沉默CXCR4基因表达抑制胰腺癌增殖和侵袭的体外研究.docVIP

沉默CXCR4基因表达抑制胰腺癌增殖和侵袭的体外研究 王 铮，李徐奇，徐 军，仵 正，张 东，沙焕臣，马清涌 (710061 西安，西安交通大学医学院第一附属医院肝胆外科) [摘要] 目的 利用RNAi技术构建CXCR4 shRNA表达载体，观察CXCR4基因干扰后胰腺癌细胞增殖及侵袭能力的变化。方法 在不同分化程度胰腺癌细胞株中筛选出CXCR4高表达胰腺癌细胞株，RNAi技术构建CXCR4 shRNA表达载体，转染胰腺癌CXCR4高表达细胞Miapaca-2并应用G418筛选出稳定表达株，Real-time PCR、Western blot实验观察RNAi对CXCR4基因的抑制效率；MTT实验检测CXCR4基因干扰后胰腺癌细胞增殖，应用Transwell侵袭小室观察CXCR4干扰对胰腺癌细胞侵袭转移力的影响。结果 胰腺癌细胞株Miapaca-2 CXCR4表达异常增高，特异性CXCR4基因沉默能够在基因和蛋白水平稳定、高效、特异地抑制CXCR4的表达，抑制效率达70%以上。特异性CXCR4 shRNA能够抑制细胞生长，细胞达到对数生长期的时间延长(P0.05)。Transwell实验证明CXCR4基因干扰后细胞侵袭能力减低 (P0.05)，即使SDF-1的刺激也不能够使细胞侵袭能力增加。结论 成功构建了针对胰腺癌细胞CXCR4的siRNA载体；CXCR4基因沉默能抑制胰腺癌细胞增殖、侵袭和成瘤能力。 [关键词] CXCR4; 胰腺癌; 侵袭转移 [中图法分类号] R735. 9 [文献标志码] A The effects of CXCR4 inhibited by RNA interference on pancreatic cancer proliferation and in vitro Wang Zheng, Li Xuqi, Xu Jun, Wu Zheng, Zhang Dong, Sha Huanchen, Ma Qingyong (Department of Hepatobilary Surgery, the First Affiliated Hospital, Medical College of Xi’an Jiaotong University, Xi’an, Shaanxi, 710061, China) [Abstract] Objective Extra-pancreatic metastasis is a difficult problem for surgical intervention on pancreatic cancer. CXCR4 was considered as an important role in this process. The aim was to observe the influence of CXCR4 knockdown on pancreatic cancer cell growth, cell cycle, cell tumorigenesis, cell invasive ability. Methods RNAi technique was applied to construct the CXCR4 shRNA expression vector. Lipofecatime 2000 was used to transfect the CXCR4 high expression pancreatic cancer cell Miapaca-2. Realtime-PCR and Western Blot were used to observe the inhibitory effect of RNAi on CXCR4 expression. MTT assay was applied to assess the influence of specific CXCR4 shRNA on cell growth; Transwell method was to observe the cancer cell invasive ability. P0.05 was considered as statistically significant. Results Through RNAi technique, the CXCR4 shRNA expression plasmid was successfully constructed. After the transfection on CXCR4 high expression pancreatic cancer cell line Miapa