【作者】 董红宇；

【导师】 毕黎琦；

【作者基本信息】 吉林大学 ， 内科学， 2012， 博士

【摘要】 研究背景：类风湿关节炎（rheumatoid arthritis，RA）是以慢性进行性对称性关节炎为主要临床表现的疾病，早期以关节的滑膜炎、细胞浸润、血管翳形成为主，中晚期累及软骨及骨质，导致骨质疏松，甚至关节结构出现破坏而引起畸形、功能障碍，从而增加了RA致残率。RA骨代谢异常可能与多种炎症因子的作用有关，过量炎症因子不仅可以造成持续性滑膜炎症，同时还可以激活破骨细胞，使骨吸收加速，造成快速骨流失破坏骨组织微环境导致关节损害。这是RA患者病程前3个月快速骨流失、关节损害的主要原因。近年来研究表明，RA疾病早期即可发生关节端的骨质疏松及不可逆的关节骨破坏,继而出现全身的骨质疏松症（osteoporosis，OP）,有多达60%-80%的RA患者合并OP，严重影响患者生活质量，同时造成巨大的社会经济负担。糖皮质激素（Glucocorticoid，GC）因其强大的抗炎及免疫调节作用而广泛用于RA的治疗，但是，由于长期大量应用GC会导致骨质疏松症，增加骨折的发病率，因此其应用颇受争议。随着人们对小剂量糖皮质激素（low dose glucocorticoid，LGC）治疗RA认识的加深，对其疗效及对RA预后的改善有了新的期待，与GC引起骨的不良反应相比，GC可以控制RA患者炎症反应、降低疾病活动度，有减轻关节损害的作用，其积极的治疗作用可能部分抵消了它对骨的不良作用。GC的作用主要是通过与糖皮质激素受体（Glucocorticoid Receptor，GR）结合实现的，近年来的研究认为糖皮质激素受体可能参与了RA的发病。miRNA是一类内源性短小非编码RNA，能够在转录后水平上对基因的表达进行负调控。新近研究还发现miRNA表达异常可能是RA发病的分子机制之一，在RA关节滑膜、滑液、外周血中都有异常表达的miRNA，它们可能与RA炎症过程、骨流失、关节骨及软骨的破坏密切相关。本研究拟应用LGC短疗程治疗RA，通过观察RA患者骨代谢的变化、GR mRNA表达的变化、miRNA表达的变化以及NK细胞对RA患者骨代谢异常的影响，探讨LGC的作用机制，为指导临床工作提供客观依据。目的：1.研究小剂量糖皮质激素短疗程治疗对RA骨代谢的影响；2.研究LGC对人类糖皮质激素受体hGRα和hGRβmRNA表达的影响；3.研究LGC对RA miRNA表达及其靶基因的影响。4.研究RA对骨代谢的影响。通过以上研究，探讨LGC对RA骨代谢的作用机制，为指导临床工作提供客观依据。方法：选取绝经后女性早期类风湿关节炎病情活动患者为研究对象，并选取同期于体检中心体检的绝经后健康女性为对照组。入选患者随机分为小剂量糖皮质激素治疗组(LGC组)和非激素治疗组(NGC组)，在试验开始时和3个月时分别测量炎症指标（IL-6、IL-1α、TNF-α、CRP和ESR）、骨代谢标志物（骨形成标志物BGP、BAP和PICP，骨吸收标志物uDPD和CTX）和骨密度；应用逆转录实时荧光定量聚合酶链反应(RT-FQ-PCR)检测外周血单个核细胞hGRα和hGRβ mRNA的表达情况；应用基因芯片技术对LGC治疗前后的患者进行miRNA分析，并对miRNA靶基因进行鉴定，应用KEGG数据库对差异表达的miRNA聚类结果进行注释。结果：1.绝经后早期RA疾病活动患者低骨量和骨质疏松症发病率高于健康对照组（P＜0.05），差异有统计学意义。2.与健康对照组相比，RA患者外周血中炎症指标IL-6、IL-1α及TNF-α水平明显增高（P＜0.01），骨形成标志物BGP、BAP和PICP明显降低（P＜0.05），骨吸收标志物uDPD和CTX明显增高（P＜0.05）。3.基线时，LGC组与NGC组间各观察指标均无明显统计学差异（P＞0.05）。试验3个月时， NGC组的IL-6、IL-1α、CRP及ESR明显下降，与基线时相比，有统计学差异（P＜0.05）；LGC组的IL-6、IL-1α、CRP及ESR明显下降，与基线时相比，有显著统计学差异（P＜0.01）；两组相比，LGC组IL-1α明显降低，有统计学差异（P＜0.05）。试验3个月时，两组相比，LGC组的骨形成标志物检测值有所增加，骨吸收标志物检测值有所下降，但无明显统计学差异（P＞0.05）。与基线时相比，两组在3个月时BMD略有增高，但无统计学差异（P＞0.05）。4.基线时，RA患者外周血单个核细胞hGRα mRNA和hGRβ mRNA的表达均高于健康对照组（P＜0.01）。5.试验3个月时，LGC组hGRα mRNA表达水平明显降低，但仍高于健康对照组（P＜0.05），与基线时比较，差异有统计学意义（P＜0.01）；NGC组hGRα mRNA表达水平无显著降低（P＞0.05）；两治疗组间hGRα mRNA表达水平比较差异有统计学意义（P＜0.05）。6.试验3个月时，LGC组和NGC组hGRβ mRNA的表达水平均无显著变化（P＞0.05）。7.人miRNA芯片实验：根据变化倍数和差异分数两项参数得到LGC治疗前后样本差异最显著的miRNA13个（hsa-miR-34a、 hsa-miR-134、 hsa-miR-200b、hsa-miR-106b*、hsa-miR-1281、hsa-miR-188-5p、hsa-miR-202*、hsa-miR-219-1-3p、hsa-miR-455-5p、hsa-miR-513b、hsa-miR-521、hsa-miR-548f和hsa-miR-645），经LGC治疗后这些miRNA表达均上调。8.通过对外周血相关miRNA及其靶基因ULBP2和配体基因NKG2D的表达水平进行验证，LGC治疗后has-miR-34a表达水平上调（P＜0.05）,靶基因ULBP2和配体基因NKG2D表达水平均下调（P＜0.05）。9.通过对13个表达下调的miRNA靶基因聚类分析，应用KEGG数据库对聚类结果进行注释，共得到4个差异显著的KEGG通路（校正后P＜0.00001），分别是细胞黏着斑信号通路、mTOR信号通路、趋化因子信号通路和凋亡信号通路。结论：1.绝经后女性早期RA疾病活动患者低骨量、骨质疏松症发病率高于正常人。2.小剂量糖皮质激素短期治疗可以通过控制炎症使早期类风湿关节炎患者骨形成增加、抑制骨吸收、减少骨流失。3.小剂量糖皮质激素短期治疗早期类风湿关节炎对骨代谢无负性影响，不会增加骨质疏松症发病率。4. hGRα mRNA和hGRβ mRNA表达增加可能与RA发病有关，LGC可显著降低RA患者外周血单个核细胞hGRα mRNA表达。5.小剂量糖皮质激素治疗RA可以使外周血13个miRNA（hsa-miR-34a、hsa-miR-134、 hsa-miR-200b、 hsa-miR-106b*、 hsa-miR-1281、 hsa-miR-188-5p、hsa-miR-202*、 hsa-miR-219-1-3p、 hsa-miR-455-5p、 hsa-miR-513b、 hsa-miR-521、hsa-miR-548f、hsa-miR-645）表达上调，其中has-miR-34a在外周血及膝关节滑液中表达上调最明显。6.小剂量糖皮质激素可以通过上调has-miR-34a表达，负调控靶基因ULBP2，改变NKG2D活性，降低NK细胞活性。7.小剂量糖皮质激素可以上调RA患者外周血miRNA表达，对与其靶基因有关的信号通路起抑制作用。8.小剂量糖皮质激素可以降低NK细胞活性，减少RA骨流失，对关节侵蚀起保护作用。更多还原

【Abstract】 Background:Rheumatoid arthritis（RA）is a chronic, progressive, systemic, autoimmune disease. Thepathogenesis is synovitis, cellular infiltration and pannus in early RA, involvement ofcartilage and bone，destruction of joint leading to deformity and functional impairment andosteoporosis(OP) with increasing disorder in mid-term and late. Bone metabolism disordersinvolve in many kinds of inflammatory cytokines leading to persistence synovitis, activitedosteoblast and increased resorption of bone in RA, which play a key role of more rapid boneloss and joint destruction at the onset of3months of disease. Within the past few years，studies have shown that periarticular bone loss as well as irreversible damage is an earlyfeature of the disease with an increased risk of generalized OP complication of60%-80%resulting in decreased quality of life and huge economic burden. Over the past decades,glucocorticoids (GC) are the most widely used anti-inflammatory and immunomodulatoryagents in RA owing to their strong anti-inflammatory and immunosuppressor effect.However, there have been controversial for inlovement of osteoporosis and increased risk offractures.The effect of low-dose glucocorticoid (LGC) in RA includes inhibiting inflammatoryresponse, decreasing disease activity and reducing the joint damage, which brings newexpect about efficacy and prognosis of RA. The benefits anti-inflammatory effect of LGCmay partially courteract their negative effect on bone.GC exert their effects mainly through the glucocorticoid receptor (GR). Recent studiesdemonstrate that GR is involved in pathogenesis of RA. MicroRNAs (miRNAs) are a kind ofendogenous, small, noncoding single-stranded RNA molecules that negatively regulate geneexpression at the post-transcriptional level. Recent studies have uncovered dysregulatedmiRNA expression in RA in the afflicted synovium，synovial fluid and the peripheralcirculation, suggesting that abnormalities in miRNA expression may contribute to themolecular mechanisms of the inflammation，bone loss and cartilage damage.Therefore, the aim of the present study is to evaluate the bone metabolism, GR mRNAexpression, miRNA expression and the activity of NK cell in early activity RA patientsreceiving LGC short-term therapy, which will explore the mechanisms of LGC and provide data for clinic.Objective:1. To investigate the effects of short-term LGC on bone metabolism in RA;2. To investigate the effects of short-term LGC on expression levels of hGRα and hGRβmRNA in RA;3. To investigate the effects of short-term LGC on expression levels of miRNA andtarget gene in RA;4. To investigate the influence of RA disease on bone metabolism;5. To investigate the mechanisms of LGC in RA and provide data for clinic.Methods:Postmenopausal female patients of early activity RA and healthy postmenopausalfemale controls sampled at medical examination center at the same time period were studied.Patients were randomly assigned to low-dose glucocorticoid therapy group (LGC group) ornon-glucocorticoid therapy group (NGC group). Biomedical markers including inflammationmarkers (IL-6, IL-1α, TNF-α, CRP and ESR), bone metabolism markers (bone formationmarkers: BGP, BAP and PICP; bone resorption markers: uDPD and CTX) and bone massdensity (BMD) were measured for both RA groups at baseline and3-month trail. Expressionlevels of hGRα and hGRβ mRNA in peripheral blood mononuclear cells was determinedwith real time fluorescent quantitative PCR for both RA groups at baseline and3-month trail.All the detections were compared with healthy controls at baseline. microRNA expressionlevels were detected with gene microarrays for LGC group at baseline and3-month trail.Cluster analysis using microRNA expression data was also performed. And, target genes ofdifferentially expressed microRNAs were predicted and analyzed with KEGG database.Results:1. The rate of low bone mass and osteoporosis were higher in postmenopausal femalepatients of early activity RA than in healthy controls (P＜0.05).2. In both RA groups, inflammation markers (IL-6, IL-1α and TNF-α) weresignificantly higher(P＜0.01), bone formation markers (BGP, BAP and PICP) weresignificantly lower (P＜0.05), and bone resorption markers (uDPD and CTX) weresignificantly higher (P＜0.05) compared with healthy controls at baseline.3. There was no significant difference observed for all measured indexes (P＞0.05)between LGC and NGC groups at baseline. At3-month trail, inflammation markers (IL-6, IL-1α, CRP and ESR) decreased dramatically in both NGC group (P＜0.05) and LGC group(P＜0.01); while IL-1α level was even lower in LGC group than in NGC group (P＜0.05).The levels of bone formation markers were higher, while the levels of bone resorptionmarkers were lower in LGC group than in NGC group, but no significant difference werefound (P＞0.05). At3-month trail, BMD increased slightly in both group, but no significantdifference were found (P＞0.05), either.4. At baseline, both levers of hGRα and hGRβ mRNA in two RA groups weresignificantly higher than that in healthy controls (P＜0.01).5. At3-month trail, the levers of hGRα mRNA in LGC group were significantlydecreased (P＜0.01), however, it was still higher than that in healthy controls (P＜0.05).While the levers of hGRα mRNA in NGC group were not obviously decreased comparedwith baseline (P＞0.05), and there was significant difference between LGC and NGC groups(P＜0.05).6. At3-month trail, there was no significant difference was found about the levers ofhGRβ mRNA in both LGC and NGC groups compared with baseline (P＞0.05).7. Thirteen differentially expressed microRNAs were identified according to foldchange and diffscore. They are hsa-miR-34a, hsa-miR-134, hsa-miR-200b, hsa-miR-106b*,hsa-miR-1281, hsa-miR-188-5p, hsa-miR-202*, hsa-miR-219-1-3p, hsa-miR-455-5p, hsa-miR-513b, hsa-miR-521, hsa-miR-548f and hsa-miR-645. After LGC treatment, all theabove listed microRNAs were up-regulated.8. Verification experiments replicated the result that hsa-miR-34a expression level wasup-regulated after3-month LGC therapy(P＜0.05), and further proved that two target genesof hsa-miR-34a (ULBP2and NKG2D) were down-regulated(P＜0.05).9. KEGG pathway analysis of target genes of the thirteen differentially expressedmicroRNAs revealed4most relevant pathways (adj P＜0.00001), they are focal adhesionkinase signaling pathway, mTOR signaling pathway, chemokine signaling pathway andapoptosis signaling pathway.Conclusions：1. The rate of low bone mass and osteoporosis were higher in postmenopausal femalepatients of early activity RA than in healthy controls.2. LGC short-term therapy can increase bone formation, inhibit bone resorption and reduce bone loss through alleviating inflammation respones in early RA patients.3. There is no negative effect of LGC short-term therapy on bone metabolism and thereis no increase on the rate of osteoporosis in RA patients.4. Increased expression of hGRα and hGRβ mRNAs may involved in the onset of RA.LGC can decrease hGRα mRNA expression in peripheral blood mononuclear cells in RApatients.5. LGC can up-regulate the expression of thirteen microRNAs （hsa-miR-34a、hsa-miR-134、 hsa-miR-200b、 hsa-miR-106b*、 hsa-miR-1281、 hsa-miR-188-5p、hsa-miR-202*、 hsa-miR-219-1-3p、 hsa-miR-455-5p、 hsa-miR-513b、 hsa-miR-521、hsa-miR-548f、hsa-miR-645）, and the expression of hsa-miR-34a in peripheral blood andknee synovial fluid were up-regulated most in the all.6. LGC may down-regulate ULBP2expression, change NKG2D activity and reduceNK cell activity through up-regulation of hsa-miR-34a.7. LGC can up-regulate certain microRNAs in peripheral blood and inhibit signalingpathways which their target genes involved.8. LGC can decrease NK cell activity, reduce bone loss and prevent erosion of joint inRA.更多还原