摘要
粒酶B (granzymeB ,GrB)是一种重要的丝氨酸蛋白酶参与细胞毒性T淋巴细胞 (CTL)和自然杀伤细胞(NK)介导的细胞杀伤过程 .为研究粒酶B在肿瘤细胞中异位表达后能否诱导细胞死亡 ,将构建的活性型粒酶B(GrBa)基因及其酶活性中心突变型 (mGrBa)基因的真核表达载体 ,以脂质体法瞬时转染HeLa细胞 ,通过绿色荧光蛋白 (GFP)共表达、间接免疫荧光、细胞计数、MTT等方法 ,观察到GrBa蛋白的异位表达引起多核巨细胞形态异常 ,并且表达细胞的生长受到抑制 .Percoll分离多核巨细胞后 ,观察到其生长状态较差 ,是导致生长抑制的直接原因 .细胞骨架破坏和具有多极纺锤体的异常有丝分裂 ,推测是多核巨细胞不断产生的根源 .
Granzyme B (GrB) is an important serine protease involved in granule-mediated killing in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In order to study whether ectopic expression of GrB in tumor cells can induce cell. death, expression vectors encoding active GrB (GrBa) and mutant GrBa (mGrBa) gene in which serine in catalytic triad was replaced with cysteine were constructed, and transiently transfected into HeLa cells with lipofectamine. It was shown by GFP coexpression, indirect immunofluorescence, cell counting and MTT analyses that ectopic expression of GrBa genes caused increased tumor cell size and multinucleation, and the growth of cells that expressed GrBa proteins was inhibited. Retarded growth was further observed in these morphologically abnormal cells isolated by 40% percoll, which directly contributed to growth inhibitory effect on GrBa-transfectants. There was cytoskeletal breakdown and abnormal mitosis characteristic of multiple spindle poles, largely resulting in accumulation of giant multinuclear cells. These results suggest that GrBa may serve as a good candidate in tumor gene therapy.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2004年第5期470-475,共6页
Progress In Biochemistry and Biophysics
基金
国家高技术"863"计划资助项目 ( 2 0 0 1AA2 1710 1)
国家杰出青年科学基金资助项目 ( 3 992 5 0 3 6)
全军医药卫生科研基金重点项目 ( 0 1Z0 90 )~~
关键词
粒酶B
异位表达
多核巨细胞
granzyme B
ectopic expression
multinuclear giant cells
