【作者】 金旻；

【导师】 陈林；

【作者基本信息】 第三军医大学 ， 人体解剖与组织胚胎学， 2009， 博士

【摘要】 软骨发育不全(Achondroplasia, ACH)是人类侏儒最常见的类型,主要影响四肢骨、椎骨等长骨的软骨内成骨过程,尤其是软骨形成过程,包括间充质细胞集聚并分化为软骨细胞,以及随后软骨细胞的增生、肥大和凋亡,但其机制目前尚不明确。成纤维细胞生长因子受体(Fibroblast growth factor receptors, FGFRs)在骨骼发育和疾病发生中发挥重要作用。FGFR属于受体酪氨酸蛋白激酶家族,目前已经发现有4种FGFR(FGFR1-4),它们在氨基酸水平有55%-72%的同源性。其中FGFR3的十多种功能增强型点突变可引起多种人类软骨发育障碍性侏儒包括软骨发育不全(ACH)、季肋发育不全(Hypochondroplasia, HCH)、致死性发育不全(Thanatophoric dysplasia, TD)等。FGFR3在长骨生长板的静息期、增殖期及前肥大软骨细胞中表达。目前利用基因敲入及转基因技术已建立了多种模拟人软骨发育不全的FGFR3功能增强型点突变小鼠(ACH小鼠)。这些小鼠个体明显短小,头颅短圆,长骨生长板组织形态结构异常。利用ACH病人及相关小鼠模型等材料对FGFR3调控软骨发育的机制进行的系列研究发现,FGFR3可通过上调细胞周期抑制基因(p21、p16、p18和p19)、转录活化蛋白(signal transducer and activator of transcription, Stat)1、5a和5b等分子的表达来抑制软骨细胞增殖,可经细胞外信号调节蛋白激酶(extracellular signal-regulated kinase , ERK)1/2通路抑制软骨细胞肥大分化。体内软骨形成过程是在多种信号分子的密切调控下完成的, FGFs/FGFR信号除经激活其下游信号途径调控软骨发育外,还可与调控软骨发育过程的另一重要信号通路骨形成蛋白(Bone morphogenetic protein, BMPs)信号相互作用来调节软骨发育。BMPs属于TGF-β超家族成员,现已发现并鉴定的分子有BMP1-BMP15。BMPs通过BMP受体(BMP receptor, BMPR)发挥生物学功能。BMP受体(BMPR)包括I型(BMPRI)和II型受体(BMPRII)。而BMPRI又包括Activin受体样蛋白激酶2 (Activin receptor-like kinase2, ALK2)、ALK3(BMPR1a)和ALK6(BMPR1b)等亚型,其中BMPR1a在长骨的前肥大及肥大软骨细胞中高表达。与BMPs结合后,BMPR II磷酸化BMPR I的甘氨酸-丝氨酸富集结构域,并进一步将信号传递给受体调节的Sma和Mad同系物(receptor-regu lated Sma and Mad homologue proteins, RSMAD),使之磷酸化,磷酸化的RSMAD从膜受体上脱离,结合Smad4进入细胞核,调节靶基因的转录。另外,BMPs还可激活TGFβ活化激酶1(TGFβ-activated kinase 1, TAK1)或者激活ERK1/2信号。近年来研究发现软骨形成过程中FGFR3可抑制BMP4的表达;BMP2可缓解体外培养的ACH小鼠胚胎肢体异常表型;BMPR1a可抑制FGFR1信号通路调节软骨发育;在神经系统中的研究发现,促分裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)活化后可磷酸化SMAD1的连接区导致SMAD1胞质内滞留或降解,从而抑制BMP信号。这些结果提示软骨发育及ACH发生过程中FGFR3信号与BMP信号间可能有相互作用,具体作用和机制如何目前还不清楚。据此,本研究拟通过分析软骨细胞特异组成型激活BMPR1a的FGFR3功能增强型点突变小鼠(caBMPR1acol2acre-ACH)的骨骼发育情况,并结合体外原代软骨细胞实验分别从整体动物水平、细胞水平和分子水平对FGFR3与BMPR1a信号相互作用调节软骨发育的机制进行了初步探讨。主要实验方法第一部分:软骨细胞特异组成型激活BMPR1a的FGFR3功能增强型点突变小鼠软骨发育分析及相关机制研究1.利用基于Cre/LoxP系统,建立了软骨细胞特异组成型激活BMPR1a基因的FGFR3功能增强型点突变小鼠模型(caBMPR1acol2acre-ACH);2.观测小鼠生长过程中体重、体长、躯干长、尾长、胫骨和股骨的变化,并采用X线摄影、全骨架染色和头颅局部摄影,观察小鼠大体形态及颅底软骨连接的变化情况;3.用阿利新蓝及藏红固绿染色等观察出生前后(E16.5、P5)不同基因型小鼠的生长板形态;4.通过BrdU掺入后免疫组化检测,观察生长板软骨细胞增殖情况;5.采用定量PCR检测软骨分化相关基因Collagen II、Collagen X和MMP13的表达情况;钙化结节染色观察生长板软骨细胞终末分化情况;6.用免疫组织化学检测胫骨生长板SMAD1/5/8磷酸化水平(phosphorylated SMAD1/5/8,pSMAD1/5/8)、p21及pERK1/2的表达;7.用激光共聚焦显微术检测软骨组织中SMAD1蛋白连接区磷酸化水平(phosphorylate SMAD1 linker region, pSMAD1L)并进行定位观察。第二部分:组成型激活BMPR1a对FGFR3功能增强型点突变小鼠软骨细胞分化的影响及相关机制的体外研究1.培养小鼠原代软骨细胞,并对培养的原代软骨细胞进行软骨分化诱导;2.定量PCR检测诱导0d、7d软骨细胞分化相关基因的表达;3.采用Western Blot检测原代软骨细胞中pERK1/2及pSMAD1L。主要实验结果一、软骨细胞特异组成型激活BMPR1a的FGFR3功能增强型点突变小鼠(caBMPR1acol2acre-ACH小鼠)的获得利用FGFR3功能增强型点突变小鼠(Fgfr3G369C/+小鼠,即ACH小鼠),带有Cre可诱导表达的组成型激活BMPR1a元件的转基因小鼠(caBMPR1a小鼠)以及软骨细胞中特异表达Cre重组酶的转基因小鼠(Col2αCre小鼠)设计交配策略,获得了基因型为caBMPR1acol2acre-ACH的小鼠。该小鼠为软骨细胞特异组成型激活BMPR1a的FGFR3功能增强型点突变小鼠。二、软骨细胞特异组成型激活BMPR1a对FGFR3功能增强型点突变小鼠一般生长情况的影响在观测期0.5至4个月内,caBMPR1acol2acre-ACH小鼠体重较ACH小鼠明显减轻;1月龄的caBMPR1acol2acre-ACH小鼠体长、尾长明显短于ACH小鼠,但是两者胫骨、股骨长度差异不显著;caBMPR1acol2acre-ACH小鼠的颅底软骨连接闭合时间较ACH小鼠提前。三、软骨细胞特异组成型激活BMPR1a基因对FGFR3功能增强型点突变小鼠软骨内成骨的影响1.BrdU掺入实验显示,出生5d的caBMPR1acol2acre-ACH小鼠软骨细胞增殖指数较同窝ACH小鼠低,提示caBMPR1acol2acre-ACH小鼠软骨细胞增殖活性较ACH小鼠降低;2.用直接提取的不同基因型小鼠骺软骨组织RNA进行的定量PCR结果显示:出生后5d时,caBMPR1acol2acre-ACH小鼠生长板软骨中Collagen II mRNA比ACH小鼠高,Collagen X mRNA表达较ACH小鼠低,MMP13 mRNA表达较ACH小鼠高;钙化结节染色发现caBMPR1acol2acre-ACH小鼠生长板软骨细胞的染色程度较ACH小鼠加深;体外未诱导的caBMPR1acol2acre-ACH、ACH小鼠原代培养软骨细胞中Collagen X mRNA及MMP13 mRNA表达变化与在体结果一致;软骨分化诱导7d , caBMPR1acol2acre-ACH小鼠原代软骨细胞中Collagen X mRNA表达仍然较ACH小鼠低。以上结果提示与ACH小鼠相比,caBMPR1acol2acre-ACH小鼠软骨细胞肥大分化受抑制,终末分化被促进。3.caBMPR1acol2acre-ACH小鼠软骨生长板中p21表达显著高于ACH小鼠,提示软骨内BMPR1a基因特异组成型激活可能通过促进p21的表达从而抑制ACH小鼠软骨细胞增殖;caBMPR1acol2acre-ACH小鼠胫骨生长板中pERK1/2水平显著高于ACH小鼠,进一步体外研究发现,caBMPR1acol2acre-ACH小鼠原代软骨细胞中pERK1/2水平及胞质pSMAD1L水平均显著高于ACH小鼠,提示软骨细胞特异组成型激活BMPR1a可能通过激活ERK1/2抑制ACH小鼠软骨细胞的肥大分化,此过程中活化的ERK1/2可能通过上调SMAD1蛋白连接区磷酸化抑制BMPR1a/SMAD1通路,从而参与对软骨细胞肥大分化的抑制调节。主要结论1.软骨细胞特异组成型激活BMPR1a加重了FGFR3功能增强型点突变所引起的侏儒表型。2.软骨细胞特异组成型激活BMPR1a可能通过促进FGFR3功能增强型点突变小鼠软骨细胞中p21表达上调,加重了FGFR3功能增强所致的软骨细胞增殖抑制。3.软骨细胞特异组成型激活BMPR1a可能通过促进FGFR3功能增强型点突变小鼠软骨细胞中ERK1/2磷酸化水平上调,加重了FGFR3功能增强所致的软骨细胞肥大分化抑制。4. ERK1/2 MAPK通路可能通过上调SMAD1蛋白连接区的抑制磷酸化来抑制BMPR1a/SMAD1通路,参与软骨细胞特异组成型激活BMPR1a对FGFR3功能增强型小鼠软骨细胞肥大分化的抑制。更多还原

【Abstract】 Achondroplasia (ACH) is the most common type of human dwarfism, mainly affecting endochondral ossification of limb and vertebrae, especially the cartilage formation which includes the condensation and differentiation of mesenchymal cells into progenitor chondrocytes, and subsequent chondrocyte proliferation, hypertrophy and apoptosis, but little is known about its pathogenic mechanism.Fibroblast growth factor receptors (FGFRs) play important roles in skeletal development and diseases. FGFRs belong to the tyrosine kinase receptor family. Four FGFRs (FGFR1-4) have been found with the amino acid level between 55% -72% homology. A dozen or more activated mutations in FGFR3 can cause a variety of human dwarfism with developmental disorders, such as Achondroplasia (ACH), Hypochondroplasia (HCH), Thanatophoric dysplasia (TD) and so on.FGFR3 is expressed in reserve, proliferating and prehypertrophic chondrocytes. Currently knock-in and transgenic technologies are used to obtain multiple kinds of mice with activated mutations in FGFR3. These mice mimicing achondroplasia are significantly short with short round heads and abnormal morphologic structure of the growth plate of the long bone. A series of studies on the role of FGFR3 during chondrogenesis using ACH patients and mouse models have observed that FGFR3 can inhibit chondrocyte proliferation by increasing the expression of cell cycle suppressor genes (p21, p16, p18 and p19), Stats (Stat1, Stat5a and Stat5b) and inhibit chondrocyte hypertrophic differentiation via MAPK pathway. A variety of signaling molecules control the process of chondrogenesis. The FGF/FGFR signaling activates its downstream signaling pathway to play important rols in endochondral ossification, moreover, it has crosstalk with the Bone Morphogenetic Protein (BMP) singnaling to co-regulate cartilage development.BMPs belong to TGF-βsuperfamily, and BMP1-BMP15 have been identified. BMPs transduce signals through heteromeric complexes of type I and type II serine/threonine kinase receptors (BMPRI and BMPRII). BMPRI includes several subtypes such as ALK2, ALK3 (BMPR1a), ALK6 (BMPR1b) and so on, and BMPR1a is highly expressed in the prehypertrophic and hypertrophic chondrocytes. Upon BMP binding, type II receptors phosphorylate serine/threonine residues in type I receptors. The receptor complex phosphorylates receptor-regulated Smad proteins (R-Smads), including Smad1, 5 and 8. Subsequently, activated R-Smads recruit and bind the common partner Smad, Smad4. This Smad complex enters the nucleus, where it directly binds defined elements on the DNA and regulates target gene expression together with numerous other factors. BMPs can also signal by activating TGFβ-activated kinase 1 (TAK1) or ERK1/2 pathway. Recent studies have showed that FGFR3 can inhibit BMP4 expression in the growth plate of the long bone. In vitro BMP2 can rescue abnormal phenotype of the cultured embryonic limb of the ACH mouse, and BMP signals can inhibit the FGFR1 signaling pathway during chondrogenesis, but the interaction between FGFR3 and BMPR1a signalings during chondrogenesis is still unknown. During neurogenesis the ERK1/2 pathway is known to phosphorylate the linker region of Smad1, subsequently inhibiting BMP signaling.Whether this level of regulation exist in the interaction between BMP and FGFR3 signalings in chondrocytes remains to be examined.In this study,we expressd a constitutively active form of BMPR1a in chondrocytes of mice harboring activated mutation in FGFR3(caBMPR1acol2acre-ACH), and then analyzed the bone development in these mice. We also studied the differentiation phenotype of the primary chondrocytes from caBMPR1acol2acre-ACH mice and their littermate controls, and finally we preliminarily approached the crosstalk between FGFR3 and BMPR1a signalings in the pathogenesis of ACH.METHODSPart I: Analysis of the skeletal phenotype and related mechanism of mice harboring activated mutation of FGFR3 with chondrocyte-specific constitutively activated BMPR1a 1. Based on Cre/LoxP strategy, mice harboring activated mutation in FGFR3 with chondrocyte-specific constitutively activated BMPR1a (caBMPR1acol2acre-ACH) were generated, and genotyped by PCR.2. The body weight as well as length of truck, tail, body, tibia and femur were measured. X-ray radiography, whole skeleton staining and skull photography were performed. The overall shape and the cranial synchondrosis of mice were observed.3. Histological sections of mice with different genotypes at different ages (E16.5, P5) were prepared to investigate the growth plate morphologically by alcian blue staining and safranine-fast green staining.4. Chondrocyte proliferation in the growth plate were investigated by BrdU incorporation assay.5. Marker genes during chondrocyte differentiation including typeⅡcollagen, type X collagen and matrix metalloproteinase 13 mRNA expression were detected by quantitative PCR. Terminal differentiation of chondrocytes in the growth plate was determined by von kossa staining.6. The levels of phosphorylated SMAD1/5/8 (pSMAD1/5/8), p21 and pERK1/2 in the growth plate were detected by immunohistochemistry.7. The phosphorylated SMAD1 linker region (pSMAD1L) level and its location pattern in the growth plate was investigated by confocal laser scanning microscopy.Part II: In vitro study of the effect of constitutively activated BMPR1a on the proliferation and differentiation of chondrocytes in ACH mice and related mechanism1. Chondrocytes were obtained from the epiphyseal cartilage. Primary culture of chondrocytes was conducted, and then chondrogenic differentiation was induced.2. The mRNA levels of typeⅡcollagen, type X collagen and matrix metalloproteinase 13 were examined by quantitative PCR to examine the chondrogenic differentiation.3. The levels of pErk1/2 and cytoplasmtic pSMAD1L in primary chondrocytes were detected by Western Blot.RESULTS1. Generation of mice harboring activated mutation of FGFR3 with chondrocyte-specific constitutively activated BMPR1aTo activate BMPR1a constitutively in chondrocytes of mice harboring activated mutation in FGFR3, constitutively activated BMPR1a mice (caBMPR1a mice) and transgenic mice with chondrocyte specific expression of Cre recombinase (Col2aCre mice) were first intercrossed to generate caBMPR1acol2acre mice , and then caBMPR1acol2acre mice crossed with mice harboring activated mutation in FGFR3 (Fgfr3G369C/+ mice, ACH mice)to generate caBMPR1acol2acre-ACH mice.2. The effects of chondrocyte-specific activation of BMPR1a on the growth of ACH miceThere was a significant decrease of the body weight of caBMPR1acol2acre-ACH mice compared with that of ACH mice. The body and tail length of caBMPR1acol2acre-ACH mice at 1 month were significantly shorter than that of ACH mice, but the length of femur, tibia had no significant difference between ACH and caBMPR1acol2acre-ACH mice. The cranial synchondrosis of caBMPR1acol2acre-ACH mice at P5 was fused earlier than that in ACH mice.3. The effects of constitutively activated BMPR1a in chondrocytes on endochondral ossification of ACH miceThe chondrocyte proliferative index measured by using BrdU incorporation assay in caBMPR1acol2acre-ACH mice at P5 was significantly lower than that in ACH mice.Quantitative PCR using mRNA directly extracted from the epiphyseal cartilage showed that the levels of typeⅡcollagen mRNA expression and matrix metalloproteinase 13 (MMP13)mRNA in the growth plate of caBMPR1acol2acre-ACH mice at P5 were upregulated,while the expression of type X collagen mRNA was downregulated compared to that in ACH mice. Deeper von kossa staining was found in the growth plate of caBMPR1acol2acre-ACH mice with comparison to that in ACH mice, indicating that the extent of chondrocytes terminal differentiation in caBMPR1acol2acre-ACH mice was higher than ACH mice. In vitro the expression of type X collagen and MMP13 mRNA in primary chondrocytes without induction of chondrogenic differentation also had the same patterns as that found in vivo. At 7d after chondrogenic differentiation induction, the level of type X collagen in the primary chondrocytes of caBMPR1acol2acre-ACH mice were downregulated when compared with that in ACH mice. Taken together, there were significantly inhibited hypertrophic differentiation and promoted terminal differentiation in the growth plate of caBMPR1acol2acre-ACH mice than these in ACH mice. To explore the possible mechanisms for the effects of chondrocyte-specific constitutively activated BMPR1a on the chondrogenesis of ACH mice, we checked and found that the levels of p21 and pERK1/2 in the growth plate were significantly higher than that in ACH mice. In vitro studies further found that the levels of pERK1/2 and cytoplasmtic pSMAD1L in primary chondrocytes of caBMPR1acol2acre-ACH mice were significantly higher than that in ACH mice. These results suggested that the effects of chondrocyte-specific activation of BMPR1a on chondrocyte proliferation and differentiation of ACH mice may be caused by the change of p21 and pERK1/2 levels, and the pERK1/2 level may regulate the activation status of the BMPR1a/SMAD1 pathway during chondrocyte differentiation.CONCLUSIONS1. Constitutive activation of BMPR1a in chondrocytes causes more severe achondroplasia in ACH mice;2. Chondrocyte-specific constitutively activated BMPR1a enhanced the p21 expression in the chondrocytes of ACH mice, which further enhanced the inhibitory effect of activated mutation in FGFR3 on chondrocyte proliferation;3. Chondrocyte-specific constitutively activated BMPR1a enhanced the pERK1/2 level in the chondrocytes of ACH mice, leading to augmented inhibitory effect on chondrocyte hypertrophic differentiation caused by activated mutation in FGFR3;4. ERK1/2 MAPK pathway activated in chondrocytes of caBMPR1acol2acre-ACH mice may inhibit BMPR1a/SMAD1 pathway by upregulating the phosphorylation level of the linker region of SMAD1, which may be involved in more inhibited hypertrophic differentiation.更多还原