VCAM-1基因敲减对小鼠间充质干细胞免疫调节能力的影响  

Effect of Knockdown of Vascular Cell Adhesion Molecule-1 on the Immunologic Regulation Capacity of Murine Mesenchymal Stem Cells

作　　者：倪永青 刘元林[2] 朱恒[2] 汪李慧 马士凤[1] 陈秀慧[3] 郑荣秀[1] 张毅[2] 

机构地区：[1]天津医科大学总医院儿科,天津300052 [2]军事医学科学院基础医学研究所细胞生物学研究室,北京100850 [3]河北北方学院研究生部,河北张家口075000

出　　处：《中国实验血液学杂志》2015年第3期796-801,共6页Journal of Experimental Hematology

基　　金：国家重点基础研究计划(973项目2010CB833600);国家自然科学基金面上项目(31070996;31171084);天津市自然科学基金项目(12JCYBJC17100)

摘　　要：目的:利用siRNA技术建立稳定低表达血管间粘附分子1(VCAM-1)基因的小鼠间充质干细胞系(C3H10T1/2)并探讨其对小鼠间充质干细胞免疫调节能力的影响。方法:利用基因重组技术构建逆转录病毒质粒GV118-VCAM-1-RNAi,应用酶切及测序方法进行相关鉴定,转染包装细胞系T293,收集病毒上清并感染C3H10T1/2细胞(C3),应用荧光倒置显微镜及流式细胞术检测转染效率,淋巴母细胞转化实验(LTT)和混合淋巴细胞反应(MLR)检测其免疫调节能力。结果:酶切和测序鉴定表明,成功构建了低表达VCAM-1的重组逆转录病毒载体GV118-VCAM-1-RNAi,感染C3H10T1/2细胞后,应用荧光显微镜和流式细胞术分选获得稳定低表达VCAM-1的间充质干细胞GV118-VCAM-1/C3;LTT和M LR实验结果显示,低表达VCAM-1基因的M SC抑制T淋巴细胞增殖的能力显著下降,差异具有统计学意义(P<0.05)。结论:敲减VCAM-1可显著下调MSC抑制T淋巴细胞增殖的能力。这为进一步研究VCAM-1基因与MSC免疫抑制功能的相关性提供了可靠的实验基础。Objective： To establish the stably lower expression of vascular cell adhesion molecule-1 （ VCAM-1 ） in MSC cell line （C3H10T1/2） by siRNA technology, and explore the effect of knockdown of VCAM-1 on the immunologic regulation capacity of murine MSC. Methods：The mouse GVII8-VCAM-1-RNAi retrovirus vector was constructed by gene recombination technology. The recombinant plasmid was identified by restriction analysis and sequencing, and then the recombinant plasmid GV118-VCAM-1-RNAi was transfected into 293 cells by Lipofectamine, and the supernatant was collected to transfect C3H10T1/2. Moreover, the VCAM-1 lower expression on MSC was evaluated by flow cytometry and fluorescent microscopy. The knockdown VCAM-1 MSC was sorted by flow cytometry. Furthermore, the inhibitory effect of the knockdown VCAM-1 MSC on lymphocyte proliferation was tested by lymphoblast transformation assay （LTT） and mixed lymphocyte reaction assay （MLR）. Results： The recombinant retroviral vector of knockdown VCAM-1 （GVll8-VCAM-1-RNAi） was successfully constructed and transfected into mouse MSC cell line C3H10T1/2. The knockdown VCAM-1/MSC was obtained by flow cytometric sorting. The LTT and MLR assay showed that the immunosuppressive effect of MSC lower-expressing VCAM-1 dramatically decreased （ P 〈0.05）. Conclusion：Knockdown VCAM-1 in MSC can significantly down-regulate the inhibitory capability of MSC on the proliferation of T-cells. The data of this study laid an experimental foundation for studying effect of VCAM-1 transfecting into MSC on immune function.

关 键 词：血管细胞粘附分子1 小RNA干扰 基因转染 间充质干细胞 免疫调节 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]