人脂肪干细胞治疗急性肝功能衰竭大鼠疗效的探索

Human adipese-derived sterm cells treatment of actute liver failure in rats

目的探讨人脂肪干细胞(adipose-derived stem cells,hADSC)对急性肝衰竭大鼠生存和肝功能重建的影响,了解其在急性肝衰竭大鼠体内的分布、迁移及促进受体肝脏再生的作用。方法贴壁培养法分离培养hADSC;30只SD大鼠用D-氨基半乳糖(D-gal)构建急性肝衰竭模型,随机分为hADSC治疗组和磷酸盐缓冲液(PBS)对照组,治疗组经脾尾注射5×106个hADSC,对照组注射相同体积PBS,动态检测肝功能指标和肝组织学表现,观察动物生存情况。用表达ZsGreen的慢病毒感染hADSC,20只肝衰竭大鼠随机分为脾脏移植组和股静脉移植组,分别经脾尾和股静脉注入5×106个hADSC,采用免疫组织化学方法检测hADSC在大鼠心、肝、脾、肾、肺等器官的分布和迁移情况。检测肝、脾组织中Ki-67蛋白的表达,TUNEL法检测肝细胞凋亡。结果体外分离人体脂肪组织培养获得的hADSC表达间充质干细胞相关表面抗原并可分化成脂肪细胞、软骨细胞和骨细胞;肝衰竭模型中脾脏移植组大鼠总体病死率(13.3%)明显低于PBS对照组(40%),血清ALT和AST等指标显著低于PBS对照组,肝组织学明显改善,Ki-67表达和TUNNEL检测提示移植组肝细胞再生明显,凋亡受抑制;hADSC经脾脏、股静脉途径移植后,大部分细胞迁移至肝脏、脾脏及肺部,其中经股静脉移植组细胞向肝脏趋化的效率更高。结论体外分离脂肪组织通过贴壁培养可获取hADSC;经脾脏和股静脉途径移植hADSC均可促进肝细胞再生并抑制凋亡,改善肝衰竭大鼠肝功能,提高存活率。

Objective Isolate human adipose derived stem cells （hADSC） and explore its efficacy for acute liver failure rats in liver function reconstruction. Methods hADSC were isolated from fat tissue. 30 SD rats induced by D-gal were randomly divided into hADSC transplantation group and PBS control group, treatment group received hADSC through spleen route and the control group received the same volume of PBS. Moreover, 20 SD rats were randomly divided into spleen transplantation group and femoral vein transplantation group, 5 x 10^6 hADSC were injected via the spleen and femoral vein respectively, immunohistochemieal method were applied to detect the migration of hADSC infected with lenti- virus expressed ZsGreen. Results hADSC which obtained from vitro cultured human adipose tissue express mesenchymal stem cell-associated surface antigens and differentiate into adipoeytes, cartilage cells and osteoeytes. The hADSC transplan ration group showed lower mortality （13.3%） significantly compared with the PBS control group （40%）. Serum ALT and AST were significantly lower than the PBS control group after hADSC transplantation indicating the liver functional improvement. HE staining also showed significant improvement in liver tissue morphology. Moreover, Tunnel assay and Ki 67 assay showed that hADSC transplantation could reduce cell apoptosis and promote cell proliferation. We further tracked the distribution of hADSC transplantation through spleen and femoral vein. Most hADSC migrated to the liver, spleen and lung in both routes, however, more hADSC migrated to the liver via femoral vein transplantation route. Conclusion hADSC can be obtained through the adherent culture of isolated adipose tissue in vitro; hADSC transplant through spleen and femoral vein promotes regneration and inhibits apoptosis, improves liver function and survival rate of liver failure rat.