大鼠骨髓间充质干细胞内皮分化后诱导同种异体白细胞毒作用

Bone marrow mesenchymal stem cells induce allogeneic leukocytes cytotoxicity after endothelial differentiation

目的探讨骨髓间充质干细胞(mesenchymal stem cells,MSCs)内皮分化前后对同种异体白细胞的耐受能力。方法体外分离培养Wistar大鼠骨髓MSCs,传代扩增,使用含50 ng/ml VEGF和2%FBS的内皮分化诱导培养液诱导分化7 d,倒置相差显微镜观察细胞形态,并免疫组化染色方法鉴定内皮细胞特有标记物vWF和FLK-1。通过混合白细胞试验,将MSCs或内皮样分化后的d-MSCs与同种异体Lewis大鼠外周血白细胞(peripheral blood leukocytes,PBLs)共同培养3 d,检测实验组(+PBLs)和对照组(未加PBLs)上清液中LDH释放量以评价免疫反应程度。结果诱导分化后的d-MSCs细胞形态学上与未分化的MSCs没有明显差异,经免疫组化染色显示d-MSCs表达vWF和FLK-1;MSCs实验组与对照组释放LDH量无统计学差异(P>0.05),d-MSCs实验组与对照组释放LDH量有显著性差异(P<0.05)。结论向内皮细胞分化的d-MSCs诱导了同种异体白细胞毒作用。

Objective To investigate allogeneic leukocytes cytotoxicity induced by bone marrow mesenchymal stem cells（MSCs） before and after endothelial differentiation.Methods MSCs of Wistar rats were separated and purified.The cultural media containing 10 ng/ml VEGF and 2%FBS were used to induce MSCs endothelial differentiation for 7 d.The cell morphology was observed by invert microscope and endothelium specific membrane antigens vWF and FLK-1 were detected with immunohistochemistry staining.The allogeneic peripheral blood leukocytes（PBLs） from Lewis rats were cocultured with MSCs or endothelial differentiated MSCs（d-MSCs） for 3 d.The LDH release in the supernatant of experiment groups（＋PBLs） and control groups were measured to assess the immune reaction.Results The cell morphology of d-MSCs showed no difference with undifferentiated MSCs; immunohistochemistry staining showed positive vWF and FLK-1 in d-MSCs.There were no significant differences in LDH release of MSCs between experiment groups and control groups（P0.05）; however,there were significant differences for d-MSCs between two groups（P0.05）.Conclusion Endothelial differentiated MSCs induce the allogeneic leukocytes cytotoxicity.