碱性成纤维细胞生长因子对肿瘤坏死因子-α联合放线菌酮协同诱导的人软骨细胞损伤的保护作用及机制研究

Protective effect and mechanism of basic fibroblast growth factor on human chondrocyte injury induced by tumor necrosis factor-α combined with cycloheximide

目的探究碱性成纤维细胞生长因子(bFGF)对人软骨细胞在体外炎症环境下的保护作用及相关机制。方法采用50 ng/ml肿瘤坏死因子(TNF-α)联合1μmol/L放线菌酮(CHX)建立人原代软骨细胞损伤模型并评估bFGF对损伤模型的影响。bFGF低、高剂量给药组是在模型组给药的基础之上分别给予1、10 ng/ml的bFGF。在bFGF给药48 h后采用MTT法检测其对模型细胞活力的影响,bFGF处理24 h后采用流式细胞术探究其对模型细胞凋亡和线粒体膜电位的影响,采用Western bolt检测bFGF处理24 h后对模型细胞凋亡相关蛋白表达的影响。结果模型组(1μmol/L CHX+50 ng/ml TNF-α)的细胞活力明显低于对照组,差异有高度统计学意义(P<0.01);bFGF低剂量和高剂量组细胞的细胞活力高于模型组,差异有统计学意义(P<0.05)。流式结果显示,bFGF低剂量和高剂量组的凋亡率低于模型组,差异有统计学意义(P<0.05)。JC-1染色结果显示,bFGF高剂量组细胞FITC阳性比率低于模型组,差异有高度统计学意义(P<0.01)。Western bolt显示,bFGF给药组细胞Bcl-2蛋白的表达水平高于模型组,Bax蛋白的表达水平低于模型组,差异有统计学意义(P<0.05)。结论bFGF可以通过抑制线粒体膜损伤和调控凋亡相关蛋白Bax和Bcl-2的表达等机制,对炎症环境下软骨细胞的损伤产生保护作用,具有潜在的应用前景。

Objective To investigate the protective effect and related mechanisms of basic fibroblast growth factor(bFGF)on human chondrocytes in an inflammatory environment in vitro.Methods Primary human chondrocyte injury model was established using 50 ng/ml tumor necrosis factor-α(TNF-α)combined with 1μmol/L cycloheximide(CHX)and the effects of bFGF on the injury model were assessed.Low and high doses of bFGF groups were administered extra 1,10 ng/ml bFGF based on model group,respectively.The effects of bFGF on the viability of model cells were detected by MTT assay after treatment for 48 hours,the effects of bFGF on apoptosis and mitochondrial membrane potential were investigated by flow cytometry after after 24 hours treatment,while the effects of bFGF on the expression of apoptosis-related proteins in model cells were detected by Western bolt after treatment for 24 hours.Results The cell viability of the model group(1μmol/L CHX+50 ng/ml TNF-α)was significantly lower than that of the control group,and the difference was highly statistically significant(P<0.01);the cell viability of bFGF low-dose and high-dose groups were higher than that of the model group,and the differences were statistically significant(P<0.05).Flow cytometry results showed that the apoptosis rate of bFGF low-dose and high-dose groups was lower than that of the model group,and the difference was statistically significant(P<0.05).The results of JC-1 staining showed that the positive rate of FITC cells in the bFGF high-dose group was lower than that in the model group,and the difference was highly statistically significant(P<0.01).Western bolt showed that the expression level of Bcl-2 protein in the bFGF administration group was higher than that in the model group,while the expression level of Bax protein was lower than that in the model group,and the differences were statistically significant(P<0.05).Conlusion bFGF can protect chondrocytes from damage in inflammatory environment by inhibiting mitochondrial membrane damage and regulating the expression of apoptosis-related proteins Bax and Bcl-2,which has potential application prospects.