豚鼠骨髓间充质干细胞培养及体外转染神经营养因子-3的研究

发布时间：2018-11-02 15:06

【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。
[Abstract]:Aim: to establish a culture system of guinea pig bone marrow mesenchymal stem cells (BMSCs) in vitro. Liposome 2000 mediated neurotrophin-3 (NT-3) transfection into BMSCs, provides a preliminary basis for inner ear transplantation. Methods: (1) Guinea pig BMSCs, was cultured by whole bone marrow adherent method. BMSCs of P3 generation was taken as growth curve. CD29, the surface marker of BMSCs, was detected by flow cytometry. The transfection efficiency of CD90 and hematopoietic cell surface marker CD45. (2) was calculated 48 hours after transfection of P3 generation BMSCs, with pEGFP-N1-NT3 mediated by different plasmide-liposome ratios. Select the best plasmide-liposome ratio. (3) according to the best plasmide-liposome ratio, the pEGFP-N1-NT3 plasmid was transfected into P3 generation BMSCs, for 1 day, 3 days, 7 days and 14 days after transfection, and the fluorescent expression was observed. (4) the BMSCs protein was extracted 48 hours after transfection. Western Blot was used to verify the expression of NT-3 protein. Results: (1) the cells obtained by whole bone marrow adherent method were numerous and proliferative, and the cells could be purified by passage. (2) the growth curve was S-shaped, which basically accorded with the growth curve of Logistic. (3) the positive expression of CD29 and CD90 was detected by flow cytometry. The negative expression of CD45 was consistent with the characteristics of BMSCs. (4) among the different plasmide-liposome ratios, the transfection efficiency was the highest when the liposome was 4 渭 g: 12 渭 l, which was the best ratio of plasmids to liposomes. (5) the fluorescence expression was stronger at 48 hours after transfection. Fluorescence expression was observed on day 7, but weakened significantly, and no obvious fluorescence expression was found at day 14. (6) NT-3 gene was successfully expressed by) Western Blot. Conclusion: (1) BMSCs. (2) liposome with high cell purity and rapid proliferation can be successfully cultured by whole bone marrow adherent method to mediate the expression of NT-3 eukaryotic plasmid in BMSCs. BMSCs may be an ideal vector for inner ear gene therapy.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R764.43

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