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[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。
[Objective] To establish a tetracycline-regulated cyclin D1 expression system in human pancreatic cancer cell line PANC-1 to study the effects of cyclin D1 on pancreatic cancer cells. [Method] The antisense cyclin D1 plasmid was transfected into pancreatic cancer cell line PANC-1 cells by two stable transfection. The expression control system adopts Tet-Off system (tetracycline control system). The inhibitory effect of cyclin D1 expression on the growth of PANC-1 cells, the ability of colony-forming and the expression of cyclin were evaluated. The regulation and validity of this system were evaluated. [Result] The pTRE-antisense cyclin D1 plasmids were transfected by the first transfection of pTet-Off plasmid and the two best expression clones were selected, and the plasmids which could be expressed in Tet-Off system were selected by immunoblot assay Clones expressing antisense cyclin D1 most efficiently. Through the regulation of antisense cyclin D1 by Tet-Off system, it was found that the inhibition of cyclin D1 expression can significantly inhibit the growth and colony-forming ability of pancreatic cancer cells and cause the morphological changes of pancreatic cancer cells. Its inhibitory effect and tetracycline concentration and time-related. [Conclusion] This study established a highly efficient and inducible antisense cyclin D1 expression system in PANC-1 pancreatic cancer cell line. Through the establishment of this system, the effect of cyclin D1 inhibition on pancreatic cancer cells can be further studied in vivo and in vitro, and the potential therapeutic value of combination therapy in clinical application can be explored in combination with other therapeutic methods such as chemotherapy.