骨髓间充质干细胞移植修复肾损伤

Bone marrow mesenchymal stem cells transplantation for the repair of renal injury

背景:研究发现外源性骨髓间充质干细胞能够定居于肾脏并且分化为肾脏细胞,参与损伤肾组织的修复,但对其修复作用途径尚无公认。目的:探讨外源性骨髓间充质干细胞移植对肾损伤的可能治疗作用。设计、时间及地点:细胞学体内实验,于2007-03/09在南昌大学泌尿外科研究所完成。材料:清洁级SD大鼠56只。雄鼠8只用于制备骨髓间充质干细胞,雌鼠48只随机分为细胞移植组、模型对照组、假手术组,16只/组。荧光染料DAPI为Biotium Inc产品,蓖麻血凝素为Vector Laboratories产品,BrdU为Sigma产品。方法:以密度梯度离心法分离鼠骨髓间充质干细胞,加入DAPI进行标记,调整细胞数为1.5×1010L-1。细胞移植组、模型对照组建立缺血再灌注肾损伤模型,假手术组开腹后不予结扎肾血管。缺血45min后,细胞移植组经下腔静脉注入用DAPI标记的骨髓间充质干细胞悬液1mL,模型对照组、假手术组输入1mL无菌生理盐水。移植后各组每天腹腔注射BrdU,其后留取血标本和肾组织。主要观察指标:荧光显微镜及免疫组织化学染色观察移植细胞在肾组织中的分布,采用能与肾小管内腔壁特异结合的蓖麻血凝素鉴定移植细胞的分化,以免疫组化方法检测Brdu反映肾组织细胞的增殖状况,检测血清肌苷、尿素氮水平。结果:移植后第3天可见少量DAPI阳性细胞,第4天DAPI阳性细胞数明显增加(P<0.05),且多数DAPI标记细胞能结合蓖麻血凝素,DAPI阳性细胞主要分布于肾脏外髓质肾小管中,肾皮质和内髓质阳性细胞很少,而肾小球内则未见DAPI阳性细胞。假手术组肾组织细胞增殖非常弱,移植后第4天Brdu阳性细胞数明显低于模型对照组(P<0.05);移植后第2,3,4天细胞移植组Brdu阳性细胞均明显多于模型对照组(P<0.05),其分布类似于DAPI阳性细胞。与模型对照组比较,细胞移植组大鼠血清尿素氮水平移植后第1,2天明显降低(P<0.05);细胞移植组大鼠血清肌苷水平移植后第1,2,3天均显著降低(P<0.05)。结论:移植的外源性骨髓间充质干细胞能够迁移、定居于缺血再灌注损伤后的肾组织中并分化为肾小管上皮细胞;骨髓间充质干细胞移植可促进损伤肾脏本身的细胞增殖再生;对肾损伤后早期肌苷、尿素氮的升高具有一定抑制作用,这种早期对肾功能的保护与其迁移定居到肾脏局部、分化修复损伤的肾小管无关。

BACKGROUND： Previous study reveals that, exogenous bone marrow mesenchymal stem cells （MSCs） may settle down in kidney and differentiate into renal cells, as well as participate in the repair of injured renal tissues. However, the repairing pathway is unclear OBJECTIVE： To investigate the potential effect of exogenous MSCs transplantation on renal injury. DESIGN, TIME AND SETTING： An in vivo trial of cytology was performed in the Urinary Surgery Institute of Nanchang University （Nanchang, Jiangxi, China） from March to September in 2007. MATERIALS： Fifty-six SD rats of clean grade were recruited. Eight male rats of them were used to prepare MSCs, while other 48 female rats were divided into three groups at random, namely cell transplantation group, model control group and sham group. Each group contained 16 rats. Fluorochrome DAPI was a product of Biotium Inc. Ricinus communis agglutinin was produced by Vector Laboratories. BrdU was offered by Sigma. METHODS： Bone marrow-derived MSCs were collected from male SD rats, then isolated by Percoll density gradient centrifugation and labeled with DAPI at a density of 1.5 × 10^10/L. Renal injury models induced by ischemia and reperfusion were created in cell transplantation group and model control, while renal vessels were not ligated in the sham group. SD rats were subjected to clamping renal pedicles for 45 minutes. DAPI-labeled MSCs or 1 mL saline were transplanted into the rats by inferior vena cava in cell transplantation group or model control group and sham group. All animals received daily injection of BrdU, then blood and renal tissues were sampled. MAIN OUTCOME MEASURES： Immunohistochemical methods and fluorescence microscope were performed to identify transplanted MSCs and BrdU in renal tissues. To confirm if the MSCs may differentiate into renal tubular ceils, ricinus communis agglutinin that binds specifically to the luminal surface of tubules was used. Biochemistry methods were used to detect the levels of creatinine and blood urea nitrogen in serum. RESULTS： At day 3 after transplantation, the DAPI-positive cells were few, but obviously increased at day 4 （P 〈 0.05）. Most of DAPI-labeled ceils could express ricinus communis agglutinin-positive cells simultaneously and were mainly distributed in the renal tubule. There were few positive cells in renal cortex and kidney inner medullary, even absent in renal glomerulus. In sham group, the renal tissue cells proliferated slowly, and BrdU-positive cells were remarkably lower than that in model control group at day 4 （P 〈 0.05）; BrdU-positive cells were distributed identically with DAPI-positive cells in the cell transplantation and model control groups at days 2, 3 and 4, but the BrdU-positive cells in transplantation group were much more than that of the control group （P 〈 0.05） Compared with the model control group, the serum level of blood urea nitrogen in cell transplantation group was significantly decreased at days 1 and 2 （P 〈 0.05）, and the serum levels of creatinine were significantly lower at days 1, 2 and 3 （P 〈 0.05）. CONCLUSION： Exogenous MSCs can migrate and repopulate in the ischemically injured kidney, differentiate into renal tubular cells and promote cells proliferation to repair renal injury. MSCs have depressant effect on increasing degrees of blood urea nitrogen and creatinine at early stage, this protective effect has no association with location and differentiation of MSCs.