【作者】 黄俊俊；

【导师】 贾连顺；

【作者基本信息】 第二军医大学 ， 外科学， 2012， 博士

【摘要】 目的非常严重的韧带损伤无法自行修复，往往需要韧带的重建。但无论自体或异体的韧带移植物及合成材料均不能取得满意结果。组织工程学的兴起为韧带重建提供了一条新的途径。组织工程学韧带构建包括建立韧带形态的支架结构，培养种子细胞并在支架上生长和产生细胞外基质，对原来的支架进行改建，最终形成具有自我更新和修复能力的韧带组织。脱细胞韧带支架通过脱去细胞成分，基本消除了免疫源性，同时保留天然韧带的形态和结构，生物力学性能未受影响。作为一种天然支架材料受到广泛关注。骨髓间充质干细胞（bone marrow mesenchymal cells，BMSCs）是目前比较热门的种子细胞，体外能快速扩增。有实验表明碱性成纤维细胞生长因子（bFGF）可促进骨髓间充质干细胞生长和分泌韧带特异性细胞外基质。通过转基因治疗基因转导入生长因子基因并在体内改良种子细胞及改变体内微环境来促进器官组织的形成，是目前组织工程中研究的热点。本研究利用腺病毒载体将碱性成纤维细胞生长基因转入骨髓间充质干细与脱细胞韧带支架复合培养共同构建组织工程韧带。方法1．分离、培养兔BMSCs和韧带成纤维细胞（ligament fibroblast，LF）。并对BMSCs细胞进行鉴定分别通过骨髓穿刺Ficoll密度梯度离心法和组织块培养法分离兔骨髓间充质干细胞和髌韧带成纤维细胞。MTT法检测两种细胞的增殖活性。通过成骨，成软骨，成脂肪细胞多向分化能力的测定来鉴定BMSCs。2．Gateway技术构建携带人bFGF基因的重组腺病毒载体(pAd-bFGF-GFP)构建只需两步：BP反应构建入门克隆，LR反应创建表达克隆。表达克隆在293细胞包装成目标腺病毒。测定病毒滴度。扩增腺病毒载体转染293细胞后realtimeRT-PCR及westernblot检测bFGF的表达。3.pAd-bFGF-GFP转染兔骨髓间充质干细胞后测定培养液中bFGF的表达和对细胞增值及分泌细胞外基质的影响及与韧带成纤维细胞的比较携带bFGF基因的腺病毒用不同MOI值来转染兔骨髓间充质干细胞，通过流式细胞仪检测转染效率。 MTT法检测转染后骨髓间充质干细胞增殖来确定最佳MOI值。以最佳MOI值转染骨髓间充质干细胞，ELISA检测bFGF蛋白表达、分泌趋势。将携带bFGF目的基因腺病毒转染的BMSCs（pAd-bFGF-GFP.BMSCs）、LF，BMSCs，转染空病毒的BMSCs（pAd-GFP-BMSCs）进行MTT细胞增殖比较。Realtime RT-PCR检测bFGF对BMSCs表达Collagen Ⅰ和Collagen Ⅲ、波形蛋白（Vimentin）在mRNA水平的影响。4．韧带脱细胞处理，并进行组织学及生物力学检测；1%DCA法进行韧带脱细胞处理。将pAd-bFGF-GFP.BMSCs,LF， BMSCs，pAd-GFP-BMSCs种植脱细胞韧带支架，MTT法检测种子细胞在脱细胞支架上的黏附、生长、增殖情况。Realtime RT-PCR检测种子细胞Collagen Ⅲ、Collagen Ⅰ和Vimentin蛋白mRNA水平的表达。组织学切片，冰冻切片和共聚焦显微镜观察pAd-bFGF-GFP.BMSCs在支架的生长分布情况。生物力学检测体外复合培养对韧带机械性能的影响。结果1．分离培养的BMSCs与LF在大体形态学上相似，均表现为长梭形、漩涡样生长。MTT检测结果显示BMSCs的增殖活性明显优于LF。BMSCs具有向成骨，成脂肪，成软骨多向分化能力。2．成功构建了携带人bFGF基因的质粒，对重组质粒进行PCR鉴定和基因测序表明成功合成和整合了人bFGF基因。用重组质粒腺病毒载体系统成功构建了重组腺病毒pAd.bFGF-GFP。通过在293细胞内的扩增，我们得到了高滴度的病毒,滴度测定为（4.85×109ifu/ml）。realtime RT-PCR及Western-blot证实目的基因bFGF在基因水平和蛋白水平高效表达。3．腺病毒对BMSCs细胞有很高的转染效率，MTT结果显示MOI值为200时，BMSCs细胞具有最强的增值能力。ELISA检测显示：转染后48h bFGF就已经显著表达，分泌高峰出现在第7天，此后逐渐下降,2周时仍检测到。pAd-bFGF-GFP转染BMSCs与其他细胞相比具有更强的细胞增值能力。培养2周后Realtime RT-PCR检测显示Collagen Ⅰ、Collagen ⅢmRNA表达均显著高于其他组。VimentinmRNA表达与LF相当。4． MTT法检测显示种子细胞与脱细胞支架复合培养后bFGF基因转染骨髓间充质干细胞后能明显促进细胞生长。Realtime PCR显示pAd-bFGF-GFP转染BMSCs的Collagen Ⅲ、Collagen Ⅰ表达均显著高于其他组。Vimentin mRNA表达与成纤维细胞相当。HE染色和冰冻切片共聚焦显微镜观察pAd-bFGF-GFP-BMSCs在脱细胞韧带纤维间生长良好，呈梭形生长。生物力学检测显示复合培养脱细胞韧带与未培养韧带相比最大应力没有差别，弹性模量降低。结论1．成功分离培养获得兔骨髓间充质干细胞和韧带成纤维细胞，并通过分化培养鉴定确认获得具有多向分化能力的BMSCs。成纤维细胞的获取创伤较大。2．成功构建了携带人bFGF基因的高滴度缺陷型重组腺病毒。3．转染bFGF基因的BMSCs细胞能持续分泌bFGF至少两周，具有很强的增值能力和表达韧带特异性细胞外基质蛋白mRNA能力，优于韧带成纤维细胞和BMSCs细胞和转染空病毒的BMSCs细胞，4．体外转基因BMSCs能在脱细胞韧带上良好生长，并能大量表达韧带特异性的基质蛋白mRNA，优于韧带成纤维细胞和BMSCs细胞和转染空病毒的BMSCs细胞，生物力学性能基本保留。更多还原

【Abstract】 ObjectiveNatural healing fails to take effect in severe injury of ligaments.Replacement grafts are usually required. However，Synthetic replacements orautografts and allografts can’t achieve satisfactory results. Tissueengineering ligament construct including the establishment of the supportstructure of the ligament，introduction of seed cells to grow and produceextracellular matrix on the stent to carry out alterations to the originalstent and ultimately the formation of self-renewal ligament tissue. Nowadays,with the rapid development of biotechnology, tissue-engineered ligaments(TELs) have provided a new strategy for injured ligaments replacement.Acellular ligament was deprived of cellular components and eliminated off theimmunogenity while retaining the natural shape and property of the ligamentbiomechanical properties.It, as a natural scaffold material, has receivedwidespread attention. Bone marrow mesenchymal stem cells (BMSCs) arecurrently popular seed cells. Experiments show that the basic fibroblastgrowth factor (bFGF) can promote bone marrow mesenchymal stem cell growth andsecretion of ligament-specific extracellular matrix. Introducing growthfactor into cell to improve seed cell capacity and make change in vivomicroenvironment to promote the formation of organs and tissues, as a meanof gene therapy, is the hot topic. In this study, the adenovirus vector wasused to introduce basic fibroblast growth gene into bone marrow mesenchymalstem cells.Then tansfected BMSCs were cultured with acellular ligament tobuild TELs.Methods1. Isolatation and culture of the BMSCs and Ligament Fibroblast (LF).Isolation and culture of the BMSCs and LF was through femoral punctureand Ficoll density gradient centrifugation，tissue culture method separately.Proliferation activity of the two cells was assessed by MTT assay.BMSCs wereconfirmed by multiple lineage differentiation capacity. 2. Construction of the recombinant adenovirus vectors containing the bFGFgenes by Gateway technique.We constructed recombinant adenovector by gateway technique with onlytwo main steps:BP reaction to construct entry Clone and LR reaction toconstruct expression clone.Then expression clone were packaged in293cellsto form the tareget virus.We amplified the recombinant adenovirus and detectedvirus titer. We detect the expression of bFGF through RT-PCR and western-blotafter293cells were transfected.3.To investigate the expression of bFGF in culture medium after BMSCs weretransfected and the effect of bFGF on cell proliferation and production ofextracellular matrix proteins specific to ligaments in cells compared amongpAd-GFP-BMSCs,BMSCs，pAd.bFGF-GFP.BMSCs， LF.A. Transfection efficiency of adenovirus vector to BMSCs was dected byflow-cytometry analysis with different MOI valure.B.The most appropriate MOI was determined by MTT after BMSCs weretransfected with pAd-bFGF-GFP with different MOIC The secrection of bFGF was dected by ELLISA.D The effect of bFGF on cell proliferation was assessed by MTT.Productionof extracellular matrix proteins (collagen Ⅰ, collagen Ⅲ, andvimentin)specific to ligaments in cells was determined by mRNA expression ofRT-PCR compared among BMSCs， LF，BMSCs，pAd-GFP-BMSCs.4. Construction of tissure engineering ligaments by decellularizedligament scaffolds and seeded cellsRabbit patellar tendons were decellularized with1%DCA as perfomed in ourearly experiment.Then decelluarized ligament scaffolds were seeded in vitrowith pAdbFGF-GFP-BMSCs， pAd-GFP-BMSCs， BMSCs and LF to fabricate tissureengineering ligaments. The growth and proliferation of seeded cells on thescaffolds was estimated by MTT. Production of extracellular matrix proteins(collagen Ⅰ, collagen Ⅲ, and vimentin)specific to ligaments in cells wasdetermined by mRNA expression of RT-PCR compared among BMSCs， LF，BMSCs， pAd-GFP-BMSCs. In pAdbFGF-GFP-BMSCs seeded scaffolds histological analysisthrough confocal microscope and HE staining was conducted to observe cellsgrowth and distribution. Biomechanical measure was made to valuae theinfluence of coculture in vitro to decellularized ligaments.Results1.We successfully isolated and obtained the BMSCs and LF. BMSCs and LFwere similar in the general cell morphology such as both of them had spindleshape with whirlpool-like growth pattern.There were significant highproliferation in BMSCs compared to LF after MTT assay.BMSCs candifferentiate into bone cell,fat cell and chondrocyte.2.Recombinant adenovirus plasmid was constructed successfully determinedby electrophoresis after PCR amplification.The result of gene sequencing wascorrect. Recombinant adenovirus titer was4.85×109pfu/ml. bFGF was expressedafter293cells were transfected by pAd-bFGF-GFP vectors dected by real timeRT-PCR and western-blot3.BMSCs were effectively transfected in vitro. Cell proliferation wasgreatest when BMSCs were transfected at MOI200. bFGF protein was detected48h after transfection by ELISA.Expression level reached peak at7d, thendecreased gradually but bFGF was still detectable at14days after transfection.Proliferation of pAd-bFGF-GFP-BMSCs was significantly high than othergroups by MTT assay.Realtime RT-PCR showed that expression of collagen typeⅠ, collagen type Ⅲ, was higher significantly in pAd-bFGF-GFP-BMSCs than theother groups at two week. There was no significant difference in vimentinexpression between pAd-bFGF-GFP-BMSCs and LF.4． Proliferation of pAd-bFGF-GFP-BMSCs was significantly high thanother groups by MTT assay. Realtime RT-PCR showed that expression of collagentype Ⅰ, collagen type Ⅲ, was higher significantly in pAd-bFGF-GFP-BMSCsthan the other groups. There was no significant difference in vimentinexpression between pAd-bFGF-GFP-BMSCs and LF.HE stain and confocalmicroscopy showed that pAdbFGF-GFP-BMSCs infiltrated excellently intodecellularized scaffolds. Biomechanical measure showed that the maximum load of the decellularized ligament scaffolds with and without cell culture hadno significant difference. Stiffness was significantly low in cell culturegroups than decellularized ligaments scaffolds alone.Conclusions1． BMSCs were achieved easily, expanded massively to reach the demandfor tissure engineering ligaments in vitro after culture for2weeks. LF wasobtained successfully though by much more traumatic approach. BMSCs wasdetermined by multiple lineage differentiaon capacity2． pAd-bFGF-GFP vectors with high titer and purity were constructed,which can be used safely in vitro.3BMSCs were effectively transfected by pAd.bFGF-eGFP. Secrection of bFGFwas detected for at least14days. Proliferation of pAd-bFGF-GFP-BMSCs wassignificantly higher than other groups. Expression of matrix prorein wassignificantly higher in pAd-bFGF-GFP-BMSCs group than the other groups.4.Proliferation and infiltration of pAd-bFGF-GFP-BMSCs was excellent indecullarized ligaments. Expression of matrix prorein was highersignificantly in pAd-bFGF-GFP-BMSCs group than the other groups.Biomechanical property of the decellularized ligament scaffolds with cellculture was basically retained. Creating TEL by seeded transgenetic BMSCson decellularized rabbit ligament scaffolds was viable.更多还原