聚凝胺提高慢病毒载体对树突状细胞的转染率  

Application of polybrene to increase transfection efficiency of lentiviral vector in bone marrow derived dendritic cells

作　　者：孙凯伦 张季 宫念樵[1] Sun Kailun;Zhang Ji;Gong Nianqiao(Institute of Organ Transplantation,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China)

机构地区：[1]华中科技大学同济医学院附属同济医院器官移植研究所,武汉430030

出　　处：《中华实验外科杂志》2020年第1期37-39,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(81570678、81873623)。

摘　　要：目的探讨聚凝胺对小鼠骨髓源树突状细胞(BMDCs)的毒性作用以及聚凝胺促慢病毒载体转染BMDCs的最佳浓度。方法制备BMDCs悬液,随机分为实验组和对照组。实验组用不同浓度的聚凝胺处理,对照组中加入等量磷酸盐缓冲液(PBS),采用细胞计数试剂盒(CCK-8)法和膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)/碘化丙锭(PI)双标记法评价聚凝胺对BMDCs的毒性作用。向BMDCs悬液中加入慢病毒载体[感染复数(MOI)=10],实验组用不同浓度的聚凝胺处理,对照组加入等量PBS,每天采用荧光显微镜动态观察各组绿色荧光蛋白(GFP)表达。刺激BMDCs成熟后,采用流式细胞术定量检测GFP表达。各组之间两两比较采用独立样本t检验。结果聚凝胺浓度≥11 mg/L时,活细胞数目显著减少[吸光度(A)值(0.881±0.007)比(1.031±0.017),t=7.832,P<0.05],差异有统计学意义。安全浓度范围内,使用聚凝胺能显著提高GFP表达,聚凝胺浓度为8 mg/L时,GFP表达最高。结论聚凝胺对BMDCs存在剂量依赖的毒性作用,安全浓度范围内,聚凝胺可明显提高慢病毒载体对BMDCs的转染率,最适浓度为8 mg/L。Objective To investigate the toxicity and optimal concentration of polybrene on lentiviral vector transduction of bone marrow derived dendritic cells(BMDCs).Methods The BMDCs suspensions in mice were prepared and randomly divided into experimental and control groups.The BMDCs in experimental groups were treated with different concentrations of polybrene,and those in control groups were added with phosphate buffer(PBS).Cell counting kit-8(CCK-8)assay and annexin V-fluoresceine isothiocyanate(FITC)/propidium iodide(PI)staining were performed to detect cells viability and apoptosis.Add to BMDCs suspensions were added with lentiviral vector[multiplicity of infection(MOI)=10]and the mixture was divided into experimental and control groups.The mixture in experimental groups was treated with different concentrations of polybrene,and that in control groups was given PBS.The expression of green fluorescent protein(GFP)was observed by fluorescence microscope every day.After the BMDCs were stimulated to mature,the GFP expression was detected using flow cytometry.The t test was used for comparison between two groups.Results The number of living cells in experimental group was significantly less than in control group when polybrene≥11 mg/L[The absorbance(A)values were 0.881±0.007 vs.1.031±0.017,t=7.832,P<0.05].The GFP expression in experimental groups was significantly higher than in control groups.The highest GFP expression was observed when polybrene at 8 mg/L.Conclusion Polybrane shows dose-dependent toxicity on BMDCs,and in the safe range of concentration,polybrane can significantly increase the transfection efficacy of lentiviral vector to BMDCs and the optimal concentration is 8 mg/L.

关 键 词：聚凝胺 慢病毒载体 骨髓源性树突状细胞 

分 类 号：R[医药卫生]