人脐带间充质干细胞对T细胞活化介导的肺动脉平滑肌细胞增殖抑制及机制研究  

Inhibitory effect and mechanism of human umbilical cordmesenchymal stem cells on T cell activation-mediated proliferation of pulmonary artery smooth muscle cells

作　　者：吴西军 刘庆 刘俊峰[1,2] 杨小燕 何志旭[1,2] WU Xijun;LIU Qing;LIU Junfeng;YANG Xiaoyan;HE Zhixu(National Guizhou Joint Engineering Laboratory for Cell Engineering and Biomedical Technique,Guizhou Provincial Key Laboratory of Regenerative Medicine,Affiliated Hospital of Guizhou Medical University,Guiyang,Guizhou Province,550004,China;Key Laboratory of Adult Stem Cell Translational Research,Affiliated Hospital of Guizhou Medical University,Guiyang,Guizhou Province,550004,China;Department of Immunology,Affiliated Hospital of Guizhou Medical University,Guiyang,Guizhou Province,550004,China;Department of Pediatrics,Affiliated Hospital of Guizhou Medical University,Affiliated Hospital of Guizhou Medical University,Guiyang,Guizhou Province,550004,China;Department of Hematology and Oncology,Affiliated Hospital of Guizhou Medical University,Guiyang,Guizhou Province,550004,China)

机构地区：[1]贵州医科大学细胞工程生物医药技术国家地方联合工程实验室,贵州省再生医学重点实验室,贵阳550004 [2]贵州医科大学中国医学科学院成体干细胞转化研究重点实验室,贵阳550004 [3]贵州医科大学免疫学教研室,贵阳550004 [4]贵州医科大学附属医院呼吸内科,贵阳550004 [5]贵州医科大学附属医院儿童血液肿瘤科,贵阳550004

出　　处：《第三军医大学学报》2019年第22期2174-2180,共7页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81871313);中国医学科学院中央级公益性科研院所基本科研业务费专项资金(2017PT31042,2018PT31048);贵州省“十二五”计划重大科技专项[黔教合重大专项(2013-021)];贵阳市-贵州医科大学联合基金项目[筑科合同(2015)001-03]~~

摘　　要：目的阐释人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hu-MSCs)抑制T淋巴细胞活化介导的肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs)增殖的作用及可能的调节机制。方法复苏并培养hu-MSCs和大鼠来源的PASMCs、T细胞,将实验分为PASMCs对照组、T细胞刺激组(PASMCs+T淋巴细胞)、MSC干预组(PASMCs+T淋巴细胞+MSC)、英夫利西单抗干预组(PASMCs+T淋巴细胞+英夫利西单抗)、TNF-α刺激组(PASMCs+T淋巴细胞+MSC+TNF-α)。通过荧光分光光度计、RT-qPCR、倒置荧光显微镜观察等方法研究钙调磷酸酶、T淋巴细胞核因子的变化。结果 T淋巴细胞刺激组TNF-α浓度明显升高(P<0.01);MSCs与英夫利西单抗均能明显降低其TNF-α浓度(P<0.01)。与单纯PASMCs对照组比较,T淋巴细胞刺激组PASMCs的增殖能力明显增强(P<0.01),胞浆中游离钙离子的浓度明显升高(P<0.01),胞内CaN的表达(P<0.01)和活性(P<0.01)均显著上调,NFATc2的表达和活性亦显著上调(P<0.01);与T淋巴细胞刺激组相比,MSCs干预组、英夫利西单抗干预组PASMCs的增殖被明显抑制(P<0.01),胞浆中游离钙离子的浓度均明显下降(P<0.01),胞内CaN的表达(P<0.01)和活性(P<0.01)均显著下调,NFATc2的表达(P<0.01)和活性亦下调;外源TNF-α可有效逆转MSC的干预作用,其作用与T淋巴细胞刺激组几乎相当。结论 hu-MSCs可以通过改变CaN、NFATc2的表达和活化状态,抑制T淋巴细胞活化,抑制炎症因子TNF-α导致的PASMCs增殖。Objective To investigate the inhibitory effect of human umbilical cord mesenchymal stem cells(hu-MSCs) on T lymphocyte activation-mediated proliferation of pulmonary artery smooth muscle cells(PASMCs) and explore the possible regulatory mechanism. Methods Cultured rat PASMCs were stimulated with activated T lymphocytes with subsequent exposures to hu-MSCs, infliximab, or both hu-MSCs and tumor necrosis factor-α(TNF-α). The changes in calcineurin and T lymphoid nuclear factor in the cell cultures were observed using fluorescence spectrophotometer, RT-qPCR and inverted fluorescence microscopy. Results Among all the treatments, stimulation with activated T lymphocytes alone resulted in the highest concentration of TNF-α in the supernatant of PASMC culture(P<0.01). Co-culture with Hu-MSCs and treatment with infliximab both significantly decreased TNF-α concentration in PASMCs stimulated with activated T lymphocytes(P<0.01). Stimulation with T lymphocytes significantly enhanced the proliferative activity of the PASMCs, increased Ca2+ concentration in the cytoplasm, and up-regulated the expression and activity of intracellular CaN and NFATc2. Co-culture with Hu-MSCs and treatment with infliximab significantly suppressed the proliferation of PASMCs stimulated with T lymphocytes, decreased the concentration of Ca2+ in the cytoplasm, and lowered both the expression and activity of intracellular CaN and NFATc2. Exogenous TNF-α obviously reversed the effects of Hu-MSC on the PASMCs, and produced responses similar to T-lymphocyte stimulation in the PASMCs. Conclusion Hu-MSCs can promote the proliferation of PASMCs by modulating the expression and activation of CaN and NFATc2 to inhibit the activation of T lymphocytes and the PASMCs proliferation induced by inflammatory factor TNF-α.

关 键 词：间充质干细胞 肺动脉平滑肌细胞 细胞增殖 肿瘤坏死因子α 钙调磷酸酶 T淋巴细胞核因子 

分 类 号：R322.121[医药卫生—人体解剖和组织胚胎学] R329.2[医药卫生—基础医学]