La(Sal)_2(Qu)对重组Fas基因酵母促凋亡作用

Apoptosis induced by La( Sal)_2(Qu) in S. pombe with recombinant Fas gene

目的观察氯化镧水杨酸8-羟基喹啉三元稀土配合物[La(C7H5O3)2.(C9H6NO)],对重组Fas基因酵母的促凋亡作用。方法抽提Jurkat细胞RNA;逆转录-聚合酶链反应(RT-PCR)制备Jurkat细胞的cDNA;PCR扩增人Fas基因;将人Fas基因克隆到pREP3X-HA质粒中构建pREP3X-HA-Fas穿梭载体,电转化穿梭载体到野生型粟酒裂殖酵母细胞中;应用蛋白印迹法(Western blotting)鉴定蛋白表达产物;应用La(Sal)2(Qu)诱导重组Fas酵母产生凋亡应答。结果经原位末端标记(TUNEL)法显微计数检测发现,对照组细胞膜泡形成、细胞核碎裂和细胞核溶解的细胞各占计数细胞总数的2.80%、3.74%和2.80%,而处理组分别为26.67%、22.86%和13.33%,差异有统计学意义(P<0.01);流式细胞术检测发现,对照组细胞凋亡检出率为1.24%,而处理组为50.17%,差异有统计学意义(P<0.01)。结论 La(Sal)2(Qu)可能通过Fas诱导酵母细胞凋亡。

Objective To observe the apoptosis induced by La（C7H5O3） 2.（C9H6NO） in recombinant yeast.Meth-ods Total RNA of Jurkat cells was extracted by TRIZOL reagent.First-strand cDNA of Jurkat cells was synthesized by RT-PCR.The Fas genes were amplified by PCR and cloned into plasmid and formed a relevant shuttle carrier.The pREP3X-HA-Fas shuttle carriers were transformed into wild-type Schizosacharomyces pombe（S.pombe） by electroporation.The active recombination was identified by Western blotting and the apoptosis induced by La（C7H5O3） 2.（C9H6NO） was ob-served.Results The ratio of the bubble of cellular membrane,the fragmentation and caryolysis of cell nucleus induced by La（C7H5O3） 2.（C9H6NO） were 26.67%,22.86%,and 13.33%,respectively,detected with terminal deoxynucleotidyl transferase dUTP nick end labeling in treated group with significant differences compared with those of the control group （2.80%,3.74%,and 2.80%,respectively,P 0.01 for all）.The apoptosis rate increased in treated group compared with that of the control group detected by flow cytometry（50.17% vs 1.24%,control group vs treated group,P 0.01）.Conclusion La（C7H5O3） 2.（C9H6NO） could induce apoptosis by Fas in S.pombe.