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Gallbladder cancer is a high malignancy which is predisposed to invade adjacent organs and have lymph node metastasis. Gallbladder cancer is not sensitive to radiotherapy or chemotherapy with the worst prognosis among biliary tract cancers. At present, radical resection is the only possible method to cure gallbladder cancer. However, there are still many controversies about the surgical strategies, the extent of liver resection and lymph node dissection, and the treatment of incidental gallbladder cancer. In addition, under the background of the great success of immunotherapy and targeted therapy in a variety of solid tumors, it is also a question worthy of further considerations that whether the status of surgery in the treatment of advanced gallbladder cancer will be changed in the near future.

Objective To analyze the prevalence of peripheral arterial disease (PAD) among residents in Shenzhen and explore its relationship with different obesity statuses, to provide data support for understanding the prevalence of PAD in China and a basis for formulating prevention and control policies to prevent PAD. Methods From April 2021 to December 2021, a multi-stage stratified random sampling method was used to select permanent residents aged 18 years or older from 54 community neighborhood committees in Shenzhen as the study population. Data were collected using questionnaires, physical examinations, and laboratory tests. A generalized additive model was fitted and plotted using the R package, and logistic regression models were used to analyze the association between different obesity status groups and PAD. Results Among the 6 230 study subjects, the prevalence of PAD in the study population was 6.21%, and the prevalence of PAD increased with age, with the lowest rate of 4.67% in the 18-<25 age group and the highest rate of 33.33% in the ≥85 age group. In addition, the prevalence of PAD increased with increasing body mass index (BMI) or waist circumference. Analysis of multifactorial logistic regression models showed that both overall obesity (OO) classified by BMI and central obesity (CO) classified by waist circumference may increase the risk of PAD (OO statistic value OR=2.63, 95%CI: 1.97-3.50; CO statistic value OR=2.32, 95%CI:1.79-2.99). When combining BMI and waist circumference classification, compared to those who were neither OO nor CO, individuals who were both OO and CO had a significantly higher risk of PAD (OR=2.61, 95%CI: 1.99-3.44). Conclusions The prevalence of PAD among adults in Shenzhen is not optimistic, and the elderly need additional attention. Both OO based on BMI and CO based on waist circumference are independent risk factors for PAD. The joint use of BMI and waist circumference grouping helps to better classify and manage people at risk of PAD. [ABSTRACT FROM AUTHOR]

The causes for male sexual orientation are complicated, which have not yet been clarified. Recent years have witnessed fruitful progress in the field of biology, while the impact of environment has received little attention. Adverse childhood experiences (ACEs), identified as a special environment in the early stage of development, can affect the individual phenotype by DNA methylation. Given the relationships among male sexual orientation, ACEs, and DNA methylation, as well as based on the existing theory, this article proposes the model "ACEs-DNA methylation-male sexual orientation"from the perspective of environment and epigenetics, aiming to provide a theoretical basis for future research.

In recent years, Hepatitis B virus (HBV) persistent infection mouse model with recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome (rAAV8-1.3HBV) is concerned. We studied and compared the efficacy among HBV persistent infection mice models by other serotypes except AAV8. First, we prepared and purified five viruses: rAAV1-1.3HBV, rAAV2-1.3HBV, rAAV5-1.3HBV, rAAV8-1.3HBV and rAAV9-1.3HBV. Then we injected each virus into 3 C57BL/6J mice with the dose of lx 1011 vg (Viral genome, vg) per mouse. We detected HBsAg and HBeAg in sera by enzyme-linked immunosorbent assay (ELISA) at different time points post injection. We killed mice 8 weeks post injection and took blood and livers for assay. We detected copies of HBV DNA by real-time quantitative PCR in sera and livers. Meantime, we detected HBcAg in the livers of mice by immunohistochemistry and further performed pathology analysis of these livers. The five groups of mice, HBeAg and HBsAg expression sustained 8 weeks in serological detection and HBV DNA was both detected in sera and livers at the time of 8 weeks post injection. HBeAg, HBsAg, HBV DNA copies expression levels in descending order were AAV8>AAV9>AAV1>AAV5>AAV2. HBcAg expression was detected in livers as well. Varied degrees of liver damage were shown in five groups of mice. This study provides more alternative AAV vector species to establish a persistent infection with hepatitis B model.

Objective: To investigate the current status of contact tracing among pregnant women infected with syphilis and to analyze the associated factors from patients' perspective., Methods: Pregnant women who aged 18 years old, receiving prenatal care services in Shenzhen, diagnosed with syphilis according to national diagnostic criteria (WS 273-2007) from 2008 to 2011 were recruited and the total number was 3 551. Information of both pregnant women (including demographic information, laboratory results, syphilis diagnosis, and personal life history) and their partners (including results of partner tracing and laboratory examination) were collected with structured questionnaire by face-to-face interview. Multivariate non-conditional logistic regression model was applied to analyze the factors associated with partners' contract tracing. Odds ratio (OR) and its 95% confidential interval (95%CI) were calculated., Results: The average age was 28.72 among recruited 3 551 syphilis-infected pregnant women, with standard deviation of 5.21 and range of 18 to 40. Totally 2 550 partners attended antenatal clinics and received syphilis examination, with a contact tracing rate of 71.81%. The OR(95%CI) was 1.70(1.26-2.30) for pregnant women with college or above education when comparing with those with senior high school or below education. The OR(95%CI) was 0.57(0.45-0.71) for those unmarried or divorce when comparing with those married. The OR (95% CI) was 0.73(0.56-0.94) and 0.65(0.53-0.81) for those screened in 28-36 gestational weeks and those screened in >36 gestational weeks or right before delivery separately, when comparing with those screened in ≤27 gestational weeks. The OR (95% CI) was 1.45(1.14-1.84) for those having received sufficient treatment before pregnancy when comparing with those diagnosed with latent syphilis. The OR(95%CI) was 0.31 (0.24-0.39) for those having no treatment or not standardized treatment when comparing with those having standardized penicillin treatment. The OR(95%CI) was 0.53 (0.43-0.65) for those terminating the pregnancy when comparing with those giving birth to a baby. The OR(95%CI) was 1.24 (1.05-1.46) for those having two or more love histories when comparing with those having only one love history. The OR(95%CI) was 4.74 (3.54-6.35) for those intending to disclose the serostatus to their partner when comparing with those showing unwillingness to disclose the serostatus., Conclusion: The successful rate of contact tracing among pregnant women infected with syphilis was high. The implementation of contact tracing may be affected by many factors, including patients' education levels, marital status, gestational weeks of screening, stage of syphilis, treatment status, outcome of pregnancy, number of love histories, and willingness of serostatus disclosure.

Objective: To examine the differential expression of miRNAs in colon cancer tissues and matched tumor adjacent tissues., Methods: Differential expression of microRNAs in twenty paired human colon cancer tissues and adjacent tissues were detected by QPCR. miRNAs with significantly differential expression (fold change >2.4 and P<0.01) were screened to analyze their accumulation by Cluster analysis. Thus correlation of miRNA and other colon cancer-associated proteins was examined., Results: Expressions of 17 miRNAs were significantly reduced in colon cancer tissues. Cluster analysis showed that miR763-3, miR451 and miR99a had similar expression. Plasma CK20 level was negatively correlated with miR100 (r=-0.948), miR152a-5p (r=-0.948), miR125b (r=-0.949), miR145 (r=-0.949) and miR145*(r=-0.949) (all P<0.05)., Conclusion: miR145 and other 16 miRNAs may be used as diagnostic molecular markers of colon cancer, and miR100, miR125a-5p, miR125b, miR145 and miR145* may become the molecular markers of colon cancer lymph node metastasis.

Objective: To identify dysregulated microRNA(miRNA) that can act as novel biomarker for colorectal cancer screening., Methods: Real time-polymerase chain reaction was used to detect the expression profile of miRNA in tumor and paired normal tissues, and the significant dysregulated miRNA was examined by non-paired t-test. Receiver operating characteristic(ROC) curve was performed to evaluate the specificity and sensitivity of miR-363 and miR-490-5p as biomarkers to discriminate colorectal cancer patients from normal person., Results: Seven-three dysregulated miRNAs were found in male samples, of which 6 miRNAs were up-regulated and other 67 miRNAs were down-regulated, while 42 dysregulated miRNAs were found in female samples, of which 5 were up-regulated and 37 were down-regulated in tumor tissues compared with normal tissues. Among above dysregulated miRNAs, 33 miRNAs had significant differences, besides, dysregulated expression level of 10 of 33 miRNAs was over 5 folds in male and female as well as mixed samples., Conclusions: The expression pattern in female is different from that in male. miR-363 and miR-490-5p possess the potential in screening colorectal cancer patients from healthy people.

Activities of 58 miRNAs for BHK21, HEK293, and Vero cell lines were screened using the high-throughput miRNA activity profiling method. miR-206 activity was found specifically high in BHK21. Considering miR-206 was recognized as a muscle-specific miRNA, we further detected the expression and activity level of miR-206 in BHK21 cells, with myoblast cells C2C12 as positive control and HEK293 cells as negative control. Then, we induced BHK21 by culturing with medium containing 2% horse serum (HS) and tested expression level of slow skeletal myosin heavy chain (MHC), activity and expression levels of miR-206, and expression level of Connexin43 (Cx43) which was reported negatively regulated by miR-206. Results demonstrated that activity and expression levels of miR-206 were both higher in BHK21 cells than in C2C12 cells. After induction of HS, MHC expression level was increased in BHK21 cells. The activity and expression levels of miR-206 were further enhanced. The Cx43 expression level was decreased. These results suggested that BHK21 had the characters of myoblast cells. In conclusion, we firstly discovered that miR-206 activity was specifically high in BHK21 cells, suggesting that BHK21 cells were derived from interstitial cells other than parenchyma cells of kidney from miRNA point of view. Our study also indicated that BHK21 cells were able to be used as models in vitro for research of miR-206 function.

We evaluated the anti-HBV effects of nucleotide analogues, Entecavir (ETV) and Lamivudine (LAM) targeting mouse model of HBV persistent infection with recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome (rAAV8-1.3HBV). Ninety percent (27 of 30 mice) of rAAVS-treated mice were chosen as mouse model. Four groups were orally administrated with different doses of ETV (1 mg/(kgd) or 0.1 mg/(kgd)) and LAM (500 mg/(kgd) or 100 mg/(kgd)) once a day for 10 days. The other two groups were set as normal saline treated and untreated control. We detected the levels of HBV DNA, HBeAg and HBsAg in sera at different time. Results indicate that HBV DNA level decreased significantly (P < 0.05) in drug-treated groups compared with normal saline group after drug administration. Fifteen days after the drug withdrawal, HBV DNA level rebounded back obviously (P < 0.05) in groups with low doses of ETV and LAM. However, there was no apparent change of HBeAg and HBsAg in the whole process among all groups. These results showed that our model could reflect the anti-viral effect of nucleotide analogues. This model can be a useful and convenient tool for anti-HBV drug discovery.

To establish an orthotopic transplant mouse model of hepatocellular carcinoma (HCC) labeled with secretary luciferase and to study its response to anti-tumor treatment with interferon-beta gene therapy. We labeled the murine hepatoma Hepal-6 cells with secretary Gaussia princeps luciferase (Gluc), and then injected Gluc labeled Hepal-6 cells intrasplenically in C57BL/6 mice. We monitored blood Glue to evaluate the tumor development and anti-tumor effects of hydrodynamic injection with interferon-beta expressing plasmid. We successfully established the orthotopic mouse model of HCC by intrasplenic injection of Glue labeled Hepal-6 cells. The Glue blood assay could reflect the amount of cancer cells in vivo, tumor progression, as well as anti-tumor effect of interferon-beta gene therapy. In conclusion, Gluc labeled orthotopic transplant mouse model of HCC can ex vivo real-time monitor the tumor development and tumor response to treatments.

We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFalpha activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFalpha's cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.

We developed a method for monitoring of miRNA activity in live cells by a secreted luciferase gene based plasmid sensor named as Gsensor. Firstly, we constructed pAAV2neo-Gluc-MCS-polyA as "empty Gsensor", which contained multiple cloning sites (MCS) for miRNA target inserted. To detect miR142-3p activity, miR142-3p Gsensor and miR142-3p Gsensor-3 were constructed by inserting one or three complementary miR142-3p targets into pAAV2neo-Gluc-MCS-ployA. Subsequently, miR142-3p Gsensor and miR142-3p Gsensor-3 were respectively transfected into U937 cells and Gluc activity was assayed in the supernatant 48 h post transfection. Results showed that both of them effectively indicated miR142-3p activity of inhibiting Gluc expression compared with empty Gsensor. Simultaneously, miR142-3p Gsensor also demonstrated the inhibition of miR142-3p activity by Anti-miR142 when they were cotransfected into U937 cells. This implied one copy of miRNA target in Gsensor was sensitive enough for investigation of miRNA activity. We further analyzed factors affecting Gsensor function including time and dose, and found that miR142-3p activity sensed by miR142-3p Gsensor rose within 48 h post transfection and approached stable thereafter. Transfected dose varying among 0.001-0.05 pg/cell had little effect on its function. Using miR142-3p Gsensor, we further detected miR142-3p activity in HEK293, U937, K562, SP2/0 and P815 cells. Results suggested that miR142-3p activity was high in U937, K562, SP2/0 and P815 cells and almost negative in HEK293. miR142-3p activity was positively correlated with its relative copies in HEK293, U937 and K562 detected by QRT-PCR. In conclusion, Gsensor proved to be an effective tool for monitoring of miRNA activity in live cells, and provide a new method for monitoring miRNA activity in vitro.

We developed a scalable AAV5/5 vector packaging system by using replication competent recombinant herpes simplex type 1 virus as helper virus. The fragment containing rep and cap genes of AAV5 was inserted into the non-necessary gene (UL2) of HSV1 genome, resulting in the helper virus rHSV1-rep5cap5. An AAV5/5 vector pAAV5neo carrying two AAV5 ITRs was constructed by inserting a neo gene expression cassette into the plasmid backbone of pAV5CMV-GFP. pAAV5neo-EGFP was constructed by inserting EGFP gene into pAAV5neo. BHK21 cell was transfected with pAAV5neo-EGFP and cultured in the presence of G418. EGFP expression positive monoclonal cells were picked up, and one that produced rAAV5/5-EGFP with the highest efficiency under the help of rHSV1-rep5cap5 was chosen as the production cell line named as C020. rAAV5/5-EGFP was produced by infecting C020 cells with rHSV1-rep5cap5, and crudely purified by our previous method of 'chloroform treatment-PEG8000/NaCl precipitation- chloroform extract'. rAAV5/5-EGFP preparation with high purity was obtained by ultrafiltration with molecular weight cut-off value of 100 kDa. SDS-PAGE stained with Coomassie brilliant blue R250 showed clearly specific pattern of three bands of AAV capsid proteins. rAAV5/5-EGFP was also assayed using negative stain transmission electron microscopy and the majority of the virus particles were found solid. About 30% green fluorescent cells could be seen after infecting HEK293 cells with rAAV5/5-EGFP 24 h at 1 x 10(5) vg/cell. In conclusion, we have established an efficient AAV5/5 vector production system and could produce recombinant AAV5/5 virus in large amounts for gene therapy research.

In order to get soluble TNF receptor (sTNFR) II with good neutralizing activity against TNFalpha, we constructed the fusion gene sTNFRII-gAD, which encoded human sTNFR II and the globular domain of adiponectin (gAD), and then expressed it in mammalian cells and analyzed its anti-TNFalpha activity. First, sTNFRII cDNA was obtained by RT-PCR from the total RNA of human peripheral blood lymphocytes, and fused in frame with gAD gene. Then, the fusion gene sTNFRII-gAD was cloned into the expression vector pAAV2neo to result in the plasmid pAAV2neo-sTNFRII-gAD. By immunofluorescent staining with monoclonal antibody either against TNFRII or against adiponectin, we demonstrated that the pAAV2neo-s7NFRII-gAD-transiently-transfected BHK-21S cells were positive. To obtain G418-resistant BHK-21S/pAAV2neo-sTNFRII-gAD cells, we cultured the transfected BHK-21S cells above in 10% FBS containing DMEM media with 800 microg/mL G418 for 15 days, and changed the serum-containing culture media to a serum-free chemically defined media so as to change the cells culturing style from adhesion to suspension. 24 hours later, we harvested the supernatant of the culture for sTNFRII-gAD fusion protein characterization and anti-TNFalpha activity analysis. With monoclonal antibody either against TNFRII or against adiponectin, the Western blotting analysis showed that the sTNFRII-gAD fusion protein was expressed and existed as monomer, trimer and multimer forms in the supernatant. The bioactivity assay demonstrated that the sTNFRII-gAD fusion protein had the ability to neutralize TNFalpha so as to inhibit the cytotoxicity of TNFalpha on L929 cells. Put together, this study has laid the groundwork for large-scale preparation of sTNFRII-gAD fusion protein.

We chose Gaussia luciferase (Gluc), a secreted luciferase gene as reporter to real-time detect and dynamically monitor hydrodynamic injection gene expression. First, we constructed an expression vector pAAV2neo-Gluc. Then Huh7 and HepG2 cells were transfected with pAAV2neo-Gluc and the activity of Gluc in the supernatant and cell lysates were assayed. Results showed that the Gluc activity in the supernatant was about 100 higher than that in cell lysates, indicating the expressed Gluc existing mainly as a secreted form as reported. Live bioluminescence imaging of mice hydrodynamic injected pAAV2neo-Gluc showed whole body distribution, while the pAAV2neo-Fluc primarily located in the liver. Then we injected different doses of pAAV2neo-Gluc into mice by tail-vein hydrodynamic injection, took minor amount of blood from mice tails at different time points and measured the luciferase activity to investigate dynamic changes of Gluc expression and secretion in vivo. The results suggested that the time courses of Gluc expression were highly consistent among each dose groups. The luciferase activity in blood could be detected as early as 2 h after injection, reached the peak at about 10 h and gradually decreased from then on. The expression level of Glue was positively correlated with the dose of injected plasmid DNA. To further detect the assay sensitivity of the ex vivo Gluc measurement method, we investigated three additional groups of mice injected with lower doses of 0.001 microg, 0.01 microg and 0.1 microg pAAV2neo-Gluc respectively. Results revealed that activity of Gluc in blood could be detected even at dose as low as 0.001 microg DNA, suggesting the assay sensitivity was extremely high. In conclusion, a real-time ex vivo detection method of dynamically monitoring of gene expression in vivo by hydrodynamic injection can be a valuable means for the study of gene expression regulation in vivo.

A fusion gene called Ig-BDNF, in which brain-derived neurotrophic factor cDNA fused to the 3' end of signal peptide of Ig coding sequence, was constructed by PCR, digested and subcloned into shuttle plasmid pSNAV to obtain a recombinant plasmid pSNAV-Ig-BDNF. Then the plasmid encoding fusion protein was transfected into 293 cell lines and the stably transfected clones were selected with neomycin. AAV1 containing Ig-BDNF fusion gene vectors were obtained by super-infection by Herpes virus. The resultant adeno-associated virus vectors AAV-Ig-BDNF were confirmed by PCR, Western blotting and a sandwich enzyme-linked immunosorbent assay (ELISA) after infection of 293 cell lines. The results indicated that AAV-Ig-BDNF contained the target gene, and infected cells and produced the fusion protein into the supernatant. The content of BDNF in medium per 5x104 cells over a 24 h incubation period reached 1000 pg/mL. With the help of non-replicative adenovirus during AAV-Ig-BDNF infection, the expression of BDNF increased 7-8 fold, and the enhancement of BDNF gene expression was observed in a concentration-dependent manner. These results suggested that a functional AAV-Ig-BDNF was successfully constructed and it offers basis for further study for gene therapy of neural degeneration diseases.

An investigation was made on the fish resources and fish habitats of Hepu Dugong State Nature Reserve and its adjacent waters from June 2003 to May 2004. The local fish markets were surveyed, and fishermen and fishmongers were interviewed to get fishery information. A total of 57 fish species belonging to 41 families and 14 orders were recorded, among which, 55 were bony fish, and 2 were elasmobranch. There were 41 fish species belonging to warm-water fish, and 37 fish species belonging to benthonic fish. The faunal characteristic was mainly of Northern Pacific and Indo-Pacific fauna. Devastating fishing and environmental pollution were the most serious threats to the local fishery resources, and some countermeasures were proposed for conservation and management of the resources.

From May 2004 to February 2005, an investigation was made on the avian communities in four typical habitats, i.e., village and farmland, urban woodlot, floodplain, and riverine wetland in Wuhu City, with the related diversity indices analyzed. A total of 117 species belonging to 15 orders and 31 families was recorded. The avian species and G-F index in village and farmland, urban woodlot, and floodplain were higher in summer than in winter, while the species number, Shannon-Wiener index and Pielou index in riverine wetland were significantly higher in winter than in summer. The avian species and G-F index in urban woodlot were the highest, and the Shannon-Wiener index and Pielou index in floodplain were higher than those in other three habitats. The highest density was observed in village and farmland in summer and in urban woodlot in winter, being 31.1140 and 42.9160 ind x hm(-2), respectively. The avian species, density, Shannon-Wiener index and G-F index in riverine wetland were the lowest. Avian diversity could be enhanced by increasing spatial heterogeneity and reducing human disturbance.

In order to reveal the reasons of the dramatic decline of wild Chinese alligator population, an investigation on its habitat status was made across the National Natural Conservation Regions of Chinese alligator in south Anhui Province from 2002 to 2003. Water samples from 21 sites were collected, and their heavy metals (Cu, Zn, Cd and Pb) content and pH value were measured. The results showed that the pH value was 7.84 +/- 0.62, with no significant difference between the waters with and without Chinese alligator, but with significant difference (P< 0.01) among 3 Chinese alligator habitats. In the waters with and without Chinese alligator, no significant difference was observed in the contents of test metals except Pb. The possible pollution source of Pb in wild Chinese alligator habitat was analyzed, which could provide reference for Chinese alligator conservation.

Urban avian diversity is an important part of city biodiversity, which plays an important role in ecological balance of cities. During the summertime in 2004, we studied three typical avian communities at the habitats of village and farmland, urban woodlot, and floodplain in the urban and suburban areas of Wuhu City. A total of 71 species were recorded, which belonged to 12 orders, 27 families. The analysis of diversity index showed that urban woodlot had the greatest avian species number and G-F index, village and farmland had the highest avian density, and floodplain had the highest Shannon-Wiener and Pielou indices due to low human disturbance and dense vegetation. It is important to keep the avian diversity of floodplain in Wuhu City.

Microsatellite DNA has been widely used as a good genetic marker in molecular ecology. This paper reviewed its application in molecular population biology, molecular environmental genetics, and molecular adaptation. The isolation of the loci is the basis of microsatellite DNA genetic analysis. Compared with the traditional isolation method by genomic library, some novel approaches carried out recently, such as enrichment library, PIMA and FIASCO, are more efficient and timesaving. The details and compare-analysis of these approaches were introduced, which would offer good choices for researches on molecular ecology.

In June and July of 2003, a field survey on the vegetation of 22 Chinese alligator habitats distributed in Nanling county, Jingxian county, Guangde county, Langxi county and Xuancheng city of Anhui province was conducted, and the plant species of the habitats were recorded and analyzed. The results showed that the vegetation of the whole habitats was secondary, and there were 294 species vascular plants, belonging to 92 families. Pteioblastus amarus shrubbery could be found in all sites, and part differences of the vegetation consisted in different habitats. The relationship between Chinese alligator and habitat vegetation was also analyzed. All of these could offer some basic botanical data for the further protection of the Chinese alligator.

Microsatellites are a novel molecular genetic marker, and offer unusual advantages, e.g., with the using of a very few sample, they can be amplified by PCR, which are particularly appropriate in the research of conservation genetics. In this paper, the structure of microsatellites and the principle of the detection technique of microsatellites polymorphism were illustrated, and the advantages and the application of this detection technique were described briefly. For the isolation of microsatellite loci in animals, an enrichment method was introduced. And then, some practical problems of this technique were summarized.

Objective: Constructing a plasmid containing the shRNA of luciferase to suppress the expression of luciferase in BHK-21 cell., Methods: A 334 bp human U6 snRNA promoter was amplified from human genomic DNA by PCR and ligated to a 21 bp reverse repeated motif of luciferase target sequence with 9 bp spacer and AAV plasmid pSNAV. The recombinant pSNAV/U6/Luc plasmid cotransfected with pMAMneoLuc or transfected luciferase cell line to detect the effect of luciferase expression separately., Results: pSNAV/U6/luc suppresses the luciferase expression from pMAMneoLuc by 50% and luciferase cell line by 70%., Conclusions: The results showed that the short hairpin RNA of luciferase can efficiently suppress its expression in BHK-21.

Background: To construct a series of recombinant herpes simplex viruses that can provide replicating and packaging functions for recombinant adeno-associated virus (rAAV), and to select the strains possessing stronger functions for large-scale production of rAAV., Methods: A set of cosmids that represents the whole genome of HSV-1 was used to generate recombinant HSV-1 expressing rep and cap proteins of AAV-2. An ATG-to-ACG mutation in the start codon of AAV-2 rep protein was generated by site-directed mutagenesis. rep and cap genes, under control of their native promoters, with or without the ATG-to-ACG mutation in the start codon of rep, were inserted into the Xba site of UL2 or UL44 genes, respectively, resulting in the recombinant cosmids cos6-rmc/ UL2, cos56-rc/ UL44 and cos56-rmc/ UL44. Homologous recombination among the HSV-1 fragment on the recombinant cosmids and the rest fragments of HSV-1 genome resulted in three strains of recombinant HSV-1. Together with the one was constructed previously, there were four strains of recombinant HSV-1named HSV1-rc/ UL2, HSV1-rmc/ UL2, HSV1-rc/ UL44 and HSV1-rmc/ UL44 respectively., Results: PCR detection confirmed the existence of rep- gene in the genomes of all four strains of the recombinant HSV-1. Recombinant AAV was produced after infecting the AAV vector cell line that carrying the GFP expression cassette with the four strains of recombinant HSV-1 respectively. However, HSV1-rc/ UL2 and HSV1-rmc/ UL2 produced much more rAAV than HSV1-rc/ UL44 and HSV1c/ UL44 did., Conclusions: All the four strains of recombinant HSV-1 support rAAV replication and packaging. HSV1 UL2 and HSV1-rmc/ UL2 that provide much stronger functions may be useful for large-scale production of rAAV.