CAJ | 학술논문

目的寻找膀胱尿路上皮癌(BUC)患者与健康志愿者尿液蛋白质组学差异,为BUC的早期诊断奠定基础.方法采用25％乙醇沉淀法制备尿液双向电泳样品,尿液蛋白经双向凝胶电泳(2-DE)分离后行考马斯亮蓝染色,利用PDQuest8.0 2D凝胶图像分析软件进行分析,所得差异蛋白点用基质辅助激光解吸电离飞行时间串联质谱仪(MALDI-TOF-TOF-MS)进行肽序测定,结合Swiss-Prot蛋白质数据库对差异蛋白进行初步鉴定,并应用Western blot方法对差异蛋白在两组间的差异表达情况进行验证.结果获得了分辨率和重复性均较好的双向电泳图谱.BUC组与对照组凝胶的蛋白质点数分别为(789±18)个和(762± 14)个；与对照组比较,BUC组有6个蛋白质点表达明显下调,11个蛋白质点表达明显上调.经质谱分析后,初步鉴定了5种蛋白质,分别为纤维蛋白原、乳酸脱氢酶B、载脂蛋白A1、丛生蛋白和触珠蛋白,均在BUC组呈表达增高趋势.Western blot验证了双向凝胶电泳结果.结论 BUC患者与健康志愿者的尿液蛋白质组存在明显的差异,差异蛋白的鉴定为进一步寻找BUC早期诊断的分子标志物提供了研究基础和依据.
목적 심조방광뇨로상피암(BUC)환자여건강지원자뇨액단백질조학차이,위BUC적조기진단전정기출.방법 채용25％을순침정법제비뇨액쌍향전영양품,뇨액단백경쌍향응효전영(2-DE)분리후행고마사량람염색,이용PDQuest8.0 2D응효도상분석연건진행분석,소득차이단백점용기질보조격광해흡전리비행시간천련질보의(MALDI-TOF-TOF-MS)진행태서측정,결합Swiss-Prot단백질수거고대차이단백진행초보감정,병응용Western blot방법대차이단백재량조간적차이표체정황진행험증.결과 획득료분변솔화중복성균교호적쌍향전영도보.BUC조여대조조응효적단백질점수분별위(789±18)개화(762± 14)개；여대조조비교,BUC조유6개단백질점표체명현하조,11개단백질점표체명현상조.경질보분석후,초보감정료5충단백질,분별위섬유단백원、유산탈경매B、재지단백A1、총생단백화촉주단백,균재BUC조정표체증고추세.Western blot험증료쌍향응효전영결과.결론 BUC환자여건강지원자적뇨액단백질조존재명현적차이,차이단백적감정위진일보심조BUC조기진단적분자표지물제공료연구기출화의거.
Objective To investigate the difference in urinary proteome between patients with bladder urothelial carcinoma (BUC) and healthy volunteers and to provide a basis for the early diagnosis of BUC.Methods The urine samples from BUC patients and healthy volunteers (controls) were treated by 25％ethanol precipitation and two-dimensional gel electrophoresis (2-DE),and the obtained urinary proteins were subjected to Coomassie brilliant blue staining and analysis by PDQuest 8.0 (2-DE image analysis software); the differentially expressed proteins were sequenced by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry and identified using the Swiss-Prot database; the differential expression of these proteins was verified by western blot.Results High-resolution and high-reproducibility 2-DE images were obtained from the urine samples of BUC patients and controls,with 789 ±18 and 762 ±14 protein spots,respectively.Compared with the control group,the BUC grouPhad significantly decreased expression of 6 protein spots and significantly increased expression of 11 protein spots.The mass spectrometry revealed five proteins with increased expression in the BUC group,including fibrinogen,lactate dehydrogenase B,apolipoprotein A1,clusterin,and haptoglobin,and the results were confirmed by western blot.Conclusion There is significant difference in urinary proteome between BUC patients and healthy volunteers; the identification of differentially expressed proteins in urine lays the foundation for identifying potential molecular markers in early diagnosis of BUC.