大鼠源性重组天冬氨酸特异性半胱氨酸蛋白酶3基因构建及其对细胞的促凋亡效应  

Construction of rat constitutively active recombinant caspase-3 and investigation of its pro.apoptotic effect in vitro

作　　者：陈莎莎 曹菲[1] 高峰[1] 许爱军[1] 田学愎[1] 肖兴鹏[1] 杨少兵[1] 田玉科[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院麻醉学教研室,武汉430030

出　　处：《中华医学杂志》2009年第43期3074-3078,共5页National Medical Journal of China

基　　金：国家自然科学基金（30672027）

摘　　要：目的探讨大鼠源性重组天冬氨酸特异性半胱氨酸蛋白酶-3（caspase-3）基因对细胞的促凋亡作用。方法通过重组PCR的方法获得大小亚基顺序颠倒的大鼠重组caspase-3基因，构建真核表达载体，分别转染人胚肾293T细胞和大鼠永生化神经干细胞后观察细胞形态学变化，通过Annexin V-PI双染、流式细胞术、MTr法来检测其促细胞凋亡作用。结果转染后，细胞出现明显的核碎裂，死亡；MTTr法检测发现细胞增殖明显被抑制（抑制率为（48．35±0．16）％和（44．61±0．15）％（P〈0．05）；Annexin V-PI共染色流式细胞术检测两种细胞的凋亡率分别为（30．7±1．5）％和（16．0±1．0）％，较对照组细胞有显著的增加（P〈0．05）。结论大鼠源性重组caspase-3基因可在转染的人和大鼠细胞中表达，并诱导细胞发生凋亡，不仅可有效用于体内实验研究，且能用于诱导神经细胞凋亡。Objective To investigate the apoptosis induction effect by the rat recombinant caspase-3. Methods Reversed rat caspase-3 gene was gained by settling the small subunit prior to the large one through recombinant PCR, and cloned into the expression vector to transfect human 293T cells and rat immortalized neural progenitor cells. The expression and pro-apoptotic effect of recombinant caspase-3 was observed by the changes of morphology of the transfected cells through immunofluorescence and analyzed by Annexin V-FITC staining, Flowcytometry and MTF assay. Results The transfected cells presented obvious apoptosis as detected by immunofluoreseence. MTF assay showed that the proliferation of recombinant caspase-3 transfected cells was significantly inhibited [ （ 48.35 ± 0. 16 ） %, （ 44. 6±1 0. 15 ） % ] （ P 〈 0. 05 ）. Annexin V-FITC staining revealed that the percentage of apoptotic cells in the transfectants of recombinant caspase-3 gene was [ （30. 7 ± 1.5 ） %, （ 16. 0 ± 1.0） % ] （ P 〈 0. 05 ）, which was much higher than that of control cells. Conclusions Rat recombinant caspase-3 can be expressed and induce apoptosis effectively, and used safely both in vitro and in vivo. It can also cause neural cells to death.

关 键 词：CASPASE-3 凋亡 大鼠 

分 类 号：R686[医药卫生—骨科学]