目的:通过腺病毒介导puma在小鼠肝细胞BNL.CL2内过表达,观察其对周期和凋亡的影响及其分子机制.方法:用HEK293 low passage(LP)细胞包装制备重组PUMA腺病毒(Ad-puma)和GFP(Ad-GFP)腺病毒,PCR,Western Blot检测鉴定包装的腺病毒.腺病毒感染BNL.CL2细胞后,流式细胞仪检测感染Ad-PUMA后的BNL.CL2细胞的周期和凋亡.免疫共沉淀实验分析BCL-2家族成员间在PUMA介导的凋亡中的相互作用.结果:①用HEK293包装制备了目的细胞中有效高表达Ad-PUMA和Ad-GFP浓缩病毒存储液.所制备的病毒浓缩液Ad-PUMA的滴度为2.0 × 10~(11)ifu/L;Ad-GFP滴度为2.1×10~(12)ifz/L.②Ad-PUMA感染BNL CL.2细胞后24,36 h与对照组相比细胞凋亡增加;36 h S期细胞明显增多细胞周期中的S期的细胞则明显增多,G2期细胞相对减少.③小鼠肝细胞中PUMA介导的凋亡不仅可能与PUMA,Bax与Bcl-XL的竞争性结合作用有关,也存在PUMA与Bax的直接激活作用.结论:PUMA可有效地促进鼠肝细胞BNL.CL2的凋亡.可能Ad-PUMA介导的PUMA过表达后使细胞从S期到G2期时发生停留或阻滞.在BNL.CL2细胞中,PUMA促凋亡的分子机制既与PUMA,Bax与Bcl-XL的竞争性结合作用有关,也存在PUMA与Bax的直接相互作用.
Effect and mechanism of PUMA on the apoptosis of mouse liver cell in vitro
AIM: To study the influence on the biological function of BNL. CL2 such as cell cycle and apoptosis and molecule mechanism of PUMA. METHODS: ① The packaging and preparation of adenoviruses expressing PUMA ( Ad-PUMA) and adenoviruses expressing GFP ( Ad-GFP). ② The cellular biological function of BNL CL2 cells after infected by Ad-PUMA. ③Molecular mechanisms of the effection of PUMA on mouse liver cells. RESULTS: The results indicated that the packaging and preparation of Adenoviruses expressing PUMA ( Ad-PUMA) was available and effective expression in mouse liver cell BNL CL2. PUMA could induce apoptosis in these cells and the number of cells at S stage displayed increasing tendency with the infection time.Taking into account above factors, we then analyzed that PUMA induction affects the interactions between Bax and Bcl-XL, between PUMA and Bcl-XL, as well as the interactions between Bax and PUMA by immunoprecipitarion. CONCLUSION:PUMA could induce apoptosis in these cells and the number of cells at S stage displayed increasing tendency with the infection time. PUMA dissociates Bax and Bcl-XL to induce apoptosis and the interact with Bax directly.
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NSTL
万方数据
维普
