VEGF基因转染对大鼠骨髓间充质干细胞生物学功能的影响
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摘要
外伤、先天畸形、口腔颌面部肿瘤术后等原因常造成颌骨组织的缺损,严重影响患者的颜面部美观、导致咀嚼功能下降,给患者造成心理负担,从而影响患者的生活质量,给患者带来身心痛苦。在临床上,骨缺损的治疗一直是临床医师常见的、难以解决的、棘手的问题。目前主要采用自体骨、异体骨、人工合成替代物来进行修复,尽管这些方法可在一定程度上恢复颌骨的功能和外形,但都存在一定的局限性,如:来源有限、供体部位继发损伤、引起免疫排斥反应、塑形性差等,不能完全满足临床上治疗的需要。随着分子生物学、细胞生物学、组织工程和基因工程理论与实验技术的发展,组织工程骨成为颌骨修复重建的新方法,组织工程骨包含了3个要素:①间充质干细胞和骨祖细胞的分离和扩增;②合适的成骨生长因子;③模拟促进成骨的内环境的支架。但外源性的成骨生长因子存在着有半衰期、需要反复给药、易流失、效率低、价格昂贵等缺点。基因强化组织工程骨就是将外源性的编码成骨生长因子的基因转染靶细胞(种子细胞),使成骨生长因子在细胞内稳定、持续、高效地表达并分泌到局部发挥作用,从而促进颌骨的修复和重建,成为解决该问题的新思路。
组织工程骨的血管化也是影响较大体积组织工程骨是否成功的关键问题,VEGF是一个强有力的促进血管生成的细胞因子,具有内皮细胞特异性有丝分裂原活性,在体内能够刺激血管的发生,增加血管的渗透性。在骨组织的发育、骨折愈合和骨缺损的修复中通过促进血管的生成而间接发挥作用。所以VEGF可以成为组织工程血管化的候选生长因子。VEGF除了促进血管的生成而间接发挥作用外,对参与骨修复的细胞如骨髓间充质干细胞是否发挥作用以及其机制尚不清楚。
骨髓间充质干细胞是骨髓内含有的非造血干细胞,具有多向分化潜能,在特定的条件下能够向成骨、成软骨、成肌细胞等方向分化,容易获取,来源丰富,并且外源基因易于导入细胞和表达的优点,是有前景的用于骨组织工程的种子细胞。
腺病毒载体是具有良好前景的一种高效基因转移载体,腺病毒本身分子稳定,不与宿主基因组整合,没有基因毒性,不会引起插入突变,对靶细胞的病理损伤较小,分裂期和静止期细胞都可以感染,感染效率高,已经在临床和临床前研究中广泛的应用。腺病毒载体的基因表达仅维持几周或几个月,随着靶细胞的死亡而消失。这点对于骨的修复和重建是有利的,因为只希望发挥促进成骨作用的蛋白在骨愈合的特定时间里表达和保持较高的浓度。
基于以上背景,本研究拟以腺病毒为载体,用VEGF基因转染体外培养扩增的大鼠骨髓间充质干细胞,研究VEGF对大鼠骨髓间充质干细胞生物学行为的影响,并阐述其机制。
首先,采用全骨髓贴壁法从大鼠骨髓中提取、分离和培养大鼠骨髓间充质干细胞(BMSCs),细胞培养基为含体积分数10%胎牛血清、100U/ml青霉素、100μg /mL链霉素的L-DMEM培养基,在培养48小时首次换液,细胞贴壁生长,为多角形、梭形,并有3-5个细胞突起,随着培养时间延长,细胞逐渐增多,呈漩涡状、辐射状排列,并形成集落。培养至80-90%汇合时0.25%胰酶+0.02%EDTA传代。第三代细胞在成骨诱导培养基(含抗坏血酸50mg/L、β-甘油磷酸钠10mmol/L、地塞米松10~(-8)mol/L)内培养21天,茜素红染色,有矿化结节形成。在成脂诱导培养基(含IBMX0.5mmol/L、地塞米松10μmol/L、胰岛素10μmol/L、消炎痛200μmol/L)内培养14天,油红O染色有脂滴形成。结果表明体外成功培养和扩增了大鼠BMSCs,在特定条件下能够向成骨细胞分化,为以后的实验提供了细胞基础。
第二,将携增强型绿色荧光蛋白的腺病毒载体(AdCMV-EGFP)以不同的MOI(0、100、200、400、600、800、1000particle/cell)转染第3代大鼠BMSCs。结果为AdCMV-EGFP在0、100、200、400时转染效率随MOI的增加而逐渐增加,并且细胞的形态没有变化,在MOI大于400时转染效率不再增加,细胞出现变圆、脱落。在荧光倒置显微镜下观察细胞在转染后的24小时就有EGFP的表达,5-7天时达到高峰,以后逐渐减弱,在转染后28天时仍有较弱的荧光。经AdCMV-EGFP转染后的细胞在成骨诱导培养液内培养21天后,茜素红染色,矿化结节形成的数量和大小与未转染组没有明显区别。结果表明AdCMV-EGFP能够有效的转染大鼠BMSCs,并且在一定的MOI范围内对细胞的形态和增殖没有影响,而且也不影响BMSCs的成骨分化能力。
第三,按照上述确定的合适MOI(400particle/cell)携VEGF的腺病毒载体(AdCMV-VEGF)转染大鼠BMSCs,采用RT-PCR方法检测细胞内有VEGFmRNA的表达,转染后24小时采用ELISA方法在细胞培养上清中就检测到有VEGF的表达和分泌,在转染后第3天达到高峰,以后逐渐下降,并稳定表达一定时间。MTT比色法实验检测AdCMV-VEGF转染对大鼠BMSCs增殖的影响。结果表明AdCMV-VEGF能够成功转染大鼠BMSCs,并且VEGF在mRNA和蛋白水平均有表达,并能分泌到细胞外,分泌的VEGF能够促进大鼠BMSCs的增殖。
第四,按照上述确定的合适MOI(400 particle/cell)AdCMV-VEGF转染大鼠骨BMSCs,并对细胞进行成骨诱导培养,在成骨诱导后6、9、12天进行碱性磷酸酶活性检测,在第6天时没有差异,在第9、12天转染组细胞的碱性磷酸酶活性高于未转染组(p<0.05)。在3、6、9、12天采用ELISA方法检测培养上清中OCN和COL?的含量,随着时间延长两组的OCN和COL?的含量均逐渐增高,OCN在第12天和COL?在第6、9、12天转染AdCMV-VEGF组的含量高于未转染组和转染AdCMV-EGFP组。在成骨诱导后3、7、14、21天采用实时定量PCR检测细胞COL?、OCN、RUNX2和OSX的表达,在第3天时4个基因的表达在转染AdCMV-VEGF组均高于未转染对照组,表明转染AdCMV-VEGF能够促进大鼠骨BMSCs成骨相关基因的表达。
最后,骨组织发育和修复是一个多种生长因子参与的过程,因此本研究还用AdCMV-VEGF和AdCMV-BMP2共同转染大鼠BMSCs,从形态学角度观察细胞的变化,并检测VEGF和BMP2蛋白的表达情况。RT-PCR实验凝胶电泳显示有VEGF和BMP2的目的条带,细胞在转染后24小时采用ELISA方法可在培养上清液中检测到VEGF和BMP2的表达,在转染后第3天达到高峰。形态学观察共同转染组细胞聚集形成结节的数目和面积大于单独AdCMV-BMP2转染的细胞。结果表明AdCMV-VEGF和AdCMV-BMP2共同转染大鼠BMSCs,并且VEGF和BMP2基因在细胞内能够转录和表达,AdCMV-VEGF转染能够促进AdCMV-BMP2转染引起的BMSCs的成骨方向分化。
本研究采用腺病毒载体介导VEGF转染BMSCs,研究了腺病毒载体转染BMSCs的可行性,以及VEGF转染对BMSCs功能的影响,为构建血管化组织工程骨提供实验基础,本研究的创新点在于将腺病毒为载体的VEGF转染用于组织工程骨的研究,以及体外研究VEGF对BMSCS成骨分化的作用。
Cranialofacial bone regeneration needs to be enhanced for repairing large bone defects secondary to tumor, congenital malformations or trauma and for treating fracture-delayed unions or nonunions. The approach of bone regeneration includes autograft, allogenic graft, and artificial substitutes graft. But But there are some shortage such as: limited source, secondary trauma, immunological rejection, poor plasticity. With the development of the molecular biology, cellular biology and gene engineer, bone tissue engineer have become the focus to resolve the problem. Bone tissue engimmer have three factors:1)mesenchymal stem cell or osteoprogenitor cells isolated and expanded; 2)growth factor; 3)scaffold. But the administration of growth factor had been limited by the half life span, low efficiency, easy lost, high cost, etc. So the gene modified tissue engineer have become the hot spot.
Vascularization of the tissue is a key factor on the success of tissue engineering bone. VEGF, the best-characterized angiogenic factor, promotes the angiogenisis, increase the permeability of vessels. VEGF plays an indirect role in the bone development, bone fracture healing and bone repair by promoting the angionenisis.
VEGF is one of the candidate factors in the vascularization of tissue engineer. But the effect of VEGF on the bone marrow mesenchymal stem cells (BMSCs) is not known clearly.
BMSCs are the non hematopoietic stem cells in the bone marrow. The characteristic of BMSCs is multi-directional differentiation potential. BMSCs can differentiate into the osteoblasts, chondroblasts, myoblasts under the certain conditions. Due to its the easy isolation, abundance source, foreign gene import and expression easily BMSCs have become the promising seed cells in bone tissue engineer.
Adenovirus vector(Ad Vector) is a promising gene transferring vehicle. Adenovirus is stable , without being integrated into the host genome , has no genotoxicity, and cannot induce insertion mutation. Adenovirus vector has little pathologic injury ,can transfect dividing cells and nondividing cells, and has high transfection efficiency. So adenovirus vector have been used in the researches. The duration of gene expression in the AdV is from weeks to months. It is fit to the bone repair and regeneration.
On the basis of above, the aim of this study is to evaluate the effect of VEGF transfection on the rat BMSCs mediated by Ad vector in vitro.
Firstly, rat BMSCs were isolated and expanded in vitro and cultured in conditioned medium to evaluate their differentiation potential. The rat BMSCs were isolated from the bone marrow of rat femurs and tibia by total bone marrow adherence. The cells were cultured in the L-DMEM medium containing 10% fetal calf serum(FCS), 100U/ml penicillin, 100μg /mL streptomycin. When the cells were confluent to 80-90%, the cells were digested with 0.25% trypsin and 0.02% EDTA and then subcultured. The cells were polygon, spindle in shape with 3~5 processes. With the culture went on, the cells were aligned as swirl, radiated and formed colony. The 3rd passage cells were cultured in the conditioned medium containing 50mg/L ascorbic acid, 10mmol/Lβ-glycerophosphate sodium and 10~(-8)mol/L dexamethasone. After 21 days, the cells were stained with Alizarin Red. There are red nodules under the microscope. The 3~(rd) passage cells were cultured in the conditioned medium containing 0.5mmol/L IBMX,10μmol/L dexamethasone,10μmol/L insulin and 200μmol/L indomethacin. After 14 days, the cells were stained with Oil Red O. The red lipid droplets were seen under the microscope. It can be concluded that the rat BMSCs were isolated from the bone marrow and expanded in vitro and can differentiate into osteoblasts and adipocytes. This will provide abundant cells for the following researchs.
Secondly, AdCMV-EGFP was transfected into the rat BMSCs to detect the transfection efficiency and the effect of the AdCMV-EGFP transfection on the BMSCs. The 3~(rd) passage rat BMSCs were transfected with AdCMV-EGFP in different multiplicity of infection(MOI: 0,100,200,400,600,800,1000particle/cell). With the increase of MOI, the transfection efficiency was increased. When MOI was lower than 400particles/cell, the morphology of the cells didn’t change. When MOI was higher than 400particles/cell, the cells became circle and fell off the wells. There were GFP expressions in the cells 24hour after transfection. The intensity and amount of GFP became increasing as time passed. It reached its peak on the 5-7~(th) day. There were still GFP in the cells on the 28th day after transfection. After the transfected cells were cultured in the conditioned medium (containing 50mg/L ascorbic acid, 10mmol/Lβ-glycerophosphate sodium, 10~(-8)mol/L dexamethasone), there were some mineralized nodules with Alizarin Red staining. There wasn’t significant difference between the AdCMV-EGFP transfected cells and untransfected cells. So, AdCMV-EGFP transfection with a range of MOI should not impact on the BMSCs proliferation and osteogenic differentiation potential.
Thirdly, AdCMV-VEGF was transferred into rat BMSCs, then VEGF mRNA and protein expression was detected by RT-PCR and ELISA. The effect of AdCMV-VEGF transfection on the cells was evaluated by MTT assay. After transfection there were VEGF mRNA and protein expression in the cells. The OD value of the AdCMV-VEGF transfected cells is higher than the untransfected cells 3d after transfection (p<0.05). The results show that AdCMV-VEGF have been transferred into rat BMSCs successfully and VEGF gene is expressed, and that VEGF can promote the proliferation of the cells.
Fourthly, AdCMV-VEGF was transferred into rat BMSCs with the MOI of 400particles/cell. Then the transfected cells were cultured in conditioned medium (containing 50mg/L ascorbic acid, 10mmol/Lβ-glycerophosphate sodium, 10~(-8)mol/L dexamethasone). On 6, 9, 12d, ALP level of the transfected cells were higher than untransfected ones(p<0.05). OCN and COLI were detected in the supernatant. OCN on 12d and COLI on 6, 9, 12d of the transfected cells were higher than untransfected ones(p<0.05). COLI, OCN, RUNX2 and OSX were evaluated by real time PCR on 3, 7, 14 and 21d. On 3d COLI、OCN、RUNX2 and OSX of the transfected cells were higher than untransfected ones. The results show that AdCMV-VEGF transfection can promote the osteogenic differentiation of BMSCs.
Finally, many growth factors involve in bone development and bone repairing. Both VEGF and BMP2 are important factors. AdCMV-VEGF and AdCMV-BMP2 were transfected BMSCs. VEGF and BMP2 expression were detected by RT-PCR and ELISA.The mineralized nodules of AdCMV-VEGF and AdCMV-BMP2 transfected ones were more than AdCMV-BMP2 transfected ones. The results show that the foreign gene can be expressed after transfection and AdCMV-VEGF transfection can promote the BMP2 induced osteogenic differentiation.
Tissue engineering bone is a hot spot in the bone defect therapy.The innovation of this study is to study VEGF transfection mediated by adenovirus vector on the study of tissue engineering bone and the effect of VEGF on the osteogenic differentiation of BMSCs.
组织工程骨的血管化也是影响较大体积组织工程骨是否成功的关键问题,VEGF是一个强有力的促进血管生成的细胞因子,具有内皮细胞特异性有丝分裂原活性,在体内能够刺激血管的发生,增加血管的渗透性。在骨组织的发育、骨折愈合和骨缺损的修复中通过促进血管的生成而间接发挥作用。所以VEGF可以成为组织工程血管化的候选生长因子。VEGF除了促进血管的生成而间接发挥作用外,对参与骨修复的细胞如骨髓间充质干细胞是否发挥作用以及其机制尚不清楚。
骨髓间充质干细胞是骨髓内含有的非造血干细胞,具有多向分化潜能,在特定的条件下能够向成骨、成软骨、成肌细胞等方向分化,容易获取,来源丰富,并且外源基因易于导入细胞和表达的优点,是有前景的用于骨组织工程的种子细胞。
腺病毒载体是具有良好前景的一种高效基因转移载体,腺病毒本身分子稳定,不与宿主基因组整合,没有基因毒性,不会引起插入突变,对靶细胞的病理损伤较小,分裂期和静止期细胞都可以感染,感染效率高,已经在临床和临床前研究中广泛的应用。腺病毒载体的基因表达仅维持几周或几个月,随着靶细胞的死亡而消失。这点对于骨的修复和重建是有利的,因为只希望发挥促进成骨作用的蛋白在骨愈合的特定时间里表达和保持较高的浓度。
基于以上背景,本研究拟以腺病毒为载体,用VEGF基因转染体外培养扩增的大鼠骨髓间充质干细胞,研究VEGF对大鼠骨髓间充质干细胞生物学行为的影响,并阐述其机制。
首先,采用全骨髓贴壁法从大鼠骨髓中提取、分离和培养大鼠骨髓间充质干细胞(BMSCs),细胞培养基为含体积分数10%胎牛血清、100U/ml青霉素、100μg /mL链霉素的L-DMEM培养基,在培养48小时首次换液,细胞贴壁生长,为多角形、梭形,并有3-5个细胞突起,随着培养时间延长,细胞逐渐增多,呈漩涡状、辐射状排列,并形成集落。培养至80-90%汇合时0.25%胰酶+0.02%EDTA传代。第三代细胞在成骨诱导培养基(含抗坏血酸50mg/L、β-甘油磷酸钠10mmol/L、地塞米松10~(-8)mol/L)内培养21天,茜素红染色,有矿化结节形成。在成脂诱导培养基(含IBMX0.5mmol/L、地塞米松10μmol/L、胰岛素10μmol/L、消炎痛200μmol/L)内培养14天,油红O染色有脂滴形成。结果表明体外成功培养和扩增了大鼠BMSCs,在特定条件下能够向成骨细胞分化,为以后的实验提供了细胞基础。
第二,将携增强型绿色荧光蛋白的腺病毒载体(AdCMV-EGFP)以不同的MOI(0、100、200、400、600、800、1000particle/cell)转染第3代大鼠BMSCs。结果为AdCMV-EGFP在0、100、200、400时转染效率随MOI的增加而逐渐增加,并且细胞的形态没有变化,在MOI大于400时转染效率不再增加,细胞出现变圆、脱落。在荧光倒置显微镜下观察细胞在转染后的24小时就有EGFP的表达,5-7天时达到高峰,以后逐渐减弱,在转染后28天时仍有较弱的荧光。经AdCMV-EGFP转染后的细胞在成骨诱导培养液内培养21天后,茜素红染色,矿化结节形成的数量和大小与未转染组没有明显区别。结果表明AdCMV-EGFP能够有效的转染大鼠BMSCs,并且在一定的MOI范围内对细胞的形态和增殖没有影响,而且也不影响BMSCs的成骨分化能力。
第三,按照上述确定的合适MOI(400particle/cell)携VEGF的腺病毒载体(AdCMV-VEGF)转染大鼠BMSCs,采用RT-PCR方法检测细胞内有VEGFmRNA的表达,转染后24小时采用ELISA方法在细胞培养上清中就检测到有VEGF的表达和分泌,在转染后第3天达到高峰,以后逐渐下降,并稳定表达一定时间。MTT比色法实验检测AdCMV-VEGF转染对大鼠BMSCs增殖的影响。结果表明AdCMV-VEGF能够成功转染大鼠BMSCs,并且VEGF在mRNA和蛋白水平均有表达,并能分泌到细胞外,分泌的VEGF能够促进大鼠BMSCs的增殖。
第四,按照上述确定的合适MOI(400 particle/cell)AdCMV-VEGF转染大鼠骨BMSCs,并对细胞进行成骨诱导培养,在成骨诱导后6、9、12天进行碱性磷酸酶活性检测,在第6天时没有差异,在第9、12天转染组细胞的碱性磷酸酶活性高于未转染组(p<0.05)。在3、6、9、12天采用ELISA方法检测培养上清中OCN和COL?的含量,随着时间延长两组的OCN和COL?的含量均逐渐增高,OCN在第12天和COL?在第6、9、12天转染AdCMV-VEGF组的含量高于未转染组和转染AdCMV-EGFP组。在成骨诱导后3、7、14、21天采用实时定量PCR检测细胞COL?、OCN、RUNX2和OSX的表达,在第3天时4个基因的表达在转染AdCMV-VEGF组均高于未转染对照组,表明转染AdCMV-VEGF能够促进大鼠骨BMSCs成骨相关基因的表达。
最后,骨组织发育和修复是一个多种生长因子参与的过程,因此本研究还用AdCMV-VEGF和AdCMV-BMP2共同转染大鼠BMSCs,从形态学角度观察细胞的变化,并检测VEGF和BMP2蛋白的表达情况。RT-PCR实验凝胶电泳显示有VEGF和BMP2的目的条带,细胞在转染后24小时采用ELISA方法可在培养上清液中检测到VEGF和BMP2的表达,在转染后第3天达到高峰。形态学观察共同转染组细胞聚集形成结节的数目和面积大于单独AdCMV-BMP2转染的细胞。结果表明AdCMV-VEGF和AdCMV-BMP2共同转染大鼠BMSCs,并且VEGF和BMP2基因在细胞内能够转录和表达,AdCMV-VEGF转染能够促进AdCMV-BMP2转染引起的BMSCs的成骨方向分化。
本研究采用腺病毒载体介导VEGF转染BMSCs,研究了腺病毒载体转染BMSCs的可行性,以及VEGF转染对BMSCs功能的影响,为构建血管化组织工程骨提供实验基础,本研究的创新点在于将腺病毒为载体的VEGF转染用于组织工程骨的研究,以及体外研究VEGF对BMSCS成骨分化的作用。
Cranialofacial bone regeneration needs to be enhanced for repairing large bone defects secondary to tumor, congenital malformations or trauma and for treating fracture-delayed unions or nonunions. The approach of bone regeneration includes autograft, allogenic graft, and artificial substitutes graft. But But there are some shortage such as: limited source, secondary trauma, immunological rejection, poor plasticity. With the development of the molecular biology, cellular biology and gene engineer, bone tissue engineer have become the focus to resolve the problem. Bone tissue engimmer have three factors:1)mesenchymal stem cell or osteoprogenitor cells isolated and expanded; 2)growth factor; 3)scaffold. But the administration of growth factor had been limited by the half life span, low efficiency, easy lost, high cost, etc. So the gene modified tissue engineer have become the hot spot.
Vascularization of the tissue is a key factor on the success of tissue engineering bone. VEGF, the best-characterized angiogenic factor, promotes the angiogenisis, increase the permeability of vessels. VEGF plays an indirect role in the bone development, bone fracture healing and bone repair by promoting the angionenisis.
VEGF is one of the candidate factors in the vascularization of tissue engineer. But the effect of VEGF on the bone marrow mesenchymal stem cells (BMSCs) is not known clearly.
BMSCs are the non hematopoietic stem cells in the bone marrow. The characteristic of BMSCs is multi-directional differentiation potential. BMSCs can differentiate into the osteoblasts, chondroblasts, myoblasts under the certain conditions. Due to its the easy isolation, abundance source, foreign gene import and expression easily BMSCs have become the promising seed cells in bone tissue engineer.
Adenovirus vector(Ad Vector) is a promising gene transferring vehicle. Adenovirus is stable , without being integrated into the host genome , has no genotoxicity, and cannot induce insertion mutation. Adenovirus vector has little pathologic injury ,can transfect dividing cells and nondividing cells, and has high transfection efficiency. So adenovirus vector have been used in the researches. The duration of gene expression in the AdV is from weeks to months. It is fit to the bone repair and regeneration.
On the basis of above, the aim of this study is to evaluate the effect of VEGF transfection on the rat BMSCs mediated by Ad vector in vitro.
Firstly, rat BMSCs were isolated and expanded in vitro and cultured in conditioned medium to evaluate their differentiation potential. The rat BMSCs were isolated from the bone marrow of rat femurs and tibia by total bone marrow adherence. The cells were cultured in the L-DMEM medium containing 10% fetal calf serum(FCS), 100U/ml penicillin, 100μg /mL streptomycin. When the cells were confluent to 80-90%, the cells were digested with 0.25% trypsin and 0.02% EDTA and then subcultured. The cells were polygon, spindle in shape with 3~5 processes. With the culture went on, the cells were aligned as swirl, radiated and formed colony. The 3rd passage cells were cultured in the conditioned medium containing 50mg/L ascorbic acid, 10mmol/Lβ-glycerophosphate sodium and 10~(-8)mol/L dexamethasone. After 21 days, the cells were stained with Alizarin Red. There are red nodules under the microscope. The 3~(rd) passage cells were cultured in the conditioned medium containing 0.5mmol/L IBMX,10μmol/L dexamethasone,10μmol/L insulin and 200μmol/L indomethacin. After 14 days, the cells were stained with Oil Red O. The red lipid droplets were seen under the microscope. It can be concluded that the rat BMSCs were isolated from the bone marrow and expanded in vitro and can differentiate into osteoblasts and adipocytes. This will provide abundant cells for the following researchs.
Secondly, AdCMV-EGFP was transfected into the rat BMSCs to detect the transfection efficiency and the effect of the AdCMV-EGFP transfection on the BMSCs. The 3~(rd) passage rat BMSCs were transfected with AdCMV-EGFP in different multiplicity of infection(MOI: 0,100,200,400,600,800,1000particle/cell). With the increase of MOI, the transfection efficiency was increased. When MOI was lower than 400particles/cell, the morphology of the cells didn’t change. When MOI was higher than 400particles/cell, the cells became circle and fell off the wells. There were GFP expressions in the cells 24hour after transfection. The intensity and amount of GFP became increasing as time passed. It reached its peak on the 5-7~(th) day. There were still GFP in the cells on the 28th day after transfection. After the transfected cells were cultured in the conditioned medium (containing 50mg/L ascorbic acid, 10mmol/Lβ-glycerophosphate sodium, 10~(-8)mol/L dexamethasone), there were some mineralized nodules with Alizarin Red staining. There wasn’t significant difference between the AdCMV-EGFP transfected cells and untransfected cells. So, AdCMV-EGFP transfection with a range of MOI should not impact on the BMSCs proliferation and osteogenic differentiation potential.
Thirdly, AdCMV-VEGF was transferred into rat BMSCs, then VEGF mRNA and protein expression was detected by RT-PCR and ELISA. The effect of AdCMV-VEGF transfection on the cells was evaluated by MTT assay. After transfection there were VEGF mRNA and protein expression in the cells. The OD value of the AdCMV-VEGF transfected cells is higher than the untransfected cells 3d after transfection (p<0.05). The results show that AdCMV-VEGF have been transferred into rat BMSCs successfully and VEGF gene is expressed, and that VEGF can promote the proliferation of the cells.
Fourthly, AdCMV-VEGF was transferred into rat BMSCs with the MOI of 400particles/cell. Then the transfected cells were cultured in conditioned medium (containing 50mg/L ascorbic acid, 10mmol/Lβ-glycerophosphate sodium, 10~(-8)mol/L dexamethasone). On 6, 9, 12d, ALP level of the transfected cells were higher than untransfected ones(p<0.05). OCN and COLI were detected in the supernatant. OCN on 12d and COLI on 6, 9, 12d of the transfected cells were higher than untransfected ones(p<0.05). COLI, OCN, RUNX2 and OSX were evaluated by real time PCR on 3, 7, 14 and 21d. On 3d COLI、OCN、RUNX2 and OSX of the transfected cells were higher than untransfected ones. The results show that AdCMV-VEGF transfection can promote the osteogenic differentiation of BMSCs.
Finally, many growth factors involve in bone development and bone repairing. Both VEGF and BMP2 are important factors. AdCMV-VEGF and AdCMV-BMP2 were transfected BMSCs. VEGF and BMP2 expression were detected by RT-PCR and ELISA.The mineralized nodules of AdCMV-VEGF and AdCMV-BMP2 transfected ones were more than AdCMV-BMP2 transfected ones. The results show that the foreign gene can be expressed after transfection and AdCMV-VEGF transfection can promote the BMP2 induced osteogenic differentiation.
Tissue engineering bone is a hot spot in the bone defect therapy.The innovation of this study is to study VEGF transfection mediated by adenovirus vector on the study of tissue engineering bone and the effect of VEGF on the osteogenic differentiation of BMSCs.
引文
[1] Partridge K, Yang XB, Clarke NM et al.Adenoviral BMP-2 Gene Transfer in Mesenchymal Stem Cells: In Vitro and in Vivo Bone Formation on Biodegradable Polymer Scaffolds[J]. Biochemical and Biophysical Research Communications, 2002,292:144–152.
[2] Yoon ST,Boden SD.Osteoinductive molecules in orthopaedics: basic science and preclinical studies[J]. Clin Orthop Relat Res,2002,395:33-43.
[3] Egermann M,Schneider E, Evans CH,et al.The potential of gene therapy for fracture healing in osteoporosis[J]. Osteoporos Int. 2005 ,Suppl 2:S120-8.
[4]Colton C.Imlantable biohybrid artificial organs[J].Cell Transplant,1995,4:415-436.
[5] Ferrara M, Henael WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells[J]. Biochem Biophys Res Commun, 1989, 161(2):851-5.
[6] Ciulla TA, Danis RP, Criswell M,,et al, Changing therapeutic paradigms for exudative age-related macular degeneration: antiangiogenic agents and photodynamic therapy[J]. Expert Opin Investig Drugs, 1999,8(12):2173-2182.
[7] Muller YA., Christinger HW, Keyt BA., et al, The crystal structure of vascular endothelial growth factor (VEGF) refined to1.93 A resolution: multiple copy flexibility and receptor binding[J]. Structure. 1997 ,5(10):1325-38.
[8] Li X,. Eriksson U.Novel VEGF family members: VEGF-B, VEGF-C and VEGF-D[J].Int J Biochem Cell Biol. 2001,33 (4): 421-6.
[9] Yamazaki Y, Takani K, Atoda H, et al. Snake venom vascular endothelial growth factors (VEGFs) exhibit potent activity through their specific recognition of KDR (VEGF receptor 2) [J]. J Biol Chem,2003,278 (52): 51985-8.
[10] Lyttle,DJ, Fraser KM, Fleming SB., et al, Homologs of vascular endothelial growth factor are encoded by the poxvirus orf virus[J].J. Virol.,1994,68 (1) :84-92.
[11] Nagy JA, Dvorak AM, Dvorak HF.VEGF-A(164/165) and PlGF :roles in angiogenesis and arteriogenesis[J].Trends Cardiovasc. Med, 2003, 13(5)169-175.
[12] Tischer E, Mitchell R, Hartman T, et al, The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing[J], J. Biol. Chem, 1991, 266 (18) 11947-11954.
[13] Shima DT, Kuroki M, Deutsch U. et al.The mouse gene for vascular endothelial growth factor. Genomic structure, definition of the transcriptional unit, and characterizationof transcriptional and post-transcriptional regulatory sequences[J], J. Biol. Chem,1996,271 (7) :3877-83.
[14] Ferrara N,Houck K,Jakeman L, et al. Molecular and biological properties of the vascular endothelial growth factor family of proteins[J], Endocr. Rev. 1992,13 (1) :18-32.
[15] Robinson CJ, Stringer SE. The splice variants of vascular endothelial growth factor (VEGF) and their receptors[J].J. Cell Sci,2001,114(Pt 5):853-65.
[16] Park JE, Keller GA, Ferrara N,et al. The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF[J]. Mol Biol Cell, 1993, 4 (12): 1317-26.
[17] Tanaka K,Yamaguchi S,Sawano A,et al.Characterization of the extracellular domain in vascular endothelial growth factor receptor-1 (Flt-1 tyrosine kinase)[J].Jpn J Cancer Res.1997,88(9):867-876.
[18] Terman BI, Dougher-Vermazen M, Carrion ME,et al.Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor[J], Biochem. Biophys. Res. Commun. 1992,187 (3): 1579-1586.
[19] de Vries C, Escobedo JA, Ueno H,et al. The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor [J], Science 1992,255 (5047): 989-991.
[20] Shibuya M, Yamaguchi S, Yamane A, et al. Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gene (flt) closely related to the fms family[J], Oncogene 1990,5 (4) 519-524.
[21] Terman BI, Carrion ME, Kovacs E,et al. Identification of a new endothelial cell growth factor receptor tyrosine kinase [J].Oncogene, 1991,6 (7) :1677-83.
[22] Jakeman LB, Winer J, Bennett GL, et al. Binding sites for vascular endothelial growth factor are localized on endothelial cells in adult rat tissues[J]. J. Clin. Invest. 1992,89 (1) :244-53.
[23] Kim I, Ryan AM, Rohan R,et al. Constitutive expression of VEGF, VEGFR-1, and VEGFR-2 in normal eyes[J], Invest. Ophthalmol. Visual Sci. 1999,40 (9) :2115-21.
[24] Yasuhara T, Shingo T, Date I. The potential role of vascular endothelial growth factor in the central nervous system[J].Rev. Neurosci. 2004,15 (4): 293-307.
[25] Pajusola K, Aprelikova O, Korhonen J, et al. FLT4 receptor tyrosine kinase contains seven immunoglobulin-like loops and is expressed in multiple human tissues and cell lines[J].Cancer Res. 1992,52 (8) :5738-43.
[26] Finnerty H, Kelleher K, Morris GE,et al. Molecular cloning of murine FLT andFLT4[J].Oncogene ,1993,8 (8) 2293-8.
[27] Finnerty H, Kelleher K, Morris GE,et al. Vascular endothelial growth factor induction of the angiogenic phenotype requires Ras activation[J]. J. Biol. Chem, 2001,276 (52) :49289-98.
[28] Takahashi T, Yamaguchi S, Chida K, et al. A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-Adependent activation of PLC-gamma and DNA synthesis in vascular endothelial cells[J]. EMBO J. 2001, 20 (11) 2768-78.
[29] Takahashi T, Ueno H, Shibuya M. VEGF activates protein kinase C-dependent, but Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells[J]. Oncogene, 1999,18 (13): 2221-30.
[30] Guo D, Jia Q, Song HY, et al. Vascular endothelial cell growth factor promotes tyrosine phosphorylation of mediators of signal transduction that contain SH2 domains. Association with endothelial cell proliferation[J]. J. Biol. Chem,1995,270 (12): 6729-33.
[31] Kabrun N, Bühring HJ, Choi K,et al. Flk-1 expression defines a population of early embryonic hematopoietic precursors[J]. Development, 1997,124 (10):2039-48.
[32] Quinn TP, Peters KG, De Vries C,et al. Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium[J]. Proc. Natl. Acad. Sci. U. S. A,1993,90 (16) :7533-37.
[33] Shalaby F, Ho J, Stanford WL, et al. A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis[J]. Cell ,1997,89 (6) :981-90.
[34] Schuh AC, Faloon P, Hu QL, et al.In vitro hematopoietic and endothelial potential of flk-1(-/-) embryonic stem cells and embryos[J]. Proc. Natl. Acad. Sci. U. S. A. ,1999,96 (5) :2159-64.
[35] Roskoski R Jr. Vascular endothelial growth factor (VEGF) signaling during tumor progression[J]. Crit Rev Oncol Hematol. 2007, 62 (3): 179-213.
[36] Hattori K, Heissig B, Wu Y, et al, Placental growth factor reconstitutes hematopoiesis by recruiting VEGFR1(+) stem cells from bone-marrow microenvironment [J].Nat Med, 2002,8 (8): 841-9.
[37] Sawano A, Iwai S, Sakurai Y, et al, Flt-1, vascular endothelial growth factor receptor 1, is a novel cell surface marker for the lineage of monocyte-macrophages in humans[J]. Blood, 2001,97 (3) 785-791.
[38] Fong GH, Rossant J, Gertsenstein M, et al, Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium[J]. Nature, 1995,376(6535):66-70.
[39] Fong GH, Zhang L, Bryce DM,, Increased hemangioblast commitment, not vascular disorganization, is the primary defect in flt-1 knock-out mice[J]. Development, 1999 ,126(13):3015-25..
[40] Olofsson B, Korpelainen E, Pepper MS, et al, Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells[J]. Proc Natl Acad Sci U S A. 1998 ,95(20):11709-14
[41] Landgren E, Schiller P, Cao Y, et al.Placenta growth factor stimulates MAP kinase and mitogenicity but not phospholipase C-gamma and migration of endothelial cells expressing Flt 1[J]. Oncogene, 1998 ,16(3):359-67.
[42] Park JE, Chen HH, Winer J,et al. Placenta growth factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR[J]. J. Biol. Chem. 1994,269(41):25646-54.
[43] Waltenberger J, Claesson-Welsh L, Siegbahn A,et al. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor[J]. J Biol Chem, 1994 ,269(43):26988-95
[44] Gille H, Kowalski J, Yu L,et al. A repressor sequence in the juxtamembrane domain of Flt-1 (VEGFR-1) constitutively inhibits vascular endothelial growth factor-dependent phosphatidylinositol 3'-kinase activation and endothelial cell migration. [J]. EMBO J. 2000 ,19(15):4064-73.
[45] Gille H, Kowalski J, Li B,et al. Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants[J].J. Biol. Chem. 2001,276(5):3222-30.
[46] Hiratsuka S, Minowa O, Kuno J, et al, Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice[J]. Proc Natl Acad Sci U S A. 1998 ,95(16):9349-54.
[47] Kendall RL, Thomas KA. Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor[J]. Proc Natl Acad Sci U S A. 1993 ,90(22):10705-9.
[48] Kendall RL, Wang G, Thomas KA. Identification of a natural soluble form of the vascular endothelial growth factor receptor, FLT-1, and its heterodimerization with KDR[J]. Biochem Biophys Res Commun. 1996,226(2):324-8.
[49] Barleon B, Sozzani S, Zhou D,et al Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1[J]. Blood, 1996 ,87(8):3336-43.
[50] Clauss M, Weich H, Breier G, et al.The vascular endothelial growth factor receptor Flt-1 mediates biological activities. Implications for a functional role of placenta growth factor in monocyte activation and chemotaxis[J]. J Biol Chem. 1996,271(30):17629-34.
[51] Selvaraj SK, Giri RK, Perelman N, et al, Mechanism of monocyte activation and expression of proinflammatory cytochemokines by placenta growth factor[J]. Blood. 2003,102(4):1515-24.
[52] Autiero M, Waltenberger J, Communi D, et al, Role of PlGF in the intra- and intermolecular cross talk between the VEGF receptors Flt1 and Flk1[J]. Nat Med. 2003 Jul;9(7):936-43.
[53] Christinger HW, Fuh G, de Vos AM,et al. The crystal structure of placental growth factor in complex ,with domain 2 of vascular endothelial growth factor receptor-1[J]. J Biol m. 2004 ,279(11):10382-8.
[54] Kaipainen A, Korhonen J, Mustonen T, et al. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development[J].Proc. Natl. Acad. Sci. USA 1995,92 (8): 3566-3570.
[55] Dixelius J, Makinen T, Wirzenius M,et al. Claesson-Welsh, Ligand-induced vascular endothelial growth factor receptor-3 (VEGFR-3) heterodimerization with VEGFR-2 in primary lymphatic endothelial cells regulates tyrosine phosphorylation sites[J]. J. Biol. Chem. 2003,278 (42): 40973-9.
[56] Soker S, Takashima S, Miao HQ,et al.Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor[J]. Cell,1998,92 (6) :735-745.
[57] Mamluk R, Gechtman Z, Kutcher ME, et al, Neuropilin-1 binds vascular endothelial growth factor 165, placenta growth factor-2, and heparin via its b1b2 domain[J]. J. Biol. Chem. 2002,277 (27) :24818-25.
[58] Neufeld G, Cohen T, Shraga N, et al. The neuropilins: multifunctional semaphorin and VEGF receptors that modulate axon guidance and angiogenesis[J]. Trends Cardiovasc. Med. 2002,12 (1) :13-9.
[59] He Z, Tessier-Lavigne M, Neuropilin is a receptor for the axonal chemorepellent Semaphorin III[J]. Cell, 1997,90 (4) 739-51.
[60] Kolodkin AL, Levengood DV, Rowe EG,et al. Neuropilin is a semaphorin III receptor[J]. Cell, 1997,90(4):753-62.
[61] Tamagnone L, Artigiani S, Chen H, et al.Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates. [J]. Cell 1999 ,99(1):71-80.
[62] Takahashi T, Fournier A, Nakamura F, et al.Plexin-neuropilin-1 complexes form functional semaphorin-3A receptors[J].Cell ,1999,99(1):59-69.
[63] Kawasaki T, Kitsukawa T, Bekku Y, et al.A requirement for neuropilin-1 in embryonic vessel formation[J].Development ,1999,126(21):4895-902.
[64] Lee P, Goishi K, Davidson AJ, et al.Neuropilin-1 is required for vascular development and is a mediator of VEGF-dependent angiogenesis in zebrafish[J]. Proc. Natl. Acad. Sci. U. S. A. 2002,99(16):10470-5.
[65] Fuh G, Garcia KC, de Vos AM. The interaction of neuropilin-1 with vascular endothelial growth factor and its receptor flt-1[J]. J. Biol. Chem. 2000 Sep 1;275(35):26690-5.
[66] Mac Gabhann F, Popel AS. Differential binding of VEGF isoforms to VEGF receptor2 in the presence of neuropilin-1: a computational model[J]. Am J Physiol Heart Circ Physiol. 2005,288(6):H2851-60.
[67] Bagnard D, Vaillant C, Khuth ST,et al. Semaphorin 3A-vascular endothelial growth factor-165 balance mediates migration and apoptosis of neural progenitor cells by the recruitment of shared receptor[J]. J Neurosci. 2001,21(10):3332-41.
[68] Schwarz Q, Gu C, Fujisawa H, et al.Vascular endothelial growth factor controls neuronal migration and cooperates with Sema3A to pattern distinct compartments of the facial nerve[J]. Genes Dev. 2004,18(22):2822-34.
[69] Gu C, Rodriguez ER, Reimert DV, et al.Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development.[J] Dev Cell. 2003,5(1):45-57.
[70] Bernfield M, G?tte M, Park PW,et al. Functions of cell surface heparan sulfate proteoglycans[J]. Annu Rev Biochem. 1999;68:729-77.
[71] Esko JD, Lindahl U. Molecular diversity of heparan sulfate [J]. J Clin Invest. 2001 ,108(2):169-73.
[72] Bernfield M, Kokenyesi R, Kato M, et al.Biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans[J]. Annu Rev Cell Biol. 1992;8:365-93.
[73] Mertens G, Cassiman JJ, Van den Berghe H,et al. Cell surface heparan sulfate proteoglycans from human vascular endothelial cells. Core protein characterization and antithrombin III binding properties[J]. J Biol Chem. 1992,267(28):20435-43.
[74] Kinsella MG, Wight TN. Structural characterization of heparan sulfate proteoglycan subclasses isolated from bovine aortic endothelial cell cultures[J]. Biochemistry, 1988,27(6):2136-44.
[75] Kojima T, Leone CW, Marchildon GA,et al. Isolation and characterization of heparan sulfate proteoglycans produced by cloned rat microvascular endothelial cells[J].J Biol Chem. 1992,267(7):4859-69.
[76] Gengrinovitch S, Berman B, David G, et al. Glypican-1 is a VEGF165 binding proteoglycan that acts as an extracellular chaperone for VEGF165[J]. J Biol Chem. 1999 ,274(16):10816-22.
[77] Gitay-Goren H, Cohen T, Tessler S,et al.Selective binding of VEGF121 to one of the three vascular endothelial growth factor receptors of vascular endothelial cells[J]. J Biol Chem. 1996,271(10):5519-23.
[78] Gitay-Goren H, Soker S, Vlodavsky I,et al.The binding of vascular endothelial growth factor to its receptors is dependent on cell surface-associated heparin-like molecules[J]. J Biol Chem. 1992 ,267(9):6093-8.
[79] Ashikari-Hada S, Habuchi H, Kariya Y,et al. Heparin regulates vascular endothelial growth factor165-dependent mitogenic activity, tube formation, and its receptor phosphorylation of human endothelial cells. Comparison of the effects of heparin and modified heparins[J]. J Biol Chem. 2005,280(36):31508-15.
[80] Tessler S, Rockwell P, Hicklin D, et al. Heparin modulates the interaction of VEGF165 with soluble and cell associated flk-1 receptors[J]. J Biol Chem. 1994 ,269(17):12456-61.
[81] Terman B, Khandke L, Dougher-Vermazan M,et al. VEGF receptor subtypes KDR and FLT1 show different sensitivities to heparin and placenta growth factor[J].Growth Factors. 1994,11(3):187-95.
[82] Dougher AM, Wasserstrom H, Torley L,et al.Identification of a heparin binding peptide on the extracellular domain of the KDR VEGF receptor[J].Growth Factors 1997,14 (4) :257-68.
[83] Keyt BA, Nguyen HV, Berleau LT,et al. Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. Generation ofreceptor-selective VEGF variants by site-directed mutagenesis[J].J. Biol. Chem. 1996,271 (10); 5638-46.
[84] Ito N, Claesson-Welsh L. Dual effects of heparin on VEGF binding to VEGF receptor-1 and transduction of biological responses[J]. Angiogenesis, 1999,3 (3) 159-166.
[85] Cohen T, Gitay-Goren H, Sharon R, et al.VEGF121, a vascular endothelial growth factor (VEGF) isoform lacking heparin binding ability, requires cell-surface heparan sulfates for efficient binding to the VEGF receptors of human melanoma cells[J]. J. Biol. Chem. 1995 ,270 (19): 11322-6.
[86] Yl(a|¨)-Herttuala S, Rissanen TT, Vajanto I,et al. Vascular endothelial growth factors: biology and current status of clinical applications in cardiovascular medicine[J]. J Am Coll Cardiol. 2007,49(10):1015-26.
[87] Breier G, Albrecht U, Sterrer S, et al, Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation[J]. Development 1992,114 (2) :521-532.
[88] Flamme I, Breier G, Risau W. Vascular endothelial growth factor (VEGF) and VEGF receptor 2 (flk-1) are expressed during vasculogenesis and vascular differentiation in the quail embryo[J].Dev. Biol. 1995,169 (2): 699- 712.
[89] Ferrara N, Carver-Moore K, Chen H, et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene[J]. Nature, 1996,380 (6573) :439-442.
[90] Carmeliet P, Ferreira V, Breier G,et al, Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele[J].Nature, 1996, (6573) :435- 439.
[91] Ferrara N. Vascular endothelial growth factor: basic science and clinical progress[J]. Endocr Rev, 2004,25 (4):581-611.
[92] Ferrara N. Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications [J]. Semin Oncol. 2002,29(6 Suppl 16):10-4.
[93] Roccaro AM, Russo F, Cirulli T, et al, Antiangiogenesis for rheumatoid arthritis[J]. Curr Drug Targets Inflamm Allergy. 2005,4(1):27-30.
[94] L.E. Smith, Pathogenesis of retinopathy of prematurity[J]. Growth Horm IGF Res. 14 (Suppl. A) (2004) S140-S144.
[95] Lashkari K, Hirose T, Yazdany J, et al, Vascular endothelial growth factor and hepatocyte growth factor levels are differentially elevated in patients with advanced retinopathy of prematurity[J]. Am. J. Pathol. 2000,156 (4): 1337-44.
[96] Zelzer E, McLean W, Ng YS,et al. Skeletal defects in VEGF(120/120) mice reveal multiple roles for VEGF in skeletogenesis[J].Development ,2002, 129(8):1893-904.
[97] Gerber HP, Vu TH, Ryan AM,et al. VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation[J].Nat Med 1999, 5(6):623-628.
[98] Haigh JJ, Gerber HP, Ferrara N,et al. Conditional inactivation of VEGF-A in areas of collagen2A1 expression results in embryonic lethality in the heterozygous state[J].Development 2000; 127(7):1445-53.
[99] Gerstenfeld LC, Cullinane DM, Barnes GL, et al. Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation. J. Cell. Biochem. 2003,88(5), 873–84.
[100] Tatsuyama K, Maezawa Y, Baba H, et al. Expression of various growth factors for cell proliferation and cytodifferentiation during fracture repair of bone[J]. Eur. J. Histochem. 2000;44(3):269-78.
[101] Orlandini M, Spreafico A, Bardelli M ,et al.Vascular Endothelial Growth Factor-D Activates VEGFR-3 Expressed in Osteoblasts Inducing Their Differentiation[J]. J Biol Chem. 2006 ;281(26):17961-7.
[102] Street J, Bao M, deGuzman L, et al. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover[J].Proc Natl Acad Sci USA 2002; 99(15):9656- 61.
[103]初同伟,王正国,朱佩芳等。血管内皮生长因子在骨折愈合中的作用[J].中国修复重建外科杂志,2002,16(2)75-78.
[104]初同伟,王正国,朱佩芳.血管内皮生长因子对骨折愈合相关因子表达的调控[J]..中华骨科杂志2003;23(4):235-238.
[105] Wang DS, Miura M, Demura H, et al. Anabolic effects of 1, 25-dihydroxyvitamin D3 on osteoblasts are enhanced by vascular endothelial growth factor produced by osteoblasts and by growth factors produced by endothelial cells[J]. Endocrinology 1997; 138(7):2953-62.
[106] Bouletreau PJ, Warren SM, Spector JA, et al. Hypoxia and VEGF up-regulate BMP-2 mRNA and protein expression in microvascular endothelial cells: implications for fracture healing[J]. Plast Reconstr Surg ,2002,109 (7):2384-97.
[107] Niida S, Kaku M, Amano H, et al. Vascular endothelial growth factor can substitute for macrophage colony-stimulating factor in the support of osteoclastic boneresorption[J]. J Exp Med ,1999,190(2):293-8.
[108] Engsig MT, Chen QJ, Vu TH, et al. Matrix metalloproteinase9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones[J].J Cell Biol 2000; 151:879-89.
[109] Deckers MM, Karperien M, van der Bent C,et al Expression of Vascular Endothelial Growth Factors and Their Receptors during Osteoblast Differentiation[J].。Endocrinology 2000,141(5): 1667-74.
[110] Midy V, Plouet J. Vasculotropin/vascular endothelial growth factor induces differentiation in cultured osteoblasts. Biochem. Biophys. Res. Commun. 1994,199(1), 380-6.
[111] Kaigler D, Wang Z, Horger K, et al.VEGF Scaffolds Enhance Angiogenesis and Bone Regeneration inIrradiated Osseous Defects[J]. J Bone Miner Res, 2006,21(5):735-44
[112] Hiltunen MO, Ruuskanen M, Huuskonen J,et al. Adenovirus- mediated VEGF-A gene transfer induces bone formation in vivo[J]. FASEB J. 2003,17(9):1147-9.
[113] Bostrom MP, Saleh KJ, Einhorn TA. Osteoinductive growth factors in preclinical fracture and long bone defects models[J]. Orthop Clin North Am. 1999 Oct;30(4):647-58.
[114] Oliver RC, Brown LJ, L?e H. Periodontal diseases in the United States population[J]. J Periodontol 1998,69(2):269-78.
[115] Anusaksathien O, Giannobile WV. Growth factor delivery to re-engineer periodontal tissues[J]. Curr Pharm Biotechnol. 2002, 3(2):129-39.
[116] Crombleholme TM. Adenoviral-mediated gene transfer in wound healing[J]. Wound Repair Regen,2000 8(6):460-72.
[117] Albertson KS, Medoff RJ, Mitsunaga MM (). The use of periosteally vascularized autografts to augment the fixation of large segmental allografts[J]. Clin Orthop Rel Res ,1991,269:113-9.
[118] Enneking WF, Mindell ER. Observations on massive retrieved human allografts[J]. J Bone Joint Surg Am,1991, 73(8):1123-42.
[119] Cook SD, Baffes GC, Wolfe MW, et al. The effect of recombinant human osteogenic protein-1 on healing of large segmental bone defects[J]. J Bone Joint Surg Am,1994,76(6):827-38.
[120] Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells[J]. Cell and Tissue Kinetics 1970,3(4):393–403.
[121]潘华,王大伟,李红波,等.密度梯度离心与贴壁法相结合体外分离培养兔骨髓基质干细胞:1～3代细胞生长特性[J].中国组织工程研究与临床康复,2007,11(42):8487-8490.
[122]金光鑫,吴德全.脐带血间充质干细胞研究进展[J].国际移植与血液净化杂志,2006,4(5):45-48.
[123] Huang JI,Beanes SR,Zhu M,et al.Rat extramedullary adipose tissue as a source of osteochondrogenic progenitor cells[J].Plast ReconstrSurg,2002,109(3):1033.1041.
[124] Halvorsen YD, Bond A, Sen A, et al. Thiazolidinediones and glucocorticoids synergistically induce differentiation of human adipose tissue stromal cells: Biochemical, cellular, and molecular analysis[J]. Metab. Clin. Exp. 2001,50 (4) :407-13.
[125] Oligino TJ, Yao Q, Ghivizzani SC, et al. Vector systems for gene transfer to joints[J]. Clin Orthop Rel Res 2000, 379:S17-30.
[126] Lakshmipathy U, Pelacho B, Sudo K, et al.Efficient transfection of embryonic and adult stem cells[J]. Stem Cells,2004,22 (4) 531-543.
[127] Franceschi RT, Wang D, Krebsbach PH,. Gene therapy for bone formation: in vitro and in vivo osteogenic activity of an adenovirus expressing BMP7[J]. J Cell Biochem, 2000,78(3):476-86.
[128] John M. Coffin, Stephen H. Hughes, Harold E. Varmus, Retroviruses[J].Cold Spring Harbor Laboratory Press, 1997.
[129] Adam MA, Ramesh N, Miller AD,et al. Internal initiation of translation in retroviral vectors carrying picornavirus 5’nontranslated regions[J].J Virol. 1991,65 (9): 4985- 90.
[130] Apperley JF, Luskey BD, Williams DA. Retroviral gene transfer of human Adenosine deaminase in murine hematopoietic cells: effect of selectable marker sequences on longterm expression[J].Blood, 1991,78 (2): 310- 317.
[131] Danos O, Heard JM . Recombinant retroviruses as tools for gene transfer to somatic cells[J]. Bone Marrow Transplant, 1992,9(Suppl 1):131-8.
[132] Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al.A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency[J]. N Engl J Med, 2003,348(3):255-6.
[133] Noguchi P. Risks and benefits of gene therapy[J]. N Engl J Med, 2003,348(3):193-4.
[134] Neumann R, Chroboczek J, Jacrot B. Determination of the nucleotide sequencefor the penton-base gene of human adenovirus type 5[J]. Gene, 1988,69(1):153-7.
[135] Bergelson JM, Cunningham JA, Droguett G, et al. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5[J].Science, 1997, 275 (5304): 1320-3.
[136] Zhao M, Zhao Z, Koh JT,et al (2005a). Combinatorial gene therapy for bone regeneration: cooperative interactions between adenovirus vectors expressing bone morphogenetic proteins 2, 4 and 7[J]. J Cell Biochem 2005,95(1):1-16.
[137] Armentano D, Zabner J, Sacks C, et al. Effect of the E4 region on the persistence of transgene expression from adenovirus vectors[J]. J Virol, 1997,71(3):2408-16.
[138] Hartigan-O'Connor D, Amalfitano A, Chamberlain JS. Improved production of gutted adenovirus in cells expressing adenovirus preterminal protein and DNA polymerase[J]. J Virol,1999 , 73(9):7835-41.
[139] Zhang XY, La Russa VF, Bao L,et al. Lentiviral vectors for sustained transgene expression in human bone marrow-derived stromal cells[J]. Mol Ther, 2002,5(5):555-565.
[140] Vigna E, Naldini L. Lentiviral vectors: excellent tools for experimental gene transfer and promising candidates for gene therapy[J].J Gene Med 2000 ,2(5):308-16.
[141] Sugiyama O, An DS, Kung SP, et al. Lentivirus-mediated gene transfer induces long-term transgene expression of BMP-2 in vitro and new bone formation in vivo[J]. Mol Ther. 2005,11(3):390-8
[142] Büning H, Braun-Falco M, Hallek M. Progress in the use of adeno-associated viral vectors for gene therapy[J]. Cells Tissues Organs. 2004;177(3):139-50
[143] Luk KD, Chen Y, Cheung KM. Adeno-associated virus-mediated bone morphogenetic protein-4 gene therapy for in vivo bone formation[J]. Biochem Biophys Res Commun. 2003,308(3):636-45
[144] Alsberg E, Anderson KW, Albeiruti A,et al.Cell-interactive alginate hydrogels for bone tissue engineering[J]. J Dent Res. 200 ,80(11):2025-9.
[145] Tsuang YH,Lin FH,Sun JS,et al.In vitro cell behavior of osteoblasts on Pyrost bone substitute[J].Anat Rec.1997,247(2):164-9.
[146] Rocha LB,Goissis G, Rossi MA. Biocompatibility of anionic collagen matrix as scaffold for bone healing[J].Biomaterials. 2002;23(2):449-456.
[147] Sato M, Asazuma T, Ishihara M, et al. An atelocollagen honeycomb-shaped scaffold with a membrane seal (ACHMS-scaffold)for the culture of annulus fibrosus cells from an intervertebral disc[J].J Biomed Mater Res A.2003;64(2):248-256.
[148] Seol YJ,Lee JY,Park YJ,et al.Chitosan sponges as tissue engineering scaffolds for bone formation[J].Biotechnol Lett. 2004;26(13):1037-41.
[149] Klokkevold PR,Vandemark L,Kenney EB,et al.Osteogenesis enhanced by chitosan(poly-N-acetyl glucosaminoglycan) in vitro[J]. J Periodontol.1996;67(11): 1170-5.
[150] VANDEPUTTE KA, URIST MR..Experimental mineralization of collagen sponge and decalcified bone[J].Clin Orthop Rel Res 1965, 40:48-56.
[151] VANDEPUTTE KA, URIST MR.Osteogenesis in the interior of intramuscular implants of decalcified bone matrix[J].Clin Orthop Rel Res 1966,43:257-70.
[152] Urist MR,Wallace TH,Adams T.The function of fibrocartilaginous fracture callus[J].JBJS 1965; 47:304-18.
[153] Urist MR, Silverman BF, Büring K, et al.The bone induction principle[J]. Clin Orthop Relat Res. 1967,53:243-83.
[154] Urist MR, Dawson E. Intertransverse process fusion with the aid of chemosterilized autolyzed antigen-extracted allogeneic (AAA) bone[J]. Clin Orthop Rel Res 1980; 154:97-113.
[155] Harakas NK. Demineralized bone-matrix-induced osteogenesis[J]. Clin Orthop Rel Res 1984,188:239-51.
[156] Russell JL, Block JE. Clinical utility of demineralized bone matrix for osseous defects, arthrodesis, and reconstruction: impact of processing techniques and study methodology[J]. Orthopaedics 1999; 22(5):524-31.
[157] Zhu L,Liu W,Cui L,et al.Tissue-engineered bone repair of goat-femur defects with osteogenically induced bone marrow stromal cells[J].Tissue Eng.2006;12(3):423-33.
[158]张华林,陈治清.羊毛角蛋白作为骨组织工程支架材料的研究进展[J].国际口腔医学杂志,2007, 34(3): 390-394..
[159]王嫣,陈小菊,王兰,等.海藻酸钠凝胶对骨髓间充质干细胞生物学效应的初步研究[J].重庆医科大学学报,2006,31(4):478-481
[160] Hollinger JO, Battistone GC. Biodegradable bone repair materials. Synthetic polymers and ceramics. [J].Clin Orthop Rel Res 1986; 207:290-305.
[161] Friedman CD, Costantino PD, Takagi S,et al. BoneSource hydroxyapatite cement: a novel biomaterial for craniofacial skeletal tissue engineering and reconstruction[J]. J Biomed Mater Res. 1998 ,43(4):428-32.
[162] H?mmerle CH, Olah AJ, Schmid J,et al. The biological effect of natural bone mineral on bone neoformation on the rabbit skull[J]. Clin Oral Implants Res 1997;8(3):198-207.
[163] Eggli PS,Müller W,Schenk RK.Porous hydroxyapatite and tricalcium phosphate cylinders with two different pore size ranges implanted in the cancellous bone of rabbits.A comparative histomorphometric and histologic study of bony ingrowth and implant substitution[J].Clin Orthop Relat Res.1988; (232): 127-38.
[164] Levine JP, Bradley J, Turk AE,et al.Bone morphogenetic protein promotes vascularization and osteoinduction in preformed hydroxyapatite in the rabbit[J]. Ann Plast Surg. 1997,39(2):158-68.
[165] Tan KK, Tan GH, Shamsul BS, et al.Bone graft substitute using hydroxyapatite scaffold seeded with tissue engineered autologous osteoprogenitor cells in spinal fusion:early result in a sheep model[J]. Med J Malaysia. 2005,60 Suppl C:53-8.
[166] Deville S,Saiz E,Tomsia AP.Freeze casting of hydroxyapatite scaffolds for bone tissue engineering.Biomaterials[J].2006; 27(32):5480-5489.
[167] Morishita T,Honoki K,Ohgushi H,et al.Tissue engineering approach to the treatment of bone tumors:three cases of cultured bone grafts derived from patients'mesenchymal stem cells[J].Artif Organs.2006; 30(2):115-8.
[168]赵丽君,毛天球,陈富林,等.不同类型骨组织工程支架材料的比较研究[J].中国临床康复,2003,7(4):29-30.
[169] Ducheyne P,el-Ghannam A,Shapiro I.Effect of bioactive glass templates on osteoblast proliferation and in vitro synthesis of bone-like tissue[J]. J Cell Biochem. 1994; 56(2): 162-7.
[170]孙蕊,潘高峰,张丽芳,等.聚乳酸/聚乙二醇-聚乳酸新型亲水支架的制备与研究[J].生物医学工程学杂志,2007,24(1):91-93.
[171] Ren T,Ren J,Jia X,et al.The bone formation in vitro and mandibular defect repair using PLGA porous scaffolds[J].J Biomed Mater Res A.2005,74(4):562-9.
[172] Puelacher WC,Vacanti JP,Ferraro NF,et al.Femoral shaft reconstruction using tissue-engineered growth of bone[J].J Oral Maxillofac Surg.1996,25(3):223-228.
[173]胡稷杰,金丹,全大萍,等.负载不同浓度骨形态发生蛋白的组织工程骨体内成骨的量效关系[J].中华骨科杂志,2006,26(3):196-201.
[174] Zhang R, Ma PX. Porous poly(L-lactic acid)/apatite composites created by biomimetic process[J]. J Biomed Mater Res ,1999, 45(4):285-93.
[175] Zhang R, Ma PX. Poly(a-hydroxyl acids)/hydroxyapatite porous composites forbone-tissue engineering. I. Preparation and morphology[J]. J Biomed Mater Res, 1999,44(4):446-55.
[176] Roweton SL. A new approach to the formation of tailored microcellular foams and microtextured surfaces of absorbable and non-absorbable thermoplastic biomaterials. Master of Science Thesis, Department of Bioengineering, Clemson University, 1994.
[177] Elisseeff J, Anseth K, Sims D,et al. Transdermal photopolymerization for minimally invasive implantation[J].Proc Nat Acad Sci USA 1999,96 (6):3104-7.
[178] Meinig RP, Rahn B, Perren SM, Gogolewski S. Bone regeneration with resorbable polymeric membranes: treatment of diaphyseal bone defects in the rabbit radius with poly(L-lactide) membrane. A pilot study[J].J Orthop Trauma, 1996,10(3):178-90.
[179] Meinig RP, Buesing CM, Helm J, et al. Regeneration of diaphyseal bone defects using resorbable poly(L/DL-lactide) and poly(D-lactide) membranes in the Yucatan pig model[J]. J Orthop Trauma, 1997,11(8):551-8.
[180] Gugala Z, Gogolewski S. Regeneration of segmental diaphyseal defects in sheep tibiae using resorbable polymeric membranes: a preliminary study[J]. J Orthop Trauma, 1999,13(3):187-95.
[181] Ekholm M, Hietanen J, Lindqvist C, Rautavuori J, Santavirta S,Suuronen R. Histological study of tissue reactions to e-caprolactone-lactide copolymer in paste form[J]. Biomater, 1999,20(14):1257-62.
[182] Muggli DC, Lee HR, Keyser SA, et al. Photocrosslinkable polyanhydride networks for use in orthopedic applications. In:Peppas NA, Mooney DJ, Mikow AG, Brannon-Peppas L, editors.Biomaterials, carriers for drug delivery, and scaffolds for tissue engineering[J].New York: AIChE, 1997. p. 275-7.
[183]申玉芹,寡核苷酸(ODN)促骨髓间充质干细胞向成骨细胞分化作用及机制研究。吉林大学博士论文
[184] Friedenstein AJ , Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs [J]. Exp Hemator, 1976, 4(5):267-74.
[185] Bianco P, Riminucci M, Gronthos S,et al. Bone marrow stromal stem cells: nature, biology, and potential applications[J].Stem Cells 2001; 19(3): 80-192
[186]邱丽燕,王金福.骨髓间充质干细胞的研究进展[J].生物工程学报,2003,19(2)136-140
[187] Pittenger MF ,Mackay AM,Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells[J].Science, 1999,284 (5411) : 143-147.
[188] Deans RJ,Moseley AB.Mesenchymal stem cells:biology and potential clinical uses[J].Exp Hematol,2000,28(8):875-84.
[189] Shugart EC, Umek RM. Dexamethasone signaling is required to establish the postmitotic state of adipocyte development[J]. Cell Growth Differ, 1997,8(10): 91-8.
[190] Hauner H, Schmid P, Pfeiffer EF.. Glucocorticoids and insulin promote the differentiation of human adipocyte precursor cells into fat cells[J]. J Clin Endocrinol Metab ,1987,64(4):832-5.
[191] L(o|¨)ffler G, Hauner H. Adipose tissue development: the role of precursor cells and adipogenic factors. Part II: The regulation of the adipogenic conversion by hormones and serum factors[J]. Klin Wochenschr, 1987,65(17):812-7.
[192] Zhang HH, Huang J, Düvel K, et al. Insulin stimulates adipogenesis through the Akt-TSC2-mTORC1 pathway[J].PLoS One 2009; 4(7): e6189.
[193] Ringold GM, Chapman AB, Knight DM,. Hormonal control of adipogenesis[J]. Ann NY Acad Sci, 1986, 478:109-19.
[194] Kellinsalmi M, Parikka V, Risteli J, et al. Inhibition of cyclooxygenase-2 down-regulates osteoclast and osteoblast differentiation and favours adipocyte formation in vitro[J]. Eur J Pharmacol 2007,572:102-10.
[195] Lehmann JM, Lenhard JM, Oliver BB,et al. Peroxisome proliferator-activated receptors alpha and gamma are activated by indomethacin and other non-steroidal anti-inflammatory drugs[J]. J Biol Chem ,1997,272(6):3406-10.
[196] Ye H, Serrero G. Stimulation of adipose differentiation related protein (ADRP) expression by ibuprofen and indomethacin in adipocyte precursors and in adipocytes[J]. Biochem J 1998;330(Pt. 2):803-9.
[197] Otsuka E, Yamaguchi A, Hirose S, et al. Characterization of osteoblastic differentiation of stromal cell line ST2 that is induced by ascorbic acid[J]. Am J Physiol. 1999,277(1 Pt 1):C132-8.
[198] Prasher DC,Eckenrode VK,Ward WW,et al.Primary structure of the Aequorea victoria green-fluorescent protein[J].Gene, 1992, 111(2):229-33
[199] Prasher D,McCann RO,Cormier MJ.Cloning and expression of the cDNA coding for aequorin,a bioluminescent calcium-binding protein[J].Biochem Biophys Res Commun, 1985, 126(3):1259-68
[200] Hastings JW.Chemistries and colours of bioluminescent reactions: a review[J]. Gene, 1996,173:5-11.
[201] Tsien RY. The green fluorescent protein[J]. Annu Rev Bioche1998, 67: 509-544.
[202]李志勇,刘磊,田卫东,等.绿色荧光蛋白基因转染大鼠骨髓间充质干细胞的实验研究[J].中华口腔医学杂志,2005,40(2):150-153.
[203] Cancedda R,Giannoni P,Mastrogiacomo MA. tissue engineering approach to bone repair in large animal models and in clinical practice[J]. Biomaterials, 2007, 28(29): 4240-50.
[204] Nomi M,Miyake H,Sugita Y,et al.Role of growth factors and endothelial cells in therapeutic angiogenesis and tissue engineering[J].Curr Stem Cell Res Ther,2006,1(3):3353
[205] Ball SG, Shuttleworth CA, Kielty CM. Mesenchymal stem cells and neovascularization:role of platelet-derived growth factor receptors[J]. J Cell Mol Med, 2007,11(5): 1012-30.
[206] Ball SG, Shuttleworth CA, Kielty CM. Vascular endothelial growth factor can signal through platelet-derived growth factor receptors[J]. J Cell Biol ,2007,177(3):489-500.
[207] Levy O, Dvir T, Tsur-Gang O, et al. Signal transducer and activator of transcription 3-A key molecular switch for human mesenchymal stem cell proliferation[J]. Int J Biochem Cell Biol,2008, 40(11): 2606-18.
[208] Harada S,Nagy JA,Sullivan KA,et al.Induction of vascular endothelial growth factor expression by prostaglandin E2 and E1 in osteoblasts [J]. J Clin Invest,1994, 93(6):2490-6
[209] Cotter EJ,Ip HS,Powderly WG, et al. Mechanism of HIV protein induced modulation of mesenchymal stem cell osteogenic differentiation[J]. BMC Musculoskelet Disord. 2008; 9: 33.
[210] Beloti MM, de Oliveira PT, Tagliani MM.Bone cell responses to the composite of Ricinus communis polyurethane and alkaline phosphatase[J]. J Biomed Mater Res A. 2008;84(2):435-441.
[211] Schouten C, van den Beucken JJ, de Jonge LT,et al. The effect of alkaline phosphatase coated onto titanium alloys on bone responses in rats[J].Biomaterials. 2009 ,30(32):6407-17.
[212] Leonardi E, Ciapetti G, Baglìo SR,et al.Osteogenic properties of late adherent subpopulations of human bone marrow stromal cells[J]. Histochem Cell Biol. 2009 ,132(5):547-57.
[213]宋爱梅,束蓉.体外培养骨髓间充质干细胞向成骨细胞分化的影响因素研究[J].牙体牙髓牙周病学杂志,2005,15(1) :55-58.
[214] Tilgar V, Kilgas P, Viitak A,et al. The rate of bone mineralization in birdsis directly related to alkaline phosphatase activity[J]. Physiol Biochem Zool,2008, 81(1): 106-111.
[215] Sim(a|¨)o AM, Beloti MM, Rosa AL,et al. Culture of osteogenic cells from human alveolar bone: a useful source of alkaline phosphatase[J]. Cell Biol Int.2007;31(11): 1405-13.
[216]范仲凯,张元和,姚琦,卢伟,转染血管内皮生长因子基因的人成骨细胞增殖及其生物学功能[J].中国组织工程研究与临床康复, 2009,13(50): 9850-54
[217] Huh JE, Kwon NH, Baek YH, et al.Formononetin promotes early fracture healing through stimulating angiogenesis by up-regulating VEGFR-2/Flk-1 in a rat fracture model[J]. Int Immunopharmacol. 2009; 9(12):1357-65.
[218] Chu Q, Wu ZF, Wang QT,et al.Influence of transfection with human transforming growth factor-beta1 gene on the osteogenic potential of the cultured human gingival fibroblast[J].Hua Xi Kou Qiang Yi Xue Za Zhi. 2009; 27(3):264-7.
[219] Nakashima K, Zhou X, Kunkel G,et al. The Novel Zinc Finger-Containing Transcription Factor Osterix Is Required for Osteoblast Differentiation and Bone Formation[J]. Cell, 2002; 108(1): 17-29.
[220] GashlerAL, Swaminathan S, Sukhatme VP. A novel repression module, an extensive activation domain, and a bipartite nuclear localization signal defined in the immediate-early transcription factor EGR -1[J]. Mol Cell Biol, 1993, 13(8): 4556-71.
[221] Abe A, Inoue K, Tanaka T, et al. Quantitation of hepatitis B virus genomic DNA by real-time detection PCR[J].J Clin Microbiol. 1999, 37(9): 2899-2903.
[222] Heid CA, Stevens J, Livak KJ, et al. Real- time quantitativePCR[J].Genome Res, 1996, 6(10): 986-94.
[223] Livark KJ, Schmittgen TD.Analysis of relative gene expression datausing real-time quantitative PCR and the 2-ΔΔCtmethod[J].Methods,2001, 25(4): 402-408.
[224] Jaiswal RK,Jaiswal N,Bruder SP,et al.Adult human mesenchymal stem cell differentiation to the osteogenic oradipogenic lineage is regulated by mitogen activated protein kinase[J]. J Biol Chem,2000,275(13):9645-52.
[225] Urist, M. R. Bone: Formation by autoinduction[J]. Science.1965,150 (698):893-9.
[226] Wozney JM, Rosen V, Celeste AJ,et al. Noval Regulators of Bone Formation: Molecular Ctones and Activities[J]. Science, 1988 ,242(4885):1528-34.
[227] Wang EA,Rosen V,Cordes P,et al.Purification and Characterization of Other Distinct Bone-inducing Factors[J]. ProcNatl Acad Sci USA,1988,85 (24): 9484-8.
[228] Matthews SJ. Biological activity of bone morphogenetic proteins (BMP’s) [J]. Injury. 2005 ,36 (Suppl 3):S34-7.
[2] Yoon ST,Boden SD.Osteoinductive molecules in orthopaedics: basic science and preclinical studies[J]. Clin Orthop Relat Res,2002,395:33-43.
[3] Egermann M,Schneider E, Evans CH,et al.The potential of gene therapy for fracture healing in osteoporosis[J]. Osteoporos Int. 2005 ,Suppl 2:S120-8.
[4]Colton C.Imlantable biohybrid artificial organs[J].Cell Transplant,1995,4:415-436.
[5] Ferrara M, Henael WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells[J]. Biochem Biophys Res Commun, 1989, 161(2):851-5.
[6] Ciulla TA, Danis RP, Criswell M,,et al, Changing therapeutic paradigms for exudative age-related macular degeneration: antiangiogenic agents and photodynamic therapy[J]. Expert Opin Investig Drugs, 1999,8(12):2173-2182.
[7] Muller YA., Christinger HW, Keyt BA., et al, The crystal structure of vascular endothelial growth factor (VEGF) refined to1.93 A resolution: multiple copy flexibility and receptor binding[J]. Structure. 1997 ,5(10):1325-38.
[8] Li X,. Eriksson U.Novel VEGF family members: VEGF-B, VEGF-C and VEGF-D[J].Int J Biochem Cell Biol. 2001,33 (4): 421-6.
[9] Yamazaki Y, Takani K, Atoda H, et al. Snake venom vascular endothelial growth factors (VEGFs) exhibit potent activity through their specific recognition of KDR (VEGF receptor 2) [J]. J Biol Chem,2003,278 (52): 51985-8.
[10] Lyttle,DJ, Fraser KM, Fleming SB., et al, Homologs of vascular endothelial growth factor are encoded by the poxvirus orf virus[J].J. Virol.,1994,68 (1) :84-92.
[11] Nagy JA, Dvorak AM, Dvorak HF.VEGF-A(164/165) and PlGF :roles in angiogenesis and arteriogenesis[J].Trends Cardiovasc. Med, 2003, 13(5)169-175.
[12] Tischer E, Mitchell R, Hartman T, et al, The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing[J], J. Biol. Chem, 1991, 266 (18) 11947-11954.
[13] Shima DT, Kuroki M, Deutsch U. et al.The mouse gene for vascular endothelial growth factor. Genomic structure, definition of the transcriptional unit, and characterizationof transcriptional and post-transcriptional regulatory sequences[J], J. Biol. Chem,1996,271 (7) :3877-83.
[14] Ferrara N,Houck K,Jakeman L, et al. Molecular and biological properties of the vascular endothelial growth factor family of proteins[J], Endocr. Rev. 1992,13 (1) :18-32.
[15] Robinson CJ, Stringer SE. The splice variants of vascular endothelial growth factor (VEGF) and their receptors[J].J. Cell Sci,2001,114(Pt 5):853-65.
[16] Park JE, Keller GA, Ferrara N,et al. The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF[J]. Mol Biol Cell, 1993, 4 (12): 1317-26.
[17] Tanaka K,Yamaguchi S,Sawano A,et al.Characterization of the extracellular domain in vascular endothelial growth factor receptor-1 (Flt-1 tyrosine kinase)[J].Jpn J Cancer Res.1997,88(9):867-876.
[18] Terman BI, Dougher-Vermazen M, Carrion ME,et al.Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor[J], Biochem. Biophys. Res. Commun. 1992,187 (3): 1579-1586.
[19] de Vries C, Escobedo JA, Ueno H,et al. The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor [J], Science 1992,255 (5047): 989-991.
[20] Shibuya M, Yamaguchi S, Yamane A, et al. Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gene (flt) closely related to the fms family[J], Oncogene 1990,5 (4) 519-524.
[21] Terman BI, Carrion ME, Kovacs E,et al. Identification of a new endothelial cell growth factor receptor tyrosine kinase [J].Oncogene, 1991,6 (7) :1677-83.
[22] Jakeman LB, Winer J, Bennett GL, et al. Binding sites for vascular endothelial growth factor are localized on endothelial cells in adult rat tissues[J]. J. Clin. Invest. 1992,89 (1) :244-53.
[23] Kim I, Ryan AM, Rohan R,et al. Constitutive expression of VEGF, VEGFR-1, and VEGFR-2 in normal eyes[J], Invest. Ophthalmol. Visual Sci. 1999,40 (9) :2115-21.
[24] Yasuhara T, Shingo T, Date I. The potential role of vascular endothelial growth factor in the central nervous system[J].Rev. Neurosci. 2004,15 (4): 293-307.
[25] Pajusola K, Aprelikova O, Korhonen J, et al. FLT4 receptor tyrosine kinase contains seven immunoglobulin-like loops and is expressed in multiple human tissues and cell lines[J].Cancer Res. 1992,52 (8) :5738-43.
[26] Finnerty H, Kelleher K, Morris GE,et al. Molecular cloning of murine FLT andFLT4[J].Oncogene ,1993,8 (8) 2293-8.
[27] Finnerty H, Kelleher K, Morris GE,et al. Vascular endothelial growth factor induction of the angiogenic phenotype requires Ras activation[J]. J. Biol. Chem, 2001,276 (52) :49289-98.
[28] Takahashi T, Yamaguchi S, Chida K, et al. A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-Adependent activation of PLC-gamma and DNA synthesis in vascular endothelial cells[J]. EMBO J. 2001, 20 (11) 2768-78.
[29] Takahashi T, Ueno H, Shibuya M. VEGF activates protein kinase C-dependent, but Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells[J]. Oncogene, 1999,18 (13): 2221-30.
[30] Guo D, Jia Q, Song HY, et al. Vascular endothelial cell growth factor promotes tyrosine phosphorylation of mediators of signal transduction that contain SH2 domains. Association with endothelial cell proliferation[J]. J. Biol. Chem,1995,270 (12): 6729-33.
[31] Kabrun N, Bühring HJ, Choi K,et al. Flk-1 expression defines a population of early embryonic hematopoietic precursors[J]. Development, 1997,124 (10):2039-48.
[32] Quinn TP, Peters KG, De Vries C,et al. Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium[J]. Proc. Natl. Acad. Sci. U. S. A,1993,90 (16) :7533-37.
[33] Shalaby F, Ho J, Stanford WL, et al. A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis[J]. Cell ,1997,89 (6) :981-90.
[34] Schuh AC, Faloon P, Hu QL, et al.In vitro hematopoietic and endothelial potential of flk-1(-/-) embryonic stem cells and embryos[J]. Proc. Natl. Acad. Sci. U. S. A. ,1999,96 (5) :2159-64.
[35] Roskoski R Jr. Vascular endothelial growth factor (VEGF) signaling during tumor progression[J]. Crit Rev Oncol Hematol. 2007, 62 (3): 179-213.
[36] Hattori K, Heissig B, Wu Y, et al, Placental growth factor reconstitutes hematopoiesis by recruiting VEGFR1(+) stem cells from bone-marrow microenvironment [J].Nat Med, 2002,8 (8): 841-9.
[37] Sawano A, Iwai S, Sakurai Y, et al, Flt-1, vascular endothelial growth factor receptor 1, is a novel cell surface marker for the lineage of monocyte-macrophages in humans[J]. Blood, 2001,97 (3) 785-791.
[38] Fong GH, Rossant J, Gertsenstein M, et al, Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium[J]. Nature, 1995,376(6535):66-70.
[39] Fong GH, Zhang L, Bryce DM,, Increased hemangioblast commitment, not vascular disorganization, is the primary defect in flt-1 knock-out mice[J]. Development, 1999 ,126(13):3015-25..
[40] Olofsson B, Korpelainen E, Pepper MS, et al, Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells[J]. Proc Natl Acad Sci U S A. 1998 ,95(20):11709-14
[41] Landgren E, Schiller P, Cao Y, et al.Placenta growth factor stimulates MAP kinase and mitogenicity but not phospholipase C-gamma and migration of endothelial cells expressing Flt 1[J]. Oncogene, 1998 ,16(3):359-67.
[42] Park JE, Chen HH, Winer J,et al. Placenta growth factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR[J]. J. Biol. Chem. 1994,269(41):25646-54.
[43] Waltenberger J, Claesson-Welsh L, Siegbahn A,et al. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor[J]. J Biol Chem, 1994 ,269(43):26988-95
[44] Gille H, Kowalski J, Yu L,et al. A repressor sequence in the juxtamembrane domain of Flt-1 (VEGFR-1) constitutively inhibits vascular endothelial growth factor-dependent phosphatidylinositol 3'-kinase activation and endothelial cell migration. [J]. EMBO J. 2000 ,19(15):4064-73.
[45] Gille H, Kowalski J, Li B,et al. Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants[J].J. Biol. Chem. 2001,276(5):3222-30.
[46] Hiratsuka S, Minowa O, Kuno J, et al, Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice[J]. Proc Natl Acad Sci U S A. 1998 ,95(16):9349-54.
[47] Kendall RL, Thomas KA. Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor[J]. Proc Natl Acad Sci U S A. 1993 ,90(22):10705-9.
[48] Kendall RL, Wang G, Thomas KA. Identification of a natural soluble form of the vascular endothelial growth factor receptor, FLT-1, and its heterodimerization with KDR[J]. Biochem Biophys Res Commun. 1996,226(2):324-8.
[49] Barleon B, Sozzani S, Zhou D,et al Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1[J]. Blood, 1996 ,87(8):3336-43.
[50] Clauss M, Weich H, Breier G, et al.The vascular endothelial growth factor receptor Flt-1 mediates biological activities. Implications for a functional role of placenta growth factor in monocyte activation and chemotaxis[J]. J Biol Chem. 1996,271(30):17629-34.
[51] Selvaraj SK, Giri RK, Perelman N, et al, Mechanism of monocyte activation and expression of proinflammatory cytochemokines by placenta growth factor[J]. Blood. 2003,102(4):1515-24.
[52] Autiero M, Waltenberger J, Communi D, et al, Role of PlGF in the intra- and intermolecular cross talk between the VEGF receptors Flt1 and Flk1[J]. Nat Med. 2003 Jul;9(7):936-43.
[53] Christinger HW, Fuh G, de Vos AM,et al. The crystal structure of placental growth factor in complex ,with domain 2 of vascular endothelial growth factor receptor-1[J]. J Biol m. 2004 ,279(11):10382-8.
[54] Kaipainen A, Korhonen J, Mustonen T, et al. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development[J].Proc. Natl. Acad. Sci. USA 1995,92 (8): 3566-3570.
[55] Dixelius J, Makinen T, Wirzenius M,et al. Claesson-Welsh, Ligand-induced vascular endothelial growth factor receptor-3 (VEGFR-3) heterodimerization with VEGFR-2 in primary lymphatic endothelial cells regulates tyrosine phosphorylation sites[J]. J. Biol. Chem. 2003,278 (42): 40973-9.
[56] Soker S, Takashima S, Miao HQ,et al.Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor[J]. Cell,1998,92 (6) :735-745.
[57] Mamluk R, Gechtman Z, Kutcher ME, et al, Neuropilin-1 binds vascular endothelial growth factor 165, placenta growth factor-2, and heparin via its b1b2 domain[J]. J. Biol. Chem. 2002,277 (27) :24818-25.
[58] Neufeld G, Cohen T, Shraga N, et al. The neuropilins: multifunctional semaphorin and VEGF receptors that modulate axon guidance and angiogenesis[J]. Trends Cardiovasc. Med. 2002,12 (1) :13-9.
[59] He Z, Tessier-Lavigne M, Neuropilin is a receptor for the axonal chemorepellent Semaphorin III[J]. Cell, 1997,90 (4) 739-51.
[60] Kolodkin AL, Levengood DV, Rowe EG,et al. Neuropilin is a semaphorin III receptor[J]. Cell, 1997,90(4):753-62.
[61] Tamagnone L, Artigiani S, Chen H, et al.Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates. [J]. Cell 1999 ,99(1):71-80.
[62] Takahashi T, Fournier A, Nakamura F, et al.Plexin-neuropilin-1 complexes form functional semaphorin-3A receptors[J].Cell ,1999,99(1):59-69.
[63] Kawasaki T, Kitsukawa T, Bekku Y, et al.A requirement for neuropilin-1 in embryonic vessel formation[J].Development ,1999,126(21):4895-902.
[64] Lee P, Goishi K, Davidson AJ, et al.Neuropilin-1 is required for vascular development and is a mediator of VEGF-dependent angiogenesis in zebrafish[J]. Proc. Natl. Acad. Sci. U. S. A. 2002,99(16):10470-5.
[65] Fuh G, Garcia KC, de Vos AM. The interaction of neuropilin-1 with vascular endothelial growth factor and its receptor flt-1[J]. J. Biol. Chem. 2000 Sep 1;275(35):26690-5.
[66] Mac Gabhann F, Popel AS. Differential binding of VEGF isoforms to VEGF receptor2 in the presence of neuropilin-1: a computational model[J]. Am J Physiol Heart Circ Physiol. 2005,288(6):H2851-60.
[67] Bagnard D, Vaillant C, Khuth ST,et al. Semaphorin 3A-vascular endothelial growth factor-165 balance mediates migration and apoptosis of neural progenitor cells by the recruitment of shared receptor[J]. J Neurosci. 2001,21(10):3332-41.
[68] Schwarz Q, Gu C, Fujisawa H, et al.Vascular endothelial growth factor controls neuronal migration and cooperates with Sema3A to pattern distinct compartments of the facial nerve[J]. Genes Dev. 2004,18(22):2822-34.
[69] Gu C, Rodriguez ER, Reimert DV, et al.Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development.[J] Dev Cell. 2003,5(1):45-57.
[70] Bernfield M, G?tte M, Park PW,et al. Functions of cell surface heparan sulfate proteoglycans[J]. Annu Rev Biochem. 1999;68:729-77.
[71] Esko JD, Lindahl U. Molecular diversity of heparan sulfate [J]. J Clin Invest. 2001 ,108(2):169-73.
[72] Bernfield M, Kokenyesi R, Kato M, et al.Biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans[J]. Annu Rev Cell Biol. 1992;8:365-93.
[73] Mertens G, Cassiman JJ, Van den Berghe H,et al. Cell surface heparan sulfate proteoglycans from human vascular endothelial cells. Core protein characterization and antithrombin III binding properties[J]. J Biol Chem. 1992,267(28):20435-43.
[74] Kinsella MG, Wight TN. Structural characterization of heparan sulfate proteoglycan subclasses isolated from bovine aortic endothelial cell cultures[J]. Biochemistry, 1988,27(6):2136-44.
[75] Kojima T, Leone CW, Marchildon GA,et al. Isolation and characterization of heparan sulfate proteoglycans produced by cloned rat microvascular endothelial cells[J].J Biol Chem. 1992,267(7):4859-69.
[76] Gengrinovitch S, Berman B, David G, et al. Glypican-1 is a VEGF165 binding proteoglycan that acts as an extracellular chaperone for VEGF165[J]. J Biol Chem. 1999 ,274(16):10816-22.
[77] Gitay-Goren H, Cohen T, Tessler S,et al.Selective binding of VEGF121 to one of the three vascular endothelial growth factor receptors of vascular endothelial cells[J]. J Biol Chem. 1996,271(10):5519-23.
[78] Gitay-Goren H, Soker S, Vlodavsky I,et al.The binding of vascular endothelial growth factor to its receptors is dependent on cell surface-associated heparin-like molecules[J]. J Biol Chem. 1992 ,267(9):6093-8.
[79] Ashikari-Hada S, Habuchi H, Kariya Y,et al. Heparin regulates vascular endothelial growth factor165-dependent mitogenic activity, tube formation, and its receptor phosphorylation of human endothelial cells. Comparison of the effects of heparin and modified heparins[J]. J Biol Chem. 2005,280(36):31508-15.
[80] Tessler S, Rockwell P, Hicklin D, et al. Heparin modulates the interaction of VEGF165 with soluble and cell associated flk-1 receptors[J]. J Biol Chem. 1994 ,269(17):12456-61.
[81] Terman B, Khandke L, Dougher-Vermazan M,et al. VEGF receptor subtypes KDR and FLT1 show different sensitivities to heparin and placenta growth factor[J].Growth Factors. 1994,11(3):187-95.
[82] Dougher AM, Wasserstrom H, Torley L,et al.Identification of a heparin binding peptide on the extracellular domain of the KDR VEGF receptor[J].Growth Factors 1997,14 (4) :257-68.
[83] Keyt BA, Nguyen HV, Berleau LT,et al. Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. Generation ofreceptor-selective VEGF variants by site-directed mutagenesis[J].J. Biol. Chem. 1996,271 (10); 5638-46.
[84] Ito N, Claesson-Welsh L. Dual effects of heparin on VEGF binding to VEGF receptor-1 and transduction of biological responses[J]. Angiogenesis, 1999,3 (3) 159-166.
[85] Cohen T, Gitay-Goren H, Sharon R, et al.VEGF121, a vascular endothelial growth factor (VEGF) isoform lacking heparin binding ability, requires cell-surface heparan sulfates for efficient binding to the VEGF receptors of human melanoma cells[J]. J. Biol. Chem. 1995 ,270 (19): 11322-6.
[86] Yl(a|¨)-Herttuala S, Rissanen TT, Vajanto I,et al. Vascular endothelial growth factors: biology and current status of clinical applications in cardiovascular medicine[J]. J Am Coll Cardiol. 2007,49(10):1015-26.
[87] Breier G, Albrecht U, Sterrer S, et al, Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation[J]. Development 1992,114 (2) :521-532.
[88] Flamme I, Breier G, Risau W. Vascular endothelial growth factor (VEGF) and VEGF receptor 2 (flk-1) are expressed during vasculogenesis and vascular differentiation in the quail embryo[J].Dev. Biol. 1995,169 (2): 699- 712.
[89] Ferrara N, Carver-Moore K, Chen H, et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene[J]. Nature, 1996,380 (6573) :439-442.
[90] Carmeliet P, Ferreira V, Breier G,et al, Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele[J].Nature, 1996, (6573) :435- 439.
[91] Ferrara N. Vascular endothelial growth factor: basic science and clinical progress[J]. Endocr Rev, 2004,25 (4):581-611.
[92] Ferrara N. Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications [J]. Semin Oncol. 2002,29(6 Suppl 16):10-4.
[93] Roccaro AM, Russo F, Cirulli T, et al, Antiangiogenesis for rheumatoid arthritis[J]. Curr Drug Targets Inflamm Allergy. 2005,4(1):27-30.
[94] L.E. Smith, Pathogenesis of retinopathy of prematurity[J]. Growth Horm IGF Res. 14 (Suppl. A) (2004) S140-S144.
[95] Lashkari K, Hirose T, Yazdany J, et al, Vascular endothelial growth factor and hepatocyte growth factor levels are differentially elevated in patients with advanced retinopathy of prematurity[J]. Am. J. Pathol. 2000,156 (4): 1337-44.
[96] Zelzer E, McLean W, Ng YS,et al. Skeletal defects in VEGF(120/120) mice reveal multiple roles for VEGF in skeletogenesis[J].Development ,2002, 129(8):1893-904.
[97] Gerber HP, Vu TH, Ryan AM,et al. VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation[J].Nat Med 1999, 5(6):623-628.
[98] Haigh JJ, Gerber HP, Ferrara N,et al. Conditional inactivation of VEGF-A in areas of collagen2A1 expression results in embryonic lethality in the heterozygous state[J].Development 2000; 127(7):1445-53.
[99] Gerstenfeld LC, Cullinane DM, Barnes GL, et al. Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation. J. Cell. Biochem. 2003,88(5), 873–84.
[100] Tatsuyama K, Maezawa Y, Baba H, et al. Expression of various growth factors for cell proliferation and cytodifferentiation during fracture repair of bone[J]. Eur. J. Histochem. 2000;44(3):269-78.
[101] Orlandini M, Spreafico A, Bardelli M ,et al.Vascular Endothelial Growth Factor-D Activates VEGFR-3 Expressed in Osteoblasts Inducing Their Differentiation[J]. J Biol Chem. 2006 ;281(26):17961-7.
[102] Street J, Bao M, deGuzman L, et al. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover[J].Proc Natl Acad Sci USA 2002; 99(15):9656- 61.
[103]初同伟,王正国,朱佩芳等。血管内皮生长因子在骨折愈合中的作用[J].中国修复重建外科杂志,2002,16(2)75-78.
[104]初同伟,王正国,朱佩芳.血管内皮生长因子对骨折愈合相关因子表达的调控[J]..中华骨科杂志2003;23(4):235-238.
[105] Wang DS, Miura M, Demura H, et al. Anabolic effects of 1, 25-dihydroxyvitamin D3 on osteoblasts are enhanced by vascular endothelial growth factor produced by osteoblasts and by growth factors produced by endothelial cells[J]. Endocrinology 1997; 138(7):2953-62.
[106] Bouletreau PJ, Warren SM, Spector JA, et al. Hypoxia and VEGF up-regulate BMP-2 mRNA and protein expression in microvascular endothelial cells: implications for fracture healing[J]. Plast Reconstr Surg ,2002,109 (7):2384-97.
[107] Niida S, Kaku M, Amano H, et al. Vascular endothelial growth factor can substitute for macrophage colony-stimulating factor in the support of osteoclastic boneresorption[J]. J Exp Med ,1999,190(2):293-8.
[108] Engsig MT, Chen QJ, Vu TH, et al. Matrix metalloproteinase9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones[J].J Cell Biol 2000; 151:879-89.
[109] Deckers MM, Karperien M, van der Bent C,et al Expression of Vascular Endothelial Growth Factors and Their Receptors during Osteoblast Differentiation[J].。Endocrinology 2000,141(5): 1667-74.
[110] Midy V, Plouet J. Vasculotropin/vascular endothelial growth factor induces differentiation in cultured osteoblasts. Biochem. Biophys. Res. Commun. 1994,199(1), 380-6.
[111] Kaigler D, Wang Z, Horger K, et al.VEGF Scaffolds Enhance Angiogenesis and Bone Regeneration inIrradiated Osseous Defects[J]. J Bone Miner Res, 2006,21(5):735-44
[112] Hiltunen MO, Ruuskanen M, Huuskonen J,et al. Adenovirus- mediated VEGF-A gene transfer induces bone formation in vivo[J]. FASEB J. 2003,17(9):1147-9.
[113] Bostrom MP, Saleh KJ, Einhorn TA. Osteoinductive growth factors in preclinical fracture and long bone defects models[J]. Orthop Clin North Am. 1999 Oct;30(4):647-58.
[114] Oliver RC, Brown LJ, L?e H. Periodontal diseases in the United States population[J]. J Periodontol 1998,69(2):269-78.
[115] Anusaksathien O, Giannobile WV. Growth factor delivery to re-engineer periodontal tissues[J]. Curr Pharm Biotechnol. 2002, 3(2):129-39.
[116] Crombleholme TM. Adenoviral-mediated gene transfer in wound healing[J]. Wound Repair Regen,2000 8(6):460-72.
[117] Albertson KS, Medoff RJ, Mitsunaga MM (). The use of periosteally vascularized autografts to augment the fixation of large segmental allografts[J]. Clin Orthop Rel Res ,1991,269:113-9.
[118] Enneking WF, Mindell ER. Observations on massive retrieved human allografts[J]. J Bone Joint Surg Am,1991, 73(8):1123-42.
[119] Cook SD, Baffes GC, Wolfe MW, et al. The effect of recombinant human osteogenic protein-1 on healing of large segmental bone defects[J]. J Bone Joint Surg Am,1994,76(6):827-38.
[120] Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells[J]. Cell and Tissue Kinetics 1970,3(4):393–403.
[121]潘华,王大伟,李红波,等.密度梯度离心与贴壁法相结合体外分离培养兔骨髓基质干细胞:1～3代细胞生长特性[J].中国组织工程研究与临床康复,2007,11(42):8487-8490.
[122]金光鑫,吴德全.脐带血间充质干细胞研究进展[J].国际移植与血液净化杂志,2006,4(5):45-48.
[123] Huang JI,Beanes SR,Zhu M,et al.Rat extramedullary adipose tissue as a source of osteochondrogenic progenitor cells[J].Plast ReconstrSurg,2002,109(3):1033.1041.
[124] Halvorsen YD, Bond A, Sen A, et al. Thiazolidinediones and glucocorticoids synergistically induce differentiation of human adipose tissue stromal cells: Biochemical, cellular, and molecular analysis[J]. Metab. Clin. Exp. 2001,50 (4) :407-13.
[125] Oligino TJ, Yao Q, Ghivizzani SC, et al. Vector systems for gene transfer to joints[J]. Clin Orthop Rel Res 2000, 379:S17-30.
[126] Lakshmipathy U, Pelacho B, Sudo K, et al.Efficient transfection of embryonic and adult stem cells[J]. Stem Cells,2004,22 (4) 531-543.
[127] Franceschi RT, Wang D, Krebsbach PH,. Gene therapy for bone formation: in vitro and in vivo osteogenic activity of an adenovirus expressing BMP7[J]. J Cell Biochem, 2000,78(3):476-86.
[128] John M. Coffin, Stephen H. Hughes, Harold E. Varmus, Retroviruses[J].Cold Spring Harbor Laboratory Press, 1997.
[129] Adam MA, Ramesh N, Miller AD,et al. Internal initiation of translation in retroviral vectors carrying picornavirus 5’nontranslated regions[J].J Virol. 1991,65 (9): 4985- 90.
[130] Apperley JF, Luskey BD, Williams DA. Retroviral gene transfer of human Adenosine deaminase in murine hematopoietic cells: effect of selectable marker sequences on longterm expression[J].Blood, 1991,78 (2): 310- 317.
[131] Danos O, Heard JM . Recombinant retroviruses as tools for gene transfer to somatic cells[J]. Bone Marrow Transplant, 1992,9(Suppl 1):131-8.
[132] Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al.A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency[J]. N Engl J Med, 2003,348(3):255-6.
[133] Noguchi P. Risks and benefits of gene therapy[J]. N Engl J Med, 2003,348(3):193-4.
[134] Neumann R, Chroboczek J, Jacrot B. Determination of the nucleotide sequencefor the penton-base gene of human adenovirus type 5[J]. Gene, 1988,69(1):153-7.
[135] Bergelson JM, Cunningham JA, Droguett G, et al. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5[J].Science, 1997, 275 (5304): 1320-3.
[136] Zhao M, Zhao Z, Koh JT,et al (2005a). Combinatorial gene therapy for bone regeneration: cooperative interactions between adenovirus vectors expressing bone morphogenetic proteins 2, 4 and 7[J]. J Cell Biochem 2005,95(1):1-16.
[137] Armentano D, Zabner J, Sacks C, et al. Effect of the E4 region on the persistence of transgene expression from adenovirus vectors[J]. J Virol, 1997,71(3):2408-16.
[138] Hartigan-O'Connor D, Amalfitano A, Chamberlain JS. Improved production of gutted adenovirus in cells expressing adenovirus preterminal protein and DNA polymerase[J]. J Virol,1999 , 73(9):7835-41.
[139] Zhang XY, La Russa VF, Bao L,et al. Lentiviral vectors for sustained transgene expression in human bone marrow-derived stromal cells[J]. Mol Ther, 2002,5(5):555-565.
[140] Vigna E, Naldini L. Lentiviral vectors: excellent tools for experimental gene transfer and promising candidates for gene therapy[J].J Gene Med 2000 ,2(5):308-16.
[141] Sugiyama O, An DS, Kung SP, et al. Lentivirus-mediated gene transfer induces long-term transgene expression of BMP-2 in vitro and new bone formation in vivo[J]. Mol Ther. 2005,11(3):390-8
[142] Büning H, Braun-Falco M, Hallek M. Progress in the use of adeno-associated viral vectors for gene therapy[J]. Cells Tissues Organs. 2004;177(3):139-50
[143] Luk KD, Chen Y, Cheung KM. Adeno-associated virus-mediated bone morphogenetic protein-4 gene therapy for in vivo bone formation[J]. Biochem Biophys Res Commun. 2003,308(3):636-45
[144] Alsberg E, Anderson KW, Albeiruti A,et al.Cell-interactive alginate hydrogels for bone tissue engineering[J]. J Dent Res. 200 ,80(11):2025-9.
[145] Tsuang YH,Lin FH,Sun JS,et al.In vitro cell behavior of osteoblasts on Pyrost bone substitute[J].Anat Rec.1997,247(2):164-9.
[146] Rocha LB,Goissis G, Rossi MA. Biocompatibility of anionic collagen matrix as scaffold for bone healing[J].Biomaterials. 2002;23(2):449-456.
[147] Sato M, Asazuma T, Ishihara M, et al. An atelocollagen honeycomb-shaped scaffold with a membrane seal (ACHMS-scaffold)for the culture of annulus fibrosus cells from an intervertebral disc[J].J Biomed Mater Res A.2003;64(2):248-256.
[148] Seol YJ,Lee JY,Park YJ,et al.Chitosan sponges as tissue engineering scaffolds for bone formation[J].Biotechnol Lett. 2004;26(13):1037-41.
[149] Klokkevold PR,Vandemark L,Kenney EB,et al.Osteogenesis enhanced by chitosan(poly-N-acetyl glucosaminoglycan) in vitro[J]. J Periodontol.1996;67(11): 1170-5.
[150] VANDEPUTTE KA, URIST MR..Experimental mineralization of collagen sponge and decalcified bone[J].Clin Orthop Rel Res 1965, 40:48-56.
[151] VANDEPUTTE KA, URIST MR.Osteogenesis in the interior of intramuscular implants of decalcified bone matrix[J].Clin Orthop Rel Res 1966,43:257-70.
[152] Urist MR,Wallace TH,Adams T.The function of fibrocartilaginous fracture callus[J].JBJS 1965; 47:304-18.
[153] Urist MR, Silverman BF, Büring K, et al.The bone induction principle[J]. Clin Orthop Relat Res. 1967,53:243-83.
[154] Urist MR, Dawson E. Intertransverse process fusion with the aid of chemosterilized autolyzed antigen-extracted allogeneic (AAA) bone[J]. Clin Orthop Rel Res 1980; 154:97-113.
[155] Harakas NK. Demineralized bone-matrix-induced osteogenesis[J]. Clin Orthop Rel Res 1984,188:239-51.
[156] Russell JL, Block JE. Clinical utility of demineralized bone matrix for osseous defects, arthrodesis, and reconstruction: impact of processing techniques and study methodology[J]. Orthopaedics 1999; 22(5):524-31.
[157] Zhu L,Liu W,Cui L,et al.Tissue-engineered bone repair of goat-femur defects with osteogenically induced bone marrow stromal cells[J].Tissue Eng.2006;12(3):423-33.
[158]张华林,陈治清.羊毛角蛋白作为骨组织工程支架材料的研究进展[J].国际口腔医学杂志,2007, 34(3): 390-394..
[159]王嫣,陈小菊,王兰,等.海藻酸钠凝胶对骨髓间充质干细胞生物学效应的初步研究[J].重庆医科大学学报,2006,31(4):478-481
[160] Hollinger JO, Battistone GC. Biodegradable bone repair materials. Synthetic polymers and ceramics. [J].Clin Orthop Rel Res 1986; 207:290-305.
[161] Friedman CD, Costantino PD, Takagi S,et al. BoneSource hydroxyapatite cement: a novel biomaterial for craniofacial skeletal tissue engineering and reconstruction[J]. J Biomed Mater Res. 1998 ,43(4):428-32.
[162] H?mmerle CH, Olah AJ, Schmid J,et al. The biological effect of natural bone mineral on bone neoformation on the rabbit skull[J]. Clin Oral Implants Res 1997;8(3):198-207.
[163] Eggli PS,Müller W,Schenk RK.Porous hydroxyapatite and tricalcium phosphate cylinders with two different pore size ranges implanted in the cancellous bone of rabbits.A comparative histomorphometric and histologic study of bony ingrowth and implant substitution[J].Clin Orthop Relat Res.1988; (232): 127-38.
[164] Levine JP, Bradley J, Turk AE,et al.Bone morphogenetic protein promotes vascularization and osteoinduction in preformed hydroxyapatite in the rabbit[J]. Ann Plast Surg. 1997,39(2):158-68.
[165] Tan KK, Tan GH, Shamsul BS, et al.Bone graft substitute using hydroxyapatite scaffold seeded with tissue engineered autologous osteoprogenitor cells in spinal fusion:early result in a sheep model[J]. Med J Malaysia. 2005,60 Suppl C:53-8.
[166] Deville S,Saiz E,Tomsia AP.Freeze casting of hydroxyapatite scaffolds for bone tissue engineering.Biomaterials[J].2006; 27(32):5480-5489.
[167] Morishita T,Honoki K,Ohgushi H,et al.Tissue engineering approach to the treatment of bone tumors:three cases of cultured bone grafts derived from patients'mesenchymal stem cells[J].Artif Organs.2006; 30(2):115-8.
[168]赵丽君,毛天球,陈富林,等.不同类型骨组织工程支架材料的比较研究[J].中国临床康复,2003,7(4):29-30.
[169] Ducheyne P,el-Ghannam A,Shapiro I.Effect of bioactive glass templates on osteoblast proliferation and in vitro synthesis of bone-like tissue[J]. J Cell Biochem. 1994; 56(2): 162-7.
[170]孙蕊,潘高峰,张丽芳,等.聚乳酸/聚乙二醇-聚乳酸新型亲水支架的制备与研究[J].生物医学工程学杂志,2007,24(1):91-93.
[171] Ren T,Ren J,Jia X,et al.The bone formation in vitro and mandibular defect repair using PLGA porous scaffolds[J].J Biomed Mater Res A.2005,74(4):562-9.
[172] Puelacher WC,Vacanti JP,Ferraro NF,et al.Femoral shaft reconstruction using tissue-engineered growth of bone[J].J Oral Maxillofac Surg.1996,25(3):223-228.
[173]胡稷杰,金丹,全大萍,等.负载不同浓度骨形态发生蛋白的组织工程骨体内成骨的量效关系[J].中华骨科杂志,2006,26(3):196-201.
[174] Zhang R, Ma PX. Porous poly(L-lactic acid)/apatite composites created by biomimetic process[J]. J Biomed Mater Res ,1999, 45(4):285-93.
[175] Zhang R, Ma PX. Poly(a-hydroxyl acids)/hydroxyapatite porous composites forbone-tissue engineering. I. Preparation and morphology[J]. J Biomed Mater Res, 1999,44(4):446-55.
[176] Roweton SL. A new approach to the formation of tailored microcellular foams and microtextured surfaces of absorbable and non-absorbable thermoplastic biomaterials. Master of Science Thesis, Department of Bioengineering, Clemson University, 1994.
[177] Elisseeff J, Anseth K, Sims D,et al. Transdermal photopolymerization for minimally invasive implantation[J].Proc Nat Acad Sci USA 1999,96 (6):3104-7.
[178] Meinig RP, Rahn B, Perren SM, Gogolewski S. Bone regeneration with resorbable polymeric membranes: treatment of diaphyseal bone defects in the rabbit radius with poly(L-lactide) membrane. A pilot study[J].J Orthop Trauma, 1996,10(3):178-90.
[179] Meinig RP, Buesing CM, Helm J, et al. Regeneration of diaphyseal bone defects using resorbable poly(L/DL-lactide) and poly(D-lactide) membranes in the Yucatan pig model[J]. J Orthop Trauma, 1997,11(8):551-8.
[180] Gugala Z, Gogolewski S. Regeneration of segmental diaphyseal defects in sheep tibiae using resorbable polymeric membranes: a preliminary study[J]. J Orthop Trauma, 1999,13(3):187-95.
[181] Ekholm M, Hietanen J, Lindqvist C, Rautavuori J, Santavirta S,Suuronen R. Histological study of tissue reactions to e-caprolactone-lactide copolymer in paste form[J]. Biomater, 1999,20(14):1257-62.
[182] Muggli DC, Lee HR, Keyser SA, et al. Photocrosslinkable polyanhydride networks for use in orthopedic applications. In:Peppas NA, Mooney DJ, Mikow AG, Brannon-Peppas L, editors.Biomaterials, carriers for drug delivery, and scaffolds for tissue engineering[J].New York: AIChE, 1997. p. 275-7.
[183]申玉芹,寡核苷酸(ODN)促骨髓间充质干细胞向成骨细胞分化作用及机制研究。吉林大学博士论文
[184] Friedenstein AJ , Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs [J]. Exp Hemator, 1976, 4(5):267-74.
[185] Bianco P, Riminucci M, Gronthos S,et al. Bone marrow stromal stem cells: nature, biology, and potential applications[J].Stem Cells 2001; 19(3): 80-192
[186]邱丽燕,王金福.骨髓间充质干细胞的研究进展[J].生物工程学报,2003,19(2)136-140
[187] Pittenger MF ,Mackay AM,Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells[J].Science, 1999,284 (5411) : 143-147.
[188] Deans RJ,Moseley AB.Mesenchymal stem cells:biology and potential clinical uses[J].Exp Hematol,2000,28(8):875-84.
[189] Shugart EC, Umek RM. Dexamethasone signaling is required to establish the postmitotic state of adipocyte development[J]. Cell Growth Differ, 1997,8(10): 91-8.
[190] Hauner H, Schmid P, Pfeiffer EF.. Glucocorticoids and insulin promote the differentiation of human adipocyte precursor cells into fat cells[J]. J Clin Endocrinol Metab ,1987,64(4):832-5.
[191] L(o|¨)ffler G, Hauner H. Adipose tissue development: the role of precursor cells and adipogenic factors. Part II: The regulation of the adipogenic conversion by hormones and serum factors[J]. Klin Wochenschr, 1987,65(17):812-7.
[192] Zhang HH, Huang J, Düvel K, et al. Insulin stimulates adipogenesis through the Akt-TSC2-mTORC1 pathway[J].PLoS One 2009; 4(7): e6189.
[193] Ringold GM, Chapman AB, Knight DM,. Hormonal control of adipogenesis[J]. Ann NY Acad Sci, 1986, 478:109-19.
[194] Kellinsalmi M, Parikka V, Risteli J, et al. Inhibition of cyclooxygenase-2 down-regulates osteoclast and osteoblast differentiation and favours adipocyte formation in vitro[J]. Eur J Pharmacol 2007,572:102-10.
[195] Lehmann JM, Lenhard JM, Oliver BB,et al. Peroxisome proliferator-activated receptors alpha and gamma are activated by indomethacin and other non-steroidal anti-inflammatory drugs[J]. J Biol Chem ,1997,272(6):3406-10.
[196] Ye H, Serrero G. Stimulation of adipose differentiation related protein (ADRP) expression by ibuprofen and indomethacin in adipocyte precursors and in adipocytes[J]. Biochem J 1998;330(Pt. 2):803-9.
[197] Otsuka E, Yamaguchi A, Hirose S, et al. Characterization of osteoblastic differentiation of stromal cell line ST2 that is induced by ascorbic acid[J]. Am J Physiol. 1999,277(1 Pt 1):C132-8.
[198] Prasher DC,Eckenrode VK,Ward WW,et al.Primary structure of the Aequorea victoria green-fluorescent protein[J].Gene, 1992, 111(2):229-33
[199] Prasher D,McCann RO,Cormier MJ.Cloning and expression of the cDNA coding for aequorin,a bioluminescent calcium-binding protein[J].Biochem Biophys Res Commun, 1985, 126(3):1259-68
[200] Hastings JW.Chemistries and colours of bioluminescent reactions: a review[J]. Gene, 1996,173:5-11.
[201] Tsien RY. The green fluorescent protein[J]. Annu Rev Bioche1998, 67: 509-544.
[202]李志勇,刘磊,田卫东,等.绿色荧光蛋白基因转染大鼠骨髓间充质干细胞的实验研究[J].中华口腔医学杂志,2005,40(2):150-153.
[203] Cancedda R,Giannoni P,Mastrogiacomo MA. tissue engineering approach to bone repair in large animal models and in clinical practice[J]. Biomaterials, 2007, 28(29): 4240-50.
[204] Nomi M,Miyake H,Sugita Y,et al.Role of growth factors and endothelial cells in therapeutic angiogenesis and tissue engineering[J].Curr Stem Cell Res Ther,2006,1(3):3353
[205] Ball SG, Shuttleworth CA, Kielty CM. Mesenchymal stem cells and neovascularization:role of platelet-derived growth factor receptors[J]. J Cell Mol Med, 2007,11(5): 1012-30.
[206] Ball SG, Shuttleworth CA, Kielty CM. Vascular endothelial growth factor can signal through platelet-derived growth factor receptors[J]. J Cell Biol ,2007,177(3):489-500.
[207] Levy O, Dvir T, Tsur-Gang O, et al. Signal transducer and activator of transcription 3-A key molecular switch for human mesenchymal stem cell proliferation[J]. Int J Biochem Cell Biol,2008, 40(11): 2606-18.
[208] Harada S,Nagy JA,Sullivan KA,et al.Induction of vascular endothelial growth factor expression by prostaglandin E2 and E1 in osteoblasts [J]. J Clin Invest,1994, 93(6):2490-6
[209] Cotter EJ,Ip HS,Powderly WG, et al. Mechanism of HIV protein induced modulation of mesenchymal stem cell osteogenic differentiation[J]. BMC Musculoskelet Disord. 2008; 9: 33.
[210] Beloti MM, de Oliveira PT, Tagliani MM.Bone cell responses to the composite of Ricinus communis polyurethane and alkaline phosphatase[J]. J Biomed Mater Res A. 2008;84(2):435-441.
[211] Schouten C, van den Beucken JJ, de Jonge LT,et al. The effect of alkaline phosphatase coated onto titanium alloys on bone responses in rats[J].Biomaterials. 2009 ,30(32):6407-17.
[212] Leonardi E, Ciapetti G, Baglìo SR,et al.Osteogenic properties of late adherent subpopulations of human bone marrow stromal cells[J]. Histochem Cell Biol. 2009 ,132(5):547-57.
[213]宋爱梅,束蓉.体外培养骨髓间充质干细胞向成骨细胞分化的影响因素研究[J].牙体牙髓牙周病学杂志,2005,15(1) :55-58.
[214] Tilgar V, Kilgas P, Viitak A,et al. The rate of bone mineralization in birdsis directly related to alkaline phosphatase activity[J]. Physiol Biochem Zool,2008, 81(1): 106-111.
[215] Sim(a|¨)o AM, Beloti MM, Rosa AL,et al. Culture of osteogenic cells from human alveolar bone: a useful source of alkaline phosphatase[J]. Cell Biol Int.2007;31(11): 1405-13.
[216]范仲凯,张元和,姚琦,卢伟,转染血管内皮生长因子基因的人成骨细胞增殖及其生物学功能[J].中国组织工程研究与临床康复, 2009,13(50): 9850-54
[217] Huh JE, Kwon NH, Baek YH, et al.Formononetin promotes early fracture healing through stimulating angiogenesis by up-regulating VEGFR-2/Flk-1 in a rat fracture model[J]. Int Immunopharmacol. 2009; 9(12):1357-65.
[218] Chu Q, Wu ZF, Wang QT,et al.Influence of transfection with human transforming growth factor-beta1 gene on the osteogenic potential of the cultured human gingival fibroblast[J].Hua Xi Kou Qiang Yi Xue Za Zhi. 2009; 27(3):264-7.
[219] Nakashima K, Zhou X, Kunkel G,et al. The Novel Zinc Finger-Containing Transcription Factor Osterix Is Required for Osteoblast Differentiation and Bone Formation[J]. Cell, 2002; 108(1): 17-29.
[220] GashlerAL, Swaminathan S, Sukhatme VP. A novel repression module, an extensive activation domain, and a bipartite nuclear localization signal defined in the immediate-early transcription factor EGR -1[J]. Mol Cell Biol, 1993, 13(8): 4556-71.
[221] Abe A, Inoue K, Tanaka T, et al. Quantitation of hepatitis B virus genomic DNA by real-time detection PCR[J].J Clin Microbiol. 1999, 37(9): 2899-2903.
[222] Heid CA, Stevens J, Livak KJ, et al. Real- time quantitativePCR[J].Genome Res, 1996, 6(10): 986-94.
[223] Livark KJ, Schmittgen TD.Analysis of relative gene expression datausing real-time quantitative PCR and the 2-ΔΔCtmethod[J].Methods,2001, 25(4): 402-408.
[224] Jaiswal RK,Jaiswal N,Bruder SP,et al.Adult human mesenchymal stem cell differentiation to the osteogenic oradipogenic lineage is regulated by mitogen activated protein kinase[J]. J Biol Chem,2000,275(13):9645-52.
[225] Urist, M. R. Bone: Formation by autoinduction[J]. Science.1965,150 (698):893-9.
[226] Wozney JM, Rosen V, Celeste AJ,et al. Noval Regulators of Bone Formation: Molecular Ctones and Activities[J]. Science, 1988 ,242(4885):1528-34.
[227] Wang EA,Rosen V,Cordes P,et al.Purification and Characterization of Other Distinct Bone-inducing Factors[J]. ProcNatl Acad Sci USA,1988,85 (24): 9484-8.
[228] Matthews SJ. Biological activity of bone morphogenetic proteins (BMP’s) [J]. Injury. 2005 ,36 (Suppl 3):S34-7.