本文選題：顳下頜關(guān)節(jié)　切入點(diǎn)：骨關(guān)節(jié)炎　出處：《第四軍醫(yī)大學(xué)》2014年博士論文

【摘要】：骨關(guān)節(jié)炎（osteoarthritis，OA）是最常見(jiàn)的關(guān)節(jié)疾病，以關(guān)節(jié)軟骨進(jìn)行性的退變和最終的關(guān)節(jié)破壞為特征[1]。骨關(guān)節(jié)炎可造成患者疼痛和運(yùn)動(dòng)受限，給患者帶來(lái)極大的壓力。OA的病理機(jī)制尚不清楚，多種因素與OA的發(fā)生發(fā)展有關(guān)：創(chuàng)傷、遺傳因素、關(guān)節(jié)位置異常、年齡、營(yíng)養(yǎng)不良、生物力異常等。OA好發(fā)于膝關(guān)節(jié)、肘關(guān)節(jié)等負(fù)重關(guān)節(jié)，其中顳下頜關(guān)節(jié)（temporomandibular joint，TMJ）也是OA的好發(fā)部位之一。由于早期OA患者的標(biāo)本難以獲得，動(dòng)物模型研究成為研究OA進(jìn)展的有效手段。關(guān)節(jié)軟骨的自我修復(fù)能力有限，而自我再生的多為纖維軟骨，其力學(xué)性能較健康的透明軟骨差。目前尚缺少能有效逆轉(zhuǎn)或終止OA進(jìn)展的藥物，臨床治療策略仍以止痛、物理治療、控制炎癥反應(yīng)、減輕病人痛苦、減緩疾病進(jìn)程等為主。干細(xì)胞治療是近些年備受關(guān)注的治療方法。有關(guān)OA軟骨的干細(xì)胞治療的臨床和動(dòng)物實(shí)驗(yàn)研究主要是應(yīng)用影像學(xué)、功能學(xué)、形態(tài)學(xué)等指標(biāo)進(jìn)行簡(jiǎn)單的療效評(píng)價(jià)，尚未見(jiàn)有關(guān)于其療效的系統(tǒng)研究和其機(jī)理研究的系統(tǒng)報(bào)道。進(jìn)行干細(xì)胞治療的關(guān)鍵決定因素是干細(xì)胞能被受損組織趨化從而遷移至靶組織。靶組織中表達(dá)的趨化因子在干細(xì)胞的遷移中起了關(guān)鍵作用。趨化因子基質(zhì)細(xì)胞衍生因子-1（Stromal cell-derived factor-1，SDF-1）和RANTES是主要的介導(dǎo)干細(xì)胞遷移的分子，但關(guān)于SDF-1和RANTES在OA的干細(xì)胞治療中趨化作用的研究還未見(jiàn)報(bào)導(dǎo)。本研究在建立了致病作用確切的咬合源性的顳下頜關(guān)節(jié)骨關(guān)節(jié)炎小鼠模型的基礎(chǔ)上，系統(tǒng)全面地論證了外源性骨髓間充質(zhì)干細(xì)胞（bone marrow-derivedmesenchymal stem cells，BMSCs）局部注射對(duì)顳下頜關(guān)節(jié)骨關(guān)節(jié)炎的治療作用，并從干細(xì)胞存活、分化和趨化等方面探索其治療作用的機(jī)制，發(fā)現(xiàn)外源性BMSCs局部注射對(duì)咬合源性的TMJ OA有積極的治療效果，且該治療效果的關(guān)鍵機(jī)制是病變髁突高表達(dá)趨化因子SDF-1和RANTES，誘導(dǎo)外源性BMSCs遷移并定植于病變髁突中。整個(gè)研究分為以下三個(gè)部分： 第一部分：?jiǎn)蝹?cè)前牙反牙合致小鼠顳下頜關(guān)節(jié)骨關(guān)節(jié)炎及其機(jī)制的實(shí)驗(yàn)研究 背景：顳下頜關(guān)節(jié)紊亂�。═emporomandibular joint disorders，TMD）被認(rèn)為有多種致病因素，其中咬合因素備受關(guān)注。目前，關(guān)于咬合是TMD的致病因素還存在很大爭(zhēng)議。骨關(guān)節(jié)炎是TMD的嚴(yán)重的病理變化形式，以軟骨退變和軟骨下骨異常改建為主要特征。最近，Semaphorin4D（Sema4D）和Plexin-B1被發(fā)現(xiàn)可抑制成骨活動(dòng)，但Sema4D/Plexin-B1在TMJ改建中的表達(dá)變化還未見(jiàn)報(bào)道。 目的：本研究的目的是建立一個(gè)原創(chuàng)性的改變小鼠咬合關(guān)系的動(dòng)物模型，并觀(guān)察TMJ軟骨和軟骨下骨的組織學(xué)和相關(guān)因子的變化，以及Sema4D和Plexin-B1的表達(dá)變化。 方法：在小鼠的左側(cè)下頜切牙或上下頜切牙粘接不良修復(fù)體，造成左側(cè)切牙的反牙合關(guān)系。用HE和甲苯胺藍(lán)染色方法觀(guān)察TMJ髁突的形態(tài)學(xué)改變。用組織化學(xué)和realtime-PCR手段檢測(cè)TRAP陽(yáng)性細(xì)胞和M-CSF表達(dá)水平，作為破骨活動(dòng)的指標(biāo)，成骨活動(dòng)水平用骨鈣素（osteocalcin，OCN）的表達(dá)水平來(lái)觀(guān)察。用免疫組織化學(xué)和realtime-PCR檢測(cè)Sema4D和Plexin-B1的表達(dá)變化。用雙因素的方差分析來(lái)比較組間差異。 結(jié)果：粘接不良修復(fù)體1周和3周后，組織學(xué)上觀(guān)察到軟骨的退變和軟骨下骨丟失。破骨活動(dòng)的指標(biāo)（TRAP陽(yáng)性細(xì)胞數(shù)和M-CSF表達(dá)水平）在軟骨和骨中均增高。OCN在軟骨中的表達(dá)在3周時(shí)升高，但在軟骨下骨中在1周時(shí)升高，3周時(shí)降低。Sema4D在軟骨和軟骨下骨中的表達(dá)在1周時(shí)降低，在3周時(shí)升高，而Plexin-B1在軟骨下骨3周時(shí)升高。 結(jié)論：?jiǎn)蝹?cè)前牙反牙合的咬合關(guān)系可造成顳下頜關(guān)節(jié)髁突的異常改建，此過(guò)程涉及到軟骨退變、成骨和破骨活動(dòng)的改變，以及Sema4D/Plexin-B1的參與。 第二部分：外源性骨髓間充質(zhì)干細(xì)胞對(duì)顳下頜關(guān)節(jié)骨關(guān)節(jié)炎的治療作用 背景：干細(xì)胞治療是目前備受關(guān)注的治療骨關(guān)節(jié)炎的新方法。骨髓間充質(zhì)干細(xì)胞具有多向分化潛能，是治療骨關(guān)節(jié)炎的理想種子細(xì)胞。盡管有臨床和動(dòng)物研究報(bào)道干細(xì)胞對(duì)骨關(guān)節(jié)炎有治療效果，但目前尚沒(méi)有資料系統(tǒng)全面地評(píng)價(jià)骨髓間充質(zhì)干細(xì)胞局部注射對(duì)骨關(guān)節(jié)炎的治療作用，及其相關(guān)分子的表達(dá)。 目的：本研究擬系統(tǒng)全面地論證外源性骨髓間充質(zhì)干細(xì)胞對(duì)顳下頜關(guān)節(jié)骨關(guān)節(jié)炎的治療作用。 方法：觀(guān)察拆除不良修復(fù)體后，顳下頜關(guān)節(jié)髁突的變化，以確定髁突病變的咬合源性。在造模3周時(shí)，開(kāi)始每周在關(guān)節(jié)局部注射外源性的GFP-BMSCs，用HE和micro-CT的方法分析軟骨和軟骨下骨的形態(tài)學(xué)變化，用番紅O染色觀(guān)察軟骨基質(zhì)的變化，用TRAP染色觀(guān)察軟骨下骨破骨活動(dòng)的變化，用GFP免疫組化染色觀(guān)察外源性GFP-BMSCs在髁突內(nèi)的定植情況，用GFP和Col-II、Col-I及OCN的免疫熒光雙標(biāo)的方法觀(guān)察GFP-BMSCs在髁突內(nèi)的分化情況，用realtime-PCR檢測(cè)注射后髁突內(nèi)相關(guān)因子的表達(dá)。 結(jié)果：拆除不良修復(fù)體可部分逆轉(zhuǎn)髁突的病變，說(shuō)明髁突的病變是咬合源性的。關(guān)節(jié)局部注射GFP-BMSCs可有效改善軟骨的厚度，逆轉(zhuǎn)軟骨下骨的丟失，且軟骨基質(zhì)得到了恢復(fù)，破骨活動(dòng)減少。注射的GFP-BMSCs可定植于病變髁突軟骨中，并表達(dá)Col-II，但不表達(dá)Col-I和OCN。軟骨內(nèi)的降低的Col-II、aggrecan、Col-X和升高的Col-I、OCN、MMP13、TNF-和IL1β的表達(dá)被有效逆轉(zhuǎn)，，纖維化指標(biāo)Col-III和Tenascin-C的表達(dá)下降。 結(jié)論：關(guān)節(jié)腔局部注射外源性BMSCs對(duì)咬合源性的顳下頜關(guān)節(jié)骨關(guān)節(jié)炎有積極的治療作用，且該治療作用是基于BMSCs可定植于髁突軟骨，并向軟骨細(xì)胞分化。 第三部分：外源性骨髓間充質(zhì)干細(xì)胞治療顳下頜關(guān)節(jié)骨關(guān)節(jié)炎的機(jī)制研究 背景：干細(xì)胞在組織局部的存活、分化、遷移和定植是進(jìn)行干細(xì)胞治療的基礎(chǔ)，也是進(jìn)行干細(xì)胞治療的前提，但BMSCs對(duì)骨關(guān)節(jié)炎治療作用的機(jī)制還不甚明了。 目的：本研究擬從干細(xì)胞存活、分化、定植和趨化等方面探索外源性BMSCs治療顳下頜關(guān)節(jié)骨關(guān)節(jié)炎的機(jī)制。 方法：應(yīng)用Transwell體外共培養(yǎng)的方法觀(guān)察病變髁突對(duì)BMSCs的誘導(dǎo)分化和定植作用；應(yīng)用生物發(fā)光技術(shù)觀(guān)察BMSCs在體內(nèi)的存活狀態(tài)；用免疫組化和realtime-PCR檢測(cè)病變髁突中SDF-1和CXCR4的表達(dá)水平，應(yīng)用體外阻斷CXCR4和CCR1信號(hào)通路的方法檢測(cè)趨化信號(hào)SDF-1/CXCR4和RANTES/CCR1在病變髁突誘導(dǎo)BMSCs遷移中的作用，并用體內(nèi)阻斷CXCR4和CCR1的方法檢測(cè)SDF-1/CXCR4和RANTES/CCR1在BMSCs治療顳下頜關(guān)節(jié)骨關(guān)節(jié)炎中的關(guān)鍵作用。 結(jié)果：Transwell共培養(yǎng)結(jié)果顯示病變和正常髁突均可誘導(dǎo)BMSCs陽(yáng)性表達(dá)Col-II，但病變髁突可誘導(dǎo)BMSCs定植于髁突軟骨中。生物發(fā)光結(jié)果顯示BMSCs在顳下頜關(guān)節(jié)局部的存活能力高峰在注射后第7天，且實(shí)驗(yàn)組的發(fā)光強(qiáng)度低于對(duì)照組，而軟骨細(xì)胞的存活能力在實(shí)驗(yàn)組和對(duì)照組均較低。病變髁突高表達(dá)SDF-1和RANTES，病變髁突在體外誘導(dǎo)BMSCs遷移的能力可被CXCR4和CCR1抑制劑所阻斷，且BMSCs在體內(nèi)對(duì)病變髁突的治療效果可被CXCR4和CCR1抑制劑所抑制。 結(jié)論：趨化信號(hào)SDF-1/CXCR4和RANTES/CCR1是BMSCs治療顳下頜關(guān)節(jié)骨關(guān)節(jié)炎的關(guān)鍵信號(hào)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 .

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R782.63

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相關(guān)期刊論文 前2條

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本文編號(hào)：1719891