同时表达4-1BBL B7.1及B7.2三个共刺激分子基因小鼠肝癌细胞系的建立和意义  

The Studies on Construction and Significance of the Mouse HCC Cell Line Simultaneously Expressing 4-1BBL,B7.1 and B7.2 Genes

作　　者：李国强[1] 王学浩[1] 印洁[1] 俞悦[1] 

机构地区：[1]南京医科大学第一附属医院肝脏外科活体肝移植研究所,南京市210029

出　　处：《中国肿瘤临床》2006年第22期1261-1264,共4页Chinese Journal of Clinical Oncology

基　　金：国家自然科学基金(编号:30271236);江苏省医学重点学科建设基金资助(编号:135-10)

摘　　要：目的:建立同时表达4-1BBL、B7.1及B7.2三个共刺激分子基因小鼠原发性肝细胞癌(hepatocellularcarcinoma,HCC)细胞系。方法:将B7.1和B7.2全长cDNA的质粒酶切,构建pcDNA3.1-B7.1-IRES-B7.2重组子,酶切法鉴定。用阳离子脂质体(LipofectamineReagent)将重组子转染H22,经均霉素(Hygromycin,300!g/ml)筛选,阳性克隆命名为H22-CD80/CD86+细胞。逆转录-聚合酶链反应(reversetranscription-polymerasechainreaction,RT-PCR)检测目的基因在H22-CD80/CD86+变异株的表达。采用同样方法将pCI-neo-4-1BBL质粒转入H22-CD80/CD86+细胞,G418筛选,阳性克隆命名为H22-CD80/CD86/CD137L+细胞。流式细胞仪检测三种基因在细胞克隆中的表达。结果:pcDNA3.1-B7.1-IRES-B7.2重组子经酶切鉴定,同时获得B7.1(862bp)和B7.2(984bp)目的基因片段和5.6kbp线性化pcDNA3.1载体片段;重组子测序结果与Genebank中B7.1和B7.2序列相符,证实构建成功。RT-PCR及FCM检测结果显示B7.1、B7.2及4-1BBL基因分别在H22-CD80/CD86+细胞及H22-CD80/CD86/CD137L+细胞中获得稳定、高效联合表达。结论:pcDNA3.1-B7.1-IRES-B7.2重组子构建正确,H22-CD80/CD86/CD137L+变异株可同时稳定表达B7.1、B7.2和4-1BBL三个共刺激分子基因。Objective： To study the construction of pcDNA3.1-B7.1-IRES-B7.2 and its stable expression in mouse HCC cell line H22. Methods： The murine full-length cDNA of B7.1 and B7.2 genes were obtained and subcloned into pcDNA3.1-IRES. The recombinant named pcDNA3.1-B7.1- IRES-B7.2 was constructed and identified with restriction enzyme digestion. Subsequently, the recombinant was transfected into H22 with Lipofectamine reagent, then Hygromycin-resistence colonies were acquired, and named H22-CD80/CD86^＋. RT-PCR was applied to determine whether there were stable mRNA expressions of objective genes in variants. The H22-CD80/CD86/CD137L^＋ cell line was set up by transfecting PCI-neo-CD137L into H22-CD80/CD86^＋ FCM was applied to determine whether there were stable mRNA expressions of objective genes in variants. Results： pcDNA3.1-B7.1- IRES-B7.2 was digested, then electrophoresis of the digested products showed fragments of 862bp （mB7.1） and 984bp （mB7.2）, which indicated the construction was successful. The DNA sequence analysis is identical to the sequence of mB7.1/B7.2 in Genebank and did not reveal any mutation. RT- PCR showed dual expressions of mB7.1/B7.2 gene with strong intensity in H22-CD80/CD86^＋. FCM showed co-expression of mB7.1/B7.2/4-1BBL gene with strong intensity in H22-CD80/CD86/CD137L^＋. Conclusions： pcDNA3.1-B7.1- IRES-B7.2 has been constructed successfully, and stable and effective expressions of B7.1, B7.2 and 4-1BBL genes also have been obtained in H22 variant.

关 键 词：基因重组 癌肝细胞 B7.1 B7.2 4-1BBL 

分 类 号：R735.7[医药卫生—肿瘤] R456[医药卫生—临床医学]