上调人1型大麻素受体表达诱导人宫颈癌细胞凋亡的实验研究

目的通过构建基因真核表达载体,探讨人1型大麻素受体(hCB1R)对人宫颈癌HeLa细胞体外凋亡的作用及机制。方法构建GV230-hCB1R质粒,经双酶切、测序鉴定后,转染HeLa细胞,免疫印迹(Western blot)法及免疫荧光细胞化学染色(ECL)联合激光扫描共聚焦显微镜技术检测hCB1R表达及细胞定位;流式细胞术检测细胞凋亡,Western blot法及实时荧光定量PCR(qRT-PCR)检测hCB1R、Bcl-2、Bax、Bad的表达。结果 hCB1R转染HeLa细胞后表达相对分子质量为40 000的hCB1R蛋白,细胞膜和细胞质中均有hCB1R表达;过表达的hCB1R,可上调Bax、Bad的表达,抑制Bcl-2的表达,流式细胞术检测结果显示HeLa细胞株呈现凋亡,hCB1R对宫颈癌HeLa细胞株的生长表现出明显的抑制作用,促进宫颈癌HeLa细胞凋亡。结论上调HeLa细胞hCB1R表达可增强Bax、Bad表达,抑制Bcl-2表达,诱导细胞凋亡。

Experimental study on up-regulating expression of hCB1R for inducing apoptosis of HeLa cells

Objective To investigate the effect and mechanism of human type 1 cannabinoid receptor (hCB1R) on the apoptosis of cervical cancer HeLa cell by constructing the gene eukaryotic expression vector .Methods The GV230‐hCB1R plasmid was con‐structed and identified by the double enzyme digestion and DNA sequencing .Then it was transfected into HeLa cells .The expres‐sion and cellular localization(ECL) of hCB1R was detected by Western blot and immunofluorescence cellular chemical staining com‐bined with laser scanning confocal microscope;the apoptosis rate was tested by flow cytometry ;the mRNA and protein expression of hCB1R ,Bcl‐2 ,Bax and Bad was examined by real‐time fluorescent quantitative PCR(qRT‐PCR) .Results After transfection into HeLa cells ,hCB1R protein with the relative molecular mass of 40 000 was expressed in both cytoplasm and cellular membrane of cells .The over expression of hCB1R could up‐regulate the apoptosis of Bax and Bad ,and inhibited the Bcl‐2 expression .The flow cytometry results displayed that HeLa cell line showed apoptosis ,hCBIR had obvious inhibiting effect on the growth of cervical cancer HeLa cell line and promoted apoptosis .Conclusion Up‐regulating expression of hCB1R in Hela cells could enhance the ex‐pression of Bax and Bad inhibit the expression of Bcl‐2 ,thus inducing the cellular apoptosis .