【作者】 贺斌；

【导师】 赵柏程；

【作者基本信息】 中南大学 ， 烧伤整形科， 2010， 硕士

【摘要】 目的：造成皮肤组织缺损后创面难以愈合的主要原因包括修复细胞缺乏和促进创伤修复的各种生物活性因子的减少。如何在无足够自体皮肤进行移植等限制下,及时有效的修复难愈创面以尽可能达到恢复皮肤正常功能的愈合,是目前创面临床治疗的难点。因此本研究拟通过AAV介导EGF基因修饰骨髓MSCs,探讨其对于促进创面修复的可能性,为皮肤组织工程种子细胞的来源及转基因技术用于促进创面愈合提供一定的理论基础及实践依据。方法与结果：1.构建携带EGF基因的重组腺相关病毒载体通过RT-PCR从胎盘组织中扩增出175bp大小的EGF基因,将其克隆入pMD18-T载体,转化感受态细胞。提取此重组质粒DNA并用限制性内切酶EcoRI和Sal I酶切,获得两端分别带有EcoRI和Sal I不同酶切位点的EGF基因片段,最终将其克隆入pAAV-IRES-hrGFP质粒中,通过酶切和测序鉴定,证明成功构建了pAAV-EGF-GFP质粒。2. AAV-EGF病毒的包装、纯化与检测将pAAV-EGF-GFP、pAAV-RC、pHelper 3种质粒共转染HEK293细胞,包装含有EGF基因的AAV-EGF病毒载体；通过“氯仿处理-PEG/NaCl沉淀-氯仿抽提”法纯化AAV-EGF;用SDS-PAGE胶电泳检测AAV的纯度,以点杂交检测AAV的滴度。最终证明获得高纯度高滴度的AAV-EGF病毒。3.骨髓间充质干细胞的分离与培养采用密度梯度离心法结合贴壁法体外培养获取小鼠骨髓MSCs,并诱导MSCs向成骨、成软骨和成脂肪三个方向分化,分别采用Von Kossa法、阿尔新蓝丽春红染色和油红0染色方法进行检测。结果表明通过体外原代和传代培养获得的纯化的小鼠MSCs具有向多个方向分化的能力,我们所培养的细胞为骨髓间充质干细胞。4.AAV-EGF转染MSCs细胞以获取的AAV-EGF病毒转染MSCs细胞,分别于培养3d、14d、27d后提取细胞的总RNA,采用RT-PCR方法检测EGF基因在小鼠MSCs细胞中的分泌表达。结果表明AAV转染了MSCs细胞,介导了EGF基因的体外表达,且EGF基因的表达随着时间的延长而增加。结论：本研究在体外成功地分离培养出具有分化潜能的小鼠骨髓MSCs细胞；构建了携带有人EGF基因的pAAV-EGF-GFP质粒重组体；获得的AAV-EGF病毒可介导EGF基因在小鼠骨髓MSCs中的表达。更多还原

【Abstract】 Objective:Refractory wounds following skin tissue defect are mainly caused by the lack of repair cells and the reduce of biological activity facors involved in would healing process. In the absence of sufficient autologous skin, it remains a difficulty for clinic treatment to decide how to restore effective wound repair and obtain the physiological healing of the skin timely and effectively. Therefore, we try to modify bone marrow MSCs with recombinant AAV (adeno-associated virus)-mediated EGF gene as a way to explore its potential for promoting wound healing, which may provide some theoretical and practical basis for the source of cells for skin tissue engineering and transgenic technology in wound healing.Methods and Results:1. Construction of pAAV-EGF-GFPIn this study, we amplified the 175bp fragment of EGF gene from the placenta by RT-PCR and cloned the cDNA into vectors. The pMD18-T-EGF vectors were then digested with EcoRI and SaiI and cloned into the multiple cloning site of plasmid AAV-IRES-GFP. And the successful construction of pAAV-EGF-GFP was identified by restriction enzyme and sequencing analysis.2. The packaging and purification of AAV-EGFThe calcium phosphate precipitate is formed by mixing pAAV-RC, pHelper and pAAV-EGF-GFP. The recombinant AAV-EGF was purified and condensed as the method including chloroform treatment, PEG/NaCl preciptation and chloroform extraction. The etiterof AAV was was detected by SDS-PAGE, and the purity by DNA dot-blot.3. Isolation and culture of bone merrow mesenchymal stem cells Mouse bone marrow-derived MSCs were isolated and purified by dsnsity gradient centrifugation and direct adherence separately. The MSCs cells were induced to osteoblasts, chondroblasts and adipocytes, identified by Von Kossa, Alcian Ponceau and oil red O staining methods. The results showed that MSCs that had multiple differentiation potentials could be obtained by primary and passage culture.4. Transduction of MSCs with recombinant AAV-EGF3d,14d,27d post-transduction, the total RNA were extracted from the transduced MSCs and detected by RT-PCR method using the specific primer for EFG gene. The results showed that AAV-EGF vectors successfully transfected MSCs and mediated the expression of EGF gene in vitro.ConclusionMSCs that have the proliferative activity and potential to differentiate were successfully isolated and cultured in vitro. EGF cDNA was cloned into plasmid pAAV-EGF-GFP. Recombinant AAV-EGF vectors could mediate the expression of EGF gene in MSCs in vitro.更多还原