论文总字数：32636字

摘 要

骨骼的形态发生和重建这个动态的生理过程包括成骨细胞形成新骨的骨形成作用，即合成骨基质并矿化；以及破骨细胞吸收旧骨的骨吸收作用，这两种作用间有着微妙的动态平衡并以此来维持骨的生理作用。

近年来不少研究发现，miRNAs作为重要的的转录后调节因素，广泛参与成骨细胞的整个分化过程。我们在试验中也发现，分化程度不同的成骨细胞，其部分miRNA表达水平也具有明显的差异性。

催产素作为一种多功能细胞因子，能够诱导髓间充质干细胞向成骨细胞分化，并促进成骨细胞的骨形成能力。本实验小组筛选出在催产素诱导成骨细胞分化过程中特异性变化的miRNAS: miR-181a-5p、miR-193b-3p、miR-205-5p、miR-22-3p、miR-29a-3p、miR-669b-3p,并以miR-22-3p为例，希望通过研究尝试阐述miR-22-3p在成骨细胞分化过程中的作用机制。

通过使用TargetScan、NCBI等预测软件及文献调研，我对miR-22-3p进行了靶基因预测。最终决定以HDAC6作为miR-22-3p的靶基因进行研究。借助构建双荧光素酶报告载体的实验方法，成功验证了HDAC6是miR-22-3p的靶基因。同时，通过转染miR-22-3p来调控体外培养的MSCs的异位miRNA表达水平，发现在成骨分化早期以5nm为转染终浓度对MSCs进行miR-22-3p转染，导致成骨细胞增殖受阻，成骨分化能力下降；而将miR-22-3p（终浓度1nm）转染分化较为成熟的成骨细胞，使其成骨分化能力受到了促进。根据后续的RNA检测报告与大量调研后，我推测miR-22通过抑制其靶基因HDAC6翻译，来达到促进成骨细胞分化的作用；同时，miR-22与β-连环蛋白的编码基因相互作用，抑制MSCs增殖。

关键词：成骨分化；miR-22-3p；作用机制；催产素

Mechanism of action on osteoblast differentiation and function regulated by miR-22

41113117 Liang Zhou Tutor：Xuan Liu
abstract

Bone morphogenesis and reconstruction of this dynamic physiological processes include osteoblast formation of new bone formation, that is, synthetic bone matrix and mineralization; and osteoclasts to absorb the old bone bone resorption, these two effects have a delicate dynamic balance and in order to maintain the physiological role of bone.

In recent years, many studies have found that miRNAs as an important post-transcriptional regulator, widely involved in the entire differentiation process of osteoblasts. We also found in the experiment, the degree of differentiation of different osteoblasts, some of its miRNA expression levels are also significantly different.

Oxytocin, as a multifunctional cytokine, can induce differentiation of mesenchymal stem cells into osteoblasts and promote bone formation in osteoblasts. The experimental group screened in the oxytocin induced osteoblast differentiation process specific changes in miRNAS: miR-181a-5p、miR-193b-3p、miR-205-5p、miR-22-3p、miR-29a-3p、miR-669b-3p. And miR-22-3p as an example, we hope to study the mechanism of miR-22-3p in osteoblast differentiation process.

Through the use of TargetScan, NCBI and other predictive software and literature research, members of this group of miR-22-3p were target gene prediction. The final decision to HDAC6 as miR-22-3p target gene were studied. The target gene of miR-22-3p was successfully confirmed by the experimental method of constructing double luciferase reporter vector. At the same time, miR-22-3p was transfected to control the expression of ectopic miRNAs in MSCs cultured in vitro, It was found that miR-22-3p was transfected into MSCs at the initial concentration of 5nm for osteoblast differentiation, leading to the proliferation of osteoblasts and the decrease of osteogenic differentiation. Simultaneously miR-22-3p (final concentration of 1nm) transfected into more mature osteoblasts, so that osteogenic differentiation ability has been promoted. According to the subsequent RNA test report and a large number of studies, I speculated that miR-22 by inhibiting the target gene HDAC6 translation, to promote the promotion of osteoblast differentiation effect; the same, miR-22 and β-catenin encoding gene interaction , inhibit the proliferation of MSCs.

KEY WORDS: Osteogenic differentiation，miR-22，Mechanism，Oxytocin

目 录

第一章 绪论 6

1.1骨形成过程成骨细胞 6

1.2催产素 7

1.3miRNA 7

1.4miRNAs参与调控成骨分化 7

1.5本课题的研究意义 8

第二章 实验材料与实验方法 9

2.1实验材料 9

2.1.1实验试剂 9

2.1.2实验器具 10

2.1.3实验所用细胞及动物 11

2.2实验方法 12

2.2.1间充质干细胞的提取、分离与体外成骨诱导培养 12

2.2.2成骨分化能力鉴定 12

2.2.3RNA提取 13

2.2.4RT-PCR检测miRNA表达量 14

2.2.5miR-22转染效率验证 14

2.2.6miRNAs预转染 15

2.2.7靶基因预测 15

2.2.8靶基因验证 16

2.2.9转染miR-22调节MSCs内miR-22表达水平 19

2.2.10标志性RNA RT-PCR 20

第三章 实验结果 22

3.1成骨细胞的体外培养以及成骨能力鉴定 22

3.1.1原代MSCs的成骨诱导培养 22

3.1.2碱性磷酸酶染色 22

3.1.3Von Kossa染色 23

3.2成骨细胞差异性分化时miR-22的表达量变化 23

3.2.1miR-22 RT-PCR 23

3.2.2转染效率验证 24

3.2.3miRNA预转染 25

3.2.4讨论 26

3.3 miR-22与HDAC6的靶向关系 27

3.3.1靶基因预测 27

3.3.2靶基因验证 27

3.4 miRNAs调节hMSCs成骨分化能力 29

3.4.1分化早期成骨细胞miR-22转染 29

3.4.2分化晚期成骨细胞miR-22转染 30

3.4.3讨论 32

结 论 34

参考文献： 37

致 谢 40

第一章 绪论

1.1骨形成过程成骨细胞

成骨细胞起源于间充质干细胞，由其行使的骨形成功能在骨代谢中至关重要，其功能的抑制甚至缺失是形成骨质疏松症的原因之一^[1]。骨骼在动态平衡中不断改造，成骨细胞产生新骨组织，而破骨细胞破坏和吸收骨组织^[2]。作为成骨的主要成分，成骨细胞对于维持骨骼结构和调节骨微环境的稳态至关重要。众所周知，成骨细胞产生各种细胞外基质蛋白，包括OCN，ALP，OPN和I型胶原蛋白^[3]。这些细胞外基质蛋白是维持骨稳态的基础，骨基质沉积的破坏将最终导致许多骨疾病如骨质疏松和成骨不完全^[4]。因此，对成骨细胞分化的分子调控网络的理解对于开发用于治疗骨疾病的治疗工具是至关重要的。

作为成骨细胞的重要起源，髓间充质干细胞（hMSCs）是具有多能性的基质细胞，其可以完全不同于各种细胞类型，例如成骨细胞，软骨细胞和脂肪细胞^[5]。在成骨细胞分化过程中，已经发现许多细胞因子和生长因子在调节成骨细胞复制和细胞分化中起重要作用。例如，几种BMP，特别是BMP2可以通过提供细胞外信号并随后触发矿物质沉积来启动成骨^[6]。除BMPs外，TGF-β1在调节骨量方面也发挥重要作用，发现TGF-β1缺陷小鼠表现出骨生长减少和矿化能力受损^[7]。

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