骨髓间充质干细胞移植对缺血再灌注肾损伤的保护作用

Protective effects of bone marrow mesenchymal stem cells on renal ischemic injury in rats

目的探讨外源性骨髓间充质干细胞(MSCs)移植对缺血再灌注肾损伤的可能保护作用。方法取SD雄性大鼠骨髓,梯度离心法分离MSCs,采用4,6-联脒-2-苯基吲哚(DAPI)标记。32只雌性SD大鼠建立缺血再灌注肾损伤模型后随机分为移植组和对照组,移植组于缺血45min后经下腔静脉注入用DAPI标记的MSCs,对照组注入等量生理盐水。术后每天经腹腔注射5-溴脱氧尿苷嘧啶(BrdU),并分别于术后1、2、3、4d处死大鼠,留取血标本,观察血清肌酐(Cr)和尿素氮(BUN)水平。留取肾组织,荧光显微镜及免疫组化染色方法观察移植MSCs在肾组织中的分布及肾细胞增殖状况,并采用蓖麻血凝素(RCA)对迁移入肾脏的MSCs分化状况进行鉴定。结果术后第1、2天,移植组大鼠血清BUN水平(31.93±1.49、19.58±1.58mmol/L)明显低于对照组(41.03±2.14、26.45±1.74mmol/L,P<0.05);第3、4天2组间BUN水平差异无统计学意义(P>0.05);第1、2、3天移植组大鼠血清Cr水平(77.47±4.77、57.45±2.41、44.93±3.97μmol/L)均显著低于对照组(102.37±3.59、67.50±2.92、53.25±2.73μmol/L),差异有统计学意义(P<0.05)。细胞增殖状况:术后第1天偶见BrdU阳性细胞,第2天2组大鼠肾组织中均可见较多BrdU阳性细胞,并随时间延长阳性细胞逐渐增多,且移植组阳性细胞数(33.71±8.50、57.60±4.88、116.29±6.99)明显多于对照组(19.67±5.20、26.88±5.89、71.71±8.44),差异有统计学意义(P<0.05)。移植组术后第3天肾组织内可见DAPI标记细胞,第4天DAPI标记细胞明显增多,且多数标记细胞能结合RCA。结论移植的外源性骨髓MSCs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;MSCs移植可促进损伤肾组织的细胞再生,对缺血再灌注肾损伤具有一定的保护作用。

Objective To study the protective effects of bone marrow mesenchymal stem cells （MSCs） on renal ischemic injury in rats. Methods Bone marrow MSCs were collected from femurs and tibias of 8 male SD rats, then isolated by percoll density gradient centrifugation and labeled with 4,6-diamidino-2 -phenylindole （ DAPI）. Thirty-two female SD rats were subjected to clamping renal pedicles for 45min and divided randomly into the transplantation group and control group. DAPI-labeled MSCs or saline were injected into the rats by inferior vena cava. All the rats received daily injection of bromodeoxyuridine （ BrdU, 50 mg/kg, ip） for identification of newly synthesized DNA. After operation the animals were sacrificed at clays 1 , 2, 3 and 4, respectively. Biochemistry methods were used to detect the serum levels of creatinine （Cr） and blood urea nitrogen （BUN）. Immunohistochemical methods and fluorescence microscope were used to identify transplanted MSCs and BrdU in renal tissues. Ricinus communis agglurinin （RCA） ,which binds specifically to the luminal surface of tubules, was used to confirm if the MSCs may differentiate into renal tubular cells. Results Compared with control group（41.03±2.14,26.45±1.74 mmol/L） , the serum BUN level of the rats in transplantation group （31.93±1.49, 19.58±1.58mmol/L） were significantly lower on the 1 st and 2nd day （P 〈 0.05） after operation, but not significantly different on the 3rd and 4th day （P 〉0.05）. On the 1st, 2nd and 3rd day the serum Cr levels of transplantation group（77.47±4.77, 57.450±2.41,44.93 ±3.97 mmol/L） were significantly lower than those of control group（ 102.37±3.59,67.50±2.92, 53.25±2.73 mmol/L, P 〈 0.05 ）. BrdU-positive ceils were found in renal tissues in 2 groups on the 2nd,3rd and 4th day, but the Brd U-positive ceils in transplantation group （ 33.71±8.50,57.60±4.88,116.29±6.99） were much more than those in control group （19.67±5.20,26.88±5.89,71.71±8.44 ;P 〈 0.05 ）. On the 3rd day ,the DAPI-positive ceils which partly expressed RCA-positive simultaneously could be found in the renal tissue in transplantation group and they significantly increased （ P 〈 0.05 ） on the 4th day. Conclusions MSCs can migrate and repopulate in the ischemically injured kidney, differentiate into renal tubular ceils and promote ceils proliferation for repair of renal injury.