ndr2基因在肺腺癌细胞GLC-82中的诱导表达  

Research of inducible expression of ndr2 gene in lung adenocarcinoma GLC-82 cells

作　　者：李树钧[1] 王华[2] 戚好文[3] 张健[1] 何鹏[1] 张文红[1] 季少平[1] 刘新平[1] 药立波[1] 

机构地区：[1]第四军医大学基础部生物化学与分子生物学教研室,西京医院呼吸内科陕西西安710033 [2]第四军医大学西京医院超声诊断科,陕西西安710033 [3]第四军医大学西京医院呼吸内科,陕西西安710033

出　　处：《第四军医大学学报》2002年第13期1209-1213,共5页Journal of the Fourth Military Medical University

基　　金：国家杰出青年科学基金资助 (3 982 5 113 ) ;国家重点基础研究发展规划 (973 )项目资助 (J19990 75 6

摘　　要：目的　构建蜕皮素诱导的抑癌基因 ndr2哺乳动物真核表达载体 (p IND- ndr2 ) ,转染肺腺癌细胞 GL C- 82 ,观察ndr2基因表达 ,为进一步研究 ndr2的生物学功能打下基础 .方法　以 RT- PCR方法确定正常人肺组织和肺腺癌 GL C- 82细胞 ndr2的表达高低 . PCR方法扩增 ndr2 ,以 Bam HI+Eco RI双酶切连接入 p U C19载体中 ,测序正确后 ,插入到蜕皮素诱导的真核表达载体 p IND中 .连有 ndr2基因的载体(p IND- ndr2 )用脂质体方法转染到培养的肺腺癌细胞 GL C-82中 .经 G4 18和 zeocin双抗生素筛选后 ,挑取单克隆进行培养 ,用蜕皮素诱导 ndr2表达 ,用 RT- PCR,western印迹和免疫组织化学方法验证获得表达的细胞株 .结果　 PCR的方法扩增获得大小约 12 0 0 bp的片段 ,连接入预先插入 HA- tag的 p UC19载体的 ndr2基因经 H ind +Eco RI酶切后 ,构建到真核表达载体 p IND中 ,酶切鉴定后证明连接片段正确 .通过双载体转染和双抗生素筛选后 ,培养的细胞经诱导后检测到实验组 ndr2的高表达 ,而对照组和未诱导的实验组 ndr2表达均较低 .结论　 ndr2基因成功转染培养的肺腺癌 GL C-82细胞 ,建立了AIM To Construction a recombinant pIND ndr2 expression vector, to observe its steady expression in transfected human lung adenocarcinoma cell line GLC 82 and its role in further study of biological function of ndr2 for lung adenocarcinoma. METHODS ndr2 gene of human normal lung tissue and human lung adenocarcinoma cell line GLC 82 was amplified by PCR RT. PCR amplification was performed by using primers based on the known gene sequence of ndr2, and fragments of DNA was cloned into vector pUC19. The sequence of the fusion gene was analyzed. Ecdysone inducible mammalian expression system was used. A recombinant pIND ndr2 vector was constructed at its Hin dⅢ+ Eco RI sites. The recombinant pIND ndr2 vector and pIND vector were tranfected into GLC 82 cells respectively, and the cell clones were screened with G418 and zeocin. Expression of ndr2 gene was detected by RT PCR, and expression of ndr2 protein was detected by western blot and immunohistochemical method after the cells were induced with ponasterone A. RESULTS The sequence of ndr2 gene that was cloned into vector pUC19 was correct. ndr2 gene was correctly transfected into GLC 82 cells and was appropriately expressed with induction of panosterone A. The expression of ndr2 of lung adenocarcinoma cell GLC 82 transfected by pIND ndr2 with induction of panosterone A was high. CONCLUSION Recombinant pIND ndr2 expression vector was successfully constructed and expressed steadily in human lung adenocarcinoma cell line GLC 82.

关 键 词：肺腺癌 ndr2基因 细胞转染 基因表达 肺肿瘤 GLC-82细胞 

分 类 号：R734.2[医药卫生—肿瘤]