【摘要】： 目的：衛(wèi)星細胞(muscle satellite cells)認為是保留在成年骨骼肌內具有增殖分化能力的肌源性細胞。建立簡便快速的鑒定和篩選骨骼肌衛(wèi)星細胞的方法對于衛(wèi)星細胞培養(yǎng)、研究和應用都具有實際的意義。Desmin是目前知道的表達開始于骨骼肌衛(wèi)星細胞并在肌纖維中高表達的標志蛋白。因此以Desmin基因的啟動子元件控制的報告基因可望建立鑒定和篩選骨骼肌衛(wèi)星細胞的簡便方法。本課題目的是構建逆轉錄病毒報告基因載體，特異性表達于骨骼肌細胞中，并通過檢測其綠色熒光蛋白表達，驗證其在骨骼肌細胞中表達的特異性。并在此基礎上將本實驗所構建重組載體制備成逆轉錄病毒懸液，檢測其在分化的P19細胞中的表達。 方法：(1)重組載體的構建與鑒定，首先用PCR法擴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啟動子和增強子(pro+enh)，然后利用限制性內切酶和T4 DNA連接酶，將目的基因連接到經(jīng)NcoⅠ，BamHⅠ，EcoRⅠ和SalⅠ酶切后回收的逆轉錄病毒載體psuper線性片段上。(2)重組載體的骨骼肌衛(wèi)星細胞表達特異性驗證：將psuper和重組載體分別轉染CHO和C2C12細胞，，24h后，倒置熒光顯微鏡下觀察綠色熒光蛋白的表達，采集圖片。(3)逆轉錄病毒懸液制備及其在分化P19細胞中的應用，逆轉錄病毒懸液轉染C2C12和DMSO誘導分化的P19細胞，倒置熒光顯微鏡下觀察綠色熒光蛋白的表達，采集圖片。 結果：(1)通過對構建過程的每一步進行電泳分析，證明了所構建重組載體是正確的。(2)重組載體和psuper轉染CHO和C2C12細胞后，在C2C12細胞表達水平相當，說明重組載體在C2C12中能夠表達，而在CHO細胞中重組載體看不到表達，psuper則具有很高的表達。這就說明了重組載體具有骨骼肌衛(wèi)星細胞表達特異性。(3)制備的逆轉錄病毒懸液轉染C2C12后，綠色熒光表達效率有所提高但是并不非常明顯。在轉染了誘導分化的P19細胞中，發(fā)現(xiàn)了綠色熒光蛋白的表達，據(jù)此可以定性判斷DMSO誘導P19細胞分化為骨骼肌細胞。 結論：通過電泳分析方法鑒定了所構建載體是正確的，另外在轉染CHO和C2C12細胞的特異性驗證過程中，通過綠色熒光蛋白表達結果分析說明了所構建載體具有骨骼肌衛(wèi)星細胞特異性。這為骨骼肌細胞的純化提供了基礎，也為骨骼肌衛(wèi)星細胞用于組織工程和基因治療提供了基礎。
[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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