血管紧张素Ⅱ2型受体抑制小鼠骨髓源树突状细胞的成熟及活化  

Angiotensin Ⅱ Type 2 Receptor Gene Transfection Inhibit Maturation of Bone Marrow Derived Dendritic Cells from Mice in Vitro

作　　者：唐兵[1] 速晓华[1] 陈劲松[1] 李刚[1] 杨大春[1] 杨永健[1] 朱峻[1] 李兴科 李德[1] 

机构地区：[1]成都军区总医院心血管内科,四川省成都市610038 [2]重庆大江医院检验科,重庆市401321

出　　处：《中国动脉硬化杂志》2015年第9期881-886,共6页Chinese Journal of Arteriosclerosis

摘　　要：目的研究血管紧张素Ⅱ2型受体（AT2R）体外基因转染对小鼠骨髓源树突状细胞（BMDC）成熟活化及部分免疫功能的影响,探讨AT2R参与动脉粥样硬化斑块进展的免疫机制。方法取C57BL/6J小鼠骨髓,经分离、纯化、分化为BMDC,先转染带有绿色荧光蛋白报告基因的AT2R基因重组腺病毒载体（p Ad CMV/AT2R）或空病毒载体（p Ad-GFP）,再用脂多糖刺激为成熟BMDC,分PBS对照组、脂多糖组、p Ad-GFP组、p Ad CMV/AT2R组及p Ad CMV/AT2R＋PD123319组。采用RT-PCR、免疫荧光和激光共聚焦技术方法检测BMDC中AT2R mRNA及蛋白表达;流式细胞术检测BMDC表面标志CD86和MHCⅡ的表达率;液体闪烁计数检测BMDC刺激同源T淋巴细胞增殖的能力;ELISA法检测培养基中白细胞介素12（IL-12）和γ干扰素（IFNγ）的水平。结果 p Ad CMV/AT2R转染BMDC后48~72 h有AT2R mRNA表达,在激光共聚焦显微镜下胞浆及胞核有绿色荧光表达;同时在胞浆有红色荧光AT2R表达。与PBS对照组比较,p Ad CMV/AT2R组和p Ad CMV/AT2R＋PD123319组CD86和MHCⅡ的表达、同源T淋巴细胞增殖和细胞因子分泌量都显著升高（P〈0.01）,但p Ad CMV/AT2R组与脂多糖组比较显著降低（P〈0.01或P〈0.05）,而p Ad CMV/AT2R＋PD123319组与脂多糖组比较差异无显著性（P〉0.05）。结论体外基因转染BMDC能表达AT2R,AT2R能抑制BMDC成熟诱导的局部免疫炎性反应,且AT2R拮抗剂能阻断此效应,推测AT2R可能介导稳定斑块及抑制动脉粥样斑块进展的作用。Aim To investigate the influence of angiotensin Ⅱ type 2 receptor（ AT2R） gene transfection on the maturation and some immune functions of bone marrow derived dendritic cells（ BMDC） from mice and explore the mechanism of AT2R-induced immunologic function in atherogenesis. Methods The cell suspension was made from C57 BL /6J mice marrow,purified and differentiated to BMDC. First the BMDC were transfected with p Ad CMV / AT2 R or control virus p Ad-GFP,containing green fluorescent protein reporter gene; Then were stimulated by 100 μg / L lipopolysaccharide（ LPS） into mature DC; In the same time were added to antagonist of AT2 R as p Ad CMV/AT2 R ＋ PD123319 group; PBS as negative control and LPS as positive control. The expressions of AT2 R in DC were evaluated by RT-PCR,immunofluorescence staining and confocal microscope. The CD86 and MHCⅡ expressing rates were detected by fluorescence-activated cell sorting（ FACS）. Liquid scintillation counting（ LSC） was used in mixed lymphocyte reactions（ MLR） to reflect the ability of BMDC to stimulating homologous T cells proliferation. Cytokines IL-12 and IFN-γ were detected by ELISA.Results After p Ad CMV / AT2 R was transduced into BMDC,the expressions of AT2 R mRNA significantly increased at 48 hours and 72 hours,green fluorescence localized to cell nuclei and plasm,red fluorescence labling AT2 R was present in cell plasm. Compared with PBS negative control,the expressions of CD86,MHC Ⅱ was significantly up-regulated in p Ad CMV / AT2 R group and p Ad CMV / AT2 R ＋ PD123319 group（ P〈 0. 01）,the stimulating capacity of BMDC obviously enhanced（ P 〈0. 01）,levels of IFN-γ and IL-12 in the supernatant increased（ P 〈0. 01）; But compared with LPS positive control,the changes of p Ad CMV / AT2 R group were significantly decreased（ P〈 0. 01 or P〈 0. 05）,the changes of p Ad CMV / AT2 R ＋ PD123319 group were not significantly different（ P 〉0. 05）. Conclusion BMDC can express AT2 R stably in vitro,AT2 R can inhibit t

关 键 词：血管紧张素Ⅱ2型受体 基因转染 树突状细胞 细胞成熟 动脉粥样硬化 

分 类 号：R363[医药卫生—病理学]