人2型大麻素受体过表达诱导宫颈癌Caski细胞凋亡

发布时间：2018-12-15 00:18

【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。
[Abstract]:Objective to construct the eukaryotic expression vector of human type 2 cannabinoid receptor (h CB2R) gene and to investigate the expression and localization of h CB2R and the effect of h CB2R on the growth of cervical cancer Caski cells and its mechanism. Methods GV230-h CB2R plasmids were constructed and identified by double enzyme digestion and sequencing. The expression and location of CB2R were detected by, Western blot method and immunofluorescence cytochemical staining combined with laser scanning confocal microscopy (LSCM) in HEK293 cells and Caski cells. The expression of h CB2R,Bcl-2,Bax,Bad was detected by flow cytometry (FCM) and real time fluorescence quantitative PCR (PCR). Results the target fragment of 1128 bp was obtained by double enzyme digestion. The homology of the sequence with NM_001841.2 of h CB2R gene was 99%. After transfection of HEK293 cells, h CB2R protein with relative molecular weight of (Mr) 40 000 was expressed, and CB2R was expressed in both cell membrane and cytoplasm of HEK293. Overexpression of CB2R, could up-regulate the expression of Bax,Bad, inhibit the expression of Bcl-2 and promote the apoptosis of Caski cells. Conclusion upregulation of h CB2R expression in Caski cells can enhance the expression of Bax,Bad, inhibit the expression of Bcl-2 and induce apoptosis.
【作者单位】：重庆医科大学实验动物中心;重庆三峡医药高等专科学校;
【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)
【分类号】：R737.33

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