IGF-1过表达的骨髓间充质干细胞保护软骨细胞免受IL-1β诱导的损伤

IGF-1 overexpressed bone marrow mesenchymal stem cells protect chondrocytes from IL-1β-induced injury

目的探讨胰岛素样生长因子-1(IGF-1)过表达的骨髓间充质干细胞(BMSCs)对白细胞介素1β(IL-1β)诱导的软骨细胞损伤的影响及相关机制。方法慢病毒转染构建IGF-1过表达的BMSCs,使用倒置显微镜观察细胞形态,流式细胞仪鉴定骨髓间充质干细胞表面抗原表达,实时定量PCR和Western blot法检测IGF-1 mRNA及蛋白表达情况。将IGF-1慢病毒转染的BMSCs、空载体慢病毒转染的BMSCs、BMSCs分别与5 ng/mL IL-1β诱导的大鼠软骨细胞共同培养24 h后,CCK-8检测软骨增殖情况;Tunel法检测软骨细胞凋亡率;Western blot法检测Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、细胞色素C(Cytc)蛋白表达;酶联免疫吸附试验(ELISA)检测细胞提取液中IL-1β、白细胞介素6(IL-6)和肿瘤坏死因子-α(TNF-α)水平。结果IGF-1过表达的BMSCs提高IL-1β处理的软骨细胞增殖活性,抑制IL-1β诱导的软骨细胞凋亡,并降低细胞提取液中IL-1β、IL-6和TNF-α水平(P<0.05),下调软骨细胞中Bax、Caspase-3、Cytc等凋亡蛋白的表达水平,上调抗凋亡蛋白Bcl-2的表达水平(P<0.05)。结论IGF-1过表达的骨髓间充质干细胞可抑制IL-1β诱导的软骨细胞凋亡及炎性因子的表达,提高软骨细胞增殖活性。

Objective Exploring the effects of IGF-1 overexpression on IL-1βin bone marrow mesenchymal stem cells(BMSCs)the effects and related mechanisms of induced chondrocyte damage.Methods Overexpression BMSCs of IGF-1 was constructed by transfection of lentivirus.The cell morphology was observed by inverted microscope,the surface antigen expression of bone marrow mesenchymal stem cells was identified by flow cytometry,the mRNA and protein expression of IGF-1 gene was detected by real-time quantitative PCR and Western blot,and the BMSCs and BMSCs transfected with empty body lentivirus were compared with 5 ng/mL IL-1βinduced rat chondrocytes were co-cultured for 24 hours.CCK-8 was used to detect chondrocyte proliferation,Tunel method was used to detect chondrocyte apoptosis rate.Western blot was used to detect protein expression of Bax,Bcl-2,Caspase-3,and Cytc,and ELISA was used to detect the level of IL-1β,IL-6 and TNF-α.Results BMSCs overexpressing IGF-1 was increased the proliferation activity of chondrocytes treated with IL-1β,inhibited the apoptosis of chondrocytes induced by IL-1β,and it was decreased the levels of IL-1β,IL-6 and TNF-αin the cell extract(P<0.05),down-regulated the expression levels of apoptosis proteins such as Bax,Caspase-3 and Cytc,and up-regulated the expression level of anti-apoptosis protein Bcl-2(P<0.05).Conclusion The apoptosis of chondrocytes and the expression of inflammatory factors induced by IL-1βin bone marrow mesenchymal stem cells may play a role in improving the proliferation of chondrocytes by regulating IGF-1.