本文關鍵詞：脂質體介導轉染的綠色熒光蛋白基因在骨髓基質細胞內表達　出處：《山東大學》2005年碩士論文　論文類型：學位論文

  更多相關文章： 細胞培養(yǎng) 骨髓基質細胞 綠色熒光蛋白 基因轉染 脂質體

【摘要】：[目的]骨髓基質細胞(bone marrow stromal cells,BMSC),也被稱為骨髓間充質干細胞(bone marrow mesenchymal cells),是存在于骨髓基質中一種成體干細胞,來源于中胚層,具有很強的自我復制和多向分化功能,可以分化為多種不同的組織細胞,是細胞工程學和組織工程學研究中的一種理想種子細胞。如果BMSC容易接受外源性基因而且穩(wěn)定表達,結合其多向分化能力,BMSC將會在組織工程、細胞移植、基因治療等領域得到十分廣闊的應用。本實驗的主要目的為: 1.建立一種體外分離、培養(yǎng)擴增大鼠骨髓基質細胞的方法,為進一步研究和利用該細胞奠定基礎; 2.研究脂質體轉染法對骨髓基質細胞進行基因修飾的可行性和有效性,為研究骨髓基質細胞在組織工程和基因治療中的應用提供。 [方法]用淋巴細胞分離液分離出大鼠骨髓基質細胞,并用含20%(體積分數(shù))胎牛血清的DMEM培養(yǎng)液培養(yǎng),胰酶消化傳代,擴增大鼠骨髓基質細胞。取第3～5代BMSC,用脂質體轉染法將外源基因綠色熒光蛋白基因介導進入到骨髓基質細胞內,然后在熒光顯微鏡下觀察,計數(shù)表達熒光蛋白細胞的數(shù)量,并用臺盼蘭排斥試驗檢測轉染細胞的活力。 [結果] 1 大鼠BMSC的分離培養(yǎng) 用Percoll淋巴細胞分離液對大鼠骨髓作密度梯度離心,所得細胞為比較一致的球形單個核細胞,臺盼蘭染色細胞活力達99%。接種24小時后,倒置顯微
[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
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R346

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