论文部分内容阅读
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 eukaryotic expression vector of human CB2R gene and investigate the effect of h CB2R on the cell proliferation and the mechanism of h CB2R on the growth of cervical cancer cell line Caski. Methods GV230-h CB2R plasmid was constructed and identified by double enzyme digestion and sequencing. The recombinant plasmid was transfected into HEK293 cells and Caski cells. Western blot and immunofluorescence cytochemistry combined with laser scanning confocal microscopy were used to detect CB2R expression and cell location. Flow cytometry Cell apoptosis rate was detected by cytometry. The expression of CB2R, Bcl-2, Bax and Bad were detected by Western blot and real-time fluorescence quantitative PCR. Results The fragment of 1128 bp was obtained by double enzyme digestion. The sequence was 99% identical to the sequence of h CB2R gene (NM_001841.2). After transfected into HEK293 cells, h CB2R protein, HEK293 cell membrane and cytoplasm both CB2R expression; over-expression of CB2R can up-regulate the expression of Bax, Bad, inhibit the expression of Bcl-2 and promote cervical cancer Caski cell apoptosis. Conclusion Upregulation of h CB2R expression in Caski cells can enhance the expression of Bax and Bad, inhibit the expression of Bcl-2 and induce apoptosis.