人体胰腺癌细胞株PANC.docVIP

　　人体胰腺癌细胞株PANC 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 origenesis[2]. A study of 82 pancreatic cancers demonstrated overexpression of cyclin D1 protEin in 65% of the tumors, and this RNA had a median survival of 6.5 months, edian survival of 15.5 months[4]. The aim of this project an 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 an 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 QIAprepSpin 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 horseradish peroxidase linked sheep), anti-β-actin antibody; electrochemiluminescence (ECL) solution and Hybond-P membranes from Amersham Biosciences Germany; DNA ladder (1 Kb plus), pH5αTM chemically petent cells from Invitrogen. 1.2 Methods 1.2.1 Cell Culture PANC-1 cells ented , penicillin G (100 units/ml), and streptomycin (100 mg/ml) termed plete medium. Cells aintained in monolayer culture at 37℃ in humidified air edium for cell lines containig a neomycin resistance gene ented g/ml G418 and 0.2 mg/ml for containing a hygromycin resi