骨髓间充质干细胞在慢性马兜铃酸肾病大鼠肾脏中向肾小管周毛细血管丛内皮细胞的分化

目的 探讨Wistar大鼠骨髓间充质干细胞(MSC)是否具有在慢性马兜铃酸肾病(CAAN)大鼠肾脏向肾小管周毛细血管丛(PTC)血管内皮细胞(EC)的分化潜能.观察MSC对CAAN的治疗作用.方法 经密度梯度离心分离和贴壁培养相结合的方法,从大鼠骨髓中提纯单核细胞,体外扩增、鉴定为MSC.将30只雌性大鼠随机分为3组:①非移植组;②MSC移植组;③正常对照组,每组10只大鼠.非移植组和MSC移植组经关木通水煎剂灌胃12周制作CAAN大鼠模型.第12周,MSC移植组经尾静脉输入1 ml MSC悬液;非移植组和正常对照组经尾静脉输入1 ml生理盐水.第16周留取血、尿、肾组织标本.肾组织连续切片,应用Y染色体荧光原位杂交、免疫荧光染色等技术并作生化、病理、免疫组织化学、RT-PCR等检查.结果 第16周,MSC移植组肾脏可加Y染色体和EC标志抗原CD34双阳性细胞.非移植组与MSC移植组比较结果分别为:PTC密度(5.3±0.8)/0.13 mm2、(26.5±1.6)/0.13 mm2(P＜0.01),血管内皮细胞生长因子(VEGF)积分光密度值为(2.8±0.4)×103、(14.7±1.7)×103(P＜0.01).MSC移植组VEGF mRNA表达高于MSC移植组,肾间质纤维化相对面积小,生化指标明显低(均P＜0.01).结论 MSC可分化为EC,对PTC具有修复作用,对CAAN具有治疗作用.

Differentiation of mesenchymal stem cells into vascular endothelial cells in treatment of chronic aristolochic acid nephropathy: experiment with rats

OBJECTIVE: To investigate the potentiality of mesenchymal stem cells (MSCs) to differentiate into vascular endothelia cells (ECs) in peritubular capillary (PTC) in chronic aristolochic acid nephropathy (CAAN). METHODS: MSCs were isolated from a male Wistar rat. The surface markers were identified with flow cytometry. Thirty female Wistar rats were randomly divided into 3 equal groups: Group A, perfused intragastrically with decoction of Caulis Aristolochiae manchuriensis for 12 weeks to establish CAAN models, Group B, perfused intragastrically with decoction of Caulis Aristolochiae manchuriensis for 12 weeks to establish CAAN models and injected with the MSCs by caudal vein in the 12th week, and Group C, perfused intragastrically with drinking water for 12 weeks and then injected with normal saline by caudal vein to be used as normal controls. At week 16, specimens of blood and urine were collected to detect the blood urea nitrogen (BUN), serum creatinie (Scr) and urine protein, and then the rats were killed with their kidneys taken out. Sex-determining region of the Y chromosome-fluorescence in situ hybridization (SRY-FISH) test with carboxyfluorescine (FAM)- was used to detect the cells originated from the source of the male donors. Immunohistochemestry was used to detect CD34, marker antigen pf EC. HE and Masson staining and electron microscope were used to observe the pathology of the kidney. Immunohistochemistry and RT-PCR were used to detect the expression of vascular endothelial growth factor (VEGF). Correlation analysis was conducted to study the relationships among these indices. RESULTS: Y chromosome and CD34 double positive cells could be seen in the renal tissue of Group B. At week 16, the density of PTC and integrated optical density of VEGF of Group A were (5.3 +/- 0.8)/0.13 mm2 and (2.8 +/- 0.4) x 10(3) respectively, both significantly lower than those of Group B [(26.5 +/- 1.6)/0.13 mm2 and (14.7 +/- 1.7) x 10(3) respectively, both P < 0.011]. The Scr and urine protein of Group A were significantly higher than those of Group B. The expression of VEGF mRNA of Group A was significantly lower than that of Group B. CONCLUSION: MSCs can differentiate into ECs. MSCs transplantation has beneficial effects on CAAN, which is possibly related with the reduction of PTC.