發(fā)布時(shí)間：2018-06-13 12:47

  本文選題：間充質(zhì)干細(xì)胞 + 軟骨�。� 參考：《大連醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 目的:本試驗(yàn)應(yīng)用基因芯片技術(shù)篩選大鼠骨髓間充質(zhì)干細(xì)胞及彈性軟骨的差異表達(dá)基因,由此尋找刺激差異基因表達(dá)的生物因子以促進(jìn)骨髓間充質(zhì)干細(xì)胞向彈性軟骨分化以得到組織工程化軟骨。 方法: (1)通過(guò)密度梯度離心法和貼壁篩選法的聯(lián)合應(yīng)用提取SD大鼠的骨髓間充質(zhì)干細(xì)胞,酶消化法得到耳部彈性軟骨細(xì)胞,分別于加入10%FCS的DMEM和DMEM/F12培養(yǎng)基中進(jìn)行體外培養(yǎng)。 (2)取培養(yǎng)2-3代后的細(xì)胞用一步法提取兩種細(xì)胞的RNA并純化,將兩者RNA反轉(zhuǎn)錄為雙鏈CDNA,用Cy5-dCTP,Cy3-dCTP分別標(biāo)記骨髓間充質(zhì)干細(xì)胞和軟骨細(xì)胞的CDNA探針,雜交并嚴(yán)格清洗,用LuxScan 10KA雙通道激光掃描儀進(jìn)行掃描熒光信號(hào)圖像,每點(diǎn)上兩種熒光信號(hào)的強(qiáng)度分別代表Cy5-dCTP和Cy3-dCTP的量,獲得的熒光信號(hào)圖像用計(jì)算機(jī)GenePix Pro 4.0圖像分析軟件進(jìn)行分析。 結(jié)果: (1)軟骨細(xì)胞和MSCs樣品RNA吸光度A260/A280比值1.79;軟骨細(xì)胞總RNA量為79.2μg,MSCs總RNA量為22.5μg。兩種細(xì)胞RNA電泳條帶清晰,28S比18S rRNA條帶亮度接近2:1. (2)按差異顯著性標(biāo)準(zhǔn),cy5/cy30.5為表達(dá)上調(diào)基因,cy5/cy32為表達(dá)下調(diào)基因,從28800條基因中篩選出差異表達(dá)基因2114條。以骨髓間充質(zhì)干細(xì)胞為對(duì)照,在軟骨中有1226條基因上調(diào),888條基因下調(diào)。由于差異基因數(shù)量較多,我們重點(diǎn)研究了差別在20倍以上的基因,即cy5/cy30.05或者cy5/cy320的基因,我們將其定義為顯著差別基因。 (3)在顯著差別基因中,包括細(xì)胞骨架和運(yùn)動(dòng)蛋白相關(guān)基因,細(xì)胞周期蛋白相關(guān)基因,免疫相關(guān)基因,細(xì)胞信號(hào)和傳遞蛋白相關(guān)基因,細(xì)胞外基質(zhì)類基因,轉(zhuǎn)錄因子類基因,蛋白翻譯和合成相關(guān)基因、代謝相關(guān)基因等。我們發(fā)現(xiàn)了兩種生長(zhǎng)因子在軟骨中高表達(dá):軟骨調(diào)節(jié)素和結(jié)締組織因子。細(xì)胞外基質(zhì)中軟骨相關(guān)基因,例如Ⅱ型膠原和彈性膠原在軟骨中高表達(dá),而在骨髓間充質(zhì)干細(xì)胞中高表達(dá)基因的多是酶類,證明了其幼稚性,有向多種組織分化的能力。 結(jié)論: (1)骨髓間充質(zhì)干細(xì)胞和軟骨細(xì)胞的基因表達(dá)譜存在顯著差異。 (2)誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向軟骨分化可能是多種因子的協(xié)同作用,上調(diào)或者下調(diào)某些關(guān)鍵基因的表達(dá)的結(jié)果。 (3)本試驗(yàn)發(fā)現(xiàn)以軟骨調(diào)節(jié)素和結(jié)締組織因子為代表的1226條基因在軟骨中高表達(dá),可能與骨髓間充質(zhì)干細(xì)胞向軟骨分化有關(guān)。 (4)本試驗(yàn)骨髓還發(fā)現(xiàn)以基質(zhì)金屬蛋白酶9為代表的888條基因相對(duì)于軟骨在骨髓間充質(zhì)干細(xì)胞中高表達(dá),要使骨髓間充質(zhì)干細(xì)胞分化成軟骨,必須抑制其表達(dá)。
[Abstract]:Objective: to screen differentially expressed genes in rat bone marrow mesenchymal stem cells and elastic cartilage by gene chip technique. In order to promote the differentiation of bone marrow mesenchymal stem cells into elastic cartilage, we can find the biological factors that stimulate the differentially expressed genes to obtain tissue engineered cartilage. Methods: bone marrow mesenchymal stem cells from SD rats were extracted by density gradient centrifugation and adherent screening. The elastic chondrocytes were obtained by enzyme digestion. In vitro culture was carried out on 10 S DMEM and DMEM / F12 medium. The RNA was reverse transcribed as double-stranded CDNA.Cy5-dCTP Cy3-dCTP was used to label CDNA probes of bone marrow mesenchymal stem cells and chondrocytes respectively, hybridization and strict cleaning. The fluorescence images were scanned by LuxScan 10KA dual-channel laser scanner. The intensity of two fluorescence signals at each point represents the quantities of Cy5-dCTP and Cy3-dCTP, respectively. The obtained fluorescence signal images are analyzed by the computer GenePix Pro 4.0 image analysis software. Results: the ratio of A260/A280 absorbance of chondrocytes to MSCs was 1.79, and the total RNA content of chondrocytes was 79.2 渭 g. The brightness of the two cell RNA bands was close to 2: 1. 2) according to the standard of difference significance, the expression of cy5 / cy30.5 was up-regulated gene, cy5 / cy32 was down-regulated gene. 2114 differentially expressed genes were screened out of 28800 genes. Compared with bone marrow mesenchymal stem cells, 1226 genes were up-regulated in cartilage. Because of the large number of differentially expressed genes, we have focused on genes that are more than 20 times different, that is, genes that are cy5/cy30.05 or cy5/cy320. We define it as a significantly different gene. We define it as a significantly different gene, including cytoskeleton and motion protein-associated genes, cyclin related genes, and cytoskeleton related genes. Immune related genes, cell signal and transfer protein related genes, extracellular matrix genes, transcription factor genes, protein translation and synthesis related genes, metabolism-related genes and so on. We found that two growth factors were highly expressed in cartilage: chondromodulin and connective tissue factor. Cartilage related genes, such as type 鈪，

本文編號(hào)：2014075