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Mastitis is a common disease of the dairy cattle, which affects the development of the dairy industry and leads to huge economic losses. Forsythoside A (FTA) has anti-inflammatory, antioxidant, antiviral and anti-apoptotic effects. However, the therapeutic effect and molecular mechanism of FTA on dairy cow mastitis remain unclear. In this study, bovine mammary epithelial cells (BMECs) were stimulated with lipoteichoic acid (LTA), a key virulence factor of Staphylococcus aureus (S. aureus), to construct in vitro models, and then treated with FTA. Subsequently, the differentially expressed genes (DEGs) in different groups were determined by RNA sequencing (RNA-Seq) analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyse the possible function of the DEGs, real-time quantitative PCR (RT-qPCR) was used to verify whether the expression levels of these DEGs were consistent with RNA-Seq results. The results showed that cell division cycle 20B (CDC20B), endothelial cell surface expressed chemotaxis and apoptosis regulator (ECSCR), complement factor H-related 5 (CFHR5) and phospholipase A2 group IVA (PLA2G4A) were down-regulated after FTA treatment. In contrast, Kruppel-like factor 15 (KLF15) and Metallothionein 1E (MT1E) were up-regulated. These DEGs are involved in processes such as apoptosis, inflammation and development of cancer. This study provides valuable insights into the transcriptome changes in BMECs after FTA treatment. Further analysis may help identify the underlying molecular mechanisms.

Mastitis is one of the most common diseases of dairy cattle and can be caused by physical stress, chemicals and microbial infection. Staphylococcus aureus is the most common pathogens that induce mastitis in dairy cattle. In this study, bovine mammary epithelial cells (BMECs) were treated either with lipoteichoic acid (LTA, 30 µg/ml) or 1 × phosphate-buffer saline (PBS, control) and RNA-Seq was applied to explore the effect of LTA on the expression microRNAs (miRNAs) in BMECs. Compared to the control group, 43 miRNAs were significantly up-regulated and eight miRNAs were significantly down-regulated. Additionally, 724 genes were significantly up-regulated and 13 genes were significantly down-regulated in LTA group relative to the control group. Bta-miR-196a, bta-miR-2285aj-5p, bta-miR-143, bta-miR-2433, bta-miR-2284f and bta-miR-2368-3p were selected from 51 differentially expressed miRNAs and are discussed in this manuscript. Target gene prediction revealed that the target genes of these six miRNAs were all differentially expressed, including MT1E, SPDYA, FGL1, TLR2, PAPOLG, ZDHHC17 and SMC4. Subsequently, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the target genes with differentially expressed miRNAs were enriched in mitogen-activated protein kinase (MAPK) signalling pathway, rheumatoid arthritis and cancer. Therefore, the results of this study provided new evidences for the molecular mechanism of LTA-induced mastitis, which may provide new targets for the diagnosis and treatment of mastitis in dairy cattle.

Purpose To build a model that utilizes clinical and blood parameters to predict the recurrence or progression of pituitary tumors after surgery. Methods A training group (67,70%) and a validation group (29,30%) were formed from 96 individuals with recurrent pituitary tumors. The training group was screened for blood parameters and a blood-related risk score (BRS) was established. Clinical-related prognostic factors were also assessed through Cox regression analysis, which was used alongside the BRS to construct a clinical prognostic model. In the validation group to assess the stability and accuracy of the BRS and the clinical prognostic model. Additionally, a clinical and blood-based nomogram was developed. Result The preoperative blood parameters K+, cholinesterase (CHE), and 5-nucleotidase (NT5E) were found to be correlated with progression-free survival (PFS). The area under the curve (AUC) for the BRS was 0.788 (95% CI: 0.657-0.919) in the training group and 0.852 (95% CI: 0.706-0.997) in the validation group. For 1-, 3-, and 5-year intervals in the validation set, the clinical model's AUC was 0.718, 0.852, and 0.864, respectively. While in the test group, the AUCs were 0.600, 0.889, and 0.660. The nomogram had C indices of 0.732 and 0.704 in the training and validation groups, respectively, and its calibration curves and clinical decision curves (DCA) indicated good calibrations and clinical utility. ConclusionThis is a new non-invasive tool that provides a tool for predicting prognosis and risk stratification.

Gliomas are common intracranial tumors with high morbidity and mortality in adults. Transmembrane protein 2 (TMEM2) is involved in the malignant behavior of solid tumors. TMEM2 regulates cell adhesion and metastasis as well as intercellular communication by degrading nonprotein components of the extracellular matrix. This study aimed to evaluate the relationship between TMEM2 expression levels and glioma subtypes or patient prognosis. Our findings revealed that TMEM2 expression was abnormally upregulated in high-grade glioma. Moreover, combining TMEM2, the status of isocitrate dehydrogenase (IDH) and 1p19q, we subdivided molecular subtypes with significant differences in survival. Patients in the MT-codel-low subgroup had better prognosis than those in the WT-no-codel-high subgroup, who fared the worst. Additionally, correlation analysis of TMEM2 and immune cell infiltration indicated an altered tumor microenvironment (TME) and cell redistribution in the TMEM2 high-expression subtype. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that focal adhesion and PI3K-Akt signaling pathways were enriched in the TMEM2-expressing group. In conclusion, aberrant TMEM2 expression can be used as an independent prognostic marker for refining glioma molecular subtyping and accurate prognosis. These findings will improve rational decision making to provide individualized therapy for patients with glioma.

Purpose To explore the lncRNA-mRNA network based on the IDH1 mutation status and construct a prognostic model for GBM. Methods The data of expression, somatic mutation, methylation, immune microenvironment were obtained from TCGA database, CGGA database and GTEx database. The common differential expressed genes (DEGs) between IDH1 mutant GBM and IDH1 wildtype GBM were used to perform a series of bioinformatics analysis to identify the hub gene. LASSO cox regression was used to screen a risk signature and nomogram was used to construct a prognostic model for GBM. Results LncRNA HCP5/MICA axis was downregulated in IDH1 mutant GBM and associated with the overall survival (OS) of GBM patients. Further analysis revealed that MICA expression was positively correlated with immune infiltration as well as the expression of several immune checkpoint gene in GBM. We screened a risk signature using MICA-associated genes by LASSO cox regression and the risk score performed well in predicting the OS. Furthermore, the risk score was used to construct a nomogram prognostic model combined with age, gender, IDH mutation, TP53 mutation, radiation and chemotherapy to predict the OS at 1-year, 2-year and 5-year. Conclusions This study constructed a prognostic model for GBM with MICA and clinical data for the first time and provided an insight into the molecular therapy of GBM. Further studies are required to uncover the mechanism of regulation between HCP5 and MICA in GBM.

To study the effect of triptolide (TP) on radiosensitivity of human glioma U251 cells and its mechanism, so as to provide new ideas and methods for the radiotherapy of glioma.U251 cells were treated with 10, 50, 100 nmol/L TP at different concentrations and irradiated with 0, 2, 4, 6, 8 Gy X-ray. The radiosensitivity of cells in each group were detected by MTT. U251 cells were then divided into control group, 10 nmol/L TP group, 4 Gy radiation group, 10 nmol/L TP + 4 Gy radiation group. The formation ability of U251 cells in each group was detected by colony formation assay. Flow cytometry was used to detect cell cycle and apoptosis in each group. Western blot was used to detect the changes of PI3K/Akt signal pathway in each group.When 10, 50, 100 nmol/L TP were combined with 2, 4, 6, 8 Gy X-ray, the proliferation inhibition rate of U251 cells in each group increased significantly (p0.05); compared with 10 nmol/L TP alone group and 4 Gy radiation alone group, the colony formation ability rate of U251 cells in 10 nmol/L TP + 4Gy radiation combined group decreased significantly (p0.05), the cell cycle was blocked in G1 phase, and the apoptosis rate was significantly reduced (p0.05). The level of p-pi3k and p-Akt decreased significantly (p0.05).Triptolide could significantly increase the radiosensitivity of human glioma U251 cells and play a role by inhibiting the PI3K/Akt signal pathway.

Background: Glioma is the primary malignant tumor of the central nervous system and presents high mortality and disability rates under existing treatment measures. Thioredoxin domain-containing 12 (TXNDC12) has been shown to play an important role in various malignant tumors. Therefore, we explored the clinicopathological characteristics of TXNDC12 in glioma to bring to light new ideas in its treatment.Methods: We obtained data packages related to TXNDC12 expression status in gliomas from public databases. We analyzed glioma TXNDC12 expression and patient survival status and validated the above results using glioma specimens from our institution. Next, we analyzed the value of TXNDC12 in combination with 1p19q and isocitrate dehydrogenase (IDH) on the prognosis of glioma by regression model and receiver operating characteristic curve (ROC). Finally, we explored the function of related genes by GO analysis and KEGG analysis.Results: Compared with normal brain tissue, the expression of TXNDC12 in glioma cells, regarding both mRNA and protein levels, was significantly upregulated. The survival time of patients with high-expression of TXNDC12 in glioma cells was shortened. In the World Health Organization pathological classification, IDH status, 1p19q status, and IDH combined with 1p19q subgroups, the expression of TXNDC12 increased with the deterioration of the above indicators. Tumor local immune analysis showed that the immune cell infiltration in TXNDC12 high-expressing glioma tissue increased, the tumor purity was reduced. GO and KEGG analyses indicated that TXNDC12 may be involved in the malignant prognosis of glioma through glycosylation and antigen processing and presentation.Conclusion: We showed that TXNDC12 is significantly highly expressed in gliomas. This high expression predicts the poor prognosis of glioma patients and is related to the gliomas’ local immune microenvironment. As a tumor-related gene, TXNDC12 may be used as a new prognostic judgment molecule.

Angiotensin II (AngII) regulates pigment synthesis by tyrosinase in melanocytes. To evaluate the association between AngII and coat color formation, we detected the expression distribution of AngII in white and black sheep skins by LC-ESI-MS/MS, western blot, quantitative real-time-PCR (qPCR) and distribution of AngII by immunohistochemistry.Liquid chromatography-electrospray ionization tandem MS (LC-ESI-MS/MS) results showed that AngII was found in white and black skin tissues of sheep. Western blot results verified the LC-ESI-MS/MS results and suggested that AngII was expressed at significantly higher levels in black sheep skins compared with the white sheep skins. Quantitative real time PCR (qRT-PCR) results also revealed that the expression level of AngII mRNA was higher in black sheep skins than that in white sheep skins. Immunohistochemical analysis further demonstrated that AngII protein was localized in the hair bulb and outer root sheath of hair follicle in sheep. In summary, protein and transcripts exhibited the same expression pattern in white and black sheep skins. Furthermore, the expressions of AngII in the hair bulb and outer root sheath of black sheep were stronger than those in white sheep. These results suggested that AngII functions in sheep coat color regulation and offer a novel insight for further investigation on the role of AngII in the coat color formation in sheep.

The tumor microenvironment plays an important role in tumor progression. Hyaluronic acid (HA), an important component of the extracellular matrix in the tumor microenvironment, abnormally accumulates in a variety of tumors. However, the role of abnormal HA accumulation in glioma remains unclear. The present study indicated that HA, hyaluronic acid synthase 3 (HAS3), and a receptor of HA named CD44 were expressed at high levels in human glioma tissues and negatively correlated with the prognosis of patients with glioma. Silencing HAS3 expression or blocking CD44 inhibited glioma cell proliferation in vitro and in vivo. The underlying mechanism was attributed to the inhibition of autophagy flux and maintaining glioma cell cycle arrest in G1 phase. More importantly, 4-methylumbelliferone (4-MU), a small competitive inhibitor of Uridine diphosphate (UDP) with the ability to penetrate the blood-brain barrier (BBB), also inhibited glioma cell proliferation in vitro and in vivo. Thus, approaches that interfere with HA metabolism by altering the expression of HAS3 and CD44 and the administration of 4-MU potentially represent effective strategies for glioma treatment.

Purpose: Brain gliomas are the most common primary malignant tumors of the central nervous system and one of the leading causes of death in patients with intracranial tumors. The lncRNA RPL34-AS1 is significantly upregulated in glioma tissues. However, the biological function of RPL34-AS1, especially in proliferation in glioma, remains unclear. Methods: The role of RPL34-AS1 in proliferation and angiogenesis in glioma cells was investigated using the LN229, U87, and U251 glioma cell lines. The levels of RPL34-AS1 were detected using real-time quantitative reverse transcription polymerase chain reaction. CCK-8 and colony formation assays were performed to determine the role of RPL34-AS1 in proliferation and survival, and its role in angiogenesis was assessed by an endothelial tube formation assay. Changes in protein levels were assessed by western blotting. Results: RPL34-AS1 was upregulated in glioma tissues and was correlated with tumor grade. RPL34-AS1 expression was also higher in glioma cells than in normal astrocytes. Knockdown of RPL34-AS1 blocked glioma cell proliferation by inhibiting angiogenesis. This effect occurred through decreased ERK/AKT signaling. Conclusions: This study suggests that RPL34-AS1 affects cell proliferation and angiogenesis in glioma and therefore may potentially serve as a valuable diagnostic and prognostic biomarker and therapeutic target in patients with glioma.

BackgroundThe tumor microenvironment plays an important role in tumor progression. Hyaluronic acid (HA), an important component of the extracellular matrix in the tumor microenvironment, abnormally accumulates in a variety of tumors. Whereas the role of abnormal HA metabolism in glioma remains unclear. MethodsThe expression level of hyaluronic acid (HA) was analyzed by ELISA assay and proteins such as HAS3, CD44, P62, LC3, CCND1 and CCNB1 were measured with Western blot analysis. The cell viability and proliferation were measured by MTT and KI67 immunofluorescence staining respectively. Autophagic vesicles and autophagosomes were quantified by transmission electron microscopy (TEM) and GFP-RFP-LC3 fluorescence analysis respectively. Cell cycle was analyzed by flowcytometry and Western blot analysis. Immunohistochemical (IHC) staining was used to detect expression levels of HA, Ki67, HAS3 and CD44 in human and mouse tumor tissues. Lentivirus constructed HAS3 and CD44 knockout stable glioma cells were transplanted to BALB/C nude mice for in vivo experiments. 4-Methylumbelliferone (4MU) was also used to treat glioma bearing mice for verifing its anti-tumor ability. The expression curve of HAS3, CD44 and the disease-free survival (DFS) curves for HAS3, CD44 in patients with LGG and GBM was performed based on TCGA database. ResultsAs shown in the present study, HA, hyaluronic acid synthase 3 (HAS3) and a receptor of HA named CD44 are expressed at high levels in human glioma tissues and negatively correlated with the prognosis of patients with glioma. Silencing HAS3 or blocking CD44 inhibited the proliferation of glioma cells in vitro and in vivo. The underlying mechanism was attributed to the inhibition of autophagy flux and further maintaining glioma cell cycle arrest in G1 phase. More importantly, 4-Methylumbelliferone (4-MU), a small competitive inhibitor of UDP with the ability to penetrate the blood-brain barrier (BBB), also inhibited the proliferation of glioma cells in vitro and in vivo. ConclusionApproaches that interfere with HA metabolism by altering the expression of HAS3 and CD44 and the administration of 4-MU potentially represent effective strategies for glioma treatment.

Background Tumors are the second most common cause of death in humans worldwide, second only to cardiovascular and cerebrovascular diseases. Although methods and techniques for the treatment of tumors continue to improve, the effect is not satisfactory. These may lack effective therapeutic targets. This study aimed to evaluate the value of SNHG12 as a biomarker in the prognosis and clinical characteristics of various cancer patients. Methods We analyzed SNHG12 expression and plotted the survival curves of all cancer samples in the TCGA database using the GEPIA tool. Then, we searched for eligible papers up to April 1, 2019, in databases. Next, the data were extracted from studies examining SNHG12 expression, overall survival and clinicopathological features in patients with malignant tumors. We used Review Manager 5.3 and Stata 15 software to analyze the statistical data. Results In the TCGA database, abnormally high expression of SNHG12 in tumor samples indicates that the patient has a poor prognosis. Results of meta-analysis is that SNHG12 high expression is related to low overall survival (HR = 2.72, 95% CI = 1.95–3.8, P < 0.00001), high tumor stage (OR = 3.94, 95% CI = 2.80–5.53, P < 0.00001), high grade (OR = 2.04, 95% CI = 1.18–3.51, P = 0.01), distant metastasis (OR = 2.20, 95% CI = 1.40–3.46, P = 0.0006), tumor size (OR = 2.79, 95% CI = 1.89–4.14, P < 0.00001), and lymph node metastasis (OR = 2.66, 95% CI = 1.65–4.29, P < 0.0001). Conclusions Our study confirmed that the high expression level of SNHG12 is closely related to the clinicopathological characteristics and prognosis of patients and is a new predictive biomarker for various cancer patients.

After parturition, bovine uterine stromal cells are often exposed to complex bacterial and viral stimuli owing to epithelial cell rupture, resulting in an inflammatory response. In this study, we used an in vitro model to study the response of bovine endometrial stromal cells to inflammatory mediators and the associated regulated microRNAs in response to lipopolysaccharide. Lipopolysaccharide (LPS) is a bacterial wall component in gram-negative bacteria that causes inflammation upon immune recognition, which is used to create in vitro inflammation models. Thus, we used high-throughput RNA sequencing to identify miRNAs that may have an anti-inflammatory role in the LPS-induced inflammatory response. Two groups of bovine uterine cells were treated with phosphate buffer saline (PBS) and LPS, respectively. Compared with the control (PBS) group, the LPS-treated group had 219 differentially expressed miRNAs, of which 113 were upregulated, and 106 were downregulated. Gene ontology enrichment analysis revealed that the target genes of differentially expressed miRNAs were significantly enriched in several activities, such as transferase activity, small molecule binding, and protein binding. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that the target genes of differential miRNAs were significantly enriched in fluid shear stress and atherosclerosis, MAPK signaling pathway, TNF signaling pathway. By analyzing differentially expressed miRNAs, we found that miR-200c, miR-1247-3p, and let-7b are directly related to the inflammatory response. For instance, miR-200c target genes (MAP3K1, MAP4K3, MAPKAPK5, MAP3K8, MAP3K5) and let-7b target genes (CASP3, IL13, MAPK8, CXCL10) were significantly enriched in the MAPK and IL-17 signaling pathways, respectively. In summary, our research provides insight into the molecular mechanism underlying LPS-induced inflammation in vitro, which may unveil new targets for the treatment of endometritis.

Background Medulloblastoma (MB) is the most common intracranial malignant tumor in children. The genes and pathways involved in the pathogenesis of MB are relatively unknown. We aimed to identify potential biomarkers and small-molecule drugs for MB. Methods Gene expression profile data sets were obtained from the Gene Expression Omnibus (GEO) database and the differentially expressed genes (DEGs) were identified using the Limma package in R. Functional annotation, and cell signaling pathway analysis of DEGs was carried out using DAVID and Kobas. A protein-protein interaction network was generated using STRING. Potential small-molecule drugs were identified using CMap. Result We identified 104 DEGs (29 upregulated; 75 downregulated). Gene ontology analysis showed enrichment in the mitotic cell cycle, cell cycle, spindle, and DNA binding. Cell signaling pathway analysis identified cell cycle, HIF-1 signaling pathway, and phospholipase D signaling pathway as key pathways. SYN1, CNTN2, FAIM2, MT3, and SH3GL2 were the prominent hub genes and their expression level were verified by RT-qPCR. Vorinostat, resveratrol, trichostatin A, pyrvinium, and prochlorperazine were identified as potential drugs for MB. The five hub genes may be targets for diagnosis and treatment of MB, and the small-molecule compounds are promising drugs for effective treatment of MB. Conclusion In this study we obtained five hub genes of MB, SYN1, CNTN2, FAIM2, MT3, and SH3GL2 were confirmed as hub genes. Meanwhile, Vorinostat, resveratrol, trichostatin A, pyrvinium, and prochlorperazine were identified as potential drugs for MB.

OBJECTIVEIt has been reported that microRNA-195 (miR-195) protects against chronic brain injury induced by chronic brain hypoperfusion. However, neither the expression profile of miR-195 nor its potential role during acute ischemic stroke has been investigated. In this study, the authors’ aim was to verify the mechanism of miR-195 in acute ischemic stroke.METHODSThe plasma levels of miR-195 expression were assessed using real-time PCR in 96 patients with acute ischemic stroke, and the correlation with the National Institutes of Health Stroke Scale score was evaluated. In addition, cerebral infarct volume, neurological score, and levels of miR-195 and CX3CL1/CX3CR1 mRNA and protein expression were assessed in mice subjected to middle cerebral artery occlusion (MCAO) with or without intra-cerebroventricular infusion of lentiviral vector. The inflammatory cytokines tumor necrosis factor–α (TNFα), interleukin (IL)–1β, and IL-6 of mouse brains after MCAO and BV2 cells treated with oxygen-glucose deprivation were measured using enzyme-linked immunosorbent assay, and apoptotic proteins were examined by Western blotting. Direct targeting of CX3CL1/CX3CR1 by miR-195 was determined by immunoblotting and dual luciferase assay.RESULTSIn ischemic stroke patients, miR-195 was significantly downregulated and expression levels of miR-195 in these patients negatively correlated with the National Institutes of Health Stroke Scale score. In mice after MCAO, miR-195 overexpression decreased infarct volume, alleviated neurological deficits, and most importantly, suppressed an inflammatory response. Meanwhile, miR-195 suppressed the expression of the inflammatory cytokines TNFα, IL-1β, and IL-6 in vitro and in vivo. The authors further discovered that both CX3CL1 and CX3CR1 are direct targets of miR-195, but miR-195 exerts neuroprotective roles mainly through inhibiting CX3CR1-mediated neuroinflammation and subsequent neuronal cell apoptosis.CONCLUSIONSTaken together, these findings suggest that miR-195 promotes neuronal cell survival against chronic cerebral ischemic damage by inhibiting CX3CR1-mediated neuroinflammation. This indicates that miR-195 may represent a novel target that regulates neuroinflammation and brain injury, thus offering a new treatment strategy for cerebral ischemic disorders.

Angiopoietin-like protein 4 (Angptl4) is a secretory protein associated with lipid and energy metabolism. In this study, the expression profile of the bovine Angptl4 gene was detected in different Nanyang cattle tissues by quantitative real-time PCR which was found highly expressed in back fat and abdominal adipose. Meanwhile, three SNPs (snp1145, snp1422 and snp5095) were found by DNA sequencing. Pairs linkage disequilibrium analysis and haplotype construction of the Angptl4 gene were conducted in 424 Chinese native cattle. Four different haplotypes (CA, CT, TA and TT) were derived from snp1422 and snp5095. The association analysis between genetic variants identified and partial growth traits showed that the individual with CCTT combined genotype of snp1422 and snp5095 had significantly higher height at withers, body length, hip width and height at hip cross than those with TTTT. The association analysis of snp1422 demonstrated that the individuals of CC genotype were higher in terms of height at withers, body length, heart girth, hip width, height at hip cross and body weight than those with TT. The effect of the combined genotype on conformation traits was consistent with the snp1422. These results suggest that Angptl4 gene might be an important gene of lipid metabolism and may provide basic, useful molecular information for further studies on growth traits in Chinese indigenous yellow cattle.

Along with the advance of market economy, the deepening of economic globalization, it makes the uncertainty of market demand increase, the competition among enterprises intensifying. The small and medium-sized enterprises (SMEs) have to carry on the organization and the system form innovation for enhancing the international competitive power themselves. Cluster economy in China has developed rapidly. They greatly promote the local economy in China, which has developed rapidly. They greatly promote the local economy development enormously. In fact, the industrial cluster in the low level of repeated construction and low efficiency operation have become the bottleneck of the development of local economy. Thus, the cluster supply chain has been proposed as a new management philosophy. Based on the background, this paper presents an analytic framework for cluster supply chain. The framework includes five layers. Each Agent can correspond to the actual operation of the entity, based on the messages between Agent interactions.

In this research, we use mouse embryonic fibroblasts as feeder layers. To eliminate the influence of serum and mouse embryonic stem cells (ESCs) conditioned medium (ESCCM) on self-renewal of sheep embryonic stem-like cells, knockout serum replacement (KSR) was used to replace serum, then supplanted with ESCCM for the isolation and cloning of sheep embryonic stem-like cells. We found when inner cell masses (ICMs) cultured in the control group with medium supplanted with fetal bovine serum (FBS), sheep ES-like cells could not survive for more than 3 passages. However, sheep embryonic stem-like cells could remain undifferentiated for 5 passages when cultured in the medium that FBS was substituted by KSR. The result indicates that KSR culture system was more suitable for the isolation and cloning of sheep embryonic stem-like cells compared to FBS culture system. Finally we applied medium with 15% KSR as basic medium supplanted with 40% ESCCM as a new culture system to isolate sheep embryonic stem-like cells, we found one embryonic stem-like cell line still maintained undifferentiating for 8 passages, which characterized with a normal and stable karyotype and high expression of alkaline phosphatase. These results suggest that it is suitable to culture sheep ICM in the new culture system with 15% KSR as basic medium and supplanted with 40% ESCCM, which indicated that mouse ES cells might secrete factors playing important roles in promoting sheep ES-like cells' self-renewal.