大鼠骨髓间充质干细胞和弹性软骨细胞基因差异表达分析

发布时间：2018-06-13 12:47

  本文选题：间充质干细胞 + 软骨　； 参考：《大连医科大学》2007年硕士论文

【摘要】： 目的:本试验应用基因芯片技术筛选大鼠骨髓间充质干细胞及弹性软骨的差异表达基因,由此寻找刺激差异基因表达的生物因子以促进骨髓间充质干细胞向弹性软骨分化以得到组织工程化软骨。 方法: (1)通过密度梯度离心法和贴壁筛选法的联合应用提取SD大鼠的骨髓间充质干细胞,酶消化法得到耳部弹性软骨细胞,分别于加入10%FCS的DMEM和DMEM/F12培养基中进行体外培养。 (2)取培养2-3代后的细胞用一步法提取两种细胞的RNA并纯化,将两者RNA反转录为双链CDNA,用Cy5-dCTP,Cy3-dCTP分别标记骨髓间充质干细胞和软骨细胞的CDNA探针,杂交并严格清洗,用LuxScan 10KA双通道激光扫描仪进行扫描荧光信号图像,每点上两种荧光信号的强度分别代表Cy5-dCTP和Cy3-dCTP的量,获得的荧光信号图像用计算机GenePix Pro 4.0图像分析软件进行分析。 结果: (1)软骨细胞和MSCs样品RNA吸光度A260/A280比值1.79;软骨细胞总RNA量为79.2μg,MSCs总RNA量为22.5μg。两种细胞RNA电泳条带清晰,28S比18S rRNA条带亮度接近2:1. (2)按差异显著性标准,cy5/cy30.5为表达上调基因,cy5/cy32为表达下调基因,从28800条基因中筛选出差异表达基因2114条。以骨髓间充质干细胞为对照,在软骨中有1226条基因上调,888条基因下调。由于差异基因数量较多,我们重点研究了差别在20倍以上的基因,即cy5/cy30.05或者cy5/cy320的基因,我们将其定义为显著差别基因。 (3)在显著差别基因中,包括细胞骨架和运动蛋白相关基因,细胞周期蛋白相关基因,免疫相关基因,细胞信号和传递蛋白相关基因,细胞外基质类基因,转录因子类基因,蛋白翻译和合成相关基因、代谢相关基因等。我们发现了两种生长因子在软骨中高表达:软骨调节素和结缔组织因子。细胞外基质中软骨相关基因,例如Ⅱ型胶原和弹性胶原在软骨中高表达,而在骨髓间充质干细胞中高表达基因的多是酶类,证明了其幼稚性,有向多种组织分化的能力。 结论: (1)骨髓间充质干细胞和软骨细胞的基因表达谱存在显著差异。 (2)诱导骨髓间充质干细胞向软骨分化可能是多种因子的协同作用,上调或者下调某些关键基因的表达的结果。 (3)本试验发现以软骨调节素和结缔组织因子为代表的1226条基因在软骨中高表达,可能与骨髓间充质干细胞向软骨分化有关。 (4)本试验骨髓还发现以基质金属蛋白酶9为代表的888条基因相对于软骨在骨髓间充质干细胞中高表达,要使骨髓间充质干细胞分化成软骨,必须抑制其表达。
[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，

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