摘要
目的探讨过表达miR-155对强直性脊柱炎滑膜细胞凋亡的影响及作用机制。方法选择2018年5-7月四川省遂宁市中心医院风湿免疫科收治的强直性脊柱炎患者,取其滑膜细胞进行试验。构建miR-155慢病毒表达载体,并在293 T细胞中获得重组慢病毒,经感染NP细胞得到稳定过表达细胞系GV369-miR-155-NP(GV369-miR-155-NP组),同时设置空载体GV369-NP组和空白组。荧光显微镜观察慢病毒载体的标签蛋白GFP的表达,RT-qPCR方法检测miR-155的表达情况,流式细胞术检测细胞凋亡,荧光素酶报告基因分析验证miR-155与FasL的靶向关系,Western-blot法检测细胞中凋亡相关蛋白FADD、Caspase-3、Bcl-2及Bax的表达,试剂盒检测细胞线粒体膜电位的变化情况。结果在荧光显微镜下观察,经慢病毒感染的过表达细胞系和空载体细胞系均出现绿色荧光,而空白组细胞未见绿色荧光。与空白组比较,GV369-miR-155-NP组中miR-155、Bcl-2表达水平和线粒体膜电位明显升高,而细胞凋亡率、FADD、Caspase-3和Bax的表达水平均明显下降,差异均有统计学意义(t/P=12.399/0.000、8.658/0.000、4.879/0.002、12.612/0.000、8.450/0.000、7.711/0.000、6.977/0.001);GV369-NP组与空白组比较差异无统计学意义(P>0.05)。荧光素酶报告基因分析证实miR-155与FasL存在靶向关系。结论miR-155可抑制强直性脊柱炎滑膜细胞发生凋亡,既可通过靶向调控外源性FasL/Fas通路参与Caspase-3和FADD介导的细胞凋亡,也可通过线粒体途径对细胞凋亡发挥作用。
Objective To investigate the effect and mechanism of overexpression of miR-155 on apoptosis of synovial cells in ankylosing spondylitis.Methods From May to July 2018,the synovial cells of patients with ankylosing spondylitis who were admitted to the Rheumatology and Immunology Department of Suining Central Hospital in Sichuan Province were selected and tested.To construct miR-155 Lentivirus Expression Vector,and obtain recombinant lentivirus in 293 T cells.After infecting NP cells,stable over expression cell line gv369 miR-155 NP(gv369 miR-155 NP group)was obtained.At the same time,empty vector gv369-NP group and blank group were set up.The expression of GFP,miR-155,apoptosis were detected by RT-qPCR,the targeting relationship between miR-155 and FasL was verified by luciferase reporter gene analysis,FADD,caspase 3,Bcl-2 and Bax were detected by Western-blot,and mitochondrial membrane was detected by kit Change of potential.Results Observation under a fluorescence microscope showed that both the over-expressing cell line and the empty vector cell line infected with lentivirus appeared green fluorescence,but no green fluorescence was observed in the blank group cells.Compared with the blank group,the expression levels of miR-155,Bcl-2 and mitochondrial membrane potential in the GV369 miR-155 NP group were significantly increased,while the apoptosis rate,FADD,Caspase-3 and Bax expression levels were significantly reduced,and the differences were statistically significant Significance(t/P=12.399/0.000,8.658/0.000,4.879/0.002,12.612/0.000,8.450/0.000,7.711/0.000,6.977/0.001).There was no significant difference between the GV369-NP group and the blank group(P>0.05).Analysis of the luciferase reporter gene confirmed that miR-155 has a targeting relationship with FasL.Conclusion The miR-155 can inhibit apoptosis of synovial cells in ankylosing spondylitis.It can not only participate in Caspase-3 and FADD mediated apoptosis by targeting and regulating exogenous FasL/Fas pathway,but also play a role in apoptosis through mitochondrial pathway.
作者
苟玉萧
吴霞
陈龙
汪佳利
Gou Yuxiao;Wu Xia;Chen Long;Wang Jiali(Department of Rheumatology and Immunology,Suining Central Hospital,Sichuan Province,Suining 629000,China)
出处
《疑难病杂志》
CAS
2020年第3期285-291,共7页
Chinese Journal of Difficult and Complicated Cases
