【作者】 刘志君；

【导师】 陈林；

【作者基本信息】 第三军医大学 ， 野战外科学， 2007， 博士

【摘要】 背景与目的骨骼的形成和重建是一个由多种生长因子、细胞外基质以及细胞之间相互作用的生理发育过程。其主要通过软骨内成骨和膜内成骨两种方式来完成。在软骨内成骨过程中,首先是间充质干细胞的密集,然后依次分化为骨祖细胞和软骨细胞,形成软骨样骨胚,破骨细胞侵入吞噬,血管延伸进入,最后被含有成骨细胞的骨组织取代;但在膜内成骨时,间充质干细胞则经骨祖细胞直接分化为成骨细胞,没有经过软骨形成阶段而直接形成骨。目前普遍认为骨形成和重建实际上是软骨内成骨和膜内成骨这两种方式相互协调、相互依赖、相互辅助的过程。在此过程中,有许多因子参与并调控着骨细胞的增殖、分化与迁移。近年来的研究表明,成纤维细胞生长因子受体3(Fibroblast growth factor receptor 3,FGFR3)是骨骼形成和重建过程中的重要调控因子。FGFR3属于酪氨酸激酶受体家族成员,FGFR3基因突变导致软骨发育不全或低下、骨关节病及易骨折等骨骼疾病。目前有关FGFR3对软骨细胞的调节、影响的研究,已有较多报道,如FGFR3基因敲除小鼠骨骼过度增长、生长区内软骨细胞的增殖活性增高,提示FGFR3抑制软骨细胞增殖和分化。但其对成骨细胞的形成,以及在成骨细胞的分化与增殖过程中的作用,尚有待进一步阐明。Chen等人在研究中发现FGFR3功能增强小鼠颅缝提前钙化、膝关节茜素红染色显示骨领增加、成骨细胞分化相关基因骨桥蛋白(osteopontin,OP)、骨钙素(osteocalcin,OC)与骨连素(osteonectin,ON)在长骨骨小梁处表达增强,提示FGFR3参与成骨细胞的分化。最近Valverde-Franco等人在研究中发现FGFR3基因敲除小鼠的骨皮质变薄,比正常小鼠更易于骨折,且骨髓间充质干细胞(Mesenchymal stem cells,MSCs)体外成骨能力下降。针对FGFR3相对特异性配体FGF2,FGF9,FGF18的研究亦发现,FGF9或FGF18基因敲除小鼠胚胎发育中期出现钙化延迟;FGF2基因敲除小鼠成年后可出现小梁骨量的下降,体外分离培养其MSCs,发现其矿化能力显著下降。提示FGFR3在成骨细胞分化过程中起着重要的调节作用。MSCs是骨组织发育形成和重建过程中成骨细胞分化的主要来源,它们首先分化为成骨祖细胞,再渐次分化为前成骨细胞与成骨细胞。通过干预FGFR3探讨其对MSCs成骨分化的影响,明确FGFR3在骨形成和重建过程中作用,有望为临床通过干预FGFR3治疗相关疾病提供理论基础。因此,本研究在成功获得pAdE-FGFR3、pAd-DNR3(Dominant-negative,DN)重组腺病毒载体和FGFR3 RNAi载体的基础上,将pAdE-FGFR3、pAd-DNR3与FGFR3 RNAi分别转染入MSCs,从正反两方面观察FGFR3对成骨分化标志基因CbfaⅠ与CollagenⅠ的影响;应用FGFR3功能增强型小鼠模型,分离培养FGFR3功能增强型小鼠MSCs,观察其体外成骨分化能力;并将MSCs复合DBM诱导培养后移植裸鼠皮下,观察FGFR3功能增强对MSCs体内成骨分化的影响。研究方法与结果本研究采用过表达、显性负调节以及RNAi干扰技术干预FGFR3的表达,从正反两方面观察其对MSCs成骨分化标志基因CbfaⅠ与CollagenⅠ的表达影响。同时利用FGFR3功能增强型小鼠模型,研究FGFR3功能增强对MSCs的体内和体外成骨分化能力的影响,探讨FGFR3在MSCs成骨分化中的作用,为阐明FGFR3在成骨细胞分化过程中的作用以及应用FGFR3治疗骨骼疾病打下基础。主要结果如下:1.成功构建并包装了pAdE-FGFR3与pAd-DNR3重组腺病毒表达载体。通过酶切与PCR方法分别获取FGFR3 cDNA与显性负调节FGFR3 cDNA,测序后将其亚克隆至穿梭质粒pAdTrack-CMV的多克隆位点,重组后进行酶切鉴定。PmeⅠ线性化并纯化后,将其转化入感受态细胞BJ5183,扩增后提取质粒。采用PacⅠ酶切进行鉴定并测序鉴定重组腺病毒质粒构建正确。将测序正确的重组腺病毒质粒经PacⅠ线性化、纯化,在脂质体Lipofectamin 2000的介导下,转染HEK293细胞进行包装。并分别将pAdE-FGFR3与pAdE-FGFR3D腺病毒成功感染HT29与HCT116细胞,鉴定二者的表达。结果表明:本实验成功构建并包装了pAdE-FGFR3与pAd-DNR3重组腺病毒表达载体。2.观察到FGFR3可促进MSCs成骨分化标志基因CbfaⅠ与CollagenⅠ的表达分别将重组腺病毒pAdE-FGFR3、pAd-DNR3与FGFR3 RNAi载体转染入骨髓间充质干细胞MSCs。荧光显微镜下可见转染腺病毒后的MSCs胞内呈现报告基因产物的绿色荧光;半定量RT-PCR检测FGFR3 mRNA的表达结果显示:FGFR3 RNAi组细胞无FGFR3 mRNA的表达;pAdE-FGFR3与pAd-DNR3组MSCs表达丰度是对照组3倍。成骨分化标志基因CbfaⅠ与CollagenⅠmRNA的检测结果表明:pAdE-FGFR3组MSCs CbfaⅠ与CollagenⅠmRNA的表达水平均比其他各组高(P＜0.05);Western blot分析结果同样显示转染pAdE-FGFR3组CbfaⅠ与CollagenⅠ蛋白的表达水平显著高于其他各组(P＜0.05)。FGFR3 RNAi组RT-PCR与免疫荧光检测以及Western blot分析均未见CollagenⅠmRNA与蛋白的表达;pAd-DNR3组RT-PCR与Western blot检测虽可检测到CbfaⅠ与CollagenⅠmRNA和蛋白的表达,但都比对照组低。从正反两方面观察到FGFR3不仅可显著提高MSCs CbfaⅠ与CollagenⅠ的转录水平,而且可显著提高MSCs CbfaⅠ与CollagenⅠ的翻译水平。3.FGFR3功能增强可促进MSCs的体内、体外成骨分化能力分离培养FGFR3功能增强小鼠的MSCs,扩增后进行生长曲线描绘。生长曲线显示FGFR3功能增强小鼠MSCs潜伏期较同窝野生鼠MSCs稍长。在成骨诱导实验检测中,钙钴法染色与ALP表达检测均表明FGFR3功能增强小鼠MSCs ALP的表达与活性均较对照组高(P＜0.05);茜素红矿化结节染色结果显示相同的趋势:FGFR3功能增强鼠MSCs的矿化结节较对照组多而大。RT-PCR结果均显示:在诱导后第7d,FGFR3功能增强小鼠MSCs CbfaⅠmRNA的表达与对照组无显著性差异(P＞0.05):在第14d,FGFR3功能增强小鼠MSCs CbfaⅠmRNA的表达显著高于对照组(P＜0.05)。为进一步观察FGFR3功能增强对MSCs成骨的影响,本实验将扩增后的FGFR3功能增强小鼠MSCs与脱钙骨基质DBM复合培养后,移植裸鼠皮下,观察其体内成骨能力。X摄片结果显示:FGFR3功能增强组细胞复合物形成的新生骨组织比对照组稍大,切片HE染色结果亦表明FGFR3功能增强组形成的新生骨组织较多,甚至可见典型的膜内成骨现象。本研究成功构建了pAdE-FGFR3、pAd-DNR3重组腺病毒质粒,并将他们转染入MSCs,使之高表达FGFR3或DNFGFR3。首次从正反两方面观察了干预FGFR3对MSCs成骨分化的影响,同时利用FGFR3功能增强型小鼠模型,研究FGFR3功能增强对MSCs的体内和体外成骨分化的影响,探讨FGFR3在MSCs成骨分化中的作用。实验结果提示FGFR3过表达和功能增强可能通过提高MSCs成骨分化标志基因CbfaⅠ与(或)CollagenⅠ的表达并促进MSCs的成骨分化,为阐明FGFR3在成骨细胞分化过程中的作用以及应用FGFR3治疗骨骼疾病提供了实验资料。更多还原

【Abstract】 Backgound and ObjectiveThe skeleton is generated by two distinct mechanisms,intramembranous and endochondral ossification;either pathway is modulated by the secreting signaling molecules including FGF,TGF-βand IGF,and cell and extracellular matrix^[1].In endochondral ossification,mesenchymal stem cells differentiate into chondrocytes,and then convert to cartilage,invaded by blood vessels and osteoclasts.Osteoblasts remodel the newly generated cartilage thus forming bone tissue.This mechanism accounts for the formation of the vertebrate and long bones^[2].During intramembranous ossification, mesenchymal stem cells convert directly into bone-forming osteoblasts,which secrete bone matrix proteins.This pathway is responsible for the generation of skull flat bones^[3,4]These two pathways often interact during the bone development.For example,in the process of long bone development,the growth mainly consists of a series of chondrocytes proliferation,differentiation and apoptosis,while the thickness of cortical bone depends on intramembranous ossification.On the contrary,cranial growth mainly depends on intramembranous ossification.Fibroblast growth factor receptor 3（FGFR3） is a member of transmembrane tyrosine kinase receptors family（FGFR1-5）,which play an important role in skeletal development. Evidence from human and mouse genetics indicates the role of FGFR3 in bone development.Mutant of the transmembrane domain of FGFR3 results in heritable skeletal dysplasias in human,such as dwarfing chondrodysplasias（hypochondroplasia, achondroplasia,severe achondroplasia with developmental delay and acanthosis nigricans—also known as ’SADDAN’—and thanatophoric dysplasia）.Based on mutant models,Several reports confirmed that FGFR3 has negative effects on signal transduction regulating growth,differentiation,migration and apoptosis of chondrocyte.Mouse lacking FGFR3（FGFR3-/-） showed the skeletal overgrowth accompanied by increased chondrocyte proliferation,and hypertrophic chondrocyte zone^[5-7].These results suggest that FGFR3 regulates bone growth by inhibiting the growth of chondrocytes.In 2004,investigations demonstrated FGFR3 gene disruption in adult mice resulted in osteopenic due to the reduction of cortical bone thickness and defective trabecular bone mineralization.FGFR3-/- mice MSCs expressed the markers of differentiated osteoblasts but fewer mineralized nodules developed.Studies on FGFR3 Gly369Cys mutant mouse model revealed that genes relating to osteoblast differentiation, such as osteopontin,osteonectin,and osteocalcin are upregulated,moreover,alizarin Red-S staining exhibited an advanced bone collar in mutant mice.These findings suggest the important role of FGFR3 in the process of osteoblasts differentiation^[8].Other researches from ligand（FGF2,FGF9,and FGF18） knockout mice demonstrated that FGFR3 participates in osteoblast differentiation.Mice lacking FGF9 or FGF18 showed the delayed ossification ^[9-12].FGF2 disruption led to the loss of trabecular bone volume and MSCs defective mineralization.^[13].Inactivating FGFR1 in osteo-chondro-progenitor cells increased the proliferation,and delayed osteoblast differentiation,but accelerated the differentiation of osteoblasts,accompanied with an increased expression of FGFR3^[14].All these findings manifest that FGFR3 signaling impacts on osteoblasts differentiation. However,the function of FGFR3 on osteoblast differentiation remains unclear.Mesenchymal stem cells（MSCs）,deriving from mesoderm,have the potential of differentiation into osteoblasts,chondrocytes etc.During bone development and the process of bone repair,MSCs differentiate into precursor,pre-osteoblast and osteoblast by turns.At present,MSCs have been regarded as the main source of ostoblast cells.To explore the role of FGFR3 in differentiation of MSCs to osteoblast will better understand the role of FGFR3 in bone development and remodeling and clarify the function of FGFR3 on osteoblast differentiation.Therefore,adenovirus pAdE-FGFR3 and pAd-DNR3（Dominant-negative FGFR3）were constructed in this study and their expressions were verified using RT-PCR and Western blotting analysis.The transcriptional and translational level of the osteoblast marker genes CbfaⅠand CollagenⅠwere detected after pAdE-FGFR3,pAd-DNR3 and FGFR3RNAi transfected into MSCs.MSCs from gain of function FGFR3 mutant mice were separated and cultured,and the differentiation capability of MSCs to osteoblast was investigated in vitro and in vivo. Methods and ResultsFGFR3 and DNR3 recombinant adenovirus were constructed and then transferred into MSCs.Using the FGFR3 Gly369Cys mutant mouse model,the primary MSCs were cultured and differentiate into osteoblast in vivo and in vitro.The main results are as follows.1.FGFR3 and DNR3 recombinant adenovirus were constructed using the pAdEasy system.FGFR3 and DNR3 were cleaved by restrictive enzyme and inserted into pAdtrack-CMV shuttle vector.Recombinant vector was constructed and transferred into E. coli BJ5183.HEK 293 cell were transfected to pack viral particles.Viral particles infected HT-29/HCT 116 cell lines.The expressions of FGFR3 and DNR3 were analyzed by semi-quantify RT-PCR and western blotting.The titer of virus was detected by TCID50. GFP report gene showed the titer of virus was 3×10⁹ pfu/ml and 3.8×10⁹ pfu/ml, respectively.2.Effect of FGFR3 on the transcriptional and translational level of CbfaⅠand CollagenⅠin MSCs.FGFR3,DNR3 adenovirus and FGFR3RNAi were transfected into MSCs,the expression of FGFR3 was determined by RT-PCR.The transcriptional and translational level of the osteoblast marker genes,collagenⅠand CbfaⅠ,were detected using semiquantify RT-PCR,immunofluorescent and western blot.The overexpression of FGFR3 obviously increased the transcriptional and translational level of collagenⅠand CbfaⅠin MSCs.However,DNR3 adenovirus and FGFR3 RNAi significantly decreased the transcriptional and translational level of collagenⅠand CbfaⅠin MSCs.These findings indicated that FGFR3 can upregulate the expression of Cbfa 1 and collagenⅠin MSCs.3.The gain of function mutation of FGFR3 accelerated the osteoblast differentiation of MSCs in vitro and in vivo.Mutant mice MSCs were isolated and cultured,the 3th passage（passage 3） cell growth curve was drawed.Compared with the control MSCs,the latency of mutant MSCs was longer,suggesting that mutant cells grew more slowly.Cells with FGFR3 mutation cultured in differentiation medium for 7,14 and 21 days stained more intensely for ALP and contained more alizarin Red-S positive mineralized nodules.Semi-quantitative RT-PCR analysis showed CbfaⅠexpressed at a similar level at day 7（P＞0.05）,but at a remarkable higher level at day 14（P＜0.05）.Mutant MSCs combined with DBM were implanted into the back subcutaneouly of nude mice.After 8 weeks,X-ray images showed that the formation of bone,histology examination manifested trabecular bone formation.These results indicate that gain of function mutation of FGFR3 accelerated the differentiation of MSCs to osteoblast in vivo.Conclusion1.FGFR3 and DNFGFR3 recombinant adenovirus were prepared and could efficiently infect cells.2.FGFR3 and DNFGFR3 recombinant adenovirus and FGFR3 RNAi were successfully transferred into MSCs3.The overexpression of FGFR3 in MSCs upregulated the transcriptional and translational level of the osteoblast marker genes,collagenⅠand CbfaⅠ.4.The gain of function mutation of FGFR3 accelerated the differentiation of MSCs to osteoblast in vitro and in vivo.This study provides a critical data to clarify the role of FGFR3 signaling in osteoblast differentiation and lay an experimental foundation for gene therapy.更多还原