骨髓间充质干细胞治疗肝硬化的动物实验研究

Treatment of liver cirrhosis by bone marrow mesenchymal stem cell transplantation in rats

目的了解肝硬化大鼠在输注骨髓间充质干细胞(BM-MSCs)后肝功能生化指标、AFP的变化情况,为临床应用提供实验依据。方法将健康雌性Wistar大鼠随机分为正常组、对照组和实验组;对照组和实验组腹腔注15%的四氯化碳石蜡油溶液建立肝硬化模型。实验组经尾静脉输入用BrdU(Bromodeoxyuridine)标记的健康雄性Wistar大鼠BM-MSCs,于1/2h、1wk、2wk、3wk、4wk处死对照组和实验组大鼠,用PCR检测雄性性别决定基因SRY、免疫组化检测BrdU以了解BM-MSCs的整合情况;用全自动生化分析仪检测血清肝功能指标、放免法检测AFP。结果PCR和BrdU免疫组化初步分析对照组在不同时期均无雄性BM-MSCs,实验组在1/2h处死的5例中均阴性,在第1wk处死的5只中有2例阳性,在第2wk处死组5只中有3只阳性,在第3、4wk处死组5只中均阳性。ALT、AST随着时间推移有所改善,在第4wk时与对照组有显著性差异(28.0±3.74,84.6±36.38vs56.0±26.66,161.2±55.43,P<0.005),ALB仅在第3kw时对照组和实验组有显著性差异(30.3±2.18vs34.4±2.49,P<0.005),但与正常组无显著性差异;GLO在不同时间点均无显著性差异;AFP虽无显著性差异,但有上升的趋势。结论BM-MSCs可以植入肝脏汇管区、肝细胞索,植入细胞最早见于移植后约7d;植入的BM-MSCs能够部分替代肝细胞的功能。

Objective To investigate changes of liver biochemical indexes and AFP in cirrhosis rats after transplantation of BM-MSCs. Methods Healthy female Wistar rats were randomly divided into three groups： Normal group （N） in which rats were injected with liquid paraffine into rat abdominal cavity, Control group （C） and Experimental group （E） in which rats were administrated 15% CC14（disloved in liquid parffin） intra-abdominally. Male Wistar rats BM-MSCs（1 -2 × 10^5/ml） labeled with BrdU were transfused into rats of E group by tail vein and 0.9% physiological saline was transfused into rats of other groups. Five female Wistar rat were killed at different time point of 1/2h, lwk, 2wk, 3wk, 4wk after transplantation, SRY gene was detected by PCR amplification and BrdU antigen in the liver cells of recipients were detected by PCR and immunohistochemistry respec tively. Liver function indexes and AFP were inspected by automated biochemical analyzer and radioimmunoassay. Results No SRY-positive was detected in the N, C group all the time and the E group after transfusion at 1/2h. Two of 5 cases were SRY-positive in the E group at 1 wk ; 3 of 5 cases were SRY-positive in the E group at 2wk ; all recipients were SRY-positive in the E group at 3wk, 4wk. ALT, AST values in .E group was significantly lower than those in C group at 4wk （ P 〈 0. 005）. ALB value in E group was significantly higher than that in C group ; but was not significantly different comparedwith N .group. Conclusion BM-MSCs can engraft into the hepatic cord at earlier time of 7d. The engrafted BM-MSCs can partly improve liver function.