Smad4基因沉默促进PanIN裸鼠移植瘤增殖和微血管形成的研究
Smad4기인침묵촉진PanIN라서이식류증식화미혈관형성적연구
Effect of Smad4 silencing on the growth and vascularization of pancreatic cancer transplantation tumor in nude mice
  • 万方数据
    中国癌症杂志 중국암증잡지
    CHINA ONCOLOGY
    2009年 7期 485-490 ,共6页

    齐晓光%慎睿哲%王立夫%曹海霞%朱黎明%董文杰%孙萍胡%章永平%张本炎%Tuveson DA

    제효광%신예철%왕립부%조해하%주려명%동문걸%손평호%장영평%장본염%Tuveson DA

    siRNA干扰%Smad4基因%胰腺腺管内上皮瘤%胰腺肿瘤%细胞增殖%血管形成 siRNA榦擾%Smad4基因%胰腺腺管內上皮瘤%胰腺腫瘤%細胞增殖%血管形成
    siRNA간우%Smad4기인%이선선관내상피류%이선종류%세포증식%혈관형성
    RNA interference%Smad4 gene%pancreatic intraepithelial neoplasia%pancreatic ductal adenocareinoma%cell proliferation%vascularization
    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一胰腺腺管内上皮瘤(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.
    배경여목적:유이건립적소서기인타파모형증실,K-ras돌변계동료이선암전병변일이선선관내상피류(pancreatic intraepithelial neoplasia,PanIN),p53혹p16실활균가단독촉진소서PanIN발전위침윤성이선암.작위인이선암중령일실활빈솔고발적억암기인Smad4,기실활대PanIN적전화작용급시부가단독촉진PanIN발전위이선암목전잉불청초.기우차목적,재이성공분리건립K-ras돌변계동적PanIN세포주기출상,본연구의진일보응용RNA간우기술침묵PanIN세포주중내원성Smad4표체,이탐토siRNA간우Smad4기인대PanIN세포악성전화작용.방법:구건Smad4기인침묵만병독질립,사선Smad4침묵은전세포병명명위PanIN-S세포;분별용PanIN화PanIN-S세포병채용피하접충적방법,구건획득PanIN급PanIN-S세포조라서이식류모형(매조5지),2주후측정각조종류체적화질량;응용면역조직화학SP법검측병비교각조증식세포핵항원(PCNA)화CD31적표체급기차이.결과:성공구건료Smad4기인SiRNA체계;여미간우PanIN세포조상비,PanIN-S조라서종류체적화질량현저증가(P<0.05);조직병이학검사,부합이선암(선암);면역조직화학결과현시PCNA화CD31표체현저증고(P<0.05).결론:Smad4기인침묵가촉사재K-ras돌변기출상적소서PanIN세포적악성전화;라서이식류중Smad4실활가현저촉진소서이식류증식화종류미혈관형성,저사가능시기치류악성전화적중요작용궤제.
    Background and purpose: Pancreatic intraepithelial neoplasia (PanIN) is thought 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 transfeetion 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 PanlN-S cells subcutaneously. Two weeks after tumor cells inoculation, tumor volume and weight were estimated. 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 PanlN cells was confirmed by RT-PCR and Western blot. Compared with PanlN groups, there was a dramatic increase in tumor volume and weight in PanIN-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 PanlN-S groups compared to the PanlN groups (P<0.05). Conclusion: Silencing of Smad4 in PanlN cells with endogenous expression of K-ras G12D, enhanced progression to invasive adenocarcinomas. Cell proliferation and vascularization may be its important mechanisms.
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