miRNA-146a对原发性干燥综合征不同中医证型外周血B淋巴细胞调控机制的研究

Our former research found that Qi and Yin deficiency syndrome are the main syndrome in patients with primary sjogren's syndrome, and miR-146a is over expression in peripheral blood, and other scholars proved that B cell hyperactivation was a prominent feature and microRNAs involved in B cell differentiation in pSS patients, so we speculate that there are correlation among miR-146a and mature of peripheral blood B cell and function regulation in pSS. In present study, we intend to separate the peripheral blood B cell in pSS patients with different traditional chinese medicine (TCM) syndromes and healthy controls, detect the expression of miR-146a and target genes TRAF6, IRAK-1 in peripheral blood B cell from pSS patients with different TCM syndromes and healthy controls by RT-PCR, and analyze the relationship between them.The effect of miR-146a on the activation, differentiation, proliferation and apoptosis of peripheral blood B cells in patients with different TCM syndromes and healthy controls was detected by flow cytometry. We observe the regulation function of peripheral blood B cell and changes of target gene expression by miR-146a over expression and silent expression in pSS with different TCM syndromes, and prove the possible molecular mechanism of miR-146a for peripheral blood B lymphocytes in pSS, provide the basis for a new method of treatment for pSS.

课题组前期研究发现，原发性干燥综合征患者多见气阴两虚证，其外周血miR-146a表达上调，而其他学者证明pSS突出特点是B细胞高度活化，miRNA参与B细胞分化，因此推测miR-146a和pSS外周血B细胞的发育成熟及功能调控存在关联。本课题拟对气阴两虚证、湿热浸渍证、脾气虚弱证pSS患者和健康对照者外周血B细胞分离，通过RT-PCR法检测不同中医证型pSS患者和健康对照者外周血B细胞miR-146a和靶基因TRAF6、IRAK-1表达，分析二者之间关系。采用流式细胞术检测不同中医证型pSS患者和健康对照者外周血B细胞活化、分化、增殖及凋亡水平，观察miR-146a对其影响。研究miR-146a过表达和沉默表达后对不同中医证型pSS外周血B细胞的调节作用及靶基因表达改变，揭示miR-146a对pSS外周血B淋巴细胞的可能分子机制，为pSS治疗的新方法提供依据。

前期研究发现pSS患者外周血miR-146a表达上调，而其他学者证明pSS突出特点是B细胞高度活化，miRNA参与B细胞分化，因此本研究对miR-146a过表达或沉默表达后对CD19+B淋巴细胞增殖、凋亡、活化及靶基因TRAF6和IRAK1的影响进行相关研究。本研究采用免疫磁珠分选pSS患者及正常人新鲜外周血CD19+B淋巴细胞并进行体外细胞培养，分别设置对照组、模拟物对照组、miR-146a-5p模拟物组、抑制物对照组、miR-146a-5p抑制物组。用PCR法检测各组细胞miR-146a及靶基因IRAK1和TRAF6表达情况，采用CCK8法检测细胞增殖情况，流式细胞仪检测细胞凋亡率、活化及分化情况。.研究结果表明：在pSS患者和正常人分离的B淋巴细胞中分别转染miR-146a模拟物或抑制剂后，B淋巴细胞的增殖、凋亡及TRAF6表达差异不显著，但影响B细胞的活化和分化。pSS患者B淋巴细胞中miR-146a过表达后CD23+ 显著降低而 CD80+ B细胞显著升高，IRAK1表达显著降低(P<0.05)；正常人B淋巴细胞中miR-146a过表达后CD23+ 、CD80+ B细胞比例显著升高，IRAK1表达显著降低(P<0.05)。转染miR-146a抑制剂后，pSS患者和正常B淋巴细胞中miR-146a表达均下调，CD23+、CD80+B细胞比例显著降低而CD20-CD138+B细胞比例显著升高，IRAK1表达显著增加(P<0.05)。虽然pSS患者miR-146a过表达、沉默表达时CD23+B细胞比例都出现下降，但pSS患者miR-146a过表达时CD23+B细胞比例明显高于miR-146a沉默表达时(P<0.05)。总之，miR-146a可促进pSS患者B淋巴细胞的活化和分化，影响CD23+、CD80+细胞比例，靶向调控IRAK1表达。.根据研究结果，初步认为通过调控CD23、CD80抗原的表达或许可以抑制SS患者B淋巴细胞的过度活化，或能成为纠正患者免疫紊乱的潜在治疗途径之一。如果敲除SS患者B淋巴细胞中miR-146a，则会提高IRAK1表达，或许可以为SS干预治疗提供新的有潜在研究价值的治疗方法。对今后基因敲除及转基因动物模型深入研究将有助于明确miR-146a在SS中的具体作用，为SS的靶向治疗以及中药有效成分的作用机制研究提供参考依据。