人体胰腺癌细胞株PANC-1中四环素可诱导周期素D1表达抑制系统.docVIP

人体胰腺癌细胞株PANC-1中四环素可诱导周期素D1表达抑制系统 　 作者：王建承　张卓　林谋斌　梁鲁 【摘要】 　　［目的］ 在人体胰腺癌细胞株PANC-1中建立一个四环素可调控周期素D1表达系统，研究抑制周期素D1对胰腺癌细胞的影响。［方法］ 反义周期素D1质粒通过两次稳定转染进入胰腺癌细胞株PANC-1细胞中，其表达调控系统采用Tet-Off系统（四环素调控系统）。通过抑制周期素D1表达对PANC-1细胞生长、集落形成能力以及周期素蛋白表达的影响，评价此系统的可调控性和有效性。［结果］ 通过第一次转染pTet-Off质粒，选择两个最佳表达克隆行第二次转染pTRE-反义周期素D1质粒，并通过免疫印记测定挑选出能在Tet-Off系统中最有效地表达反义周期素D1的克隆。通过Tet-Off系统对反义周期素D1的调控，发现周期素D1的表达抑制可明显地抑制胰腺癌细胞生长和集落能力，并可导致胰腺癌细胞形态学改变。其抑制作用与四环素调控浓度和时间有关。 ［结论］ 此研究在PANC-1胰腺癌细胞株中建立了一个高效、可诱导的反义周期素D1的表达系统。通过这个系统的建立，可进一步在体内和体外研究周期素D1的抑制对胰腺癌细胞的影响，并可结合其它治疗手段如化疗来探讨联合治疗在临床的潜在应用价值。 【关键词】 周期素D1　反义Tet-Off系统　胰腺肿瘤　PANC-1 　　Pancreatic cancer is the fourth to fifth leading cause of cancer death in the world with an overall 5-year survival of only 0.4%[1]. Overexpression of growth factors in conjunction with deregulated cyclin D1 expression has been proposed to contribute to the loss of cell cycle control and to enhance tumorigenesis[2]. A study of 82 pancreatic cancers demonstrated overexpression of cyclin D1 protein in 65% of the tumors, and this was associated with shorter survival for these patients[3]. In another study patients with high levels of cyclin D1 mRNA had a median survival of 6.5 months, whereas patients with low levels had a median survival of 15.5 months[4]. The aim of this project was to establish human pancreatic cancer cells that harbor CD1 antisense cDNA in a tetracycline inducible vector system that can be turned on and off depending on the surrounding tetracycline concentration. And with these cells the effects of inhibition of expression of CD1 were also explored. 1 MATERIAL AND METHODS 1.1 Material PANC-1 human pancreatic cancer cells from American Type Culture Collection; pTet-Off, pTRE-Luc and pTK-Hyg from Clontech Laboratories Inc U.S.; plasmid maxi kit and QIAprep@Spin mini prep Kit from Qiagen; lipofectamine from Gibco; luminometer and the dual-luciferase reporter assay system from Promega; Mouse nonoclonal anti-cyclin D1 antibody (DCS-6) from Neomarker Inc; anti-mouse IgG hor