肿瘤坏死因子α在血管内皮细胞向间质细胞转化中的作用  

Role of tumor necrosis factor α in endothelial-mesenchymal transition in vitro

作　　者：代卉[1] 黄宏[3] 王莎莉[1] 徐祥[3] 简宇[3] 崔文慧[2] 张猛[3] 张波[3] 蒋建新[3] 

机构地区：[1]重庆医科大学基础医学院神经科学研究中心,400016 [2]重庆医科大学基础医学院生理教研室,400016 [3]第三军医大学大坪医院野战外科研究所,创伤、烧伤与复合伤国家重点实验室

出　　处：《中华烧伤杂志》2012年第1期19-24,共6页Chinese Journal of Burns

基　　金：基金项目：国家重点基础研究发展计划（2012CB518105）;国家自然科学基金（30871025）;创伤、烧伤与复合伤国家重点实验室自主研究课题（SKLZZ200907）;重庆市自然科学基金（CSTC,2010BB5036）

摘　　要：目的 观察TNF-α在血管内皮细胞向间质细胞转化中的作用,探讨纤维化疾病发生的机制. 方法 取健康胎儿脐带,体外酶消化法分离培养人脐静脉内皮细胞（HUVEC）,用免疫荧光法进行鉴定.取第3～5代对数生长期HUVEC分别接种于12孔板和6孔板中,均按随机数字表法分为6组：对照组,培养液中不加任何刺激因素；5、10、25、50、100 ng/mL TNF-α组,分别在培养液中添加相应终浓度TNF-α,每组3个样本.培养72 h后倒置相差显微镜下观察各组细胞形态；免疫荧光法检测12孔板中各组细胞凝血因子Ⅷ和α平滑肌肌动蛋白（α-SMA）的表达,计算2种因子双阳性细胞数比值及吸光度值比值；RT-PCR检测6孔板中各组细胞钙黏蛋白、α-SMA和Ⅰ型胶原mRNA的表达（以灰度值比值表示）.对数据行单因素方差分析及LSD检验. 结果（1）原代培养HUVEC为圆形、短梭形或扁平形,传代后细胞呈铺路石样旺盛生长.经第1、2、3、4、5次传代后HUVEC凝血因子Ⅷ阳性表达率分别为（85.5±1.8）％、（88 1±5 0）％、（93.6±3.7）％、（92.9±4 8）％、（89.5±1.1）％,明显高于原代培养HUVEC的（81.4±3.8）％,F值均为7.481,P值均小于0.05.（2）对照组细胞呈圆形、短梭形或扁平形,细胞间连接紧密；5、10、25、50、100 ng/mL TNF-α组随着TNF-α浓度的增加,细胞形态逐渐向长梭形转变,细胞间连接减少、间隙变大.（3）对照组中凝血因子Ⅷ、α-SMA双阳性细胞数比值和吸光度值比值分别为0.055±0 015、0.078±0 017,均显著低于5、10、25、50、100 ng/mL TNF-α组的0 257±0.106、0 280±0.129、0.505±0 059、0.817±0.035、0.929±0.101和0.437±0 040、0 456±0.097、0 496±0.082、0.787±0 131、0.885±0 087,F值分别为45 009、50.099,P值均小于0.01.（4）5、10、25、50、100 ng/mL TNF-α组细胞中钙黏蛋白mRNA水平分别为0.70±0.05、0.63±0 06、0 60±0.10、0.45±0.16、0 26±0.14,后4组显著低于对照组的0 8Objective To observe the role of tumor necrosis factor α（TNF-α）in endothelial-mesenchymal transition（EnMT）,and to explore the mechanism of fibrosis disease. Methods Human umbilical vein endothelial cells（HUVEC）from umbilical cord of healthy fetus were isolated by enzymatic digestion and identified by immunofluorescence assay.The third to fifth generations of cultured HUVEC in logarithmic phase were harvested and seeded in 12-well plates and 6-well plates,and they were divided into control group（ordinary culture without any stimulation）,5,10,25,50,and 100 ng/mL TNF-α groups （5,10,25,50,100 ng/mL of TNF-α was respectively added into the nutrient solution）according to the random number table,with three samples in each group.After being cultured for 72 hours,the cell morphology was observed under inverted phase-contrast microscope; the expression levels of coagulation factor Ⅷ and α smooth muscle actin（α-SMA）were detected by immunofluorescence assay,and the ratios of numbers （absorbance values）of cells with expression of both factors were calculated.The mRNA expression levels of cadherin,α-SMA,and type Ⅰ collagen were detected by RT-PCR（denoted as gray value ratio）.Data were processed with one-way analysis of variance and LSD test. Results （ 1 ）The shape of primary HUVEC was round,short-spindle,or flat,and ceils grew vigorously in cobblestone appearance after passages.After being subcultured for 1,2,3,4,5 passage（s）,the positive rate of coagulation factor Ⅷ of HUVEC was respectively（85.5 ±1.8）%,（88.1 ±5.0）%,（93.6 ±3.7）%,（92.9 ±4.8）%,（89.5 ±1.1）%,and they were significantly higher than that of primary HUVEC [（ 81.4 ± 3.8）%,with F values all equal to 7.481,P values all below 0.05 ].（2）As compared with that in control group,the appearance of cells in 5,10,25,50,and 100 ng/mL TNF-α groups was gradually transformed from round,short-spindle,or flat shape to long-spindle shape with reduced intercellular junction and larger intercellular

关 键 词：肿瘤坏死因子Α 内皮细胞 间质细胞 细胞转分化 肌成纤维细胞 

分 类 号：R363[医药卫生—病理学]