人乙型肝炎病毒DNA阳性血清对人骨髓间充质干细胞向肝细胞分化的影响

Impact of hepatitis B virus infected serum on the hepatic differentiation of human bone marrow mesenchymal stem cells

目的:探索人乙型肝炎病毒感染血清对间充质干细胞向肝细胞分化的影响,为肝细胞移植提供实验基础。方法:体外分离和纯化骨髓间充质干细胞,接种在肝细胞诱导培养基中,刺激干细胞向肝细胞分化。设立4个实验组加入不同血清:A组,5%(体积分数)胎牛血清(FBS);B组,2.5%(体积分数)FBS+2.5%(体积分数)乙型肝炎感染血清;C组,2.5%FBS+2.5%(体积分数)正常人血清;D组,以含10%(体积分数)FBS的LG-DMEM培养液作为对照。诱导培养后,利用免疫组织化学染色检测肝细胞特异性标志,以及感染细胞是否表达乙型肝炎特异性蛋白。应用糖原染色技术检测细胞合成和储存糖原的功能。结果:诱导组的间充质干细胞表达白蛋白(ALB)和甲胎球蛋白(AFP),并具备合成和储存糖原的功能。B组细胞加入感染血清后,扩增能力受到抑制,少量细胞可表达乙型肝炎表面抗原(HBsAg),但ALB和AFP的表达阳性率以及糖原储存能力与C组相似。荧光共聚焦技术检查可见部分细胞同时表达ALB和HBsAg。结论:骨髓间充质干细胞在特定的肝细胞诱导条件下具有向肝细胞分化的能力;乙型肝炎感染血清可以明显抑制间充质干细胞的体外扩增,但对其向肝细胞分化没有明显影响。

Objective：To study the effects of hepatitis B virus （HBV） infection in vitro on differentiation of mesenchymal stem cells （MSCs） to liver cells. Methods： MSCs were isolated from human bone marrow by density gradient centrifugation, and expanded by adherent culture. MSCs were cultured under liver-stimulating condition, and different composition of serum was added to the induced medium ： Group A： 5% fetal bovine serum （FBS）; Group B： 2.5% FBS ＋2.5% HBV-containing serum; Group C： 2.5% FBS ＋ 2.5% serum from healthy volunteers; Group D： the undifferentiated MSCs cultured in LG- DMEM ＋ 10% FBS. The expressions of a variety of hepatic lineage markers were analyzed by immunocytochemistry and immunofluorescence. The functionality of differentiated cells was assessed by their ablility to store glycogen. After 2 weeks of exposure to HBV infected serum, HBV-specific protein was also detected by immunocytochemistry. Results： As a result of our hepatic induction, the expressions of albumin （ALB） and alpha-fetoprotein （AFP） by MSCs were observed by immunocytochemical and immunofluorescence techniques. Moreover, MSCs had acquired the ability of glycogen storage which was characteristic of liver cells. Compared with the control group, the proliferation of MSCs was inhibited greatly by the virus-containing serum. After 2 weeks of exposure to HBV infected serum, the surface antigen （HBsAg） was detected in some induced MSCs. However, after immunocytochemical stain for ALB and AFP, there was not much difference between the Group B and C. The ability of glycogen storage of two groups were almost the same. Using confocal microscopy, we found the co-expressions of ALB and HBsAg in the same differentiated cells. Conclusion： The bone marrow MSCs have the ability to trans-differentiate into functional hepatocyte-like cells, hence may serve as a cell source for tissue engineering and cell therapy of hepatic diseases. HBV infected serum could inhibit the proliferation of MSCs in culture, but it seemed that the hepatic differentiation of the cell was unsuppressed.