【摘要】 目的探讨晚期糖基化终末产物受体(receptor for advanced glycation end products,RAGE)对胰腺癌PANC-1细胞增殖及转染后接种裸鼠在成瘤过程中的作用。方法构建RAGE短发夹RNA(shRNA),即shRNARAGE-1、-2、-3质粒,转染胰腺癌PANC-1细胞后,采用八肽胆囊收缩素(CCK-8)法、逆转录聚合酶链反应(RT-PCR)和Western blot检测shRNA RAGE对细胞增殖、RAGE表达的影响,筛选干扰效果最好的shRNA RAGE质粒;将转染了shRNARAGE质粒的PANC-1细胞接种于裸鼠皮下,观察成瘤时间,并测量计算肿瘤体积;采用RT-PCR和Western blot检测shRNARAGE对RAGE、基质金属蛋白酶(matrix metalloproteinase,MMP)2和9(MMP-2和MMP-9)、核因子-κB(nuclear factor kappa-light-chain-enhancer of activated B cells,NF-κB)、血管内皮生长因子(vascular endothelial growth factor,VEGF)的mRNA及蛋白表达的影响,并采用免疫组化方法对肿瘤进行微血管计数,计算微血管密度。结果转染shRNARAGE24h后CCK-8检测细胞的吸光度(A)值低于对照组(P<0.05),且在转染48h时最明显。RT-PCR和Western blot检测显示,shRNA RAGE-2质粒转染后下调RAGE表达效果最好。转染shRNA RAGE-2质粒的PANC-1细胞接种于裸鼠后,裸鼠成瘤时间、肿瘤体积低于shRNARAGE-1,-3,裸鼠肿瘤组织RAGE、MMP-2、NF-κB、MMP-9、VEGF mRNA及蛋白表达低于shRNA RAGE-1,-3(P<0.05),其微血管密度也低于shRNARAGE-1,-3(P<0.001)。结论 RAGE参与了胰腺癌的发生发展过程。RAGE可影响胰腺癌肿瘤生长及微血管形成。更多还原

【Abstract】 Objective To investigate the effect of receptor for advanced glycation end products(RAGE)on cell proliferation and tumor growth in nude mice with pancreatic cancer.Methods PANC-1cells were transfected with shRNA RAGE-1,-2,-3to down-regulate the expression of RAGE.Cholecystokinin octopeptide-8(CCK-8),real-time PCR and Western blot were performed to test the impact of shRNARAGEon the expressions of mRNAs and proteins of RAGE,matrix metalloproteinase-2(MMP-2),MMP-9,nuclear factor kappa-light-chain-enhancer of activated B cells(NF-κB),and vascular endothelial growth factor(VEGF).Tumor growth and microvessel density in the nude mice implanted with shRNA RAGE transfected PANC-1 cells were observed using immunohistochemistry.Results The shRNARAGE-1,-2,-3transfected cells had lower absorbance values than the controls 24 hafter transfection,and the absorbance value reached the lowest at 48 h.The specific shRNA sequences significantly inhibited the expressions of mRNA and protein of RAGE.The mice implanted with shRNA RAGE-2had lower tumor volume and microvessel density than shRNARAGE-1,-3.The expressions of mRNAs and proteins of RAGE,MMP-2,NF-κB,MMP-9and VEGF were lower in the cells transfected with shRNARAGE-2compared with shRNARAGE-1,-3.Conclusion RAGE is involved in the progression of pancreatic cancer in vitro and in vivo.The RAGE expression could influence the process of tumor angiogenesis.更多还原