CTGF siRNA对类风湿关节炎成纤维样滑膜细胞凋亡的影响

摘要

目的：小干扰RNA技术沉默结缔组织生长因子（CTGF）表达，并观察对类风湿关节炎（RA）成纤维样滑膜细胞（FLS）凋亡及对部分凋亡相关基因的影响。方法脂质体转染CTGF siRNA至FLS，real time PCR筛选出有效的CTGF siRNA。使FLS CTGF表达沉默后，采用FITC⁃PI双染法通过流式细胞仪检测对FLS凋亡的影响。Real⁃time PCR检测CTGF基因沉默对bax、bcl⁃xl和survivin等凋亡相关基因表达的影响。结果筛选出H⁃1序列是针对人CTGF的特异性SiRNA，抑制率>70％。在外源性CTGF作用下，由去血清导致的FLS凋亡明显减少；沉默FLS中的CTGF基因表达，FLS的凋亡明显增加。在FLS中CTGF基因表达沉默后，Bax基因表达上调，bcl⁃xl基因表达下调，但差异没有统计学意义（ P>0.05）。survivin基因表达出现明显下调（P<0.01）。结论 CTGF减少RA FLS凋亡的保护性作用很可能是通过维持survivin表达来实现的。%Objective To silence the expression of CTGF by small interfering RNA technology,to observe the influence on fibroblast⁃like synovial cell apoptosis and several apoptosis⁃related genes,and to explore the mechanism of action of CTGF in rheumatoid arthritis synovial lesions. Methods Effective CTGF siRNA was screened through real⁃time PCR. The influence of CTGF siRNA on FLS apoptosis was detected with FITC⁃PI double staining by flow cytometry. bax,bcl⁃xl and survivin were detected using real⁃time PCR when CTGF mRNA has been silenced. Results Compared with other 2 groups of oligo and NC oligo,H1 oligo exhibited the strongest interfering action to CTGF(inhibition ratio>70%),so that it is selected as the effective target gene sequence for the following experiment. Apoptosis of FLS induced by serum deprivation was significantly decreased in the presence of exogenous CTGF. When expression of the CTGFgene was knocked down in FLS,FLS apoptosis was significantly increased,and expres⁃sion levels of survivin mRNA were decreased significantly(P<0.01). Conclusion FLS survival is positively regulated by CTGF,which may through the sustaining the expression of survivin.