本文關(guān)鍵詞：軟骨源性形態(tài)發(fā)生蛋白2基因轉(zhuǎn)染小鼠成肌細(xì)胞表達(dá)軟骨細(xì)胞表型的實(shí)驗(yàn)研究　出處：《遼寧醫(yī)學(xué)院》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 成肌細(xì)胞 軟骨分化 軟骨源性形態(tài)發(fā)生蛋白-2

【摘要】：目的 1.研究軟骨形態(tài)發(fā)生蛋白-2(cartilage-derived morphogenetic protein-2)體外對(duì)小鼠成肌細(xì)胞向軟骨細(xì)胞分化的可行性。 2.構(gòu)建軟骨形態(tài)發(fā)生蛋白-2基因轉(zhuǎn)染小鼠成肌細(xì)胞，檢測(cè)轉(zhuǎn)染后小鼠成肌細(xì)胞的特異性表達(dá)，探討成肌細(xì)胞作為軟骨組織工程種子細(xì)胞的可行性。 方法 對(duì)小鼠成肌細(xì)胞進(jìn)行體外培養(yǎng)傳代，取第3代細(xì)胞進(jìn)行實(shí)驗(yàn)處理，對(duì)照組(A組)為無(wú)血清L-DMEM培養(yǎng)液+小鼠成肌細(xì)胞；實(shí)驗(yàn)組（B組）為無(wú)血清L-DMEM培養(yǎng)液+透明質(zhì)酸鈉（10g/L）+小鼠成肌細(xì)胞；實(shí)驗(yàn)組C組為無(wú)血清L-DMEM培養(yǎng)液+CDMP-2(100ng/ml)+小鼠成肌細(xì)胞；實(shí)驗(yàn)組D組為無(wú)血清L-DMEM培養(yǎng)液+CDMP-2基因轉(zhuǎn)染的小鼠成肌細(xì)胞。16天后終止細(xì)胞培養(yǎng)，應(yīng)用倒置相差顯微鏡觀察細(xì)胞形態(tài)，進(jìn)行甲苯胺藍(lán)糖胺聚糖(GAGs)染色，行Ⅱ型膠原免疫組織化學(xué)染色、灰度值分析和Western blot檢測(cè)。 結(jié)果 細(xì)胞培養(yǎng)16天后可見轉(zhuǎn)染組和誘導(dǎo)組的成肌細(xì)胞形態(tài)由長(zhǎng)梭形向軟骨細(xì)胞的多角形轉(zhuǎn)變。甲苯胺藍(lán)染色顯示遇甲苯胺藍(lán)變?yōu)樗{(lán)色的糖胺聚糖(GAGs)均勻分布于細(xì)胞基質(zhì)中；免疫組織化學(xué)染色顯示經(jīng)SP法處理后變?yōu)樽睾稚蘑蛐湍z原蛋白均勻分布于鏡下，對(duì)照組與透明質(zhì)酸鈉組未見著色。Western blot顯示轉(zhuǎn)染組和誘導(dǎo)組Ⅱ型膠原蛋白表達(dá)陽(yáng)性，且CDMP-2基因轉(zhuǎn)染組細(xì)胞表達(dá)Ⅱ型膠原蛋白水平高于CDMP-2誘導(dǎo)組，具有統(tǒng)計(jì)學(xué)意義；對(duì)照組和透明質(zhì)酸鈉組陰性表達(dá)。 結(jié)論 CDMP-2體外能夠誘導(dǎo)成肌細(xì)胞向軟骨細(xì)胞分化，，CDMP-2基因轉(zhuǎn)染的小鼠成肌細(xì)胞能夠分泌軟骨細(xì)胞分泌的特異性蛋白及多糖，且CDMP-2基因轉(zhuǎn)染的小鼠成肌細(xì)胞能夠更好的表達(dá)軟骨特異性Ⅱ型膠原及糖胺多糖。
[Abstract]:Purpose 1. Study on cartilage morphogenetic protein -2cartilage-derived morphogenetic protein-2. Feasibility of differentiation of mouse myoblasts into chondrocytes in vitro. 2. Construction of chondromorphogenetic protein-2 gene transfected mouse myoblasts, detection of the specific expression of mouse myoblasts after transfection, to explore the feasibility of myoblast as seed cells for cartilage tissue engineering. Method Mouse myoblasts were cultured in vitro and treated with the third passage. The control group (group A) was serum-free L-DMEM medium. The experimental group (group B) was divided into serum-free L-DMEM medium (sodium hyaluronate 10 g / L) mouse myoblasts. Experimental group C was murine myoblast in serum-free L-DMEM medium (CDMP-2ng-1 / ml). Group D was treated with serum-free L-DMEM culture medium CDMP-2 gene transfected mouse myoblasts. After 16 days, the cultured cells were terminated, and the morphology of the cells was observed by inverted phase contrast microscope. Toluidine blue glycosaminoglycan (GAGs) staining, type 鈪

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