炎性预处理的脂肪干细胞条件培养基对骨性关节炎软骨细胞表型的调节作用

Regulatory effect of inflammatory preconditioned adipose stem cell conditioned medium on phenotype of osteoarthritis chondrocytes

目的探讨炎性因子TNF-α、IL-1β和IFN-γ预处理脂肪间充质干细胞(Adipose mesenchymal stem cells,ADMSCs)是否可以改变其分泌组的生物学特性并分析其对人骨性关节炎软骨细胞的表型产生影响。方法获取并培养AD-MSCs和人骨性关节炎软骨细胞,分别使用工作浓度为10 ng/mL的TNF-α、IL-1β、IFN-γ的条件培养液和对照培养液(DMEM-F12+2%FBS)预处理AD-MSCs并收集对应的条件培养基(CM-1组、CM-2组、CM-3组和CM-0组),各组分别培养骨性关节炎软骨细胞72 h后,通过免疫荧光染色、RT-PCR和Western Blot法检测软骨细胞SOX-9、COL2、RUNX2、MMP13的表达情况,所有细胞实验均重复3次,比较4组SOX9、COL2、MMP13、RUNX2表达水平。结果CM-1组、CM-2组、CM-3组AD-MSCs较CM-0组细胞明显变得扁平、粗大。CM-1组、CM-2组、CM-3组SOX9、COL2的表达水平较CM-0组高,MMP13表达水平较CM-0组低,差异有统计学意义(P<0.05)。在基因水平上4组RUNX2的表达水平差异无统计学意义(P>0.05)。结论采用炎性因子预处理MSCs,形成的条件培养基可以明显增强骨性关节炎软骨细胞的成软骨能力,并降低骨性关节炎软骨细胞的肥大化和降解代谢水平。

ObjectiveTo investigate whether pretreatment of adipose mesenchymal stem cells(AD-MSCs)with inflammatory factors TNF-α,IL-1βand IFN-γcan change the biological characteristics of their secretome and analyze their effect on human osteoarthritis phenotype of chondrocytes.MethodsAD-MSCs and human osteoarthritis chondrocytes were obtained and cultured,the TNF-α,IL-1β,and IFN-γconditioned medium with a working concentration of 10 ng/mL and the control medium(DMEM-F12+2%FBS)were used respectively to pretreat AD-MSCs and the corresponding conditioned medium(CM-1 group,CM-2 group,CM-3 group and CM-0 group)were collected.After culturing osteoarthritis chondrocytes for 72 h in each group,the expressions of SOX-9,COL2,RUNX2,and MMP13 in chondrocytes were detected by immunofluorescence staining,RTPCR and Western Blot.All cell experiments were repeated three times,and the expression levels of SOX9,COL2,MMP13,and RUNX2 in the four groups were compared.ResultsCompared with CM-0 group,AD-MSCs in CM-1 group,CM-2 group and CM-3 group became flat and thick.The expression levels of SOX9 and COL2 in CM-1 group,CM-2 group and CM-3 group were higher than those in CM-0 group,and the expression level of MMP13 was lower than that in CM-0 group,and the difference was statistically significant(P<0.05).There was no significant difference in the expression levels of RUNX2 among the four groups at the gene level(P>0.05).ConclusionThe conditioned medium after pretreatment of MSCs with inflammatory factors can significantly enhance the chondrogenic capacity of osteoarthritis chondrocytes,and reduce the hypertrophy and degradation metabolism of osteoarthritis chondrocytes.