, 7 postoperatively (each n = 6). The quantitative variations of the cells with stem cell markers in the bone marrow, including beta2m-/Thy-1.1+, CD45+/CD34+, Flt2/3+ and c-kit+ markers, were detected using flow cytometry. Sry gene positive cells in donor livers were detected by fluorescent in situ hybridization (FISH), and the expressions of CD34, c-kit and Thy-1.1 were detected by immunohistochemistry technique., Results: Compared with the WLTx and sham operation groups, beta2m-/Thy-1.1+, CD45+/CD34+ cells in bone marrows in the PLTx group increased on the first postoperative day and decreased on the following days. The CD34, c-kit and Thy-1.1 positive cells detected in portal tract areas peaked during the 3-5 postoperative days. CD34+/CD45+ positive cells could be detected. The expressions of CD34, c-kit and Thy-1.1 positive cells were rare in the WLTx and sham operation groups. Sry+ cells could be detected in portal tract areas and few Sry+/CD34+ and Sry+/Thy-1.1+cells were detected., Conclusion: In the PLTx group, the stem cells in the bone marrow were mobilized and stem cells in the liver were activated.

Objective: To investigate the inhibitory effects of interferon-alpha (IFN-alpha) on HCV replicon and evaluate the expressing levels of signal transducer and activator of transcription genes (STAT1 and STAT2) and IFN-alpha stimulated genes (ISGs) which may mediate the inhibitory effects of IFN-alpha on HCV., Methods: Firstly HCV replicon cell culture system was established by transfecting HCV replicon RNA transcribed in vitro into Huh7 cells and screening with G418. Secondly, the established HCV replicon cells were treated with various concentrations of IFN-alpha (0, 10, 25, 50, 100, 250, 500, 750, 1000, 2500, and 5000 IU/ml) for 72 h or treated with 1000 IU/ml of IFN-alpha for different lengths of time (0, 24, 48, 72, 96 h), then the levels of HCV RNA and NS5A protein in these cells were examined by semi-quantitative RT-PCR and Western blot respectively., Results: IFN-alpha could effectively inhibit HCV RNA replication. 10 IU/ml or 25 IU/ml of IFN-alpha could lead to about 68% and 75% of HCV-RNA reduction respectively. The cells treated with 1000 IU/ml IFN-alpha for 24 h or 96 h had about 75% and 88% of HCV RNA reduction compared with the cells of control, demonstrated that the inhibitory effects of IFN-alpha on HCV replicon are in dose and time dependent manners. The expressions of antiviral ISGs-PKR, 2'5'OAS, G1P3, ISG20 and ISGF3gamma are strongly induced by IFN-alpha., Conclusions: HCV replicons were sensitive to IFN-alpha treatment. The inhibitory effects of IFN-alpha on HCV RNA and NS5A are both dose and time dependents. PKR, 2'5'OAS, G1P3, ISG20 and ISGF3gamma may mediate the inhibitory effects of IFN-alpha on HCV replicon replication.

Objectives: To investigate the correlation between MAGE-A1 mRNA expression and genic demethylation in hepatoma cell lines., Methods: Total RNA and genomic DNA were prepared from 10 human hepatoma cell lines. MAGE-1 mRNA expression was determined with RT-PCR and the level of genome-wide demethylation was evaluated by enzyme digestion and Southern blot assay. The genomic DNA was digested by HpaII, then the promoter of MAGE-A1 gene was amplified with primers CDS21, EDP4 and CDS20. EDP4 and the PCR products were further hybridized with a probe to detect the methylation in the promoter of the MAGE-A1 gene. HLA-A locus was typed using SSP kit., Results: In cell lines QGY-7703, SMMC-7721, HLE, BEL-7402, BEL-7404 and BEL-7405, MAGE-A1 mRNA expression was positive and cell differentiation was moderate or low. In cell lines of HepG2 2.2.15, HepG2, QGY-7701 and Huh7, MAGE-A1 mRNA expression was negative and cell differentiation was well or moderate. The level of genomic demethylation in MAGE-A1 mRNA positive cell lines was much higher than that in MAGE-A1 mRNA negative cell lines (t = 2.896, P = 0.02). The methylation analysis showed that methylation in the promoters of MAGE-A1 gene of HepG2 2.2.15, HepG2, QGY-7701 and Huh7 was high, and that methylation in those of SMMC-7721, HLE, BEL-7402, BEL-7404, and BEL-7405 was low., Conclusion: The results suggest that MAGE-A1 mRNA expression in the human hepatoma cell lines is associated with genic hypomethylation.

Objective: To construct a series of recombinant replication competent plasmids of hepatitis B virus (HBV) full-length genome with varying mutations of precore and basic core promoter., Methods: The plasmid containing the entire HBV (1.2 copies) genome was used to construct objective plasmids. The objective competent vectors were constructed by molecular cloning and PCR-based site-directed mutagenesis in vitro and confirmed with sequence analysis. After introducing the plasmids into hepatocellular carcinoma cell line (Huh7) by calcium phosphate transfection, the culture supernatant was collected to detect HBsAg and HBV DNA to analyze viral replication and expression., Results: Ten competent vectors with varying precore and basic core promoter mutations were constructed. After transfecting into Huh7, the vectors replicated and secreted viral particles., Conclusions: Ten full-length competent HBV recombinants were obtained, which were harbouring varying mutations within precore and basic core promoter.

Background: To investigate natural history of hepatitis C virus (HCV) infection and related factors in China., Methods: Totally 41 women infected with HCV 10-15 years ago were evaluated in this study. Their clinical manifestations were recorded and sera tested for alamine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (gamma-GT), alkaline phosphatase (ALP), total bilirubin (TBIL), direct bilirubin (DBIL), total protein (TP), albumin (ALB), and alpha-fetoprotein (alpha-FP), as well as virus markers. The B-type ultrasound examination was performed on the liver, gall bladder, spleen and pancreas. For virus markers, HCV RNA was detected with fluorogenic-quantitative polymerase-chain-reaction, HBsAg and anti-HCV antibody, with enzyme-linked immunosorbent assay (ELISA)., Results: Ten to 15 years after infection, the infection was cleared spontaneously in 8 cases (19.51%); 73%(30/41) cases had signs at present, 32% (13/41) cases had mild ALT and/or AST elevation. On ultrasound examination, 83%(34/41) had mild degree 17%(7/41) moderate degree changes of the viral hepatitis., Conclusion: Women with HCV infection for 13 +/- 1 (10-15) years after transfusion had slight hepatic inflammation. A part of them had cleared the virus spontaneously.

Objectives: To investigate the expression of melanoma-associated antigen 1 (MAGE-1) in Chinese hepatocellular carcinoma (HCC) patients and to determine the existence and distribution of single nucleotide polymorphisms (SNP) of MAGE-1 gene., Methods: Total RNA was extracted from cancer tissues and adjacent tissues from 19 HCC patients and the expression of MAGE-1 mRNA was examined by using RT-PCR. The PCR products were sequenced to analysis the gene variation. Genomic DNA was extracted from cancer tissues, adjacent tissues and peripheral blood cells of 19 HCC patients, as well as from peripheral blood cells of 23 healthy donors. The PCR product of MAGE-1 DNA was sequenced to determine the existence and distribution of SNPs of MAGE-1 gene., Results: 9 of 19 (47.4%) tumor tissues and none of adjacent tissue from HCC patients expressed MAGE-1 mRNA. There were three kinds of gene variations of cDNA in 9 MAGE-1 mRNA positive patients. One type was named type I including 1 patient, which sequence is as same as that of the GenBank M77481. The other was named TGA type including 5 patients, which involved three nucleotide changes (C159T, A272G and G393A) and result in two amino acid changes (T32A and R72Q). Another one was named GTG type including 3 patients, which involved three nucleotide changes (A272G, C991T, A1125G) and result in only one amino acid changes (T32A). According to the analysis of genomic DNA, above three types were not specific mutations of tumor tissue, but were SNPs. These SNPs types were distributed in HCC patients and normal donors with the frequencies of 26.3% (5/19) and 60.9% (14/23) for type I, 57.9% (11/19) and 47.8% (11/23) for TGA type, and 21.1% (4/19) and 21.7% (5/23) for GTG type, respectively. The sequences of two new SNPs had been deposited in GenBank with the accession numbers of AF463515 and AY148486. In male population, the distributions of SNPs were not correlated to HCC suffering or MAGE-1 expression. Several new HLA-restricted epitopes were probably resulted from SNPs existing. The three-dimensional models of MAGE-1 proteins of type I and TGA type was established by using computer software., Conclusion: The expression rate of MAGE-1 gene in Chinese HCC patients is high. Three SNP types of MAGE-1 gene exist in Chinese population. The three-dimensional models of MAGE-1 proteins were obtained by computer processing. These results will be helpful to developing MAGE-1 peptide-vaccine for HCC immunotherapy.

Objective: To investigate the correlation between impaired non-viral specific immune function of dendritic cell (DC) and viral clearance and cytotoxic T lymphocyte (CTL) response to HBV or HCV in patients with HBV and HCV coinfection., Methods: Twenty-five patients with HBV and HCV coinfection were investigated in this study. In 1994 and 2002, biochemical and virological markers and quantitative serum HBV DNA and HCV RNA levels were detected in these patients. According to the virus clearance status, these patients were divided into 4 groups: 14 patients with both HBV and HCV clearance (Group A), 6 patients with HCV clearance only (Group B), 3 patients with HBV clearance only (Group C), and 2 patients with persistent infection of HBV and HCV (Group D). Phenotypes and immune functions of monocyte-derived DCs were compared between these groups. 51Cr release assay were used to measure CTL response to epitopes derived from HBV, HCV or influenza virus (as positive control) in HLA-A2+ patients., Results: Impaired non-viral specific immune functions of DCs were observed in group B, C and D compared with group A and normal donors (Group N). These impaired functions included CD86 decreasing expression and lower capacity to stimulating allogenic T cells and uptaking antigen. The specific CTL response to HBV- and HCV-derived peptides could be induced in group A (12/12). The specific CTL response to HBV-derived peptides or to HCV-derived peptides could be induced in group C (3/3) or B (5/5), respectively. But the specific CTL response to both of two HBV-derived peptides or two HCV-derived peptides could not be induced in group C (0/3) or B (0/5), respectively. And no CTL response to HBV or HCV-derived peptides could be induced in groups D (0/1) and N (0/4)., Conclusion: 1. The results suggest that specific CTL response to HBV or HCV play a vital role in the viral clearance. 2. The DCs with impaired non-viral specific immune functions exist in chronic patients with HBV and/or HCV infection, but do not interfere with clearance and CTL response to HBV or HCV. It is reasonable to speculate that impaired functions of DCs result from viral infection.

Objective: To understand HBV serotypes and genotypes epidemiology in a northern city and a southern city in China., Methods: Using polymerase chain reaction (PCR) and direct sequencing of HBV DNA PCR products, the serotypes and genotypes of HBV in 530 from HBsAg positive samples. The enrolled patients were from Harbin, a northern city and Lianjiang, a southern city in China., Results: Comparison of the serotypes and genotypes of HBV between Harbin and Lianjiang showed that adrq+ was the most predominant hepatitis B virus serotype in both Harbin and Lianjiang (87.2% and 73.5%,respectively), adw2 was the next (12.0% and 25.7%, respectively); genotype C was the most frequent in Harbin and Lianjiang (87.8% and 73.2%, respectively), and genotype B was the next (12.2% and 26.1%, respectively) only 1 patient was infected by genotype D, and 1 patient was found to be co-infected by genotype B and C in Lianjiang., Conclusion: The results suggest that the percentage of HBV serotypes and genotypes between Harbin and Lianjiang was significantly different (P less than 0.001), but the main HBV serotype and genotype of the two cities were similar.

Objective: To observe the differentiation of bone marrow stem cells in rat hepatic fibrogenesis environment into hepatocytes., Methods: Rat hepatic fibrosis was induced by subcutaneous injection of CCl4. Bone marrow stem cells with Thy positive, CD3 and CD45RA negative were enriched from the bone marrow by fluorescence-activated cell sorting. The bone marrow stem cells were labeled with PKH26-GL, and then autotransplanted. After six weeks, albumin, ck8 and a-smooth muscle actin expression were determined by immunocytochemistry., Results: The PKH26-GL labeled cells expressed albumin and ck8, but did not express a-smooth muscle actin in hepatic fibrogenesis environment., Conclusion: Bone marrow stem cells in hepatic fibrogenesis environment can differentiate into hepatocytes, but can't differentiate into myofibroblasts

Objective: To study whether dendritic cells (DCs) derived from the peripheral blood in chronic hepatitis B patients can induce specific T cell immune response., Methods: (1)The subjects were divided into 3 groups: chronic hepatitis B group (CHB), acute hepatitis B group (AHB), and normal donor group (ND). The peripheral blood mononuclear cells (PBMCs) isolated from those subjects were stimulated with HBcAg 18 to 27 CTL epitope peptide, and intracellular cytokine staining (ICCS) was used for detecting IFN-gamma, IL-2 and TNF-alpha produced by CD8+ T cell. (2) DCs generated from PBMCs were pulsed with HBcAg 18 to 27 CTL epitope peptide, then were cocultured with autologous lymphocytes for 10 days to induce antigen-specific T cell, which was assessed by ICCS and cytotoxic assay., Results: (1) The memory effect of the PBMCs from AHB group to HBcAg 18 to 27 CTL epitope peptide was stronger than that from CHB or ND group (t=2.508-3.305, P<0.05). (2)After lymphocytes were cocultured with DC treated with HBcAg 18 to 27 CTL epitope peptide, antigen-specific T cell effect was induced. And the killing rates were (57.0+/-23.0)%, (49.5+/-20.2)%, (21.8+/-12.9)% at the effector/target of 30:1, 10:1, 3:1, which were higher than that in control group., Conclusions: The memory T cells against HBV antigen lacks in CHB patients. DCs from CHB patients pulsed with HBcAg 18 to 27 epitope peptide can induce HBV antigen-specific T cell, which can kill specific target cells and produce cytokines involved in virus clearance.

Aim: To prepare fluorescein sodium (FS) cationic liposomes and investigate the influence of cationic lipid (DC-chol) and polyethylene glycol (PEG) with different molecule weight (MW) on cationic liposome incorporation efficiency, cellular delivery and fluidity of liposome membrane., Methods: Using FS as a model material for encapsulation, the liposomes were prepared and separated (by sephadex G-50 1 cm x 20 cm column), and the liposome incorporation efficiencies was measured. The interaction between the FS and cationic liposomes was investigated by measuring the change of fluorescent spectrum. The cellular uptake of different liposome forms by choosing HepG2 2.2.15 as an in vitro cell culture assay model, and the influence of PEG on the fluidity of liposome membrane with the technique of fluorescence polarization were investigated., Results: Cationic lipid and different PEGs showed great effects on increasing liposome incorporation efficiency (from 0.64% to 86.57%), cellular uptake (from 2.18% to 48.46%) and fluidity of liposome membrane. The effect of PEG was MW dependent, and with the increase of MW, the incorporation efficiency and transfection was improved, and the fluidity of liposome membrane increased., Conclusion: Addition of cationic lipid and high MW PEG into cationic liposomes can enhance the cellular delivery and fluidity of cationic liposomes. Also, they can improve the incorporation efficiency of cationic liposomes.

Objective: To investigate the influences of different lipids on cellular delivery and fluidity of liposome membrane of cationic liposome., Methods: The 3 beta [N-(N',N'-dimethylaminoethane) carbamoyl] cholesterol (DC-chol), a cationic lipid was synthesized, which is approved by FDA. Using fluorescein sodium (SF) as a model material, dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE) and cholesterol (ch) as help lipids, the DPPC/ch/DC-chol cationic liposomes, DOPE/ch/DC-chol cationic liposome and soybean-derived sterylglucoside (SG) or polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) modified cationic liposomes were prepared, respectively. The neutral liposome DPPC/ch was also prepared. HepG2 2.2.15 was selected as an in vitro cell culture assay model to investigate the cellular delivery of different liposomes. The influences of different lipids on the fluidity of liposome membranes were investigated with the technique of fluorescence polarization., Results: It is showed that the DC-chol has significant influences on the incorporation efficiencies (from 0.64% to 74.84%), cellular delivery (from 1.83% to 32.29%) and the fluidity of liposome membrane; SG can significantly increase the incorporation efficiency (from 52.60% to 63.37%), cellular delivery (from 20.18% to 47.48%) and fluidity of liposome membrane; PEG-DSPE can enhance the cellular delivery of cationic liposome (from 20.18% to 28.17%)., Conclusion: DC-chol and SG can significantly increase the cellular delivery and fluidity of liposome membrane, PEG-DSPE can enhance the cellular delivery of cationic liposome.

Background: To investigate sequence of the complete E region of genotype 2a hepatitis C virus genome and to set up the basis for the further study on biological functions of envelope proteins of HCV., Methods: Two cDNA fragments of 770bp, 1,100bp long, from serum of a patients infected HCV, were amplified by reverse transcription nested polymerase chain reaction (RT-nPCR). After being digested with two restriction endonucleases, the two fragments were cloned into JM105 respectively, then sequenced., Results: The two corresponding nucleotide sequences were obtained and ligated. The complete nucleotide sequences of envelope region and corresponding deduced amino acid sequences were compared with those of HCV-1, HC-C2, HCV-BK, HC-J6 and HC-J8. The homology of nucleotide sequence of the isolated strains was 60.5%, 60.1%, 59.7%, 87.8% and 67.5% respectively; the amino acid sequence homology to the isolated strains was found to be 67?3%, 66.4%, 65.0%, 87.8%, 79.0%, respectively., Conclusions: The results indicate that the isolated strain (HCV-JS) belongs to genotype 2a, but there is heterogeneity between HCV-JS and HC-J6 strain.

Objective: (1) To investigate the expression and gene diversity of the 7 major cancer/testis (CT) antigens, MAGE-1, MAGE-3, MAGE-4, MAGE-10, NY-ESO-1, SSX-2 and SCP-1, in hepatocellular carcinoma (HCC). (2) To analyze the correlations between the clinical characters and CT antigens' expression., Methods: The cancer and para-cancer tissues were collected from 30 HCC patients. The mRNAs of seven CT antigens were detected by reverse transcription-polymerase chain reaction (RT-PCR) with the specific primers. The PCR products were sequenced to analyze the CT genes., Results: The MAGE-1, MAGE-3, MAGE-4, MAGE-10, NY-ESO-1, SSX-2 and SCP-1 were expressed in 66.7%, 70.0%, 20.0%, 36.7%, 40.0%, 33.3% and 33.3% of the tumor tissues from HCC patients respectively, however, they were not expressed in the para-cancer tissues. Among the 30 patients investigated, 90.0% expressed one CT gene at least, 70.0% expressed two CT genes, and 53.3% expressed three CT genes of the seven CT genes. The coding genes of these CT antigens were highly conserved between in Chinese patients and patients abroad. There were discernible correlations between alpha-fetoprotein level and MAGE-10 or SCP-1 expression level, as well as between average age and MAGE-3 or SSX-2 expression levels (P<0.05)., Conclusions: With a highly conserved coding gene, seven CT antigens were expressed in 20.0% - 70.0% of Chinese HCC patients. CT antigens' expression had correlations with some clinical characters.

Objective: To investigate the variations of HCV core and E2 region epitopes during transfusion of activated immune cells., Methods: Four patients receiving transfusion of activated immune cells were under continuously observation. HCV titers were measured by quantitive PCR. HCV core and E2 regions were cloned and sequences were analyzed by computer software., Results: During the follow-up the serum ALT levels and the HCV virus titers fluctuated greatly in each of these persons. After transfusion, no significant variations were observed in HCV core and E2 coding regions., Conclusions: The alteration of host immune attacks could induce the fluctuations of HCV load without any mutations in the currently observed coding genes of the epitopes.

Objective: To obtain very end full-length cDNA of hepatitis C virus (HCV) 5' untranslated region (5' UTR), and analyse its primary and secondary structure., Methods: By reverse transcription-nested polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP), a patient infected with genotype 2a HCV was found. Total RNA isolated from the serum as template, the cDNA of 5' noncoding region was amplified using rapid amplification of cDNA ends methods (RACE), the fragments were recombined by A-T clone strategy, the recombinants were confirmed by RFLP and PCR then sequenced. Secondary structures were analysed by RNA draw., Results: Very end full-length cDNA of 2a genotype HCV 5' UTR was obtained by RACE. In five clones obtained, three contained full-length 5' UTR cDNA, and A21G, G170A, T222C, T247C, C339T substitutions were found compared with HC-J6. he homologies with HCV-1,HC-J6,HC-C2, HC-J8 were 93.6%-94.4%, 92.1%-93.0%, 98.8%-99.7%, 96.2%-96.5%, respectively; however, the substitutions did not alter the secondary structure. Two out of five clones were deleted to have 53 and 144 bases at 5' terminus of HCV 5' UTR, respectively., Conclusions: RACE is rapid and effective, works well to obtain very end of virus genome. With that, Authors obtained full-length cDNA of genotype 2a of HCV 5' UTR. There are genes deleted at 5' terminus circulated in hepatitis C patients.

Objective: To compare the phenotype and function of dendritic cells (DCs) of patients with chronic hepatitis Beta with those of DCs from healthy persons., Method: Peripheral blood monocytes (PBMCs) were isolated from 10 patients with chronic hepatitis Beta and 10 healthy blood donors. DCs were generated by culturing PBMCs in 1 000 U/ml granulocyte-macrophage stimulating factor (GM-CSF) and 500 U/ml IL-4 with AIM-V in vitro. DCs and supernatant were collected 3, 5, 7, 9, 11, 13 and 15 days after culture. TNF- alpha 50 ng/ml was added into the culture of DCs from patients with hepatitis Beta and DCs were collected 48 hours after. FACS was used to detect the phenotype. The changes of cell phenotype on different days with or without TNF-alpha were tested by immunolabelling and flow cytometry analysis. IL-12 ELISA kit was applied to investigate IL-12 secretion of DCs. To perform mixed leucocyte reaction (MLR) test, DCs were cocultured with allogeneic T cells at different stimulatory ratio., Results: The expression of CD83 was higher in the group with TNF-alpha than in the group without TNF-alpha. The positive rates of CD80 and CD54 were not different between the two groups. DCs from patients and normal donors with typical morphology were harvested successfully in vitro. The IL-12 level was 103.93 +/- 12.59 pg/ml in the group with TNF-alpha, higher than that in the group without TNF-alpha (11.45 +/- 2.58 pg/ml, P < 0.05). MLR test showed that when the stimulatory ratio of allogeneic T cells was 1 : 20, 1 : 50, and 1 : 100 respectively, the results were 40 835 +/- 3 480, 37 525 +/- 6 398, and 31 814 +/- 5 521 respectively in group of patient; and 49 097 +/- 2 273, 43 049 +/- 2 833, and 39 091 +/- 6 469 respectively in the group of healthy blood donors., Conclusion: 1.DCs of patients and of healthy persons can be cultured success fully with AIM-V when induced by GM-CSF, IL-4 and TNF-alpha in vitro. 2. Functions of DCs from patients are lower than those from healthy donors. However, they are not different in amount and morphology.

Objective: To explore the possibility of MAGE-4 gene encoding protein used as a target for immunotherapy in HCC patients., Methods: The expression of MAGE-4 gene in tumor tissues and tumor adjacent non-HCC liver tissues was examined by the RT-PCR method. The relationship between positive expression rate of MAGE-4 gene and other clinical and lab data including AFP, AFU, anti-HCV, HBsAg, AFP mRNA, and the diameter of the tumors in HCC patients was also determined., Results: The positive expression rate of MAGE-4 gene was significantly higher in the tumor than in tumor surrounding tissues (38.7% vs 0%, P<0.01), while the positive expression rate of MAGE-4 gene had no relationship with the clinical and lab data (P>0.05)., Conclusions: The high frequency of MAGE-4 gene expression in HCC suggests the possibility of MAGE-4 gene encoding protein as a target for immunotherapy in HCC patients, but the expression has no relationship with the tumor metastasis and the recurrence of HCC.