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作 者:张萍[1,2] 徐敏[2] 严孝岭[2] 高德玉[2] 高文博[2] 虞伟[2] 李晓军[2]
机构地区:[1]江苏大学基础医学与医学技术学院,江苏镇江212013 [2]南京军区南京总医院临床中心实验科,南京210002
出 处:《临床检验杂志》2014年第4期287-290,共4页Chinese Journal of Clinical Laboratory Science
基 金:国家自然科学基金项目(81071419);江苏省社会发展基金(BE2011786)
摘 要:目的提纯并鉴定类风湿关节炎(RA)患者血清中免疫复合物(s—IC),并研究其对人脐静脉内皮细胞(HUVEC)增殖功能的影响。方法用G蛋白亲和层析法分别从抗瓜氨酸化蛋白抗体(ACPA)阳性(ACPA+)RA患者、SLE患者及健康人对照血清中提取IC,用SDS—PAGE对提取的s—IC进行纯度鉴定,用化学发光免疫分析法测定s—IC中ACPA含量,用westernblot法鉴定纯化Ic中的瓜氨酸化蛋白质。体外培养HUVEC,分别用RA患者血清IC(RA—IC)、SLE患者血清IC(SLE-IC)、健康人血清IC(C-IC)刺激HUVEC,培养24h后,用CCK-8细胞增殖法检测细胞的增殖水平。结果用G蛋白亲和层析法可自血清中提取出纯度较高的IC,ACPA’RA患者血清经G蛋白柱纯化后,IC中ACPA的回收率可达56.85%。westernblot结果显示,RA患者s—Ic中在相对分子质量(Mr)25000~55000间出现较多SLE—IC和c—Ic中未有的条带。用s—IC刺激体外培养的HUVEC,当s-IC浓度为50μg/mL时,RA—Ic与SLE-Ic组HUVEC的增殖水平均显著高于c-Ic组(P均〈0.01);当IC浓度为100μg/mL时,RA—IC组的HUVEC增殖水平显著高于SLE-IC与C-IC组(P均〈0.01)。结论成功提取ACPA-RA患者s-IC,RA-IC能显著促进HUVEC增殖。Objective To purify and identify the serum immune complexes (s-IC) from the patients with rheumatoid arthritis (RA) and investigate the effects of s-IC on the proliferation of human umbilical vein endothelial cells (HUVEC). Methods s-ICs from the RA pa- tients with anti-citndlinated protein antibody (ACPA) (ACPA + ), SLE patients and healthy controls were isolated by the affinity chroma- tography with immobilized protein G. The purity of ICs was identified by SDS-PAGE and the ACPA content in s-IC was detected by chem- iluminescence immunoassay. The citrullinated protein in s-IC was identified by Western blot. HUVECs were cultured in vitro and stimula- ted with s-IC from RA patients (RA-IC), SLE patients (SLE-IC) and healthy controls (C-IC), respectively. After 24 hours of incuba- tion, the proliferation rates of HUVECs were detected by CCK-8 test. Results Highly purified ICs could be obtained from sera by the af- finity chromatography with immobilized protein G and the recovery of ACPA in ICs from ACPA+ RA patients was up to 56.85%. Western blot analysis revealed many bands with molecular weight between 25 000 and 55 000 in RA-IC from ACPA + RA patients but not in SLE- IC and C-IC. When HUVECs were stimulated with 50 μg/mL of s-IC in vitro, the proliferation rates of HUVECs stimulated with RA-IC and SLE-IC were significantly higher than that with C-IC (P 〈0.01 ). When the concentration of s-IC was 100 μg/mL, the proliferation rate of HUVECs stimulated with RA-IC was significantly higher than that with SLE-IC and C-IC (P 〈 0.01 ). Conclusion The s-IC from ACPA ~ RA patients was purified successfully and it could significantly promote the proliferation of HUVECs.
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