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
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Angiotensin Ⅱ Type 2 Receptor Gene Transfection Inhibit Maturation of Bone Marrow Derived Dendritic Cells from Mice in Vitro
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    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(pAdCMV/AT2R)或空病毒载体(pAd-GFP),再用脂多糖刺激为成熟BMDC,分PBS对照组、脂多糖组、pAd-GFP组、pAdCMV/AT2R组及pAdCMV/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 pAdCMV/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,pAdCMV/AT2R组和pAdCMV/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pAdCMV/AT2R组与脂多糖组比较显著降低(P<0.01或P<0.05),而pAdCMV/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。

    Abstract:

    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 C57BL/6J mice marrow,purified and differentiated to BMDC. First the BMDC were transfected with pAdCMV/AT2R or control virus pAd-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 AT2R as pAdCMV/AT2R+PD123319 group PBS as negative control and LPS as positive control. The expressions of AT2R 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 pAdCMV/AT2R was transduced into BMDC,the expressions of AT2R mRNA significantly increased at 48 hours and 72 hours,green fluorescence localized to cell nuclei and plasm,red fluorescence labling AT2R was present in cell plasm. Compared with PBS negative control,the expressions of CD86,MHCⅡ was significantly up-regulated in pAdCMV/AT2R group and pAdCMV/AT2R+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 pAdCMV/AT2R group were significantly decreased(P<0.01 or P<0.05), the changes of pAdCMV/AT2R+PD123319 group were not significantly different(P>0.05). Conclusion BMDC can express AT2R stably in vitro,AT2R can inhibit the maturation and immunologic action of BMDC and antagonist of AT2R can hold up the effect,so AT2R may induce inhibition of atherosclerosis.

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唐 兵,速晓华,陈劲松,李 刚,杨大春,杨永健,朱 峻,李兴科,李 德.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[J].中国动脉硬化杂志,2015,23(09):881~886.

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  • 收稿日期:2014-11-14
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  • 在线发布日期: 2015-07-21