摘要
目的 构建低氧诱导因子1α（hypoxia-inducible factor 1α,HIF-1α）过表达重组慢病毒质粒并包装病毒,将病毒感染乳鼠心肌细胞,分析其对Notch配体Jagged1表达的影响。方法 从cDNA文库中PCR扩增人HIF-1α编码序列并克隆入酶切线性化慢病毒载体GV218,转化感受态大肠杆菌细胞筛选出阳性克隆并测序鉴定。将HIF-1α过表达慢病毒质粒与病毒包装辅助质粒共转染工具细胞HEK293T,包装出慢病毒颗粒。原代分离SD乳大鼠心肌细胞,将HIF-1α过表达慢病毒感染心肌细胞,定量PCR分析Jagged1mRNA水平的变化。结果 成功扩增HIF-1α编码序列并正确连接入线性化载体GV218,获得阳性转化子并测序无误。将转化子质粒与病毒包装辅助质粒共转染HEK293T后,获得滴度为1×108TU/ml的慢病毒颗粒,Western blot法检测到HIF-1α-GFP融合蛋白。重组慢病毒感染乳鼠心肌细胞后,胞核中见GFP信号。与对照组相比,HIF-1α过表达5天后显著上调Jagged1mRNA的水平。结论 心肌细胞中HIF-1α可诱导Jagged1的表达。HIF-1α过表达重组慢病毒载体的成功构建,为研究缺血缺氧性心肌损伤反应的分子机制奠定了基础。
Purpose To construct the hypoxia-inducible factor 1 ct （ HIF-1α） overexpressing recombinant lentiviral vectors and package the virus and to analyze the expression of Notch ligand Jaggedl in HIF-1α lentivirus-infected neonatal rat cardiomyocytes. Methods Human HIF-1α coding sequence （CDS） was amplified by PCR using the cDNA library and cloned into the linearized vector GV218. After transduction the competent cells with HIF-1α recombinant vectors, the positive clones were selected and sequenced. After cotransfection the HEK293T cells with HIF-1α overexpressing plasmids and lentviral packaging plasmids, the HIF-1α overexpressing lentivirus were packaged. The changes in Jaggedl mRNA levels were assessed by quantitative PCR post infection the primary neonatal rat cardiomyocytes with HIF-1α lentivirus. Results HIF-1α CDS was successfully amplified and properly cloned into the linearized GV218. The positive clones were obtained and further confirmed by sequencing. With the help of packaging plasmids, the HIF-1α overexpressing plasmids-transfeeted HEK293T cells successfully produced the lentiviral particles with the titer of 1× 10^8 TU/ml, and the HIF-1α-GFP fusion protein was detected by Western blot in virus-infected HEK293T cells. After infection the neonatal rat eardio-myoeytes with HIF-1α lentivirus, Jaggedl mRNA levels were significantly upregulated. Conclusion HIF-1α induces Jaggedl expression in eardiomyoeytes. The eonstruetion of HIF-1α recombinant lentiviral vector makes a strong foundation for the investigation of molecular mechanisms regarding the cardiomyoeyte＇ s response for hypoxic-isehemic injury.
出处
《临床与实验病理学杂志》
CAS
CSCD
北大核心
2016年第4期416-420,共5页
Chinese Journal of Clinical and Experimental Pathology
基金
国家自然科学基金(81170121
81541004)
广东医学院优硕培育基金(YS2014013
YS2015004
YS2015005)
