CAJ | 학술논문

目的构建大鼠早期黏膜下浸润结直肠癌( SICRC)与非黏膜下浸润结直肠癌(SNICRC)模型并鉴定两者的差异蛋白,以筛选早期SICRC的生物学标记.方法应用N-甲基-N-亚硝基脲( MNU)诱导建立大鼠SICRC与SNICRC模型.二维荧光差异定量双向电泳技术(2D-DIGE)和基质辅助激光解吸/电离-飞行时间质谱鉴定出SICRC与SNICRC的差异蛋白.荧光定量Real-time PCR和Westem blot实验验证所选蛋白的鉴定结果.结果成功建立大鼠SICRC与SNICRC模型.2D-DIGE发现SICRC和SNICRC之间有5个差异表达蛋白(P值均＜0.01).质谱分析鉴定这5个蛋白发现,与SNICRC和正常对照(NC)比较,转凝蛋白(Transgelin)、肽基脯氨酰异构酶A和原肌球蛋白1在SICRC表达上调,而碳酸酐酶Ⅱ(CAⅡ)与一种未命名蛋白表达下调.Real-time PCR和Westemblot验证结果显示,Transgelin mRNA和蛋白水平在SICRC组(33.05±0.75、86.63±1.83)高于SNICRC( 22.68±0.89、67.93±2.39)和NC组(18.07 ±0.55、44.25±1.55)(P值均＜0.05),而CAⅡmRNA和蛋白水平在SICRC组(18.01±0.53、41.55±1.89)低于SNICRC组(26.28±1.08、61.11±1.57)和NC组(33.08±0.76、83.43 ±1.61)(P值均＜0.05),变化趋势与蛋白质组学一致.结论 2D-DIGE是筛选早期SICRC与SNICRC差异表达蛋白的有效手段,鉴定的差异蛋白有可能成为临床早期黏膜下浸润结直肠癌筛选和诊断的生物学标记.
목적 구건대서조기점막하침윤결직장암( SICRC)여비점막하침윤결직장암(SNICRC)모형병감정량자적차이단백,이사선조기SICRC적생물학표기.방법 응용N-갑기-N-아초기뇨( MNU)유도건립대서SICRC여SNICRC모형.이유형광차이정량쌍향전영기술(2D-DIGE)화기질보조격광해흡/전리-비행시간질보감정출SICRC여SNICRC적차이단백.형광정량Real-time PCR화Westem blot실험험증소선단백적감정결과.결과 성공건립대서SICRC여SNICRC모형.2D-DIGE발현SICRC화SNICRC지간유5개차이표체단백(P치균＜0.01).질보분석감정저5개단백발현,여SNICRC화정상대조(NC)비교,전응단백(Transgelin)、태기포안선이구매A화원기구단백1재SICRC표체상조,이탄산항매Ⅱ(CAⅡ)여일충미명명단백표체하조.Real-time PCR화Westemblot험증결과현시,Transgelin mRNA화단백수평재SICRC조(33.05±0.75、86.63±1.83)고우SNICRC( 22.68±0.89、67.93±2.39)화NC조(18.07 ±0.55、44.25±1.55)(P치균＜0.05),이CAⅡmRNA화단백수평재SICRC조(18.01±0.53、41.55±1.89)저우SNICRC조(26.28±1.08、61.11±1.57)화NC조(33.08±0.76、83.43 ±1.61)(P치균＜0.05),변화추세여단백질조학일치.결론 2D-DIGE시사선조기SICRC여SNICRC차이표체단백적유효수단,감정적차이단백유가능성위림상조기점막하침윤결직장암사선화진단적생물학표기.
Objective To construct tumor models of early submucosal invasive colorectal cancer (SICRC) and submucosal non-invasive colorectal cancer (SNICRC) in rats,identify the differential proteins between SICRC,SNICRC and normal control (NC) and screen the biomarkers for early detection and diagnosis of SICRC.Methods N-methyl-N-nitrosourea (MNU) was used to induce tumor models of SICRC and SNICRC in rats.2D-DIGE and MOLDI-TOF-MS/MS were applied to identify the differential proteins between SICRC,SNICRC and NC.Quantitative real-time polymerase chain reaction (PCR) and Western blotting assays confirmed the validation of 2D-DIGE analysis.Results MNU-induced tumor models of SICRC and SNICRC in rats were successfully constructed and five differential proteins were found between SICRC,SNICRC and NC by 2D-DIGE (each P ＜ 0.01 ).The identified results using mass spectrometry showed that Transgelin,Peptidylprolyl isomerase A and Tropomyosin 1 were up-regulated,while carbonic anhydrase 2 ( CA Ⅱ ) and an unnamed protein were down-regulated in SICRC compared with SNICRC and NC.Furthermore,consistent with the proteomics,real-time PCR and Western blottoing assays demonstrated that the relative mRNA and protein levels of Transgelin were highly expressed in SICRC (33.05 ± 0.75,86.63 ± 1.83 ) as compared with SNICRC (22.68 ± 0.89,67.93 ± 2.39) and NC ( 18.07 ± 0.55,44.25 ± 1.55 ) ( each P ＜ 0.05 ),while those of CA Ⅱ were weakly expressed in SICRC (18.01 ±0.53,41.55 ± 1.89) as compared with SNICRC (26.28 ± 1.08,61.11 ± 1.57) and NC (33.08 ±0.76,83.43 ± 1.61 ) (each P ＜0.05).Conclusion 2D-DIGE technique is an effective way to screen the differential proteins between early SICRC and SNICRC and identified proteins such as Transgelin and CA Ⅱ may be the potential biomarkers for early detection and diagnosis of SICRC.