RNA干扰环氧合酶2对人腰椎退变终板软骨细胞生长增殖及凋亡的影响

目的 观察沉默环氧合酶2(COX-2)基因对人腰椎退变软骨终板细胞增殖、凋亡的影响.方法 根据COX-2mRNA的序列,分别设计合成4对不同的且能干扰COX-2基因表达的siRNA并筛选出最高效抑制序列,构建以COX-2基因为靶点的RNA干扰质粒并将其转染腰椎退变软骨终板细胞.分为空白对照组(不予任何处理)、阴性对照siRNA组(30 nmol/L阴性siRNA)、siRNA1组(15 nmol/L COX-2 siRNA)和siRNA2组(30 nmol/L COX-2 siRNA).siRNA转染48 h后,采用荧光定量PCR法检测COX-2基因表达;WST-8检测细胞增殖情况;实时荧光定量聚合酶链反应(qPCR)法检测细胞凋亡相关基因survivin、bcl-x1、bax基因表达的情况.结果 构建的4组重组体插入片段的碱基序列完全正确.转染COX-2siRNA 48 h后人腰椎退变软骨细胞中COX-2mRNA表达量在siRNA1组和siRNA2组分别为(1.3±7.2)%和(35.4±3.6)%,均显著低于空白对照组(100±5.7)%和阴性对照组siRNA(83.4±2.8)%,P＜0.05.免疫印迹实验显示COX-2蛋白表达量明显下降,以siRNA2组表达量最低(P＜0.05＝.WST-8法检测显示4组细胞存活率分别为(100.0±8.3)%、(84.9±4.2)%、(52.5±6.7)%、(48.9±5.4)%,siRNA1组和siRNA2组与其他各组相比,差异有统计学意义(P＜0.05＝.实时荧光定量聚合酶链反应(qPCR)检测显示,随着转染浓度的递增,退变软骨细胞中survivin、bcl-2基因表达较对照组显著降低,bax基因表达则较对照组逐渐升高.结论 RNA干扰COX-2基因能明显抑制人腰椎终板软骨细胞COX-2的表达,抑制细胞生长增殖,并可通过下调抗凋亡基因survivin和bcl-2的表达,上调促凋亡基因bax的表达,从而诱导退变软骨终板细胞凋亡.

Abstract：

Objective To investigate the influence of siRNA-COX-2 gene upon the growth inhibition and apoptosis of cartilage endplate chondrocytes and provide new methods and evidence for siRNA in gene therapy of cartilage endplate chondrocytes. Methods According to the sequence of COX-2 mRNA,COX-2 siRNA was designed, synthesized, cloned into the GFP reporter pcDNA6. 2GW/EmGFPmiR vector and transfected into Hep cell line. The integrity of inset fragment was detected by colony PCR ( polymerase chain reaction) and sequencing analysis. The cultured cartilage endplate chondrocytes were divided into 4 groups: control group (untreated), negative siRNA group (treatment with 30 nmol/L negative control siRNA), siRNA1 group (treatment with 15 nmol/L COX-2 siRNA) and siRNA2 group (treatment with 30 nmol/L COX-2 siRNA). The biological activity of recombinants was identified with the interference efficiency of COX-2 siRNA recombinant by real-time PCR and Western blot. And the effects of COX-2 inhibitor on the growth of chondrocytes were detected by WST-8 and the mRNA expressions of survivin, bcl-2 and bax genes measured by real-time PCR. Results The sequences of inset fragment in 4 siRNA expressing recombinants were correct After COX-2 transfection, the expression of COX-2 mRNA in chondrocytes was 51.3% ±7. 2%in the siRNA1 group and 35.4% ±3.6% in the siRNA2 group. Western blot showed that the expression of COX-2 protein decreased, especially in siRNA2 group (P ＜ 0.05 ). And the cell survival rate was 100.0% ±8.3% in the control group, 84.9% ±4.2% in the negative control siRNA group, 52.5% ±6. 7% in the siRNA1 group and 48.9% ± 5.4% in the siRNA2 group ( P ＜ 0. 05 ). Meanwhile, the expressions of mRNA of survivin and bcl-2 decreased while the expression of bax mRNA increased in degenerative cartilage endplate chondrocytes transfected with COX-2 siRNA ( P ＜ 0. 05 ). Conclusion COX-2-targeting siRNA inhibits the expression of COX-2, suppresses the proliferation of chondrocytes and induces the cell apoptosis. These effects may be attributable to the up-regulation of survivin and bcl-2 and the downregulation of bax.

Effect of siRNA-Cox-2 on the growth inhibition and apoptosis of cartilage endplate chondrocytes

Objective To investigate the influence of siRNA-COX-2 gene upon the growth inhibition and apoptosis of cartilage endplate chondrocytes and provide new methods and evidence for siRNA in gene therapy of cartilage endplate chondrocytes. Methods According to the sequence of COX-2 mRNA,COX-2 siRNA was designed, synthesized, cloned into the GFP reporter pcDNA6. 2GW/EmGFPmiR vector and transfected into Hep cell line. The integrity of inset fragment was detected by colony PCR ( polymerase chain reaction) and sequencing analysis. The cultured cartilage endplate chondrocytes were divided into 4 groups: control group (untreated), negative siRNA group (treatment with 30 nmol/L negative control siRNA), siRNA1 group (treatment with 15 nmol/L COX-2 siRNA) and siRNA2 group (treatment with 30 nmol/L COX-2 siRNA). The biological activity of recombinants was identified with the interference efficiency of COX-2 siRNA recombinant by real-time PCR and Western blot. And the effects of COX-2 inhibitor on the growth of chondrocytes were detected by WST-8 and the mRNA expressions of survivin, bcl-2 and bax genes measured by real-time PCR. Results The sequences of inset fragment in 4 siRNA expressing recombinants were correct After COX-2 transfection, the expression of COX-2 mRNA in chondrocytes was 51.3% ±7. 2%in the siRNA1 group and 35.4% ±3.6% in the siRNA2 group. Western blot showed that the expression of COX-2 protein decreased, especially in siRNA2 group (P ＜ 0.05 ). And the cell survival rate was 100.0% ±8.3% in the control group, 84.9% ±4.2% in the negative control siRNA group, 52.5% ±6. 7% in the siRNA1 group and 48.9% ± 5.4% in the siRNA2 group ( P ＜ 0. 05 ). Meanwhile, the expressions of mRNA of survivin and bcl-2 decreased while the expression of bax mRNA increased in degenerative cartilage endplate chondrocytes transfected with COX-2 siRNA ( P ＜ 0. 05 ). Conclusion COX-2-targeting siRNA inhibits the expression of COX-2, suppresses the proliferation of chondrocytes and induces the cell apoptosis. These effects may be attributable to the up-regulation of survivin and bcl-2 and the downregulation of bax.