人2型大麻素受体过表达诱导宫颈癌Caski细胞凋亡  

Over-expression of cannabinoid receptor 2 induces the apoptosis of cervical carcinoma Caski cells

作　　者：严磊[1] 李晶[2] 赵婷婷[1] 王会娟[1] 赖国旗[1] 

机构地区：[1]重庆医科大学实验动物中心,重庆400016 [2]重庆三峡医药高等专科学校,重庆404120

出　　处：《细胞与分子免疫学杂志》2015年第6期758-762,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：重庆市教委自然科学基金(KJ111802);重庆市卫生局医学科研项目(2012-1-096);重庆市高等教育教学改革研究重点项目(132128;133309);重庆万州区科技计划项目(201203055)

摘　　要：目的构建人2型大麻素受体(h CB2R)基因真核表达载体,探讨h CB2R在细胞中的表达、定位以及h CB2R对宫颈癌Caski细胞的生长的影响及机制。方法构建GV230-h CB2R质粒,经双酶切、测序鉴定后,转染HEK293细胞和Caski细胞,Western blot法及免疫荧光细胞化学染色联合激光扫描共聚焦显微镜技术检测CB2R表达及细胞定位;流式细胞术检测细胞凋亡率,Western blot法及实时荧光定量PCR检测h CB2R、Bcl-2、Bax、Bad的表达。结果双酶切获得1128 bp的目的片段,测序结果与h CB2R基因注册序列(NM_001841.2)的同源性为99%;转染HEK293细胞后,可表达相对分子质量(Mr)40 000的h CB2R蛋白,HEK293细胞膜和细胞质中均有CB2R表达;过表达的CB2R,可上调Bax、Bad的表达,抑制Bcl-2的表达,促进宫颈癌Caski细胞凋亡。结论上调Caski细胞h CB2R表达可增强Bax、Bad表达,抑制Bcl-2表达,诱导细胞凋亡。Objective To construct a eukaryotic expression vector containing human cannabinoid receptor 2 （hCB2R） gene and investigate its expression, location and the influence on the apoptosis of cervical cancer Caski cells. Methods The eukaryotic expression vector GV230-hCB2R was constructed and identified by double enzyme digestion and DNA sequencing analysis. Then it was transfected into HEK293 cells and Caski cells by LipofectamineTM 2000. The expression and cellular localization of hCB2R protein were detected by Western blotting and immunofluorescent cytochemistry combined with laser scanning confocal microscopy, the apoptosis rate was tested by flow cytometry. The mRNA and protein expressions of hCB2R, Bcl-2, Bax and Bad were examined by real-time fluorescent quantitative PCR （qRT-PCR） and Western blotting, respectively. Resells The gene fragment of 1128 bp was obtained by double enzyme digestion, it had 99% homology with human hCB2R gene nucleic acid sequence reported （NM_001841.2）. After transfected into HEK293 cells, hCB2R protein, with the relative molecular mass （ Mr ） being 40 000, was expressed in both cytoplasm and cellular membrane. The over-expression of hCB2R promoted apoptosis of Caski cells via up-regulating the Bax, Bad expressions and down-regulating the Bcl-2 expression. Coaclasioa The up-regulated expression of hCB2R could induce cell apoptosis by enhancing the expressions of Bax, Bad and suppressing the expression of Bcl-2 in Caski cells.

关 键 词：人2型大麻素受体 HEK293细胞 宫颈癌细胞 细胞凋亡 

分 类 号：R711.74[医药卫生—妇产科学] R392.33[医药卫生—临床医学]