连接蛋白43在子宫颈癌HeLa细胞线粒体中的定位及分布

Localization and distribution of connexin 43 in mitochondria of cervical cancer HeLa cells

目的 研究连接蛋白43 （Cx43）在子宫颈癌HeLa细胞亚细胞结构中的分布,并确认其在线粒体中的定位.方法 通过匀浆及离心的方法将HeLa细胞分离为细胞质、细胞核、线粒体和分离后上清,用免疫电子显微镜观察HeLa细胞线粒体的超微形态学及Cx43在分离的线粒体中的分布和定位,电压依赖性阴离子通道（VDAC1）用于线粒体定位确定.用免疫荧光法对HeLa细胞进行Cx43和线粒体生物标志物VDAC1共染色,确定Cx43在子宫颈癌HeLa细胞线粒体中的定位,进一步用蛋白质印迹法定量分析Cx43在各细胞分离成分中的分布情况,线粒体标志物为细胞色素C氧化酶亚基Ⅳ（COXⅣ）和VDAC1,细胞膜黄体生成素受体（LHR）用于定位确定.结果 免疫电子显微镜观察结果显示,在完整的HeLa细胞及分离的HeLa细胞线粒体中均可见到正常的线粒体及囊状肿胀的线粒体,且Cx43和VDAC1均分布在分离的线粒体中.免疫荧光结果显示,Cx43与VDAC1共定位;Cx43在HeLa细胞各亚细胞结构中的表达差异有统计学意义（细胞质：1.23±0.11,细胞核：0.39±0.09,线粒体：3.67±0.59,分离后上清：0.16±0.06,F=84.17,P＜0.05）,Cx43在线粒体中富集.结论 Cx43在子宫颈癌HeLa细胞的线粒体中富集,因此,线粒体Cx43很可能成为子宫颈癌诊断、治疗和判断预后的新靶点.

Objective To study the distribution of connexin 43 （Cx43） in cervical cancer HeLa cells, and to verify the localization of Cx43 in mitochondria. Methods HeLa cells were segregated into cytoplasm, cell nucleus, mitochondria and supernatant after segregation by using the method of homogenate and centrifuge. Immunoelectron microscope was used to observe the morphology of mitochondria and the localization as well as the distribution of Cx43 in HeLa cells. Voltage-dependent anion channel 1 （VDAC1） was used to confirm the localization of mitochondria. Immunofluorescence was used to costain HeLa cells with Cx43 and mitochondrial marker VDAC1 to verify mitochondria localization of Cx43 in cervical cancer HeLa cells. Then Western blot was used to quantify the expression of Cx43 in fractions （cytoplasmic fraction,nuclear, mitochondria and post mitochondrial supernatant）. Mitochondrial markers including VDAC1 and cytochrome c oxidaseⅣ（COXⅣ） were used to confirm mitochondria. Plasma membrane marker （LHR） was used to confirm plasma membrane. Results Immunoelectron microscope confirmed that the normal mitochondria or cystic swollen one could be seen in the complete HeLa cells and the detached HeLa cells mitochondria, with the presence of Cx43 and VDAC1 in detached mitochondria. Immunofluorescence showed Cx43 colocalized with VDAC1. There was a significant difference in the Cx43 expressions of the subcellular structure in the HeLa cells [cytoplasm （1.23±0.11）, cell nucleus （0.39±0.09）, mitochondria （3.67±0.59）, supernatant after segregation （0.16±0.06）; F =84.17, P 〈0.05]. It also showed that the relative amount of Cx43 in mitochondria was enriched. Conclusions Cx43 is enriched in mitochondria in cervical cancer HeLa cells. Therefore, Cx43 in mitochondria might be a potential target in diagnosis, therapy and prognosis of cervical cancer.