【作者】 熊卉；

【导师】 龙星；

【作者基本信息】 武汉大学 ， 口腔临床医学， 2010， 博士

【摘要】 第一部分携带TGF-β1基因重组腺相关病毒载体的构建目的构建携带TGF-β1基因的重组腺相关病毒载体,制备表达TGF-β1基因的重组腺相关病毒rAAV-TGF-β1,检测重组病毒的滴度,为TGF-β1基因的真核表达及临床应用提供实验基础。方法含TGF-β1 cDNA的质粒pcDNA3-TGF-β1和质粒pAAV-MCS用EcoRⅠ+XbaⅠ进行双酶切后连接,转化大肠杆菌DH5a感受态细胞,获得重组质粒pAAV-MCS-TGF-β1。通过酶切和DNA测序鉴定重组质粒的正确性。采用磷酸钙共沉淀法,以重组质粒pAAV-MCS-TGF-β1和pAAV-RC、pHelper共转染AAV-293细胞,产生具有感染性的病毒颗粒rAAV-TGF-β1并检测重组病毒的滴度。结果成功构建携带目的基因TGF-β1的重组质粒pAAV-MCS-TGF-β1,并获得携带TGF-β1基因的重组腺相关病毒rAAV-TGF-β1。重组质粒pAAV-MCS—TGF-β1经双酶切和测序证实TGF-β1基因正确插入载体且序列正确。重组腺相关病毒rAAV-TGF-β1经PCR检测,可见1.2kb大小的TGF-β1基因片段。重组病毒的滴度为3×108pfu／ml。结论成功构建了携带目的基因TGF-β1的重组腺相关病毒载体(rAAV-TGF-β1),为下一步组织工程种子细胞转染目的基因TGF-β1奠定了基础。第二部分兔颞下颌关节滑膜间充质干细胞的分离与培养目的探讨兔颞下颌关节滑膜间充质干细胞(SMSCs)的分离纯化及其在体外培养条件下的基本生物学特性,为颞下颌关节组织工程选择合适的种子细胞。方法在无菌条件下,取3月龄健康新西兰大白兔颞下颌关节腔内面平滑光亮的滑膜组织,采用酶消化法体外培养,有限稀释法进行单细胞克隆,筛选出SMSCs,观察其增殖和生长特性,绘制生长曲线,测定贴壁率。取第3代SMSCs采用不同诱导液进行成脂肪、成骨、成软骨诱导等多向分化,观察细胞的形态变化,在诱导第14d进行油红染色和茜素红染色以鉴定SMSCs成脂肪和成骨分化潜能；在诱导第21d用番红O染色、Ⅱ型胶原免疫组化鉴定SMSCs成软骨分化潜能。结果SMSCs生物性状稳定,为均一的成纤维样细胞。原代细胞24 h后贴壁,48 h贴壁细胞逐渐增多。第5-6 d细胞数目最多；传代后10-12 h贴壁率高达95%,细胞传至第10代细胞形态无明显变化。不同诱导液诱导后细胞形态发生变化。成骨诱导后可检测到钙化结节形成；成脂肪诱导后可检测到脂滴形成。成软骨诱导番红0染色可见胞浆内有明显的特异性蛋白多糖着色,Ⅱ型胶原免疫组化胞浆内可见棕黄色颗粒。结论经体外分离纯化的单克隆SMSCs可以表现为成纤维细胞的特性,与其他来源的MSCs具有类似的生物学特性及多向分化潜能,有可能成为颞下颌关节组织工程种子细胞的来源。第三部分重组腺相关病毒介导TGF-β1基因体外转染SMSCs的实验研究目的应用重组腺相关病毒rAAV-TGF-β1转染体外培养的SMSCs,检测转染后SMSCs中TGF-β1 mRNA和蛋白的表达。并通过检测Ⅰ型胶原、II型胶原、X型胶原、Sox 9、aggrecan基因及Ⅰ、Ⅱ型胶原蛋白,对TGF-β1诱导SMSCs向纤维软骨细胞分化的表型进行鉴定。方法将收集好的重组腺相关病毒rAAV-TGF-β1转染SMSCs,按不同处理因素分为3组：转染rAAV-TGF-β1组(实验组),转染空载体组和未转染组(对照组)。于转染后1-7d,应用MTT法检测3组SMSCs的增殖情况。转染后进行SMSCs细胞球团培养,分别于2d、7d、21d收集细胞,应用RT-PCR检测3组细胞TGF-β1 mRNA表达情况,Western blot法检测培养液上清中TGF-β1蛋白含量。并分别于7d、21d采用RT-PCR检测软骨基质Ⅰ、Ⅱ、Ⅹ型胶原、Sox 9、Aggrecan mRNA的表达,同时用免疫组化检测SMSCs及周围基质中Ⅰ、Ⅱ型胶原蛋白的表达。结果TGF-β1 mRNA及蛋白在基因转染细胞得到正确表达,且随时间的延长,表达量逐渐增加,组间比较有显著性差异(p<0.01)；转染后SMSCs目的基因CollagenⅠ、CollagenⅡ、Sox 9及Aggrecan的表达和软骨特异性基质Ⅰ、Ⅱ型胶原蛋白的分泌增强,且Ⅰ型胶原蛋白的染色明显强于Ⅱ型胶原蛋白。未见X型胶原mRNA的表达。表明SMSCs被诱导向纤维软骨方向分化。结论重组腺相关病毒介导的外源性基因TGF-β1能够安全有效转染SMSCs,转染后细胞的TGF-β1 mRNA表达和蛋白分泌水平明显增加,高表达的TGF-β1可促进SMSCs向纤维软骨方向分化。第四部分TGF-B1基因转染滑膜间充质干细胞复合支架构建纤维软骨组织的体外三维培养目的：研究在体外三维培养条件下,经重组腺相关病毒rAAV-TGF-β1转染的兔滑膜间充质干细胞(SMSCs)复合壳聚糖／Ⅰ型胶原支架培养向纤维软骨分化的生物学特性。方法：将rAAV-TGF-β1转染的SMSCs以两次沉降法接种到壳聚糖／Ⅰ型胶原支架复合物上,进行三维培养。HE染色观察细胞在复合支架上的生长状况；番红O染色和Ⅱ型胶原免疫组化了解转染后细胞外软骨基质的分泌；扫描电镜观察细胞在材料上的附着和生长情况。MTT检测细胞在支架上的增殖情况。结果：rAAV-TGF-β1转染的SMSCs在支架中生长黏附良好,有较多的基质形成。番红O染色可见红染的细胞外软骨基质,细胞和基质成分填充于支架材料的孔隙内。免疫组化染色显示胞浆及细胞外均有Ⅱ型胶原蛋白分泌。扫描电镜下可见细胞紧贴支架复合物生长并彼此间有纤维交联,胞体周围有基质样物质分泌。MTT检测细胞具有良好的增殖特性。结论：壳聚糖／Ⅰ型胶原复合支架材料可为rAAV-TGF-β1转染SMSCs生长分化提供一个良好的环境,建立了较为稳定的SMSCs体外三维培养体系。更多还原

【Abstract】 PartⅠConstruction of Recombinant Adeno-associated Virus with TGF-β1Objective To construct and confirm the recombinant adeno-associated virus with transforming growth factorβ1 (TGF-β1).Methods Both plasmids of pcDNA3-TGF-β1 and pAAV-MCS were linked after being digested by enzyme of EcoR I+Xba I, and the recombinant plasmids of pAVV-MCS-TGF-β1 were collected from the colon bacillus DH5a transformed by the linked plasmids. The recombinant plasmids were confirmed by enzyme digestion and DNA sequence examination. The recombinant virus rAAV-TGF-β1 were collected from the transfected AAV-293 cells which were co-transfected by pAAV-MCS-TGF-β1, pAAV-RC and pHelper through the process of calcium-phosphate coprecipitation. The titers of recombinant virus were detected.Results The recombinant plasmid pAAV-MCS-TGF-β1 with TGF-β1 were reconstructed, and the recombinant adeno-associated virus with TGF-β1 were also achieved. After being treated by enzyme digestion and sequence analysis, the recombinant pAAV-MCS-TGF-β1 was confirmed that TGF-β1 was inserted correctly into the vector and the sequence of this gene was also corrected. The titers of the recombinant virus (rAAV-TGF-β1) were detected as 3×108pfu/ml and TGF-β1 of rAAV-TGF-β1 was detected by PCR. Conclusion The recombinant adeno-associated virus vector with TGF-β1 (rAAV-TGF-β1) were reconstructed successfully and might be employed to transduce the synovial mesenchymal stem cells in the tissue engineering of fibrocartilage in the following experiments.PartⅡIsolation and culture of synovial mesenchymal stem cells of rabbit temporomandibular jointObjective To explore the isolation and culture of synovial mesenchymal stem cells(SMSCs) for the tissue engineering of temporomandibular joint.Methods Synovium membrane was harvested from New-Zealand white rabbits (3-month old) under the condition of asepsis. SMSCs were harvested through the method of enzyme digestion and isolated by limited dilution. The proliferation and viability of the isolated SMSCs were detected. For evaluation of the multipotential differentiation of SMSCs, the passage 3 SMSCs were cultured in the defined medium to be differentiated into adipose cells, osteoblasts and chondrocytes. After being cultured in defined medium for 14d, the differentiation of SMSCs into adipose cells and osteoblasts were tested by Oil red O staining or Alizarin red staining. Being induced by TGF-β1 for 21d in vitro, the differentiation of SMSCs into chondrocyte were evaluated by Safranin O staining and collagen typeⅡimmunohistochemical staining.Results Rabbit SMSCs maintained the fibroblast-like morphology after being cultured 10 passages. Primary SMSCs began attaching on the floor of the flash after 24h, and the attachment ability of SMSCs reached to 80% after 48h. It was found that calcified nodules and lipid drops in the SMSCs after SMSCs being induced and differentiated into osteogenic cells and adipogenic cells. And special extracellular matrix of cartilage could be found in the SMSCs stained by Safranin O and collagen typeⅡstained positive immunohistochemically after the chondrogenic differentiation of SMSCs.Conclusion It was found that the SMSCs could maintain the fibroblast-like morphology after being cultured several passages, and had multipotential differentiation cultured in the defined medium. Therefore, it suggested that SMSCs might be a choice of seeding cells for temporomandibular joint tissue engineering.PartⅢStudy on transduction of SMSCs in vitro using recombinant adeno-associated virus with TGF-β1Objective To detect the expression of TGF-β1 mRNA and protein of the SMSCs transduced by recombinant adeno-associated virus with TGF-β1 (rAAV-TGF-β1) in vitro and investigate the ability of the transduced SMSCs differentiation into fibrocartilage.Methods Three groups were divided as experimental group of SMSCs transduced by rAAV-TGF-β1, control group of SMSCs transduced by empty vector of adeno-associated virus, and blank control group of SMSCs non-transduced. MTT assay was employed to detect the proliferation of SMSCs after being transduced 1～7d. RT-PCR was applied to explore the expression of TGF-β1, and Western blot was used to evaluate the protein of TGF-β1 of the three groups. Pellet culture of tansduced SMSCs was adopted to detect the expression of mRNA of collagen typeⅠ,ⅡandⅩ, sox9 and aggrecan using RT-PCR, and the collagen typeⅠproteins and collagen typeⅡprotein were investigated by the method of immunocytochemistry staining.Results Both TGF-β1 mRNA and protein could express in the experimental group of SMSCs transduced by rAAV-TGF-β1; the mRNA expression of collagen typeⅠ,Ⅱ, Sox9 and aggrecan were detected, and the expression of collagen typeⅠprotein is stronger than collagen typeⅡby the method of immunocytochemistry staining. No collagen typeⅩexpressed in transduced SMSCs at any time point during the experiment. In the conrol group, the secretion of cartilage matrixes were not detected. These date suggest that the SMSCs transfected by rAAV-TGF-β1 could differentiate into fibrocartilage.Conclusion It could be concluded from this study that TGF-β1 could be transduced by adeno-associated virus vector into SMSCs; the transduced cells could express more TGF-β1 mRNA and protein than that of non-transduced cells; and transduced SMSCs could differentiate into fibrocartilage.PartⅣIn vitro study on fibrocartilage tissue engineering using gene TGF-β1 modified SMSCs and chitosan/collagen typeⅠscaffoldObjective To investigate the potential of the SMSCs transduced by recombinant adeno-associated virus rAAV-TGF-β1 cultured in the fabricated chitosan/collagen typeⅠ(CS/COL-I) scaffold for fibrocartilage tissue engineering.Methods The SMSCs were transduced by rAAV-TGF-β1 and cultured onto the fabricated chitosan/collagen typeⅠscaffold. After being cultured in defined medium, histological HE staining was applied to examine the cell morphology of SMSCs-CS/COL-I constructs. Safranin O/Fast green staining and immunocyto-chemistry staining were used to detect the the cartilage matrix secreted by transduced SMSCs. The cell morphology and attachment of the SMSCs-CS/COL-I constructs were examined through scanning electronic microscope (SEM). And the proliferation of SMSCs on scaffold was detected by MTT.Results The SMSCs transduced by rAAV-TGF-β1 attached onto the scaffolds and secreted much extracellular matrix around the cells. The cartilage-like matrix filled in the pores of the scaffolds and was stained red by Safranin O/Fast green. It was found that collagen typeⅡwas immunohistochemically positive in the transduced SMSCs or around the cells. The cells in the constructs communicated each other in the pores of the scaffolds, and matrix surrounded the cells examined through SEM. And the transduced SMSCs cultured in the scaffolds presented good viability and proliferation indicated by MTT assay.Conclusion The fabricated CS/COL-I coploymer could be a proper three-dimensional scaffold for the SMSCs transduced by rAAV-TGF-β1 to proliferate and differentiate.更多还原