| Background and aim:At present, the treatment for acute liver failure and terminal stage liver disease are tough problems of worldwide. Orthotopic liver transplantation is the most effective method , but there are still some difficulties with widely clinical application due to insufficient liver donor, high cost , immunity rejection and other reasons. Hepatocellular transplantation, bioartificial liver and hepatic tissue engineering, which have shown the potential for the treatment of liver diseases, are limited by the lack of liver cells. The status encourage the search for new therapies.The development of stem cells provides new insights into therapies of liver disease now. Diverse studies have suggested that Mesenchymal stem cells (MSCs), one of adult stem cells, are not only have the ability to differentiate into mature cells of multiple mesenchymal tissues, but also might have the ability to differentiate into cell types other than those of the tissues in which they reside or derive during embryonic development. For instance, MSCs are known to differentiate into hepatic-like or hepatic cells under in vitro and in vivo experiment by a lot of researchers. The potential of MSC differentiate into hepatocytic cells provides alternative cells source for hepatocellular transplantation, bioartificial liver and hepatic tissue engineering, and open new possibilities for therapies of liver disease.Although the studies about MSCs on liver disease have obtained an enormous development, there are many questions remain with regard to their potential therapeutic use. The first problem is how to separate, culture ,cryopreserved and proliferate MSCs in vitro? Secondly, how to establish the appropriate experimental conditions inducing differentiation of MSCs into hepatocyte in vitro. Then, how to determine the number of cells and route of transplantion, how to repair liver injury and so on.This study aim to establish a method of isolation and expandation mesenchymal stem cells, to optimize and standardize the condition of inducing MSCs to differentiate into hepatocyte-like cells in vitro, and to investigate MSCs transplantation for the treatment of acute and chronic liver injury treated with CCl4. The exploration will be beneficical for clinical application.Methods:1. MSCs were isolated, expanded, purified and cryopreserved by culture of tissue adherence.The growth curves of different passages MSCs were drawn by MTT. Cell cycle and the surface antigen expressions of MSCs were detected by flow cytometry. The ultrastructure of MSCs were observed by transmission electron microscope. MSCs were detected osteogenic and adipogenic differentiation respectively by treated with osteogenic induction medium , as well as the adipogenic induction medium.2. MSCs of P7 were induced to differentiate into hepatocyte-like cells which divided by experiment and control groups (group C) by chance, experiment groups were subdivided by A which treat with DMEM/F12 medium, containing 10% FBS, 20ng/mlHGF, 10ng/ml bFGF, and B which treat with DMEM/F12 medium, containing 10% FBS, 20ng/mlHGF, 10ng/ml bFGF and 20ng/ml OSM respectively. group C treated without growth factors. on 7d, 14d , 18d and 21d after induction, the markers of hepatocyte lineage such as AFP, ALB and CK18 were examined by immunocytechemistry. The content of AFP, ALB , urea was examined and the glycogen deposits were examined by PAS at the same time. The ultrastructure of MSCs was observed by transmission electron microscope.3. The Wistar rats were treated with 10% CCl4 olive oil mixture by intraperitoneal injection (0.7ml/100g) to establish acute liver injury model. The model rats were randomly divided into control (C1, C2, C3) and treatment (T1, T2, T3) groups. Hochest33342-labelled rat bone marrow MSCs (1×106/0.5ml) were transplanted into the rats by tail vein, portal vein and liver injection in treament group respectively. PBS was transfused into rats in various control groups. On day 3 and 7 after transplantation, The distribution of Hochest33342- labelled cells were observed by fluorescence microscopy. The liver function indexes and pathologic structure of liver were detected at same time. 4. The Wistar rats were treated with 40% CCl4 olive oil mixture by intraperitoneal injection (0.1ml/100g, 2 times one week, for 8 weeks) to establish chronic liver injury model. The model rats were randomly divided into control groups (C1, C2), and treatment groups (T1, T2). Hochest33342-labelled rat bone marrow MSCs (1×106/0.5ml) were transplanted into the rats by portal vein (T1 group) and liver injection (T2 group) respectively. PBS was transfused into rats in various control groups. On day 7 and 14 after transplantation, The distribution of the Hochest33342-labelled cells were observed by fluorescence microscopy. The liver function indexes and pathologic structure of liver were detected at same time.5. One-way analysis of variance and t test were adopted to compare every index values by SPSS 12.0 statistic analysis software.Results:1. The hUCMSCs of P3 were homogenenously long fusiformshaped, fibroblast-like by culture of tissue adherence. The MSCs inoculated as 5×105/ml which exhibited the same morphological and growth characteristic as before cryoperserved . The grow curves of P3, P5 and P7 MSCs were"S"shape. Flow cytometry analysis revealed that the hUCMSCs were normal diploid cells , whose cell cycle showed the percentage of G0/G1 was more than 70%. CD44, CD90, CD105 were highly expressed, but there was negative for CD34, CD31, CD45 respectively. The ultrastructure of MSCs indicated that there are two types. One at quiescent state, with high nuclear-cytoplasmic ratio and few cytoplasmic organelles; other at active state, with microvilli on the surface, low nuclear-cytoplasmic ratio and many cytoplasmic organelles. The assays in vitro demonstrated the cells exhibited multipotential differentiation into osteogenic and adipogenic cells.2. The morphology of MSCs in experiment groups transformed gradually into round, polygonal endothelial cell shape after induction, resembling as hepatocytes . The positive expression ratio of AFP protein on 7th day was higher than that on 14th day (P<0.05), but there was no diversity between group A and B, so did the concentration of AFP in the supernatant. The positive expression ratio of ALB protein and the concentration of the supernatant were higher in group B than in group A on 14, 18 d and 21 day after induction, so did the positive expression ratio of CK18 protein. The markers of hepatocyte lineage did not appear in group C. urea concentration of the supernatant didn't change on 7d,14d in group A and B, but ascended on 18,21d in group B than group A. The positive cells of PAS on 21d were more in group B than group A (P<0.05) The ultratructure of group B on 21d indicated that cells grew bigger in size with similar round nucelus, large nucleoli, and a lot of endoplasmic reticulum, mitochondrion, lysosome, glycogen granule. So did the ultratructure in group A,but less organelles than in group B. The MSCs ultrastructure of group C only found few cytoplasmic organelles3. The levels of serum ALT and AST in rats treated with with CCl4 for 24h were significantly elevated compared with those in normal (P<0.01). Compared with control group, the levels of serum ALT and AST were decreased on day 3 and day 7 after transplantation in groupT2, T3 (P<0.01), but were not change in groupT1. Hochest33342 labelled cells were found in group T2, T3, the days were more, the cells were more. But not found in T1 and group C1, C 2, C 3. Pathological structure of liver displayed multiple necrosis of hepatocyte, diffuse degeneration of hepatocyte and infiltration of inflammatory cells in rats treated with CCl4 for 24h. The severity of pathological structure of liver wasn't alleviated in group T1 on day 3 and day 7 after transplantation, but was alleviated in T2, T3 group, group T3 was better than group T2.4. The levels of serum ALT, AST, GGT in rats treated with CCl4 for 8 weeks were significantly elevated,but the level of ALB was significantly decreased compared with those in normal. Compared with control group, the levels of serum ALT, AST, GGT were decreased on day 7and day 14 after transplantation, ALB were increased . Few Hochest33342 labelled cells were found in the liver in groupT1 on day 7, a few labelled cells were found on day 14, but a lot of labelled cells were found in the liver in groupT2. Hochest33342 labelled cells were not found in control group. Pathological structure of liver displayed significant necrosis, steatosis, inflammatory cell infiltration and fibrosis in rats treated with CCl4 for 8 weeks. Compared with control group, The severity of pathological structure of liver was alleviated in various treament group on day 7 and day 14 after transplantation. The pathologial scores of chronic liver injury on 14d after transplantation of MSCs had statistically difference between group T1 and T2 (P<0.05).Conclusions:1. It is a simple and practical method to separate MSCs from the human umbilical cord by means of tissue culture method. Flow cytometry analysis revealed that the hUCMSCs were normal diploid cells, whose cell cycle showed the percentage of G0/G1 was more than 70%. CD44, CD90, CD105 were highly expressed, but there was negative for CD34, CD31, CD45 respectively. The assays in vitro demonstrated the cells exhibited self-renewal and multipotential differentiation into osteogenic and adipogenic cells.2. DMEM/F12 medium supplemented with 10% fetal bovine serum is suitable for culture of hUCMSCs. fetal bovine serum supplemented with 10% DSMO is suitable for cryopreservation of hUCMSCs, whose morphological and growth characteristic are as same as before.3. The hUCMSCs could be differentiated into heaptocyte-like cells by DMEM/F12 medium supplemented with 10% FBS, 20ng/ml HGF, 10ng/ml bFGF and 20ng/ml OSM . the induced efficiency is better than by DMEM/F12 medium supplemented with 10% FBS, 20ng/ml HGF, 10ng/ml bFGF.4. Acute liver injury model in rats could be established by intraperitoneal injection with 10% CCl4 olive oil mixture (0.7ml/100g). Rat bone marrow MSCs could survive, settle down in liver after transfusion. Intraportal vein, intraliver injection transplantation of bone marrow MSCs ameliorate liver function of acute hepatic injury. The route of intraliver injection transplantation is better than intraportal vein transplantation .5. Chronic liver injury model in rats could be established by intraperitoneal injection with 40% CCl4 olive oil mixture (0.1 ml/100g, 2 times/week, 8 weeks). MSCs could survive, settle down in liver after intraportal vein and intraliver injection transplantation, that ameliorate liver function of chronic hepatic injury. The route of intraliver injection transplantation is better than intraportal vein transplantation.
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