肝细胞生长因子基因修饰的人脐带间充质干细胞对大鼠慢性肝损伤的治疗研究

目的：研究肝细胞生长因子（HGF）基因修饰的人脐带间充质干细胞（MSC）对Wistar大鼠慢性肝损伤的治疗作用。方法：利用携带人HGF基因的重组腺病毒（Ad～HGF）对MSC进行基因修饰；通过皮下注射CCl4-橄榄油溶液建立大鼠肝损伤模型；56只Wistar雄性大鼠随机分为正常对照组、模型组、MSC组、HGF组和HGF／MSC组，分别尾静脉注射生理盐水、MSC、Ad-HGF或HGF／MSC，通过血清肝功能检测及肝组织的生化指标、病理切片等评价治疗效果。结果：利用腺病毒将HGF基因转入MSC并确定了最佳感染条件。CCl4-橄榄油诱导4周后，动物肝脏外观出现明显的脂肪变性和血清转氨酶明显升高，表明成功诱导了大鼠慢性肝损伤模型。经过4周治疗，治疗组动物的体重明显升高、肝指数明显降低，动物存活率高于模型组（模型组：50％；MSC组：70％；Ad-HGF组：70％；HGF／MSC组：90％）。肝功能指标测定结果显示，与模型组比较，HGF／MSC组动物的天门冬氨酸氨基转移酶（406.75±35.98 vs.513.75±12.71U／L，P〈0.01）、丙氨酸氨基转移酶（124.6±10.6 vs.169.67＋15.38 U／L，P〈0.05）和总胆红素（14.6±2.08 vs.19.25±1.38g／dL，P〈0.01）均明显降低，白蛋白含量（29.1±1.3vs.22.05±2.61g／L，P〈0.05）明显升高；Ad-HGF组只有天门冬氨酸氨基转移酶（436.0±18.40vs.513.75±12.71U／L，P〈0.05）明显降低；而单纯MSC治疗对肝功能改善不明显。丙二醛测定结果显示，治疗后动物肝组织中的过氧化反应均较模型组显著减弱。HGF／MSC组和MSC组动物肝组织中的羟脯氨酸含量与模型组比较明显降低（69.27±14.58，63.23±13.23 vs.96.59±15.05mg／mL，P〈0.01）。上述实验结果提示3种治疗方式对CCl4-橄榄油引起的大鼠肝损伤均有一定的治疗效果，可减轻CCl。导致的肝损伤程度，增加动物体重，降低肝脏指数，减轻肝组织的过氧化反应，提高动物的存活率，其中HGF／MSC治疗效果最为明显，而且细胞注射没有引起动物的不良反应。结论：3种治疗方式均可改善CCl。导致的肝损伤，其中以HGF／MSC对肝损伤的治疗效果最好。本实验为HGF／MSC的临床研究提供了新的实验数据，为治疗肝损伤提供了新的治疗措施。

Effect of Human Umbilical Mesenchymal Stem Cells Modified with HGF Gene on Chronic Liver Injury in Rats

Objective： To evaluate the therapeutic effects of human umbilical cord mesenchymal stem cells（MSC） modified with hepatocyte growth factor（HGF） gene on liver injury of rat. Methods： MSC were modified with recombinant adenovirus（Ad） carrying human HGF gene and liver injury model of rat was developed by subcutaneous injection of CCl4-olive oil solution. 56 Wistar rats were divided into five groups randomly and were administered with normal saline, MSC, Ad-HGF or HGF/MSC respectively by tail vein injection. The therapeutic effects were evaluated by biochemical indicators of serum and liver tissue, and tissue sections of liver as well as. Resuits： MSC were modified with HGF-gene successfully through recombinant adenovirus and the optimal multiplicity of infection was determined. The results of fatty degeneration and function of liver showed that rat liver injury model was developed after induced with CCl4-olive oil solution for 4 weeks. Any side effect was not observed in rats after injection with cells. However, compared with the model group, the body weight of rats in three treatmentgroups increased significantly after therapy for 4 weeks and the liver index decreased, moreover, the survival rate obviously increased（Model： 50%; MSC： 70%; Ad-HGF： 70%; HGF/MSC： 90%）. The liver function of rats in HGF/ MSC group was improved markedly compared with model group, indication by the increase of alanine aminotransferase（406.75±35.98 vs. 513.75±12.71 U/L, P〈0.O1） and aspartate aminotransferase（124.6±10.6 vs. 169.67±15.38 U/ L, P〈0.05） activities, the decrease of serum total bilirubin（14.6±2.08 vs. 19.25±1.38 g/dL, P〈0.01）, and the increase of serum albumin（29.1±1.3 vs. 22.05±2.61 g/L, P〈0.05） as well. In contrast, only the aspartate aminotransferase activity in MSC group was lower than it in model group. Moreover, the quantities of malondialdehyde indicated that the peroxidatic reaction in the liver tissues of treatment group was ameliorated significantly compared with model group. Furthermore, the results of hydroxyproline demonstrated that the treatment with HGF/MSC and MSC may attenuate the fibrosis of liver. Conclusion： The liver injury induced by CCl4-olive oil solution was ameliorated with administration with MSC, Ad-HGF or HGF/MSC and the therapeutic effect of HGF/MSC was better. The results of this study may provide a new treatment for live injury.