MicroRNA-34a检测方法的建立及其在类风湿性关节炎中的应用  

Establish a Stem-loop RT-PCR for microRNA-34a Detection and Applied for Rheumatoid Arthritis Patients Assay

作　　者：陈晓旦[1,2] 刘秀雯[1] 吴琳[1] 成静[1] 阴晴[3] 李晶[4] 吴忠[4] 钱晖[1] 许文荣[1] 邵启祥[1] 

机构地区：[1]江苏大学基础医学与医学技术学院免疫学与免疫学检验系,江苏镇江212013 [2]上海浦东新区光明中医医院检验科,上海200120 [3]江苏大学附属医院检验科,江苏镇江212013 [4]江苏大学附属医院风湿免疫科,江苏镇江212013

出　　处：《现代检验医学杂志》2010年第1期22-24,共3页Journal of Modern Laboratory Medicine

基　　金：国家自然科学基金资助项目（30671984）;江苏大学科技创新团队项目（2008-018-02）;江苏大学大学生科技立项（07A132,07A133）.

摘　　要：目的采用自行设计的颈环结构引物，建立外周血检测microRNA-34a（miR-34a）的检测方法，并初步应用于类风湿性关节炎（rheumatoid arthritis，RA）患者外周血单个核细胞（peripheral blood monocytes，PBMCs）样品的检测。方法用TRlzol提取外周血单个核细胞的总RNA，以颈环结构的RT引物逆转录出相应的cDNA，然后以miR-34a的PCR引物进行PCR检测，电泳观察结果。结果建立的颈环RT—PCR能正确检测miR-34a。在RA患者外周血中检测到miR-34a的表达，其中初发者栓出率较高（2／6），复发者检出率为（1／9）；而健康志愿者外周血和治疗好转者未见表达。结论成功建立检测miR-34a的方法，miR-34a检测可能对RA的诊断有一定意义，但尚需进一步深入研究。Objective Established a stem-loop RT-PCR for detection microRNA-34a （miR-34a）,and applied for detecting the expression of miR-34a in rheumatoid arthritis （RA） patients. Methods Total RNA was isolated from peripheral blood monocytes （PBMCs） of rheumatoid arthritis patients and healthy volunteer donors by TRzol reagent. First,cDNA was reverse transcripted by using the stem-loop RT primer base on the mRNA. Then,the RT product-cDNA was amplified using PCR by miR-34a PCR primer. The products were analyzed by DNA electrophoresis. Results The method was worked well in detecting miR-34a,and found that miR-34a levels were up-regulated in RA patients＇PBMCs compared with that of volunteer donors＇. The positive ratio was 2/6 in patients in acute phase with newly diagnosed,and 1/9 in that of patients in reactive phase,but there no positive in that of improving patients and healthy volunteer donors. Conclusion The stemloop RT-PCR succeeded to amplify the miR-34a,and the miR-34a may have some relationship with the RA,but it need to do some further research work to confirm and valuate it.

关 键 词：MIR-34A 逆转录聚合酶链反应 类风湿性关节炎 

分 类 号：Q503[生物学—生物化学] R593.22[医药卫生—内科学]