摘要
目的 研究信号转导和转录激活子（STAT）信号通路在高糖诱导的心肌成纤维细胞增殖和胶原沉积中的表达及其作用.方法 出生1～3d的SD幼鼠,从其心尖组织中获取第2～4代心肌成纤维细胞.将心肌成纤维细胞分为7组,即高渗组、正常糖组、正常糖＋氟达拉滨（FLU）组、正常糖＋ S3I-201组、高糖组、高糖＋FLU组和高糖＋S3I-201组.正常糖＋FLU组和高糖＋FLU组中加入STAT1抑制剂FLU干预,正常糖＋S3I-201组和高糖＋S3I-201组中加入STAT3抑制剂S3I-201干预,高渗组细胞放置于含5.5 mmol/L葡萄糖和19.5 mmol/L甘露糖的DMEM培养基中培养.MTT比色法检测心肌成纤维细胞的增殖情况.定量聚合酶链反应法检测Ⅰ、Ⅲ型前胶原mRNA的表达水平.ELISA法检测Ⅰ、Ⅲ型胶原蛋白的表达水平.蛋白印迹法检测磷酸化STAT1（p-STAT1）和磷酸化STAT3（p-STAT3）的表达水平.结果 （1）各组心肌成纤维细胞增殖情况的检测结果：培养24和48 h,高糖组心肌成纤维细胞增殖水平均明显高于正常糖组和高渗组（P均＜0.05）.而高糖＋FLU组和高糖＋ S3I-201组心肌成纤维细胞的增殖水平则均明显低于高糖组（P均＜0.05）.（2）各组心肌成纤维细胞Ⅰ、Ⅲ型前胶原mRNA和蛋白表达水平的检测结果：培养12和24h,高糖组Ⅰ、Ⅲ型前胶原mRNA的表达水平均明显高于正常糖组和高渗组（P均＜0.05）.而高糖＋FLU组和高糖＋S3I-201组Ⅰ、Ⅲ型前胶原mRNA的表达水平则均明显低于高糖组（P均＜0.05）,且以高糖＋S3I-201组为甚.培养24和72 h,高糖组Ⅰ、Ⅲ型前胶原蛋白表达水平均明显高于正常糖组和高渗组（P均＜0.05）,而高糖＋FLU组和高糖＋S3I-201组Ⅰ、Ⅲ型前胶原蛋白表达水平则均明显低于高糖组（P均＜0.05）.（3）高糖组心肌成纤维细胞中p-STAT1和p-STAT3蛋白表达水平的检测结果：高糖组刺激后0和30 min心肌成纤维细胞中p-STAT1表达水平没有明显
Objective To observe the signal transducers and activator of transcriptions （STATs） protein expression changes and investigate the functional role of STATs pathway in case of high glucoseinduced cardiac fibroblasts （CFs） proliferation and collagen deposition in vitro.Methods Rat cardiac fibroblasts were isolated from 1-to 3-day-old SD rats,cells from the second to fourth passages were used for the experiment.CFs were cultured in Dulbecco＇s modified Eagle＇s medium,supplemented with 5.5 mmol/L glucose（NG）,5.5 mmol/L glucose plus 19.4 mmol/L mannose（OC） or 25 mmol/L glucose（HG） in the presence of absence of STAT1 inhibitor （fludarabine,FLU） and STAT3 inhibitor （S3I-201）.After 24 h and 48 h culture in vitro,the proliferation of CFs was measured by 3-（4,5-dimethyl-2 thiazoyl）-2,5-diphenyl2H-tetrazolium bromide （MTT） assay.After 12 h and 24 h culture in vitro,the production of type Ⅰ and Ⅲ collagen was evaluated using real-time quantitative PCR and ELISA.After 0,30,60 and 120 min culture in vitro,the phosphorylated expression of STAT1 and STAT3 was analyzed by Western blot.Results CFs proliferation was significantly enhanced post 24 h and 48 h HG stimulation,and procollagen Ⅰ and Ⅲ mRNA expression was significantly upregulated post 12 h and 24 h HG stimulation.Deposition of collagen Ⅰ and Ⅲ was also significantly increased post 24 h and 72 h HG stimulation.STAT1 phosphorylation in CFs was increased after 120 min HG stimulation and STAT3 phosphorylation in CFs was increased post 60 min and 120 min HG stimulation.FLU and S3I-201 could inhibit HG-induced CFs proliferation and suppress of which was stimulated by FLU and S3I-201 could both suppress upregulated procollagen Ⅰ and Ⅲ mRNA expression and the deposition of collagen types Ⅰ and Ⅲ post HG stimulation.STAT1 phosphorylation inhibition resulted in less mRNA downregulation of procollagen type Ⅱ than that of procollagen type Ⅰ post 12 h HG stimulation.The STAT3 phosphorylation inhibition resulted in more si
出处
《中华心血管病杂志》
CAS
CSCD
北大核心
2015年第5期442-447,共6页
Chinese Journal of Cardiology
关键词
心肌
成纤维细胞
细胞增殖
胶原
信号转导和转录激活子
Myocardium
Fibroblasts
Cell proliferation
Collagen
Signal transducers and activator of transcriptions
