力生长因子E肽（MGF-Ct24E）与应力作用对成骨细胞基因表达影响的基因芯片分析

发布时间：2018-06-28 14:17

本文选题：成骨细胞 + 力生长因子　；参考：《重庆大学》2011年硕士论文

【摘要】：最近力生长因子(Mechano growth factor, MGF)是生物力学及生长因子研究方面的新热点。MGF是Igf-1基因的选择性剪接变异体,在拉伸刺激的骨骼肌和损伤的神经、肌肉等组织中高表达。进一步研究发现,MGF及其E肽(MGF-Ct24E)能够促进肌肉肥大,损伤修复,并且具有神经保护和心肌保护等功能。本实验室的研究发现在拉伸刺激的成骨细胞中也有MGF mRNA的高表达,提示MGF也可能是骨组织中响应力信号的效应因子,还发现MGF能够调控成骨细胞的增殖分化过程,促进骨缺损修复作用。应力在骨组织的生长、重建过程中发挥了重要的调控作用,骨折损伤部位往往因为缺乏应力而导致愈合后的骨质疏松甚至再骨折现象。因此,本课题比较分析应力与MGF-Ct24E作用后成骨细胞的基因表达谱,寻找两者在骨代谢调控方面的异同,设想以MGF代替应力解决骨损伤修复过程因固定医疗措施使得应力严重降低的问题。主要内容和结论如下: (1)运用组织块培养法培养出新生大鼠颅骨成骨细胞,利用差时黏附法对培养出的细胞进行纯化。通过形态学观察、碱性磷酸酶(ALP)染色、基质前体染色、矿化结节染色等鉴定方法证实该组织块法培养的细胞具有典型的成骨细胞生物学特征,符合后续实验要求。将传代培养至第三代的成骨细胞用于后续实验研究。 (2)对细胞施加12%的周期性拉伸刺激和50ng/mL MGF-Ct24E直接作用8h,以静态培养的细胞作为对照组,提取细胞总RNA,进行基因芯片杂交实验分析各组细胞的差异表达基因。以对照组为参照,在力学加载组的细胞中共发现差异表达基因1866条,其中上调1113条,下调753条;在MGF-Ct24E处理组的细胞中共发现差异表达基因1178条,其中上调基因796条,下调基因382条。以力学加载组为参照,分析MGF-Ct24E处理组的细胞发现差异表达基因共602条,其中上调基因290条,下调基因312条。 (3)应用Gene Ontology(GO)数据库对筛选出的差异表达基因进行基因本体论分析,分类分析各差异表达基因的功能类型。应力和MGF-Ct24E作用成骨细胞后,得到的差异表达基因所参与调控的GO类型有众多相似之处。这些共同之处主要是关于细胞增殖和分化、细胞周期、骨发育和骨化的调节,细胞迁移及对应力刺激的响应等。除了两者共同调节的表达类型外,应力作用后的差异表达基因还参与调控了肌动蛋白细胞骨架组织、细胞生长、蛋白区域特异性结合和蛋白质二聚化活性等;MGF-Ct24E作用后的差异表达基因还参与调控了软骨发育、细胞骨架、生长因子结合、蛋白激酶结合和受体结合等。以力学加载组为参照,分析MGF-Ct24E处理组的基因表达谱,发现MGF-Ct24E处理组中的差异表达基因主要与胚胎和组织发育、转录与基因表达的正调节等有关。MGF-Ct24E处理组与力学加载组的差异表达基因在调控骨代谢方面有很高的相似性,其差异基因表达的变化趋势与差异表达倍数也基本一致。 (4)通过细胞增殖、细胞周期分析和细胞分化检测等实验进一步分析了MGF-Ct24E对成骨细胞生物学活性的影响。结果显示:MGF-Ct24E在成骨细胞生长早期促进了细胞的增殖,抑制了ALP活性;细胞周期分析发现MGF-Ct24E作用组在第一天和第三天S期的细胞所占比例显著高于对照组,细胞增殖指数显著提高,随着培养时间的延长增殖速度逐渐减缓,促增殖效率逐渐降低;在细胞分化晚期,MGF-Ct24E促进了胶原的表达和矿化结节的形成,促进了成骨细胞的分化。说明MGF-Ct24E并非是抑制成骨细胞的分化,而是促进成骨细胞早期的增殖活性,延迟了其分化活性。细胞实验所得结果与基因芯片分析结果一致。综上所述,MGF-Ct24E和应力作用在骨代谢基因表达的调控方面有着极为类似的效应,细胞实验分析再一次证实了MGF-Ct24E促进成骨细胞的增殖,延迟其分化,最终促进骨形成作用。因此,MGF-Ct24E有望能代替应力刺激用于骨损伤修复的临床治疗。
[Abstract]:Recently, Mechano growth factor (MGF) is a new hot spot in the study of biomechanics and growth factors.MGF is a selective splicing variant of the Igf-1 gene, which is highly expressed in the skeletal muscles and injured nerves and muscles. Further studies have found that MGF and its E peptide (MGF-Ct24E) can promote muscle hypertrophy and loss of muscle. This laboratory study found that there is a high expression of MGF mRNA in the osteoblasts stimulated by tension, suggesting that MGF may also be an effector factor of response signal in bone tissue. It is also found that MGF can regulate the proliferation and differentiation process of osteoblasts and promote the repair of bone defects. Force plays an important regulatory role in the process of bone tissue growth and reconstruction. The fracture site often causes osteoporosis and even refracture after the lack of stress. Therefore, we compare and analyze the gene table of the osteoblasts after the stress and MGF-Ct24E action, and find the similarities and differences in the regulation of bone metabolism. It is envisaged that MGF should be used instead of stress to solve the problem of serious stress reduction due to fixed medical measures in the process of bone repair.
(1) the cultured rat calvarial osteoblasts were cultured by tissue mass culture, and the cultured cells were purified by differential time adhesion method. By morphological observation, alkaline phosphatase (ALP) staining, matrix precursor staining, mineralized nodule staining and other identification methods confirmed that the cells cultured in this tissue block have typical osteoblast biological characteristics. The results were consistent with the requirements of follow-up experiment. The osteoblasts from third generations were used for subsequent experimental studies.
(2) 12% periodic stretching stimulation and direct action of 50ng/mL MGF-Ct24E on 8h were applied to the cells. The static cultured cells were used as the control group, and the total RNA was extracted. The differential expression genes of each cell were analyzed by gene chip hybridization experiment. In the control group, 1866 differentially expressed genes were found in the cells of the mechanical loading group. There were 1113 up-regulated and 753 down regulation. In the MGF-Ct24E treatment group, there were 1178 differentially expressed genes, among which 796 were up-regulated, and 382 down regulated genes. A total of 602 differentially expressed genes were found in the MGF-Ct24E treatment group. Among them, 290 and 312 down regulated genes were up-regulated.
(3) using the Gene Ontology (GO) database to carry out the gene ontology analysis of the selected differentially expressed genes and classify the functional types of the differentially expressed genes. After the action of stress and MGF-Ct24E into osteoblasts, there are many similarities in the GO types involved in the regulation of differentially expressed genes. These similarities are mainly about the cells. Proliferation and differentiation, cell cycle, regulation of bone development and ossification, cell migration and response to stress stimulation are also involved in the regulation of actin cytoskeleton, cell growth, protein region specific binding and protein dimerization activity, in addition to the expression types that are co regulated by both. The differentially expressed genes after the action of MGF-Ct24E also regulate the development of cartilage, cytoskeleton, growth factor binding, protein kinase binding and receptor binding. Based on the mechanical loading group, the gene expression profiles in the MGF-Ct24E treatment group are analyzed, and the differential expression genes in the MGF-Ct24E treatment group are mainly developed with the embryo and tissue, and the transcriptional and transcriptional genes are found. The positive regulation of gene expression related to the differential expression genes of the.MGF-Ct24E treatment group and the mechanical loading group have high similarity in the regulation of bone metabolism, and the variation trend of the differential gene expression is also basically consistent with the differential expression multiplier.
(4) the effects of MGF-Ct24E on the biological activity of osteoblasts were further analyzed by cell proliferation, cell cycle analysis and cell differentiation test. The results showed that MGF-Ct24E promoted cell proliferation and inhibited ALP activity in the early stage of osteoblast growth, and the cell cycle analysis found that the MGF-Ct24E action group was in the first and third days S. The proportion of cells in the stage was significantly higher than that in the control group, and the proliferation index increased significantly. The proliferation rate gradually slowed down with the incubation time, and the proliferation efficiency gradually decreased. In the late differentiation of the cells, MGF-Ct24E promoted the expression of collagen and the formation of mineralized nodules, and promoted the differentiation of osteoblasts. It showed that MGF-Ct24E was not a inhibition. The differentiation of osteoblasts, but the early proliferation activity of osteoblasts, delayed the differentiation activity. The results of cell experiments were in accordance with the results of gene chip analysis.
In summary, MGF-Ct24E and stress play a very similar effect on the regulation of gene expression in bone metabolism. Cell experimental analysis again confirms that MGF-Ct24E promotes the proliferation of osteoblasts, delays its differentiation, and ultimately promotes bone formation. Therefore, MGF-Ct24E is expected to replace stress stimulation in the clinical treatment of bone damage repair. Therapy.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

【引证文献】