大鼠骨髓间充质干细胞转染Sox9基因后成软骨情况的研究

发布时间：2018-07-07 15:38

  本文选题：Sox9-eGFP质粒 + BMSCs　； 参考：《暨南大学》2015年硕士论文

【摘要】：目的:随着人类平均寿命的延长及生活方式的变化,椎间盘退行性变导致的疾病无时不刻地影响着人类的健康和生活质量。椎间盘组织之细胞表型类似软骨细胞。本实验研究运用的基因转染技术是慢病毒介导的基因转染,整体思路是将Sox9基因通过上述基因转染技术导入到SD大鼠骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs),培养后检测其基因、蛋白表达等相关指标,从而研究其成软骨的效果,为后续人工生物椎间盘再造的研究提供种子细胞。方法:从SD大鼠骨髓腔内提取BMSCs,随即进行传代培养。观察骨髓间充质干细胞形态、测定其贴壁率。用慢病毒及Sox9基因构建Sox9-e GFP质粒,此病毒包装是通过转染293T细胞来实现的,在此步试验中我们会测定病毒的最佳感染复数(multiplicity of infection,MOI)值。然后将有目的基因之慢病毒转染SD大鼠的BMSCs。用骨髓间充质干细胞完全培养基常规培养转染病毒的BMSCs,每天在细胞显微镜下密切关注细胞的生长状态。经过14天培养后,用q RT-PCR的方法检测目的基因的相对表达量,用Westem Blot的方法测定目的基因蛋白(Sox9蛋白及Ⅱ型胶原蛋白)表达的情况。结果:从SD大鼠骨髓中提取的BMSCs贴壁状态好,细胞形态是长条梭形、呈极性排列。细胞传代培养后活力良好,细胞体积正常,细胞核清晰,细胞质内无大量颗粒产生、细胞贴壁生长、生长能力旺盛。传至第9代时出现衰老,传至12代时衰老征象十分明显。q RT-PCR结果示:在对照组、空载组、实验组均见表达,三组样品Sox9 m RNA的相对表达量:1.064ug/ul,1.434ug/ul,1.456ug/ul,三组样品间两两比较的结果均具统计学意义(P0.01)。Westem Blot结果示:通过检测,Sox9蛋白的表达在三组细胞中都可以检测的到,其相对表达量分别是空白组5351.69,空载组5536.347,实验组11103.782,同样通过统计学组间进行两两比较,结果有统计学意义(P0.05);II型胶原蛋白的表达在转染Sox9组细胞中开始出现,其余两组此蛋白无表达。结论:综上所述,BMSCs可以做为较为理想的种子细胞而在软骨组织工程运用。其提取方法容易,且易于培养及进行体外扩增;以慢病毒做为载体转染SD大鼠BMSCs的方法,可使目的基因得到高效而稳定地表达,且通过实验证明了这种方法可使BMSCs向软骨细胞分化。转染Sox9的骨髓间充质干细胞可作为制备人工生物椎间盘的种子细胞。
[Abstract]:Objective: with the prolongation of life expectancy and the change of life style, the disease caused by intervertebral disc degeneration affects the health and quality of life. The cell phenotype of intervertebral disc tissue is similar to that of chondrocytes. The gene transfection technique used in this study was lentivirus-mediated gene transfection. The whole idea was to transfer Sox9 gene into SD rat bone marrow mesenchymal stem cells (BMSCs) and detect its gene after culture. Protein expression and other related indicators, so as to study the effect of cartilage formation, and provide seed cells for the subsequent study of artificial disc reconstruction. Methods: BMSCs were extracted from the medullary cavity of SD rats and then subcultured. The morphology of bone marrow mesenchymal stem cells (BMSCs) was observed and the adherence rate was measured. Sox9-e GFP plasmids were constructed with lentivirus and Sox9 gene. The virus packaging was carried out by transfection of 293T cells. In this step test, we will determine the optimal infection complex (multiplicity of infection moi of the virus. Then the lentivirus with the target gene was transfected into BMSCs of SD rats. BMSCs transfected with BMSCs were cultured on the complete culture medium of bone marrow mesenchymal stem cells (BMSCs). After 14 days of culture, the relative expression of the target gene was detected by qRT-PCR, and the expression of Sox9 protein and type 鈪，

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