心肌梗死大鼠单个核细胞分泌TNF-α对间充质干细胞迁移的影响  

Effect of mononuclear cell secreting TNF-α on the migration of mesenchymal stem cells in myocardial infarction in rats

作　　者：廖礼强[1] 张晓刚[1] 史若飞[2] 王丁琼[1] 戴树人[1] 

机构地区：[1]重庆医科大学附属第一医院心血管内科,重庆400016 [2]重庆市急救医疗中心心血管内科,重庆400014

出　　处：《解放军医学杂志》2010年第7期807-810,共4页Medical Journal of Chinese People's Liberation Army

摘　　要：目的探讨心肌梗死(MI)大鼠循环血单个核细胞分泌的TNF-α对间充质干细胞(MSCs)迁移的影响。方法 14只SD大鼠中8只结扎左冠状动脉前降支建立MI模型,其余6只行假手术处理(只穿线不结扎),于第7天时分离大鼠循环血单个核细胞。自制体外三室共培养模型,将4,6-二脒基-2苯基吲哚(DAPI)-MSCs、心肌细胞和单个核细胞分别培养于三室模型的上、中、下室内。按下室所加成分的不同,设立MI组(只加入2×106个MI大鼠单个核细胞)、TNF-α中和抗体组(TNF-α-NA组,加入2×106个MI大鼠单个核细胞及终浓度为50ng/L的TNF-α中和抗体)、假手术组(只加入2×106个假手术大鼠单个核细胞)和空白对照组(只加入培养液)。共培养48h后,对MSCs迁移细胞进行CD44及CD34免疫组化鉴定和荧光显微镜下计数,ELISA法检测上清液TNF-α含量,Westernblotting检测MSCs表面血管细胞黏附分子1(VCAM-1)表达情况,并分析TNF-α含量与MSCs迁移数目的相关性。结果迁移细胞CD44呈阳性表达,而CD34呈阴性表达,符合MSCs的特点。与假手术组比较,MI组迁移细胞数量显著增多(P<0.05),TNF-α含量明显升高(P<0.05),而中和TNF-α后,TNF-α-NA组迁移细胞数明显减少(P<0.05)。MSCs迁移数目与TNF-α含量呈明显正相关(r=0.969,P<0.05)。与假手术组比较,MI组MSCs表面VCAM-1的表达量显著升高(P<0.05),而中和TNF-α后,TNF-α-NA组VCAM-1的表达量明显下降(P<0.05)。结论 MI大鼠单个核细胞可促进MSCs迁移,其机制可能与单个核细胞分泌的TNF-α增强MSCs黏附性有关。Objective To investigate the effect of tumor necrosis factor-alpha （TNF-α） released by circulating mononuclear cells （MNCs） on the migration of mesenchymal stem cells （MSCs） in myocardial infarction （MI） in rats.Methods Eight of 14 SD rats were selected for reproduction of MI model by ligation of left anterior descending coronary artery,the rest 6 rats underwent sham operation （with no ligation）,and circulating MNCs were isolated on the 7th day.A tri-chamber co-culture model in vitro was established with self-made Transwell chambers.4,6-diamidino-2-phenyl-indole（DAPI）-labeling MSCs,myocardial cells and MNCs were planted into the upper,middle and lower compartment of the tri-chamber co-culture model,respectively.According to the different additions into the lower compartments,the whole experiment was divided into MI group （ 2× 10^6 MNCs from MI rats were added）,TNF-α neutralizing antibody group （TNF-α-NA group,2×10^6 MNCs from MI rats and TNF-α in final concentration of 50ng/L were added）,sham operation group （2×10^6 MNCs from sham rats were added） and blank control group （culture medium only was added）.After 48h cultivation,migrated cells were identified by CD44 and CD34 immunocytochemistry staining and counted under the fluoroscope.Concentration of TNF-α in the supernatant was measured by ELISA,and the correlation between the concentration and the number of migrated MSCs was analyzed.The expression of vascular cell adhesion molecule-1 （VCAM-1） in MSCs was evaluated by immunofluorescent staining and Western blotting.Results CD44 was positively,while CD34 negatively expressed in all the migrated cells,indicating they were consistent with MSCs.More migrated cells and higher TNF-α concentration were found in MI group than in sham operation group （P〈0.05）,while the number of migrated cells was significantly reduced in TNF-α-NA group after neutralization of TNT-α （P〈0.05）.A positive correlation was observed between TNF-α concentration and the number o

关 键 词：心肌梗死 单个核细胞 肿瘤坏死因子α 间质干细胞 

分 类 号：R542.22[医药卫生—心血管疾病]