摘要
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 explore the relationship between epidermal growth factor receptor（EGFR） nuclear internalization regulated by Tat interacting protein 30（Tip30） and intranuclear EGFR target molecule or gene to provide a basis for targeted therapy directing to EGFR molecule.Methods The wild type Balb / c mice（Tip30 ＋ / ＋,control group） and knockout gene and Balb / c pure background（Tip30-/-） mice（n = 6 each） were adopted as animal model.All experimental mice were killed 18 months after feeding,and the lung tissues were taken for preparing tissue section via formalin fixation,Alcohol dehydration and paraffin embedding.The histological examination,immunohistochemistry assay,immunofluorescence assay,cultured cells in vitro immunofluorescence / laser confocal detection,Western blotting,CCK8 method for cell inhibition assay in vitro,fluorescence quantitative PCR detection and gene chip database analysis were made.Results Knockout of Tip30 gene can promote proliferation of lung epithelial cells and up-regulate the expression of cyclin D1.After transfection,Tip30-SH1 and Tip30-SH2 cells inhibited the expression of Tip30 gene.Tip gene inhibition induced EGFR retention in early endosomes.The epidermal growth factor（EGF） treatment could induce nuclear internalization of Tip30 cells.Tip30 gene inhibition may promote EGF-induced EGFR cell nuclear internalization.Tip30 protein can be gathered in the early endosomes.Conclusions Tip30 gene inhibits can interfere EGFR sorting,inhibited EGFR degradation induced by EGF,delay EGFR downstream signaling molecule activation time,promote EGF-induced EGFR cell nuclear internalization,promote the growth of lung cancer cells,but its association with the activation of EGFR signaling passage is weak.
出处
《中国临床研究》
CAS
2015年第1期7-10,共4页
Chinese Journal of Clinical Research
基金
广东省自然科学基金(S20120009392)
