山药及干细胞对肾缺血再灌注损伤的保护作用

Protective effect of rhizoma dioscoreae and bone marrow mesenchymal stem cells in rats with renal ischemia-reperfusion injury

目的研究山药灌胃预处理及移植骨髓间充质干细胞(MSCs)对大鼠肾缺血再灌注损伤的保护作用。方法4～6周龄雌性SD大鼠60只,随机分正常对照(A)组(n=6),再灌注损伤(B)组(n=18),再灌注损伤移植MSCs(C)组(n=18)和再灌注损伤山药灌胃移植MSCs(D)组(n=18)。D组损伤前山药灌胃5d,C、D组再灌注1h输注BrdU标记的MSCs,B、C、D组再灌注6、48h及2周各采集6只鼠的血并取肾。4组标本进行血清尿素氮(BUN)及肌酐(SCr)、肾匀浆内皮素(ET)测定,常规病理切片,免疫组化检测增殖细胞核抗原(PCNA)、5-溴脱氧尿嘧啶核苷(BrdU)标记MSCs、Ⅷ因子相关抗原(FⅧ-RAg)阳性细胞情况。结果再灌注6h,C、D组BUN、SCr低于B组,D组低于C组;再灌注48h,仅B组高于A组;2周4组间无统计学意义。再灌注6h,B、C组ET较A组高,D组与A组无统计学意义,C、D组较B组低;48h4组间无统计学意义。再灌注6、48hC、D组于肾小球、肾间质微血管见散在BrdU标记的MSCs,2周则见较多。再灌注6、48h及2周PCNA及FⅧ-RAg阳性细胞,C、D组较B组多,D组较C组多。结论肾缺血再灌注损伤时移植MSCs有保护作用,山药灌胃预处理联合MSCs移植有协同治疗作用。

Objective To investigate the protective effect of rhizoma dioscoreac and bone marrow mesenchymal stem cells in rat with renal ischemia-reperfusion（I-R） injury. Methods Sixty female SD rats （4-6 weeks old） were randomly assigned into normal control（group A,n= 6）, I-R injury （group B,n=18）, bone marrow mesenchymal stem cell （MACs） treatment（group C,n= 18）, and treated with rhizoma dioscoreae and MSCs （group D, n= 18）. The rats in group D were pretreated with rhizoma dioscoreae for five days before being subjected to left renal vascular occlusion for 60 rain,those in group C and D were injected BrdU positive MSCs （1×10^6/ml） 1 ml at 60 rain after reperfusion. Rats in group B, C, and D were randomly euthanized for blood sampling and kidney harvesting at 6,48 h and 2 w after reperfusion. The blood urea nitrogen （BUN） and serum creatinine （SCr） were measured. The renal tissue homogenate content of endothelin（ET） was determined with radioimmunoassay. Renal morphologic changes were studied on hematoxylin and eosin （H＆E） stained sections. PCNA positive cells of renal tubular epithelia and glomeruruls were detected immunohistochemically as proliferation index. The distribution of BrdU positive MSCs and FⅧ-Rag positive cells were also identified immunohistochemically. Results BUN and SCr were significantly lower in group C,D than those in group B at 6 h after reperfusion. At 48 h after reperfusion,BUN and SCr were elevated only in group B and no difference was observed among the four groups 2 w after reperfusion. At 6 h after reperfusion, ET in group B,C was significantly higher than that in group A. ET was significantly lower in group C and D than that in group B, and no difference was observed between group D and group A. At 48 h after repcrfusion, there were no significant differences in ET levels among the four groups. At 6 h and 48 h after reperfusion,BrdU positive MSCs were located in renal interstitium and glomerulus in group C and D. At 2 w after reperfusion, more BrdU positive MSCs were located at peritubular capillary and glomerulus. At 6 h, 48 h and 2 w after reperfusion, PCNA positive cells and FⅧ-Rag positive cells were much more in group C,D than those in group B, which were more in group D than those in group C. Conclusion MSCs are of protective effect on renal LR injury. Rhizoma dioscoreae and MSCs have synergistic action in ameliorating renal I-R injury.