Objective:To construct a monoclone cell line expressing M-HBsAg.Methods:Transfected SP2/0 with pRc/CMV-S 2S by Calcium Phosphate Method,selected the cells by G418,detected HBsAg by Dot-blot、ELISA and Western-blot hybridization.Results:By selection of G418,G418 resistent cells were obtained;tests of Western-blot hybridization and ELISA demonstrated their expression of aimed protein.Conclusion:Monoclone Cell Lines of SP2/0-S 2S were obtained.

To explore the possibility of mouse bone marrow mesenchymal stem cells (BMSCs) differentiating into hepatocyte in vivo, the BMSCs isolated from donor male BALB/C mice at age of 6-8 weeks were transplanted to female BALB/C mice as recipients by transfusion via tail vein or direct liver injection. By the end of 1st, 2nd, 4th weeks after transplantation, the Y chromosome positive and albumin expression positive hepatocytes were detected from the recipient female mouse liver tissue by fluorescence in situ hybridization (FISH ) and immunohistochemistry (IHC). In the two groups of direct liver injection, no matter whether the donor cells are fresh isolated bane marrow cells or passage 3 cells, the Y chromosome positive cells with albumin expression simultaneously were detected by the end of the 1st week after transplantation. Similar result has been observed in the other subgroup of tail vein injection, but only by the end of the 2nd week after transplantation. BMSCs are capable of differentiating into hepatocyte in vivo.
Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Female ; Hepatocytes ; cytology ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred BALB C

It has been suggested that non-structural protein 5A (NS5A) of hepatitis C virus (HCV) may have a regulatory role similar to other RNA viruses in RNA replication. In order to investigate the replication function of NS5A, we tried to purify recombinant His(6) tagged NS5A expressed in Escherichia coli by a denature-renaturing method. The native lysis buffer was used to remove most of the soluble non-specific proteins. His-NS5A protein was solublized with the denaturing lysis buffer containing 8 mol/L Urea, and then bound to Ni2+ -NTA resin. The protein bound resin was successively washed with buffer containing reducing concentrations of Urea in the presence of NaCl and DTT to renature the protein. The renatured His-NS5A protein was eluted from the resin and it was capable of interacting with glutathione S-transferase fused form NS5Bt (GST-NS5Bt). The purified His-NS5A exhibited an inhibitory effect on RNA-dependent RNA Polymerase (RdRP) activity of GST-NS5Bt in vitro. Conclusively, in this study, we have established a purification method of bacterial recombinant HCV NS5A, and the results support the notion that NS5A may involve in the regulation of HCV replication through direct interaction with NS5B.
DNA Primers ; Escherichia coli ; genetics ; Hepacivirus ; genetics ; physiology ; Plasmids ; Protein Binding ; RNA Replicase ; metabolism ; Recombinant Proteins ; metabolism ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; Virus Replication

To establish the best condition of isolation and cultivation of mouse bone marrow mesenchymal stem cells (BMSCs) in vitro, we isolated BMSCs from BALB/C mouse using density gradient centrifugation and adherent selecting. The effects of different centrifuge power, adherent time, serum concentration and cell density on the isolation and cultivation of BMSCs were investigated. The best isolating condition is: 500g x 30min, 24 hours adhering. The best cell density is 12-20 x 10(5)/ml of primary cells and 6.4-25.6 x 10(4)/ml of secondary cells. The best serum concentration is 10%. More than 90% of subcultured cells were adhesive in 8 hours. Thus we have established a cell biological method of isolation and cultivation of BMSCs.
Animals ; Bone Marrow Cells ; cytology ; Cell Culture Techniques ; methods ; Cell Separation ; Culture Media ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred BALB C ; Serum

Objective To investigate the prediction of maternal HBV transmission by breast milk of postpartum women with chronic HBV infection.Methods HBV DNA levels in serum and breast milk weredetected by fluorescent quantitative polymerase chain reaction in 64 postpartum women with chronic HBV infection.HBV DNA≥1.0×103copies/ml was defined as positive,and correlation analysis was conducted.Results HBV DNA positive rate was 78.1%and 62.5%in serum and breast milk respectively,with a HBV DNA range of 1.05×103～3.87 ×104copies/ml in breast milk.When HBV DNA in serum was 1.0×105～1.0×107copies/ml,the HBV DNA positive rate in breast milk reached to 94.9%;however,when HBV DNA in serum was 1.0×103～1.0×104copies/ml,the positive rate in breast milk was only 18.2%.Conclusion The HBV DNA positive rate of breast milk in postpartum women with chronic HBV infection is correlated with the HBV DNA levels in serum;and breast-feeding should be avoided for postpartum women with HBV DNA≥1.0×105copies/ml in the serum.So serum HBV DNA detection is necessary in antenatal care for women with chronic HBV infection.

Adult ; Aged ; Carcinoma, Hepatocellular ; pathology ; Female ; Humans ; Immunohistochemistry ; Kupffer Cells ; pathology ; Liver Neoplasms ; pathology ; Male ; Middle Aged

To evaluate the feasibility of using human embryonic fibroblast(HEF) as feeder layer in the culture of human embryonic stem(ES) cells in vitro, we investigated the morphology, the sensitivity to 0.25% trypsin, the growth curve and cell cycle of HEF with DMEM(low glucose) +10% FBS used as culture medium, and then we compared HEF with mouse embryonic fibroblast (MEF). The results showed that both HEF and MEF are adherent cells in vitro, and HEF has longer life span and better growth ability than MEF. In room temperature, HEF is more sensitive to 0.25% trypsin. Our research suggested that HEF can be used as feeder layer in culture of ES cells. HEF has longer service life than MEF and is worthy to be studied further.
Animals ; Cell Cycle ; Cell Differentiation ; Cell Division ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Fibroblasts ; cytology ; physiology ; Humans ; Mice

CD44+ CD29+ and CD44- CD29+ bone marrow mesenchymal stem cells (BMSCs) were isolated from bone marrow of BALB/C male donor mouse by adherence selecting in DMEM media with low glucose and 20% fetal bovine serum. Then P5 cells were injected into the liver remnants of hepatectomined female mouse. Simultaneous assays were performed on the injected liver lobe and other liver lobe for detecting the Y-chromosome by fluorescence in situ hybridization and for defecting the albumin or cytokeratin 18 (CK18) by fluorescence immunoassays. Both Y chromosome and albumin (or CK18) positive cell could be detected at 5 days and 14 days after transplantation. At 14 days after transplantation, the liver weight in transplanted mouse was higher than that in model mouse. These results suggest that BMSCs could be induced to differentiate into hepatocytes and participate in the regeneration of liver.
Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Cells, Cultured ; Female ; Hepatectomy ; Hepatocytes ; cytology ; Liver Regeneration ; physiology ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred BALB C

To evaluate the value of two histological quantitative methods of hepatic fibrosis: semiquantative scoring system (SSS) and image analysis by computer. The prophylactic and therapeutic effect of Ganzhifu on hepatic fibrosis induced by CCl4 were studied on a total 73 of specimens from liver tissue of rats. All specimen were analyzed quantatively by two methods of SSS marks and image analysis respectively. Difference between groups was compared and hydroxyproline (Hyp) content of each liver tissue was examined. Correlation analysis was done between SSS marks, image analysis and Hyp content. Both prophylactic and therapeutic study showed the same information. Results of SSS marks, image analysis and Hyp content were coincidence. It suggest that both SSS marks and image analysis were interrelated well with Hyp content (P < 0.01). The result suggests that both SSS marks of hepatic fibrosis and image analysis by computer can be taken as reliable histological quantitative method of hepatic fibrosis.
Animals ; Carbon Tetrachloride ; Carbon Tetrachloride Poisoning ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Hydroxyproline ; analysis ; Image Processing, Computer-Assisted ; Liver ; chemistry ; pathology ; Liver Cirrhosis, Experimental ; chemically induced ; drug therapy ; pathology ; Male ; Phytotherapy ; Rats ; Rats, Wistar