【作者】 刘霞；

【导师】 孙宏晨；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2011， 博士

【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^-8mol/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。更多还原

【Abstract】 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 3⁵ 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^-8mol/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^-8mol/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^-8mol/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.更多还原