Smad4基因沉默促进PanIN裸鼠移植瘤增殖和微血管形成的研究

Effect of Smad4 silencing on the growth and vascularization of pancreatic cancer transplantation tumor in nude mice

背景与目的:由已建立的小鼠基因打靶模型证实,K-ras突变启动了胰腺癌前病变—胰腺腺管内上皮瘤(pancreatic intraepithelial neoplasia,PanIN),p53或p16失活均可单独促进小鼠PanIN发展为浸润性胰腺癌。作为人胰腺癌中另一失活频率高发的抑癌基因Smad4,其失活对PanIN的转化作用及是否可单独促进PanIN发展为胰腺癌目前仍不清楚。基于此目的,在已成功分离建立K-ras突变启动的PanIN细胞株基础上,本研究拟进一步应用RNA干扰技术沉默PanIN细胞株中内源性Smad4表达,以探讨siRNA干扰Smad4基因对PanIN细胞恶性转化作用。方法:构建Smad4基因沉默慢病毒质粒,筛选Smad4沉默稳转细胞并命名为PanIN-S细胞;分别用PanIN和PanIN-S细胞并采用皮下接种的方法,构建获得PanIN及PanIN-S细胞组裸鼠移植瘤模型(每组5只),2周后测定各组肿瘤体积和质量;应用免疫组织化学SP法检测并比较各组增殖细胞核抗原(PCNA)和CD31的表达及其差异。结果:成功构建了Smad4基因siRNA体系;与未干扰PanIN细胞组相比,PanIN-S组裸鼠肿瘤体积和质量显著增加(P<0.05);组织病理学检查,符合胰腺癌(腺癌);免疫组织化学结果显示PCNA和CD31表达显著增高(P<0.05)。结论:Smad4基因沉默可促使在K-ras突变基础上的小鼠PanIN细胞的恶性转化;裸鼠移植瘤中Smad4失活可显著促进小鼠移植瘤增殖和肿瘤微血管形成,这些可能是其致瘤恶性转化的重要作用机制。

Background and purpose： Pancreatic lntraepitlaehal neoplasla （panIN） is thouglat to be a precursor lesion of infiltrating pancreatic ductal adenocarcinoma. The mutation of the phenotypic impact of K-ras G12D alone, silencing of p53 and p16 could promote this process. The role of Smad4 in this progression was poorly understood. In the present study, we investigated the role of Smad4 in the development of pancreatic tumor, based on PanIN cell line from mice with K-ras G12D mutation in order to investigate the effect of Smad4 silencing on PanIN cells in the development and malignant transformation in nude mice. Methods： Smad4 knock-down PanIN cells （PanIN-S） were established by stable transfection with lentiviral-mediated Smad4 RNA interference （RNAi）. In xenograft model experiments, BALB/c nude mice were randomly divided into 2 groups （5 mice per group） implanted with PanIN or PanIN-S cells subcutaneously. Two weeks after tumor cells inoculation, tumor volume and weight wereestimated. PCNA staining was used to evaluate cell proliferation and CD31 polyclonal antibody was used to assess micro-vessel density （MVD） in tumors. Results： Effect of siRNA of Smad4 gene in PanIN cells was confirmed by RT-PCR and Western blot. Compared with PanIN groups, there was a dramatic increase in tumor volume and weight in PanlN-S groups （P〈0.05）. Furthermore, immunohistochemical analysis of the harvested tumors suggested that Smad4 silencing was associated with increased tumor cell proliferation （PCNA reactivity） and angiogenesis （micro-vessel density, MVD）. The percentage of PCNA-positive cells in the PanlN-S groups were significantly increased than PanIN groups （P〈0.05）. CD31 staining revealed a significant increase in the PanIN-S groups compared to the PanlN groups （P〈0.05）. Conclusion： Silencing of Smad4 in PanIN cells with endogenous expression of K-ras G12D, enhanced progression to invasive adenocarcinomas. Cell proliferation and vascularization may be its important mechanisms.