RNA干扰沉默信号转导和转录激活因子3对类风湿关节炎成纤维样滑膜细胞增殖的影响

Effects of siRNA-STAT3-mediated Gene Silencing on the Proliferation of Fibroblast- like Synoviocytes in Patients with Rheumatoid Arthritis

目的探讨RNA干扰沉默信号转导和转录激活因子3(STAT3)对类风湿关节炎(RA)成纤维样滑膜细胞(FLS)增殖的影响。方法取本院2012年2—12月行关节镜滑膜切除的RA患者的滑膜组织,先进行RA FLS培养。将FLS分为5组:空白组(正常培养的FLS)、阴性对照组(加入空白质粒的FLS)、RNAi-1组、RNAi-2组和RNAi-3组,后3个组均加入靶向小干扰RNA(siRNA)的重组质粒,分别针对3个不同的靶点。然后用反转录聚合酶链反应和免疫印迹法分别检测各组FLS中STAT3 mRNA及其蛋白的表达情况;用四甲基偶氮唑蓝(MTT)法检测FLS增殖情况〔以490 nm处的吸光度(A)值代表活细胞数量〕。结果 (1)在转染RA FLS后,空白组、阴性对照组、RNAi-1组、RNAi-2组和RNAi-3组STAT3 mRNA表达抑制率分别为(27.32±2.06)%、(30.61±2.47)%、(76.99±2.05)%、(72.75±1.74)%和(66.50±2.47)%,STAT3表达抑制率分别为(44.35±2.04)%、(43.10±2.59)%、(83.57±1.56)%、(77.05±0.54)%和(69.44±2.76)%,5组STAT3 mRNA及其蛋白表达抑制率间差异均有统计学意义(F值分别为362.92和248.08;P<0.01);其中RNAi-1组、RNAi-2组和RNAi-3组STAT3 mRNA及其蛋白表达抑制率均高于空白组、阴性对照组(P<0.05)。(2)空白组、阴性对照组以及RNAi组(即RNAi-1组)A值分别为(0.553 3±0.343 2)、(0.535 0±0.337 3)和(0.158 3±0.079 4),3组间差异有统计学意义(F=3.761,P=0.047);其中空白组A值与阴性对照组比较,差异无统计学意义(P=0.912);RNAi组A值均低于空白组、阴性对照组(P值分别为0.028和0.035)。结论 RNA干扰沉默STAT3能够有效抑制RA FLS中STAT3 mRNA及其蛋白的表达,并能明显抑制FLS的增殖。

Objective To explore the effects of siRNA - STAT3 - mediated gene silencing on the proliferation of fibro- blast - like synoviocytes （FLS） in patients with rheumatoid arthritis. Methods The synovial tissue which was obtained from arthroscopie synovectomy in patients with RA from February to December in 2012 was used to culture RA FLS. The FLS was divided into 5 groups： the blank group （ normally cultured FLS）, the negative control group （with FLS blank plasmids）, the RNAi - 1 group, the RNAi -2 group and the RNAi -3 group. The last three groups were transfected with three different targeted recombinant plasmids respectively. Then RT - PCR and Western blotting methods were used to detect the expression levels of both STAT3 - mRNA and its protein. The proliferation of RA FLS was examined by MTI＇. Results The expressions of both STAT3 - mRNA and its protein inhibit rate of the blank group, the control group, the RNAi - 1 group, the RNAi - 2 group and the RNAi - 3 group were （27.32 ±2. 06）% and （44. 35±2. 04）%, （30. 61 ±2.47）% and （43.10 ±2. 59）%, （76.99 ±2. 05）% and （83.57±1.56） %, （72. 75 ± 1.74） % and （77.05 ± 0. 54） %, （66. 50 ± 2.47 ） % and （69. 44 ± 2. 76） %, respectively, showing statistically significant differences （ F = 362. 92, 248.08 ; P 〈 0.01 ） . There was no statistically significant difference between the blank group and the negative control group （ P 〉 0.05 ） . Compared with the blank group and the negative control group, there were statistically significant difference in the RNAi - 1 group, the RNAi - 2 group and the RNAi - 3 group （ P 〈 0.05） . The absorbanee at 490 nm of the blank group, the negative control group and the RNAi group （ RNAi - 1 group） were （0. 553 3 S0. 343 2）, （0. 535 0 s0. 337 3 ） and （0. 158 3 ±0. 079 4）, respectively, showing statistically significant differ ences （F = 3. 761, P = 0. 047 ） . And there was no statistical significance between the blank group and the negative control group （P 〉 0.05） . Compared with the blank group and the negative control group, there was statistically significant difference in RNAi group （P 〈 0.05） . Conclusion The plasmid of siRNA - STAT3 can inhibit the expression of STAT3 in RA FLS and inhibit the RA FLS proliferation significantly.