KAT2A对二氧化钛纳米管形貌促进颌骨骨髓间充质干细胞成骨分化的作用研究

发布时间：2018-05-05 11:55

本文选题：KAT2A + 二氧化钛　；参考：《广州医科大学》2017年硕士论文

【摘要】：研究背景生物医学材料的性能强烈依赖于第一个发生相互作用时,材料表面接触的生物环境[1];同时。在骨组织工程种子细胞发挥功能和分化过程中,由于骨头本身最基本的结构层次纳米范围内,而纳米级别材料可以通过人体仿生来精确控制[2,3],因此,种子细胞与纳米级别材料的研究受到人们研究和应用的青睐。在口腔临床治疗应用领域,种植体材料是最为常见的生物材料之一,用于牙齿缺失和骨组织缺损的修复[4];对于种植体的表面的处理技术,研究所和企业也在不断探索和改进。我们已知表观遗传学和微环境的改变有着密切的联系[5],而种植体材料表面的形貌也为细胞的粘附、生长、增殖、分化提供了一个微环境,这两者存在的联系值得我们更为深入的研究。2012年叶玲等[6]研究显示组蛋白去甲基化转移酶KDM4B和KDM6B促进人间充质干细胞成骨分化。2015年周永胜等[7]研究表明二氧化钛纳米管通过上调启动子区域里成骨基因Runx2和OCN的组蛋白H3K4甲基化水平以及抑制去甲基化的RBP2表达,从而促进脂肪间充质干细胞的成骨分化。然而对于表观遗传乙酰转移酶在TiO_2纳米管形貌与颌骨骨髓间充质干细胞的成骨分化过程中发挥的作用还未做相关的报道。因此,明确表观遗传学在纳米形貌与干细胞之间的相互作用有利于我们对口腔修复材料与细胞作用机制有着更加深入的认识,并为患者的临床治疗选择提供新的思路。研究目的通过观察体外实验研究表观遗传酶对TiO_2纳米管形貌与JMMSCs成骨分化作用的影响,探究表观遗传学在对种植体表面形态生物学性能是否起到作用,以及如何在种植体表面微观形态与在颌骨骨髓干细胞成骨方向上作用的内在机制,从而为种植体表面电化学处理技术提供相应的理论支持,为改善种植体骨结合能力提供新的研究方向,同时为表观遗传酶在种植体修复中骨结合作用途径提供一定的依据。方法1、JMMSCs分离、纯化、培养及鉴定通过体外组织块培养法和有限稀释法培养颌骨来源骨髓间充质干细胞(JMMSCs);流式细胞仪检测细胞表面特定标记;噻唑蓝(methylthiazolyl tetrazolium,MTT)检测干细胞增殖能力;成骨、成脂分化诱导检测干细胞多向分化能力。2、TiO_2纳米管形貌对JMMSCs成骨分化能力的影响利用电化学阳极氧化的方法制备均匀有序的TiO_2纳米管(NTs),通过扫描电子显微镜(SEM)进行材料表面形态的观察,以确定纳米级微观形貌的获取;电镜下观察TiO_2纳米管上JMMSCs形态,通过Cell count 8(CCK-8)法检测与TiO_2纳米管共培养的JMMSCs自我增殖能力;成骨诱导后通过茜素红染色比较研究TiO_2纳米管对JMMSCs成骨分化的作用。3、表观遗传酶在TiO_2纳米管形貌对JMMSCs成骨分化方向上的影响先是通过NTs与JMMSCs的体外共培养,利用qRT-PCR的方法在不同时间点下筛选出特异性表达的表观遗传酶,应用小干扰RNA对筛选出来的特定基因的进行干扰,通过两种方式:一种是应用小干扰RNA后的与NTs共培养的JMMSCs进行常规成骨分化诱导7d、14d,对其进行碱性磷酸酶(ALP)的染色;一种是应用小干扰RNA后与NTs共培养的JMMSCs进行常规成骨分化诱导14d后对细胞的成骨基因的表达进行qRT-PCR的检测,探讨特定基因干扰后JMMSCs成骨分化能力的变化。4、统计学分析所有数据采用SPSS 17.0软件系统进行统计学分析。两组间比较使用两样本t检验;多组间比较采用单因素方差分析。计量资料采用均数±标准差表示,以双侧P0.05为差异具有统计学意义。实验结果1、所有样本都能成功获得原代JMMSCs;细胞在显微镜下呈长梭形;流式细胞仪检测结果表明JMMSCs中间充质干细胞表面特定标记(CD105、CD90)均为阳性表达,而造血干细胞表面特定标记(CD34、CD14、CD45)均为阴性表达;MTT实验结果表明JMMSCs具有自我增殖能力;成骨分化诱导检测可见到钙化结节沉积,成脂分化诱导检测可见脂滴形成。结果说明,JMMSCs具有干细胞多向分化的潜能。2、利用阳极氧化法在一定浓度的电解溶液的条件下,扫描电子显微镜(SEM)观察材料成功制备出TiO_2纳米管形貌,通过CCK8试剂盒检测发现TiO_2纳米管对JMMSCs的增殖能力有一定的促进作用,成骨诱导28d以后茜素红结果显示NTs组的对JMMSCs的成骨分化能力促进作用更显著。结果说明,TiO_2纳米管形貌具有促进JMMSCs的成骨分化的作用。3、根据qRT-PCR在3d、7d检测乙酰基转移酶基因表达结果,综合对比分析得出,在乙酰基转移酶家族中KAT2A在TiO_2纳米管与JMMSCs共培养过程中特异性的表达;应用小干扰RNA后,相比于未干扰组,干扰组NTs上的JMMSCs的成骨基因ALP、COL-1、OCN表达相应的出现下调;同时碱性磷酸酶染色结果表示,干扰后与NTs共培养的JMMSCs相比不干扰组成骨分化能力下降。结果说明,KAT2A在纳米管形貌促进JMMSCs成骨分化能力起着关键作用。结论1、体外培养获取JMMSCs具有干细胞的特性,可以应用于体外实验。2、TiO_2纳米管形貌促进JMMSCs的成骨分化。3、KAT2A在TiO_2纳米管促进细胞成骨分化的过程中起着促进作用。
[Abstract]:Background the performance of biomedical materials is strongly dependent on the first biological environment, [1], on the surface of the material when the first interaction occurs. At the same time, in the process of function and differentiation of the bone tissue engineering seed cells, the nanoscale material can be refined through the bionics of the human body because of the most basic structural level of the bone itself. In the field of oral clinical treatment, implant materials are one of the most common biomaterials, which are used in dental defects and bone tissue defects to repair [4], and for the treatment of implant surfaces, research institutes and enterprises, in the field of oral clinical treatment. We have been exploring and improving. We have known that epigenetics and microenvironment changes have a close relationship with [5], and the surface morphology of the implant material also provides a microenvironment for cell adhesion, growth, proliferation and differentiation. The relationship between the two is worthy of further study in the study of [6] and other [6] studies in.2012. Methylation transferase KDM4B and KDM6B promote osteogenic differentiation of human mesenchymal stem cells for.2015 Zhou Yongsheng and other [7] studies, suggesting that TiO2 nanotubes promote osteogenic differentiation of adipose mesenchymal stem cells by up regulation of the level of histone H3K4 methylation and the inhibition of demethylation of RBP2 expression in the promoter region of the promoter region of Runx2 and OCN. However, the role of epigenetic acetyltransferase in the osteogenesis of TiO_2 nanotube and bone marrow mesenchymal stem cells in bone marrow mesenchymal stem cells has not been reported. Therefore, it is clear that the interaction between epigenetics and stem cells is beneficial to the mechanism of oral repair materials and cell action. The purpose of this study is to explore the effects of epigenetic enzymes on the morphology of TiO_2 nanotubes and the osteogenic differentiation of JMMSCs in vitro, and to explore whether epigenetics can play a role in the morphological biological ability of the implant surface and how to be in the implant. The internal mechanism of the surface micromorphology and the osteogenesis in the bone marrow stem cells of the jaw bone marrow stem cells can provide the corresponding theoretical support for the electrochemical treatment of implant surface, and provide a new direction for improving the bone binding ability of the implant. At the same time, it provides a certain basis for the epigenetic enzyme in the repair of bone binding in implant. Method 1, JMMSCs isolation, purification, culture and identification of bone marrow mesenchymal stem cells (JMMSCs) derived from bone marrow by tissue block culture and finite dilution method; flow cytometer was used to detect cell surface specific markers; methylthiazolyl tetrazolium (MTT) was used to detect the proliferation of stem cells; osteogenesis and lipid differentiation were used to detect stem cells. The multidirectional differentiation capacity.2, the effect of TiO_2 nanotube morphology on the ability of JMMSCs osteogenesis, using electrochemical anodizing method to prepare uniform and ordered TiO_2 nanotubes (NTs), and observe the surface morphology of the material by scanning electron microscope (SEM), to determine the nanoscale micromorphology, and to observe JMMS on the TiO_2 nanotube under the electron microscope. Cs morphology, Cell count 8 (CCK-8) method was used to detect the self proliferation ability of JMMSCs co cultured with TiO_2 nanotubes. After osteogenesis, the effect of TiO_2 nanotubes on JMMSCs osteogenesis differentiation was compared by alizarin red staining. The effect of epigenetic enzyme on TiO_2 nanotube morphology on the direction of JMMSCs osteogenesis was first through NTs and the bodies of JMMSCs. The epigenetic enzyme of specific expression was screened by qRT-PCR method at different time points, using small interference RNA to interfere with the selected specific genes, and through two ways: one was to induce 7d, 14d, and alkaline phosphorous by NTs co cultured JMMSCs after the application of small interference RNA. The staining of acid enzyme (ALP); one is to detect the expression of the osteogenic gene of the cell after the routine osteogenic differentiation induced by JMMSCs after the use of small interference RNA and NTs co culture to detect the qRT-PCR of the expression of the osteogenic gene of the cells, and to explore the changes of the bone differentiation ability of JMMSCs after specific gene interference, and all data are statistically analyzed by the SPSS 17 software system for statistics. Analysis. The two groups were compared with two samples t test; the multiple groups were compared with single factor variance analysis. The measurement data were expressed with mean mean difference of standard deviation. The difference of bilateral P0.05 was statistically significant. The experimental results were 1, all the samples were successful to obtain the original JMMSCs; the cells were long shuttle shape under the microscope; the flow cytometry results showed that the results showed that The specific surface markers (CD105, CD90) of JMMSCs mesenchyme stem cells were all positive, while the specific markers (CD34, CD14, CD45) of hematopoietic stem cells were negative expression. The results of MTT experiment showed that JMMSCs had the ability of self proliferation, and the osteogenic differentiation induction detection could see the deposition of calcified nodules, lipid differentiation induction detection of lipid droplets formation. The result shows that JMMSCs has the potential of multiple differentiation of stem cells,.2. Under the condition of a certain concentration of electrolysis solution, the morphology of TiO_2 nanotubes was successfully prepared by scanning electron microscope (SEM) by anodic oxidation method. The CCK8 kit detection found that the TiO_2 nanotube had a certain promoting effect on the proliferation of JMMSCs, and the osteogenic induction was induced. The results of alizarin red after 28d showed that the NTs group had a more significant effect on the osteogenic differentiation of JMMSCs. The results showed that the morphology of the TiO_2 nanotube had the effect of promoting the osteogenic differentiation of JMMSCs,.3, according to qRT-PCR in 3D, 7d detection of the gene expression of acetyltransferase gene, and a comprehensive comparison analysis showed that KAT2A in the acetyltransferase family was in TiO. The specific expression of _2 nanotube and JMMSCs co culture process; after the use of small interference RNA, compared to the undisturbed group, the osteogenic gene ALP, COL-1, OCN on the JMMSCs on the NTs of the interference group decreased, and the alkaline phosphatase staining results indicated that the interference with JMMSCs of the NTs co culture did not interfere with the decrease of the bone differentiation ability. The results show that KAT2A plays a key role in promoting the ability of JMMSCs osteogenesis in nanotube morphology. Conclusion 1, the culture of JMMSCs has the characteristics of stem cells in vitro, and can be applied to.2 in vitro. The morphology of TiO_2 nanotubes promotes the osteogenic differentiation of JMMSCs in.3, and KAT2A plays an important role in the process of promoting the osteogenic differentiation of the cells in TiO_2 nanotubes.

【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R783.6

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