趋化因子SDF-1和RANTES在外源性骨髓间充质干细胞治疗颞下颌关节骨关节炎中的作用

发布时间：2018-04-07 16:07

本文选题：颞下颌关节　切入点：骨关节炎　出处：《第四军医大学》2014年博士论文

【摘要】：骨关节炎（osteoarthritis，OA）是最常见的关节疾病，以关节软骨进行性的退变和最终的关节破坏为特征[1]。骨关节炎可造成患者疼痛和运动受限，给患者带来极大的压力。OA的病理机制尚不清楚，多种因素与OA的发生发展有关：创伤、遗传因素、关节位置异常、年龄、营养不良、生物力异常等。OA好发于膝关节、肘关节等负重关节，其中颞下颌关节（temporomandibular joint，TMJ）也是OA的好发部位之一。由于早期OA患者的标本难以获得，动物模型研究成为研究OA进展的有效手段。关节软骨的自我修复能力有限，而自我再生的多为纤维软骨，其力学性能较健康的透明软骨差。目前尚缺少能有效逆转或终止OA进展的药物，临床治疗策略仍以止痛、物理治疗、控制炎症反应、减轻病人痛苦、减缓疾病进程等为主。干细胞治疗是近些年备受关注的治疗方法。有关OA软骨的干细胞治疗的临床和动物实验研究主要是应用影像学、功能学、形态学等指标进行简单的疗效评价，尚未见有关于其疗效的系统研究和其机理研究的系统报道。进行干细胞治疗的关键决定因素是干细胞能被受损组织趋化从而迁移至靶组织。靶组织中表达的趋化因子在干细胞的迁移中起了关键作用。趋化因子基质细胞衍生因子-1（Stromal cell-derived factor-1，SDF-1）和RANTES是主要的介导干细胞迁移的分子，但关于SDF-1和RANTES在OA的干细胞治疗中趋化作用的研究还未见报导。本研究在建立了致病作用确切的咬合源性的颞下颌关节骨关节炎小鼠模型的基础上，系统全面地论证了外源性骨髓间充质干细胞（bone marrow-derivedmesenchymal stem cells，BMSCs）局部注射对颞下颌关节骨关节炎的治疗作用，并从干细胞存活、分化和趋化等方面探索其治疗作用的机制，发现外源性BMSCs局部注射对咬合源性的TMJ OA有积极的治疗效果，且该治疗效果的关键机制是病变髁突高表达趋化因子SDF-1和RANTES，诱导外源性BMSCs迁移并定植于病变髁突中。整个研究分为以下三个部分：第一部分：单侧前牙反牙合致小鼠颞下颌关节骨关节炎及其机制的实验研究背景：颞下颌关节紊乱病（Temporomandibular joint disorders，TMD）被认为有多种致病因素，其中咬合因素备受关注。目前，关于咬合是TMD的致病因素还存在很大争议。骨关节炎是TMD的严重的病理变化形式，以软骨退变和软骨下骨异常改建为主要特征。最近，Semaphorin4D（Sema4D）和Plexin-B1被发现可抑制成骨活动，但Sema4D/Plexin-B1在TMJ改建中的表达变化还未见报道。目的：本研究的目的是建立一个原创性的改变小鼠咬合关系的动物模型，并观察TMJ软骨和软骨下骨的组织学和相关因子的变化，以及Sema4D和Plexin-B1的表达变化。方法：在小鼠的左侧下颌切牙或上下颌切牙粘接不良修复体，造成左侧切牙的反牙合关系。用HE和甲苯胺蓝染色方法观察TMJ髁突的形态学改变。用组织化学和realtime-PCR手段检测TRAP阳性细胞和M-CSF表达水平，作为破骨活动的指标，成骨活动水平用骨钙素（osteocalcin，OCN）的表达水平来观察。用免疫组织化学和realtime-PCR检测Sema4D和Plexin-B1的表达变化。用双因素的方差分析来比较组间差异。结果：粘接不良修复体1周和3周后，组织学上观察到软骨的退变和软骨下骨丢失。破骨活动的指标（TRAP阳性细胞数和M-CSF表达水平）在软骨和骨中均增高。OCN在软骨中的表达在3周时升高，但在软骨下骨中在1周时升高，3周时降低。Sema4D在软骨和软骨下骨中的表达在1周时降低，在3周时升高，而Plexin-B1在软骨下骨3周时升高。结论：单侧前牙反牙合的咬合关系可造成颞下颌关节髁突的异常改建，此过程涉及到软骨退变、成骨和破骨活动的改变，以及Sema4D/Plexin-B1的参与。第二部分：外源性骨髓间充质干细胞对颞下颌关节骨关节炎的治疗作用背景：干细胞治疗是目前备受关注的治疗骨关节炎的新方法。骨髓间充质干细胞具有多向分化潜能，是治疗骨关节炎的理想种子细胞。尽管有临床和动物研究报道干细胞对骨关节炎有治疗效果，但目前尚没有资料系统全面地评价骨髓间充质干细胞局部注射对骨关节炎的治疗作用，及其相关分子的表达。目的：本研究拟系统全面地论证外源性骨髓间充质干细胞对颞下颌关节骨关节炎的治疗作用。方法：观察拆除不良修复体后，颞下颌关节髁突的变化，以确定髁突病变的咬合源性。在造模3周时，开始每周在关节局部注射外源性的GFP-BMSCs，用HE和micro-CT的方法分析软骨和软骨下骨的形态学变化，用番红O染色观察软骨基质的变化，用TRAP染色观察软骨下骨破骨活动的变化，用GFP免疫组化染色观察外源性GFP-BMSCs在髁突内的定植情况，用GFP和Col-II、Col-I及OCN的免疫荧光双标的方法观察GFP-BMSCs在髁突内的分化情况，用realtime-PCR检测注射后髁突内相关因子的表达。结果：拆除不良修复体可部分逆转髁突的病变，说明髁突的病变是咬合源性的。关节局部注射GFP-BMSCs可有效改善软骨的厚度，逆转软骨下骨的丢失，且软骨基质得到了恢复，破骨活动减少。注射的GFP-BMSCs可定植于病变髁突软骨中，并表达Col-II，但不表达Col-I和OCN。软骨内的降低的Col-II、aggrecan、Col-X和升高的Col-I、OCN、MMP13、TNF-和IL1β的表达被有效逆转，，纤维化指标Col-III和Tenascin-C的表达下降。结论：关节腔局部注射外源性BMSCs对咬合源性的颞下颌关节骨关节炎有积极的治疗作用，且该治疗作用是基于BMSCs可定植于髁突软骨，并向软骨细胞分化。第三部分：外源性骨髓间充质干细胞治疗颞下颌关节骨关节炎的机制研究背景：干细胞在组织局部的存活、分化、迁移和定植是进行干细胞治疗的基础，也是进行干细胞治疗的前提，但BMSCs对骨关节炎治疗作用的机制还不甚明了。目的：本研究拟从干细胞存活、分化、定植和趋化等方面探索外源性BMSCs治疗颞下颌关节骨关节炎的机制。方法：应用Transwell体外共培养的方法观察病变髁突对BMSCs的诱导分化和定植作用；应用生物发光技术观察BMSCs在体内的存活状态；用免疫组化和realtime-PCR检测病变髁突中SDF-1和CXCR4的表达水平，应用体外阻断CXCR4和CCR1信号通路的方法检测趋化信号SDF-1/CXCR4和RANTES/CCR1在病变髁突诱导BMSCs迁移中的作用，并用体内阻断CXCR4和CCR1的方法检测SDF-1/CXCR4和RANTES/CCR1在BMSCs治疗颞下颌关节骨关节炎中的关键作用。结果：Transwell共培养结果显示病变和正常髁突均可诱导BMSCs阳性表达Col-II，但病变髁突可诱导BMSCs定植于髁突软骨中。生物发光结果显示BMSCs在颞下颌关节局部的存活能力高峰在注射后第7天，且实验组的发光强度低于对照组，而软骨细胞的存活能力在实验组和对照组均较低。病变髁突高表达SDF-1和RANTES，病变髁突在体外诱导BMSCs迁移的能力可被CXCR4和CCR1抑制剂所阻断，且BMSCs在体内对病变髁突的治疗效果可被CXCR4和CCR1抑制剂所抑制。结论：趋化信号SDF-1/CXCR4和RANTES/CCR1是BMSCs治疗颞下颌关节骨关节炎的关键信号分子。
[Abstract]:osteoarthritis ( OA ) is the most common joint disease characterized by degeneration of articular cartilage and eventual joint destruction . Objective : To study the effects of exogenous bone marrow - derived factor - 1 ( SDF - 1 ) on bone - derived mesenchymal stem cells ( OA ) .

The first part : experimental study of osteoarthritis and its mechanism of unilateral anterior teeth reverse occlusion in mice

BACKGROUND : Templar joint disorders ( TMD ) are considered to be a variety of pathogenic factors , among which the bite factors are concerned . At present , there is a great deal of controversy about the pathogenesis of TMD . In recent years , Sema4D / Plexin - B1 has been found to inhibit bone formation , but the expression of Sema4D / Plexin - B1 in the reconstruction of the joint is not reported .

Objective : The aim of this study was to establish an original animal model to change the occlusal relationship between mouse and mouse , and to observe the changes of histological and related factors in the cartilage and subcostal bone , as well as the expression of Sema4D and Plexin - B1 .

Methods : To observe the morphological changes of condylar process in the left flank of mice . The morphological changes of the condylar process were observed by HE and methylene blue staining . The expression level of TRAP positive cells and M - CSF was detected by histochemical and immunohistochemical staining . The expression of Sema4D and Plexin - B1 was detected by immunohistochemistry and PCR - PCR . The differences of expression were compared by means of variance analysis of two factors .

Results : After 1 week and 3 weeks of repair of articular cartilage , the degeneration of cartilage and bone loss in cartilage were observed . The expression of TRAP positive cells and M - CSF was increased in cartilage and bone . The expression of OCN in cartilage increased at 3 weeks , but decreased at 3 weeks .

Conclusion : The occlusal relationship between unilateral anterior and posterior teeth may result in abnormal alteration of the condyle of the temporo - mandibular joint . This process involves the changes of cartilage degeneration , osteogenesis and bone - breaking activity , and the participation of Sema4D / Plexin - B1 .

The second part : the effect of exogenous bone marrow mesenchymal stem cells on the osteoarthritis of the temporo - mandibular joint

BACKGROUND : Stem cell therapy is a new method for the treatment of osteoarthritis . Bone marrow mesenchymal stem cells have multi - directional differentiation potential and are ideal seed cells for osteoarthritis . Although clinical and animal studies have reported that stem cells have therapeutic effects on osteoarthritis , there is no data system to comprehensively evaluate the therapeutic effects of bone marrow mesenchymal stem cells on osteoarthritis and the expression of related molecules .

Objective : To systematically demonstrate the therapeutic effect of exogenous bone marrow mesenchymal stem cells ( MSCs ) on osteoarthritis .

Methods : The changes of condylar process were observed in the condylar process of articular cartilage . After 3 weeks of molding , the changes of cartilage matrix were observed by means of HE and micro - CT , and the changes of bone breaking activity were observed with HE and micro - CT .

Results : The lesions of condylar process can be partially reversed by the removal of defective repair bodies . The local injection of GFP - MSCs can effectively improve the thickness of cartilage , reverse the loss of bone in cartilage , and decrease the activity of cartilage . The injected GFP - BMSC can be implanted in the condylar cartilage of the lesion , and the expression of Col - II , aggrecan , TNF - and IL1尾 in cartilage is reversed . The expression of Col - III and Tenascin - C decreases .

Conclusion : The local injection of exogenous bone marrow into the articular cavity has a positive therapeutic effect on the intercondylar osteoarthritis , and the therapeutic effect is based on the implantation of bone marrow into the condylar cartilage and the differentiation of the chondrocytes into the chondrocytes .

The third part : the mechanism of exogenous bone marrow mesenchymal stem cells in the treatment of osteoarthritis

Background : The survival , differentiation , migration and colonization of stem cells in tissue are the basis for the treatment of stem cells , as well as the premise of stem cell therapy , but the mechanism for the treatment of osteoarthritis is not very clear .

Objective : To explore the mechanism of exogenous bone marrow stromal cells ( MSCs ) in the treatment of osteoarthritis in vitro from the aspects of stem cell survival , differentiation , colonization and chemotropism .

Methods : Transwell in vitro co - culture method was used to observe the effect of condylar process on the differentiation and colonization of bone marrow cells .
To observe the survival status of bone marrow cells in vivo by means of biotechnology .
The expression level of SDF - 1 / CXCR4 and CXCR4 in the condylar process of the lesion was detected by immunohistochemistry and PCR - PCR . The role of SDF - 1 / CXCR4 in vitro and CCR1 signaling pathway were used to detect the role of SDF - 1 / CXCR4 and IL - 1 / CCR1 in the migration of the lesion condylar process , and the key role of SDF - 1 / CXCR4 and IL - 1 / CCR1 in the treatment of osteoarthritis of the mandibular joint was detected by means of blocking CXCR4 and CCR1 in vivo .

Results : The results showed that both the lesion and the normal condylar process could induce the positive expression of Col - II , but the condylar process of the lesion could induce the bone to be implanted in the condylar cartilage . The results showed that the survival ability of the rabbits was lower in the experimental group and the control group .

Conclusion : Chemotactic signal SDF - 1 / CXCR4 and IL - 1 / CCR1 are the key signal molecules in the treatment of osteoarthritis of the temporo - mandibular joint .

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R782.63

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