OPN反义真核表达载体的构建及其对肾癌细胞增殖侵袭的影响  

Influence of antisense osteopontin gene eucaryotic expression vector on cell proliferation and invasion of human renal cancer cells

作　　者：柳昊[1] 陈安民[1] 郭风劲[1] 黄晖[1] 解杰[1] 胡伟华[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院骨科,湖北武汉430030

出　　处：《中国癌症杂志》2009年第9期649-655,共7页China Oncology

基　　金：国家973重点基础研究发展规划资助项目(项目编号:2002CB513100-7)

摘　　要：背景与目的:骨桥蛋白(osteopontin,OPN)的过表达和多种恶性肿瘤的发展和转移密切相关,然而,在肾癌中OPN所扮演的角色尚未被清楚地阐明。为验证其作为肾癌基因治疗靶点的可行性,本研究通过构建获得OPN反义基因真核表达载体转染人肾癌ACHN细胞,观察其对人肾癌细胞增殖及侵袭的影响。方法:提取ACHN细胞总RNA,RT-PCR法扩增获得OPN目的片段,将其反向克隆到pcDNA3.1(-)质粒中构建OPN反义真核表达载体,并转染人肾癌ACHN细胞。采用半定量RT-PCR法和Western印迹法检测OPN mRNA及蛋白的表达情况;MTT法检测对细胞的生长抑制作用;流式细胞术检测对细胞周期和凋亡率的影响;软琼脂克隆形成实验和Transwell法分别检测细胞的增殖和侵袭能力。结果:RT-PCR检测OPN mRNA表达量结果显示,转染重组质粒细胞中的OPN mRNA的表达量为0.29±0.04,与转染空质粒细胞的0.86±0.05和未转染细胞的0.88±0.04相比,差异有统计学意义(P<0.05)。MTT法检测结果显示ACHN/OPN细胞的生长速度受到抑制;流式细胞检测结果显示ACHN/OPN细胞生长停滞于S期且凋亡率明显升高;软琼脂克隆形成实验及Transwell实验结果显示细胞的增殖和侵袭能力下降(P<0.05)。结论:OPN在肾癌ACHN细胞的增殖和侵袭过程中发挥了重要作用,反义OPN基因能抑制肾癌细胞的恶性表型。Background and purpose： Overexpression of osteopontin （OPN） is correlated with cell proliferation, invasion, and metastasis in a variety of malignant tumors. However, the role of OPN in human renal cancer has not been fully understood. To verify the feasibility of OPN in renal cancer gene therapy, we constructed antisense OPN gene eucaryotic expression vectors and transfected it into human renal cancer ACHN cells. The impacts of the vector on the proliferation and invasion of ACHN cells were studied. Methods： The total RNA were extracted from ACHN cells. The target fragments of OPN were amplified by RT-PCR method and the fragments were inserted into eucaryotic expression vector pcDNA3.1 （-）. Antisense OPN gene reconstructed plasmids were constructed and transfected into ACHN cells with LipofactamineTM 2000. The mRNA and protein expressions of OPN were analyzed by RT-PCR and Western blot; MTT method was used to analyze cell growth; the cell cycle and ratios of apoptotic cells were assessed by flow cytometry; the abilities of proliferation and invasion of cells were detected by soft agar clone formation experiments and Transwell chamber invasive assays. Results： The OPN expression （0.29±0.04） in cells transfected with recombinant vector was significantly inhibited, compared with the cells transfected with empty vector （0.86±0.05） and the control groups （0.88±0.04）（P〈0.05）. The growth velocity of ACHN/OPN cells was obviously slowed down. Flow cytometry showed that ACHN/OPN cells were blocked in the S phase and apoptotic ratio increased significantly （P〈0.05）. The results through transwell assays and soft agar clone formation experiment suggested that the abilities of invasion and proliferation of ACHN/OPN cells decreased significantly （P〈0.05）, compared with the control groups. Conclusion： OPN plays an important role in the proliferation and invasion of human renal cancer ACHN ceils. The malignant phenotype of ACHN ceils can be partly inhibited by antisense OPN gene.

关 键 词：骨桥蛋白基因 肾癌细胞 反义基因 基因转染 

分 类 号：R737.11[医药卫生—肿瘤]