骨髓间充质干细胞在慢性马兜铃酸肾病肾组织动态分布的实验研究

Dynamical Distribution of Bone Marrow Mesenchymal Stem Cells in Rat Model of Chronic Aristolochic Acid Nephropathy

目的研究雄性大鼠骨髓间充质干细胞(MSCs)经尾静脉移植到慢性马兜铃酸肾病(CAAN)雌性大鼠后,在CAAN肾脏的分布路线和增殖规律。方法取出雄性Wistar大鼠双侧股骨,制成骨髓单细胞悬液后,培养、纯化并鉴定为MSCs。将60只雌性CAAN模型大鼠随机分为2组:(1)MSCs移植组(n=50);(2)假移植组(n=10)。MSCs移植组经尾静脉输入MSCs,分别在此后第6h、48h、10天、30天、60天各时间点随机处死10只大鼠;假移植组于经尾静脉输入生理盐水后30天处死10只大鼠,取肾脏组织。应用SRY基因荧光原位杂交(SRYFISH)和SRY基因聚合酶链式反应(SRYPCR)技术检测各时间点MSCs在肾脏的分布和数量变化。结果MSCs移植后第6、48h,MSCs主要分布在肾小球毛细血管中,MSCs移植后第10、30、60天可见肾小球毛细血管中MSCs数目逐渐减少,肾间质逐渐增多并最终趋于稳态;肾小管MSC呈现稳定的增加态势。MSCs移植后第30、60天肾间质可见MSCs集落分布,MSCs增殖速度逐渐减慢,肾小管MSCs稳定增加。结论雄性大鼠MSCs沿毛细血管、肾间质、肾小管这一路线向CAAN雌性大鼠肾脏迁徙分布。MSCs可在CAAN肾脏以长期定居、增殖。

Objective To investigate the distributive path and proliferative rule of marrow mesenchymal stem cells （MSCs） in the rat transplanted via caudal vein from male rat to female rats model of chronic aristolochic acid nephropathy （CAAN）. Methods Cells taken from femoral bone marrow of male Wistar rats were made into single cell suspension, cultured, purified and identified as MSCs. MSCs were transplanted via caudal vein into 50 female Wistar CAAN model rats allocated in the test group, they were killed, 10 rats in a batch, at various time points （6 h, 48 h, 10 d, 30 d and 60 d after transplantation）. Besides, 10 rats allocated in the control group were killed on the 30th day after received sham-transplantation. Kidney tissue of all rats was taken for detecting cells originated from the donors by fluorescence in situ hybridization test with FAM-labeled sex determining region of Y chromosome （SRY FISH） probe, and their number in SRY was counted using SRY PCR. Results MSCs were mainly distributed in the glomerular capillaries at the time points of 6 h and 48 h, but the number of MSCs in glomerular capillaries decreased and those in renal mesenchyma increased at the time points from 10 d to 60 d gradually, then tended to a steady state, meanwhile it showed a stable increasing trend in renal tubule. Cell colony of MSCs could be found in mesenchyma with a slowed down increasing between 30 d to 60 d, but the increasing in tubule was still steady. Conclusion MSCs originated from the donor can enter the kidney of acceptor and distribute from blood capillary to renal mesenchyma and tubule, and they can long time inhabit there and make propagation.