CAJ | 학술논문

目的运用蛋白质组学的方法,比较正常人及早期活动性类风湿关节炎(RA)患者外周血单个核细胞(PBMCs)蛋白质的差异表达,寻找RA疾病相关致病蛋白质.方法选取9例早期活动期RA患者以及9名健康成年志愿者,用淋巴细胞分离液分离PBMCs,抽提PBMCs中的蛋白,采用同相pH梯度(IPG)双向凝胶电泳(2-DE)分离正常人及RA患者PBMCs总蛋白质.凝胶经考马斯亮蓝染色显色后,PDQuest图像分析软件进行比较分析、识别差异表达的蛋白质,对差异蛋白质点用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)进行鉴定,运用反转录-聚合酶链反应(RT-PCR)方法验证部分差异蛋白质.结果获得RA患者及正常人PBMCs蛋白质双向凝胶电泳图谱,平均蛋白质点数分别为556和579,匹配率分别为89.4%和 88.5%,通过比较分析,差异表达蛋白质点数为23,选取18个点进行质谱鉴定,成功鉴定14个蛋白质,其中a-肌动蛋白、纤维蛋白素原a-链、载脂蛋白A-I(ApoA-I)等9个蛋白质点在RA中表达上调,硫氧还蛋白-2、谷胱甘肽S-转移酶等6个蛋白质点在RA中表达下调,这些差异蛋白质的功能涉及物质代谢、抗氧化、信号传导、能量产生及细胞骨架.并用RT-PCR方法验证差异蛋白质ApoA-I.其结果与上述蛋白质差异表达结果相符.结论在RA患者PBMCs中存在着差异表达蛋白质,这些差异蛋白质可能是RA发病的内在因素.其RT-PCR结果与蛋白质差异表达相符,证明蛋白质组研究的可靠性.
목적 운용단백질조학적방법,비교정상인급조기활동성류풍습관절염(RA)환자외주혈단개핵세포(PBMCs)단백질적차이표체,심조RA질병상관치병단백질.방법 선취9례조기활동기RA환자이급9명건강성년지원자,용림파세포분리액분리PBMCs,추제PBMCs중적단백,채용동상pH제도(IPG)쌍향응효전영(2-DE)분리정상인급RA환자PBMCs총단백질.응효경고마사량람염색현색후,PDQuest도상분석연건진행비교분석、식별차이표체적단백질,대차이단백질점용기질보조격광해석전리비행시간질보(MALDI-TOF-MS)진행감정,운용반전록-취합매련반응(RT-PCR)방법험증부분차이단백질.결과 획득RA환자급정상인PBMCs단백질쌍향응효전영도보,평균단백질점수분별위556화579,필배솔분별위89.4%화 88.5%,통과비교분석,차이표체단백질점수위23,선취18개점진행질보감정,성공감정14개단백질,기중a-기동단백、섬유단백소원a-련、재지단백A-I(ApoA-I)등9개단백질점재RA중표체상조,류양환단백-2、곡광감태S-전이매등6개단백질점재RA중표체하조,저사차이단백질적공능섭급물질대사、항양화、신호전도、능양산생급세포골가.병용RT-PCR방법험증차이단백질ApoA-I.기결과여상술단백질차이표체결과상부.결론 재RA환자PBMCs중존재착차이표체단백질,저사차이단백질가능시RA발병적내재인소.기RT-PCR결과여단백질차이표체상부,증명단백질조연구적가고성.
Objective To explore the related protein which lead to rheumatoid arthritis (RA) and to find different proteins associated with active RA by comparing the expression levels of proteins in the peripheral blood mononuclear cells (PBMCs) of healthy individuals to patients with rheumatoid arthritis using a proteomics approach. Methods Samples of peripheral blood were collected from 9 patients diagnosed as active RA and 9 healthy individuals. PBMCs were isolated from blood using lymphoeytes separation medium. The total protein was extracted from the peripheral blood mononuclear cells. The total protein from either RA patients or normal controls was prepared by means of immobilized pH gradient based on two-dimensional gel eleetrophoresis. After Coomassie brilliant blue G250 staining, gel-image analysis was performed by using PDQuest.The differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALD I-TOF-MS). Then APOA-I was validated by RT-PCR. Results 2-DE patterns of PBMCs from controls and RA patients were presented. The results showed that the average number of protein spots was 556 and 579 respectively, and the corresponding average matching rate was 89.4％ and 88.5％ respectively. Gel-image analysis revealed that there were 23 differential protein spots. Fourteen of total 18 differential protein spots were successfully identified by MALD I-TOF-MS, of which 8 proteins were upregulated such as actin beta, fibrinogen beta chain, ApoA-I ; and 6 proteins such as peroxiredoxin-2, glu-tathione S-transferase omega 1 were down-regulated when compared with controls. The result of ApoA-I by RT-PCR was consistent with the proteomics results. Conclusion Some differentially expressed proteins are observed in the PBMCs of patients with rheumatoid arthritis, which may play a potential role in the pathogenesis of RA.