SiRNA干扰技术稳定下调基因osterix在骨髓基质干细胞中的表达

发布时间：2018-06-22 22:25

本文选题：骨髓间充质干细胞 + Osterix　；参考：《浙江大学》2011年硕士论文

【摘要】：目的：检测特异性的转录因子osterix对骨髓间充质干细胞诱导分化成骨细胞以及软骨细胞途径的影响,且与外源性BMPs长期培养对骨髓间充质干细胞诱导分化各途径的效应相比较。同时,BMP-2能代表性的诱导种子细胞分化为成骨细胞或软骨细胞。因此该实验合成并构建特异性的细胞系,可稳定下调基因osterix的表达,且抑制骨髓间充质干细胞诱导分化软骨细胞系后期出现的肥大变性时基因指标的表达,从而促进软骨组织工程临床应用和推广。方法：构建特异性的慢病毒载体,其带有攻击目的基因osterix的shRNA序列,通过转染液介导后可转染骨髓间充质干细胞,干扰并致基因osterix沉默效应,稳定下调基因osterix的表达。同时挑选已公开的不针对基因osterix的靶基因,构建慢病毒载体行阴性对照。最后另一组为BMP-2为主要诱导剂的诱导液,促进骨髓间充质干细胞诱导分化。分别于不同时间点观察并行ELISA检测各组碱性磷酸酶表达水平,组织染色(阿尔新蓝染色以及茜素红染色)检测成骨细胞系以及软骨细胞水平,行实时定量PCR检测各组目的基因表达水平,行Western blot检测各组目的蛋白表达水平。结果：基因osterix下调组基因osterix表达降低,多数成骨型指标如Runx2,10型胶原蛋白下降,同时成软骨性指标如2型胶原蛋白等基因以及蛋白表达量增加。BMP-2诱导组多数基因以及蛋白表达情况基本与osterix下调组相反。结论：BMP-2对骨髓间充质干细胞成骨分化诱导增强,效果明显；对软骨途径诱导分化时,效果较osterix基因沉默组效果差。基因osterix下调二十一天内可明显抑制骨髓间充质干细胞成骨细胞系诱导分化的途径,且显著增强骨髓间充质干细胞向软骨细胞诱导分化的途径,且诱导骨髓间充质干细胞分化成软骨细胞时后期出现的类软骨细胞肥大变性现象通过对成骨关键性基因osterix的下调可显著抑制。稳定下调基因osterix的表达水平可明确增强骨髓基质干细胞表面软骨细胞系显型表达,将有效阻抑长期培养后软骨细胞系肥大性显型基因的表达水平,从而对解决广泛大量培养的骨髓基质干细胞诱导分化为软骨细胞后期出现的肥大变性的问题提示了方向。
[Abstract]:Aim: to investigate the effects of specific transcription factor osterix on osteoblast differentiation and chondrocyte pathway induced by bone marrow mesenchymal stem cells (MSCs), and to compare the effects of long-term culture of exogenous BMPs on the differentiation pathways of bone marrow mesenchymal stem cells (BMSCs). BMP-2 can induce seed cells to differentiate into osteoblasts or chondrocytes. Therefore, the synthesis and construction of specific cell lines can stably down-regulate the expression of gene osterix and inhibit the expression of gene markers during hypertrophic degeneration in the later stage of differentiation of chondrocytes induced by bone marrow mesenchymal stem cells. So as to promote the clinical application and promotion of cartilage tissue engineering. Methods: a specific lentivirus vector containing shRNA sequence of target gene osterix was constructed and transfected into bone marrow mesenchymal stem cells mediated by transfection medium. The silencing effect of gene osterix was interfered with and gene osterix expression was steadily down-regulated. At the same time, the target genes which were not specific to gene osterix were selected, and the lentivirus vector was constructed for negative control. The last group was treated with BMP-2 as the main inducer to promote the differentiation of bone marrow mesenchymal stem cells. The expression of alkaline phosphatase was detected by Elisa at different time points, and the osteoblasts and chondrocytes were detected by tissue staining (Alxin blue staining and alizarin red staining). The expression level of target gene in each group was detected by real-time quantitative PCR, and the expression level of target protein was detected by Western blot. Results: the expression of gene osterix was decreased in the down-regulated group of gene osterix, and the expression of most osteogenic markers such as Runx2O10 collagen was decreased. At the same time, the expression of most genes and proteins in the chondrogenic index such as collagen type 2 and protein increased. The expression of most genes and proteins in BMP-2 induced group was basically opposite to that in osterix down-regulated group. Conclusion the osteogenic differentiation of bone marrow mesenchymal stem cells induced by bone morphogenetic protein (BMP-2) was significantly enhanced, and the effect on cartilage pathway was worse than that in osterix gene silencing group. The down-regulation of osterix could significantly inhibit the differentiation of bone marrow mesenchymal stem cells in osteoblasts within 21 days, and enhance the differentiation of bone marrow mesenchymal stem cells into chondrocytes. The hypertrophic denaturation of chondrocytes induced by bone marrow mesenchymal stem cells differentiation into chondrocytes was significantly inhibited by down-regulation of osteoblast key gene osterix. The stable down-regulation of the expression of osterix gene can obviously enhance the expression of chondrocytes on the surface of bone marrow stromal cells, and can effectively inhibit the expression of hypertrophic explicit genes in long-term cultured chondrocytes. Therefore, it suggests the direction to solve the problem of hypertrophic degeneration induced by a large number of cultured bone marrow stromal cells into chondrocytes.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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