miR-30a-5p在人骨髓间充质干细胞体外向成骨细胞分化中的生物学功能验证

发布时间：2018-01-10 01:18

本文关键词：miR-30a-5p在人骨髓间充质干细胞体外向成骨细胞分化中的生物学功能验证　出处：《第四军医大学》2011年硕士论文　论文类型：学位论文

【摘要】：背景组织工程干细胞(SC)的研究作为生物医学领域的研究热点,为组织器官缺损的修复重建提供了一条全新而极具临床应用前景的途径。成体干细胞(ASC)作为早期未分化细胞具有强大的增殖更新及定向分化为成骨细胞、软骨细胞、神经细胞、脂肪细胞、骨骼肌细胞、真皮层细胞、肝细胞等的潜能。因其还具有来源广泛、易于体外扩增且可行个体化治疗从而避免排斥反应等众多优点而被密切关注并于多层面展开进行深入研究。多项研究证实骨髓间充质干细胞(BMSCs)的增殖、分化受到各种细胞因子、信号通路、细胞内外微环境等多方面因素的联合作用影响。同时大量研究还证实microRNA(miRNA)也参与了干细胞的各种生物学行为的调控。 miRNA是一类平均含有22nt、主要于基因转录后水平行使调控功能的小分子非蛋白编码RNA。近年来大量研究表明在多个物种中存在着一个异常庞大的miRNA家族,不同物种中其类型、数量及表达时序性均具有差异性。另外,在生物个体的生长发育、信号转导、组织器官分化、疾病的发生发展等过程中miRNA也参与了重要调控,并决定了该生物体的生理、病理和行为等的变化。本课题组前期实验通过高通量基因芯片检测技术检测BMSCs及其诱导分化的成骨细胞中的miRNA基因表达谱,分析筛选出一系列成骨诱导分化前后差异性表达miRNA,本实验从该结果中选取成骨性表达量显著增高的miRNA-30a-5p,来验证其在成骨中的生物学作用,并对其产生的该生物学作用的靶基因进行预测。目的探讨并验证重组miRNA-30a-5p体外转染对人骨髓间充质干细胞向成骨细胞分化的生物学作用。并通过生物信息学研究方法来预测其可能的作用靶基因。方法人工重组合成从人骨髓MSCs定向成骨分化差异性表达miRNA基因芯片结果中筛选成骨性表达量显著增高的miRNA-30a-5p(3组样品结果筛选结果参见附录2)。从人体骨髓中分离培养BMSCs,并于体外转染重组miRNA-30a-5p后继续行成骨诱导培养,第14、21天时分别取细胞爬片通过茜素红S法染色检测钙盐沉积、碱性磷酸酶(ALP)钙钴法染色及ALP比色法定量检测成骨细胞ALP活性,对比观察研究重组miRNA-30a-5p在人骨髓MSCs定向成骨诱导分化过程中的生物学功能。结果成功分离培养人骨髓MSCs并诱导其成骨定向分化。经体外向人BMSCs转染miRNA-30a-5p并诱导其成骨定向分化,转染效率为37.32%±2.43%。分别于诱导培养第14天、21天行ALP染色观察、ALP活性测定、茜素红钙盐结节染色检测各组细胞成骨活性,结果显示miRNA-30a-5p mimics转染组MSCs成骨分化特性显著性增强(P0.05)。结论 (1)用percoll密度梯度离心法结合全骨髓贴壁培养法可以从新鲜骨髓液中分离得到数量充足、纯度较高的人BMSCS,并可在体外诱导培养促使其向成骨细胞分化。(2)miRNA-30a-5p在MSCs定向成骨分化的过程中具有一定的促进增强作用,被转染的MSCs向成骨细胞定向分化表达有所增加,为进一步阐明人骨髓MSCs定向成骨分化的分子生化机制,细胞移植修复治疗骨缺损奠定了理论基础。
[Abstract]:background
Tissue engineering stem cells (SC) as a research focus in the field of biomedical research, provides a way to a new and very prospect of clinical application for repair and reconstruction of tissue and organ defects. Adult stem cells (ASC) as early undifferentiated cells have strong self-renewal and proliferation and differentiated into osteoblasts, cartilage cells. Nerve cells, fat cells, skeletal muscle cells, dermal cells, liver cells and other potential. Because it also has a wide range of sources, easy to amplify in vitro and individual treatment to avoid possible rejection by many advantages closely and conduct research on many levels. Studies have shown that bone marrow mesenchymal stem cells (BMSCs) proliferation and differentiation by various cytokines, signaling pathway, the combined effects of extracellular micro environment and other factors. At the same time, the study confirmed that a large number of microRNA (miRNA) are also involved in The regulation of various biological behavior of stem cells.
MiRNA is a small molecule containing an average of 22nt, mainly in the post transcriptional gene regulates the non protein encoding RNA. in recent years a large number of studies show that in many species there is an unusually large miRNA family in different species of the type, quantity and time expressions are different. In addition, signal transduction in individuals'growth,, tissue and organ differentiation, the occurrence and development process of disease in miRNA also play an important role in regulation, and determines the organism's physiological, pathological changes and behavior.
Our previous studies by high-throughput gene chip detection technology BMSCs and its differentiation into bone cells in miRNA gene expression profiles analysis screened a series of osteogenic differentiation and expression of miRNA, the experimental results from the selected osteogenic significantly increased expression of miRNA-30a-5p, to verify the in the biological effects of bone in the target gene of the biological function and the forecast.
objective
Objective to investigate and verify the biological function of recombinant miRNA-30a-5p transfection on human bone marrow mesenchymal stem cells to differentiate into osteoblasts, and to predict the possible target genes by bioinformatics.
Method
The artificial synthesis of recombinant human bone marrow MSCs from osteoblast specific expression of miRNA gene chip results in screening of osteogenic significantly increased expression of miRNA-30a-5p (3 samples the screening results see Appendix 2). BMSCs was isolated from human bone marrow, and continue to osteoblast induced in vitro after transfected with recombinant miRNA-30a-5p, 14,21 when the days were taken cell sheets by alizarin red S staining method to detect calcium deposition, alkaline phosphatase (ALP) calcium cobalt staining and ALP colorimetric method for quantitative detection of ALP activity in osteoblasts, a comparative study of biological function of recombinant miRNA-30a-5p in human bone marrow MSCs osteogenic differentiation process.
Result
Culture of human bone marrow MSCs and induce osteogenic differentiation. The separation successfully transfected to human BMSCs in vitro by miRNA-30a-5p and induce osteogenic differentiation, the transfection efficiency was 37.32% + 2.43%. were induced for fourteenth days, 21 days were observed by ALP staining, ALP activity, alizarin red calcium nodules staining cells were detected with osteogenic activity the results showed that miRNA-30a-5p mimics transfection group, MSCs osteogenic differentiation increased significantly (P0.05).
conclusion
(1) by Percoll density gradient centrifugation combined with whole bone marrow adherent culture method can obtain a sufficient number of isolated from fresh bone marrow, high purity of BMSCS, and can be cultured to promote its osteogenic differentiation in vitro. (2) miRNA-30a-5p in the MSCs process oriented differentiation has certain promotion enhanced expression of MSCs transfected into osteoblasts differentiation increased, in order to further clarify the orientation of human bone marrow MSCs into molecular and biochemical mechanisms of osteogenic differentiation, cell transplantation of bone defect repair treatment has laid a theoretical foundation.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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