本文選題：綿羊 + 分離培養(yǎng)��； 參考：《內(nèi)蒙古大學(xué)》2012年碩士論文

【摘要】：骨骼肌衛(wèi)星細(xì)胞是成體肌源性干細(xì)胞,在各種成體干細(xì)胞研究中越來越受到人們的關(guān)注。雖然從一些物種(如小鼠、大鼠和人)的骨骼肌中已經(jīng)成功分離出衛(wèi)星細(xì)胞,但對于家畜(如牛、綿羊)的研究卻很少。在家畜轉(zhuǎn)基因的研究和生產(chǎn)過程中,需要在動(dòng)物生產(chǎn)之前對轉(zhuǎn)基因細(xì)胞系進(jìn)行基因表達(dá)的鑒定和驗(yàn)證。因此,家畜骨骼肌衛(wèi)星細(xì)胞體外培養(yǎng)模型的建立就顯得特別迫切和重要。 本研究以綿羊骨骼肌衛(wèi)星細(xì)胞作為研究對象,重點(diǎn)探討衛(wèi)星細(xì)胞的分離、純化及體外培養(yǎng)方法,并對得到的細(xì)胞進(jìn)行鑒定,旨在建立可穩(wěn)定傳代的綿羊衛(wèi)星細(xì)胞系及其鑒定方法,為今后綿羊骨骼肌衛(wèi)星細(xì)胞在家畜繁育和再生醫(yī)學(xué)等相關(guān)方面的研究和應(yīng)用提供實(shí)驗(yàn)依據(jù)。 用兩步酶消化法和差速貼壁法分離和純化綿羊骨骼肌衛(wèi)星細(xì)胞,并對其進(jìn)行鑒定和誘導(dǎo)分化,分析其多能性。結(jié)果表明,1.1%Ⅰ型膠原酶和0.25%胰蛋白酶兩步消化法分離綿羊骨骼肌衛(wèi)星細(xì)胞效率較高,含20%FBS+10%馬血清的培養(yǎng)基更有利于綿羊骨骼肌衛(wèi)星細(xì)胞的體外培養(yǎng)。 用免疫組化和流式細(xì)胞術(shù)檢測綿羊骨骼肌衛(wèi)星細(xì)胞中幾個(gè)標(biāo)記基因Desmin、α-SarcOmeric Actinin、MyOD1、Myf5和PAX7的表達(dá)情況,本研究得到的細(xì)胞中這5個(gè)基因表達(dá)均呈陽性,說明分離得到的細(xì)胞為綿羊骨骼肌衛(wèi)星細(xì)胞。 為驗(yàn)證其多能性,將分離得到的衛(wèi)星細(xì)胞向成肌、成脂和成骨三個(gè)方向誘導(dǎo)。成肌誘導(dǎo)后形成明顯的多核肌管細(xì)胞,標(biāo)記基因MyoG表達(dá)陽性,快肌肌球蛋白細(xì)胞免疫熒光染色呈陽性；成骨誘導(dǎo)后經(jīng)茜素紅和ALP染色細(xì)胞均呈陽性,成骨細(xì)胞特異性基因Osteocalcin表達(dá)陽性；成脂誘導(dǎo)后細(xì)胞周圍有明顯的脂滴出現(xiàn),經(jīng)0-油紅染色呈陽性,且PPARy2表達(dá)呈陽性。說明本實(shí)驗(yàn)得到的細(xì)胞具有一定的多能性。 本研究初步建立了綿羊骨骼肌衛(wèi)星細(xì)胞體外分離、純化、培養(yǎng)和鑒定的方法。
[Abstract]:Skeletal muscle satellite cells are adult myogenic stem cells.Although satellite cells have been successfully isolated from skeletal muscles of some species, such as mice, rats and humans, little has been done on livestock (such as cattle and sheep).It is necessary to identify and verify the gene expression of transgenic cell lines before animal production.Therefore, the establishment of animal skeletal muscle satellite cells in vitro culture model is particularly urgent and important.In this study, sheep skeletal muscle satellite cells were selected as the research object. The isolation, purification and in vitro culture of the satellite cells were discussed, and the obtained cells were identified.The purpose of this study was to establish a stable passage sheep satellite cell line and its identification method, and to provide experimental evidence for the future research and application of sheep skeletal muscle satellite cells in animal breeding and regenerative medicine.Sheep skeletal muscle satellite cells were isolated and purified by two-step enzyme digestion method and differential adherence method.The results showed that the separation efficiency of sheep skeletal muscle satellite cells by two-step digestion of 1. 1% collagenase and 0.25% trypsin was higher, and the culture medium containing 20s 10% horse serum was more favorable for the in vitro culture of sheep skeletal muscle satellite cells.The expression of several marker genes Desmin, 偽 -SarcOmeric Actinin 1 (MyOD1) Myf5 and PAX7 in sheep skeletal muscle satellite cells were detected by immunohistochemistry and flow cytometry.The results showed that the isolated cells were sheep skeletal muscle satellite cells.To verify its pluripotency, the isolated satellite cells were induced in three directions: myoblast, fat-forming and osteogenesis.After induction of myogenesis, the myoblast cells were formed, the labeled gene MyoG was positive, the fast myosin cells were positive by immunofluorescence staining, and the cells were positive by alizarin red and ALP staining after osteogenesis induction.The expression of specific gene Osteocalcin in osteoblasts was positive, and there were obvious lipid droplets around the cells after fat-forming induction, and the expression of PPARy2 was positive after 0-oil red staining.The results show that the cells obtained in this experiment have certain pluripotency.In this study, a method for isolation, purification, culture and identification of sheep skeletal muscle satellite cells in vitro was established.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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