Fas和DcR3在垂体腺瘤的表达及FasL与RGD-FasL诱导的细胞凋亡机制

Expressions of Fas/DcR3 and FasL/RGD-FasL mediated apoptosis in pituitary adenoma cells

目的研究RGD-FasL诱导垂体腺瘤细胞株GH3、MMQ、AtT20所产生的细胞毒性效应,并探讨其机制。方法应用RT-PCR法和流式细胞仪检测肿瘤细胞上Fas、DcR3的表达。应用MTT测定法检测FasL、RGD-FasL对肿瘤细胞所产生的细胞毒性效应,并经琼脂糖凝胶电泳证实是否为该配体所诱导的凋亡。通过流式细胞仪PI染色法评价细胞周期的变化和凋亡分析。通过Western蛋白印迹法检测Caspase 8、Caspase 9、Caspase 3、Bcl-2、RANKL和JNK2的表达。结果垂体腺瘤细胞株GH3、MMQ、AtT20均表达Fas和DcR3。FasL和RGD-FasL诱导肿瘤细胞所产生的细胞毒性效应均呈现剂量依赖关系。细胞周期分析表明RGD-FasL能诱导细胞周期的停滞。经RGD-FasL或FasL干预的肿瘤细胞的凋亡指数差异不明显。经RGD-FasL干预后,Caspase 8、Caspase 9、Caspase 3、RANKL和JNK2的表达均增加,而Bcl-2的表达减少。结论RGD-FasL能通过Caspase途径诱导垂体腺瘤细胞的凋亡。RGD-FasL的研制很可能为垂体腺瘤的生物靶向治疗提供一种新的途径。

Objective To investigate the cytotoxic effects of pituitary adenoma cell lines GH3, MMQ, ART20 induced by RGD-FasL and the underlying mechanism. Methods Fas and DcR3 mRNAs were detected by RT-PCR and their surface expression was measured by flow cytometry. Cytotoxicity exerted by RGD-FasL on tumor cells was measured with MTT assay and the induced apoptosis was determined by agarose gel electrophoresis. The cell cycle and apoptosis was assessed by flow cytometry with PI staining. The expressions of Caspase 8 , Caspase 9 , Caspase 3, Bcl- 2, RANKL and JNK 2 were detected by Western Blot. Results GH3, MMQ and AtT20 cells expressed Fas and DcR 3. The cytotoxic effects of FasL and RGD-FasL on tumor cells were dose-dependent. The cell cycle analysis indicated that RGD-FasL could induce cell cycle stagnation. In pituitary tumor cells treated with RGD-FasL, the apoptostic index was insignificantly different from that treated with FasL. The expression of CaspaseS, Caspase9, Caspase3, RANKL and JNK2 increased while that of Bcl-2 decreased after treated with RGD-FasL. Conclusion RGD-FasL induces apoptosis of pituitary adenoma cells through Caspase activation, and may be a novel therapeutical candidate for the treatment of pituitary adenomas.