摘要
目的:通过慢病毒系统将HRE-VEGF整合基因转染至MSCs细胞,评价该新型MSCs用于缺血性心肌病的治疗的可行性。方法:运用同尾酶法构建9拷贝缺氧反应元件,并构建9HRE-VEGF表达载体。使用慢病毒系统将目的基因9HRE-VEGF转染干细胞建立转基因MSCs,检测其在不同缺氧、复氧时间VEGF的表达。结果:慢病毒系统转染MSCs具有较高的转染效率,成功构建了整合有9HRE-VEGF的新型转基因MSCs,在低氧下表达VEGF,而在常氧状态下VEGF表达关闭。结论:HRE对转导的VEGF基因表达具有调控作用,缺氧状态下VEGF稳定表达,此低氧诱导调控方式将更有利于缺血性心脏疾病的治疗。
Objective： To explore the feasibility of regulating VEGF expression activity by combining VEGF gene with hypoxia response element （HRE）, and to evaluate the function of transgenetic MSCs transfected by lentivirus mediated HRE-VEGF gene. Methods： Isocaudarner technique was used to construct 9 copy HRE （9HRE）, and 9HRE-VEGF expression vector were re- combined, which were then transfected into rat MSCs by pPACKH1- Lentivector Packaging Kit composed by 3 plasmids. Transgenetic MSCs were incubated under different condition of hypoxia of normoxia at different time set. Methods including immunofluorescence, RT-PCR and ELISA were deployed to detected expression level of VEGF proteinum, VEGF-mRNA and VEGF protein, respectively. Results： MSCs were successfully transfected with 9HRE-VEGF gene via lentivirus trasnfection system, which had high transfection efficiency in this process and facilitated the construction of transgenetic MSCs. VEGF-mRNA and VEGF protein were detected in hypoxia group, which were greatly inhibited in the group under re oxygenation, as both expression level of VEGF- mRNA and VEGF protein were significantly higher under hypoxic condition, compared with these under re- oxygenation state. Conclusion： 130 HRE were proved to regulate the expression of VEGF in the current study, as stable expression of VEGF were detected during hypoxia condition, which might be a promising and favorable gene modification method in treating ischemic heart disease.
出处
《国际心血管病杂志》
2012年第6期368-371,376,共5页
International Journal of Cardiovascular Disease
基金
上海市科学技术委员会纳米技术专项(1052nm05300)
关键词
骨髓间充质干细胞
血管内皮细胞生长因子
缺氧反应元件
基因治疗
Bone marrow mesenchymal stem cells（MSCs）
Vascular endothelium growth factor（VEGF）
Hypoxic response element（HRE）
Gene therapy
