Human Umbilical Cord Mesenchymal Stem Cell Differentiation Direction Of Hep3B Liver Cancer Cells In Vitro Microenvironment And The Impact Of The Proliferation Of Hep3B Cells

bjective:Through separation, culture and identification of human umbilical cord mesenchymal stem cells (hUCS-MSCs), discuss the differentiation direction of human umbilical cord stroma-derived mesenchymal stem cells(hUCS-MSCs) co-cultured the microenvironme with liver cancer cells and umbilical cord mesenchymal stem cells on the proliferation of hepatoma cells. Methods:1. Isolation and co-ulture of human umbilical cord mesenchymal stem cells. With adherentmethod, Sterile collection of the umbilical cord, Immersedin PBS containi-ng1%pairs of anti-(Penicillin, streptomycin) glass containers, about2cm of the umbilical cord in superclean stage scissor, cut open the arteriovenous,with PBS wash bloodstained, umbilical cord was cut into the meat-like,Transferred to a centrifuge tube, add4ml of digestive juice, and mix, at37℃to digest one hour, every10minutes to shaking. Absorb the digestion supernatant was diluted centrifugation, cells were collected andtransferred to T25cm2cell culture flasks。adding L-DMEM medium3mL (containing10%fetal bovine serum),37℃,5%CO2incubator during the don’t shake, On the third day half of the medium was changed, the seventh day the whole medium was changed, When the cells grew to80%confluence with0.25%trypsin digestion and passage alternate。2. Flow cytometry detection of umbilical cord mesenchymal stem cell surface marker。 Take the fourth generation of the amplified cell culture flasks hUCS-MSCs like cells with0.25%trypsin solution,37℃, dig-estive cells3minutes, centrifugation, cells were collected. Washed with PBS, made the concentration of a single cell suspension of2×105/500μL each tube, and make a mark, each tube were added different anti-human of CD34-FITC, CD29-PE, of CD105-APC monoclonal antibody2μL and join anti-mouse IgGl-FITC monoclonal antibody10μl, incubated at room temperature30min, detected by flow cytometry.3. discuss the differentiation direction of human umbilical cord stroma-derived mesenchymal stem cells(hUCS-MSCs) co-cultured the microenvironme with liver cancer cells In vitro and umbilical cord mesenchymal stem cells on the proliferation of hepatoma cells. Through RT-PCR detection of liver cell gene AFP mRNA, ALB mRNA and CK19mRNA level were detected and glycogen synthesis function wasdetected by glycogen staining when cells had been co-cultured for7th days,14th days and21th days, respectively. By MTT assay hUCS-MSCs with Hep3B cells co-cultured after Hep3B cell proliferation. Results:1. MSCs were isolated from hu-man umbilical cord sucessfully, showing fibroblastic morphology and adh-erent cell characterization. Among these cells we isolated,96.02%cells are CD29positive cells and96.6%cells are CD105positive cells. The percentage of CD34negative cells is99.65%. The percentage of CD105+CD29+double positive cells is94.84%.2. The only AFP mRNA positive expression was found on the7th day after these cells had been co-clutured with hepatoma cells Hep3B experiment group and the normal human liver cell LO2control group; On the14th day Hep3B experiment group expression of CK19mRNA and the LO2group expression of ALBmRNA;21th days, Hep3B and hU-MSCs co-clutured experiment group there was a consistent positive expression of AFP mRNA, CK19mRNA, while LO2and hUCS-MSCs co-cultured control group ALB mRNA positive expression, but AFP mRNA; In the above-mentioned time points, the negative control group was not detected in the positive expression. Glycogen staining in Hep3B experimental group and the LO2control group, cultured positive (some cells could be detected in the glycogen staining positive cells, cytoplasm pink) for21days,7th,14th and the negative control group, no significant red particles, were negative.3. the hUCS-MSCs and Hep3B co-culture can inhibit the proliferation of Hep3B, and increase with the hUCS-MSCs increase in the number and processing time, the inhibition rate increase.24h,48h and72h, hUCS-MSCs:of Hep3B=10:1and hUCS-MSCs: Hep3B=5:1Hep3B roups of Hep3B cell proliferative capacity weakened, with the control group with significant differences (P<0.01); hUCS-MSCs:of Hep3B=1:1groups of Hep3B cell proliferation diminished capacity, a significant difference (P<0.05) and the control group; hUCS-MSCs:Hep3B=1:5group of Hep3B cells with proliferative capacity compared with the control group did not differ significantly (P>0.05). Conclusion:1.Through Double enzyme digestion were isolated, expanded cells and identification of mesenchymal stem cell markers by flow cytometry, was successfully isolated from umbilical cord mesenchymal stem cells and for in vitro culture.2. Human umbilical cord mesenchymal stem cells and human hepatoma Hep3B cells co-cultured in vitro microenvironment, the umbilical cord mesenchymal stem cells may differentiate into immature liver cells.3. Human umbilical cord mesenchymal stem cells and normal human liver cell LO2cells co-cultured in vitro microenvironment, the umbilical cord mesenchymal stem cells may differentiate toward the mature liver cells (hepatocyte-like cells).4. liver cancer cells and human umbilical cord mesenchymal stem cells co-cultured directly under the conditions of liver cancer cell proliferation was significantly inhibited.