【作者】 李福兵；

【导师】 陈林；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 成纤维细胞因子Ⅰ型受体(FGF ReceptorⅠ,FGFRⅠ)在骨骼发育和人类遗传疾病中具有重要作用。人FGFR1 P252A功能增强性点突变引起Pfeiffer综合征和OD综合征,主要引起人类颅缝早闭和趾骨发育异常。FGFR1功能丧失性点突变引起人类Kallmann综合征,但不直接调节骨骼发育。FGFR1敲除导致小鼠胚胎期死亡,条件性基因敲除技术使得研究FGFR1在骨骼发育中的作用成为可能。条件性基因敲除技术必须依赖于组织或细胞特异性Cre转基因小鼠的制备,已有研究报告指出OC-Cre转基因小鼠可以在成熟成骨细胞中特异性表达CRE重组酶,这使得研究成熟成骨细胞中FGFR1在骨骼发育中功能成为可能。已有实验室利用基因条件性敲除技术研究FGFR1在小鼠骨骼肢芽发育中的作用,结果FGFR1是肢芽(limb bud)发生、发育和趾骨图式发育(patterning)所必需,Jacob等的研究结果表明FGFR1促进间充质细胞增生,抑制前成骨细胞增生和成熟成骨细胞矿化,但FGFR1对出生后骨发育和重建的影响和分子机制还不完全清楚,同时FGFR1对出生后骨骼质量的影响还未见报道。。BMP受体ALK3是骨骼发育和重建过程中的负性调节分子。Mishina实验室发现成骨细胞敲除ALK3后,成骨细胞成骨功能下降,破骨细胞骨吸收功能受抑制。在骨骼发育过程中,FGF信号通路和BMP信号通路之间关系复杂:BMP和FGF信号相互抑制共同调控软骨生成;而在成骨过程中,Warren等发现FGF信号通路可以通过抑制BMP阻断分子noggin来调节BMP信号通路。Matsushita等最新的研究结果显示FGFR3激活性突变可以通过FGF信号依赖的BMP信号通路促进成骨。目前,成骨细胞中BMP信号通路与FGFR1的关系还不明确。本课题利用fgfr1CKO小鼠和OC-Cre转基因小鼠制备fgfr1OC-CKO小鼠,实现在小鼠成熟成骨细胞敲除FGFR1,从而研究FGFR1对骨骼发育、重建、出生后骨骼质量的维持和钙和磷代谢的功能。为研究FGFR1和ALK3信号通路的关系,本研究利用条件性组成性激活ALK3转基因小鼠(caALK3小鼠)和fgfr1OC-CKO小鼠交配研究BMP信号通路和FGFR1信号通路在成骨细胞中的作用。本课题包括以下两部分:1.探讨FGFR1对骨骼发育和骨骼重建的影响及其分子机制;2.激活fgfr1OC-CKO小鼠成骨细胞中ALK3来研究FGFR1和ALK3信号通路的关系。主要实验内容和方法如下:第一部分FGFR1信号通路在小鼠骨骼发育和重建中的作用及其分子机制实验动物:成熟成骨细胞特异性fgfr1敲除(fgfr1OC-CKO)小鼠;对照(fgfr1CKO)小鼠。实验方法(一)体内实验1. fgfr1OC-CKO小鼠整体表型分析利用X光摄片和骨密度测定对3月、4月和8月龄fgfr1OC-CKO小鼠和对照小鼠骨骼进行整体表型分析。此外,还对3月龄小鼠骨架进行茜素红染色。2.骨骼生物力学测定用Instron(5565)力学测定仪通过3点折断法(three point bending)检测3月龄和8月龄小鼠股骨力学性能。3.钙和磷测定采用Beckman DXC800全自动生化分析仪测定6周、4月和8月龄小鼠血清中钙和磷含量。用能谱仪测定骨皮质中钙和磷百分含量。4.使用micro CT对3月龄和8月龄fgfr1OC-CKO小鼠和对照小鼠股骨进行扫描利用Scanco公司Micro CT(μCT 80)对3月和8月龄小鼠右侧股骨进行扫描。5.组织形态学研究和破骨细胞功能评价对3月龄fgfr1OC-CKO小鼠和对照小鼠股骨进行病理切片,然后利用HE染色和TRAP染色分别对骨骼形态学分析和破骨细胞功能进行检测;利用扫描电镜对骨骼及其成骨细胞形态进行观察。6.骨组织中类骨质、钙和骨形成率检测3月龄小鼠股骨树脂包埋后行硬组织切片,然后采用Von Kossa染色检测骨骼中类骨质、钙盐,荧光显微镜直接观察钙黄绿素和四环素标记的新骨形成,并测定计算骨沉积率。(二)体外实验1.成骨细胞增生、凋亡和矿化检测新生小鼠颅骨来源的成骨细胞培养3天后计数检测成骨细胞增生;诱导矿化1周后用TUNEL法进行凋亡检测;诱导矿化3周后行矿化能力检测。2.成骨细胞中与分化和成骨相关功能基因的转录水平检测颅骨成骨细胞诱导矿化2周,提取成骨细胞总RNA,然后利用real time PCR检测osteocalcin、osteopontin、cbfa1、noggin、bmp4、bmpr1a和fgfr2的转录水平。3.成骨细胞中与分化和成骨相关功能基因的蛋白水平检测新生小鼠颅骨来源成骨细胞诱导矿化2周,提取细胞总蛋白;利用western blot检测phospho-p38、phospho-ERK、ERK、phospho-SAPK/JNK、p85、ALK3和内参β-actin。第二部分激活成熟成骨细胞中ALK3可恢复fgfr1OC-CKO小鼠部分骨骼异常表型实验动物:成熟成骨细胞中特异性敲除fgfr1并激活ALK3小鼠(fgfr1OC-CKO-caALK3小鼠);fgfr1OC-CKO小鼠和对照小鼠(fgfr1CKO小鼠)。实验方法实验方法同于第一部分。主要实验结果:一、Fgfr1OC-CKO小鼠骨量增加X光片和BMD测定结果显示8月龄fgfr1OC-CKO小鼠长骨、颅骨和椎骨等的骨量显著高于对照小鼠,8月龄fgfr1OC-CKO小鼠胫骨和股骨BMD显著高于对照小鼠。股骨micro CT数据显示:和对照小鼠相比,3月和8月龄fgfr1OC-CKO小鼠股骨骨干单位体积骨密度(体密度)显著降低;8月龄fgfr1OC-CKO小鼠骨小梁显著增加,骨皮质厚度和BV/TV极显著增高;3月龄fgfr1OC-CKO小鼠骨皮质厚度无显著改变,骨小梁多于对照小鼠。HE染色结果显示fgfr1OC-CKO小鼠骨细胞数量极显著减少,骨皮质小孔隙显著增加。钙黄绿素和四环素标记新骨形成结果表明:fgfr1OC-CKO小鼠骨形成率显著增加。体外成骨细胞矿化实验结果显示fgfr1OC-CKO小鼠成骨细胞诱导产生矿物含量显著高于对照小鼠。二、FGFR1经ALK3信号通路调节骨骼质量3月龄fgfr1OC-CKO小鼠部分(28/140)出现自发骨折现象,生物力学测定结果显示fgfr1OC-CKO小鼠股骨杨氏弹性模量和最大载荷均显著小鼠低于对照小鼠(P<0.01);Von Kossa染色结果显示fgfr1OC-CKO小鼠股骨骨皮质中央存在大量的红染区域,并且着色较对照小鼠浅,提示fgfr1OC-CKO小鼠骨皮质钙和磷含量较低,骨皮质中存在较多不成熟的类骨质;能谱测定结果显示骨皮质中单位面积中钙和磷百分含量显著低于对照小鼠(P<0.001)。激活fgfr1OC-CKO小鼠成骨细胞中ALK3后,3月龄小鼠股骨和胫骨生物力学测定结果显示fgfr1OC-CKO-caALK3小鼠胫骨杨氏弹性模量和最大加载与对照小鼠无显著差异,股骨最大加载与fgfr1CKO小鼠无显著差异,但杨氏弹性模量显著低于fgfr1CKO小鼠,高于fgfr1OC-CKO小鼠。三、FGFR1经ALK3调节骨骼和血液的钙和磷代谢股骨中段骨皮质能谱检测结果显示fgfr1OC-CKO小鼠股骨皮质中钙和磷百分含量极显著低于对照小鼠(P<0.01);4月和8月龄fgfr1OC-CKO小鼠血清钙和磷含量显著高于对照小鼠(分别高30.1%和27.4%);激活成熟成骨细胞中ALK3可以恢复fgfr1OC-CKO小鼠血清钙和磷水平到正常。四、激活成熟成骨细胞中ALK3可以恢复fgfr1OC-CKO小鼠破骨细胞活性。股骨石蜡切片TRAP染色结果显示,fgfr1OC-CKO小鼠破骨细胞数量减少,铺展欠佳;而fgfr1OC-CKO-caALK3小鼠破骨细胞数量和形态均恢复正常。五、FGFR1通过抑制PI3K和促进MAPK信号通路调节成骨细胞增生和凋亡新生小鼠颅骨成骨细胞增生检测结果显示,敲除FGFR1后成骨细胞增生加快。TUNEL法检测诱导一周的fgfr1OC-CKO小鼠和对照小鼠颅骨来源成骨细胞凋亡结果:fgfr1OC-CKO小鼠成骨细胞凋亡显著减少。诱导一周的fgfr1OC-CKO小鼠成骨细胞PI3K亚基p85的表达明显上调,MAPK信号通路分子ERK,p38和SAPK/JNK的磷酸化水平下调。六、成骨细胞中FGFR1可以调节ALK3信号通路的蛋白表达定量PCR结果显示fgfr1OC-CKO小鼠颅骨成骨细胞bmp4和其高亲和受体alk3转录水平下调;Western blot结果显示fgfr1OC-CKO小鼠颅骨成骨细胞LK3蛋白表达水平也明显下调。七、FGFR1经MAPK信号通路影响骨形成Western blot结果显示fgfr1OC-CKO小鼠颅骨成骨细胞MAPK信号通路蛋白分子ERK、p38和SAPK/JNK的磷酸化水平均显著下调。ERK表达水平无明显改变。主要结论:1. FGFR1抑制成骨细胞矿化和增生,通过PI3K和MAPK信号通路促进成骨细胞凋亡。2.成熟成骨细胞中FGFR1可能经ALK3信号通路调节血清钙和磷代谢,敲除成熟成骨细胞中FGFR1后导致血清钙和磷增高,但同时激活ALK3可恢复其血清钙和磷水平。3. FGFR1缺乏会导致小鼠股骨机械性能下降,成骨细胞成骨能力异常,激活ALK3可以恢复由于缺乏FGFR1导致的骨骼异常表型。4.成骨细胞FGFR1缺乏导致fgfr1OC-CKO小鼠破骨细胞受损,激活成骨细胞中ALK3可以恢复由于缺乏FGFR1导致的破骨细胞活性下降。更多还原

【Abstract】 FGF receptor 1 has an important role in bone development and congenital diseases in human. Fgfr1 (P252R) mutation causes Pfeiffer syndrome. A rare mutation has been identified that causes osteoglophonic dysplasia (OD), a disease characterized by craniosynostosis, prominent supraorbital ridge, and depressed nasal bridge, as well as the rhizomelic dwarfism and nonossifying bone lesions. Loss of function mutations in fgfr1 is associated with one form of Kalmann syndrome (KS), a disease that does not directly affect skeletal development. Fgfr1 knockout leads to embryonic lethality shortly after gastrulation, necessitating a conditional knockout approaches to address fgfr1 function in later development. Conditional knockout technology makes it possible to investigate FGFR1 function in bone devemopment. Osteocalcin-Cre (OC-Cre) transgenic mice are suitable for studying fgfr1 function in differentiated osteoblast, which highly express Cre at 4 to 8 week after birth. Fgfr1 has recently been shown to be pivotal for early limb bud development and distal skeleton patterning. How FGFR1 regulates osteoblast and bone quality are not clear yet. FGFR1 is one of FGF23’s receptor, which implies a potential role of FGFR1 on calcium and phosphate metabolism.ALK3, one of BMP type I receptor, is a negative regulator of bone mass. Bone mass was increased in Alk3OC-CKO mice. Crosstalk between BMP signaling and FGF signaling are complicated and controversial in bone development. Conditionally Inactivating BMPRIA and BMPRIB showed that BMP and FGF coordinately regulate chondrogenesis, BMP signaling inhibits FGF signaling. FGF promotes BMP signaling by inhibiting BMP antagonist noggin. Crosstalk between FGFR1 and ALK3 is not clear. The aim of this project is to study the role of FGFR1 in bone development and the crosstalk between FGFR1 and ALK3 signaling.To explore the role of FGFR1 in differentiated osteoblast in bone development after birth and its mechanism, OC-Cre transgenic mice and fgfr1 floxed mice were used. And conditional caALK3 transgenic mice were used for constitutively activated ALK3 in fgfr1OC-CKO mice. Two parts of this project were included: Part one: To investigation the role of FGFR1 in osteoblast in bone development and remodeling and its mechanism.Part two: To investigation the relationship between FGFR1 and ALK3 in differentiated osteoblast.Main experiments show below:Part one: Role of FGFR1 signaling in bone development and remodeling Experiment animal: fgfr1OC-CKO mice and fgfr1CKO miceMethods:In vivo assay1. Obtaining of loss of FGFR1 function in differentiated osteoblast mice (fgfr1OC-CKO mice) and general phenotype analysis Fgfr1OC-CKO mice were obtained according to what we reported. X-Ray photograph was taken for different ages. Whole skeleton prepared according to litterature.2. Analysis of fgfr1OC-CKO mice and control mice femurFemur of 3 and 8 month-old mice were scanned byμCT 80 (scanco). Condition: 70KVp, 113μA, resolution: 20μm. Integration time: 400msec.3. Histology analysisFemurs were collected from 3 month-old mice, 24 hrs fixation, and paraffin embedding for section. H&E staining and TRAP staining were taken. SEM was applied for bone and osteoblast morphology analysis.4. Osteoid, phosphate and bone formation rate analysisUndecalcified sections were used for Von Kossa staining. Calcein was used for bone formation rate detection. EDS was applied for Ca and P content and percentage analysis.5. Serum Ca and PO4 detectionSerum calcium and phosphate were analyzed by Beckman DXC800 auto biochemistry analysis machine and kit. 6. Mechanical testThree bending approaches were applied to analyze 3 and 8 month-old mice femur mechanical characteristics. Speed 6 mm/min, Maximal load 5 kN load cell.In vivo assay1. Primary calvarial osteoblast proliferation, mineralization, and apoptosis detectionPrimary calvarial osteoblasts were separated from neonatal mice. Osteoblast proliferation, mineralization, and apoptosis were detected.2. Osteoblast gene transcription analysis by real time PCRPrimary calvarial osteoblast was maintained in mineralization medium for two weeks, and total RNA was extracted. Osteoblast gene transcription of osteocalcin、osteopontin、cbfa1、noggin、bmp4、bmpr1a and fgfr2 were detected by real time PCR using SYBR.3. Osteoblast protein expression detectionTotal protein was extracted from primary calvarial osteoblast maintained in mineralization medium for two weeks. Phospho-p38, phospho-ERK, ERK, phospho-SAPK/JNK, p85, BMPRIA and internal control beta actin expression were detected by western blot.Part two: Conditionally constitutively activated ALK3 in differentiated osteoblast can partially rescue fgfr1OC-CKO mice bone abnormalityAnimal: fgfr1OC-CKO-caALK3 mice, fgfr1OC-CKO mice and fgfr1CKO mice (control)Methods: all the methods in this part were described in part one.Main results:1. Bone formation was increased after deleting FGFR1 in differentiated osteoblastLoss of FGFR1 signaling led to significantly increased BMD, cortical bone thickness BV/TV and trabecular bone at 8 month-old mice, though 3 month mice had no difference in cortical bone thickness compare with control mice, but had much more trabecular bone. HE staining results showed that osteocytes were impaired in FGFR1 conditional knockout mice. In vitro osteoblast mineralization test also showed that lacking of FGFR1 in differentiated osteoblast led to increased mineralization. In vivo double labeling results showed increase bone formation, which was consistent with in vitro data. 2. FGFR1 regulates bone quality partially through ALK3 signalingAlthough bone formation was significantly increased at 8 month-old mice, part of 3 month-old mice displayed spontaneous fracture. And the mechanical properties were decreased, in the middle of cortical bone, some osteoid bone was found by Von Kossa staining and counterstained with Van Gieson’s staining. To our surprise, low mechanical properties of fgfr1OC-CKO mice could be partially rescued by constitutively activated ALK3 in differentiated osteoblast.3. FGFR1 signaling and BMP signaling can coregulate serum calcium and phosphateFgfr1OC-CKO mouse showed increase serum calcium and phosphate, especial the serum phosphate level. At 4 and 8 month-old mice, serum phosphate was increased 30.1% and 27.4% separately. Calcium and phosphate disorder of fgfr1OC-CKO mouse could be rescued by constitutively activated ALK3 in differentiated osteoblast.4. FGFR1 indirectly promotes osteoclast function, ALK3 active osteoclast function.Compared with control mice, fgfr1OC-CKO mice showed decreased osteoclast number and osteoclast size. Activation of ALK3 could turn over this situation.5. FGFR1 can regulate osteoblast proliferation and apoptosis through PI3KWestern blot results showed that p85 expression, the p-ERK, p-p38 and p-SAPK-JNK level were increased in fgfr1OC-CKO mice primary calvarial osteoblast. And the apoptosis of osteoblast lacking FGFR1 was decreased by TUNEl, the proliferation was in creased.6. FGFR1 can regulate BMPRIA and BMP4 expression in osteoblastGene expression and transcription detection results showed that BMP4 transcription and BMPRIA transcription and expression were down regulated in the osteoblast lacking of FGFR1.7. FGFR1 signaling regulate bone formation through MAPK signalingWestern blot results showed that phospho-p38, phospho-ERK and phospho-SAPK/JN were deceased, but the ERK level did not changed in fgfr1OC-CKO mice primary calvarial osteoblast. This suggested that FGFR1 can affect bone formation through MAPK signaling.Conclusions:1. Disruption of FGFR1 in differentiated osteoblast-specific led to mice tibia spontaneous fracture, decreased biomechanical properties, and could be rescued by constitutively activated ALK3 in differentiated osteoblast. 2. FGFR1 promotes apoptosis through PI3K and MAPK, and FGFR1 inhibits osteoblast proliferation and mineralization.3. FGFR1 inhibits serum phosphate and calcium metabolism by promoting ALK3 signaling.4. FGFR1 deficiency in osteoblast led to decreased osteoclast activity, and could be rescued by constitutively activated ALK3 in differentiated osteoblast.更多还原