自体骨髓干细胞经肝动脉移植治疗急性肝损伤的实验研究

Transplantation of autologous bone marrow stem cells via hepatic artery for the treatment of acute hepatic injury:an experimental study in rabbits

目的评价自体骨髓干细胞经肝动脉移植对兔急性肝损伤模型的治疗效果,了解促肝细胞生长素(pHGF)在干细胞移植治疗中的协同作用。方法新西兰雄性大白兔15只,皮下注射50%CCl4橄榄油溶液0.8ml/kg连续4d制作急性肝损伤模型。模型动物随机分为实验组A(干细胞移植组),实验组B(干细胞移植+pHGF组)和对照组(各5只)。实验组经兔双侧胫骨抽取骨髓约5ml,采用密度梯度离心法分离骨髓干细胞,制成5ml细胞悬液,透视下经股动脉插管将骨髓干细胞经肝动脉注入肝脏,对照组注入生理盐水。实验组B在干细胞移植后隔日静脉注射pHGF2.0mg/kg共20d。干细胞移植后2、4、8周抽血进行肝功能检测,8周后取兔肝脏组织标本进行病理学观察。结果骨髓干细胞肝动脉移植后肝功能逐渐改善,至移植后8周,各组血清AST、ALT活性和TBIL、ALB含量均显著低于术前指标,而球蛋白含量高于术前(P均<0.05),而且各组之间仍有明显差异。病理组织学观察显示实验组肝纤维组织增生程度和肝细胞变性较对照组明显减轻。免疫组化染色可见肝组织内散在分布有CD34阳性肝样细胞,实验组A较对照组多,实验组B最多。结论自体骨髓干细胞经肝动脉移植对肝损伤有明显治疗作用,可改善肝功能,促进肝组织结构重建,抑制纤维组织增生;pHGF能加速肝组织的修复和肝功能的改善。

Objective To evaluate the transplantation of autologous bone marrow stem cells via hepatic artery in treating acute hepatic injury in experimental rabbit models and to clarify the synergistic effect of hepatocyte growth-promoting factor （pHGF） in stem cell transplantation therapy for liver injury. Methods Acute hepatic injury models were established in 15 experimental rabbits by daily subcutaneous injection of CCl4 olive oil solution with the dose of 0.8 mL/kg for 4 days in succession. The experimental rabbits were randomly and equally divided into three groups： study group A （stem cell transplant, n = 5）, study group B （stem cell transplant ＋ pFHG, n = 5）, and control group （n = 5）. Bone marrow of 5 ml was drawn from the tibia in all rabbits of both study groups, from which bone marrow stem cells were isolated by using density gradient centrifugation, and 5 ml cellular suspension was prepared. Under fluoroscopic guidance, catheterization through the femoral artery was performed and the cellular suspension was infused into the liver via the hepatic artery. Only injection of saline was carried out in the rabbits of control group. For the rabbits in group B, pFHG （2.0 mg/kg） was administered intravenously every other day for 20 days. At 2, 4 and 8 weeks after stem cell transplantation, hepatic function was determined. Eight weeks after the transplantation all the rabbits were sacrificed and the liver specimens were collected and sent for pathological examination. Results After stem cell transplantation, the hepatic function was gradually improved. Eight weeks after the transplantation, the activity of AST, ALT and the content of ALB, TBIL were significantly lower than that before the procedure, while the content of GOLB was markedly increased in all rabbits. In addition, the difference in the above parameters between three groups was statistically significant （P 〈 0.05）. Pathologically, the bepatocyte degeneration and the fiberous hyperplasia in the study groups were obviously not as severe as in the control group. Moreover, the amount of CD34＋ hepatic cells within liver tissue in the study group A was more than that in the control group, and the amount of cells was the largest in the study group B. Conclusion Transplantation of autologous bone marrow stem cells via hepatic artery is very effective for the treatment of acute hepatic injury in experimental animals. It can improve liver function, promote hepatic reconstruction and inhibit fibrous hyperplasia. The hepatocyte growth-promoting factor is helpful in repairing liver tissue and in improving liver function.