人羊水间充质干细胞的分离鉴定及其对抗Thy-1肾炎的治疗作用

Isolation and identification of human amniotic fluid mesenchymal stem cell and its therapeutic effect in anti-Thy-1 nephritis

探讨羊水间充质干细胞(amniotic fluid mesenchymal stem cells,AF-MSCs)对抗Thy-1肾炎模型的治疗作用。方法采用差异性贴壁与机械性分离法从人孕中期羊水中分离出羊水间充质干细胞,流式细胞术鉴定其表面标志物,成脂、成骨诱导分化检测其分化能力。利用尾静脉注射抗Thy-1抗体建立SD大鼠抗Thy-1系膜增生性肾炎模型;将第5代羊水间充质干细胞通过尾静脉注射治疗抗Thy-1系膜增生性肾炎大鼠,检测大鼠尿蛋白的变化,PAS染色观察大鼠肾脏病理改变。结果羊水间充质干细胞顺利分离,流式结果显示其表达间充质干细胞标志物(CD29、CD44、CD73、CD90、CD105)和胚胎干细胞标志物(SSEA-4)而不表达造血干细胞标志物(CD34、CD45、CD133),且在适当条件下能够被诱导分化为脂肪细胞和骨细胞。羊水间充质干细胞治疗后,与模型组相比,治疗组在第7、12天时24 h尿蛋白明显下降(P<0.01),且肾脏病理结果表明系膜细胞增殖减轻,系膜区细胞外基质积聚减少。结论成功的从人孕中期羊水中分离出羊水间充质干细胞,羊水间充质干细胞治疗系膜增生性肾小球肾炎有效。

Objective To explore the therapeutic effect of amniotic fluid mesenchymal stem cells(AF-MSCs)in anti-Thy-1 mesangial proliferative glomerulonephritis.Methods AF-MSCs were isolated from human amniotic fluid during the second trimester by differential adherence and mechanical separation methods,and their surface markers were identified by flow cytometry,while their differentiation ability was detected by lipogenesis and osteogenesis induction methods.The anti-Thy-1 mesangial proliferative nephritis model in SD rats was established by injecting anti-Thy-1 antibody into tail vein.The rats with anti-Thy-1 mesangial proliferative nephritis were treated with the 5th-generation AF-MSCs through tail vein injection.Changes of urine protein of the rats were detected,and pathological changes of the rats kidneys were observed after PAS staining.Results The AF-MSCs were successfully isolated,and flow cytometry results showed that they expressed mesenchymal stem cell markers(CD29,CD44,CD73,CD90,CD105)and embryonic stem cell markers(SSEA-4)but not hematopoietic stem cell markers(CD34,CD45,CD133).The AF-MSCs could be induced to differentiate into adipocytes and bone cells under appropriate conditions.The urinary protein level of the AF-MSCs treatment group was significantly lower than that of the model group at 24 h,on day 7,and on day 12(P<0.01).And renal pathology examination showed that the mesangial cell proliferation and mesangial extracellular matrix accumulation were both reduced.Conclusion AF-MSCs were successfully isolated from human amniotic fluid during the second trimester,and were effective in treating the anti-Thy-1 mesangial proliferative glomerulonephritis of rats.