【摘要】： 目的：衛(wèi)星細(xì)胞(muscle satellite cells)認(rèn)為是保留在成年骨骼肌內(nèi)具有增殖分化能力的肌源性細(xì)胞。建立簡(jiǎn)便快速的鑒定和篩選骨骼肌衛(wèi)星細(xì)胞的方法對(duì)于衛(wèi)星細(xì)胞培養(yǎng)、研究和應(yīng)用都具有實(shí)際的意義。Desmin是目前知道的表達(dá)開始于骨骼肌衛(wèi)星細(xì)胞并在肌纖維中高表達(dá)的標(biāo)志蛋白。因此以Desmin基因的啟動(dòng)子元件控制的報(bào)告基因可望建立鑒定和篩選骨骼肌衛(wèi)星細(xì)胞的簡(jiǎn)便方法。本課題目的是構(gòu)建逆轉(zhuǎn)錄病毒報(bào)告基因載體，特異性表達(dá)于骨骼肌細(xì)胞中，并通過檢測(cè)其綠色熒光蛋白表達(dá)，驗(yàn)證其在骨骼肌細(xì)胞中表達(dá)的特異性。并在此基礎(chǔ)上將本實(shí)驗(yàn)所構(gòu)建重組載體制備成逆轉(zhuǎn)錄病毒懸液，檢測(cè)其在分化的P19細(xì)胞中的表達(dá)。 方法：(1)重組載體的構(gòu)建與鑒定，首先用PCR法擴(kuò)增所需要的目的片段，綠色熒光蛋白和新霉素基因(GFP+NEO)和desmin啟動(dòng)子和增強(qiáng)子(pro+enh)，然后利用限制性內(nèi)切酶和T4 DNA連接酶，將目的基因連接到經(jīng)NcoⅠ，BamHⅠ，EcoRⅠ和SalⅠ酶切后回收的逆轉(zhuǎn)錄病毒載體psuper線性片段上。(2)重組載體的骨骼肌衛(wèi)星細(xì)胞表達(dá)特異性驗(yàn)證：將psuper和重組載體分別轉(zhuǎn)染CHO和C2C12細(xì)胞，，24h后，倒置熒光顯微鏡下觀察綠色熒光蛋白的表達(dá)，采集圖片。(3)逆轉(zhuǎn)錄病毒懸液制備及其在分化P19細(xì)胞中的應(yīng)用，逆轉(zhuǎn)錄病毒懸液轉(zhuǎn)染C2C12和DMSO誘導(dǎo)分化的P19細(xì)胞，倒置熒光顯微鏡下觀察綠色熒光蛋白的表達(dá)，采集圖片。 結(jié)果：(1)通過對(duì)構(gòu)建過程的每一步進(jìn)行電泳分析，證明了所構(gòu)建重組載體是正確的。(2)重組載體和psuper轉(zhuǎn)染CHO和C2C12細(xì)胞后，在C2C12細(xì)胞表達(dá)水平相當(dāng)，說明重組載體在C2C12中能夠表達(dá)，而在CHO細(xì)胞中重組載體看不到表達(dá)，psuper則具有很高的表達(dá)。這就說明了重組載體具有骨骼肌衛(wèi)星細(xì)胞表達(dá)特異性。(3)制備的逆轉(zhuǎn)錄病毒懸液轉(zhuǎn)染C2C12后，綠色熒光表達(dá)效率有所提高但是并不非常明顯。在轉(zhuǎn)染了誘導(dǎo)分化的P19細(xì)胞中，發(fā)現(xiàn)了綠色熒光蛋白的表達(dá)，據(jù)此可以定性判斷DMSO誘導(dǎo)P19細(xì)胞分化為骨骼肌細(xì)胞。 結(jié)論：通過電泳分析方法鑒定了所構(gòu)建載體是正確的，另外在轉(zhuǎn)染CHO和C2C12細(xì)胞的特異性驗(yàn)證過程中，通過綠色熒光蛋白表達(dá)結(jié)果分析說明了所構(gòu)建載體具有骨骼肌衛(wèi)星細(xì)胞特異性。這為骨骼肌細(xì)胞的純化提供了基礎(chǔ)，也為骨骼肌衛(wèi)星細(xì)胞用于組織工程和基因治療提供了基礎(chǔ)。
[Abstract]:Aim: satellite cell (muscle satellite cells) is thought to be a myogenic cell reserved in adult skeletal muscle with proliferative and differentiation ability. To establish a simple and rapid method for the identification and screening of skeletal muscle satellite cells for satellite cell culture, Research and application are of practical significance. Desmin is now known as a marker protein expressed in skeletal muscle satellite cells and highly expressed in muscle fibers. Therefore, the reporter gene controlled by promoter element of Desmin gene can be used to identify and screen skeletal muscle satellite cells. The purpose of this study was to construct a retroviral reporter gene vector, which was specifically expressed in skeletal muscle cells, and to test the expression of green fluorescent protein in skeletal muscle cells. On this basis, the recombinant vector was prepared into retroviral suspension and its expression in differentiated P19 cells was detected. Methods: (1) Construction and identification of the recombinant vector. First, the desired target fragments, (GFP NEO) and neomycin gene (GFP NEO) and desmin promoter and enhancer (pro enh), were amplified by PCR method. Using restriction endonuclease and T4 DNA ligase, the target gene was linked to Nco 鈪

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