LRP5对雄性DBA/1小鼠异位骨化机制的研究

Ankylosing spondylitis (AS) is a common and debilitating inflammatory rheumatic disease characterized by sacroiliitis, pathological bone formation, low bone mineral density (BMD), peripheral arthritis, enthesis and iritis. AS is a highly heritable and polygenic trait.Pathological bone formation associated with AS is a major health issue and produce a significant economic and social burden for AS patients. So far, the susceptible gene studies of AS primarily are present in the field of inflammation and immunization. However, few experiments have been performed in the field of ossification, which is an important cause of disability. TNF-α antagonists provide long-term control of the clinical symptoms and systemic in?ammation in most patients with AS. But neither etanercept nor in?iximab signi?cantly slowed the radiological progression of AS over a 2-year period treatment.So,exploring the target point related to ossification will certainly be the subject of intense investigations in treatment of AS. In our previous experiments, we selected 15 candidate genes(BMP2、BMP4、LRP5、SOX9、RUNX2、ACVR1、ANTXR2、COL11A2、MGP、DMP1、IHH、PTGS2、DKK1、BMP6、ENPP1) related to ossification in order to determining genetic associations with AS in cohorts including 300 Chinese Han AS patients and 180 controls via illumina goldengate genetype flatform. Finally, we demonstrated a signi?cant association between LRP5 and AS in the Chinese Han population.This is the reason why we have chosen the gene LRP5 for target intervention study. Using the molecular biology techology such as Talen,west blot, we should observed the changes of the signal transduction pathway, ectopic bone formation, joint activities and so on in the DBA/1 male SpA mice model, cell model after intervention. The new perspective of the subject is to clarfy the pathogenesis of ankylosing spondylitis, provide a new target for heterotopic ossification treatment of ankylosing spondylitis, reducing patient morbidity. We will provide experimental evidence and treatment reference for the disease of heterotopic ossification or mineralization such as heterotopic ossification after surgery, the intravascular ossification, kidney stone and so on.

强直性脊柱炎(AS)发病过程包括发病初期的炎症反应和后期的异位骨化，而异位骨化是患者致残的主要原因。TNF-α阻断剂能够有效抑制AS的炎性症状，但却不能阻止异位骨化的发展。因此,AS的炎症和骨化可能是两个独立的过程，各有自己独立的遗传学基础。目前，对AS易感基因的研究主要集中在炎症免疫领域，在成骨领域的研究则鲜见报道。在前期的研究工作中，我们发现Wnt信号通路的LRP5与AS的发病呈强相关。为此我们提出假说，LRP5可能通过对Wnt信号通路及基质矿化的调控，独立于炎症而促进AS异位骨化的发展。因此，我们拟利用DBA/1雄性SpA小鼠模型，采用real time PCR、Western blot、慢病毒载体转染、talen等分子生物学技术，从分子、细胞、以及动物整体水平探讨LRP5在AS异位骨化中的作用机制。本研究将从LRP5这个新视点为揭示AS的异位骨化奠定基础，从而减少AS患者的致残率。

强直性脊柱炎是高度遗传的风湿免疫性疾病，发病率大约为0.3%。目前，强直性脊柱炎易感基因并不明确，以及缺乏研究AS机制的动物模型。这些都限制了AS异位骨化机制研究的进展。.1.收集300个病人，180个对照，筛选到DMP1的rs10019009 SNP位点与AS相关，并通过了620个病人和683个对照的实验验证。细胞实验发现rs10019009风险型等位基因增强ALP的酶活性。DMP1可能参与了AS的异位骨化机制。为后续对AS异位骨化的研究奠定了遗传学基础。获得锦州市学术成果奖2等奖。.2.应用二型胶原对BALB/c和DBA/1杂交F1代小鼠进行免疫，应用钼靶照相和HE染色等技术，发现二型胶原促进了F1代小鼠外周关节炎、中轴关节炎的出现。异位骨化性质表现为软骨内骨化。培养硕士研究生1名。.3.培养MC3T3细胞，应用矿化诱导液培养，siRNA干扰，qRT-PCR，同时检测了基质小泡内外离子变化，以及蛋白酶的酶活性，矿化结节表达情况，收集55个病人和65个对照，研究了血清离子变化。DMP1通过TNAP/ANK/ENPP1轴参与基质矿化；DMP1调节TNAP活性可能是通过滞后的转录调节和早期的离子通道快速调节两种方式；而对ENPP1和ANK的调节可能只是通过转录调节的方式；DMP1可能作为催化剂或钙离子募集剂，参与了胶原矿化的过程；DMP1沉默后，MC3T3细胞矿化能力的降低，可能是因为细胞外基质内Pi浓度的降低，阻滞了Pi/PPi-TNAP正反馈过程；局部的离子失衡失调导致了AS异位骨化。培养硕士研究生1名。.4.通过纯化非聚合蛋白聚糖，免疫BALB/c小鼠，形成典型的外周及中轴关节炎表现。同时 ELISA检测特异性IgG抗体和流式细胞术检测CD4+T细胞，二者都明显的降低了。本实验是首次用非聚合蛋白聚糖免疫诱导成关节炎动物模型，与完整的蛋白聚糖相比，非聚合蛋白聚糖可能是关节炎的自身抗原。.5.收集10例AS病人和10例健康人空腹血液，进行白细胞分离，流式细胞术检测TR2、TR4、TR7、TR9，IL18、IL18R、IL18BP，CD4+、IFNr+、IL4+、IL17A+、CD25+FOXP+、CD8+、CD14+、CD16+、CD19+以及嗜碱性粒细胞等，后续实验分离病人及健康人外周血CD4+T细胞，体外扩增培养，用IL17A和TNF干预后，检测细胞的表型变化。