3D黏附下DISH骨化相关特异性microRNA分析

发布时间：2018-06-16 00:22

本文选题：miRNA + 弥漫性特发性骨质增生症　；参考：《天津医科大学》2011年硕士论文

【摘要】：目的：在自然衍发基质(naturally occurring matrix, NOM),即人完全脱细胞羊膜(human acellular amniotic membrane, HAAM)上培养弥漫性特发性骨质增生症(Diffuse idiopathic skeletal hyperostosis, DISH)病人骨化黄韧带与正常病人黄韧带来源的成纤维细胞,实现3D黏附(three dimensional adhesion),获得并分析DISH骨化相关的特异性microRNA (miRNA)。方法：使用本课题组研发的羊膜无酸碱脱细胞专利技术制取HAAM,制成直径为9.0cm大小的圆形培养基质。分别获取4份DISH病人颈椎/胸椎来源的骨化黄韧带组织块与颈椎/胸椎骨折病人来源的正常黄韧带组织块,分为骨化组和正常对照组。采用胶原酶消化法分离组织块中成纤维细胞,接种于制备好的铺有HAAM的培养皿中。待细胞生长至约80%满度时收获细胞,提取细胞总RNA并检测其质量。通过YM-100(Millipore)微离心过滤柱得到片段小于300nt的小RNA。采用u ParafloTM MicroRNA微阵列基因表达实验和分析技术分析miRNA表达谱,筛选差异miRNAs;采用PicTar2005、miRanda v5、TargetScan5.1软件预测差异miRNAs的靶基因；采用Gene Ontology(GO)数据库分析靶基因；使用KEGG pathway数据库分析靶基因所参与的信号传导通路。结果： DISH组与正常组来源的黄韧带经过酶消化法分离获得的细胞均为典型的长梭形成纤维细胞,光镜下未见其他形状细胞,平均需要14天细胞生长至HAAM培养基的80%满度。细胞主要以成簇聚集的方式分布,呈现复层生长；细胞的胞突相互接触,建立起了细胞之间的联系。μ ParafloTM MicroRNA微阵列基因表达实验和分析共发现6种差异miRNAs,其中2种为下调(let-7c、miR-21),4种为上调(let-7b、miR-214、miR-4298、miR-1975)。靶基因预测和分析发现差异miRNAs靶基因参与分化,黏附,细胞增殖等多个生理过程。信号途径预测靶基因参与MAPK信号途径、Jak-STAT信号途径、NOTCH信号途径等。结论：利用HAAM可以实现3D黏附下培养DISH病人骨化黄韧带与正常病人黄韧带来源的成纤维细胞,有利于消除细胞培养时黏附因素引起的miRNA表达差异；实验得到了DISH骨化相关特异性miRNAs,为进一步在基因水平研究DISH的发病机制获得了突破口,为DISH的基因水平诊断、治疗奠定了基础,也为研究3D黏附对细胞基因表达的影响提供了科学数据。
[Abstract]:Objective: to culture fibroblasts derived from the ossified ligamentum flavum of diffuse idiopathic skeletal hyperostosis, hyperplasia (Dish) and normal patients on the naturally derived occurring matrix (NOMX), that is, human acellular amniotic membrane, amniotic membrane (Ham). Methods: the fibroblasts derived from the ossified ligamentum flavum were cultured in the patients with diffuse idiopathic skeletal hyperostosis, dysplasia (Dish), and the fibroblasts derived from the ligamentum flavum from the normal patients were cultured. Three dimensional adhesions were realized to obtain and analyze the specific microRNAs related to ossification of fish. Methods: the amniotic membrane was prepared by amniotic membrane without acid-base acellular patent technology developed by our research group, and the circular culture substrate with diameter of 9.0cm was prepared. Four specimens of ossified ligamentum flavum from cervical vertebrae / thoracic vertebrae and normal ligamentum flavum from patients with cervical / thoracic vertebrae fracture were obtained and divided into ossification group and normal control group. Fibroblasts from tissue blocks were isolated by collagenase digestion and inoculated into a prepared dish coated with HAAM. When the cells grow to about 80% full, the cells are harvested, the total RNA of the cells is extracted and its quality is measured. Small RNAs with a fragment smaller than 300nt were obtained by using YM-100 Millipore-based microcentrifugation column. U ParafloTM microRNA microarray gene expression experiment and analysis technique were used to analyze miRNA expression profile to screen differential miRNAs. PicTar2005miRanda v5 TargetScan5.1 software was used to predict the target genes of differential miRNAs, Gene Ontology GOdatabase was used to analyze target genes. KEGG pathway database was used to analyze the signal transduction pathway involved in target gene. Results: the cells isolated from ligamentum flavum from dish group and normal group were all typical fusiform fibroblasts, but no other shape cells were found under light microscope. It takes an average of 14 days for cells to grow to 80% of HAAM medium. The cells are mainly distributed in clusters, showing laminated growth, and the processes of the cells are in contact with each other. Six different miRNAss were found in 渭 ParafloTM microRNA microarray gene expression experiment, of which 2 were down-regulated and 4 were upregulated to upregulate the expression of miR-214miR-4298miR-1975. Target gene prediction and analysis showed that different miRNAs target genes were involved in many physiological processes, such as differentiation, adhesion and cell proliferation. Target genes are involved in MAPK signaling pathway Jak-STAT signaling pathway and NOTCH signaling pathway. Conclusion: HAAM can be used to culture fibroblasts derived from the ossified ligamentum flavum of the patients with ish and normal patients, which is helpful to eliminate the difference of miRNA expression caused by adhesion factors in cell culture. The specific miRNAs-related ossification of fish was obtained, which provided a breakthrough for the further study of the pathogenesis of dish at the gene level, and laid a foundation for the gene level diagnosis and treatment of dish. It also provides scientific data for studying the effect of 3D adhesion on cell gene expression.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R3416

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