【作者】 宫伟雁；

【导师】 林守清；

【作者基本信息】 中国协和医科大学 ， 妇产科学， 2007， 博士

【摘要】 背景:骨膜下成骨作用能够使长骨外径增粗,皮质骨量增加,对增强骨强度,减少骨折发生具有重要意义;并且骨膜成骨细胞对雌激素、甲状旁腺素、机械应力等调节骨形成的因素具有独特的反应性。以骨膜下成骨细胞为靶点的药物,可能对发现新的抗骨质疏松症治疗方案具有重要意义。但长久以来,骨膜下成骨细胞分化、参与骨形成的分子机制尚不清楚,对其在抗骨质疏松症药物治疗中的作用关注甚少。本实验室一直关注骨膜成骨细胞在骨形成中的作用。前期建立了骨外膜细胞的体外培养模型,研究了雌激素对骨膜成骨细胞的作用特点;构建了骨膜cDNA文库和雌激素作用前后骨外膜细胞差异表达基因文库,并完成了两个文库的大规模的测序。目的:本研究利用骨膜cDNA文库和雌激素作用前后骨外膜细胞差异表达基因文库的基因序列信息,对骨膜成骨细胞中表达的未知功能基因进行了克隆和筛选,并详细研究感兴趣基因在骨组织中的表达规律,初步探索其在成骨细胞骨形成中的作用,从而可以深入了解骨膜下成骨细胞骨形成机制、寻找新的促进骨形成的药物打下基础。方法:第一部分、骨膜成骨细胞中新功能基因高通量的筛选(1)基因克隆:目的基因序列来源于骨膜cDNA文库和雌激素作用前后骨膜细胞差减库,以PCR方法扩增未知功能基因蛋白编码区并构建真核表达载体(2)利用双荧光素酶报告基因系统筛选影响信号通路的基因:利用共转染技术,将萤火虫荧光素酶报告基因载体(pAP1-luc/pNFAT-luc/pCRE-luc/pNFkB-luc/pStat-luc)、内参照水母荧光素表达载体、未知功能基因表达载体转染入工程细胞293T细胞中,检测无信号通路刺激物和有信号通路刺激物时,未知功能基因对萤火虫荧光素酶报告基因转录活性的影响第二部分、METRNL生物信息学分析对上一步发现的能够影响信号通路的基因METRNL进行启动子结构、蛋白结构、保守序列比对等信息分析第三部分、METRNL细胞及组织表达定位(1) METRNL基因原核蛋白表达、纯化及多克隆抗体制备(2)蛋白细胞定位:通过pEGFP荧光报告基因法和间接免疫荧光检测METRNL蛋白在MG63细胞中的定位;通过western blot检测转染METRNL的CHO细胞培养上清中METRNL蛋白的表达情况(3)组织表达:利用northern blot检测多种组织中METRNL mRNA的表达情况第四部分、METRNL在骨组织表达特点及其对成骨功能影响(1)骨组织表达:利用免疫组织化学法,检测不同年龄大鼠骨组织中METRNL蛋白的表达规律(2)成骨功能影响:建立稳定过表达METRNL的MG63细胞,检测在成骨诱导条件下矿化结节形成、成骨相关标记物ALP、OPG的表达情况。结果:第一部分:成功建立5个未知功能基因编码区的真核表达载体;通过双荧光素酶报告基因系统筛选发现METRNL号基因即METRNL与报告基因共转染后,未加刺激物时AP1的转录活性与对照组相比下降达74%,加入信号通路刺激物后,同样能够显著抑制AP1的转录活性:另外,METRNL也可以显著抑制转录因子NFAT和CRE的转录活性。第二部分:信息分析显示,METRNL蛋白有311氨基酸组成,N端45个氨基酸可能是信号肽,METRNL可能是一种分泌性蛋白;蛋白功能预测METRNL不存在与已知蛋白模体或序列相似的结构第三部分:(1)利用原核表达体系成功纯化了METRNL蛋白83—311位氨基酸组成的多肽片段,免疫大白兔后获得多克隆抗体,抗体效价达1:5×10~4。(2)细胞定位研究显示MG63细胞中,METRNL主要出现于细胞浆中,胞核无表达;转染METRNL的CHO细胞培养上清,经过浓缩后能够检测到METRNL蛋白的存在,提示METRNL是分泌性蛋白。(3) Northern blot结果显示,METRNL的mRNA约长1500bp,可在成人脑、心、肝脏、肾脏、脾脏、卵巢、子宫、骨骼肌等8个组织中表达,但表达量较低第四部分:(1)骨组织免疫组化显示,METRNL皮质骨和松质骨骨表面的成骨细胞中,METRNL呈阳性表达。随着成骨细胞分化成熟,被骨基质包埋,成为骨细胞后,METRNL表达降低,甚至呈阴性表达;生长板肥大软骨细胞中METRNL染色阳性,而增殖软骨细胞无表达。不同年龄段大鼠相比较发现,随着年龄增加,METRNL表达呈下降趋势。提示METRNL主要表达于活跃骨形成部位,可能参与调控骨形成(2)过表达METRNL的MG63在成骨诱导培养液条件下,骨结节形成数较转染空载体的对照组降低;成骨细胞早期标记物ALP的量在诱导后持续增加,不随时间延长而降低;而成熟成骨细胞标记物OPG,则较对照组明显降低。提示METRNL基因过表达使MG63处于活跃的骨基质形成期,但未能进一步向骨基质矿化期进展。结论:(1) METRNL是成骨细胞分泌的一种新的功能蛋白(2) METRNL可能通过AP—1、NFAT、CRE等多条信号通路发挥作用(3) METRNL主要表达于活跃骨形成部位,可能参与调控骨形成(4) METRNL可能促进成骨细胞分泌骨基质,表达过度时能抑制成骨细胞的进一步成熟(5)推测METRNL在成骨细胞、软骨细胞前体向成熟分化这一阶段发挥调节作用;有可能作为新的药物作用靶点。更多还原

【Abstract】 Bankgroud:periosteal bone apposition increase the bone diameter,cortical bone thickness,and is important for the bone strength,reducing the bone fracture;and Compared to endosteal osteoblasts,periosteal osteoblasts exhibit greater mechanosensitivity to strain,a lower threshold of responsiveness to parathyroid hormone,higher levels of expression of estrogenαreceptors.So the new drug targeting the periosteum may be an alternate strategy to protect human bones from fracture.But for a long time,the molecular mechanism of periosteal osteoblasts differentiation and function is unclear.The response of periosteam to anti-osteoporosis drug is unknown.We have paid attention to the periosteal osteoblast for a long time.Previous we established establish the in vitro culture model ofperiosteal cells,and studied the response of periosteal osteoblasts to estrogen.We construct periosteum cDNA library and the. differently expressing genes library(SSH library) of human periosteal cells between estrogen and control treatment.Objective:Now we used Dual-luciferase reporter system to screen for unknown function genes expression in periosteum,and studied the expression pattern of the interesting gene in bone and explored the gene’s function in osteoblasts.Methods:Part 1:high-throughput screening of novel gene expressing in perioteal cells that could influence the signal pathway(1) Gene clone:The target gene sequence originates from the periosteum eDNA library and the SSH library.PCR methods were used to expand the ORFs area and cloned into eukaryotic expression vector pCDNA3.1(2) Using dual luciferase reporter gene system to screen new function gene that c ould influence signal pathway:the Reporter plasmid(pAP1-luc/pNFAT-luc/pCRE -luc/pNFkB-luc/pStat-luc),which codes a firefly luciferase under the control of pAP1-luc/pNFAT-luc/pCRE-luc/pNFkB-luc/pStat-luc consensus sequence in the p romoter region:an internal control vector pRL-TK and the target gene in pCD NA3.1 were co-transfected into 293T cell,activity of both Firefly and Renilla luciferase are measured to identify the new gene that could influence the sig nal pathwayPart 2:Bioinformatics analysis of METRNLPart 3:the location in MG63 and the expression pattern of METRNL(1)Prokaryotic expression of the polypeptide 83-311 and its antibody preparation(2) cell localization:the pEGFP fluorescence reported gene and Indirect Immunofluorescence methods were used to study the localization of METRNL in MG63; western blot was used to exam the protein in CHO cell transfected with METRNL expression vector(3)using northern blot to exam the mRNA level of METRNL in multiple tissue membranePart 4:the expression pattern of METRNL in bone and its function in osteoblast(1)Immunohistochemistry was used to identify the location and distribution of METRNL in the bone of different age SD rat(2)MG63 cells were stable transfected with pEGFP-METRNL to overexpress the METRNL,the number of mineralization node in osteogenic supplement was compaired with control.And the difference of ALP,OPG was also compared.Result:part 1:we succeeded in cloning five new genes expressing in periosteum osteoblasts into pCDNA3.1.After screening with Dual-luciferase reporter system,we find that METRNL cotransfected with cis-reporter vector can significantly inhibit the transcription activity of AP-1 by 74%when without stimulating as compared with control;after stimulated with PMA and ionomycin,the activity of AP-1 are also inhibited.In addition,the transcription activity of CRE and NFAT are also inhibitedpart 2:Bioinformatics analysis show that human METRNL mRNA contains an open reading frame that encodes a protein of 311 amino acids.The encoded amino-acid sequence showed no significant homology to the sequence of any other previously described protein except the meteorin with 42%homology,suggesting that METRNL is an novel protein.The predicted protein contains a putative signal peptide in its NH-terminal region.Part 3:(1)both the fusion protein EGFP-METRNL and the Indirect Immunofluorescence result shows that the METRNL located in cytoplasm of MG63.After purified the protein from CHO culture medium,we can detected the METRNL by western blot,so METRNL is a secreted protein.(2)Northen blot show that the METRNL mRNA is about 1500 bp,expressing in adult brain,heart,liver,kidney,spleed,ovary,muscle and uterus,but the expression level is very lowPart 4:(1)osteoblasts lining the trabecular and periosteal bone surface express high level of METRNL.In bone,expression of METRNL is highest in embryos and neonates,and decreasing steadily with age to very low levels in 12 month-old long bones.(2) the MG63 cells overexpressing METRNL shows lower mineralization node after 14 days inducing by osteogenic supplement,but the ALP level is higher and the OPG level is lower as compared with control after inducedConclusion:METRNL is a novel secreted proteinMETRNL maybe display its function through AP1,NFAT and CRE.METRNL mainly express in actcive osteoblasts,maybe involve in controlling bone formation.METRNL can significantly enhance the matrix production,but overexpression can inhibit the further differentiation更多还原