CAJ | 학술논문

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(DC)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)×106/孔,第8天为(2.27±0.19)×106/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)×106/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前体细胞Blimp1基因的有效手段;Blimp1基因的下调不影响DC前体细胞的定向分化,但能够抑制DC的成熟.
목적 탐토이만병독위재체전염B림파세포유도단백-1 (Blimp1)-단발협RNA대소서골수원대세포향수돌상세포(DC)유도분화적영향.방법 구건Blimp1-단발협RNA.재Balb/c소서골수원대세포정향분화위DC적8d배양체계중,우배양제2천용만병독재체장Blimp1-단발협RNA전염입세포중.실험분위3조:공백대조조배양체계중불가입임하만병독재체,공재체대조조배양체계중진행공재체만병독간예,실험조배양체계중진행lenti-Blimp1-단발협RNA간예.전염1주내관찰전염효솔,관찰세포적형태변화,기록세포적생장곡선;우배양제4、5천수집세포,실시정량역전록취합매련반응화단백질인적법검측각조Blimp1신사RNA화Blimp1적표체정황;검측배양제8천CD11c+、CD86+급주요조직상용성복합물Ⅱ(MHC-Ⅱ)+세포적백분솔.결과 병독전염후제3천세포출현형광,기후형광지속존재;배양과정중출현전형DC형태,접수병독전염적세포유형태수손표현.공백대조조세포재배양적전3d내정대수증장,제4천세포수목체도봉치,위(2.45±0.26)×106/공,제8천위(2.27±0.19)×106/공;공재체대조조화실험조세포재병독침염후세포증식정체,세포수목은정재(1.69±0.39)×106/공.공재체대조조화실험조세포중Blimp1신사RNA급Blimp1단백적표체량분별위공백대조조적76％화1％이급105％화74％.재공백대조조、공재체대조조화실험조중CD11c+세포분별점(69.2±5.0)％、(68.6±5.9)％화(72.8±5.5)％(P＞0.05);CD86+세포분별점(51.1±4.9)％、(49.5±4.3)％화(50.2±6.0)％(P＞0.05);MHC-Ⅱ+세포분별점(56.3±7.3)％、(69.4±4.5)％화(46.5±5.7)％,3조간적차이유통계학의의(P＜0.05).결론 만병독개도적단발협RNA기인치료시조공골수원성DC전체세포Blimp1기인적유효수단;Blimp1기인적하조불영향DC전체세포적정향분화,단능구억제DC적성숙.
Objective To investigate the effect of down-regulated Blimp1 gene expression on differenetiation of bone marrow cells into dendritic cells (DCs).Methods Blimp1-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-blimp1-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,CD11 c and CD86/MHC-Ⅱ were quatitified using flow cytometry.Results GFP fluorescence emerged 3 days after transfection and was continuously expressed.Classic DC morphology was shown in no treatment cells,while damaged morphology presented in the cells with lentivirus transfection.The empty control cells proliferated from day 3,peaked as (2.45 ± 0.26) 106/well at day 4,and kept at (2.27 ± 0.19) 106/ well at day 8,The cells receiving lentivirus presented (1.69 ± 0.39) 106/well.The expression of 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,CD11c,CD86 and MHC-Ⅱ expression in the empty control group was (69.2 ±5.0)％,(51.1± 4.9) ％ and (56.3 ± 7.3) ％,while (68.6±5.9)％,(49.5±4.3)％ and (69.4±4.5)％ in the lenti-control group,and (72.8 ± 5.5)％,(50.2 ± 6.0)％ and (46.5 ± 5.7)％ in the lenti Blimp1 group.Conclusion Lentivirus-mediated Blimp1-shRNA gene therapy modulates blimp1 expression of DC precursors.Down-regulation of Blimp1 fails to interrupt the differentiation of DCs but inhibits the maturation.