人干细胞白血病基因重组慢病毒载体的构建及其在Cajal样间质细胞中的表达

发布时间：2018-02-05 04:19

本文关键词： 干细胞白血病基因慢病毒载体转染 Cajal样间质细胞　出处：《中国现代医学杂志》2017年06期 　论文类型：期刊论文

【摘要】：目的构建含人干细胞白血病(SCL)基因的重组慢病毒载体,并转染体外培养的Cajal样间质细胞(ICC),为进一步利用慢病毒表达载体行体内实验奠定基础。方法通过聚合酶链反应(PCR)将人SCL基质粒内的遗传物质扩增,与慢病毒载体质粒GV287-EGFP结合,构建重组质粒GV287-EGFP/SCL,通过Western blot检测及基因测序对阳性克隆进行鉴定,并测定病毒滴度。体外分离、培养及鉴定ICC,重组慢病毒载体以感染复数(MOI)值为0.5、1.0、5.0、10.0、50.0和100.0时,分别转染ICC,通过激光共聚焦显微镜观察,计算不同MOI值的转染效果,确定最佳MOI值。重组慢病毒载体以最佳MOI值感染ICC作为实验组,以空载体转染的ICC(空载体组)及未转染的ICC(空白组)作为对照,通过逆转录聚合酶链反应(RT-PCR)检测SCL基因在ICC中的表达。结果经Western blot检测及基因测序鉴定SCL重组慢病毒表达载体构建成功,并成功转染ICC,激光共聚焦显微镜下可观察到强绿色荧光。MOI为10时转染效果最佳,转染效率85%;RT-PCR结果表明,实验组SCL基因的表达量高于空载体组和空白组。结论成功制备人SCL基因重组慢病毒载体,并能高效转染ICC,介导SCL基因在ICC中表达。
[Abstract]:Objective to construct a recombinant lentivirus vector containing human stem cell leukemia (hSCL) gene and transfect it into Cajal like stromal cells in vitro. Methods Polymerase chain reaction (PCR) was used to amplify the genetic material in human SCL matrix. The recombinant plasmid GV287-EGFP/SCL was constructed by binding with lentivirus vector plasmid GV287-EGFP. The positive clones were identified by Western blot detection and gene sequencing, and virus titer was determined. ICC was isolated, cultured and identified in vitro. The recombinant lentivirus vector was transfected with ICC by laser confocal microscopy when the moi value of the recombinant lentivirus vector was 0.5 ~ 1.0 ~ 5.0 ~ 10.0 ~ 50.0 and 100.0 respectively. The transfection effect of different MOI values was calculated and the optimal MOI value was determined. The recombinant lentivirus vector infected ICC with the best MOI value was used as the experimental group. ICC- (empty vector group) and untransfected ICC- (blank group) were used as control group. Reverse transcriptase polymerase chain reaction (RT-PCR). Results the recombinant lentivirus expression vector of SCL was successfully constructed by Western blot detection and gene sequencing. The transfection efficiency was 85% under laser confocal microscope. RT-PCR results showed that the expression of SCL gene in experimental group was higher than that in blank vector group and blank group. Conclusion Recombinant lentivirus vector of human SCL gene was successfully prepared and highly transfected into ICC. To mediate the expression of SCL gene in ICC.
【作者单位】：石河子大学医学院附属第一医院泌尿外科;
【基金】：国家自然科学基金(No:81360120)
【分类号】：R587.2;R694.5
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