慢病毒介导的自杀基因对树突状细胞的杀伤作用  

Cytotoxicity Effect of Lentivirus- mediated Suicide Genes on Dendritic Cells

作　　者：程婷婷[1] 徐希[2] 葛杭萍[1] 陈枫煜[1] 梁彬[2] 俞康[2] 

机构地区：[1]温州医科大学附属第一医院,325000 [2]温州医科大学附属第一医院血液科,325000

出　　处：《医学研究杂志》2014年第4期71-75,共5页Journal of Medical Research

基　　金：浙江省自然科学基金资助项目(Y2101069;LY12H08002)

摘　　要：目的探讨慢病毒介导的自杀基因胸腺激酶(TK)对小鼠树突状细胞(DC)的杀伤作用。方法构建含自杀基因TK的慢病毒载体质粒,转染树突状细胞后通过更昔洛韦激活自杀基因使细胞死亡。结果阳性重组质粒酶切及测序鉴定与预期结果相符,成功构建其真核表达载体。收集含自杀基因的慢病毒,测其效价为2×109TU/ml,达到实验要求。慢病毒转染树突状细胞后,观察树突状细胞的形态、大小及数量与对照组比较无明显差异,经流式细胞仪检测绿色荧光蛋白(GFP)表达率为96.43%。CCK-8法检测发现,树突状细胞随着更昔洛韦药物浓度的增加,细胞凋亡率逐渐上升。药物浓度100μg/ml作用时间48h时,为药物对细胞的最佳杀伤作用时间;更昔洛韦该药物对正常的树突状细胞未发现有毒性作用。结论慢病毒介导的自杀基因TK对小鼠树突状细胞具有显著的杀伤作用。Objective To explore the killing effect of lentivirus-mediated suicide gene thymidine kinase （TK） on dendritic cells （DC） in mice.Methods The lentiviral vector plasmid containing suicide gene TK was constructed and transfected into the dendritic cells.Dendritic cells was leading to death by activating suicide gene through ganciclovir treatment.Results The outcome of restriction endonuclease digestion of positive recombinant plasmids and DNA sequencing were in consistence with the expected.The TK gene was successfully cloned and eukaryotic expression vector were constructed successfully.Titer lentivirus which contain the suicide gene was harvested and concentrated.Adenoviral titer was 2 × 109 TU/ml which achieved the requirement of our experiment.After lentiviral vector transferred into the dendritic cells,we observed the morphology,size and number of dendritic cells,and found there was no significant differences between the control group and experiment group.Dendritic cells were infected by lentivirals with a Green Fluorescent Protein （GFP） of 96.43％,which was confirmed by flow cytometry.CCK-8 assays showed that the death rates of the dendritic cells increased with the increasing ganciclovir concentration.With the drug concentration of 100μg/ml for 48 hours,it could get the maximum drug effects.In the meantime we found ganciclovir had no toxic effects on the normal dendritic cells.Conclusion The lentiviral vector-mediated suicide gene thymidine kinase has marked cytotoxicity effects against dendritic cells in mice.

关 键 词：树突状细胞 自杀基因 慢病毒 胸腺激酶 更昔洛韦 

分 类 号：R733[医药卫生—肿瘤]