miR-125b调控人骨髓间充质干细胞成骨机制的研究
发布时间：2019-05-17 00:54
【摘要】：研究背景和目的随着国内人口年龄结构逐渐步入老龄化,许多老龄化相关的疾病日渐引起医学界的广泛重视。骨质疏松作为一种老年人群高发的疾病,尤其重度骨质疏松具有较高的骨折风险,同时此类骨折发生后容易迁延不愈,是严重影响老年人健康和生活质量的一类骨科门急诊常见病。世界卫生组织已将骨质疏松、糖尿病与心血管病一起列为影响中老年人健康的“三大杀手”。此外,临床上因严重暴力所致的大段骨缺损、骨折后不愈合以及骨不连一直以来都是创伤骨科中的治疗难点。以上疾病的发生和发展均与机体内骨代谢失衡,成骨能力受损密切相关。因此,对成骨机制的研究和探索,将可能为这些临床问题的解决提供支持。人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,hBMSCs)作为成骨细胞重要的一种前体细胞,在骨的形成和成骨分化上都起着重要的作用。随着对间充质干细胞成骨机理的研究不断深入,将有助于我们更好地理解多种骨病的病理过程,为此类疾病的治疗寻找新的思路。此外,hBMSCs具有较强的增殖能力和多向分化潜能,是骨组织工程领域最重要的种子细胞来源,同时在干细胞移植领域也具有广泛的应用前景。microRNA(miRNAs)是一类广泛存在于真核生物细胞内的非编码单链小分子RNA,它能够与靶基因的3’非翻译区部分碱基互补结合,继而调控基因的表达,在生物体内众多基本的生理和病理过程中都扮演着重要的角色。近年来,越来越多的研究表明miRNAs在成骨细胞和破骨细胞的动态平衡中起着重要的调控作用,并且直接参与了诸多骨病如骨质疏松、关节炎等疾病的发生和发展过程。有研究发现,miR-125b在成骨过程中下调,但具体的调控机制尚不完全明了。本研究选定miR-125b作为研究对象,希望通过对miR-125b调控人骨髓间充质干细胞成骨分化的机制研究。进一步丰富干细胞成骨定向分化的理论体系,同时可加深对骨骼发生、发育的分子机制认识,为骨质疏松防治,骨折治疗,骨缺损修复寻找新的治疗思路并提供理论支持。研究方法1.人骨髓间充质干细胞的分离、培养和鉴定使用密度梯度离心的方法,从健康成年志愿者捐献的骨髓中分离获取hBMSCs,通过传代、培养、纯化,获取稳定的干细胞来源。培养过程中注意观察贴壁细胞形态变化,使用MTT法检测其增殖活力。经流式细胞术检测细胞表面抗原对细胞属性进行鉴定,通过成骨、成脂、成软骨诱导分化,对hBMSCs的多向分化能力即干性进行鉴定。2.miR-125b对hBMSCs成骨分化的影响用慢病毒转染的方式对细胞进行干预。首先将人工合成的miR-125-pre和miR-125b-inhibitor核酸序列构建到慢病毒载体并转染hBMSCs。对转染后的细胞转染效率和增殖能力进行观察检测。然后,采用Real Time RT-PCR技术检测转染后细胞内miR-125b表达量的变化。最后,使用Real Time RT-PCR和Western Blot对间充质干细胞成骨分化过程中重要的成骨因子包括ALP,Runx2,OSX以及OCN的表达情况进行核酸和蛋白水平上的检测,观察其表达变化情况。采用碱性磷酸酶染色和茜素红染色的方法对转染后的细胞进行染色观察,通过细胞显色情况鉴定其成骨分化能力。3.miR-125b调控hBMSCs的作用靶点预测及验证首先,选用生物信息学的方法,使用Target Scan,miRanda及Pic Tar作为靶基因预测工具,对可能与miR-125b结合的靶基因序列进行筛选预测,选择可能与其结合的潜在靶点BMPR1b作为研究对象。随后,构建BMPR1b的荧光素酶报告载体,使用双荧光素酶报告的方法对所预测的靶基因进行初步验证。最后,将hBMSCs分别转染miR-125b的上调和下调慢病毒载体,使用Real Time RT-PCR和Western Blot检测当细胞内miR-125b表达水平变化时,BMPR1b的mRNA和蛋白表达变化。4.miR-125b抑制hBMSCs成骨分化的机制首先使用Real Time RT-PCR和Western Blot检测hBMSCs成骨过程中BMPR1b的表达变化。随后,采用RNAi技术,将BMPR1b的siRNA载体转染hBMSCs,使用Real Time RT-PCR和Western Blot检测Runx2,OSX以及OCN的mRNA和蛋白表达变化。采用碱性磷酸酶染色和茜素红染色的方法对转染后的细胞进行染色观察,鉴定成骨分化能力。最后,将miR-125b-inhibitor与si-BMPR1b共转染hBMSCs,使用Real Time RT-PCR和Western Blot检测Runx2,OSX以及OCN的mRNA和蛋白表达变化。采用碱性磷酸酶染色和茜素红染色的方法对转染后的细胞进行染色观察,鉴定成骨分化能力。5.miR-125b调控hBMSCs成骨分化的体内研究首先,建立裸鼠股骨原位骨缺损模型。随后,将miR-125b-inhibitor转染hBMSCs,将细胞复合至DBM后进行成骨诱导,再通过手术的方式将DBM移植至裸鼠体内,利用micro CT观测新骨生成状况,采用组织切片,HE染色和Masson染色,从细胞的微观角度评估成骨能力。研究结果1.本实验所采用的密度梯度离心法分离获取hBMSCs,培养过程中细胞各项指标表明hBMSCs细胞形态正常,具有较强的增殖活力。hBMSCs的阳性表面抗原CD73,CD90,和CD105阳性率均在95%以上,而阴性抗原CD34,CD45,CD14,CD19,HLA-DR阳性率在5%以下。符合hBMSCs鉴定标准。此外,分离培养的hBMSCs具备成骨、成软骨和成脂的多向分化能力,具备良好的干性。2.在hBMSCs向成骨分化的过程中,miR-125b处于低水平表达的状态。慢病毒载体对hBMSCs转染效果较好,且对细胞的增殖不产生影响。在hBMSCs成骨分化过程中,上调miR-125b表达能够抑制成骨。相反地,下调miR-125b则能够促进hBMSCs的成骨分化。3.生物信息学预测出BMPR1b基因可能是miR-125的作用靶点。双荧光素酶报告实验证实miR-125b在细胞内能与BMPR1b的3’UTR部分碱基序列存在结合位点。在hBMSCs中,上调miR-125b能够抑制BMPR1b的mRNA和蛋白表达。相反,下调miR-125b则能够增加BMPR1b的mRNA和蛋白表达量。4.hBMSCs成骨分化过程中,BMPR1b呈高表达状态。沉默BMPR1b的基因后,hBMSCs的成骨能力下降。miR-125b-inhibitor与si-BMPR1b共转染hBMSCs后,hBMSCs的成骨分化能力同样受到抑制。5.对miR-125b调节成骨分化的体内研究发现,转染miR-125b-inhibitor后的hBMSCs,micro CT和组织切片染色其体内成骨能力强于阴性对照组。结论1.本实验所分离培养的hBMSCs增殖活跃,符合间充质干细胞的一般形态学特点。干细胞特性明显,经不同条件诱导,能向成骨、成脂以及成软骨细胞分化。纯度较高,干细胞来源稳定可靠。2.miR-125b能够负向调控hBMSCs成骨分化。3.miR-125b通过与靶基因BMPR1b的3’UTR部分碱基互补结合,从而抑制BMPR1b的表达。4.miR-125b通过抑制其靶基因BMPR1b的表达,进而抑制hBMSCs的成骨分化。5.复合hBMSCs的DBM骨移植材料,能够在体内较好地修复骨缺损。下调miR-125b的表达后,能够进一步提高h BMSC在体内的成骨能力。
[Abstract]:The background and purpose of the study, as the age structure of the population of the country has gradually entered the age of aging, many of the aging-related diseases are increasing the attention of the medical community. Osteoporosis is a high-incidence disease of the old people, especially the severe osteoporosis has a higher risk of fracture, and at the same time, it is easy to move after the fracture, which is a kind of common diseases in the department of orthopedics that seriously affects the health and quality of life of the old people. The World Health Organization has included osteoporosis, diabetes and cardiovascular disease as a "the three killers" that affects the health of the middle-aged and the elderly. In addition, the clinical experience of the large-segment bone defect due to the severe violence, the non-union after the fracture, and the non-union of the bone all the time are the treatment difficulties in the trauma orthopedics. The occurrence and development of the above diseases are closely related to the imbalance of bone metabolism and the damage of osteogenesis in the body. Therefore, the research and exploration of the osteogenic mechanism will provide support for the solution of these clinical problems. Human bone marrow mesenchymal stem cells (hBMSCs) play an important role in the formation of bone and the differentiation of osteogenesis. With the development of the bone formation mechanism of the mesenchymal stem cells, it will help us to better understand the pathological process of various bone diseases, and find a new way for the treatment of such diseases. In addition, hBMSCs have a strong proliferation ability and multi-directional differentiation potential, which are the most important seed cell sources in the field of bone tissue engineering, and have a wide application prospect in the field of stem cell transplantation. The microRNA (miRNAs) is a kind of non-coding single-chain small-molecule RNA which is widely present in eukaryotes, which can be combined with the base of the 3 'non-translation region of the target gene, and then regulate the expression of the gene, and plays an important role in many basic physiological and pathological processes in the organism. In recent years, more and more studies have shown that miRNAs play an important role in the dynamic balance of osteoblasts and osteoclasts, and are directly involved in the occurrence and development of many bone diseases such as osteoporosis and arthritis. It has been found that miR-125b is down-regulated in the process of osteogenesis, but the specific regulatory mechanism is not entirely clear. In this study, miR-125b was selected as the subject of study, and it was hoped to study the mechanism of human bone marrow-derived mesenchymal stem cells in human bone marrow by the mechanism of miR-125b. The theory system of stem cell osteogenesis and orientation differentiation is further enriched, and the molecular mechanism of bone formation and development can be enhanced, and a new treatment idea and theoretical support are provided for the prevention and treatment of osteoporosis, fracture treatment and bone defect repair. Study Method 1. The method for separating, culturing and identifying human bone marrow mesenchymal stem cells using density gradient centrifugation is to separate and obtain hBMSCs from the bone marrow donated by healthy adult volunteers, and to obtain stable stem cell sources by passage, culture and purification. In the process of culture, the morphological changes of the adherent cells were observed, and the proliferative activity of the adherent cells was detected by the MTT method. The cell surface antigen was identified by flow cytometry. The differentiation of hBMSCs was induced by osteogenic, adipogenic and chondrogenic differentiation, and the multi-directional differentiation ability of hBMSCs was identified. The synthetic miR-125-pre and miR-125b-inhitor nucleic acid sequences are first constructed to a lentiviral vector and transfected with hBMSCs. And the transfection efficiency and the proliferation ability of the transfected cells are observed and detected. Then, the expression of miR-125b in the transfected cells was detected by Real Time RT-PCR. Finally, the expression of ALP, Runx2, OSX and OCN was detected by using Real Time RT-PCR and Western Blot to detect the expression of the nucleic acid and protein. The method of using alkaline phosphatase staining and fluorescein red staining to dye and observe the transfected cells, and the bone differentiation capacity of the transfected cells is identified by the color development of the cells.3. The method for predicting and verifying the action target of the hBMSCs by using the miR-125b is first, the method of bioinformatics is selected, the target Scan is used, MiRANDa and Pic Tar are used as target gene prediction tools to screen and predict target gene sequences that may be combined with miR-125b to select potential targets BMPR1b that may be combined with them as the study target. Subsequently, the luciferase reporter vector of BMPR1b was constructed, and the predicted target gene was initially validated using a double luciferase reporter method. Finally, hBMSCs were transfected with the up-regulated and down-regulated lentiviral vector of miR-125b, respectively, and the expression level of miR-125b in the cell was detected by Real Time RT-PCR and Western Blot. 4. The expression of BMPR1b was detected by using Real Time RT-PCR and Western Blot, and the expression of BMPR1b was detected by using Real Time RT-PCR and Western Blot. Subsequently, the siRNA vector of BMPR1b was transfected into hBMSCs by using the RNAi technology, and the mRNA and protein expression of the Runx2, OSX and OCN were detected by Real Time RT-PCR and Western Blot. The cells of the transfected cells were stained with alkaline phosphatase and fluorescein red staining, and the osteogenic differentiation ability was identified. Finally, hBMSCs were cotransfected with miR-125b-inhitor and si-BMPR1b, and the mRNA and protein expression of Runx2, OSX and OCN were detected by Real Time RT-PCR and Western Blot. 5. The in vivo study of the osteogenic differentiation of hBMSCs by miR-125b was first established, and the in-situ bone defect model of the femur in nude mice was established. Subsequently, the hBMSCs were transfected with miR-125b-inhitor, the cells were combined to DBM, and the bone formation was induced, and the DBM was transplanted into the nude mice by operation. The new bone formation was observed by microCT, and the osteogenic ability was assessed from the micro-angle of the cells by using tissue sections, HE staining and Masson staining. Study Results 1. In this experiment, hBMSCs were obtained by density gradient centrifugation, and the indexes of the cells in the culture process showed that the morphology of hBMSCs was normal and the proliferation of hBMSCs was strong. The positive rate of CD73, CD90 and CD105 of hBMSCs was more than 95%, while the positive rate of negative antigen CD34, CD45, CD14, CD19 and HLA-DR was below 5%. The criteria for the identification of hBMSCs were met. In addition, the cultured hBMSCs have the ability of osteogenesis, chondrogenesis and adipocyte differentiation, and have a good dry.2. MiR-125b is at a low level of expression during the differentiation of hBMSCs into osteogenesis. The effect of lentiviral vector on hBMSCs was better, and the effect of lentiviral vector on the proliferation of cells was not affected. The up-regulation of the expression of miR-125b in the osteogenesis of hBMSCs can inhibit the formation of bone. In contrast, down-regulation of miR-125b can promote the osteogenic differentiation of hBMSCs. Bioinformatics predicts that the BMPR1b gene may be the target of miR-125. The double luciferase reporter assay demonstrated that miR-125b can bind to the 3 'UTR partial base sequence of BMPR1b in the cell. In hBMSCs, up-regulation of miR-125b can inhibit the mRNA and protein expression of BMPR1b. In contrast, down-regulation of miR-125b can increase the mRNA and protein expression of BMPR1b. After the gene of BMPR1b was silent, the osteogenic ability of hBMSCs decreased. The osteogenic differentiation of hBMSCs was also inhibited by the co-transfection of hBMSCs with miR-125b-inhitor and si-BMPR1b. In vivo study of the regulation of the osteogenic differentiation of miR-125b, the ability of hBMSCs, micro-CT and tissue sections transfected with miR-125b-inhitor was stronger than that of the negative control group. Conclusion 1. The proliferation of hBMSCs isolated and cultured in this experiment is in accordance with the general morphological characteristics of the mesenchymal stem cells. The characteristics of stem cells are distinct and can be induced by different conditions, and can be differentiated into osteogenesis, adipogenesis and chondrocyte differentiation. 3. miR-125b can inhibit the expression of BMPR1b by combining with the base of the 3 'UTR part of the target gene BMPR1b, thereby inhibiting the expression of the BMPR1b. The DBM bone graft material of the composite hBMSCs can be used for repairing the bone defect in vivo. After the expression of miR-125b is down-regulated, the osteogenic ability of the h BMSC in the body can be further improved.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R580
本文编号：2478679
[Abstract]:The background and purpose of the study, as the age structure of the population of the country has gradually entered the age of aging, many of the aging-related diseases are increasing the attention of the medical community. Osteoporosis is a high-incidence disease of the old people, especially the severe osteoporosis has a higher risk of fracture, and at the same time, it is easy to move after the fracture, which is a kind of common diseases in the department of orthopedics that seriously affects the health and quality of life of the old people. The World Health Organization has included osteoporosis, diabetes and cardiovascular disease as a "the three killers" that affects the health of the middle-aged and the elderly. In addition, the clinical experience of the large-segment bone defect due to the severe violence, the non-union after the fracture, and the non-union of the bone all the time are the treatment difficulties in the trauma orthopedics. The occurrence and development of the above diseases are closely related to the imbalance of bone metabolism and the damage of osteogenesis in the body. Therefore, the research and exploration of the osteogenic mechanism will provide support for the solution of these clinical problems. Human bone marrow mesenchymal stem cells (hBMSCs) play an important role in the formation of bone and the differentiation of osteogenesis. With the development of the bone formation mechanism of the mesenchymal stem cells, it will help us to better understand the pathological process of various bone diseases, and find a new way for the treatment of such diseases. In addition, hBMSCs have a strong proliferation ability and multi-directional differentiation potential, which are the most important seed cell sources in the field of bone tissue engineering, and have a wide application prospect in the field of stem cell transplantation. The microRNA (miRNAs) is a kind of non-coding single-chain small-molecule RNA which is widely present in eukaryotes, which can be combined with the base of the 3 'non-translation region of the target gene, and then regulate the expression of the gene, and plays an important role in many basic physiological and pathological processes in the organism. In recent years, more and more studies have shown that miRNAs play an important role in the dynamic balance of osteoblasts and osteoclasts, and are directly involved in the occurrence and development of many bone diseases such as osteoporosis and arthritis. It has been found that miR-125b is down-regulated in the process of osteogenesis, but the specific regulatory mechanism is not entirely clear. In this study, miR-125b was selected as the subject of study, and it was hoped to study the mechanism of human bone marrow-derived mesenchymal stem cells in human bone marrow by the mechanism of miR-125b. The theory system of stem cell osteogenesis and orientation differentiation is further enriched, and the molecular mechanism of bone formation and development can be enhanced, and a new treatment idea and theoretical support are provided for the prevention and treatment of osteoporosis, fracture treatment and bone defect repair. Study Method 1. The method for separating, culturing and identifying human bone marrow mesenchymal stem cells using density gradient centrifugation is to separate and obtain hBMSCs from the bone marrow donated by healthy adult volunteers, and to obtain stable stem cell sources by passage, culture and purification. In the process of culture, the morphological changes of the adherent cells were observed, and the proliferative activity of the adherent cells was detected by the MTT method. The cell surface antigen was identified by flow cytometry. The differentiation of hBMSCs was induced by osteogenic, adipogenic and chondrogenic differentiation, and the multi-directional differentiation ability of hBMSCs was identified. The synthetic miR-125-pre and miR-125b-inhitor nucleic acid sequences are first constructed to a lentiviral vector and transfected with hBMSCs. And the transfection efficiency and the proliferation ability of the transfected cells are observed and detected. Then, the expression of miR-125b in the transfected cells was detected by Real Time RT-PCR. Finally, the expression of ALP, Runx2, OSX and OCN was detected by using Real Time RT-PCR and Western Blot to detect the expression of the nucleic acid and protein. The method of using alkaline phosphatase staining and fluorescein red staining to dye and observe the transfected cells, and the bone differentiation capacity of the transfected cells is identified by the color development of the cells.3. The method for predicting and verifying the action target of the hBMSCs by using the miR-125b is first, the method of bioinformatics is selected, the target Scan is used, MiRANDa and Pic Tar are used as target gene prediction tools to screen and predict target gene sequences that may be combined with miR-125b to select potential targets BMPR1b that may be combined with them as the study target. Subsequently, the luciferase reporter vector of BMPR1b was constructed, and the predicted target gene was initially validated using a double luciferase reporter method. Finally, hBMSCs were transfected with the up-regulated and down-regulated lentiviral vector of miR-125b, respectively, and the expression level of miR-125b in the cell was detected by Real Time RT-PCR and Western Blot. 4. The expression of BMPR1b was detected by using Real Time RT-PCR and Western Blot, and the expression of BMPR1b was detected by using Real Time RT-PCR and Western Blot. Subsequently, the siRNA vector of BMPR1b was transfected into hBMSCs by using the RNAi technology, and the mRNA and protein expression of the Runx2, OSX and OCN were detected by Real Time RT-PCR and Western Blot. The cells of the transfected cells were stained with alkaline phosphatase and fluorescein red staining, and the osteogenic differentiation ability was identified. Finally, hBMSCs were cotransfected with miR-125b-inhitor and si-BMPR1b, and the mRNA and protein expression of Runx2, OSX and OCN were detected by Real Time RT-PCR and Western Blot. 5. The in vivo study of the osteogenic differentiation of hBMSCs by miR-125b was first established, and the in-situ bone defect model of the femur in nude mice was established. Subsequently, the hBMSCs were transfected with miR-125b-inhitor, the cells were combined to DBM, and the bone formation was induced, and the DBM was transplanted into the nude mice by operation. The new bone formation was observed by microCT, and the osteogenic ability was assessed from the micro-angle of the cells by using tissue sections, HE staining and Masson staining. Study Results 1. In this experiment, hBMSCs were obtained by density gradient centrifugation, and the indexes of the cells in the culture process showed that the morphology of hBMSCs was normal and the proliferation of hBMSCs was strong. The positive rate of CD73, CD90 and CD105 of hBMSCs was more than 95%, while the positive rate of negative antigen CD34, CD45, CD14, CD19 and HLA-DR was below 5%. The criteria for the identification of hBMSCs were met. In addition, the cultured hBMSCs have the ability of osteogenesis, chondrogenesis and adipocyte differentiation, and have a good dry.2. MiR-125b is at a low level of expression during the differentiation of hBMSCs into osteogenesis. The effect of lentiviral vector on hBMSCs was better, and the effect of lentiviral vector on the proliferation of cells was not affected. The up-regulation of the expression of miR-125b in the osteogenesis of hBMSCs can inhibit the formation of bone. In contrast, down-regulation of miR-125b can promote the osteogenic differentiation of hBMSCs. Bioinformatics predicts that the BMPR1b gene may be the target of miR-125. The double luciferase reporter assay demonstrated that miR-125b can bind to the 3 'UTR partial base sequence of BMPR1b in the cell. In hBMSCs, up-regulation of miR-125b can inhibit the mRNA and protein expression of BMPR1b. In contrast, down-regulation of miR-125b can increase the mRNA and protein expression of BMPR1b. After the gene of BMPR1b was silent, the osteogenic ability of hBMSCs decreased. The osteogenic differentiation of hBMSCs was also inhibited by the co-transfection of hBMSCs with miR-125b-inhitor and si-BMPR1b. In vivo study of the regulation of the osteogenic differentiation of miR-125b, the ability of hBMSCs, micro-CT and tissue sections transfected with miR-125b-inhitor was stronger than that of the negative control group. Conclusion 1. The proliferation of hBMSCs isolated and cultured in this experiment is in accordance with the general morphological characteristics of the mesenchymal stem cells. The characteristics of stem cells are distinct and can be induced by different conditions, and can be differentiated into osteogenesis, adipogenesis and chondrocyte differentiation. 3. miR-125b can inhibit the expression of BMPR1b by combining with the base of the 3 'UTR part of the target gene BMPR1b, thereby inhibiting the expression of the BMPR1b. The DBM bone graft material of the composite hBMSCs can be used for repairing the bone defect in vivo. After the expression of miR-125b is down-regulated, the osteogenic ability of the h BMSC in the body can be further improved.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R580
【参考文献】
相关期刊论文 前5条
1 程燕;陈琳;曹忻;哈斯其美格;谢小冬;;Hsa-miR-125b在人胃癌耐药细胞株中的表达及其靶基因的功能分析[J];遗传;2014年02期
2 周名亮;李谧;余希杰;;微小RNA对成骨细胞分化成熟的调控及临床意义[J];中国修复重建外科杂志;2012年06期
3 高杰;杨彤涛;裘秀春;范清宇;马保安;;microRNA和干细胞的研究进展[J];中国修复重建外科杂志;2007年09期
4 黄晓兵;刘霆;孟文彤;智伟;;骨髓间充质干细胞分化的成骨细胞支持脐血造血干祖细胞的研究[J];中国实验血液学杂志;2006年03期
5 王军,赵志河,罗颂椒,樊瑜波;骨髓间充质干细胞骨向诱导分化过程中破骨细胞分化因子和细胞间粘附分子-1的表达变化[J];华西口腔医学杂志;2005年03期
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