RNA干扰HIF-1α对类风湿关节炎成纤维样滑膜细胞的VEGF表达及增殖的影响  

Effects of shRNA-HIF-1α-mediated gene silencing on VEGF expression and proliferation of fibroblast-like synoviocytes in patients with rheumatoid arthritis

作　　者：陈勇[1] 彭勇[1] 于静雅[1] 邬秀娣[1] 罗晶[2] 张振[1] 干敏芝[1] 黄娴倩[1] 章海均[1] 

机构地区：[1]宁波市第二医院风湿免疫科,315010 [2]宁波市第六医院风湿免疫科

出　　处：《浙江医学》2014年第14期1205-1209,共5页Zhejiang Medical Journal

基　　金：宁波市社会发展科研项目基金资助(2012C50010);宁波市自然基金项目(2013A610259)

摘　　要：目的探讨shRNA特异性干扰沉默缺氧诱导因子-1α[(HIF-1α)对类风湿关节炎(RA)成纤维样滑膜细胞(FLS)的血管内皮生长因子(VEGF)表达及其增殖的影响。方法获取9例RA患者滑膜组织进行RA-FLS的分离培养及传代。将FLS分为4组:常氧组、缺氧组、阴性质粒组(转染阴性质粒)和阳性质粒组(转染HIF-1α阳性质粒),常氧组进行常规培养,后3组进行缺氧培养12h;采用Western blot和qRT-PCR法分别检测各组FLS中HIF-1α和VEGF以及HIF-1α和VEGF mRNA表达情况。将缺氧组、阴性质粒组和阳性质粒组的FLS分别于缺氧条件下培养6、12、24、48h,应用MTT比色法检测各组细胞增殖情况。结果与常氧组相比,缺氧组FLS的HIF-1α(和VEGF以及HIF-1α和VEGF mRNA表达明显升高,差异均有统计学意义(均P<0.05),而阴性质粒组和缺氧组FLS的HIF-1α和VEGF以及HIF-1α和VEGF mRNA表达水平无明显差异(P>0.05);阳性质粒组FLS的HIF-1α和VEGF以及HIF-1α和VEGF mRNA较缺氧组下调,差异均有统计学意义(均P<0.05)。4组FLS的HIF-1α和VEGF以及HIF-1α和VEGFmRNA表达呈正相关(P<0.01)。缺氧培养12h后阳性质粒组的FLS生长较缺氧对照组及阴性质粒组减慢,差异有统计学意义(P<0.05),而后两组FLS生长无统计学差异(P>0.05)。结论 RNA干扰HIF-1 oc表达能有效下调RA-FLS中HIF-1α和VEGF以及HIF-1α和VEGF mRNA水平,并且能抑制FLS的异常增殖。Objective To investigate the effects of shRNA-HIF-1 α-mediated gene silencing on VEGF expression and proliferation of fibroblast-like synoviocytes （FLS） in patients with rheumatoid arthritis in vitro. Methods The fibroblast-like synoviocytes were isolated and cultured from synovial tissue of 9 patients with rheumatoid arthritis. The cultured FLSs were divided into 4 groups： hypoxic group, normoxic group, the negative control group （transfected with blank plasmids） and the positive group （transfected with HIF-1 α-shRNA）. The cells in normoxic group were incubated under normoxia, and cells in other three groups were incubated under hypoxia. The expression of HIF-1α and VEGF protein and mRNA were detected by Western blot and qRT-PCR, respectively. The cells in hypoxia, negative control and positive groups were incubated under hypoxia for 6, 12, 24 and 48 hours. The proliferation of RA-FLS was examined by MTT. Results Higher expressions of HIF-1α and VEGF protein and mRNA were detected in hypoxic group compared with those in normoxia group （P〈0.05）. There were no significant differences in protein and mRNA expression of HIF-1α and VEGF between normoxic group and negative control group （P 〉0.05）. Compared with the normoxic group and the negative control group, the expression of HIF-1α and VEGF protein and mRNA in positive group were significantly lower （P〈0.05）. The expression of HIF-1α were positively correlated with VEGF in all 4 groups （P〈0.05）. MTT assays showed that the FLS proliferation in positive group were slower than that in normoxic and negative control groups （P〈0.05）. Conclusion The plasmids of HIF-1α-shRNA can inhibit HIF-1α and VEGF expression and cell proliferations of fibroblast-like synoviocytes in patients with rheumatoid arthritis .

关 键 词：类风湿关节炎 成纤维样滑膜细胞 RNA干扰 细胞增殖 缺氧诱导因子-1α 血管内皮生长因子 Hypoxia-inducible factor-1α 

分 类 号：R593.22[医药卫生—内科学]