本文選題：腺病毒　切入點(diǎn)：骨形成蛋白2　出處：《山東大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】：目的： 1．探討骨骼肌衛(wèi)星細(xì)胞體外分離培養(yǎng)、鑒定的方法，研究骨骼肌衛(wèi)星細(xì)胞的生物學(xué)特性，為后續(xù)的實(shí)驗(yàn)奠定基礎(chǔ)。 2．評價腺病毒介導(dǎo)的人骨形態(tài)發(fā)生蛋白2(Ad-hBMP-2)基因體外轉(zhuǎn)染骨骼肌衛(wèi)星細(xì)胞后，肌衛(wèi)星細(xì)胞分化為成骨樣細(xì)胞的能力；并測定基因轉(zhuǎn)染后目的基因體外表達(dá)的情況。應(yīng)用Ad-GFP基因轉(zhuǎn)染肌衛(wèi)星細(xì)胞，測定腺病毒轉(zhuǎn)染的效率。為應(yīng)用骨骼肌衛(wèi)星細(xì)胞修復(fù)骨缺損提供實(shí)驗(yàn)依據(jù)。 方法： 1．采用Ⅰ型膠原酶和胰蛋白酶二步消化法獲取大鼠骨骼肌衛(wèi)星細(xì)胞，進(jìn)行體外原代和傳代培養(yǎng)。觀察細(xì)胞的形態(tài)，通過生長曲線、細(xì)胞融合率等指標(biāo)研究骨骼肌衛(wèi)星細(xì)胞的增殖與分化能力，利用免疫細(xì)胞化學(xué)染色對所獲得的細(xì)胞進(jìn)行鑒定。 2．Ad-hBMP-2基因體外轉(zhuǎn)染骨骼肌衛(wèi)星細(xì)胞后，，通過形態(tài)學(xué)觀察、生長曲線的測定、堿性磷酸酶(ALP)細(xì)胞化學(xué)染色及活性檢測、骨鈣素(OC)及Ⅰ型膠原的免疫細(xì)胞化學(xué)染色、礦化結(jié)節(jié)形成實(shí)驗(yàn)和Western—blot，觀察Ad-hBMP-2基因轉(zhuǎn)染后骨骼肌衛(wèi)星細(xì)胞生物學(xué)行為的變化和目的基因表達(dá)的情況。Ad-GFP基因轉(zhuǎn)染骨骼肌衛(wèi)星細(xì)胞后2天，用熒光顯微鏡測定腺病毒轉(zhuǎn)染效率，并觀測Ad-GFP基因轉(zhuǎn)染的骨骼肌衛(wèi)星細(xì)胞傳代后綠色熒光蛋白的表達(dá)情況。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R329.2

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