人胰岛素样生长因子1基因质粒载体的构建及其在人脐血源性神经干细胞中的表达  

Construction of Plasmid Vector Containing Human Insulin-Like Growth Factor 1 and Its Expression in Human Umbilical Cord Blood-Derived Neural Stem Cells

作　　者：王军[1] 吴值荣[1] 朱登纳[1] 高峰 侯艳艳[1] 陈海[1] 王留霞[1] 

机构地区：[1]郑州大学第三附属医院小儿脑瘫康复治疗中心,郑州450052 [2]河南省医药科学研究院神经免疫实验室,郑州450052

出　　处：《实用儿科临床杂志》2011年第6期448-451,共4页Journal of Applied Clinical Pediatrics

基　　金：河南省教育厅自然科学研究计划项目(2009A320039)

摘　　要：目的构建人胰岛素样生长因子1(IGF-1)质粒表达载体,并观察重组体pcDNA3.1-IGF-1转染后的脐血源性神经干细胞(NSCs)中IGF-1基因的表达情况。方法通过反转录-PCR(RT-PCR)方法从胎肝中提取IGF-1基因,胶回收方法分别纯化PCR产物(IGF-1基因)和质粒pcDNA3.1,二者分别由DNA限制性内切酶BamHⅠ与HindⅢ双酶切后,经T4 DNA Ligase连接的方法将IGF-1基因克隆到质粒表达载体pcDNA3.1中,采用测序方法及DNA限制性内切酶BamHⅠ与HindⅢ双酶切方法鉴定重组质粒,脂质体转染法将重组体pcDNA3.1-IGF-1及空质粒pcDNA3.1分别转染至脐血源性NSCs内,经G418抗性筛选后,利用免疫细胞化学法和RT-PCR法检测IGF-1基因在基因转染脐血源性NSCs内的表达情况。结果 IGF-1基因从胎肝中成功提取。重组体pcD-NA3.1-IGF-1经基因测序及DNA限制性内切酶BamHⅠ与HindⅢ双酶切证实质粒表达载体pcDNA3.1-IGF-1构建正确。重组体经脂质体法转染脐血源性NSCs24 h后,经G418筛选2周得到细胞抗性克隆,免疫细胞化学法检测到IGF-1基因在重组质粒表达载体pcDNA3.1-IGF-1转染的脐血源性NSCs中成功表达。RT-PCR方法检测到重组质粒pcDNA3.1-IGF-1转染的脐血源性NSCs中IGF-1mRNA表达阳性,而空质粒pcDNA3.1转染的脐血源性NSCs中IGF-1 mRNA表达阴性。结论 IGF-1基因可在重组质粒表达载体pcDNA3.1-IGF-1转染的脐血源性NSCs内成功表达。Objective To construct the plasmid expression vector containing human insulin-like growth factor 1（IGF-1）,and exa-mine the expression of IGF-1 gene in human umbilical cord blood-derived neural stem cells（NSCs）.MethodsThe IGF-1 gene was extracted from the fetal liver via reverse transcription polymerase chain reaction（RT-PCR）,then the product of PCR and plasmid pcDNA3.1 were purified by gel extraction.After enzyme digestion of DNA restriction enzyme-BamH Ⅰ and Hind Ⅲ,purified IGF-1 gene was cloned into expression plasmid vector pcDNA3.1 by T4 DNA Ligase.Recombinant plasmid was identified by DNA sequencing method and enzyme digestion of DNA restriction enzyme-BamH Ⅰ and Hind Ⅲ.Recombinant pcDNA3.1-IGF-1 and empty plasmid were transfected into human umbilical cord blood-derived NSCs through lipofectin transfection.After transfection,transfected umbilical cord blood-derived NSCs were filtered with neomycin（G418）.The expression of IGF-1 gene in the gene transfected umbilical cord blood-derived was examined by immunocytochemical method and RT-PCR.ResultsIGF-1 gene was successfully extracted from the fetal liver.Recombinants pcDNA3.1-IGF-1 was proved accurate by restriction enzyme digestion and sequencing.Recombinant was transfected into human umbilical cord blood-derived NSCs by liposome for 24 hours,then selection cell clones appeared after 2 weeks for G418 filtering.In umbilical cord blood-derived NSCs transfected by recombinant plasmid vector,the expression of IGF-1 gene was successful,which was detected by immunocyto-chemical method and the expression of IGF-1 mRNA was also positive while the other was negative compared with controls,which was examined by RT-PCR.ConclusionIGF-1 gene can expressed in umbilical cord blood-derived NSCs transfected by recombinant plasmid vector.J Appl Clin Pediatr,2011,26（6）： 448-451

关 键 词：质粒pcDNA3.1 人胰岛素样生长因子1 转染 脐血 神经干细胞 

分 类 号：R741[医药卫生—神经病学与精神病学]