后肾间充质细胞对大鼠急性肾小管损伤的防护作用

Protective effects of metanephric mesenchymal cells on acute renal tubular damage： experiment with rats

目的观察外源性后肾间充质细胞（MMCs）对缺血再灌注（I／R）后急性肾小管损伤的防护作用，并探讨其可能的机制。方法取孕13d大鼠胚胎分离培养后肾间充质细胞，采用5-溴脱氧尿嘧啶核苷（BrdU）标记。建立缺血再灌注急性肾小管损伤大鼠模型，SD大鼠随机分为MMCs组、I／R组和假手术对照组（sham），MMCs组大鼠在成功建立肾脏缺血再灌注损伤后，即经下腔静脉输注BrdU标记后的大鼠后肾间充质细胞，分别于术后24、48、72、96h处死各组大鼠，自动生化仪检测血清尿素氮（BUN）及血清肌酐（Scr）水平，常规HE染色观察肾脏病理改变，原位末端标记法（TUNEL）检测肾小管上皮细胞凋亡情况，免疫组织化学染色法观察凋亡调控蛋白Bcl-2、Bax在肾脏表达，荧光显微镜观察标记的MMCs在肾组织中的定位分布。结果MMCs组各时相点大鼠BUN、Scr水平均显著低于同时相点I／R组（均P〈0．05），TUNEL结果显示，术后48h凋亡的肾小管上皮细胞数量MMCs组[（13．4±3．2）／HPF]明显少于同时相点I／R组[（25．4±5．2）／HPF]（P〈0．05），免疫组化结果显示I／R组Bcl-2、Bax蛋白表达水平均高于sham组，48h达高峰，MMCs组Bcl-2表达水平高于同时相点I／R组，而Bax表达水平则低于同时相点I／R组，Bcl-2／Bax比值明显升高（均P〈0．05）。MMCs组术后72h肾组织内可见BrdU阳性细胞[（60±6）／HP]，术后96h阳性细胞明显增多，为[（143±8）／HP]。结论外源性后肾间充质细胞能够迁移、定居于缺血再灌注损伤后的肾脏小管上皮，MMCs输注后可抑制肾小管上皮细胞凋亡、坏死及炎症细胞浸润，对缺血再灌注急性肾小管损伤具有防护作用。

Objective To study the protective effects of metanephric mesenchymal ceils （MMCs） on acute renal tubular damage and explore its possible mechanism. Methods MMCs were isolated and cultured from 13-day-old embryonic rats and labeled with 5-bromodeoxyuridine. Seventy-two male SD rats were randomly divided into 3 equal groups： MMC group, receiving MMC injection instantaneously when ischemia/reperfusion （I/R） renal injury was induced, I/R group, undergoing I/R to establish acute renal tubular damage models, and sham operation group. Six rats from each group were killed at different time points： 24 h, 48 h, 72 h, and 96 h later. Blood sample was collected from the vena cava inferior, to examine the serum creatinine （SCr） and blood urea nitrogen （BUN）. Specimens of kidney underwent microscopy. Apoptosis was conformed by TUNEL assay. Immunohistochemistry was used to detect the protein expression of Bcl-2 and Bax. The distribution of MMCs labeled with 5-bromodeoxyuridine in kidney was observed by immunofluorescence technique. Results The SCr and BUN levels in different time points of the MMC group were both significantly lower than those of the I/R group （ both P 〈 0.05 ）, HE staining showed that pathological damage of the MMC group was less than that of the I/R group （ P 〈 0.05 ）. TUNEL results investigated that the number of apoptosis renal tubular epithelial ceils of the MMC group was （ 13.4 ± 3.2/HPF） , significantly less than that of the I/R group [ （25.4 ± 5.2/HPF） ]. In comparison with the I/R group, there were more Bcl-2 positive cells and fewer Bax positive cells in the MMC group. BrdU-labeled MMCs began to occur in the renal tissue （60 ±6/HP） In the 72 h subgroup of MMC group, and number of BrdU-labeled MMCs, the 96 h subgroup was （ 143 ± 8/HP）, significantly higher than that of the 72 h subgroup （P 〈 0.05）. Conclusion MMCs have the ability to protect renal function in acute renal tubular damage in rats, migrate and repopulate in the I/R injured renal tubules, and inhibits renal tubular epithelial cell apoptosis. The mechanism may be involved in regulating the expression of Bcl-2 and Bax.