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目的探讨肿瘤转移抑制基因(Tip30)调节表皮生长因子受体(EGFR)核内化及与核内化EGFR靶分子或基因的关联性,为针对EGFR分子靶向治疗提供依据。方法采用野生型Balb/c小鼠(Tip30+/+小鼠,对照组)和Tip30基因敲除、Balb/c纯背景的小鼠(Tip30-/-小鼠)作为动物模型(每组6只),饲养18个月后处死,取肺组织,福尔马林固定,酒精脱水,石蜡包埋,组织切片后,进行组织学检查、免疫组化检测、组织免疫荧光检测、体外培养细胞免疫荧光/激光共聚焦检测、免疫印迹检测、细胞计数试剂盒-8方法体外细胞抑制实验、荧光定量PCR检测和基因芯片数据库分析。结果 Tip30基因敲除促进肺上皮细胞增殖、上调细胞周期蛋白cyclin D1表达;基因转染后Tip30-SH1、Tip30-SH2细胞抑制Tip30表达;Tip基因抑制致EGFR在早期内涵体滞留;表皮生长因子(EGF)处理可诱导Tip30细胞核内化;Tip30基因抑制可促进EGF诱导EGFR细胞核内化,Tip30蛋白可在早期内涵体内聚集。结论 Tip30基因抑制干扰EGFR分选,抑制EGF诱导EGFR降解,延迟EGFR下游信号分子激活时间,同时促进EGF诱导的EGFR细胞核内化,促进肺癌细胞生长,而与细胞膜或细胞浆内EGFR信号通路激活的关联性较小。
Objective To investigate the relationship between the expression of Tip30 and the internalization of EGFR target molecules or genes in epidermal growth factor receptor (EGFR) in order to provide a basis for targeted therapy of EGFR. Methods The wild type Balb / c mice (Tip30 + / + mice, control group) and Tip30 knockout mice (Tip30 - / - mice) with Balb / c pure background were used as animal models , Were sacrificed 18 months after the death, take lung tissue, formalin fixed, alcohol dehydration, paraffin embedding, tissue sections, histological examination, immunohistochemistry, tissue immunofluorescence, cultured in vitro immunofluorescence / Confocal laser scanning microscopy, western blotting, cell counting kit-8 in vitro cytostatic assay, real-time quantitative PCR and gene chip database analysis. Results Tip30 knockdown promoted the proliferation of lung epithelial cells and up-regulated the expression of cyclin D1. Tip30-SH1 and Tip30-SH2 cells inhibited the expression of Tip30 after transfection. The Tip gene inhibited the retention of EGFR in early endosomes. The expression of epidermal growth factor EGF) could induce the endocytosis of Tip30 cells. Tip30 gene knockdown promoted EGF-induced nuclear internalization of EGFR cells and Tip30 protein could accumulate in early endosomes. Conclusion The Tip30 gene inhibits the EGFR sorting, inhibits the EGF-induced degradation of EGFR, delays the activation of EGFR downstream signaling molecules, promotes the EGF-induced nuclear internalization of EGFR cells and promotes the growth of lung cancer cells, but not with the activation of EGFR signal pathway in the cell membrane or cytoplasm Less relevant.