CD14稳定沉默胃癌细胞系的建立及其侵袭能力研究  

The research on the construction of gastric cancer cells with CD14 silencing and it's invasion ability

作　　者：李康[1] 旦增[1] 王中华[1] 德吉[1] 陈晓红[1] 刘明花[1] 

机构地区：[1]西藏自治区人民医院消化内科,西藏拉萨850000

出　　处：《中国癌症杂志》2013年第4期254-259,共6页China Oncology

基　　金：国家自然科学基金资助项目(No:81060165)

摘　　要：背景与目的:白细胞分化抗原14(cluster of differentiation antigen 14,CD14)为脂多糖(lipopolysaccharide,LPS)的高亲和受体,能够识别革兰阴性菌、真菌以及结核杆菌并介导机体的炎性反应,为病菌引发多种细胞信号转导的第一步,在肿瘤组织以及癌细胞中常呈异常表达。近来有研究指出,CD14启动子区的功能性突变能够影响CD14的表达,并增加幽门螺杆菌感染后罹患胃癌的风险,据此推测CD14与胃癌的发生、发展密切相关。本研究拟构建CD14-shRNA干扰载体,建立CD14稳定沉默的胃癌细胞系,初步探讨CD14对胃癌细胞侵袭能力的影响,以期为胃癌发病机制的研究奠定实验基础。方法:根据shRNA引物设计原则设计并合成4条CD14-shRNA序列,构建sh-CD14表达载体,转染MGC-803细胞,G418筛选稳转细胞系;RT-PCR检测CD14 mRNA的表达;蛋白质印迹法(Western blot)检测CD14蛋白的表达;Transwell小室模型检测细胞的侵袭能力。结果:测序结果表明,CD14-shRNA表达载体构建成功并筛选得到了CD14稳定沉默的胃癌细胞系,其CD14 mRNA以及蛋白的表达分别降低了71.7%%和63.4%;与对照组相比CD14-shRNA组细胞侵袭能力显著降低,差异有显著统计学意义(P<0.01)。结论:成功构建了CD14-shRNA干扰载体并获得了CD14稳定沉默的胃癌细胞系,初步证实CD14的沉默能够影响胃癌细胞的侵袭能力。Background and purpose： Cluster of differentiation antigen 14（CD14）, a high-affinity receptor for the lipopolysaccharide （LPS）, could identify the gram-negative bacteria, fungi and mycobacterium tuberculosis, and mediate the inflammatory response of infected body, which is the first step in bacteria induced signal transduction. Expression of CD14 often turned to be abnormal in the tumor tissue and cancer cells. Recent studies have observed that functional CD 14 polymorphisms, especially in the promoter motifs, are associated with a higher risk of H.pylori-related gastric carcinoma, indicated that CD14 is closely associated with the development of gastric cancer. This study aimed to construct CD14-shRNA expression vector and gastric cancer cells with CD14 silencing and to discuss the influence of CD14 on the invasion ability of gastric cancer cells and to lay a experimental basis for the study of the pathogenesis of gastric cancer. Methods： Four CD14-shRNA sequences were synthesized and CD14-shRNA expression vector was constructed to transfect cells and gastric cancer cells with CD14 silencing were screened by G418. RT-PCR and Western blot was used to detect the CD14 mRNA and protein level. The invasion ability of gastric cancer cells was detected by Transwell chamber. Results： CD14-shRNA expression vector was successfully constructed and screened to obtain gastric cancer cells with CD 14 silencing, of which the CD 14 mRNA and protein silencing efficiency were 71.7% and 63.4% respectively. Compared with the control group, the invasion ability of CD14-shRNA group was decreased obviously with statistical differences （P〈0.01）. Conclusion： Gastric cancer cells with CD14 silencing are constructed successfully and the influence of CD 14 on the invasion ability of gastric cancer cells is preliminarily confirmed.

关 键 词：白细胞分化抗原14 shRNA载体构建 胃癌细胞 侵袭 

分 类 号：R735.2[医药卫生—肿瘤]