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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 Due to the difficulty of pancreatic islet cell separation technology, the current cell transplantation for diabetes still lacks an ideal source of cells. Objective To construct retroviral expression vector of Glucokinase (GK) gene and stable virus-producing cell line, and to lay the foundation for the construction of pancreatic islet-derived cells with glucose-responsive insulin secretion, which is expected to provide a more physiological basis for diabetes mellitus Endogenous insulin replacement therapy. Design non-randomized non-controlled experimental study. Location, Materials and Interventions The study was conducted at the Xinqiao Hospital Central Laboratory, Third Military Medical University of PLA. GK plasmid (pCMV4-GKZ1) was digested with EcoR1 / BamH1 and subcloned into retroviral vector PLXSN to construct retroviral expression vector PLX-GK. The recombinants were identified by restriction enzyme digestion and sequencing. Then, the liposome-mediated retrovirus expression vector PLX-GK was transferred into the packaging cell PA317, and the stable virus-producing cell line with high virus titer was selected for PCR identification. MAIN OUTCOME MEASURES ① Construction and identification of recombinant retroviral vector. ② virus titer identification and PCR results. Results The retroviral expression vector of GK gene was constructed successfully. The insertion site and the reading frame of the target gene were confirmed by enzyme digestion and sequencing. The average virus titer of the producer cells was 6.8 × 108CFU / L, Strains of virus titer of 1.8 109 CFU / L stable toxin-producing cell line PA317 / GK, PCR confirmed GK gene integration into the cell genome. Conclusion The recombinant retroviral vector containing GK gene and high titer virus producing cell line were successfully constructed.