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目的:研究人孤儿核受体生殖细胞核因子(hGCNF)对H9C2心肌细胞的影响并初探其机制。方法:通过感染Ad-hGCNF腺病毒使H9C2中的GCNF基因表达上调,利用间接免疫荧光化学技术确定其亚细胞定位,通过MTT法研究GCNF表达对细胞活力的影响,DAPI染色检测细胞凋亡水平,实时定量PCR及Western blot技术检测受体结合蛋白激酶2(RIPK2)和受体结合蛋白激酶3(RIPK3)的表达水平。结果:Ad-hGCNF腺病毒感染后,H9C2心肌细胞GCNF表达上调,亚细胞定位位于细胞核,细胞活力降低,凋亡水平显著增加,RIPK2和RIPK3表达水平均升高。结论:感染Ad-hGCNF腺病毒表达载体降低H9C2细胞活力,增加其凋亡水平,机制与RIPK2和RIPK3的表达上调有关。
AIM: To investigate the effect of orphan nuclear receptor-derived germ cell nuclear factor (hGCNF) on H9C2 cardiomyocytes and to explore its mechanism. Methods: The expression of GCNF gene in H9C2 was induced by Ad-hGCNF infection. The subcellular localization of GCNF was determined by indirect immunofluorescence staining. The effect of GCNF expression on cell viability was investigated by MTT assay. The level of apoptosis was detected by DAPI staining. Real-time quantitative PCR and Western blot were used to detect the expression of receptor-bound protein kinase 2 (RIPK2) and receptor-bound protein kinase 3 (RIPK3). Results: After Ad-hGCNF adenovirus infection, the expression of GCNF in H9C2 myocardial cells was up-regulated, the subcellular localization was located in the nucleus, the cell viability was decreased, the apoptosis level was significantly increased, and the expression levels of RIPK2 and RIPK3 were increased. CONCLUSION: Ad-hGCNF adenovirus expression vector reduces the viability of H9C2 cells and increases the apoptosis of H9C2 cells. The mechanism is related to the upregulation of RIPK2 and RIPK3.