| Objective To establish and optimize the culture system, markers in vitro, tracing in vivo system of the human umbilical cord blood mesenchymal stem cells (hUCBMSCs). To observe the migration and differentiation of human umbilical cord blood mesenchymal stem cells marked with green fluorescent protein (GFP) after being transplanted into rat model of acute hepatic necrosis. To evaluate the effects of transplantation on the outcome of rat model of acute hepatic necrosis, and probe its underlying mechanism.Method Human umbilical cord blood mesenchymal stem cells had been cultured with density centrifugation and adherent culture method. Different culture result had been compared between the condition of low serum medium and serum-rich medium,the ways of single sample culture and three-sample mixed culture of human umbilical cord blood mesenchymal mesenchymal stem cells. Green fluorescent protein gene was transfected to human umbilical cord blood mesenchymal stem cells, using lentivirus vector. GFP-hUCBMSCs were transplanted into rat modles through the ways of liver injection, CC14 was injected into abdominal to manufacture model of acute hepatic necrosis. Observe the survival rate of rat model of acute liver necrosis after transplantation. Migration of GFP-hUCBMSCs was traced through detecting fluorescent signal in liver slices 48 h, 72 h,1 week,2 week,4 week post-transplantation. The trans-differentiation status of hUCBMSCs was evaluated using immunohistochemistry and real-time PCR.Results:Serum-rich medium and mix-components methods contribute to culture result. Green fluorescent protein transfected cells exhibited uniform green fluorescence under fluorescence microscope images. The rat model of acute hepatic necrosis transplanted with hUCBMSCs had significantly less death rates than the group without transplantation of hUCBMSCs 48h post transplantation.24 hours after the transplantation of GFP-hUCBMSCs to rat model with acute hepatic necrosis, the fluorescent signal can be seen in the portal area of liver,48 hours after the transplantation, stronger fluorescence signal with disperse pattern can be observed along the portal area; one week after the transplantation, fluorescent signal of transplanted cells dispersed in liver necrosis area; four weeks after the transplantation, fluorescent signal was still persistent. Control group had the similar result before the time point of 48h post transplantation, from then on, fluorescence signal were distributed randomly, and became more and more weak. Immunohistochemistry analysis showed positive staining of human liver tissue-specific marker AFP, ALB, Hep par I in rat model with transplantation of hUCBMSCs. Real-time PCR analysis also showed the expression of human liver cell-specific gene AFP, ALB,CK19and CK18 in rat model with transplantation of hUCBMSCs.Conclusion:This study established the optimization of human umbilical cord blood stem cells culture system and the marker in vitro, tracing in vivo system. Transplantation of hUCBMSCs can improve the survival of rat model of acute hepatic necrosis. HUCBMSCs transplantation can prolong the rat model of acute hepatic necrosis survival. Transplanted cells in model mice experience the course of twice migration patterns " from the Pinhole to portal area", "from portal area-necrotic region".Transplanted cells in model mice showed liver-like cell phenotype. The efficacy was caused by the transplanted cells migration to the target lesion, directed differentiation, but also on immune regulation and cell fusion related.
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