脂质体介导转染的绿色荧光蛋白基因在骨髓基质细胞内表达

发布时间：2018-01-18 00:17

本文关键词：脂质体介导转染的绿色荧光蛋白基因在骨髓基质细胞内表达　出处：《山东大学》2005年硕士论文　论文类型：学位论文

【摘要】：[目的]骨髓基质细胞(bone marrow stromal cells,BMSC),也被称为骨髓间充质干细胞(bone marrow mesenchymal cells),是存在于骨髓基质中一种成体干细胞,来源于中胚层,具有很强的自我复制和多向分化功能,可以分化为多种不同的组织细胞,是细胞工程学和组织工程学研究中的一种理想种子细胞。如果BMSC容易接受外源性基因而且稳定表达,结合其多向分化能力,BMSC将会在组织工程、细胞移植、基因治疗等领域得到十分广阔的应用。本实验的主要目的为: 1.建立一种体外分离、培养扩增大鼠骨髓基质细胞的方法,为进一步研究和利用该细胞奠定基础; 2.研究脂质体转染法对骨髓基质细胞进行基因修饰的可行性和有效性,为研究骨髓基质细胞在组织工程和基因治疗中的应用提供。 [方法]用淋巴细胞分离液分离出大鼠骨髓基质细胞,并用含20%(体积分数)胎牛血清的DMEM培养液培养,胰酶消化传代,扩增大鼠骨髓基质细胞。取第3～5代BMSC,用脂质体转染法将外源基因绿色荧光蛋白基因介导进入到骨髓基质细胞内,然后在荧光显微镜下观察,计数表达荧光蛋白细胞的数量,并用台盼兰排斥试验检测转染细胞的活力。 [结果] 1 大鼠BMSC的分离培养用Percoll淋巴细胞分离液对大鼠骨髓作密度梯度离心,所得细胞为比较一致的球形单个核细胞,台盼兰染色细胞活力达99%。接种24小时后,倒置显微
[Abstract]:[Objective to study the bone marrow stromal cells (BMSCs) of bone marrow stromal cells (BMSCs). Bone marrow mesenchymal cells, also known as bone marrow mesenchymal stem cells, is a kind of adult stem cells existing in bone marrow stromal cells, derived from mesoderm. It has a strong function of self-replication and multi-differentiation, and can differentiate into a variety of different tissue cells. It is an ideal seed cell in cell engineering and tissue engineering. If BMSC is easy to accept exogenous gene and express stably, it will be in tissue engineering if combined with its multidirectional differentiation ability. Cell transplantation, gene therapy and other fields have been widely used. 1. To establish a method of isolating and amplifying rat bone marrow stromal cells in vitro, so as to lay a foundation for further study and utilization of the cells. 2. To study the feasibility and effectiveness of gene modification of bone marrow stromal cells by liposome transfection, and to study the application of bone marrow stromal cells in tissue engineering and gene therapy. [Methods Rat bone marrow stromal cells were isolated from lymphocytes and cultured with DMEM medium containing 20% fetal bovine serum for trypsin digestion and passage. Rat bone marrow stromal cells were amplified. The third passage BMSCs were used to transfect the green fluorescent protein gene into the bone marrow stromal cells by lipofectamine transfection, and then observed by fluorescence microscope. The number of cells expressing fluorescent protein was counted and the viability of transfected cells was detected by trypan blue exclusion test. [Results] 1 isolation and culture of rat BMSC Percoll lymphocyte isolate was used to centrifuge rat bone marrow with density gradient centrifugation. The cells were spherical mononuclear cells. The activity of Trypan blue stained cells was 99%. 24 hours after inoculation. Inverted microscope
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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