| Human placenta mesenchymal stem cells (hPMSCs) are useful for the treatment of fulminant hepatic failure (FRF). CD200positive mesenchymal stem cells (CD200+hMSCs) can down-regulate several immunocyte activities and suppress TNF-a secretion from macrophages via the CD200-CD200R axis in vivo and in vitro.At present.there are plenty of reseraches reported that hPMSCs can promote liver’s regeneration and have been applied in clincal therapy. However, the influence of CD200positive placenta chorionic mesenchymal stem cells(CD200+hPCMSCs) on human normal hepatocytes regeneration is lack of research.Objective:To detect the influence of CD200positive placenta chorionic mesenchymal stem cells(CD20(ThPCMSCs) on human normal hepatocytes regeneration in coculture system.Methods:Human placenta chorionic mesenchymal stem cells were isolated from placenta chorion with type Ⅱ collagenase digestion method. CD200’hPCMSCs was sorted from hPCMSCs. Human primary hepatocytes were separated by two-step perfusion method. Experiment includes control group (0.5%FBS medium, hPCMSCs’supernatant and supernatant of mono hepatocytes) and coculture group (CD200+hPCMSCs:hepatocytes=1:3,1:1and3:1). Urea and albumin were detected by urea assay kit and albumin assay kit to evaluate hepatocellular function influenced by CD200+hPCMSCs. HL7702hepatocytes proliferation cocultured with CD200+hPCMSCs was detected by MTT method to evaluate hepatocellular proliferation influenced by CD200’hPCMSCs. Moreover, HL7702hepatocytes were induced by TNF-a/Act-D. Another three experimental group was set up including negative control,apoptosis group and anti-apoptosis group. The influence of hPCMSCs on HL-7702hepatocytes apoptosis was assayed with AnnexinV-FTTC/P1. Effects of CD200’hPCMSCs on Bcl-xL, Bax and Bcl-2protein levels in HL7702hepatocytes was detected by western blot procedure.Results:Compared with the quantity of urea (1.93±0.42mg/dl) and albumin(85.63±4.02ng/ml) in control group (0:3×105), when cell ratio was1:3, the co-cultural group of urea and albumin were without statistical difference (urea 2.53±0.26mg/dl, P=0.418;albumin69.15±2.38ng/ml, P=0.926); when ratio were1:1, quantities were higher (urea2.99±0.74mg/dl, P=0.063; albumin172.52±45.19ng/ml, P=0.012) and3:1, the quantity of urea and albumin were also higher (urea3.35±0.24mg/dl, P=0.012; albumin249.25±49.83ng/ml, P=0.000). After co-culture for24h, the growth percentage of human HL7702hepatocytes was higher at1:1(114.10±2.75%, P<0.01) and3:1(127.16±5.15%, P<0.01) than control group (100±0.00%), however, it was not significant at1:3(97.201:1.85%, P=0.391). Alter co-culture for48h, the proliferation of hepatocytes was also higher at1:3(133.36±0.70%, P<0.05),1:1(133.86±3.30%, P<0.05) and3:1(142.50±12.30%. P <0.05) than control group (130.03±0.55%). And after co-culture for72h,(he proliferative rates of HL7702hepatocyles was highest at1:3(161.73±1.96%,P <0.01),1:1(164.90±0.75%, P<0.01) and3:1(171.30±5.40%, P<0.01) than control group (139.40±1.85%). Moreover, After24h, microscope showed that anti-apoptosis group had a less apoptosis rate than apoptosis group. Flow cytometry showed, after12h, that apoptotic rate of anti-apoptosis group was less than apoptosis group (14.30±0.1414%VS20.40±1.4142%, P<0.01). Western blot results show Bcl-xl. relative expression (Bcl-xL/(3-actin) in the anti-apoptosis group was markedly increased at12hours and weakly increased at24hours compared with the apoptosis group. Contrary to Bcl-xL expression, the relative expression level of Caspase-3protein (Caspase-3/p-actin) was down-regulated by CD200+hPCMSCs at12and24hours in the anti-apoptosis group compared with the apoptosis group.Conclusion:CD200+hPCMSCs contributes to human normal hepatocytes’synthesis metabolism,regeneration and decreases hepatocytes’apoptosis by increasing expression of Bcl-xL protein and lowering level of Caspase-3proteins. |
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