乌梢蛇蛋白对滑膜细胞增殖、凋亡及wt-p53/bcl-2 mRNA表达的影响

Effects of Proteins from Zaocys Dhumnades on Apoptosis,Proliferation and the Expression of wt-p53 and bcl-2 mRNA in Vitro Cultured FLS

目的研究乌梢蛇蛋白对体外培养的类风湿关节炎患者成纤维样滑膜细胞(fibroblast-like synoviocytes,FLS)生长的抑制情况,探讨乌梢蛇蛋白影响FLS凋亡的可能分子机制。方法常规方法提取乌梢蛇总蛋白,组织贴块法培养FLS;采用不同剂量(高、中、低剂量)乌梢蛇蛋白作用FLS后,应用MTT法检测乌梢蛇蛋白对细胞增殖的影响,流式细胞仪技术检测细胞的凋亡情况,RT-PCR方法检测细胞wt-p53/bcl-2mRNA的变化。结果乌梢蛇蛋白中高剂量组FLS增殖率与空白对照组比较明显减少,细胞凋亡率明显增加,差异均有统计学意义(P<0.01);乌梢蛇蛋白中高剂量组FLS的wt-p53mRNA表达量增加,Bcl-2mRNA表达量降低,与空白对照组比较差异均有统计学意义(P<0.01)。结论乌梢蛇蛋白可抑制FLS增殖和促进其凋亡,其作用机制可能是通过调节wt-p53/bcl-2基因表达来实现。

Objective To investigate the growth inhibition and apoptosis of fibroblast-like synoviocytes （FLSs） induced by the proteins extracted from Zaocys dhumnades, and to explore the underlying molecular mechanisms. Method Proteins from Zaocys dhumnades were obtained using the traditional protein extraction methods. Primary FLSs were obtained from synovium in patients with RA. Various doses （low, medium and high doses） of snake proteins were used to treat FLSs. Cell growth was determined by the MTT assay. Flow cytometry was employed to determine the apoptosis of FLS. Expression of wt-p53 and bcl-2 mRNA were determined using RT-PCR. Result The proliferation of FLS was significantly inhibited by the medium and high doses of proteins extracted from Zaocys dhumnades（P〈0.01 ）, and the apoptotic rate of FLS was also increased（P〈0.01）. The expression levels of wt- p53 in FLS treated with medium and high doses of the snake proteins were significantly higher than their corresponding negative control groups （P〈0.01）, and the expression levels of bcl2-mRNA were also significantly decreased （P〈0.01）. Conclusion The growth inhibition and induction of apoptosis were probably achieved via the regulation of expression of the wt-p53 and bcl-2 genes.