BMSCs移植对单侧输尿管结扎大鼠CD4^+CD25^+Treg的影响

Effects of BMSCs on expression of CD4^+CD25^+Treg in rats with unilateral ureter obstruction

目的研究骨髓间充质干细胞(BMSCs)移植对单侧输尿管结扎(UUO)梗阻性肾病模型大鼠外周血调节性T细胞CD4+CD25+Treg以及肾脏组织CD4+CD25+Treg特异性标志物FoxP3表达影响。方法自Wistar大鼠分离BMSCs,经体外传代培养、成脂诱导及鉴定后制备细胞悬液。54只Wistar大鼠随机分为假手术组、BMSCs干预组和生理盐水注射组,每组18只。BMSCs干预组和生理盐水注射组大鼠经UUO建立梗阻性肾病模型,并于造模同时经下腔静脉分别注入BMSCs和等体积生理盐水。分别于造模后第3、7和14天,采集外周血后分批处死各组大鼠(每组6只),留取肾脏组织标本。流式细胞仪检测外周血CD4+CD25+Treg占CD4+Treg的百分比(CD4+CD25+Treg/CD4+Treg);HE染色光学显微镜观察肾脏组织病理学改变;免疫荧光组织化学方法检测肾脏组织CD4+CD25+Treg特异性标志物FoxP3表达。结果 BMSCs干预组和生理盐水注射组大鼠造模后各时点外周血CD4+CD25+Treg/CD4+Treg均明显高于假手术组(P<0.05),BMSCs干预组与生理盐水注射组比较差异无统计学意义(P>0.05)。造模后第3、7天,生理盐水注射组大鼠肾间质水肿、肾小管扩张进行性加重,肾间质淋巴细胞和巨噬细胞浸润逐渐加重;相应时点BMSCs干预组大鼠肾脏组织结构较生理盐水注射组明显改善。建模后第3、7天,BMSCs干预组大鼠肾脏组织FoxP3表达显著高于假手术组和生理盐水注射组(P<0.05)。结论静脉注入BMSCs对梗阻性肾病模型大鼠肾脏的早期间质纤维化具有改善作用,可能与上调FoxP3在组织局部的表达而诱导CD4+CD25+Treg产生有关。

Objective To investigate the effects of bone mesenchymal stem cells(BMSCs) on expression of CD4+CD25+ Treg in peripheral blood in rats with unilateral ureter obstruction(UUO).Methods BMSCs were isolated from Wistar rats,and were cultured and passaged in vitro.Cell suspension was prepared after identification of adipocytes differentiated from BMSCs.Fifty-four Wistar rats were randomly divided into sham operation group,BMSCs intervention group and normal saline infusion group(n=18).Obstructive nephropathy models were established in rats of BMSCs intervention group and normal saline infusion group by UUO,and were injected with BMSCs and normal saline respectively through inferior vena cava.Three days,7 d and 14 d after model establishment,peripheral blood was taken,and renal tissue samples were obtained after sacrifice of rats(n=6 on each day in each group).The percents of CD4+CD25+Treg in CD4+T cells(CD4+CD25+Treg/CD4+Treg) were detected by flow cytometry,the pathological changes of renal tissues were observed with HE staining by light microscopy,and the expression of CD4+CD25+Treg specific marker FoxP3 in renal tissues was detected by immunohistochemical method.Results CD4+CD25+Treg/CD4+Treg in peripheral blood in BMSCs intervention group and normal saline infusion group were significantly higher than that in sham operation group at each time point after model establishment(P<0.05),while there was no significant difference between BMSCs intervention group and normal saline infusion group(P>0.05).Three days and 7 d after model establishment,rats in normal saline infusion group experienced renal interstitial edema,progressive development of renal and renal interstitial lymphocyte and macrophage infiltration,and rats in BMSCs intervention group had better renal tissue structure than those in normal saline infusion group.Three days and 7 d after model establishment,the expression of FoxP3 in renal tissues in BMSCs intervention group was significantly higher than that in sham operation group and normal saline infusion group(P<0.05).Conclusion Renal interstitial fibrosis of early stage can be improved by BMSCs injection in rats with obstructive nephropathy,which may be related to the increase of local expression of FoxP3 and generation of CD4+CD25+Treg.