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目的:探讨核受体亚家族6A1(NR6A1)对血管平滑肌细胞凋亡的影响及可能的分子机制。方法:将腺病毒Ad-NR6A1感染大鼠血管平滑肌细胞,分别在感染后0 h、24 h和48 h时进行MTT实验,以时间为横坐标,A570为纵坐标绘制细胞生长曲线,观察NR6A1对细胞生长的影响;进行DAPI染色、TUNEL染色及caspase活性检测,观察细胞凋亡情况;进一步通过基因芯片技术,寻找NR6A1的靶基因;采用siRNA介导的基因沉默技术,观察受体相互作用丝氨酸/苏氨酸蛋白激酶3(RIPK3)基因沉默对NR6A1诱导的血管平滑肌细胞凋亡的影响。结果:腺病毒Ad-NR6A1感染细胞48 h时,NR6A1过表达组细胞数量较对照组(重组腺病毒载体Ad-Lac Z)明显减少;DAPI染色显示NR6A1过表达诱导血管平滑肌细胞出现核浓缩和核碎裂的凋亡表型,TUNEL染色显示NR6A1过表达引起细胞凋亡,caspase活性检测结果显示NR6A1过表达细胞内caspase-3、caspase-8和caspase-9活性均较对照组高;基因芯片技术检测发现,NR6A1过表达上调血管平滑肌细胞中RIPK3基因表达;RIPK3基因沉默可以显著抑制NR6A1诱导的平滑肌细胞凋亡。结论:NR6A1通过上调RIPK3基因表达诱导血管平滑肌细胞凋亡。
Aims: To investigate the effect of nuclear receptor subfamily 6A1 (NR6A1) on the apoptosis of vascular smooth muscle cells and its possible molecular mechanism. Methods: Ad-NR6A1 cells were infected with Ad-NR6A1 and MTT assay was performed at 0 h, 24 h and 48 h after infection. The growth curve was plotted on the time axis and A570 as the ordinate. The expression of NR6A1 Cell growth; DAPI staining, TUNEL staining and caspase activity assay to observe the cell apoptosis; further through gene chip technology, looking for NR6A1 target gene; using siRNA-mediated gene silencing technology to observe the receptor interaction serine / Effects of Threonine Protein Kinase 3 (RIPK3) Gene Silencing on Apoptosis of Vascular Smooth Muscle Cells Induced by NR6A1. Results: The number of cells in NR6A1 overexpression group was significantly decreased compared with that in control group (Ad-Lac Z recombinant adenovirus) after Ad-NR6A1 infection for 48 h. DAPI staining showed that overexpression of NR6A1 induced nuclear condensation and nuclear condensation in vascular smooth muscle cells Cell apoptosis was detected by TUNEL staining and overexpression of NR6A1. The results of caspase activity assay showed that the activity of caspase-3, caspase-8 and caspase-9 in NR6A1 overexpression cells were higher than those in control group. Gene chip technology The results showed that NR6A1 overexpression up-regulated the expression of RIPK3 in vascular smooth muscle cells. RIPK3 silencing could significantly inhibit NR6A1-induced smooth muscle cell apoptosis. Conclusion: NR6A1 induces apoptosis of vascular smooth muscle cells by up-regulating RIPK3 gene expression.