缺氧诱导的肿瘤特异性基因治疗载体的靶向性鉴定  

Targeting validation of a hypoxia-induced tumor-specific gene therapy vector

作　　者：贺赛[1] 郑见宝[1] 孙学军[1] 陈南征[1] 周培华[1] 贾蓬勃[1] 魏光兵[1] 王晖[1] 姚建锋[1] 禄韶英[1] 

机构地区：[1]西安交通大学医学院第一附属医院普通外科,陕西西安710061

出　　处：《西安交通大学学报（医学版）》2014年第4期465-469,共5页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金资助项目(No.81101874;81172362;81172359);陕西省科学技术研究发展计划项目(No.2011-K12-19)~~

摘　　要：目的检测本课题组已构建的缺氧诱导的hTERT基因启动子(5HRE-hTERTp)调控的基因表达载体调控元件的肿瘤特异性和对缺氧诱导的反应性。方法 MTT法检测不同浓度(100、200、300、400、500μmol/L)CoCl2对LoVo细胞活力以及增殖的影响。收集前期已包装成功的缺氧反应元件(hypoxia-response elements,HRE)和人端粒酶催化亚单位启动子hTERTp联合调控CDX2表达的慢病毒表达载体pLVX-5HRE-hTERTp-CDX2-3FLAG(5HhC)病毒液,感染hTERT+LoVo细胞及hTERT-HK-2细胞,细胞免疫组化法验证该治疗载体的靶向性;复苏前期筛选的稳定表达5HhC的LoVo细胞(5HhC/LoVo),用缺氧模拟剂CoCl2模拟缺氧微环境,Western blot和RT-PCR分别检测缺氧调控下CDX2的表达水平。结果结肠癌细胞株LoVo在不同浓度CoCl2环境下随缺氧时间的延长,细胞活力及增殖均受到抑制,在高浓度(300、400、500μmol/L)作用下抑制效果更加明显;5HhC病毒成功感染hTERT+LoVo细胞,而在hTERT-HK-2细胞中不表达;Western blot和RT-PCR证实缺氧可上调5HhC/LoVo细胞中CDX2蛋白和mRNA的表达,且在300μmol/L CoCl2作用24h其蛋白表达量最高。结论缺氧微环境可明显上调hTERTp靶向性的治疗载体5HhC中抑癌基因CDX2的表达。Objective To verify the specificity and the response to hypoxia stimulation of a gene therapy vector containing the tumor suppressor gene CDX2 regulated by the hypoxia-induced enhancer （HRE） and the hTERT promoter.Methods ① The tumor cell line LoVo was exposed to CoCl2 （100,200,300,400,and 500 μmol/L） for different time periods （1,3,5,and 7 d）; cell viability and proliferation were detected by MTT method.②） The recombinant lentiviral vector pLVX-5HRE-hTERTp-CDX2-3FLAG（5HhC） containing an hTERT promoter and 5 copies of the hypoxia-response elements （HRE） enhancer,which had been constructed in our previous study,was collected.5HhC was then transfected into hTERT＋ LoVo cells and hTERT-HK-2 cells; the hTERT-specificity was verified by immunohistochemistry.The LoVo cells with stable expression of 5HhC （5HhC/ LoVo） were obtained.Hypoxia microenvironment was simulated by CoCl2,and Western blot and RT-PCR were employed to examine the expression of CDX2 regulated by hypoxia.Results As hypoxia prolonged,the viability and proliferation of the hTERT＋ LoVo cells were inhibited by CoCl2 of all concentrations,especially the higher concentrations （300,400,and 500 μmol/L） （P ＜ 0.05）.The hTERT＋ LoVo cells were infected with the recombinant lentiviral vector 5HhC while the hTERT-HK-2 cells were not infected.Western blot and RT-PCR confirmed that the expressions of CDX2 protein and mRNA in 5HhC/LoVo cells were further increased with hypoxia,especially with CoCl2 of 300μmol/L for 24 h.Conclusion Hypoxia microenvironment can upregulate the expression of CDX2 in the hTERT-specific gene therapy vector 5HhC regulated by the hypoxia-induced enhancer （HRE） and the hTERT promoter in hTERT＋ LoVo cells.

关 键 词：缺氧 hTERT靶向性 抑癌基因CDX2 基因治疗载体 缺氧反应元件(HRE) 

分 类 号：Q78[生物学—分子生物学]