人羊膜间充质干细胞对四氯化碳诱导小鼠肝损伤定位修复的研究

目的：观察活体染料羧基荧光素乙酰乙酸（CFSE）标记的人羊膜间充质干细胞对四氯化碳诱导小鼠肝损伤模型的定位修复情况。方法：采用胰蛋白酶-胶原酶消化法从羊膜组织中分离间充质干细胞，通过流式细胞术和免疫荧光等方法进行鉴定。模型组按浓度为20μl/g剂量的四氯化碳和橄榄油混合液诱导小鼠肝损伤，治疗组经小鼠尾静脉注射羧基荧光素乙酰乙酸标记的人羊膜间充质干细胞约1×10⁶个/ml。分别取模型组和细胞移植的治疗组小鼠眼球血和肝组织进行相关检测。结果：分离得到纯度较高的羊膜间充质干细胞；冰冻切片免疫荧光显示移植1周后细胞向小鼠受损肝组织定植，CFSE标记的人羊膜间充质干细胞呈绿色荧光；细胞移植后4周，与模型组比较，细胞移植组小鼠血清中天冬氨酸转移酶、丙氨酸氨基转移酶显著降低，而白蛋白明显升高（P< 0.01）；肝组织病理切片模型组小鼠细胞水肿，坏死灶多见，脂肪变性，可见不同程度的炎性细胞浸润；治疗组小鼠肝组织病理学改变和损伤程度有较明显改善；小鼠肝组织冰冻切片的免疫荧光显示移植4周后人羊膜间充质干细胞周围分泌血清白蛋白。结论：羧基荧光素乙酰乙酸标记的人羊膜间充质干细胞可有效改善肝组织的生理功能，为细胞定位移植治疗肝脏疾病的修复情况提供实验数据。

Human Amnion Mesenchymal Stem Cells Positioned Transplantation for the Treatment of Mice Liver Damage Induced by Carbon Tetrachloride

Objective: To observe the therapeutic effect of human amnion mesenchymal stem cells transplantation labeled with 5, 6, 2-carboxy fluorescein diacetate succinimidy ester(CFSE)on mice model with liver damage induced by carbon tetrachloride. Methods: The amnion was mechanically separated from the placenta obtained by cesarean section after 38~40 week-pregnancy. The amniotic mesenchymal stem cells were obtained by using two-step digestion with trypsin and collagenase type I, then the flow cytometry and immunofluorescent staining techniques were adopted to identify the surface molecular markers and stem cell characteristics. Hepatic damage model groups were established through carbon tetrachloride and oil inducing, 20 μl/g doses of the mixture were given to mice by abdominal injection. Mouse models of hepatic damage were randomly divided into two groups. The control group was injected with the same volume of PBS via the tail vein; the treatment group was injected with human amnion mesenchymal stem cells suspension labeled with CFSE, and the number of the stem cells was 1×10⁶. At 4 weeks after transplantation, animals were sacrificed. Liver tissues were obtained for the histological observation and the eyeball blood was collected to detect the liver function indicators. Results: The human amniotic mesenchymal stem cells obtained by using two-step digestion with trypsin and collagenase type I were high-purity. At 1 weeks after transplantation, the immunofluorescent staining of frozen slice showed that human amnion mesenchymal stem cell colonization could be seen in the mice liver tissues of the cell transplantation group. The labeled human amnion mesenchymal stem cell showed strong green fluorescence. At 4 weeks after cell transplantation, compared with the model group, levels of serum aspartate aminotransferase, alanine aminotransferase in the cell transplantation group were significantly decreased, while the total albumin level was increased significantly. The liver cell inflammatory necrosis, steatosis and liver fibrosis were improved significantly. After 4 weeks, the immunofluorescent staining of frozen slice result indicated that the expressions of human serum albumin could be observed in the mice liver tissue of the cell transplantation group, but no expression could be seen in the model group. It is visible that human amnion mesenchymal stem cells can improve liver function and pathological damage of liver damage mice in a certain extent. The findings may provide useful experimental datas for cell positioned transplantation with treatment liver disease.