PPCN-G与TAZ调控BMP9诱导的间充质干细胞成骨过程的研究

发布时间：2018-03-13 14:21

本文选题：BMP9　切入点：间充质干细胞　出处：《重庆大学》2016年博士论文　论文类型：学位论文

【摘要】：脊椎动物拥有的内骨骼是由矿化的细胞外基质组成的。骨骼是一种高度特殊性的连接组织,对于内部器官具有保护性,能够容纳骨髓,作为钙和磷酸盐的保存组织,还是能量代谢的重要调节因子。成骨成软骨祖细胞,成骨细胞和骨细胞是骨骼生成的三种基本细胞类型。正常的骨形成需要成骨细胞的分化,而成骨细胞又是由间充质干细胞(MSCs)分化而来。近年来,随着人们对于骨骼生长发育、骨骼疾病认识的加深以及科技的进步,形成了一门新的学科:骨组织工程学。骨组织工程是指将分离的自体高浓度成骨细胞、骨髓间充质干细胞或软骨细胞,经体外培养扩增后种植于一种天然或人工合成的、具有良好生物相容性、可被人体逐步降解吸收的细胞支架上,并加入一些诱导成骨的细胞因子。在这种生物材料支架上细胞可以按照支架的三维形态进行生长,并且进行营养物质与气体交换。然后将这种细胞、因子、材料的混合物植入骨缺损部位,在生物材料逐步降解的同时,种植的细胞不断增殖与成骨分化从而达到修复骨组织缺损的目的。骨组织工程有三要素:足够的成骨细胞,有效的诱导成骨因子,生物相容性良好的支架材料。我们之前已经证明了BMP9是一个最有效的促进间充质干细胞(MSCs)成骨分化的因子。而且我们之前也成功永生化获得了一种间充质干细胞iMEF,所以我们想找到一种材料,用BMP9诱导间充质干细胞后种植在这种材料上能够让细胞三维形态上生长成骨。这样的实验为生物材料在临床诱导成骨提供了理论依据。我们首先用一种生物可降解的温敏性大分子聚合物(PPCN)混合明胶(PPCN-G)作为一种支架材料来承载BMP9诱导的间充质干细胞,测试其是否有促进骨形成的能力。这种材料PPCN在37℃会变为固体,有利于材料在人体内的固定,在21℃变为液体有利于材料与细胞还有因子的混合。实验结果证明,在3D培养中明胶的加入能够促进间充质干细胞的粘附与生存。在小鼠背部的异位骨形成实验证明PPCN-G能够促进BMP9诱导的体内成骨分化,并且能够促骨小梁的生成以获得更好的骨样结构。将背部的包块取下做组织学分析发现PPCN-G能够促进血管的生成。相应的免疫组化结果证明PPCN-G能够促进血管内皮细胞生长因子(VEGF)的表达。所以我们的实验证明了PPCN-G是一种新型的可注射可降解的载体,并且可以和细胞与因子一起用于骨组织工程。最近的研究表明BMP9诱导的骨形成和其他的BMP蛋白相比具有不同的分子机制。一些与生长发育有关的信号通路在BMP9诱导骨形成的过程中发挥了关键作用,他们与BMP9协同作用,共同完成骨骼发育和维持的任务。尽管许多在骨骼形成中的调节因子已经被功能性地描述,比如说TAZ就具有明确的促进成骨分化抑制成脂分化的作用,但是TAZ对于BMP9诱导的成骨过程之间有什么关系,在成骨过程中它们是如何相互作用的还没有研究清楚。这样的研究对于骨组织工程中通过加入因子对成骨过程精确控制有指导意义。首先我们构建了TAZ的siRNA沉默腺病毒并使用已经得到的过表达腺病毒,然后将沉默或过表达的腺病毒与Ad-BMP9一起感染iMEF细胞,在体外进行碱性磷酸酶活性检测,测试TAZ是否有促进成骨分化的能力。其次我们将TAZ过表达和沉默的腺病毒与Ad-BMP9一起感染iMEF细胞,然后与PPCN-G混合后注射到裸鼠皮下,与Ad-GRFP与Ad-BMP9一起感染的iMEF细胞的对照组进行对照,在体内测试TAZ是否有促进成骨分化的能力。体内外的实验结果证明过表达TAZ和沉默TAZ都有促进BMP9诱导的成骨分化的能力,并且沉默TAZ之后的促进作用特别明显,于是我们又将TAZ过表达和沉默的腺病毒与Ad-BMP9一起感染iMEF细胞,并且进行体内和体外的细胞增殖检测。证明了沉默TAZ能够显著促进iMEF细胞的增殖,可能是通过增加细胞间的相互接触进而促进了iMEF细胞的成骨分化。我们用Ad-BMP9感染了iMEF和iMEF-OKmTAZ细胞后,在不同的时间点将细胞收集起来提取RNA,并反转录得到cDNA,将cDNA样本进行各个谱系标志物以及TAZ下游转录因子TEAD的qPCR检测。实验结果证明沉默TAZ的促成骨作用主要还是通过增强Runx2的表达来完成的,并且沉默TAZ对TEAD也起到了一定的抑制作用。所以我们的实验证明了可以通过沉默TAZ来促进BMP9诱导的成骨过程。综上所述,我的研究证明了通过使用温敏材料PPCN-G与沉默TAZ都能够有效促进BMP9诱导的成骨过程。
[Abstract]:The skeleton is composed of vertebrates with extracellular matrix mineralization. Bone is a highly connective tissue specific, with protection for internal organs, to accommodate the bone marrow, as calcium and phosphate metabolism or preservation of tissue, an important regulatory factor. Osteogenic chondrogenic progenitor cells, osteoblasts and bone cells are three basic cell types in skeletal formation. Normal bone formation requires the differentiation of osteoblasts and osteoblasts are composed of mesenchymal stem cells (MSCs) differentiation. In recent years, with the growth and development of bone, bone disease understanding and the progress of science and technology, the formation of a new subject: bone tissue engineering. Bone tissue engineering refers to the high concentration of autologous osteoblasts isolated from the bone marrow mesenchymal stem cells and chondrocytes, amplified and cultivated in a natural or artificial synthesis in vitro The good biocompatibility, cell scaffold can be gradually degraded and absorbed on the human body, and add some cytokines induced into bone. In this biological scaffold cells can grow in accordance with the three-dimensional morphology of scaffolds, and nutrient and gas exchange. Then the cells implanted into the bone defect site factor. A mixture of materials, in biological materials gradually degraded at the same time, the plant cell proliferation and osteogenic differentiation to repair bone tissue defect. Bone tissue engineering has three elements: sufficient osteoblasts, effective osteoinductive factor, good biocompatibility of scaffold materials. We have proved that BMP9 is one of the most effective promotion of mesenchymal stem cells (MSCs) into bone factor differentiation. And we also successfully obtained before an immortalized mesenchymal stem cells iMEF, so we want to find A material with BMP9 induced mesenchymal stem cells after planting can make 3D cell morphology growth of osteoblasts on this material. This experiment provides a theoretical basis for the biological material in clinical bone induction. We first used a temperature sensitive biodegradable high molecular polymer (PPCN) mixed gelatin (PPCN-G) as a scaffold to carry BMP9 induced mesenchymal stem cells, to test whether there is the ability to promote bone formation. This material PPCN will become solid at 37 DEG C, in favor of material fixed in the body, at 21 DEG C into a liquid material and is beneficial to the cell and the mixed factor experimental results show that, in the culture of 3D gelatin could promote mesenchymal stem cell adhesion and survival. Experiments show that PPCN-G can promote BMP9 induced osteogenic differentiation in vivo in mice heterotopic bone formation, and can promote bone trabecula The formation of bone like structure in order to get better. The back of the bag piece removed for histological analysis showed that PPCN-G can promote the formation of blood vessels. The immunohistochemical results show that PPCN-G can promote vascular endothelial growth factor (VEGF) expression. So our experiment proved that PPCN-G is a new type of carrier injection of biodegradable, and can and cell factor for bone tissue engineering. Recent studies suggest that BMP9 induced bone formation and other BMP protein compared with different molecular mechanisms. Play a key role in some growth related signaling pathways to induce bone formation in the process of BMP9, BMP9 and their synergistic effect, to complete the development and maintenance of bone task. Although many in bone formation in regulating factor has been described to function, such as TAZ has clear to promote osteogenic differentiation inhibition Made of fat differentiation, but TAZ for BMP9 induced into what is the relationship between bone formation in bone, in the process of how they interact has not been studied clearly. This research for the factors on the osteogenesis process by adding the accurate control of significance in bone tissue engineering. Firstly, we constructed a TAZ siRNA silence has been the use of adenovirus and expression of adenovirus, and then silencing or overexpression of adenovirus with Ad-BMP9 infected iMEF cells, detect alkaline phosphatase activity in vitro, to test whether TAZ can promote the osteogenic differentiation capacity. Then we will TAZ over expression and silencing of Ad-BMP9 with adenovirus and iMEF infection the cell, then mixed with PPCN-G injected into nude mice, the control group with Ad-GRFP infection and Ad-BMP9 iMEF cells were compared in vivo test whether TAZ can promote bone points The ability of the in vivo results demonstrated that overexpression of TAZ and TAZ have the ability to promote bone silence differentiation induced by BMP9, and the silence after TAZ to promote special effect, so we have the over expression of TAZ and adenovirus and Ad-BMP9 silencing with the infection of iMEF cells, and the cells in vivo and in vitro the proliferation of detection. It is proved that silencing of TAZ can significantly promote the proliferation of iMEF cells, probably by increasing the mutual contact between cells and promote the osteogenic differentiation of iMEF cells. We infected iMEF and iMEF-OKmTAZ cells by Ad-BMP9, at different time points were collected and extracted RNA, reverse transcription cDNA, will the cDNA samples for each lineage markers and TAZ downstream of the transcription factor TEAD qPCR detection. The experimental results show that the osteogenic effect of silencing TAZ mainly through enhancing the expression of Runx2 to complete the, And the silence of TAZ TEAD also has played a certain effect. So we can prove by silencing TAZ to promote bone formation induced by BMP9. In summary, our research proved that through the use of temperature sensitive materials PPCN-G and TAZ are silent can effectively promote bone formation induced by BMP9.

【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R68;R318.08

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