An Experimental Study On Inducement And Differentiation Of Fetal Hepatic Stem Cells

Background At present the mortality of acute or chronic hepatic failure is still very high and the effective treatment is orthotopic liver transplantation(OLT).But the extensive application of OLT is restricted due to clinical shortage of donor organs. BAL(Bioartif icial liver)and HCT(Hepatocyte transplantation) can replace the failed liver temporarily. Researchers have acquired some successful experience from various animal model experiments, however, most of researchers pay more attention to heterogenic animal hepatocytes. But these heterogenic hepatocytes maybe bring the infection of unknown pathogens and spread of heterogen in the crowd. If cell resource is adequate, BAL and HCT will have a good prospect in clinical application. Recently hepatic stem cells has become a hot topic in the world. The hepatic stem cells are considered to be a kind of bipotential cells which can differentiate into hepatocytes and biliary epithelial cells. If we can proliferate hepatic stem cells and differentiate them into mature hepatocytes, we will solve above-mentioned problems about cell source. In this experiment we want to prove whether the human fetal HSCs can be induced and differentiated into hepatocytes and be used in the future. We will make some preparations for the clinical application of hepatic stem cells.Part I Isolation and culture of human fetal hepatic stem cellsObjective To establish a way of isolation, purification, identification and culture of human fetal hepatic stem cells. Methods Liver cells were isolated by collagenase (type IV) digestion method, and hepatic stem cells were purified from liver cells by Percoll discontinuous gradient centrifugat ion. Human fetal HSCs were obtained from the liver of aborted fetus in the second trimester of pregnancy. Under the asepsis conditions, establish umbilical-inferior vena extrinsic circulation. Perfuse EGTA balance liquid and collegen solution one by one. Then filtrate liver tissue by filter net and gain liver cells suspension. Centrifugate the liver cells suspension in three kinds of Percoll work solutions with different densities(1. llOg/ml, 1. 070g/ml, 1. 035g/ml). Extract the cells (HSCs) between the middle layer of Percoll and the bottom one. Then adjust the density of cells and culture them in DMEM/F12 media.The viability of hepatic stem cells was checked by trypan blue excluded test. The morphologic characters of human HSCs were observed by optic and electronic microscopy. The surface markers of HSCs including albumin, CK19, AFP and CD34 were identified by immuno- cytochemical staining. Count the amount of the cells during the whole process. Compute the yield of liver cells and human HSCs respectively. Results After two-step collagenase purfusion and centrifugation in discontinuous dentity Percoll solution, the HSCs could be purified from liver cells. The mean viability of HSCs is (88.35 + 2.45)% by trypan blue excluded test. By optic microscopy we can observe that HSCs have oval appearance and are smaller than mature hepatocytes. By electronic microscopy we can find that the diameter of HSCs is about 10 in, there are some microvilli on their surface, the oval nucleus occupy the mostspace of the cell and there are few cytoplasm, endoplasmic reticula, mitochondria and other organelle. Immunocytochemical staining shows that the HSCs present the positive signal of CK19, CD34 and AFP and the negative signal of ALB. During short-term culture course the HSCs gradually adhere and begin to grow like epithelia. The mean yield of liver cells is (8. 72 + 1. 02) XlO'/liver (n = 15) , the mean yield of HSCs is (2.34+0.93) XlOVliver (n=15) or (8. 76+0. 46) X105yh/g (n=15) .Conclusions Human HSCs can be obtained by purfusion of collagenase solution and centrifugation of discontinuous dentity Percoll solution satisfactorily. We can acquire enough HSCs with considerable viability and use them for the following experiment.Part II Inducement and differentiation of human fetal hepaticstem cells with HGF and EGF in conditional culture mediaObjective Induce and differentiate human fetal HSCs into hepatocytes with HGF and EGF in conditional culture media. Methods Culture the HSCs in DMEM/F12 media with appropriate cell density as soon as they have been isolated. By MTT colorimetry observe the dose-effect relation of HGF or EGF in five groups which have different concentration of HGF or EGF except for one control group. In inducement experiment we devicle four groups, namely, negative control group, HGF group, EGF group and HGF+EGF group. Every three days replace the media and collect the specimens to be measured. Observe cells by optic microscopy and depict the growth-curves. Measure the content of urea...