【作者】 张思功；

【导师】 王国春；

【作者基本信息】 北京协和医学院 ， 内科学， 2014， 博士

【摘要】 背景间质性肺病(ILD)是皮肌炎(DM)和多发性肌炎(PM)最常见且严重影响患者预后的并发症,而其发病机制仍不十分清楚。新近的研究发现低密度粒细胞(LDGs)不同与中性粒细胞,不但可以分泌促炎因子、损伤血管内皮,而且形成中性粒细胞胞外网状陷阱(NETs)的能力比中性粒细胞更强,而NETs的调控异常与炎症性肺病和多种自身免疫病的发病机制密切相关。但是目前仍不清楚肌炎患者中LDGs的比例及NETs的调控是否正常,以及NETs是否参与在肌炎患者伴发的ILD的发病机制中。目的研究目的之一是明确LDGs与DM患者伴发的ILD的相关性及与临床特征的关系；目的之二是探究NETs是否参与在与DM/PM患者伴发的ILD的发病机制中及可能的调控机制。方法为了达到第一个研究目的,研究I中纳入了48例DM患者和19例健康对照,其中28例DM患者患有ILD。采用流式细胞术测定外周血单个核细胞(PBMCs)中LDGs的比例；采用酶联免疫吸附法(ELISA)和实时定量PCR测定PBMCs中中性粒细胞相关蛋白和mRNA表达水平：采用肌炎活动度视觉模拟评分(MYOACT)评估疾病活动度。比较伴发ILD的患者和未患ILD的患者LDGs水平的差异,分析LDGs与临床指标的相关性。为了实现第二个研究目的,研究II中纳入了72例DM患者、25例PM患者和54例健康对照,其中35例DM和7例PM患者合并有ILD。比较两组血浆诱发NETs形成和降解NETs的能力；采用辐射状酶扩散法测定血浆DNase I的活性,分析NETs降解异常的原因,并进一步在患有ILD的患者和未患ILD的患者间进行比较。结果研究I中DM患者PBMCs中LDGs的比例显著高于健康对照(9.06%±11.50%vs.1.28%±0.73%,P<0.0001)；且伴有ILD的DM患者中LDGs的比例显著高于不伴ILD的DM患者(12.29%±14.13%vs.4.54%±2.61%,P=0.0083)；LL-37、髓过氧化物酶和基质金属蛋白酶-8的mRNA表达水平和LL-37的蛋白表达水平在DM组显著高于健康对照组；LDGs比例与MYOACT肺疾病活动度评分、肌酸肌酶同工酶(CKMB)、乳酸脱氢酶(LDH)、肿瘤标记物、中性粒细胞计数、甘油三酯(TG)和高密度脂蛋白(HDL)呈显著正相关,与总三碘甲腺原氨酸(tT3)和游离三碘甲腺原氨酸(fT3)呈显著负相关。研究Ⅱ中DM/PM患者的血浆比健康对照的血浆能诱发更多的中性粒细胞形成NETs(246±93.48RFUs vs.191.6±52.88RFUs,P=0.002),显著升高的血浆细胞外游离DNA(cfDNA)水平和LL-37水平进一步印证了DM/PM患者中NETs形成异常增多；而且DM/PM患者的血浆对NETs的降解显著低于健康对照,尤其以伴发ILD的DM/PM患者降低更明显(58.58%±21.4%vs.95.07%±5.35%,P<0.0001)；血浆DNase Ⅰ的活性在PM组和DM组分别是0.1822±0.0940U/ml和0.2094±0.1112U/ml,均显著低于健康对照组(0.3933±0.1523U/m1,P<0.0001)。而且DNase Ⅰ活性在伴发ILD的DM/PM患者中显著低于不伴ILD的DM/PM患者(0.1677±0.077U/ml vs.0.2301±0.1187U/ml,P=0.0039)；抗Jo-1抗体阳性的患者和伴发亚急性ILD的患者DNase Ⅰ活性更低,糖皮质激素治疗似可改善DNase Ⅰ活性。结论DM患者PBMCs中LDGs的比例显著增高,尤其是伴发ILD的DM患者PBMCs中LDGs的比例增高更明显,而且LDGs比例与MYOACT肺疾病活动度评分呈显著正相关,提示过度增多的LDGs可能是DM患者伴发的ILD的参与因素。DM患者PBMCs中中性粒细胞相关的基因mRNA和蛋白表达水平均显著高于健康对照,进一步印证了LDGs是另一群强有力的NETs形成细胞,LDGs过度形成NETs可能是其参与在ILD发病机制中的方式之一。DM/PM患者的血浆体外诱发NETs形成显著增加,而伴发ILD的DM/PM患者因DNaseⅠ的活性受损而不能及时有效地清除大量形成的NETs,使得DM/PM患者,尤其使伴有ILD的患者暴露于大量形成的NETs中,而且DM患者体内异常增多的LDGs可能会进一步放大NETs的暴露。反复而严重的NETs暴露可能会造成肺损伤,进而诱发及加重ILD。推测NETs的调控异常可能参与在DM/PM伴发的ILD中,而且NETs通路可能是DM/PM中ILD发生发展的关键因素之一。更多还原

【Abstract】 BackgroundInterstitial lung disease (ILD) is the most common complication in Dermatomyositis (DM) and polymyosits (PM). Although ILDs are closely related to bad prognosis, its pathogeneses remain unclear. New researches indicted that low density granulocytes (LDGs) can release proinflammatory cytokines, injure vascular endothelial and form neutrophil extracellular traps (NETs). Abnormal regulation of NETs is reputed to play an important role in the pathogenesis of autoimmune diseases and inflammatory lung diseases. However, whether the percentage of LDGs and the regulation of NETs are normal and how NETs contribute to the pathogenesis of ILD in DM/PM are unknown.ObjectiveThe first aim of this study is to investigate whether LDGs is involved in pathogenesis of ILD in DM and to analyze the correlations between LDGs with clinical items.The second aim of this study is to test the hypothesis that NETs may be pathogenic in DM/PM and to explore the possible mechanisms.MethodsStudy Ⅰ. Forty eight DM patients were recruited for this study, among whom28complicated with ILD. Nineteen age-and sex-matched healthy Chinese volunteers were selected to be control subjects. LDGs percentage in peripheral blood mononuclear cells (PBMCs) was tested by flow cytometer. Neutrophil-related protein expression in PBMCs was tested by enzyme-linked immunosorbent assay (ELISA). Neutrophil-related gene expressions in PBMCs were tested by quantitative RT-PCR. Myositis Disease Activity Assessment Visual Analogue Scales (MYOACT) was used to assess the disease activity. Comparisons of LDGs and others items were performed between DM patients with ILD and without. Correlations between LDGs with other clinical items were performed with linear correlation analysis. Study Ⅱ. Plasma samples from97DM/PM patients (72DM,25PM) and54healthy controls were tested for the capacities to induce and degrade NETs. Plasma DNase I activity was tested by the radial enzyme-diffusion method to further explore possible reasons for the incomplete degradation of NETs. Results from35DM patients and seven PM patients with ILD were compared with results from DM/PM patients without ILD.ResultsStudy Ⅰ. Compared with healthy control (1.28%±0.73%of total PBMCs), DM patients showed significantly higher percentage of LDGs (9.06%±11.50%of total PBMCs, P<0.0001). Compared with DM patients without ILD, DM patients with ILD exhibited significantly higher percentage of LDGs in PBMCs (4.54%±2.61%vs.12.29%±14.13%, P=0.0083). The mRNA expression level of LL-37, MPO and MMP-8and LL-37protein levle in DM group were significantly higher than that in Control group. The percentage of LDGs positively correlated with MYOACT lung disease activity scores, CKMB, tumor markers, neutrophil count, TG and HDL, and inversely correlated with tT3and fT3.Study Ⅱ. Compared with control subjects, DM/PM patients exhibited a significantly enhanced capacity for inducing NETs (191.6±52.88RFUs vs.246±93.48RFUs, P=0.002), which was supported by elevated levels of plasma LL-37and circulating cell-free DNA (cfDNA) in DM/PM. NETs degradation in DM/PM paients was significantly lower than in control patients. Compared with DM/PM patients without ILD, DM/PM patients with ILD degraded less NETs in vitro (83.41%±12.64%vs.58.58%±21.4%, P=0.0002); Plasma DNase I activity in PM and DM were0.1822±0.0940U/ml and0.2094±0.1112U/ml, repectively, significantly lower than in control subjects (0.3933±0.1523U/ml, P<0.0001). Compared with DM/PM patients without ILD, DM/PM patients with ILD exhibited a lower DNase I activity (0.2301±0.1187U/ml vs.0.1677±0.077U/ml, P=0.0039). Patients with anti-Jo-1antibodies and patients with subacute ILD exhibited a significantly decreased DNase I activity. Glucocorticoid therapy seems to improve DNase I activity.ConclusionDM patients, especially patients with ILD, exhibited a significantly increased percentage of LDGs in PBMCs. Moveover, the percentage of LDGs positively correlated with MYOACT lung disease activity scores, suggesting that LDGs may play a role in pathogenesis of DM-associated ILD. The significantly increased mRNA expression level and protein level of neutrophils-related markers in PBMCs indicate that LDGs are another group of cells that can potently forme NETs and LDGs may contribute to ILD in DM patients by excessively forming NETs.The excessively formed NETs cannot be completely degraded because of decreased DNase I activity in DM/PM patients, especially in patients with ILD, which makes DM/PM patients expose to large amounts of NETs. Moreover, abnormally increased LDGs may contribute to abnormal regulation of NETs. All of which may cause constant and severe damage of lung and further induce ILD. It is reasonable to suppose that abnormal regulation of NETs may be involved in the pathogenesis of DM/PM and could be one of factors that initiate and aggravate ILD.更多还原