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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 effect of shRNA-specific silencing hypoxia inducible factor-1α (HIF-1α) on the expression of vascular endothelial growth factor (VEGF) and its proliferation in rheumatoid arthritis (RA) fibroblast-like synoviocytes . Methods The synovial tissue from 9 patients with RA was obtained for the isolation, culture and passage of RA-FLS. FLS were divided into 4 groups: normoxia group, hypoxia group, negative plasmid group (transfected negative plasmid) and positive plasmid group (transfected with HIF-1α positive plasmid), normoxia group were routinely cultured, Oxygen for 12 hours. The expressions of HIF-1α and VEGF, HIF-1α and VEGF mRNA in FLS were detected by Western blot and qRT-PCR respectively. The FLS of hypoxia group, negative plasmid group and positive plasmid group were cultured in hypoxia for 6, 12, 24 and 48 hours respectively. Cell proliferation was detected by MTT assay. Results Compared with normoxia group, the expression of HIF-1α (and VEGF) and HIF-1α and VEGF mRNA in FLS of hypoxia group were significantly increased (all P <0.05), but negative plasmid group HIF-1α, VEGF and HIF-1α and VEGF mRNA levels in FLS of oxygen group were not significantly different (P> 0.05). HIF-1α, VEGF, HIF-1α and VEGF mRNA in FLS of positive plasmid group were lower than those in hypoxia group, (P <0.05) .4 The expression of HIF-1α, VEGF, HIF-1α and VEGFmRNA in FLS of 4 groups were positively correlated with each other (P <0.01) Oxygen control group and negative plasmid group slowed down, the difference was statistically significant (P <0.05), but there was no significant difference in FLS growth between the latter two groups (P> 0.05) .Conclusion RNA interference of HIF-1 oc expression can effectively downregulate RA-FLS HIF-1α and VEGF as well as HIF-1α and VEGF mRNA levels, and can inhibit the abnormal proliferation of FLS.