Axl-siRNA核转染对髓源性DC前体细胞分化的影响  

Effect of Nucleofection of Axl-siRNA on Differentiation of Marrow-derived Dendritic Cell Precursors

作　　者：李幸[1] 王浩[2] 戴晓敏[1] 马俊磊 汪晶[1] 姜汉英[1] 宫念樵[1] 

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、卫生部器官移植重点实验室、器官移植教育部重点实验室,武汉430030 [2]海军工程大学海工医院,武汉430033

出　　处：《华中科技大学学报（医学版）》2013年第5期511-514,546,共5页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.81072441)

摘　　要：目的 将Axl（Anexelekto）-siRNA（small interfering RNA）序列核转染于髓源性树突状细胞（dendritic cells,DC）前体细胞中,观察转染效率、细胞生长及分化情况.方法 构建Axl-siRNA序列,用核转染法将此序列转染于小鼠骨髓原代细胞并诱导向DC分化;根据是否转染Axl-siRNA,将实验分为空白对照组和Axl-siRNA核转染组;转染后荧光显微镜观察细胞荧光水平以评估转染效率;通过锥虫蓝染色观察细胞的存活情况;观察细胞的形态变化,记录细胞的生长情况,绘制生长曲线;转染后第8天流式细胞术检测DC的表面标志CD11c＋.结果 转染后1～3 d荧光表达率分别为（70.5±6.3）%、（72.6±7.3）%、（68.8±6.5）%,而后荧光逐渐减弱并淬灭;核转染后细胞的死亡率为（21.2±5.4）%;空白对照组细胞在培养的前3d内的生长曲线呈对数增长,第4至6天,细胞数稳定在（2.38～2.47）×10^6/孔,培养后第7、8天细胞数略有下降,为（2.23～2.31）×10^6/孔;Axl-siRNA核转染组细胞生长曲线也呈现相同的趋势,但与空白对照组相比每阶段的细胞计数略有减少;细胞培养8 d后,空白对照组和Axl-siRNA核转染组CD11c＋的细胞百分比分别为（70.28±6.13）%和（67.53±7.45）%.结论 Axl-siRNA核转染法对髓源性DC前体细胞具有较高的转染效率,不影响DC的生长和分化;Axl-siRNA核转染法是研究DC功能的一种有效手段.Objective To transfer Anexelekto-small interfering RNA （Axl-siRNA） into precursors of marrow-derived dendritic cells （DC） by nucleofection,and to examine the transfection efficiency,DC growth and DC differentiation. Methods Axl- siRNA was designed,manufactured, and nucleofected into mouse hone marrow cells. The cells were then cultured in a DC differentiation system,divided into the Axl siRNA group and the the blank control group according to receiving Axl-siRNA nucleo fection or not. The nuclefection efficiency was evaluated by fluorescence examination, and the cell survival was assayed by trypan blue staining during 1 week after nucleofection. The cell morphology and growth curve were documented. At day 8 after trans fection,CD11c＋ expression was determined by flow cytometry. Results The rate of fluorescence-expression cells after nucleofection reached （70.5 ± 6.3） %, （ 72.6 ± 7.3） %and （ 68.8 ± 6.5 ） % at the first three days, respectively. Meanwhile, the cell death rate was （21.2±5.4）%. In the blank control group,the cells assumed logarithmic growth at the first 3 days and the cell number achieved （2.38-2.47）×10^6/well at day4 6 and （2. 23-2. 31） ×10^6/wellat day7 and 8, similar to that in the Axl siRNA group. Flow cytometry showed that CDllc positive cells in the vector control group and the Axl siRNA group were （70.28±6.13） % and （67.53±7.45） % ,respectively. Conclusion Axl-siRNA nucleofection presents high transfection efficiency for DC precursors, without significant interruption on DC growth and differentiation. This approach is beneficial to further investigate the DC function.

关 键 词：核转染 Axl-siRNA 树突状细胞 树突状细胞前体细胞 

分 类 号：R349.6[医药卫生—基础医学]