中国癌症杂志
中國癌癥雜誌
중국암증잡지
CHINA ONCOLOGY
2013年
7期
481-486
,共6页
齐晓光%胡毅%汪进良%谈文龙%王琪%王立夫%Tuveson DA
齊曉光%鬍毅%汪進良%談文龍%王琪%王立伕%Tuveson DA
제효광%호의%왕진량%담문룡%왕기%왕립부%Tuveson DA
siRNA干扰%Smad4基因%胰腺上皮肉瘤变细胞%细胞增殖%转移
siRNA榦擾%Smad4基因%胰腺上皮肉瘤變細胞%細胞增殖%轉移
siRNA간우%Smad4기인%이선상피육류변세포%세포증식%전이
siRNA interference%Smad4 gene%PanIN cell%Proliferation%Migration
背景与目的：由己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-胰腺上皮内瘤变(pancreatic intraepithelial neoplasia，PanIN)，p53、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，本课题组前期研究提示，应用RNA干扰技术沉默PanIN细胞株中内源性Smad4表达，促使PanIN细胞恶性转化。基于此目的，本研究进一步探讨siRNA干扰Smad 4基因对PanIN细胞体内外增殖及转移能力的影响，从而有助于阐明PanIN恶性转化的新机制。方法：构建和筛选能特异性静默PanIN细胞中Smad4表达的最佳siRNA稳定表达质粒,稳定转染最佳siRNA表达质粒进入PanIN细胞，用Zeocin筛选稳定转染克隆，挑选阳性克隆、扩增，稳定转染后细胞命名为PanIN-S。本研究运用细胞计数、克隆形成实验等分别比较两组在活细胞率和体外增殖能力的影响，同时为进一步验证Smad 4基因静默对PanIN细胞体外迁移、侵袭能力的影响，本研究采用Transwell和Mitigel assay检测其干扰前后体外运动、侵袭能力。动物实验中，建立PanIN及PanIN-S细胞组裸鼠移植瘤模型，并应用免疫组化方法检测并比较两组PCNA、VEGF和MMP-9的表达及其差异。结果：成功构建了Smad4基因siRNA体系；体外实验中与PanIN组相比， PanIN-S组细胞增殖、侵袭能力明显增强，差异有统计学意义(P<0.05)；动物模型免疫组化结果显示，与PanIN组相比，PanIN-S组PCNA、VEGF和MMP-9表达显著增高(P<0.05)。结论：K-ras突变基础上小鼠PanIN细胞Smad4基因静默促使PanIN细胞的恶性转化；PanIN基础上Smad4静默促使小鼠PanIN细胞体内外增殖及转移能力的增强，其增殖及转移可能与PCNA、VEGF和MMP-9高表达有关。
揹景與目的：由己建立的小鼠基因打靶模型證實，K-ras突變啟動瞭胰腺癌前病變-胰腺上皮內瘤變(pancreatic intraepithelial neoplasia，PanIN)，p53、p16失活均可單獨促進小鼠PanIN髮展為浸潤性胰腺癌。作為人胰腺癌中另一失活頻率高髮的抑癌基因Smad4，本課題組前期研究提示，應用RNA榦擾技術沉默PanIN細胞株中內源性Smad4錶達，促使PanIN細胞噁性轉化。基于此目的，本研究進一步探討siRNA榦擾Smad 4基因對PanIN細胞體內外增殖及轉移能力的影響，從而有助于闡明PanIN噁性轉化的新機製。方法：構建和篩選能特異性靜默PanIN細胞中Smad4錶達的最佳siRNA穩定錶達質粒,穩定轉染最佳siRNA錶達質粒進入PanIN細胞，用Zeocin篩選穩定轉染剋隆，挑選暘性剋隆、擴增，穩定轉染後細胞命名為PanIN-S。本研究運用細胞計數、剋隆形成實驗等分彆比較兩組在活細胞率和體外增殖能力的影響，同時為進一步驗證Smad 4基因靜默對PanIN細胞體外遷移、侵襲能力的影響，本研究採用Transwell和Mitigel assay檢測其榦擾前後體外運動、侵襲能力。動物實驗中，建立PanIN及PanIN-S細胞組裸鼠移植瘤模型，併應用免疫組化方法檢測併比較兩組PCNA、VEGF和MMP-9的錶達及其差異。結果：成功構建瞭Smad4基因siRNA體繫；體外實驗中與PanIN組相比， PanIN-S組細胞增殖、侵襲能力明顯增彊，差異有統計學意義(P<0.05)；動物模型免疫組化結果顯示，與PanIN組相比，PanIN-S組PCNA、VEGF和MMP-9錶達顯著增高(P<0.05)。結論：K-ras突變基礎上小鼠PanIN細胞Smad4基因靜默促使PanIN細胞的噁性轉化；PanIN基礎上Smad4靜默促使小鼠PanIN細胞體內外增殖及轉移能力的增彊，其增殖及轉移可能與PCNA、VEGF和MMP-9高錶達有關。
배경여목적：유기건립적소서기인타파모형증실，K-ras돌변계동료이선암전병변-이선상피내류변(pancreatic intraepithelial neoplasia，PanIN)，p53、p16실활균가단독촉진소서PanIN발전위침윤성이선암。작위인이선암중령일실활빈솔고발적억암기인Smad4，본과제조전기연구제시，응용RNA간우기술침묵PanIN세포주중내원성Smad4표체，촉사PanIN세포악성전화。기우차목적，본연구진일보탐토siRNA간우Smad 4기인대PanIN세포체내외증식급전이능력적영향，종이유조우천명PanIN악성전화적신궤제。방법：구건화사선능특이성정묵PanIN세포중Smad4표체적최가siRNA은정표체질립,은정전염최가siRNA표체질립진입PanIN세포，용Zeocin사선은정전염극륭，도선양성극륭、확증，은정전염후세포명명위PanIN-S。본연구운용세포계수、극륭형성실험등분별비교량조재활세포솔화체외증식능력적영향，동시위진일보험증Smad 4기인정묵대PanIN세포체외천이、침습능력적영향，본연구채용Transwell화Mitigel assay검측기간우전후체외운동、침습능력。동물실험중，건립PanIN급PanIN-S세포조라서이식류모형，병응용면역조화방법검측병비교량조PCNA、VEGF화MMP-9적표체급기차이。결과：성공구건료Smad4기인siRNA체계；체외실험중여PanIN조상비， PanIN-S조세포증식、침습능력명현증강，차이유통계학의의(P<0.05)；동물모형면역조화결과현시，여PanIN조상비，PanIN-S조PCNA、VEGF화MMP-9표체현저증고(P<0.05)。결론：K-ras돌변기출상소서PanIN세포Smad4기인정묵촉사PanIN세포적악성전화；PanIN기출상Smad4정묵촉사소서PanIN세포체내외증식급전이능력적증강，기증식급전이가능여PCNA、VEGF화MMP-9고표체유관。
Background and purpose: Pancreatic intraepithelial neoplasia (PanIN) may be a precursor lesion of inifltrating 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 our previous studies, we investigated that RNA interference silence of Smad4 to promote the PanIN cell malignant transformation. In the present study, we investigate. The further explores the siRNA interference of Smad4 expression on PanIN cells could lead to proliferation and metastasis in vitro and in vivo. Methods:Smad4 knock-down PanIN cells (PanIN-S) were established by stable transfection with lentiviral-mediated Smad4 RNA interference. In vitro,silence of Smad4 enhanced the proliferation of PanIN cells as determined by cell counting. A soft agar assay was used to assess the anchorage-independent growth ability of cells. Cell migration and invasion assays were performed using transwell chambers with or without Matrigel. In xenograft model experiments, PCNA, VEGF and MMP-9 staining was separately used to evaluate cell proliferation and angiogenesis and migration (VEGF and MMP-9). Results:Effect of siRNA of Smad4 gene in PanIN cells was conifrmed by real-time RT-PCR and western blot. In vitro, silence of Smad4 enhanced the proliferation of PanIN cells as determined by cell counting. Soft agar assay showed that there were more colony cell numbers in PanIN-S cells compared with PanIN cells (P<0.05). Using the transwell assay, we observed that PanIN-S cells migrated faster than PanIN cells and similar results were obtained by Matrigel assay (P<0.05). Furthermore, immunohistochemical analysis of the harvested tumors suggested that Smad4 silencing was associated with cell proliferation (PCNA reactivity) and angiogenesis and migration (VEGF and MMP-9), and the expressions of PCNA, VEGF and MMP-9 in PanIN-S group were signiifcantly increased (P<0.05). Conclusion:Silence of Smad4 in PanIN cells enhanced progression to invasive adenocarcinoma of the pancreas by promoting cell growth, migration and invasion. Smad4 might be a new diagnostic marker in pancreatic cancer and prove to be a feasible and novel target for therapeutic intervention.
