IL-10基因修饰的BMSCs对大鼠脑缺血再灌注损伤炎性因子及神经细胞凋亡的影响

Effects of interleukin 10 gene modified bone marrow mesenchymal stem cells on expression of inflammatory cytokines and neuronal apoptosis in rats after cerebral ischemia reperfusion injury

目的探讨IL-10基因修饰的BMSCs对大鼠脑缺血再灌注损伤(ischemia reperfusion injury,IRI)炎性因子及神经细胞凋亡的影响。方法取SD大鼠骨髓,采用贴壁培养法分离培养BMSCs,经免疫组织化学染色鉴定后,取第3代BMSCs以重组腺病毒(recombinant adenovirus,Ad)介导IL-10基因转染(Ad IL-10-BMSCs)。取40只清洁级成年SD雄性大鼠,采用线栓法制备大脑中动脉阻塞模型后随机分为4组,每组10只。模型制备后3 h于各组大鼠尾静脉注射1 m L含10%FBS的L-DMEM培养液(A组)、61.78 ng IL-10(B组)、1 m L BMSCs悬液(细胞浓度为2×10~6个/m L,C组)、1 m L Ad IL-10-BMSCs悬液(细胞浓度2×10~6个/m L,D组)。C、D组细胞移植前采用Brd U标记。7 d后大鼠经颈总动脉灌注处死取脑组织,免疫染色法检测小胶质细胞(OX42)表达,ELISA法测定TNF-α、IL-1β含量,TUNEL法检测神经细胞凋亡;取C、D组组织行Brd U免疫荧光染色观察。结果C、D组脑组织中均见呈绿色荧光的Brdu阳性细胞,主要分布于梗死区周围的纹状体、大脑皮质、皮质下等。免疫组织化学染色示,B、C、D组OX42阳性细胞数明显少于A组,D组少于B、C组,比较差异有统计学意义(P<0.05)。ELISA检测示D组大鼠脑组织中TNF-α、IL-1β含量较其余3组明显降低(P<0.05)。TUNEL法检测各组凋亡细胞(TUNEL阳性细胞)主要见于梗死灶周围的纹状体及额顶皮质下脑组织(相当于缺血半暗带),D组与A、B、C组比较差异有统计学意义(P<0.05)。结论Ad IL-10-BMSCs能通过抑制小胶质细胞分泌促炎性因子TNF-α、IL-1β,抑制梗死灶周围脑组织神经细胞凋亡,对大鼠脑IRI起保护作用。

Objective To explore the effects of interleukin 10 （IL-10） gene modified bone marrow mesenchymal stem cells （BMSCs） on the expression of inflammatory cytokines and neuronal apoptosis in rats after cerebral ischemia reperfusion injury. Methods BMSCs were cultured by whole bone marrow adherence screening method. The properties of BMSCs were identified by immunocytochemical methods. BMSCs at passage 3 were transfected with recombinant adenovirus IL-10 gene （AdIL-10-BMSCs）. The model of middle cerebral artery occlusion was made in 40 adult male Sprague Dawley rats by thread embolism method. The rats were randomly divided into 4 groups （n=10）. At 3 hours after modelling, the rats of groups A, B, C, and D received tail intravenous injection of 1 mL L-DMEM medium containing 10% FBS, 61.78 ng IL-10, 1 mL BMSCs suspension （2×10^6 cells/mL）, and 1 mL AdIL-10-BMSCs cell suspension （2×10^6 cells/mL）, respectively. The cells were labelled with BrdU before cell transplantation in groups C and D. At 7 days after reperfusion, the brain tissue was harvested to detect the expression of OX42 by immunohistochemical assay, to determine the concentration of tumor necrosis factor α （TNF-α） and IL-1 β by ELISA, and to detect the apoptosis by TUNEL assay.BrdU labelled cells were observed by immunofluorescence staining in groups C and D. Results BrdU labelled positive cells with green fluorescence were observed in the brain tissue of groups C and D, which mainly distributed in the striatum, cerebral cortex, and subcortex around the infarction area. The number of OX42 positive cells was significantly less in groups B, C, and D than group A （P〈0.05）, and in group D than groups B and C （P〈0.05）. Compared with the other 3 groups, the contents of TNF-α and IL-1βsignificantly decreased in group D （P〈0.05）. TUNEL assay showed that the apoptotic cells （TUNEL positive cells） were mainly seen in the striatum and fronto parietal subcortical tissues （equivalent to ischemic penumbra）. The number of TUNEL positive cells in group D was significantly less than that in groups A, B, and C （P〈0.05）. Conclusion AdlL-10-BMSCs can inhibit secretion of TNF-α and IL-1β from microglial cells and inhibit the nerve cell apoptosis around infarct brain tissue, which might contribute to its protective role upon cerebral ischemia reperfusion injury.