高表达β-1,6乙酰氨基葡萄糖支链的大鼠神经细胞模型构建  

Construction of Rat Nerve Cell Model with High Expression ofβ-1,6-Acetylglucosamine Branch

作　　者：王勃霏 吴红艳[2] 蔡惠丽 周耀君[5] 黄泉川[5] 贾思宇 陈海丹[1] Wang Bofei;Wu Hongyan;Cai Huili;Zhou Yaojun;Huang Quanchuan;Jia Siyu;Chen Haidan(Department of Spinal Surgery,Yichang Central People's Hospital,The First College of Clinical Medical Science,China Three Gorges University,Yichang 443003,China;Department of Immunology,Medical College of Three Gorges University,Yichang 443002,China;Department of Hematology,Yichang Central People's Hospital,The First College of Clinical Medical Science,China Three Gorges University,Yichang 443003,China;Department of Burn/Medical Cosmetology,Affiliated Renhe Hospital of Three Gorges University,Yichang 443000,China;Department of Orthopedics,Yuan'an County People's Hospital,Yuan'an 444200,China)

机构地区：[1]三峡大学第一临床医学院[宜昌市中心人民医院]脊柱外科,湖北宜昌443003 [2]三峡大学医学院免疫系,湖北宜昌443002 [3]三峡大学第一临床医学院[宜昌市中心人民医院]血液内科,湖北宜昌443003 [4]三峡大学附属仁和医院烧伤/医疗美容科,湖北宜昌443000 [5]远安县人民医院骨科,湖北远安444200

出　　处：《巴楚医学》2020年第4期11-17,共7页Bachu Medical Journal

基　　金：湖北省自然科学基金项目(No:2017CFB334);湖北省卫生健康科研资金资助项目(No:WJ2019Q015)。

摘　　要：目的:构建高表达β-1,6乙酰氨基葡萄糖支链的原代神经细胞模型。方法:构建大鼠Mgat5慢病毒载体并测定病毒滴度。提取大鼠原代背根神经节(DRG)细胞并鉴定纯度。CCK8法检测Mgat5慢病毒对神经元细胞毒性。PHA-L结合实验检测DRG细胞中β-1,6乙酰氨基葡萄糖支链表达水平。结果:测序结果显示表达Mgat5的慢病毒载体构建成功。DRG细胞鉴定纯度为90%。Mgat5慢病毒载体成功感染大鼠DRG细胞,确定MOI=10为最佳转染剂量。CCK8结果显示,MOI=10转染细胞1、3、5、7 d后,Mgat5慢病毒对神经元细胞活性均无显著影响。PHA-L结合实验证明慢病毒载体介导的Mgat5表达能够显著提高DRG细胞β-1,6乙酰氨基葡萄糖支链水平。结论:本研究成功构建了表达Mgat5的慢病毒载体,该慢病毒载体能够在体外稳定感染大鼠原代DRG细胞,对细胞生长无明显影响,且能够提高细胞中N糖基化表达水平。Objective:To construct primary neuronal cell model with high expression ofβ-1,6 acetylglucosamine branch.Methods:Rat Mgat5 lentiviral vector was constructed and virus titer was measured.The primary rat dorsal root ganglion(DRG)cells were extracted and identified.The CCK8 method was applied to detect the toxicity of Mgat5 lentiviral vector to neuron cells.The PHA-L binding experiment was used to detect the expression level ofβ-1,6 acetylglucosamine branched chains in DRG cells.Results:The sequencing results showed that the lentiviral vector expressing Mgat5 was successfully constructed.The purity of adult rat primary DRG cells was 90%.Rat DRG cells were successfully infected with lentiviral vectors carrying Mgat5,and the optimal transfection efficacy was gotten at MOI=10.CCK8 results showed that Mgat5 lentivirus had no significant effect on neuronal cell activity with MOI=10 for 1,3,5,and 7 days.PHA-L binding experiments proved that lentiviral vector-mediated Mgat5 significantly increased the level ofβ-1,6 acetylglucosamine branched chains in DRG cells.Conclusion:The lentiviral vector carrying Mgat5 has been successfully constructed,which can stably infect rat primary DRG cells to up-regulate N Glycosylation in vitro,with no significant impact on cell growth.

关 键 词：Mgat5 慢病毒 β-1 6乙酰氨基葡萄糖支链 背根神经节细胞 

分 类 号：R744.9[医药卫生—神经病学与精神病学]