DPC4基因对人胰腺癌细胞JF305增殖的影响

Influence of DPC4 gene on proliferation of human pancreatic cancer cell line JF305.

目的研究肿瘤抑制基因DPC4(deleted in pancreatic carcinoma)对人胰腺癌细胞系JF305增殖能力的影响。方法将携带DPC4基因的真核表达载体转入JF305细胞内,经G418筛选获得DPC4稳定表达细胞株,免疫细胞化学和RT-PCR法检测转染前后细胞内DPC4的表达。用MTT法、细胞计数法和流式细胞仪测定细胞生长曲线、细胞周期、细胞贴壁率和细胞克隆形成率。比较转染前后细胞增殖能力的变化。结果未转染及转染空质粒的JF305细胞无DPC4的表达,转染pBK-CMV-DPC4的JF305细胞可检测到DPC4的表达,且其细胞增殖能力较前明显下降(P<0．001),细胞倍增时间显著延长(由11．8d延长至18d),细胞贴壁率和克隆形成率均明显下降(P<0．0001),G_1期细胞所占比例明显增加(P<0．0001),G_2／M期细胞所占比例明显下降(P<0．0001)。结论无DPC4表达的胰腺癌细胞株JF305可转基因获得DPC4稳定表达,DPC4转基因后可抑制其增殖能力,细胞G_1期延长,G_2／M期缩短。DPC4有望成为胰腺癌基因治疗新的候选基因。

Objective To investigate the influence of DPC4 gene on the proliferation of human pancreatic cancer cell line JF305. Methods DPC4 cDNA was transfected into human pancreatic cancer cell line JF305 by lipofectamine transfection technique. The non transfected cells and the cells transfected with pBK-CMV were used as controls. The expression of DPC4 was detected by RT-PCR and immunocytochemistry.Cell cycle was assessed by flow cytometry. The cell growth rate was estimated by cell count and MTT method. Changes in prolileration of JF305 cells were evaluated by sticking and cloning efficiency. Results JF305 cells transfected with pBK CMV-DPC4 strongly expressed DPC4, while this expression was not detected in cells transfected with pBK-CMV and none transfected cells. The number of G1 phase JF305 cells transfected with pBK-CMV DPC4 plasmid increased, while the number of G2/M phase cells decreased significantly. The growth of JF305 cells was suppressed markedly as was demonstrated by MTT and cell count assay. The population doubling time was much longer than that in JF305 cells not transfected. The cloning and sticking efficiency was significantly different from that of JF305 cells not transfected and those transfected with pBK-CMV plasmid. Conclusions DPC4 gene could inhibit the growth and proliferation of human pancreatic cancer cell line JF305. The alteration of the tumor suppressor gene DPC4 might be an important molecular event in pancreatic carcinoma and possibly plays a crucial role in the development and progression of the neoplasm. DPC4 gene might become one of the target genes for pancreatic adenocarcinoma gene therapy.