论文总字数：49446字

摘 要

Abstract： 2

第一章 绪论 3

1.1 前言 3

1.2成骨细胞 3

1.3催产素 4

1.4 microRNA及其在成骨细胞分化过程中的作用 5

1.4.1miRNA调节成骨细胞分化过程 5

1.4.2miRNA靶基因的预测与鉴定 10

1.5本课题的意义 11

第二章 实验材料与实验方法 12

2.1实验材料 12

2.1.1实验用动物/细胞 12

2.1.2实验试剂 12

2.1.3实验器材 13

2.1.4主要试剂配制 14

2.2实验方法 15

2.2.1成骨细胞分化相关miRNA的筛选 15

2.2.2小鼠骨髓细胞的提取 15

2.2.3间充质干细胞的纯化和扩增 16

2.2.4骨髓细胞计数 16

2.2.5成骨细胞体外诱导分化 16

2.2.6成骨细胞早期分化状况鉴定（ALP染色） 17

2.2.7成熟成骨细胞矿化鉴定（Von Kossa染色） 17

2.2.8总RNA提取 18

2.2.9 Real Time-PCR检测 19

2.2.10 miRNA表达谱分析 19

2.2.11 miRNA靶基因预测 19

第三章 实验结果 20

3.1催产素调节成骨细胞分化相关miRNA的筛选 20

3.1.1成骨细胞分化相关miRNA文献调研 20

3.1.2成骨细胞早期分化相关miRNA的筛选 20

3.1.3成骨细胞晚期分化相关miRNA的筛选 23

3.1.4催产素调节成骨细胞分化相关miRNA的筛选 27

3.2原代骨髓细胞的成骨诱导分化验证 30

3.2.1成骨细胞分化最适接种数 30

3.2.2成骨细胞早晚期分化情况鉴定 30

3.2.3催产素调节成骨细胞分化情况鉴定 30

3.3实时定量PCR检测催产素调节成骨细胞早期分化相关miRNA的表达 31

3.3.1纯度浓度结果 31

3.3.2 Real-Time PCR验证miRNA结果 32

3.4 催产素调节成骨细胞早期分化相关miRNA靶基因预测 35

结论 36

参考文献 38

致谢 43

催产素调节成骨细胞分化相关miRNA的筛选及靶基因预测

41111107 陈奕含 指导教师：刘璇

摘要

骨的功能是为肌肉收缩提供附着处及保护内脏等重要生命器官，骨代谢的过程便是骨骼的自我更新及修复过程，而骨代谢的稳定是由成骨细胞的骨形成过程和破骨细胞的骨吸收过程来维持的。当骨代谢异常，骨的重建平衡被破坏，使破骨作用大于成骨作用时，将会发生骨质疏松。催产素（oxytocin，OT）是一种哺乳动物神经垂体后叶激素，通过神经轴突在下丘脑的视上核和室旁核产生，并储存在垂体后叶，目前有研究表明，催产素与骨代谢相关。 microRNA（miRNA）是近年来发现的一类长度在19-22nt，高度保守的，通过降解mRNA或抑制其靶基因表达来调节生理过程的单链非编码小RNA，几乎存在于所有的真核生物及部分病毒中，被认为是遗传调控的重要组成部分，在细胞周期、生物体发育等方面起着相当重要的作用。在本课题中，我们在先期证明催产素对骨代谢有直接调节作用的基础上，进一步从miRNA角度探讨催产素对骨代谢的调节机制。在对成骨细胞相关miRNA进行大量的文献调研的同时，利用Ion Torrent测序系统进行miRNA表达谱检测，对比分析数据，筛选得出了催产素调节成骨细胞分化相关的miRNA。进行实时定量PCR验证得出miR-146a-5p、miR-31-3p、miR-199b-5p、miR-183-5p为催产素调节成骨细胞分化关键miRNA，并利用miRanda等靶基因预测软件预测其靶基因。研究参与催产素调节骨代谢的相关miRNA及其作用机制，对于进一步阐明催产素调节骨代谢的分子机理，完善对miRNA功能的认识和开发治疗骨质疏松症新的治疗策略具有重要的意义。

关键词：miRNA，成骨细胞，催产素，靶基因

Screening potential microRNAs involved in osteoblast differentiation regulated by oxytocin and predict target genes

41111107 Yihan Chen Tutor：Xuan Liu

Abstract：

Bone function is to provide attachment of muscle contraction and the protection of internal organs, bone metabolism is the process of self-renewal and repair, the stability of bone metabolism is maintained by the process of bone formation of osteoblast and bone resorption of osteoclast. When bone metabolism abnormal, bone reconstruction is destroyed, which makes the osteoclasts role more than osteoblast, osteoporosis happens. At present, the incidence rate of osteoporosis has been the seventh common disease in the world. Osteoporosis seriously threatened the health of the elderly. Oxytocin (OT) is a mammalian neurohypophysial hormone, produced in the supraoptic and paraventricular nuclei of the hypothalamus by nerve axons, and stored in the posterior pituitary gland. Studies have indicated that oxytocin is associated with bone metabolism. MicroRNA (miRNA) is a class of length in 19-22nt, highly conserved non-coding single-stranded RNA that down-regulate expression of their target genes by either mRNA degradation or translational inhibition. It exists in almost all eukaryotic organisms and some viruses, it is considered to be an important pair of genetic regulation, plays a significative role in the cell cycle, and the development of organism. In the present study, on the basis of oxytocin has a direct regulatory effect on bone metabolism in the preliminary proof, we further investigate on the mechanism of oxytocin on bone metabolism in miRNA. As well as research osteoblast associated miRNA’s literature, we use Ion Torrent sequencing system to detect miRNA expression, analysis data, screening potential microRNAs involved in osteoblast differentiation regulated by oxytocin. Real time PCR was performed to verify miRNAs that are associated with osteoblast differentiation, such as miR-146a-5p, miR-199b-5p, miR-31-3p, and miR-183-5p. Our study also predictive their target genes by software which can prediction target genes like miRanda. To study the microRNAs involved in osteoblast differentiation regulated by oxytocin and its mechanism, would help us to understand the molecular mechanism of oxytocin regulation of bone metabolism, improve the understanding and development of miRNA function and develop promising strategies to treat osteoporosis.

KEY WORDS: microRNA, osteoblast, oxytocin, target gene

绪论

1.1 前言

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