Blimp1-短发夹RNA基因转染对骨髓源性树突状细胞前体细胞分化的影响  

Lentivirus-mediated Blimpl-shRNA gene therapy regulates the differentiation of bone marrow-derived dendritic procursors （Pre-DCs）

作　　者：李幸[1] 戴晓敏[1] 姜汉英[1] 周平[1] 陈知水[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

出　　处：《中华器官移植杂志》2013年第12期749-753,共5页Chinese Journal of Organ Transplantation

基　　金：国家自然科学基金（81072441）

摘　　要：目的探讨以慢病毒为载体转染B淋巴细胞诱导蛋白-1（Blimp1）-短发夹RNA对小鼠骨髓原代细胞向树突状细胞（DcC）诱导分化的影响。方法构建Blimp1-短发夹RNA。在Balb／c小鼠骨髓原代细胞定向分化为DC的8d培养体系中，于培养第2天用慢病毒载体将Blimp1-短发夹RNA转染入细胞中。实验分为3组：空白对照组培养体系中不加入任何慢病毒载体，空载体对照组培养体系中进行空载体慢病毒干预，实验组培养体系中进行lenti—Blimp1-短发夹RNA干预。转染1周内观察转染效率，观察细胞的形态变化，记录细胞的生长曲线；于培养第4、5天收集细胞，实时定量逆转录聚合酶链反应和蛋白质印迹法检测各组Blimp1信使RNA和Blimp1的表达情况；检测培养第8天CD11c^＋、CD86^＋及主要组织相容性复合物Ⅱ（MHC-Ⅱ）＋细胞的百分率。结果病毒转染后第3天细胞出现荧光，其后荧光持续存在；培养过程中出现典型DC形态，接受病毒转染的细胞有形态受损表现。空白对照组细胞在培养的前3d内呈对数增长，第4天细胞数目达到峰值，为（2．45±0．26）×10^6／孔，第8天为（2．27±0．19）×10^6／孔；空载体对照组和实验组细胞在病毒侵染后细胞增殖停滞，细胞数目稳定在（1．69±0．39）×10^6／孔。空载体对照组和实验组细胞中Blimp1信使RNA及Blimp1蛋白的表达量分别为空白对照组的76％和1％以及105％和74％。在空白对照组、空载体对照组和实验组中CD11c^＋细胞分别占（69．2±5．0）％、（68．6±5．9）％和（72．8±5．5）％（P〉0．05）；CD86^＋细胞分别占（51．1±4．9）％、（49．5±4．3）％和（50．2±6．0）％（P〉0．05）；MHC-Ⅱ^＋细胞分别占（56．3±7．3）％、（69．4±4．5）％和（46．5±5．7）％，3组间的差异有统计学意义（P〈0．05）。结论慢病毒介导的短发夹RNA基因治疗是调控骨髓源性DC�Objective To investigate the effect of down-regulated Blimp1 gene expression on differenetiation of bone marrow cells into dendritic cells （DCs）. Methods Blimpl-shRNA was constructed and then loaded into lentivirus vector as lenti-blimp1-shRNA. Bone marrow cells from Balb/c mice were induced differentiation to DCs in an 8-day cell culture system with GM-CSF/IL-4 incubation and LPS stimulation at day 7. The cells were divided into groups as empty control （no treatment）, lenti-control （transfected by lentivirus empty vector at day 1 ）, and lenti-Blimp （transfected by lenti-blimpl-shRNA at day 1）. The transfection efficiency was evaluated by GFP fluorescence for one week. The morphology and growth curve were analyzed. Real-time PT-PCR and Western blotting were used to evaluate mRNA and protein expression of Blimp1. At day 8, CD1 lc and CD86/MHC-Ⅱ were quatitified using flow cytometry. Results GFP fluorescence emerged 3 days after transiection and was continuously expressed. Classic DC morphology was shown in no treatment ceils, while damaged morphology presented in the cells with lentivirus transfection. The empty control cells proliferated from day 3, peaked as （2. 45 ± 0. 26） 10^6/well at day 4, and kept at （2. 27 q 0. 19） 10^6/well at day 8, The cells receiving lentivirus presented （1.69±0. 39） 10^6/well. The expresmon ot Blimp1 mRNA and protein in the lenti-Blimp1 group was 76%/1% and 1.0%/74. 0% of the empty control group. At day 8, CDllc, CD86 and MHC-Ⅱ expression in the empty control group was （69.2 ±5.0）%, （51.1±4.9）% amd （56.3±7.3）%, while （68.6±5.9）%, （49.5±4.3）% and （69.4± 4. 5）% in thelenti-control group, and （72.8±5.5）%, （50.2±6.0）% and （46.5±5.7）% in the lenti-Blimpl group. Conclusion Lentivirus-mediated Blimp1-shRNA gene therapy modulates blimpl expression of DC precursors. Down-regulation of Blimpl fails to interrupt the differentiation of DCs but inhibits the maturation.

关 键 词：树突细胞 慢病毒 载体 小鼠 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]