炎症诱导缺血缺氧环境下骨髓间充质干细胞的活性

Viability of bone marrow mesenchymal stem cells in inflammation-induced ischemia-reperfusion environment

背景:局部组织缺血缺氧和炎症微环境因素是造成骨髓间充质干细胞生存率低的主要原因。目的:探讨炎症环境下缺血缺氧对骨髓间充质干细胞活性的影响以及细胞活性与炎症因子之间的关系。方法:采用全骨髓培养法提取培养骨髓间充质干细胞,流式细胞仪对细胞进行鉴定,将第3代细胞进行不同时间氧糖剥夺(6,12,24 h)和炎症诱导(氧糖剥夺、氧糖剥夺+20μg/L肿瘤坏死因子α、氧糖剥夺+100μg/L脂多糖),倒置显微镜观察细胞生长状况,MTT法和流式细胞仪测定细胞活力,ELISA检测上清液中白细胞介素6、白细胞介素10、肿瘤坏死因子α水平,RT-PCR检测细胞中白细胞介素6、白细胞介素10、肿瘤坏死因子α、NF-κB基因表达水平。结果与结论:(1)成功分离培养大鼠骨髓间充质干细胞且纯度为95%以上;(2)氧糖剥夺6 h和12 h细胞活力明显增强,氧糖剥夺24 h细胞活力明显下降;在肿瘤坏死因子α和脂多糖诱导下氧糖剥夺6,12,24 h后细胞活力下降;(3)与正常条件比较,氧糖剥夺6,12 h后白细胞介素6、白细胞介素10水平和白细胞介素6、白细胞介素10、肿瘤坏死因子α、NF-κB基因表达下降;(4)与氧糖剥夺条件比较,在肿瘤坏死因子α和脂多糖诱导下氧糖剥夺6,12h后白细胞介素6、肿瘤坏死因子α水平和白细胞介素6、白细胞介素10、肿瘤坏死因子α、NF-κB基因表达显著升高;(5)由此可见,在肿瘤坏死因子α和脂多糖诱导下,骨髓间充质干细胞活力受炎症因子表达和分泌影响,随着炎症因子表达和分泌增加,骨髓间充质干细胞的活力降低。

BACKGROUND:Local tissue ischemia-hypoxia and inflammatory microenvironment mainly contribute to the low survival rate of bone marrow mesenchymal stem cells.OBJECTIVE:To study the effect of ischemia-hypoxia on viability of bone marrow mesenchymal stem cells in inflammatory environment and to explore the relationship between cell viability and inflammatory factors.METHODS:The whole bone marrow culture method was used to separate and culture bone marrow mesenchymal stem cells and flow cytometry was used for cell identification.The third passage cells were treated by oxygen-glucose deprivation(OGD)preconditioning for different periods(6,12 and 24 hours)as well as inflammatory induction(OGD,OGD+20μg/L tumor necrosis factor alpha(TNF-α),OGD+100μg/L lipopolysaccharide(LPS)).The treated cells were observed under inverted microscope.Cell viability was tested by MTT assay and flow cytometry.The levels of interleukin-6,interleukin-10,and TNF-αin the cell supernatant were measured by ELISA and the mRNA expression levels of interleukin-6,interleukin-10,TNF-αand nuclear factor-κB in the cells were detected by RT-PCR.RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cells with a purity of 95%or more were successfully isolated and cultured.MTT and flow cytometry results showed that the cell viability was significantly increased after 6 and 12 hours of OGD,but was significantly decreased after 24 hours of OGD.The viability of bone marrow mesenchymal stem cells decreased after 6,12,and 24 hours of OGD under the induction of TNF-αand lipopolysaccharide.The results of ELISA and RT-PCR showed that the levels of interleukin-6 and interleukin-10 in the cell supernatant were lowered after 6 and 12 hours of OGD and the gene expression of interleukin-6,interleukin-10,TNF-α,and nuclear factor-κB was lower compared with normal conditions.The levels of interleukin-6 and TNF-αin the cell supernatant increased significantly and the expression of interleukin-6,interleukin-10,TNF-αand nuclear factor-κB genes increased significantly after 6,12 hours of OGD under TNF-αand LPS induction compared with simple OGD.Therefore,under the induction of TNF-αand lipopolysaccharide,the viability of bone marrow mesenchymal stem cells is affected by the expression and secretion of inflammatory factors.With the increase of the expression and secretion of inflammatory factors,the viability of bone marrow mesenchymal stem cells shows a decreasing trend.