重组腺相关病毒介导B细胞表面分化成熟抗原269基因转染树突状细胞治疗多发性骨髓瘤的体外实验研究  

External experimental study on recombinant adeno-related virus mediated CD269 gene transfection of dendritic cells in the treatment of multiple myeloma

作　　者：陈海梅 徐运孝[1] 李清照 沈婵娟 胡国瑜 

机构地区：[1]中南大学湘雅二医院血液科,湖南长沙410011 [2]湖南省株洲市中心医院血液科

出　　处：《临床内科杂志》2019年第1期51-54,共4页Journal of Clinical Internal Medicine

基　　金：湖南省卫生与计划生育委员会科研计划课题项目C2015-075.

摘　　要：目的探讨重组腺相关病毒(rAAV)介导B细胞表面分化成熟抗原269(CD269)基因转染树突状细胞(DCs)对多发性骨髓瘤(MM)的治疗作用。方法取中南大学湘雅二医院健康人外周血单个核细胞,采用含粒细胞.巨噬细胞集落刺激因子(GM-CSF)和白细胞介素(IL)-4的培养液进行体外诱导,并设置转染组、空载组和对照组,转染组培养第1d加入rAAV/CD269病毒液,空载组培养第1d加入rAAV病毒液,对照组培养第1d加入等量磷酸缓冲盐溶液,3组均于培养第5 d加入肿瘤坏死因子(TNF)-α仪诱导树突状细胞(DCs)成熟。观察分化细胞的形态学变化,检测3组DCs的CD269表达。将3组DCs分别加入自体T淋巴细胞,获得细胞毒性T淋巴细胞(CTLs),检测CTLs中干扰素(IFN)-γ的表达,并检测3组中CTLs分别对人U266细胞、人MM原代细胞和阴性对照细胞的杀伤率和转染组不同效靶比下的杀伤率。结果细胞逐渐发生形态学改变且符合DCs形态特征。转染组DCs中CD269表达明显高于空载组和对照组(P<0.05)。转染组IFN-γ的表达率高于空载组和对照组(P<0.05)。转染组CTLs对人MM细胞株U226和人MM原代细胞的杀伤率均高于空载组和对照组(P<0.05)。当效靶比为1:1、5:1、10:1、20:1、50:1时,CTLs对人MM原代细胞的杀伤率分别为(13.24±1.29)%、(17.68±1.96)%、(25.66±2.17)%、(33.14±2.33)%、(39.74±2.56)%,差异均有统计学意义(P<0.05)。结论rAAV介导CD269基因转染DCs能够获得CTLs,上调IFN-γ表达,增强对人MM细胞株U226的杀伤作用,且效靶比越高,杀伤率也越高。Objective To investigate the therapeutic effect of recombinant adeno-related virus (rAAV) mediated B cell surface differentiation mature antigen 269( CD269) gene transfection of dendritic cells( DCs) on multiple myeloma ( MM ). Methods Peripheral blood mononuclear cells from healthy individuals in Xiangya No. 2 Hospital of Central South University were induced by granulocyte macrophage colony stimulating factor(GM-CSF) and interleukin (IL)-4 in vitro. Three groups were set up as transfection group, no-load group and the control group. rAAV/CD269 virus was added to transfection group at the 1 st day of culturing,rAAV virus was added to no-load group at the 1st day of culturing,and the same amount of phosphate buffer buffer solution was added to the control group at the 1st day of culturing. In the three groups,dendritic cells( DCs) were induced by adding tumor necrosis factor( TNF) - a at the 5th day of culturing. The morphological changes of differentiated cells were observed, and the expression of CD269 in DCs of the three groups was detected. Three groups of DCs cells were added to autologous T lymphocytes to obtain cytotoxic T lymphocytes ( CTLs) , then the interferon (IFN) -y expression in CTLs of the three groups were detected. The killing rates of CTLs on human U266 cells, primary human MM cells and negative control cells were detected in the three groups, and the killing rates of CTLs in different effective target ratios in transfection group were determined. Results The morphology of cells gradually changed and accords with the morphological characteristics of DCs. The CD269 expression in the DCs of the transfection group was significantly higher than those of no-load group and the control group(P < 0. 05 ). The expression rate of IFN--y in transfection group was higher than those in no-load group and control group (P <0.05). In the transfection group,the killing rates of CTLs on human MM cell line U226 and primary cells were all higher than those in no-load group and control group ( P < 0. 05 ). When the

关 键 词：重组腺相关病毒 B细胞表面分化成熟抗原269 基因转染 树突状细胞 多发性骨髓瘤 

分 类 号：R733.3[医药卫生—肿瘤]