人胰腺癌细胞再增殖体外模型的建立

Establishment of the in Vitro Repopulation Model of Human Pancreatic Cancer Cells

目的:细胞再增殖是导致胰腺癌放化疗失败的主要原因之一,但缺乏合适的用于研究胰腺癌再增殖的细胞模型。本研究拟建立简便、实用的胰腺癌细胞再增殖体外模型。方法:表达绿色荧光蛋白-荧光素酶(GFP-Luc)的慢病毒感染人胰腺癌细胞,经嘌呤霉素筛选,用荧光显微镜和流式细胞仪观察双标记细胞GFP表达情况,用生物成像检测双标记细胞Luc活性及分析细胞数量与Luc活性之间的关系。以X-线照射胰腺癌细胞制备饲养细胞,以相应的双标记肿瘤细胞为报告细胞进行共培养。对共培养细胞进行荧光显微镜观察和生物成像,以判断饲养细胞对报告细胞的生长促进作用。结果:通过表达GFP-Luc的慢病毒感染获得双标记人胰腺癌细胞,经荧光显微术、流式细胞术和生物成像术证实这些双标记的人胰腺癌细胞能有效地表达GFP和Luc活性,可作为报告细胞用于建立人胰腺癌再增殖细胞模型。将经X-线照射的饲养细胞和相应的报告细胞共培养,经荧光显微镜观察和生物成像分析,结果显示X-线照射过的饲养细胞对报告细胞的生长具有显著的促进作用。结论:成功建立了简便、实用的人胰腺癌再增殖体外模型,该模型能很好地模拟人体内胰腺癌细胞再增殖过程,为进一步研究胰腺癌细胞再增殖的分子机制提供了新的技术手段。

Objective： Tumor cell repopulation is one of the major causes of therapeutic failure in pancreatic cancer. However,there are no available in vitro pancreatic cancer cell repopulation models. We herein devote to establish a convenient and practicable in vitro model for pancreatic cancer cell repopulation. Methods： Human pancreatic cancer cells Bx PC-3, As PC-1 and SW1990 were infected with recombinant lentiviruses expressing double-labeling proteins GFP and luciferase（Luc） and then subjected to puromycin selection. The expression of GFP and the activity of Luc in the stable transgenic cell lines, and the linear correlation between the Luc activity and cancer cell numbers were detected by fluorescent microscopy, FACS and bioluminescence imaging, respectively. We then seeded a small number of double-labeled reporter cancer cells（living cells） onto a bed of a much larger number of unlabeled feeder cancer cells（dying cells） which had been irradiated with various doses X-ray. To determine whether the in vitro repopulation model of pancreatic cancer cells were successfully established, the growth enhancing ability of the dying feeder cells for the living reporter cells was examined by the growth of the latter monitored by fluorescent microscopy at 3 days intervals and determined by bioluminescence imaging on day 14. Results： The expression of GFP and the activity of Luc in the stable transgenic cell lines, and the linear correlation between the Luc activity and cancer cell numbers were confirmed by fluorescent microscopy, FACS and bioluminescence imaging respectively, indicating that these cell lines can be used as the reporter cells in tumor repopulation models. The results of fluorescent microscopy and bioluminescence imaging showed that reporter cells grew significantly faster when seeded onto dying feeder cells than seeded alone, indicating the growth enhancing ability of the dying feeder cells for the reporter cells. Conclusions： We established a practicable in vitro model for pancreatic cancer cell repopulation, which mimics tumor cell repopulation and has applications to the study of tumor repopulation mechanisms.