FGFR3在关节软骨稳态维持中的作用与机制研究

发布时间：2018-06-15 02:50

本文选题：成纤维细胞生长因子受体3 + 成纤维细胞生长因子受体3　；参考：《第三军医大学》2017年博士论文

【摘要】：骨性关节炎(Osteoarthritis,OA)是发生在关节中最常见的一种慢性退行性疾病。骨性关节炎会多发生在负重关节并且造成负重软骨的软骨缺损,最终可能导致关节疼痛以及关节活动受限的临床症状,病情发展到最后常常导致患者失去行走能力。目前临床上能阻止或缓解骨性关节炎的措施非常有限。因此,理解骨性关节炎发生过程中的关节软骨的稳态维持的机制,对研究治疗或者延缓骨性关节炎的发生和发展的新型防治措施有重要的意义。成纤维生长因子(Fibroblast growth factor,FGF)信号在骨形成和发育过程中的作用非常关键。成纤维生长因子(FGFs)家族由22种配体和4种受体组成。成纤维生长因子(FGFs)通过结合成纤维生长因子受体(FGFRs)来发挥作用。FGFRs在骨形成过程中有特异性的时空表达模式。成纤维生长因子受体3(FGFR3)是在软骨发育与软骨稳态维持中都发挥重要作用的一种跨膜酪氨酸激酶受体蛋白。FGFR3在骨发育早期的时候先在间充质凝集中心的软骨细胞中表达,接着在生长板软骨和关节软骨中的增殖带和肥大前带的软骨细胞中表达。人的FGFR3的基因突变会导致一系列的骨骼畸形。FGFR3的增强型点突变导致以身材矮小为临床表现的骨骼发育不良,包括致死性软骨发育不良(thanatophoric dysplasia,TD I/II),软骨发育不全(Achondroplasia,ACH)等;而人的FGFR3的失活性点突变会导致CATSHL综合征,即听力丧失、高身材和屈曲指。FGFR3被认为是软骨内成骨的发育过程中的负性调节分子,但FGFR3信号在关节软骨中的直接作用还需要进一步研究。FGFR3在成年人的关节软骨中有丰富的表达,在OA病人的关节软骨中表达下降。全身敲除FGFR3的小鼠自发发生骨性关节炎。并且,ACH病人虽然有严重的内弯腿以及肥胖等引发OA的高危因素,但是却很少出现OA症状。这些发现提示了FGFR3有延缓OA发生和发展的作用。由于ACH病人和FGFR3全敲除小鼠都有骨骼结构上的变化,因此FGFR3对维持关节软骨稳态的直接作用,还有待研究。FGF2在动物模型中可调节关节软骨的代谢,并且可在OA动物模型中延缓OA进程。但是,FGF2是否在人的关节软骨中起保护作用仍有争议。FGF18被认为在关节软骨的降解过程中起保护软骨的作用。FGF2和FGF18在OA进程中的不同作用的具体机制仍未阐述清楚。旁分泌性的FGFs,比如FGF2和FGF18,是通过结合FGFRs后激活包括MAPK,PKC-γ,PI-3K/AKT和STAT等下游信号通路,来起到其生物学作用的。不同的FGFRs在维持软骨稳态中起不同的生物学作用。FGFR1信号促进分解因子的表达,比如matrix metalloproteinase(MMP)13和a disintegrin and metalloproteinase with thrombospoindin motifs(ADAMTS)5,并抑制软骨基质生成。而激活FGFR3信号则抑制软骨肥大化和促进软骨基质合成。FGFR3在骨性关节炎病人的关节软骨中其表达下调。并且类似的在小鼠关节软骨中的结果也报道过。此外,不管是在生长板或是关节软骨,FGFR3信号被认为是调节软骨肥大化和软骨特异性的细胞外基质的生成,并且Fgfr3缺失导致小鼠关节出现早期关节炎的症状。因此,在软骨中激活FGFR3信号可能是治疗骨性关节炎的潜在治疗手段。和FGF18相似,FGF9是FGFR3的相对特异性配体,能在软骨中激活FGFR3信号,提示FGF9在关节软骨的稳态维持中发挥作用。在骨发育过程中,FGF9最先在顶端外胚层嵴表达,接着在骨膜或软骨膜、松质骨和关节中表达,但在生长板软骨几乎不表达。骨性关节炎目前治疗措施仅仅局限在解决疼痛和抗炎治疗等对症方面,最后只能控制表面症状而难以缓解OA的进展。为了寻找有效的缓解OA进程的生物因子,我们把视线放到FGFR3的相对特异性配体FGF9上。但FGF9对关节软骨损伤修复的影响和作用尚不清楚。我们构建了FGFR3软骨特异性诱导敲除和增强小鼠的DMM手术诱导的骨性关节炎模型,来研究FGFR3在关节软骨中稳态维持的作用和机制。接着,为了进一步寻找治疗骨性关节炎的生物因子,我们通过观察关节腔内注射FGF9到DMM手术诱导骨性关节炎小鼠模型中,来研究FGF9是否可能成为靶向调节FGFR3的骨性关节炎的新型治疗因子。研究方法第一部分:1.建立成年期可诱导条件性敲除或增强fgfr3小鼠,即在1月或2月龄的同窝Fgfr3f/f;Col2a1-Cre ERT2(Fgfr3 c KO)小鼠和Fgfr3f/f(Cre-negative)小鼠,或1月或2月龄的同窝Fgfr3K644E/neo;Col2a1-Cre ERT2(Fgfr3 c Act)小鼠和Fgfr3K644E/neo(Cre-negative)小鼠,同时腹腔内注射1mg/10g/天Tamoxifen(TM)腹腔内注射5天。对小鼠行DMM(Destabilization of the Medial Meniscus)手术。2.通过藏红-固绿染色法观测小鼠膝关节组织学结构,并且使用OARSI(Osteoarthritis research Society International)推荐的评测方法对关节软骨进行评分;通过免疫组织化学染色法观测膝关节软骨组织的FGFR3、Collagen II、Collagen X、MMP13、AggrecanNeo的表达情况。3.建立老年期Fgfr3ACH/+小鼠自发OA模型,并在6月龄、12月龄、20月龄分别取材,通过藏红-固绿染色法观测小鼠膝关节组织学结构,通过免疫组化染色法观察小鼠膝关节软骨组织中Collagen X和MMP13的表达情况。4.取Fgfr3f/f;Col2a1-CreERT2(Fgfr3 c KO)和Fgfr3f/f(Cre-negative)小鼠的股骨头的关节软骨进行体外组织块培养,然后用4-hydroxytamoxifen处理,接着用IHH信号阻断剂(GDC0449,SMOi)处理,并提取RNA然后反转录,利用实时荧光定量PCR检测Ihh、Gli、Runx2、Mmp13、Adamts5、Col10a1、Col2a1和Aggrecan的表达。第二部分:1.为了研究FGF9在体内对关节软骨的作用,我们在小鼠DMM关节炎模型中注射FGF9。先对小鼠行DMM手术,接着在DMM手术后2周注射FGF9。先把FGF9溶液(2.5μg粉剂溶解在5μl生理盐水中)与纤维蛋白原溶液混合(3μg/μl)后注射在关节腔中,接着注射1μl凝血酶(0.2 unit)使纤维蛋白原转变为纤维蛋白,这样就可以使FGF9缓慢释放到关节腔内。在手术后第8周和12周时处死小鼠,取小鼠膝关节进行组织切片观察;2.建立人关节软骨体外培养模型,研究了外源性FGF9的处理对白介素-1β(interleukin-1β,IL-1β)诱导的人关节软骨退变的作用,并且通过藏红固绿染色法观察人关节软骨的组织学结构,接着再通过免疫组化染色法观察人关节软骨的collagen II,collagen X和MMP13的表达情况;通过WB检测人关节软骨aggrecan和MMP13的蛋白表达变化;4.通过Micro-CT扫描重建,观察FGF9关节腔内注射后小鼠膝关节软骨下骨的变化情况,通过藏红固绿染色法以及阿尔新蓝/苏木精/伊红染色法观察小鼠的膝关节中组织学结构变化。5.通过免疫组织化学染色法观察FGF9注射后小鼠膝关节软骨中的collagen II,collagen X,MMP13以及Cleaved caspase-3的表达情况,以及在骨赘形成处Collagen II,PCNA和SOX9的表达情况。实验结果:第一部分:FGFR3抑制成年小鼠膝关节骨性关节炎的发展1.成年小鼠软骨特异性敲除Fgfr3后关节软骨降解过程加速。2.成年小鼠软骨特异性敲除Fgfr3后Collagen II表达降低,Collagen X、MMP13和AggrecanNeo表达升高。3.成年小鼠软骨特异性增强Fgfr3后关节软骨降解过程减缓。4.Fgfr3ACH/+小鼠其老年自发OA的进程减缓。5.成年小鼠软骨特异性增强Fgfr3后Collagen X、MMP13表达降低,老年Fgfr3ACH/+小鼠Collagen X、MMP13表达降低。6.体外关节软骨组织中敲除Fgfr3后,IHH信号和Runx2表达升高,而阻断IHH信号会抑制已上调表达的Runx2、Mmp13的表达。第二部分:外源性FGF9缓解关节软骨损伤后引起的骨性关节炎1.关节内注射FGF9延缓小鼠骨关节炎模型中关节软骨降解。DMM手术加关节腔内FGF9治疗组的股骨和胫骨的评分其最高分和总分都显著低于DMM手术加空白盐水的治疗组;而DMM诱导并注射FGF9后,MMP13和Collagen X的表达相比对照降低。2.FGF9对炎症环境中的人关节软骨退变有保护作用,外源性FGF9处理后能明显导致IL-1β处理后的人关节软骨细胞其MMP13的表达降低、aggrecan的表达增加。3.关节腔内注射FGF9促进DMM手术后的关节周围骨赘的形成。FGF9处理后能显著增加DMM手术诱导的关节附近形成的骨赘的大小;在小鼠8周及12周时注射FGF9能明显提高DMM诱导术后的关节周围骨赘其大小。4.FGF9促进关节周围骨赘形成过程中的细胞增殖和软骨细胞的分化。在DMM手术后的小鼠关节中,我们发现SOX9在关节周围的骨赘处有大量表达,并且FGF9治疗后会明显提高SOX9其表达水平;关节腔注射FGF9会明显上调关节骨赘处的细胞PCNA的表达。结论:一、FGFR3抑制成年小鼠膝关节骨性关节炎的发展;二、关节腔注射FGF9缓解小鼠关节软骨损伤后引起的骨性关节炎,同时可促进骨性关节炎中骨赘的形成。
[Abstract]:Osteoarthritis (OA) is the most common chronic degenerative disease occurring in the joints. Osteoarthritis occurs mostly in the heavy joint and causes the cartilage defect of the heavy cartilage. It may eventually lead to joint pain and the clinical symptoms of limited joint activity, and the development of the disease often leads to the loss of walking in the patient. At present, the measures to prevent or alleviate osteoarthritis are very limited. Therefore, understanding the mechanism of the steady state of the articular cartilage in the process of osteoarthritis is of great significance for the study of the new prevention and treatment of the occurrence and development of osteoarthritis. Fibroblast growth factor (Fibroblast growth) Factor, FGF) signal plays a key role in bone formation and development. The fibroblast growth factor (FGFs) family consists of 22 ligands and 4 receptors. Fibroblast growth factor (FGFs) plays a role in the specific spatio-temporal expression of.FGFRs in the process of bone formation by combining fibroblast growth factor receptor (FGFRs). Long factor receptor 3 (FGFR3) is a transmembrane tyrosine kinase receptor protein.FGFR3, a transmembrane tyrosine kinase receptor protein, which plays an important role in the development of cartilage development and cartilage homeostasis. It is expressed in the chondrocytes of the mesenchymal agglutination center at the early stage of bone development, followed by the proliferating zone in the growth plate cartilage and joint soft bone and the cartilaginous cells in the front of the hypertrophy. Gene mutations in human FGFR3 can lead to a series of enhanced point mutations in the skeletal deformity of.FGFR3, resulting in skeletal dysplasia in short stature, including fatal cartilage dysplasia (thanatophoric dysplasia, TD I/II), cartilage dysplasia (Achondroplasia, ACH), and other mutations in the human FGFR3 inactive point. CATSHL syndrome, such as hearing loss, high stature and flexion finger.FGFR3, is considered as a negative regulator in the development of endochondral osteogenesis, but the direct role of FGFR3 signals in articular cartilage needs further study of the rich expression of.FGFR3 in the articular cartilage of adults and the expression of the cartilage in the articular cartilage of the OA patients. Reduced FGFR3 mice spontaneously osteoarthritis. And, while ACH patients have severe internal kits and obesity and other high risk factors for OA, there are few OA symptoms. These findings suggest that FGFR3 has the effect of retarding the occurrence and development of OA. Both ACH patients and FGFR3 full knockout mice have skeletal structural changes. Therefore, the direct effect of FGFR3 on the maintenance of articular cartilage homeostasis, it remains to be studied that.FGF2 can regulate the metabolism of articular cartilage in animal models, and can delay the OA process in the OA animal model. However, it is still controversial whether FGF2 plays a protective role in human articular cartilage and.FGF18 is considered to protect the cartilage during the degradation of articular cartilage. The specific mechanisms of the effects of bone on.FGF2 and FGF18 in the OA process are still not clear. Paracrine FGFs, such as FGF2 and FGF18, plays a biological role by activating downstream signaling pathways such as MAPK, PKC- gamma, PI-3K/AKT and STAT by combining FGFRs. Different FGFRs plays different organisms in the maintenance of cartilage homeostasis. .FGFR1 signals promote the expression of decomposition factors, such as matrix metalloproteinase (MMP) 13 and a disintegrin and metalloproteinase with thrombospoindin motifs (ADAMTS) 5, and inhibit the formation of cartilage matrix. The expression in the cartilaginous cartilage is down. And similar results have been reported in the articular cartilage of the mice. In addition, the FGFR3 signal is considered to be the formation of the extracellular matrix that regulates cartilage hypertrophy and cartilage specificity, regardless of the growth plate or articular cartilage, and the absence of Fgfr3 leads to the symptoms of early arthritis in the joints of mice. Therefore, Activation of FGFR3 signal in cartilage may be a potential treatment for osteoarthritis. Similar to FGF18, FGF9 is a relative specific ligand for FGFR3, which activates FGFR3 signals in cartilage, suggesting that FGF9 plays a role in the steady state maintenance of articular cartilage. During bone development, FGF9 is first expressed in the apical ectodermal ridge and then in the periosteum. Or cartilaginous membrane, expressed in cancellous bone and joints, but almost unexpressed in the growth plate cartilage. Osteoarthritis is currently only limited to the treatment of pain and anti-inflammatory treatment. Finally, it can only control surface symptoms and difficult to alleviate the progress of OA. In order to find effective biological factors to alleviate the OA process, we put our eyes to F GFR3's relative specific ligand FGF9. But the effect and effect of FGF9 on the repair of articular cartilage injury is not clear. We constructed a FGFR3 cartilage specific induced knockout and enhanced DMM operation induced osteoarthritis model in mice to study the role and mechanism of FGFR3 in the homeostasis of articular cartilage. In the treatment of osteoarthritis, we observe whether FGF9 may be a new therapeutic factor for targeting osteoarthritis of FGFR3 by observing the intravaric injection of FGF9 to DMM to induce osteoarthritis in mice. Part 1.: the first part of the study is to induce conditional knockout or increase FGFR3 in the adult period. Mice, that is, in January or 2 month old of the same nest Fgfr3f/f; Col2a1-Cre ERT2 (Fgfr3 C KO) mice and Fgfr3f/f (Cre-negative) mice, or January or 2 month old of the same fossa Fgfr3K644E/neo; Col2a1-Cre ERT2 (Fgfr3) mice and mice, and intraperitoneal injection of peritoneal injection for 5 days. M (Destabilization of the Medial Meniscus) operation.2. was used to observe the histological structure of the knee joint by the method of red solid green staining, and the articular cartilage was scored by the evaluation method recommended by OARSI (Osteoarthritis research Society International), and the cartilage tissue of the knee was observed by immunohistochemical staining. The expression of llagen II, Collagen X, MMP13, AggrecanNeo.3. was used to establish the spontaneous OA model of senile Fgfr3ACH/+ mice, and were obtained in 6 month old, 12 month old, 20 month old respectively. The histological structure of the knee joint was observed by the method of Tibetan red solid green staining, and the Collagen X and MMP13 table in the cartilage tissue of the knee of mice was observed by immunohistochemical staining. The articular cartilage of the femoral head of Col2a1-CreERT2 (Fgfr3 C KO) and Fgfr3f/f (Cre-negative) mice was cultured in vitro, and then treated with 4-hydroxytamoxifen, then the IHH signal blocker (GDC0449, SMOi) was then treated with the IHH signal blocking agent. The expression of Adamts5, Col10a1, Col2a1 and Aggrecan. Second: 1. in order to study the role of FGF9 in the articular cartilage in the body, we injected FGF9. in the mouse DMM arthritis model before DMM surgery, and then injected FGF9 solution (2.5 micron g powder in 5 mu saline) and fibrinogen solution at 2 weeks after DMM operation. The mixture (3 g/ Mu L) was injected into the articular cavity, then 1 l thrombin (0.2 unit) was injected into the fibrinogen to transform the fibrinogen into the fibrin, so that the FGF9 could be slowly released into the articular cavity. The mice were killed at eighth and 12 weeks after the operation, and the knee joints were taken to observe the tissue section. 2. to establish the culture model of human articular cartilage in vitro. The effect of exogenous FGF9 on the degeneration of human articular cartilage induced by interleukin -1 beta (interleukin-1 beta, IL-1 beta) was investigated, and the histological structure of human articular cartilage was observed by the method of Tibetan red solid green staining, and then the expression of collagen II, collagen X and MMP13 were observed by immunohistochemical staining; WB examination was carried out by WB. The changes in the protein expression of aggrecan and MMP13 in human articular cartilage were measured. 4. the changes of the subchondral bone in the knee joint of the mice after the intra-articular injection of FGF9 were observed by Micro-CT scanning. The histological structural changes in the knee joints of the mice were observed by the Tibetan red green staining and alino blue / hematoxylin / eosin staining and.5. through the immune tissue. The expression of collagen II, collagen X, MMP13 and Cleaved caspase-3 in the knee cartilage of mice after FGF9 injection, and the expression of Collagen II, PCNA and SOX9 in the formation of osteophyte. Experimental results: the first part: inhibiting the development of osteoarthritis of the knee joint in adult mice, the cartilage specific of 1. adult mice The degradation process of articular cartilage after knockout of Fgfr3 accelerates the decrease of Collagen II expression, Collagen X, Collagen X, MMP13 and AggrecanNeo expression in adulthood mice, Collagen X, MMP13 and AggrecanNeo expression increase in.3. adult mice. The cartilage degradation in.3. adult mice slows down the process of cartilage degradation in.4.Fgfr3ACH/+ mice. Cartilage specificity enhanced Fgfr3 Collagen X, MMP13 expression decreased, Collagen X in the elderly Fgfr3ACH/+ mice, MMP13 expression reduced.6. in articular cartilage tissue after knockout Fgfr3, IHH signal and Runx2 expression increased, but blocking the signal will inhibit the expression of the expression. The second part: exogenous cartilage injury relieves articular cartilage injury The 1. intra-articular injection of FGF9 in the posterior articular osteoarthritis delayed the osteoarthritis model of mice and the articular cartilage degradation.DMM operation and the femoral and tibia score of the FGF9 group in the joint intracavitary group were significantly lower than those in the treatment group with DMM and blank saline; while DMM induced and injected FGF9, the expression of MMP13 and Collagen X was compared. The reduction of.2.FGF9 has a protective effect on the degeneration of articular cartilage in the inflammatory environment. Exogenous FGF9 can obviously lead to the decrease of the expression of MMP13 in human articular chondrocytes after IL-1 beta treatment. The expression of aggrecan is increased by.3. in the intra-articular injection of FGF9 to promote the formation of osteophyte around the joint after DMM surgery. The size of osteophyte in the vicinity of the joint induced by DMM operation; the injection of FGF9 at 8 and 12 weeks in mice can significantly increase the size of the osteophyte around the joint after DMM induction and promote the proliferation of cells and the differentiation of chondrocytes during the formation of the osteophyte around the joint. In the joints of the mice after DMM, we found that SOX9 is in the joint. There is a large amount of expression in the surrounding osteophyte, and the expression level of SOX9 can be significantly increased after FGF9 treatment; the injection of FGF9 in the articular cavity can obviously increase the expression of PCNA in the joint osteophyte. Conclusion: 1, FGFR3 inhibits the development of osteoarthritis of the knee joint in adult mice; two, the joint cavity injection of FGF9 alleviates the bone caused by the injury of articular cartilage in mice. Arthritis can also promote osteophyte formation in osteoarthritis.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R684.3

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