骨髓间充质干细胞对移植肾缺血再灌注损伤保护作用的实验研究

Effect of intravenous administered mesenchyml stem cells on ischemia-reperfusion-induced renal dysfunction of renal transplantation in a rat model

目的探讨骨髓间充质干细胞(MSCs)对移植肾缺血再灌注损伤(IRI)模型修复的保护作用及其作用机制。方法 (1)采用密度梯度离心法结合贴壁分离法分离培养纯化SD大鼠骨髓MSCs,观察其形态,流式细胞仪检测细胞表面标记,检测骨髓MSCs向成骨和成脂细胞分化的潜能;(2)成年雌性SD大鼠28只,随机分组:正常对照组(n=6),假手术对照组(n=6),移植肾IRI组(n=8),经尾静脉输注间充质干细胞(MSCs)移植肾IRI组(n=8)。检测肾功能指标血尿素氮(BUN)和肌酐(Cr)水平变化,评定肾小管的凋亡指数和增殖指数,测定肾组织起氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性及微量丙二醛(MDA)水平,以及对肾脏病理学变化进行观察。结果 (1)分离培养的骨髓MSCs纯度高、生物学特征稳定;(2)移植肾IRI组肾功能指标[BUN(36.9±4.8)mmol/L,Scr(279.9±22.6)mmol/L]、氧化应激指标明显升高,组织形态学出现肾间质水肿明显,肾小管上皮细胞空泡样变性,近曲小管管壁肿胀,管腔变小。而经尾静脉输注MSCs移植肾IRI组大鼠肾功能指标[BUN(22.6±7.8)mmol/L,Scr(223.6±26.7)μmol/L]和氧化应激指标得到明显改善(F=57.616,243.789,P<0.001),组织形态学肾小管上皮细胞细胞核固缩、碎裂和溶解等细胞坏死和变性征象明显减轻,肾小管上皮细胞增殖指数(PI)高于IRI组,肾小管上皮细胞凋亡指数(AI)低于IRI组,两组间差异有统计学意义(F=23.689,P<0.001)。结论骨髓MSCs输注能促进肾脏IRI损伤后肾脏细胞增殖,抑制肾脏细胞凋亡,降低血清Cr和BUN,在一定程度上促进IRI后肾功能的恢复,通过抑制氧自由基的生成减轻肾组织的损伤程度,改善肾功能。

[Abstract] Objective To investigate the effects of intravenous administered mesenchymal stem cells （MSCs） on ischemia/reperfusion （I/R） injury and the underlying mechanisms. Methods Transplanting kidney ischemia-reperfusion injury （IRI）was established. Immediately after visual confirmation of reflow, 1 ×10^6 MSCs were administered by intravenous injection, followed by reperfusion for 24 h. The kidney functions, tissue malondialdehyde （MDA）, superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px） levels were evaluated. Histopathological and immunohistochemistry examinations were performed. Results Pure MSCs could be obtained with stable biological properties. The functuion of IRI kidney was markedly injuried [BUN （36.9± 4.8） m mol/L, Scr （279.9 ± 22.6） m mol/L], accompanied by higher oxidation stress index.MSCs infusion significantly improved kidney function as indicated by lower urea and creatinine levels in the MSCs compared to the vehicle group （P 〈 0.05）. I/R-induced reduction in renal tissue SOD enzyme activity and GSH-Px was significantly improved by MSCs （ F = 57.616, 243.789, P 〈 0.001）. Treatment with MSCs also resulted in significant reduction in renal tissue MDA levels that were in creased by renal I/R injury （F = 23.689, P 〈 0.001）. At histological examination,the kidneys of MSCs-treated rats showed a fairly normal morphology. The percents of proliferative cell nuclear antigen （PCNA） -positive cells were higher in MSCs groups.The percents of terminal transferase-mediated dUTP nick- end labeling （TUNEL）-positive cells were lower in MSCs group. Conclusions BMSCs can promote cell proliferation and suppress apoptosis after renal IRI and decrease the serum levels of urea and creatinine, and promote the renal functional repair.MSCs protects the kidneys against I/R injury at least via their antioxidant effects.