【作者】 孙启荻；

【导师】 陈林；

【作者基本信息】 第三军医大学 ， 遗传学， 2011， 博士

【摘要】 FGFR3是受体酪氨酸激酶家族成员（RTK）之一,通过与22种FGF（FGF1-14,16-23）结合传递信号。FGFR3是软骨内成骨过程的负性调节因子。FGFR3功能增强型突变导致软骨发育缺陷包括软骨发育不全（ACH）、软骨发育不良（HCH）、致死性发育不全（TD）。FGFR3的部分缺失突变产生僵直小指、高身材、脊柱侧凸和听力丧失综合征。ACH表现为短肢侏儒、椎管狭窄、面部异形,是最常见遗传性侏儒。ACH的新生儿的发病率为1/40000-1/15000。尽管已经知道ACH中FGFR3功能增强型突变抑制软骨细胞的增殖和分化,进而导致长骨变短,可是目前FGFR3对ACH出生后骨形成和骨重建的作用还不清楚。出生后和成年阶段,骨骼通过骨形成和骨吸收的协调作用经历持续的重建。研究证实FGFR3参与骨形成。FGFR3能影响膜内成骨。FGFR3的两种功能获得型突变（P250R和A391E）导致人类Muenke综合征和Crouzon综合征。这两种病症是由于颅缝早闭引起。TD中也经常发现颅缝早闭。另外,成年FGFR3敲除小鼠骨质减少,骨基质矿化有障碍。这些研究表明FGFR3能够调节骨形成和骨重建。我们以往的研究发现模拟人ACH的FGFR3^G369C/+突变小鼠（ACH小鼠）15天时骨小梁减少、生长板中成骨细胞标记如Cbfa1、OP和OC表达增高。我们还发现FGFR3激活能够影响ACH的早期骨形成,但是成年ACH小鼠骨形成的特点还不清楚。因此,我们利用FGFR3^G369C/+突变模拟人ACH的小鼠模型深入研究FGFR3调节骨形成的机制。在利用遗传动物模型研究FGFR3功能增强后对骨形成的影响的同时,我们还进行了软骨发育不全相关的临床研究。目前认为,ACH患者主要由FGFR3基因的G380R突变引起,此外也存在其他较为少见的突变。国外对ACH及相关疾病的患者研究较多,国内对ACH的研究相对零散。本研究中,我们收集了60例侏儒患者的病例和血液样本,并在知情同意的情况下,对其中临床诊断为软骨发育不全的患者进行遗传学分析,明确了FGFR3突变类型。主要实验方法第一部分FGFR3在小鼠骨形成中的作用和机制研究1、利用组织形态学分析和定量PCR检测骨形成情况及相关标记分子的改变。血清生化分析和Elisa检测PINP等研究矿物质平衡的系统变化。2、利用体外BMSCs培养和阻断实验检测成骨细胞的分化。通过ALP活性测定和染色,茜素红染色分析矿化的成骨细胞以检测成骨细胞的分化程度。Western Blot检测Erk1/2和p38的磷酸化水平。第二部分软骨发育不全患者的FGFR3基因突变1、对患者的一般情况、病史等进行询问,测量其身高、坐高、肢体长度、头围等相关指标,对患者进行临床诊断。2、收集临床诊断为ACH的患者的血液标本,提取DNA,采用PCR测序加限制性内切酶酶切方法对其进行FGFR3突变的遗传诊断。主要实验结果:一、FGFR3功能增强后小鼠骨量减少,成骨活性改变1、2月龄FGFR3^G369C/+小鼠胫骨骨小梁稀疏,骨矿化障碍。骨小梁表面成骨细胞胞体增大,伴有类骨质增多,矿物质沉积率降低,提示该小鼠成骨细胞虽然分泌基质的功能活跃,这与血清中PINP含量增加的结果一致。但细胞外基质矿化能力存在缺陷。2、FGFR3^G369C/+突变小鼠抑制BMSCs增殖,促进其分化,但抑制矿化,这些骨髓基质细胞自我复制能力降低及成骨活性障碍可能是导致小鼠骨量降低的原因。3、培养的BMSCs中FGFR3激活Erk1/2和p38通路参与调节成骨细胞增殖、分化和骨基质矿化。FGFR3主要通过激活Erk1/2通路导致BMSCs的骨基质矿化降低,通过激活p38通路导致BMSCs的增殖降低、成骨分化增强。二、发现了17例软骨发育不全患者存在FGFR3基因突变1、17位患者有ACH相关表型,临床诊断为ACH;2、发现16例ACH患者FGFR3基因发生G380R突变,1例FGFR3 Y278C的罕见突变。.主要结论:1、FGFR3^G369C/+突变小鼠通过抑制BMSCs增殖和矿化导致骨量降低。2、FGFR3主要通过激活Erk1/2通路导致BMSCs的骨基质矿化降低,通过激活p38通路导致BMSCs的增殖降低、成骨分化增强。3、绝大部分ACH病人为FGFR3G380R突变,与国外报道一致。.更多还原

【Abstract】 Fibroblast growth factor receptor3 （FGFR3） belongs to membrane-bound receptor tyrosine kinases. FGFR3 is a negative regulator of endochondral bone development. Gain-of-function mutations in FGFR3 result in chondrodysplasias which include achondroplasia （ACH）, hypochondroplasia and thanatophoric dysplasia （TD）. In contrast, partial loss-of-function mutation of FGFR3 causes camptodactyly, tall stature, scoliosis and hearing loss （CATSHL） syndrome.ACH is characterized by the short limb dwarfism, spinal stenosis and facial dysmorphism, which is the most common hereditary dwarfism with an incidence rate between 1/15 000 and 1/40 000 live births. Although gain-of-function mutations of FGFR3 in ACH have been found to inhibit the proliferation and differentiation of chondrocytes and subsequently cause shortened long bone resulting in dwarfism, the effects of FGFR3 on the postnatal bone formation and bone remodeling in ACH have not been well elucidated.During postnatal and adult stages, bone undergoes continuous remodeling through the coordinated processes of bone formation and bone resorption. Evidence suggests that FGFR3 is involved in bone formation. FGFR3 can affect intramembranous ossification. Two gain-of-function mutations of FGFR3 （P250R and A391E） have been identified in humans resulting in Muenke syndrome, and Crouzon syndrome with Acanthosis Nigricans. Both syndromes have premature fusion of the cranial sutures. Furthermore, TD is also frequently associated with severe craniosynostosis. Additionally, adult Fgfr3 and Fgfr3IIIc null mutant mice showed osteopenia and defects in bone matrix mineralization. These studies indicate that FGFR3 could regulate bone formation and remodeling.Our previous study showed shortened trabecular bone and increased expressions of osteoblast markers such as core binding factor a1 （Cbfa1）, osteopontin （OP） and osteocalcin （OC） in the growth plate of FGFR3^G369C/+ mice （a mouse model mimicking human ACH resulting from the gain-of-function mutation, Gly375Cys, of FGFR3） on postnatal day 15. We also observed that activating mutation in FGFR3 caused decreased bone mass and compromised architecture in adult mice.These data indicate that activation of FGFR3 could influence bone formation at an early age of ACH, but the characteristics of the bone formation in adult ACH mice are not clear.Thus, we used mouse model mimicking human achondroplasia resulting from gain-of-function mutation of FGFR3 (FGFR3^G369C/+ mice) to further explore the mechanism of FGFR3 regulating bone formation.In addition to study the mechanism of FGFR3 regulating bone formation, we also did some clinical studies. More than 98% of ACH cases are caused by a single G380R mutation in the transmembrane domain of FGFR3. Multiple researches on FGFR3 mutations associated with ACH have been carried out abroad,however there are a few studies on ACH patients in China. In this study, we collected 60 subjects with dwarfism and made genetic analysis of ACH patients of them.MethodsPart I The mechanism of FGFR3 regulating bone formation1.Histomorphometric measurement and real-time quantitative RT-PCR （qRT-PCR） were used to evaluate osteoblastogenesis. Serum biochemistry and ELISA for PINP were used to investigate systemic alteration in mineral homeostasis.2. BMSCs culture and inhibitor studies in vitro were used to examine the differentiation of osteoblasts. ALP activity and staining, and alizarin red staining of mineralized osteoblast cultures were used to detect the extent of the differentiation of osteoblasts. The levels of Erk1/2 and p38 phosphorylation were detected by Western Blot.Part II Mutation analysis of ACH patients1. Sixty subjects with dwarfism were enrolled.The investigation content included: general condition and medical history. Physical examination was carried out including:body height,height of limbs, head circumference ,etc. 2. Blood samples were collected from the patients with short limbs and genomic DNA wasisolated. Direct DNA sequence analysis of all exons and exon-intron bandraries of FGFR3 was carried out. ResultsPart I FGFR3^G369C/+ mice showed decreased bone mass and changed osteoblastic function1. The adult FGFR3^G369C/+mice showed reduced trabecular number and thickness, larger and more cuboidal osteoblasts. There was a significant decrease in calcein labeling of mineralizing surfaces in FGFR3^G369C/+ mice as evidenced by reduced mineral apposition rate （MAR） .2. Compared with primary cultures of bone marrow stromal cells （BMSCs） from wild-type mice, FGFR3^G369C/+ cultures showed decreased proliferation, increased alkaline phosphatase activity, and up-regulated expressions of osteogenic differentiation related genes but fewer mineralized nodules.3. Enhanced Erk1/2 activity in FGFR3^G369C/+ BMSCs was responsible for the impaired mineralization, and that up-regulated p38 phosphorylation led to decreased proliferation and enhanced osteoblastic differentiation.Part II Mutation analysis of ACH patients1. There are 17 ACH patients according to clinical diagnostic criteria. They have signs of short limb dwarfism, spinal stenosis and facial dysmorphism.Most of them have limitation of motion and pain in joints.2. The mutation in 16 paients is a missense mutation G380R that results in a single heterozygous G to A transversion at nucleotide1138. One patient has a missense mutation Y278C that results in a single heterozygous A to G transversion at nucleotide 833.Conclusions1. A gain-of-function mutation （G369C） in FGFR3 in mice led to decreased bone mass by inhibiting the proliferation and mineralization of bone marrow stromal cells （BMSCs）. 2. Erk1/2 signaling activated by FGFR3 is mainly responsible for the decreased bone matrix mineralization of BMSCs, and the enhanced activity of p38 signaling results in the reduced replication and increased osteogenic differentiation of Fgfr3^G369C/+ BMSCs.3. Majority of ACH patients in China carrying FGFR3 G380R mutation, which is similar to data from other countries.更多还原