内源性胰岛素替代疗法中葡萄糖激酶基因逆转录病毒表达载体的构建(英文)

Construction of retroviral vector containing glucokinase gene in the replacement therapy of endogenous insulin

背景由于胰岛细胞分离技术难度较大,目前糖尿病细胞移植治疗尚缺乏理想的细胞来源。目的构建葡萄糖激酶(Glucokinase,GK)基因逆转录病毒表达载体及稳定的产毒细胞系,为构建具有葡萄糖反应性胰岛素分泌能力的胰岛代理细胞打下基础,可望为糖尿病提供一种更符合生理要求的内源性胰岛素替代疗法。设计非随机非对照的实验研究。地点、材料和干预本研究在解放军第三军医大学附属新桥医院中心实验室进行。将GK质粒(pCMV4-GKZ1)经EcoR1/BamH1双酶切后亚克隆至逆转录病毒载体PLXSN,构建逆转录病毒表达载体PLX-GK,用酶切法和测序法对重组体进行鉴定。然后脂质体介导逆转录病毒表达载体PLX-GK转入包装细胞PA317,筛选病毒滴度较高的稳定产毒细胞系,PCR鉴定。主要观察指标①重组逆转录病毒载体构建与鉴定结果。②病毒滴度鉴定及PCR结果。结果成功构建GK基因逆转录病毒表达载体,经酶切及测序证明目的基因插入位点和读码框架正确、无突变;产毒细胞系的平均病毒滴度为6.8×108CFU/L,筛选出一株病毒滴度为1.8×109CFU/L稳定产毒细胞系PA317/GK,PCR证实GK基因整合入细胞基因组。结论成功构建了携GK基因的逆转录病毒表达载体及高滴度的产毒细胞系。

BACKGROUND:There is no good cell source for cell transplant treatment of Diabetes because of the difficulty in the separation of islet cells at present.OBJECTIVE:To construct a retroviral vector containing glucokinase(GK) gene as well as a stable cell line of recombinant virus to provide a base for islet cell replacement that has insulin secretion ability reacted to glucose,which hopefully could furnish a replacement therapy of endogenous insulin that is more accorded with physiological requirement. DESIGN:Non randomised,non controlled studySETTING,MATERIALS and INTERVENTIONS:Our study was conducted in the central laboratory of Xinqiao Hospital Affiliated to the Third Military Medical University of Chinese PLA.The GK plasmid(pCMV4 GKZ1) was subcloned onto retroviral vector PLXSN after cut by EcoR1/BamH1 restriction to construct retroviral vector PLX GK.The recombinant was identified by enzymic cutting and sequencing methods.The PLX GK was transferred to PA317 cell line mediated by Lipofectamine2000.The stable recombinant virus with highest titre was screened for PCR evaluation.MAIN OUTCOME MEASURES:①The construction and identification of the recombinant retroviral vector;②The verification of viral titre and PCR evaluationRESULTS:A retroviral vector containing GK gene was successfully constructed,which was proved by enzymic cutting and sequencing analysis of accurate inserting site and coding frame with no mutation.The average titre of the recombinant virus is about 6.8×105 CFU/mL.A vector production cell line PA317/GK whose titre of the recombinant virus is about 1.8×106 CFU/mL was selected,which was proved with GK gene integration by PCR technique.CONCLUSION:A retroviral vector containing GK gene,which is a cell line of recombinant virus with high titre,has been successfully constructed.