【摘要】：第一章 大鼠肋生長板軟骨細(xì)胞的分離、培養(yǎng)及生物學(xué)特性的研究 目的:建立大鼠肋生長板軟骨細(xì)胞的體外培養(yǎng)模型,探討其在體外培養(yǎng)條件下的生物學(xué)特性。 方法:顯微解剖分離出大鼠肋生長板,酶消化法(0.05%透明質(zhì)酸酶和0.2%Ⅱ型膠原酶)獲得分散的大鼠肋生長板軟骨細(xì)胞(rat costochondral growth plate chondrocyte,RGC),常規(guī)接種,單層培養(yǎng)。對原代～第5代RGC進(jìn)行生長動力學(xué)分析,并通過免疫細(xì)胞化學(xué)、阿爾新蘭染色和Western blot檢測原代～第5代RGC中Ⅱ型膠原、α-SMA和蛋白多糖的分布和表達(dá)。 結(jié)果:原代培養(yǎng)的生長板軟骨細(xì)胞中95%以上的細(xì)胞Ⅱ型膠原陽性染色,細(xì)胞核周圍和細(xì)胞膜周邊都有Ⅱ型膠原的陽性染色,陽性染色的纖維包裹著細(xì)胞核,保持圓形的細(xì)胞強(qiáng)陽性染色。隨著傳代次數(shù)的增加,表達(dá)Ⅱ型膠原的細(xì)胞比例和表達(dá)量下降,第6代RGC中僅保持圓形的細(xì)胞仍呈陽性染色。原代培養(yǎng)的RGC表達(dá)大量的蛋白多糖,阿爾新蘭陽性染色。第2,3代阿爾新蘭染色減少,3代后阿爾新蘭染色皆呈陰性。原代RGC中,α-SMA的表達(dá)很弱,隨傳代次數(shù)增加,α-SMA的表達(dá)量逐漸增高;Ⅱ型膠原的表達(dá)剛好和α-SMA相反,原代RGC表達(dá)最強(qiáng),隨著傳代次數(shù)的增加,表達(dá)逐漸下降。 結(jié)論:成功建立大鼠肋生長板軟骨細(xì)胞的體外培養(yǎng)模型。單層培養(yǎng)的RGC在第4代后逐漸失去分化表型,α-SMA可以作為RGC去分化的一個(gè)新的標(biāo)志物。
[Abstract]:Chapter 1 isolation, culture and biological characteristics of rat costal growth plate chondrocytes objective: to establish an in vitro culture model of rat costal growth plate chondrocytes. Objective: to study the biological characteristics of its in vitro culture. Methods: the rat costal growth plates were isolated by microdissection. The scattered (rat costochondral growth plate chondrocyte chondrocytes were obtained by enzyme digestion (0.05% hyaluronidase and 0.2% collagenase). The growth kinetics of primary to fifth generation RGC was analyzed. The distribution and expression of type 鈪，

本文編號：2199152