论文部分内容阅读
目的 在培养的心脏细胞模型上，探讨胰岛素诱导心肌肥厚的作用及其机制。 方法 建立乳鼠心肌细胞和心肌成纤维细跑的培养方法，用光镜、电镜及免疫细胞化学染色进行鉴定。与空白对照比较，观察胰岛素干预后两种细胞肥大、增殖的量效关系：（１）测定细胞蛋白含量（考马斯亮蓝法）。（２）细胞计数；ＢｒｄＵ、ＷＳＴ－１ ＥＬＩＳＡ 同步测定细胞ＤＮＡ 合成与代谢活性；流式细胞仪分析细胞周期。（３）检测胰岛素（１０－ ７ｍ ｏｌ）干预后细胞纤维连接蛋白（ＦＮ）、层粘连蛋白（ＬＮ）、转化生长因子（ＴＧＦβｌ）及早期反应基因蛋白ｃ－ｆｏｓ、ｃ－ｍｙｃ的表达（免疫细胞化学染色）。 结果 （１）培养的乳鼠心肌细胞和心肌成纤维细胞纯度分别在９０％ 和９５％ 以上；（２）一定浓度胰岛素使心肌细胞蛋白含量增高，且在１０－ １０～１０－ ７ｍ ｏｌ呈量效关系，但心肌细胞数量、ＢｒｄＵ 掺入、ＷＳＴ－１代谢活性及Ｓ＋ Ｇ２＋ Ｍ 期细胞百分比无明显变化（Ｐ＞ ０．０５）；（３）一定浓度胰岛素使心肌成纤维细胞数量、ＢｒｄＵ 掺入、ＷＳＴ－１ 代谢活性及Ｓ＋ Ｇ－２＋ Ｍ 期细胞百分比均明显升高；（１０－ ７～１０－ ６）ｍ ｏｌ胰岛素尚增加其细胞蛋白含量（Ｐ＜ ０．０１ 或０
Objective To investigate the effect and mechanism of insulin-induced cardiac hypertrophy on cultured cardiac cell model. Methods The culture method of neonatal cardiomyocytes and myofibroblasts was established and identified by light microscopy, electron microscopy and immunocytochemical staining. Compared with the blank control, observe the dose-effect relationship between the two kinds of cell hypertrophy and proliferation after insulin intervention: (1) Determination of cellular protein content (Coomassie brilliant blue method). (2) Cell counting; BrdU, WST-1 ELISA simultaneous determination of DNA synthesis and metabolic activity; Cell cycle analysis by flow cytometry. (3) The expression of fibronectin (FN), laminin (LN), transforming growth factor (TGFβ1) and early response gene proteins c-fos, c-myc after 10-7mol / Cytochemical staining). Results (1) The purity of cultured cardiomyocytes and myofibroblasts were above 90% and 95%, respectively. (2) Insulin in a certain concentration increased the protein content of cardiomyocytes, and in 10-10 ~ 10-7mol (P> 0.05). (3) The number of myocardial fibroblasts, the number of BrdU incorporation, the metabolic activity of WST-1 and the percentage of cells in S + G2 + M phase did not change significantly WST-1 metabolic activity and percentage of S + G-2 + M phase cells were significantly increased; (10- 7 ~ 10-6) m ol insulin still increased the cellular protein content (P <0.01 or 0