人胰岛素样生长因子1真核细胞表达质粒的构建及其稳定表达细胞株的建立  

Construction of eukaryotic expression plasmid for human insulin-like growth factor and the establishment of its stable expression in cell strain

作　　者：付长峰[1] 刘一[1] 张绍坤[1] 付士波[2] 

机构地区：[1]吉林大学第一医院骨科,吉林省长春市130021 [2]吉林大学公共卫生学院,卫生部放射生物重点实验室,吉林省长春市130021

出　　处：《中国临床康复》2005年第47期49-51,共3页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金资助项目(30400447)~~

摘　　要：目的构建胰岛素样生长因子1(insulin growth factor1,IGF-1)的真核细胞表达质粒,转染神经胶质瘤细胞(C6细胞),建立hIGF-1稳定表达细胞株,为进一步观察胰岛素样生长因子1基因体内转染后对脊髓损伤后神经细胞凋亡的影响打下基础。方法实验于2004-05/09在吉林大学公共卫生学院放射生物实验室进行。用聚合酶链反应方法从人的肝细胞cDNA文库中克隆出IGF-1cDNA,然后定向插入真核细胞表达载体pcDNA3.1中,酶切电泳和基因测序鉴定插入质粒的hIGF-1基因序列;使用C6细胞株,用脂质体方法转染C6细胞。经G418筛选,形成阳性细胞克隆。继续培养4周,进行W estern blot检测。结果重组的真核细胞表达质粒pcDNA3.1-hIGF-1所含的IGF-1cDNA序列和插入方向均正确。W estern blot检测结果证实转染的hIGF-1基因在C6细胞内成功表达。结论实验所构建的重组真核细胞表达载体pcDNA3.1-hIGF-1能够稳定表达有活性的hIGF-1,实验结果对进一步观察脊髓损伤后胰岛素样生长因子1抑制神经细胞凋亡的作用有一定意义。AIM： To construct eukaryotic cells expression plasmid for human insulin growth factor-1 （hIGF-1）, transfect it into neuroglioma cell （C6 cell）, establish hIGF-1 stable expression cell strain, so as to lay a foundation for the further observed on the effect of hIGF-1 gene after in vivo transfection on the apoptosis of nerve cells after spinal cord injury. METHODS： The experiment was carried out in the Laboratory of Radiobiology, School of Publie Health, Jilin University from May to September 2004. hIGF-1cDNA was cloned from human liver eDNA library by polymerase ehain reaction （PCR） and then inserted into the eukaryotie expression vector of pcDNA3.1. The gene sequence of the hIGF in which plasmid was inserted was identified by fragment electrophoresis and gene sequence measurement. The C6 cell strain was used, and the C6 cells were transfected with liposome-mediated method. The positive transfected eel1 strain was selected by G418. The G418 resistance cells were cultured for 4 weeks, and then the gene expression was detected by western blot. RESULTS： The IGF-1cDNA sequence and insert direction in recombinant eukaryotic cells expression plasmid peDNA3.1-hIGF-1 were both correct. The results of western blot proved that there was expression of the transfected hIGF-1 in C6 cells. CONCLUSION： The constructed hIGF-1 expression vector for eukaryotic cells could express hIGF-1 in C6 cell stably, the result is of significance for the observation on the role of hIGF-1 in inhibiting apoptosis nerve cells after spinal cord injury.

关 键 词：脊髓损伤 细胞凋亡 胰岛素样生长因子1 转染 

分 类 号：R654.2[医药卫生—外科学]