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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 & OBJECTIVE: The established mouse gene targeting model confirmed that the K-ras mutation initiates pancreatic precancerous lesions, pancreatic intraepithelial neoplasia (PanIN). The inactivation of p53 or p16 alone can promote mice. PanIN develops invasive pancreatic cancer. As the other tumor suppressor gene Smad4 with high frequency of inactivation in human pancreatic cancer, it is still unclear whether its inactivation of PanIN and whether it can promote PanIN to pancreatic cancer alone are still unclear. Based on this purpose, based on the successful isolation and establishment of K-ras mutation-initiated PanIN cell line, the present study intends to further use RNA interference technology to silence the expression of endogenous Smad4 in PanIN cell line, in order to investigate the effect of siRNA interference Smad4 gene on PanIN cell malignant Conversion effect. METHODS: The Smad4 gene silencing lentiviral plasmid was constructed and the Smad4 silencing stable transfected cells were selected and named as PanIN-S cells. The PanIN and PanIN-S cell groups were obtained by subcutaneous inoculation with PanIN and PanIN-S cells respectively. The transplanted tumor models (5 in each group) were used to measure the volume and quality of tumors in each group after 2 weeks. Immunohistochemical SP method was used to detect and compare the expression of proliferating cell nuclear antigen (PCNA) and CD31 in each group. RESULTS: The Smad4 gene siRNA system was successfully constructed. Compared with the PanIN-S group, the tumor volume and quality of the PanIN-S group significantly increased (P<0.05). Histopathological examination was consistent with pancreatic cancer (adenocarcinoma). The results of immunohistochemistry showed that the expression of PCNA and CD31 were significantly increased (P<0.05). Conclusion: The silencing of Smad4 gene can promote the malignant transformation of mouse PanIN cells based on K-ras mutation. The inactivation of Smad4 in nude mice can significantly promote the proliferation of transplanted tumors and the formation of tumor microvessels in mice. These may be tumorigenic. The important mechanism of malignant transformation.