经兔肝动脉自体骨髓干细胞移植治疗肝硬化的研究

Treatment of chronic hepatic cirrhosis with autologous bone marrow stem cells transplantation in rabbits

目的评价自体骨髓干细胞经肝动脉移植对兔肝硬化模型的治疗作用，了解促肝细胞生长素（pHGF）在干细胞移植治疗中的作用，为干细胞移植的临床应用提供实验研究基础。方法用皮下注射四氯化碳（CCl4）橄榄油溶液的方法复制兔肝硬化模型。25只肝硬化模型兔根据治疗方法不同分为对照组（5只）、干细胞移植组（10只）和移植＋pHGF组（10只）。无菌条件下从兔胫骨上端抽取骨髓4～5ml，采用密度梯度离心法分离骨髓干细胞，在透视下将分离的骨髓干细胞经肝动脉移植于肝脏。术后移植＋pHGF组隔日静脉内注射pHGF2mg／kg共20d。然后在移植后4、8、12周分别抽血检测肝功能，12周时取肝脏行病理学检查。分别对3组12周时肝功能指标进行比较，各组间比较用方差分析。结果在移植骨髓干细胞后，动物肝功能逐渐改善，至移植治疗后12周，各组肝功能与术前比较均有明显改善。干细胞移植组术后12周，兔血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）逐渐降低，由术前（73．0±10．6）、（152．4±22．8）U／L分别降至移植12周后的（48．0±1．0）、（86．7±2．1）U／L（F值分别为72．38、78．86，P值均〈0．05）；白蛋白（ALB）和凝血酶原活动度（FrA）逐渐上升，分别由（27．5±1．8）g／L和28．3％上升至干细胞移植后12周的（33．2±0．5）g/L和44．1％（F值分别为56．37、126．47，P值均〈0．05）；但12周时干细胞移植组除PTA外，其他上述血清酶改变与对照组相比差异无统计学意义（P值均〉0．05）。而干细胞移植＋pHGF组肝功能指标改变较移植组迅速，术后12周时ALT、AST分别降至（43．3±0．6）、（78．7±4．0）U／L，而ALB、PTA上升至（35．7±0．4）g／L和50．5％，这些指标改变与对照组相比均差异均有统计学意义（F值分别为47．38、23．52、52．27、174．45，P值均〈0．05）。病理学检查显示干细胞移植组肝细胞形态较规整，无明显细胞坏死和变性，假小叶结构仍存在，但肝组织纤维化程度较对照组明显减轻；干细胞移植＋pHGF组肝结构的改善更明显。免疫组织化学染色发现肝组织内散在分布有CD34^＋肝样细胞，并且这些肝样细胞在不同组数量差异较大，干细胞移植组较对照组多，移植＋pHGF组最多。结论自体骨髓干细胞移植对肝硬化有一定的治疗作用；促肝细胞生长素可促进干细胞在肝脏的归巢与分化，与骨髓干细胞移植有协同治疗作用。

Objective To evalute the feasibility of treatment for rabbit model with hepatic cirrhosis by transplantation of autologous bone marrow-derived stem cells via the hepatic artery and evaluate the effect of hepatocyte growth-promoting factors （pHGF） in the treatment of stem cells transplantation to liver cirrhosis. To provide empirical study foundation for future clinical application. Methods Chronic hepatic cirrhosis models of rabbits were developed by subcutaneous injection with 50% CCl4 0. 2 ml/kg. Twenty-five model rabbits were randomly divided into three experimental groups, stem cells transplant group （10） ,stem cells transplant ＋ pHGF group （10） and control group （5）. Autologous bone marrow was harvested from tibia of each rabbit,and stem cells were disassociated using density gradient centrifugation and transplanted into liver via the hepatic artery under fluoroscopic guidance. In the stem cells transplant ＋ pHGF group, the hepatocyte growth-promoting factor was given via intravenous injection with 2 mg/kg every other day for 20 days. Liver function tests were monitored at 4, 8,12 weeks intervals and histopathologic examinations were performed at 12 weeks following transplantation. The data were analyzed using analysis of variance Results Following transplantation of stem cells, the liver function of rabbits improved gradually. Twelve weeks after transplantation, the activity of ALT and AST decreased from （73.0 ± 10. 6）U/L and （ 152. 4 ± 22. 8） U/L to （48. 0 ± 1.0 ） U/L and （ 86.7 ± 2. 1 ） U/L respectively ; and the level of ALB and PTA increased from （27. 5 ± 1.8 ） g/L and 28. 3 % to （ 33.2 ± 0. 5 ） g/L and 44. 1% respectively. The changes did not have statistically significant difference when compared to the control group（P 〉 0.05）. However, in the stem cellstransplant ＋ pHGF group, the activity of ALT and AST decreased to （43.3 ± 0. 6） U/L and （78. 7 ± 4.0） U/L respectively and the level of ALB and PTA increased to （35.7 ± 0. 4）g/L and 50. 5% respectively. The difference was statistically significant when compared to the control group （ F = 47. 38, 23.52,52.27,174.45 ,P 〈 0. 05）. In pathohistology, the degeneration and necrosis of hepatic cells and the degree of fibrous hyperplasia in stem cells transplant group were less obvious than that of the control group. Hepatic pseudo-lobules persisted. The improvement of liver architecture in the stem cells transplant ＋ pHGF group was more evident than that in stem cells transplant only group. In addition, there were more hepatic CD34 ^＋ ceils within liver tissue in the stem cells trasplant group when compared to the control group, and the most hepatic CD34^＋ cells were seen in the stem cells transplant ＋ pHGF group. Conclusion Autologous bone marrow stem cells transplanted via the hepatic artery for the rabbit hepatic cirrhosis model is an effective method to treat hepatic cirrhosis. The hepatocyte growth-promoting factor can help to enhance the results of treatment.