Ad-hBMP-2基因转染骨骼肌卫星细胞体外成骨分化的实验研究

发布时间：2018-03-07 08:50

本文选题：腺病毒　切入点：骨形成蛋白2　出处：《山东大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的： 1．探讨骨骼肌卫星细胞体外分离培养、鉴定的方法，研究骨骼肌卫星细胞的生物学特性，为后续的实验奠定基础。 2．评价腺病毒介导的人骨形态发生蛋白2(Ad-hBMP-2)基因体外转染骨骼肌卫星细胞后，肌卫星细胞分化为成骨样细胞的能力；并测定基因转染后目的基因体外表达的情况。应用Ad-GFP基因转染肌卫星细胞，测定腺病毒转染的效率。为应用骨骼肌卫星细胞修复骨缺损提供实验依据。方法： 1．采用Ⅰ型胶原酶和胰蛋白酶二步消化法获取大鼠骨骼肌卫星细胞，进行体外原代和传代培养。观察细胞的形态，通过生长曲线、细胞融合率等指标研究骨骼肌卫星细胞的增殖与分化能力，利用免疫细胞化学染色对所获得的细胞进行鉴定。 2．Ad-hBMP-2基因体外转染骨骼肌卫星细胞后，，通过形态学观察、生长曲线的测定、碱性磷酸酶(ALP)细胞化学染色及活性检测、骨钙素(OC)及Ⅰ型胶原的免疫细胞化学染色、矿化结节形成实验和Western—blot，观察Ad-hBMP-2基因转染后骨骼肌卫星细胞生物学行为的变化和目的基因表达的情况。Ad-GFP基因转染骨骼肌卫星细胞后2天，用荧光显微镜测定腺病毒转染效率，并观测Ad-GFP基因转染的骨骼肌卫星细胞传代后绿色荧光蛋白的表达情况。
[Abstract]:Objective:. 1. To study the methods of isolation, culture and identification of skeletal muscle satellite cells in vitro, to study the biological characteristics of skeletal muscle satellite cells, and to lay a foundation for further experiments. 2. To evaluate the ability of adenovirus-mediated human bone morphogenetic protein (2Ad-hBMP-2) gene transfection into skeletal muscle satellite cells to differentiate into osteoblast-like cells. After gene transfection, the expression of the target gene in vitro was determined. The efficiency of adenovirus transfection was measured by Ad-GFP gene transfection into muscle satellite cells, which provided experimental basis for repairing bone defect by skeletal muscle satellite cells. Methods:. 1. Rat skeletal muscle satellite cells were obtained by two-step digestion of type I collagenase and trypsin, and cultured in vitro. The morphology of the cells was observed and the growth curve was obtained. Cell fusion rate was used to study the proliferation and differentiation of skeletal muscle satellite cells, and the cells were identified by immunocytochemical staining. 2. After transfection of Ad-hBMP-2 gene into skeletal muscle satellite cells in vitro, morphological observation, growth curve determination, alkaline phosphatase (ALP) cytochemical staining and activity detection, osteocalcin OC) and type I collagen immunocytochemical staining were used. Mineralized nodule formation test and Western-blot were used to observe the changes of biological behavior and target gene expression of skeletal muscle satellite cells after transfection of Ad-hBMP-2 gene .Ad-GFP gene was transfected into skeletal muscle satellite cells 2 days after transfection. The transfection efficiency of adenovirus was measured by fluorescence microscope. The expression of green fluorescent protein (GFP) in skeletal muscle satellite cells transfected with Ad-GFP gene was observed.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2

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