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背景与目的:胰腺腺管内上皮瘤(pancreatic intraepithelial neoplasia,PanIN)是近几年提出的新术语,目前对其缺乏足够认识。在首次建立小鼠PanIN细胞系的基础上探索Kras突变小鼠PanIN细胞的生物学特性。方法:通过免疫组化分析基因打靶KrasG12D突变小鼠PanIN组织,分离和建立LoxP-Stop-LoxP-KrasG12D;Pdx1-Cre产生的小鼠PanIN细胞系,鉴定其体外贴壁依赖性生长及在软琼脂半固体培养基中集落形成,对PanIN细胞中抑癌基因Tp53、Smad4、p16进行突变分析。结果:KrasG12D激活突变产生了各期小鼠胰腺癌前病变-胰腺腺管内上皮瘤(Pa-nIN);PanIN细胞的胞浆和核中的磷酸化MAPK染色强阳性和高表达,EGFR、Her-2/Neu、和β-Catenin在PanIN细胞中也表现了高表达,Tp53在早期PanIN细胞和Smad4在各期PanIN细胞中的表达无异常;PanIN细胞在体外培养中的增殖速度与原发胰腺癌相似,并且能在软琼脂中形成光学显微镜下所见的集落,随着培养时间的延长而集落增大;PanIN细胞中除可检测到KrasG12D突变外无p16、Tp53、Smad4突变或缺失。结论:单纯Kras的激活突变足以启动PanIN,是胰腺癌发生的早期主要分子遗传学改变事件,PanIN细胞已在一定程度上具备了恶性转化的肿瘤细胞的生物学特性。
BACKGROUND & OBJECTIVE: Pancreatic intraepithelial neoplasia (PanIN) is a new term proposed in recent years and its current lack of understanding. On the basis of the first establishment of mouse PanIN cell line, the biological characteristics of PanIN cells in Kras mutant mice were explored. Methods: Pancinin (KrasG12D) mutant mice were immunostained for PanIN tissue samples. Panax LoxP-Stop-LoxP-KrasG12D, Panx1-Cre-induced mouse PanIN cell lines were isolated and identified. Semi-solid medium colony formation, PanIN cell tumor suppressor genes Tp53, Smad4, p16 mutation analysis. RESULTS: The KrasG12D activation mutation resulted in the development of pancreatic precancerous lesion (Pa-nIN) in pancreatic adenocarcinoma. The phosphorylation of MAPK in the cytoplasm and nucleus of PanIN cells was strongly and overexpressed. The expression of EGFR, Her- 2 / Neu, and β-Catenin also showed high expression in PanIN cells. There was no abnormal expression of Tp53 in early stage of PanIN cells and Smad4 in various stages of PanIN cells. The proliferation rate of PanIN cells in vitro was similar to that of primary pancreatic cancer Similar colonies were found in soft agar under light microscope. Colonies increased with incubation time. No mutation or deletion of p16, Tp53 and Smad4 was found in PanIN cells except for KrasG12D mutation. CONCLUSIONS: Activation of Kras alone is sufficient to initiate PanIN, an early major molecular genetic event in the development of pancreatic cancer. PanIN cells, to a certain extent, have the biological characteristics of malignantly transformed tumor cells.