人羊膜间充质干细胞移植联合百令胶囊改善大鼠肾功能及血液高凝状态

Effect of corbrin capsule combined with human amniotic mesenchymal stem cell transplantation on rat renal function and hypercoagulability

背景:百令胶囊的主成分是虫草酸,主要功效为免疫调节、改善血脂和保护肾脏等。人羊膜间充质干细胞是一种比骨髓间充质干细胞分化潜能更强的干细胞,但是目前其对肾损伤后的修复及改善尚缺乏研究。目的:观察人羊膜间充质干细胞移植联合百令胶囊对肾病综合征大鼠大鼠肾功能及凝血的影响。方法:体外培养人羊膜间充质干细胞,并制备其细胞悬液并进行CM-Dil标记。50只SD大鼠随机分为5组(n=10)。正常组不予任何处理;余40只大鼠静脉注射阿霉素建立阿霉素肾病大鼠模型,均分为4组,模型组不作处理,人羊膜间充质干细胞组连续3 d尾部静脉注射干细胞、百令胶囊组连续12周灌胃百令胶囊,联合组连续3 d尾静脉注射人羊膜间充质干细胞,同时连续12周灌胃百令胶囊。实验12周时检测各组大鼠血肌酐、尿素氮、凝血指标、24 h尿蛋白水平;苏木精-伊红染色观察肾组织病理变化;荧光显微镜观测CM-Dil标记人羊膜间充质干细胞的存活及分布;TUNEL法检测肾脏细胞凋亡情况。结果与结论:(1)人羊膜间充质干细胞组、百令胶囊组血肌酐和尿素氮、24 h尿蛋白水平高于联合组,明显低于模型组,大鼠的血液高凝状态有所缓解,差异有显著性意义(P<0.05);联合组凝血指标低于人羊膜间充质干细胞组、百令胶囊组(P<0.05);(2)模型组的肾脏病理没有改善甚至恶化,联合组的病理改善情况优于人羊膜间充质干细胞组和百令胶囊组;(3)荧光显微镜观测CM-Dil标记的人羊膜间充质干细胞,联合组多于人羊膜间充质干细胞组;(4)模型组凋亡细胞面积最大,人羊膜间充质干细胞组、百令胶囊组可见细胞凋亡,联合组细胞凋亡面积较小,组间比较差异均有显著性意义(P<0.05);(5)结果提示,人羊膜间充质干细胞移植联合百令胶囊能够改善肾病综合征大鼠的肾功能及血液高凝状态,减少蛋白尿程度,改善组织病理损伤程度,减少肾组织细胞凋亡。

BACKGROUND： As the main component of cordycepic acid, corbrin capsule mainly functions to improve blood lipids and protect the kidneys. Human amniotic mesenchymal stem cells are a kind of stem cells that have stronger differentiation potential than bone marrow mesenchymal stem cells, However, there are less reports on renal injury repair and post-repair functional improvement. OBJECTIVE： To observe the effect of human amniotic mesenchymal stem cell transplantation with corbrin capsule on blood coagulation and renal functions of rats with nephrotic syndrome. METHODS： Human amniotic mesenchymal stem cells were cultured in vitro and made into cell suspension labeled with with CM-Dil. Fifty Sprague-Dawley were randomly divided into five groups （n=10 per group）： normal control group with no intervention, model group, cell transplantation group, corbrin capsule group and combined treatment group. Except the normal control groups, rats in all the groups were used to make nephropathy models by rats by intravenous injection of adriamycin. After modeling, rats in the latter three groups were given intravenous injection of human amniotic mesenchymal stem cells for 3 days, intragastric administration of corbrin capsule for 12 weeks, and the combination of cell transplantation and corbrin capsule, respectively. Twelve weeks later, serum creatinine, blood urea nitrogen, blood coagulation index, and 24-hour urine protein levels were detected. Pathological changes of the renal tissues were observed by hematoxylin-eosin staining. Survival and distribution of CM-Dil labeled human amniotic mesenchymal stem cells were observed by fluorescence microscope. Apcptosis in renal tissues was detected using TUNEL method. RESULTS AND CONCLUSION： Serum creatinine, blood urea nitrogen and 24-hour urine protein levels in the cell transplantation and corbrin capsule groups were significantly decreased compared with the model group, but still higher than that in the combined treatment group （P 〈 0.05）. Coagulation indexes in the combined treatment group were significantly lower than those in the cell transplantation and corbrin capsule groups （P 〈 0.05）. The hypercoagulable state of rats was relieved in the three treatment groups. Pathological changes of the renal tissues were deteriorated in the model group, and improved in the three treatment groups, especially in the combined treatment group. Under the florescence microscope, the number of CM-Dil-labeled human amniotic mesenchymal stem cells was higher in the combined treatment group than the cell transplantation and corbrin capsule groups. Compared with the model group, the cell apoptosis in the renal tissue was relieved in the three treatment groups, especially in the combined treatment （P 〈 0.05）. To conclude, human amniotic mesenchymal stem cell transplantation with corbrin capsule can improve renal function and hyperooagulable state of nephrotic syndrome rats, by reducing the degree of proteinuria, improving the degree of pathological damage and reducing renal cell apoptosis,