本文選題：關(guān)節(jié)軟骨細(xì)胞 + 細(xì)胞培養(yǎng)　； 參考：《大連醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的：優(yōu)化兔關(guān)節(jié)軟骨細(xì)胞分離方法,探索其體外單層培養(yǎng)過程條件及端粒、生物學(xué)功能學(xué)特性的變化。 方法：選用4周齡雄性、健康、新西蘭大白兔的關(guān)節(jié)軟骨,采用機(jī)械、雙酶消化法進(jìn)行分離得到關(guān)節(jié)軟骨細(xì)胞。測定細(xì)胞存活率,并在含有10%胎牛血清的DMEM高糖培養(yǎng)基中進(jìn)行體外單層培養(yǎng)。待細(xì)胞融合率達(dá)到85%至90%時,用胰蛋白酶消化收集并以相同密度鋪板傳代。逐代觀察細(xì)胞形態(tài)及應(yīng)用MTT法繪制細(xì)胞生長曲線；同時采用甲苯胺藍(lán)染色和RT-PCR方法動態(tài)研究各代關(guān)節(jié)軟骨細(xì)胞的蛋白多糖,Ⅰ、Ⅱ型膠原蛋白與端粒酶因水平的表達(dá)變化。 結(jié)果：本方法兔關(guān)節(jié)軟骨細(xì)胞收集過程簡便、高效,平均每克軟骨可得到2×106個細(xì)胞。在體外單層培養(yǎng)的過程中,隨傳代次數(shù)增加,軟骨細(xì)胞傳代時間變長,細(xì)胞形態(tài)逐漸向“纖維樣”細(xì)胞轉(zhuǎn)化。Ⅰ型膠原蛋白基因水平的表達(dá)呈遞增趨勢,Ⅱ型膠原蛋白與端�；蛩降谋磉_(dá)呈遞增趨勢,細(xì)胞外基質(zhì)蛋白多糖逐漸減少。前三代細(xì)胞具有較高的生物學(xué)及功能學(xué)特性表達(dá),四代后主要表現(xiàn)為Ⅰ型膠原的高表達(dá),而軟骨細(xì)胞所特有的Ⅱ型膠原及蛋白多糖基本消失。整個趨勢呈現(xiàn)去分化。 結(jié)論：1關(guān)節(jié)軟骨細(xì)胞機(jī)械、雙酶獲取方法簡便易操作,可獲取大量存活率高的軟骨細(xì)胞。 2.體外單層培養(yǎng)的兔關(guān)節(jié)軟骨細(xì)胞前三代表型穩(wěn)定,生物學(xué)及功能學(xué)特性表達(dá)良好。
[Abstract]:Aim: to optimize the separation method of rabbit articular chondrocytes, and to explore the changes of telomere and biological function in monolayer culture of rabbit articular chondrocytes. Methods: articular cartilage of 4 week old male healthy New Zealand rabbits was isolated by mechanical and double enzyme digestion. Cell survival rate was measured and monolayer culture was carried out in DMEM medium containing 10% fetal bovine serum in vitro. When the cell fusion rate reached 85% to 90%, trypsin was used to digest and collect the cells. Cell morphology was observed generation by generation and cell growth curve was plotted by MTT method. Meanwhile, the expression of proteoglycan, type 鈪，

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