【作者】 王文博；

【导师】 曹英林；

【作者基本信息】 山东大学 ， 医学免疫学， 2006， 硕士

【摘要】 研究目的： 通过运用基因重组技术构建hTERT启动子调控的canstatin基因分泌型真核表达载体phTERT-canstatin，并通过RT-PCR、Western blotting的方法检测其在MCF-7／ADR细胞中的肿瘤特异性表达；及通过MTT、形态学观察、FCM的PI染色法和AnnexinV-FITC／PI双标法检测其诱导人脐静脉内皮细胞ECV204的凋亡情况；继而研究canstatin基因联合hTERT启动子调控的TRAIL基因治疗人乳腺癌裸鼠皮下移植瘤的协同作用。 研究方法： 1．分泌型真核表达载体phtert-canstatin的构建及鉴定 设计包含NheⅠ和HindⅢ酶切位点的canstatin特异性引物，以质粒pETC为模板，PCR扩增带有人IgG γ链信号肽序列的canstatin基因，经NheⅠ和HindⅢ双酶切PCR产物，回收后连接入经相同酶切的phTERTluc1载体中，连接产物转化大肠杆菌DH5 α，LA平板筛选挑取阳性重组子，并经酶切、PCR、DNA测序分析鉴定。 2．hTERT启动子调控的canstatin基因靶向MCF-7／ADR细胞表达的检测 设立未转染组、phTERTluc1转染组和phtert-canstatin转染组，分别检测各组细胞canstatin的表达情况。 2．1 半定量RT-PCR检测各组细胞canstatin mRNA水平的表达转染后48h，收集各组2×10~6个细胞，提取细胞总RNA。利用反转录得到的cDNA为模板，PCR扩增canstatin基因，PCR产物经琼脂糖凝胶电泳并拍照，光密度分析软件半定量测定各组细胞canstatin基因的表达情况。 2．2 Western blotting检测各组细胞canstatin蛋白水平的表达 转染后48h，收集各组5×10~6个细胞，以含1 mmoL／L PMSF的细胞裂解液裂解细胞，提取总蛋白质，经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、转膜，经canstatin特异性一抗、相应二抗孵育后，DAB显色分析各组细胞中更多还原

【Abstract】 Objective:To construct the canstatin gene secretory eukaryotic expression vector under the hTERT promoter by recombinant DNA technology. Then to observe its tumor-specific expression in MCF-7/ADR cell by RT-PCR and Western blotting analysis and to detect its specific apoptosis-inducing effects in ECV204 cells by MTT、morphologic changes、 PI stained flow cytometry (FCM) and AnnexinV-FITC/PI stained FCM. To observe the effects of canstatin gene combined with TRAIL gene on implanted human breast cancer of nude mice.Methods:1. Construction of the secretory eukaryotic expression vector phTERT-canstatinSpecific forward and reverse primers for the canstatin gene were designed and both of them contained restrictive enzyme site (NheI and HindIII, respectively) at the 5’ end. Then the canstatin gene(684bp) with signal sequence of human IgG γ chain was specifically amplified using pETC as a template by PCR, the product was then extracted and orientationly ligated into phTERTlucl digested with the same restrictive enzymes, ligation product was transformed into E. coli. expression host DH5α, to screen and identify the positive recombinants, the positive clones were digested by restrictive enzymes、 PCR、 automatic DNA sequencing after plasmid isolation.2. Expression of the secretory plasmid phtert-canstatin in MCF-7/ADR cells更多还原