人体胰腺癌细胞株PANC-1中四环素可诱导的周期素D1表达抑制系统的建立

[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DTRE-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。

Inhibition of Cyclin D1 Expression Using a Tetracycline Inducible Vector System in PANC-1 Human Pancreatic Cancer Cells

[Purpose ] To establish an inducible Tetracycline Vector System in PANC-1 cells to evaluate the effects of inhibition of cyclin D1 on pancreatic cancer cells. [Methods] PANC-1 cells were transfected with pTRE-cyclin D1 antisense in two separate steps using lipofeetamine. And inducibility and efficiency of this system was evaluated by inhibition of expression of cyclin D1 on cell growth, colony formation and protein level. [Results] By first transfeetion with pTet-Off, 2 clones were selected for further study. By second transfection with pTRE-cyclin D1 antisense, one clone was selected which was confirmed by im- munoblotting when inducing expressing of cyclin DI antisense inhibited expression of cyclin D1. By anchorage dependent and independent study inhibition of cyclin D1 using Tet-Off system had showed marked suppression on pancreatic cell growth and colony formation. Cell progression from G0-l to S phase was prevented and deformity of cells also occurred when cyclin D1 antisense expression was induced. [Conclusion] A highly inducible and efficient pTRE-eyclin D1 antisense system was well established in PANC-1 cells. With this further study could be conducted both in vivo and in vitro to determine the effects of inhibition of cyclin DI on pancreatic cancer cells, and its potential usage in clinical usage with other modality such as chemotherapy.