雷公藤内酯醇对Bcl-2基因转染细胞凋亡作用的研究

EFFECTS OF TRIPTOLIDE ON APOPTOSIS IN Bcl 2 TRANSFECTED T CELL HYBRIDOMA

目的：通过比较转染ｂｃｌ－２（＋／＋）及未转染ｂｃｌ－２（－／－）原癌基因的２Ｂ４细胞，在雷公藤内酯醇作用下细胞凋亡和细胞增殖的差别，探讨该化合物诱导淋巴细胞凋亡的机制。方法：以ｂｃｌ－２（＋／＋）２Ｂ４细胞及ｂｃｌ－２（－／－）２Ｂ４细胞为研究对象，结合流式细胞仪，ＤＮＡ凝胶电泳，及３Ｈ－ＴｄＲ掺入等方法，评价雷公藤内酯醇对此两种细胞的作用。结果：①雷公藤内酯醇只能促使ｂｃｌ－２（－／－）２Ｂ４细胞凋亡，且呈浓度依赖关系；但是不能使ｂｃｌ－２（＋／＋）２Ｂ４细胞发生凋亡；②无论２Ｂ４细胞是否转染ｂｃｌ－２原癌基因，雷公藤内酯醇均能够抑制其掺入３Ｈ－ＴｄＲ，并呈浓度依赖关系；③无论是转染及未转染ｂｃｌ－２原癌基因的２Ｂ４细胞，雷公藤内酯醇均能够使其Ｇ０／Ｇ１期细胞数增多，Ｓ＋Ｇ２／Ｍ期细胞数减少。结论：雷公藤内酯醇不能造成过度表达Ｂｃｌ－２蛋白的ｂｃｌ－２（＋／＋）２Ｂ４细胞凋亡。同时，实验结果还说明雷公藤内酯醇可能通过独立的作用途径诱导细胞凋亡并阻止其增殖。

OBJECTIVE In order to explored the mechanisms of triptolide induced apoptosis,the effects of triptolide on the apoptosis of bcl 2 transferred(bcl 2+/+)or non transferred(bcl 2-/-)2B4 cell were studied. METHODOLOGY For measurement of apoptosis and cell cycle analysis,bcl 2 transferred (bcl 2+/+)or non transferred(bcl 2-/-)2B4 cell were collected and stained with propidium iodide,then analyzed with a flow cytometer(EPICS X,Coulter).In addition,apoptosis was confirmed by DNA fragmentation and membrane damage. RESULTS (1)Triptolide induced apoptosis in bcl 2(-/-)2B4 cell,while not in bcl 2(+/+)2B4 cell;(2)Triptolide decreased 3H thymidine incorporation in both bcl 2(+/+)and bcl 2(-/-)2B4 cell;(3)The proportions of G0/G1 phase in both bcl 2(+/+)and bcl 2(-/-)2B4 cells were increased 48 hours after triptolide was added. CONCLUSION Triptolide can arrest cell growth but not induce apoptosis of bcl 2 transfected T cell hybridoma that Bcl 2 protein overexpressed.It is indicated that the anti proliferative and apoptotic action of triptolide are two separable events.