缝隙连接蛋白26在肺腺癌相关成纤维细胞中的表达及临床意义

Expression and clinical significance of connexin 26 in lung adenocarcinoma associated fibroblasts

目的探讨缝隙连接蛋白26(connexin 26,Cx26)在肺腺癌相关成纤维细胞中的表达及其临床意义。方法收集2018年11月至2019年5月于广西医科大学附属肿瘤医院6例手术切除肺腺癌癌组织及相应癌旁组织,分离原代肿瘤相关成纤维细胞(cancer-associated fibroblasts,CAFs)和正常肺成纤维细胞(normal lung fibroblasts,NLFs);采用光学显微镜观察细胞形态,并结合Western blot和细胞免疫荧光染色检测CAFs标志物α-SMA、E-cadherin和Vimentin的表达,鉴定原代成纤维细胞;qRT-PCR检测Cx26 mRNA表达水平。组织免疫荧光染色法检测Cx26在2013年1月至2018年12月收集的22例肺腺癌癌及癌旁组织中的分布及表达情况。结果成功分离原代CAFs和NLFs,镜下可见细胞呈纺锤形,两者均表达α-SMA、Vimentin,但仅微量表达E-cadherin,且CAFs较NLFs高表达α-SMA(P<0.001)。qRT-PCR检测结果显示,Cx26表达水平在CAFs中随肿瘤分期增加呈持续下调趋势(t=5.684,P=0.011);组织免疫荧光染色检测结果显示,Cx26在肺腺癌组织中的表达低于癌旁组织(t=2.609,P=0.016)。结论肿瘤微环境中CAFs的Cx26表达下调可能与肺腺癌更高分期及恶性进展有关。

ObjectiveTo investigate the expression of connexin 26(Cx26)in lung adenocarcinoma associated fibroblasts and its clinical significance.MethodsThe 6 cases of lung adenocarcinoma tissues and adjacent normal lung tissues that were surgically resected in Guangxi Medical University Cancer Hospital from November 2018 to May 2019 were collected,and the primary cancerassociated fibroblasts(CAFs)and normal lung fibroblasts(NLFs)were isolated.The primary fibroblasts were identified through the cell morphology observed by optical microscope,the expression level of cell biomarkersα-SMA,E-cadherin and Vimentin detected by the Western blot and immunofluorescence staining;the level of Cx26 mRNA was detected by qRT-PCR.The distribution and expression of Cx26 in 22 cases of lung adenocarcinoma and adjacent tissues collected from January 2013 to December 2018 were detected by immunofluorescence staining.ResultsThe primary CAFs and NLFs were successfully isolated.The microscopy reported that CAFs and NLFs were spindle-shaped cells,both of which expressedα-SMA and Vimentin,but only slightly expressed E-cadherin,and the expression level ofα-SMA in CAFs was higher than that in NLFs(P<0.001).The qRT-PCR results showed that the expression level of Cx26 in CAFs showed a continuous downward trend with the increase of tumor stage(t=5.684,P=0.011).The immunofluorescence staining results showed that the expression of Cx26 in lung adenocarcinoma tissues was lower than that in normal lung tissues(t=2.609,P=0.016).ConclusionsDownregulation of Cx26 expression in CAFs within tumor microenvironment may be associated with higher stage and progressive malignancy of lung adenocarcinoma.