目的探究皮层肌动蛋白结合蛋白(Cortactin)对异丙肾上腺素(isoprenaline,ISO)诱导的病理性心肌肥大的调控作用及其机制。方法采用ISO刺激新生大鼠心肌细胞(neonatal rat cardiomyocytes,NRCMs)24 h,在细胞水平建立心肌肥大模型;C57BL/6...目的探究皮层肌动蛋白结合蛋白(Cortactin)对异丙肾上腺素(isoprenaline,ISO)诱导的病理性心肌肥大的调控作用及其机制。方法采用ISO刺激新生大鼠心肌细胞(neonatal rat cardiomyocytes,NRCMs)24 h,在细胞水平建立心肌肥大模型;C57BL/6小鼠皮下注射ISO 1周,在动物水平建立心肌肥大模型。采用RT-qPCR检测mRNA的变化;免疫印迹法检测相应蛋白含量的变化;免疫荧光法检测Cortactin的亚细胞定位及表达量的变化;采用腺病毒感染的方法过表达Cortactin,通过转染小干扰RNA敲低Cortactin。结果在细胞和动物水平上,成功建立ISO诱导的心肌肥大模型,均观察到ISO引起Cortactin和N型钙黏连蛋白(N-cadherin)水平降低;过表达Cortactin可逆转ISO导致的N-cadherin蛋白水平的降低及心肌细胞肥大反应;敲低Cortactin则显示相反的效应。结论Cortactin可能联合N-cadherin通过增强心肌细胞之间的连接,发挥抗心肌肥大的作用。展开更多
AIM: To study the effects of cortactin on the tumor biology of SGC-7901 cells and identify the mechanism involved in the process. METHODS: Cell lines in which cortactin was stably overexpressed or knocked down as well...AIM: To study the effects of cortactin on the tumor biology of SGC-7901 cells and identify the mechanism involved in the process. METHODS: Cell lines in which cortactin was stably overexpressed or knocked down as well as the respective control cell lines were established by standard molecular methods. The effects of cortactin on the proliferation, migration and invasion capacity of SGC-7901 cells were assessed by the MTT assay, colony formation, flow cytometry, transwell migration and matrigel invasion. Nude mouse models were also used to assess the role of cortactin in the growth and metastasis of SGC-7901 cells in vivo. Western blotting analysis was performed to detect the expression of epidermal growth factor receptor (EGFR) and downstream molecules. RESULTS: Cell lines in which cortactin was stably overexpressed or knocked down as well as control cell lines were successfully established and designated as LV5-cortactin-SGC, LV5-SGC, LV3-shRNA-SGC and LV3SGC. Cortactin overexpression promoted SGC-7901 cell migration (340.7 +/- 12.6 vs 229.1 +/- 23.2, P < 0.01) and invasion (71.6 +/- 5.2 vs 48.4 +/- 3.6, P < 0.01). Cortactin downregulation impaired SGC-7901 cell migration (136.2 +/- 19.8 vs 225 +/- 17) and invasion (29.2 +/- 5.2 vs 49.6 +/- 3.8, P < 0.01). The results from the MTT and colony formation assays results indicated increased LV5-cortactin-SGC cell proliferation and decreased LV3shRNA-SGC cell proliferation compared to the control cells. Flow cytometry analysis demonstrated that cortactin overexpression promoted the proliferation index of SGC-7901 cells, and the results were reversed when cortactin was downregulated. Mouse tumor models confirmed that cortactin expression increased SGC-7901 cell proliferation and metastasis in vivo. Western blotting analysis revealed that cortactin elevated EGFR expression and activated the downstream molecules. CONCLUSION: Cortactin expression promoted the migration, invasion and proliferation of SGC-7901 cells both in vivo and in vitro. The EGFR signaling pathway is mechanistically involved. (c) 2014 Baishideng Publishing Group Co., Limited. All rights reserved.展开更多
文摘目的探究皮层肌动蛋白结合蛋白(Cortactin)对异丙肾上腺素(isoprenaline,ISO)诱导的病理性心肌肥大的调控作用及其机制。方法采用ISO刺激新生大鼠心肌细胞(neonatal rat cardiomyocytes,NRCMs)24 h,在细胞水平建立心肌肥大模型;C57BL/6小鼠皮下注射ISO 1周,在动物水平建立心肌肥大模型。采用RT-qPCR检测mRNA的变化;免疫印迹法检测相应蛋白含量的变化;免疫荧光法检测Cortactin的亚细胞定位及表达量的变化;采用腺病毒感染的方法过表达Cortactin,通过转染小干扰RNA敲低Cortactin。结果在细胞和动物水平上,成功建立ISO诱导的心肌肥大模型,均观察到ISO引起Cortactin和N型钙黏连蛋白(N-cadherin)水平降低;过表达Cortactin可逆转ISO导致的N-cadherin蛋白水平的降低及心肌细胞肥大反应;敲低Cortactin则显示相反的效应。结论Cortactin可能联合N-cadherin通过增强心肌细胞之间的连接,发挥抗心肌肥大的作用。
基金Supported by Grants from the Project of Shanghai Science and Technology Commission of China,No.10411968400
文摘AIM: To study the effects of cortactin on the tumor biology of SGC-7901 cells and identify the mechanism involved in the process. METHODS: Cell lines in which cortactin was stably overexpressed or knocked down as well as the respective control cell lines were established by standard molecular methods. The effects of cortactin on the proliferation, migration and invasion capacity of SGC-7901 cells were assessed by the MTT assay, colony formation, flow cytometry, transwell migration and matrigel invasion. Nude mouse models were also used to assess the role of cortactin in the growth and metastasis of SGC-7901 cells in vivo. Western blotting analysis was performed to detect the expression of epidermal growth factor receptor (EGFR) and downstream molecules. RESULTS: Cell lines in which cortactin was stably overexpressed or knocked down as well as control cell lines were successfully established and designated as LV5-cortactin-SGC, LV5-SGC, LV3-shRNA-SGC and LV3SGC. Cortactin overexpression promoted SGC-7901 cell migration (340.7 +/- 12.6 vs 229.1 +/- 23.2, P < 0.01) and invasion (71.6 +/- 5.2 vs 48.4 +/- 3.6, P < 0.01). Cortactin downregulation impaired SGC-7901 cell migration (136.2 +/- 19.8 vs 225 +/- 17) and invasion (29.2 +/- 5.2 vs 49.6 +/- 3.8, P < 0.01). The results from the MTT and colony formation assays results indicated increased LV5-cortactin-SGC cell proliferation and decreased LV3shRNA-SGC cell proliferation compared to the control cells. Flow cytometry analysis demonstrated that cortactin overexpression promoted the proliferation index of SGC-7901 cells, and the results were reversed when cortactin was downregulated. Mouse tumor models confirmed that cortactin expression increased SGC-7901 cell proliferation and metastasis in vivo. Western blotting analysis revealed that cortactin elevated EGFR expression and activated the downstream molecules. CONCLUSION: Cortactin expression promoted the migration, invasion and proliferation of SGC-7901 cells both in vivo and in vitro. The EGFR signaling pathway is mechanistically involved. (c) 2014 Baishideng Publishing Group Co., Limited. All rights reserved.
