本文選題：miRNA + 彌漫性特發(fā)性骨質(zhì)增生癥��； 參考：《天津醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的： 在自然衍發(fā)基質(zhì)(naturally occurring matrix, NOM),即人完全脫細(xì)胞羊膜(human acellular amniotic membrane, HAAM)上培養(yǎng)彌漫性特發(fā)性骨質(zhì)增生癥(Diffuse idiopathic skeletal hyperostosis, DISH)病人骨化黃韌帶與正常病人黃韌帶來源的成纖維細(xì)胞,實(shí)現(xiàn)3D黏附(three dimensional adhesion),獲得并分析DISH骨化相關(guān)的特異性microRNA (miRNA)。 方法： 使用本課題組研發(fā)的羊膜無酸堿脫細(xì)胞專利技術(shù)制取HAAM,制成直徑為9.0cm大小的圓形培養(yǎng)基質(zhì)。分別獲取4份DISH病人頸椎/胸椎來源的骨化黃韌帶組織塊與頸椎/胸椎骨折病人來源的正常黃韌帶組織塊,分為骨化組和正常對(duì)照組。采用膠原酶消化法分離組織塊中成纖維細(xì)胞,接種于制備好的鋪有HAAM的培養(yǎng)皿中。待細(xì)胞生長至約80%滿度時(shí)收獲細(xì)胞,提取細(xì)胞總RNA并檢測(cè)其質(zhì)量。通過YM-100(Millipore)微離心過濾柱得到片段小于300nt的小RNA。采用u ParafloTM MicroRNA微陣列基因表達(dá)實(shí)驗(yàn)和分析技術(shù)分析miRNA表達(dá)譜,篩選差異miRNAs;采用PicTar2005、miRanda v5、TargetScan5.1軟件預(yù)測(cè)差異miRNAs的靶基因；采用Gene Ontology(GO)數(shù)據(jù)庫分析靶基因；使用KEGG pathway數(shù)據(jù)庫分析靶基因所參與的信號(hào)傳導(dǎo)通路。 結(jié)果： DISH組與正常組來源的黃韌帶經(jīng)過酶消化法分離獲得的細(xì)胞均為典型的長梭形成纖維細(xì)胞,光鏡下未見其他形狀細(xì)胞,平均需要14天細(xì)胞生長至HAAM培養(yǎng)基的80%滿度。細(xì)胞主要以成簇聚集的方式分布,呈現(xiàn)復(fù)層生長；細(xì)胞的胞突相互接觸,建立起了細(xì)胞之間的聯(lián)系。μ ParafloTM MicroRNA微陣列基因表達(dá)實(shí)驗(yàn)和分析共發(fā)現(xiàn)6種差異miRNAs,其中2種為下調(diào)(let-7c、miR-21),4種為上調(diào)(let-7b、miR-214、miR-4298、miR-1975)。靶基因預(yù)測(cè)和分析發(fā)現(xiàn)差異miRNAs靶基因參與分化,黏附,細(xì)胞增殖等多個(gè)生理過程。信號(hào)途徑預(yù)測(cè)靶基因參與MAPK信號(hào)途徑、Jak-STAT信號(hào)途徑、NOTCH信號(hào)途徑等。 結(jié)論： 利用HAAM可以實(shí)現(xiàn)3D黏附下培養(yǎng)DISH病人骨化黃韌帶與正常病人黃韌帶來源的成纖維細(xì)胞,有利于消除細(xì)胞培養(yǎng)時(shí)黏附因素引起的miRNA表達(dá)差異；實(shí)驗(yàn)得到了DISH骨化相關(guān)特異性miRNAs,為進(jìn)一步在基因水平研究DISH的發(fā)病機(jī)制獲得了突破口,為DISH的基因水平診斷、治療奠定了基礎(chǔ),也為研究3D黏附對(duì)細(xì)胞基因表達(dá)的影響提供了科學(xué)數(shù)據(jù)。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R3416

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相關(guān)期刊論文 前2條

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本文編號(hào)：2024334