【摘要】 为评价抗caspase 3核酶在阻抑细胞凋亡发生中的潜在价值 ,以RNaseP催化亚基M1RNA为模板 ,设计合成 3个特异性针对人caspase 3的核酶pM1 GS716、pM1 GS337和pM1 GS2 35 ,并对它们的体内外切割活性进行探讨 .3 2 P标记的caspase 3基因片段体外转录物作为靶RNA ,体外切割实验表明 ,pM1 GS716和pM1 GS337均有切割活性 ,其中pM1 GS716的切割效率可达到 93% .3个核酶转染HeLa细胞 ,评价其在体内的切割活性 .在TNF α作用下 ,转染pM1 GS716的HeLa细胞内caspase 3mRNA下降了 75 % ,蛋白含量下降了 6 9% ,caspase 3蛋白酶活性下降了 5 2 % .Hoechst 332 5 8染色表明 ,细胞凋亡率较对照明显下降 (分别为 2 1 6± 0 7%和 4 9 4± 0 2 % ,P <0 0 1) .提示体外制备的pM1 GS716具有良好的特异催化切割活性 ,有望通过切割caspase 3而抑制细胞凋亡 .更多还原

【Abstract】 To assess the potential value of ribozyme mediated inhibition of caspase 3 in protection against apoptotic cell death, three M1RNA GSs against human caspase 3 mRNA were designed and synthesized by PCR using the catalytic RNA subunit of RNase P as a template, and their cleavage activities in vitro and in vivo were determined. The 32 P labled caspase 3 transcript being target RNA, cleavage reaction in vitro showed that pM1 GS716 and pM1 GS337 were active, the extent of pM1 GS716 cleavage was 93%. The ribozyme pM1 GS716 constructed could downregulate the expression of caspase 3 in vivo and protect HeLa cells from apoptosis induced by TNF α. Caspase 3 mRNA expression was reduced by 75% and caspase 3 protein was reduced by 69% in HeLa cells transfected with pM1 GS716 by quantitative RT PCR and Western blot analyses, respectively. Caspase 3 protease activity was reduced by 52% by ELISA. Moreover, similar decreased level of apoptotic cells was assessed by staining with Hoechst 33258 compared with control exposure to TNF α(21.6±0 7% vs 49 4±0 2%, P <0 01). These results confirm that pM1 GS716 prepared in vitro possesses the perfect specific catalytic cleavage activity, and is hopeful to effectively inhibit apoptosis in vivo through cleaving the key gene, caspase 3.更多还原