pAd-bFGF-GFP转染兔骨髓间充质干细胞与脱细胞支架体外构建组织工程韧带的实验研究

发布时间：2018-07-12 12:36

本文选题：韧带 + 骨髓间充质干细胞　；参考：《第二军医大学》2012年博士论文

【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。
[Abstract]:objective
A very serious ligament injury can not be repaired by itself and often requires the reconstruction of ligaments. However, neither autologous or allograft ligaments and synthetic materials can achieve satisfactory results. The rise of tissue engineering provides a new way for the reconstruction of ligaments. Tissue engineering ligament construction includes the scaffolding of the form of ligaments. The seed cells grow and produce the extracellular matrix on the scaffold, and the original scaffold is rebuilt to form the ligamentous tissue with the ability of self renewal and repair. The acellular ligament scaffold eliminates the immune origin by removing the cell components, while retaining the morphology and structure of natural toughened bands, and the biomechanical properties are not affected. As a natural scaffold material, bone marrow mesenchymal cells (BMSCs) is one of the most popular seed cells and can be rapidly expanded in vitro. Some experiments have shown that basic fibroblast growth factor (bFGF) can promote the growth of bone marrow mesenchymal stem cells and the secretion of ligamentous extracellular matrix. It is a hot spot in tissue engineering to promote the formation of organ tissue by transferring gene transfected gene into the growth factor gene and improving the seed cells in the body and changing the microenvironment of the body in the body. This study uses adenovirus vector to transfer the basic fibroblast growth gene into the bone marrow mesenchymal stem and decellular ligaments. The structure of tissue engineering ligament was constructed by composite culture.
Method
1. isolation, culture of rabbit BMSCs and ligament fibroblast (LF) and identification of BMSCs cells.
The proliferation activity of two cells was detected by.MTT method of bone marrow mesenchymal stem cells and patellar ligament fibroblasts by Ficoll density gradient centrifugation and tissue mass culture method. BMSCs. was identified by bone formation, cartilage formation, and adipocyte differentiation ability.
Construction of recombinant adenovirus vector carrying human bFGF gene (pAd-bFGF-GFP) by 2.Gateway Technology
The construction needed only two steps: the BP reaction was used to construct an introductory clone, the LR reaction was used to create the expression clone. The expression of the clone was packed into the target adenovirus in 293 cells. The virus titer was measured. The expression of bFGF was detected by realtimeRT-PCR and Westernblot after the adenovirus vector was transfected into 293 cells.
Expression of bFGF in cultured rabbit bone marrow mesenchymal stem cells after transfection of 3.pAd-bFGF-GFP and its effect on cell proliferation and extracellular matrix and comparison with ligamentous fibroblasts
The adenovirus carrying the bFGF gene transfected rabbit bone marrow mesenchymal stem cells with different MOI values. The transfection efficiency was detected by flow cytometry. The optimal MOI value was determined by MTT assay. The optimal MOI value was used to transfect bone marrow mesenchymal stem cells. ELISA was used to detect the expression of bFGF protein and the secretion trend. BFGF will be carried with the order of bFGF. Gene adenovirus transfected with BMSCs (pAd-bFGF-GFP.BMSCs), LF, BMSCs, BMSCs (pAd-GFP-BMSCs) transfected with empty virus (pAd-GFP-BMSCs), MTT cell proliferation compared.Realtime RT-PCR detection bFGF to BMSCs expression Collagen I, Collagen III, and vimentin at the level of response.
4. the acellular ligament was treated with histological and biomechanical examination.
1%DCA method for acellular disposal of ligaments. PAd-bFGF-GFP.BMSCs, LF, BMSCs, pAd-GFP-BMSCs were planted with acellular ligament scaffolds. MTT method was used to detect the adhesion, growth and proliferation of seed cells on the decellular scaffold..Realtime RT-PCR was used to detect the expression of Collagen III, Collagen I and Vimentin protein mRNA level. Tissue section, ice The growth and distribution of pAd-bFGF-GFP.BMSCs on the scaffold were observed by the frozen section and confocal microscope. The effects of the combined culture on the mechanical properties of the ligaments were examined by biomechanics.
Result
1. the BMSCs and LF were similar in morphology, and all showed long spindle shape. The.MTT detection results of vortex like growth showed that the proliferation activity of BMSCs was obviously superior to that of LF.BMSCs, which had the ability to differentiate into osteogenesis, adipose and cartilage.
2. the plasmid carrying human bFGF gene was successfully constructed. PCR identification and gene sequencing of the recombinant plasmid showed that the human bFGF gene was successfully synthesized and integrated. The recombinant adenovirus vector system successfully constructed the recombinant adenovirus pAd.bFGF-GFP. by amplification in 293 cells, and we got a high titer virus, and the titer determination was (4.). 85 * 109ifu/ml).Realtime RT-PCR and Western-blot confirmed that the target gene bFGF was highly expressed at the gene level and protein level.
3. adenovirus has high transfection efficiency to BMSCs cells. MTT results show that when MOI value is 200, BMSCs cells have the strongest increment ability.ELISA detection: 48h bFGF has been expressed significantly after transfection, the peak of secretion appears at seventh days, then gradually decreases, and.PAd-bFGF-GFP transfected BMSCs is still compared with other cells at 2 weeks. After 2 weeks of culture, Realtime RT-PCR detection showed that the expression of Collagen I and Collagen III mRNA were significantly higher than those of other groups, and the expression of.VimentinmRNA was equivalent to that of LF.
The 4.MTT assay showed that after the bFGF gene was transfected into the bone marrow mesenchymal stem cells, the cell growth was obviously promoted by the transfection of the bone marrow mesenchymal stem cells, and the.Realtime PCR showed the Collagen III of the pAd-bFGF-GFP transfected BMSCs. The expression of Collagen I was significantly higher than the.Vimentin mRNA expression of other groups and the equivalent.HE dyeing and ice of the fibroblasts. The frozen section confocal microscope observed that pAd-bFGF-GFP-BMSCs grew well between the acellular ligament fibers, and showed spindle growth. The biomechanical test showed that the maximum stress of the ACL was not different from that of the uncultured ligaments, and the modulus of elasticity decreased.
conclusion
1. the rabbit bone marrow mesenchymal stem cells and ligamentous fibroblasts were successfully isolated and cultured. Through differentiation and culture identification, the acquisition of BMSCs. fibroblasts with multiple differentiation ability was confirmed.
2. a recombinant adenovirus with high titer defect was successfully constructed with human bFGF gene.
3. BMSCs cells transfected with bFGF gene can continuously secrete bFGF for at least two weeks, and have strong value-added ability and expression of ligamentous extracellular matrix protein mRNA, which are superior to ligamentum fibroblasts and BMSCs cells and BMSCs cells transfected with empty viruses.
4. in vitro transgenic BMSCs can grow well on the acellular ligament, and can express a large number of ligamentous specific matrix protein mRNA, which is superior to the ligamentum fibroblasts and BMSCs cells and the BMSCs cells transfected with null virus, and the biomechanical properties are basically retained.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.17

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