【作者】 张杰；

【导师】 蒋明；

【作者基本信息】 中南大学 ， 临床医学， 2014， 硕士

【摘要】 目的：建立豚鼠骨髓间充质干细胞(BMSCs)的体外培养体系,体外脂质体2000介导神经营养因子-3(NT-3)转染BMSCs,为内耳移植提供前期研究基础。方法：(1)采用全骨髓贴壁法培养豚鼠BMSCs,传代培养,取P3代BMSCs做生长曲线图,在流式细胞仪上检测BMSCs的表面标志物CD29,CD90以及造血细胞表面标志物CD45。(2)按照不同质粒-脂质体配比介导pEGFP-N1-NT3转染P3代BMSCs,转染后48h后计算转染效率,选择最佳质粒-脂质体配比。(3)按照最佳质粒-脂质体配比将pEGFP-N1-NT3质粒转染P3代BMSCs,转染后1d,3d,7d,14d,观察其荧光表达情况。(4)转染后48h的BMSCs提取蛋白,采用Western Blot来验证NT-3蛋白表达。结果：(1)全骨髓贴壁法获得细胞数量多,增殖快,通过传代可以纯化细胞。(2)生长曲线呈S型,基本符合Logistic生长曲线。(3)流式细胞仪检测细胞CD29和CD90阳性表达,CD45阴性表达,符合BMSCs特征。(4)不同质粒-脂质体配比中,质粒：脂质体为4μg：12μl时转染率最高,是最佳质粒-脂质体配比。(5)转染后48小时荧光表达较强,7d可见荧光表达,但较前明显减弱,到14d基本未见明显荧光表达。(6)Western Blot验证NT-3基因成功表达。结论：(1)全骨髓贴壁法可以成功培养出增殖快,细胞纯度高的BMSCs。(2)脂质体法成功介导NT-3真核质粒在BMSCs内表达,BMSCs可能作为内耳基因治疗的理想载体。更多还原

【Abstract】 Objective:Establishing the culture system of guinea bone marrow-derived mesenchymal stem cells (BMSCs) in vitro, and transfecting neurotrophic factor-3(NT-3) by Lipofectamin2000into BMSCs, so as to provide the foundation for the next experiments of inner ear transplantation.Methods:(1) Guinea BMSCs were extracted,cultured and subcultured by whole bone marrow directly adhering in flasks, the third generation were used for flow cytometric detection about the surface markers CD29, CD90of BMSCs and the surface antigen CD45of hematopoietic cell.(2) BMSCs of P3were transfected with pEGFP-N1-NT3by plasmid-liposome mixture which were mixed at different ratios, then calculated the transfection efficiency in48h after transfection, chose the best ratio of plasmid-liposome.(3) BMSCs of P3were transfected with pEGFP-Nl-NT3by liposome at the best ration of plasmid and liposome, then observing its fluorescent expression in1d,3D,7d,14d after transfection.(4) Extracting the protein of BMSCs in48h after transfection, validating the expression of NT-3by Western Blot.Results:(1)We got many BMSCs proliferating quickly by the method of whole bone marrow directly adhering in flasks.(2)BMSCs grew in an s-shaped curve type, largely in line with Logistic growth curve.(3)The flow cytometric detection showed the positive expression of CD29and CD90, negative expression of CD45, matching the characteristics of BMSCs.(4)The transfection efficiency was the highest with the ratio of plasmid-liposome (μg:μl) at4:12, making it the best ratio of plasmid-liposome.(5)Fluorescence was expressed strongly in48hours after transfection,and was visible but significantly decreased in7d, and could not been seen to14d.(6)NT-3gene were validated successfully expressed in BMSCs by Western Blot Conclusion:(1) BMSCs of Guinea Pig cultured by the method of whole bone marrow directly adhering in flasks high purity cells can be highly purified and proliferate quickly.(2) Eukaryotic plasmid of NT-3can be successfully mediated by Lipofectamin2000and express in BMSCs and BMSCs can be a desirable vector for gene therapy in the inner ear.更多还原