Tbx3对Runx2活性与功能的影响的研究
发布时间：2018-09-05 15:13
【摘要】：骨骼是人体内的重要组成成分,具有一定的形状,结构和硬度。骨骼由骨组成,在生物体的生命过程中不断的进行着生长和改建的复杂演变。骨的发生方式有两种,膜内骨化成骨(间充质细胞直接成骨)和软骨内骨化成骨(先形成软骨后再成骨)。这两个过程决定着生物体的存亡,并在转录水平上被严格的调控。在这个过程中有很多转录因子和蛋白质起着重要作用。例如,Runx2是成骨细胞分化的重要的转录因子之一;最近Tbx3(Tbox包含因子)被报道,在生长激素的诱导下可以成骨过程中,负调控Runx2和骨形成。本学位论文的研究工作主要集中在阐明Tbx3抑制Runx2和骨形成的分子机制。 成骨细胞中Runx2是主要的调控因子。我们在C3H10T1/2细胞中过表达Runx2来研究其功能以及影响Runx2功能的因子。很多报告显示Runx2招募共激活因子(如HATs)或共抑制因子(如HDACs)来调控它的靶基因。我们选择了HDAC1, 5和P300(HAT)来分析存在或不存在Runx2的情况下乙酰化酶和去乙酰化酶的核定位。免疫荧光结果显示,在存在和不存在Runx2的情况下这三个蛋白都在核内定位,不发生核质穿梭。 我们研究了Tbx3调控Runx2介导的成骨细胞分化。在C3H10T1/2细胞中共转染了Tbx3和Runx2。双报告分析结果显示,Tbx3干涉了Runx2介导的OPN启动子的转录活性,从而消除了Runx2的活性。免疫荧光结果显示,在过表达Tbx3细胞中Runx2在核质中同时出现。这些结果证实,Tbx3消除了Runx2的活性,提高Tbx3水平可以导致部分Runx2的细胞质定位。 HDACs是一个大家族,它们被很多的细胞过程重要的转录因子所招募。我们研究了HDAC1是否参与Tbx3抑制Runx2的活性。我们用RT-PCR,免于印记和ALP染色等方法研究了HDAC1的表达和对Tbx3影响。在正常培养条件下,C3H10T1/2细胞同时过表达Tbx3和Runx2与C3H10T1/2细胞过表达Runx2相比,其HDAC1的mRNA水平增加两倍;而在分化培养条件下其HDAC1的mRNA水平增加三倍。这两个细胞培养条件中HDAC1的蛋白水平也显著增加。在TSA(HDAC抑制剂)处理情况下,原本被Tbx3抑制的、Runx2介导的碱性磷酸酶活性(ALP)阳性的细胞又出现ALP阳性染色结果。 全部结果显示了Tbx3影响Runx2的功能和作用。Tbx3有效的抑制Runx2及其核定位。Tbx3上调了HDAC1从而对Runx2活性和功能产生影响,抑制HDAC1可以缓解Tbx3对Runx2的抑制作用。
[Abstract]:Bone is an important component of the human body, with a certain shape, structure and hardness. Bone is composed of bone, which is a complex evolution of growth and remodeling in the life of organism. There are two types of osteogenesis: intramembranous ossification (direct mesenchymal cell osteogenesis) and endochondral ossification (cartilage formation followed by osteogenesis). These two processes determine the survival of organisms and are strictly regulated at the transcriptional level. Many transcription factors and proteins play an important role in this process. For example, Runx2 is one of the important transcription factors in osteoblast differentiation. Recently, Tbx3 (Tbox inclusion factor) has been reported to negatively regulate Runx2 and bone formation during osteogenesis induced by growth hormone. This dissertation focuses on elucidating the molecular mechanism of Tbx3 inhibiting Runx2 and bone formation. Runx2 is the main regulatory factor in osteoblasts. We overexpressed Runx2 in C3H10T1/2 cells to study its function and the factors affecting Runx2 function. Many reports suggest that Runx2 recruits co-activators (such as HATs) or co-suppressors (such as HDACs) to regulate its target genes. We selected HDAC1, 5 and P300 (HAT) to analyze the nucleotide localization of acetylase and deacetylase in the presence or absence of Runx2. The results of immunofluorescence showed that the three proteins were located in the nucleus and no nuclear and cytoplasmic shuttle occurred in the presence or absence of Runx2. We studied the regulation of Tbx3 on Runx2 mediated osteoblast differentiation. Tbx3 and Runx2. were transfected into C3H10T1/2 cells. The results of double report analysis showed that Tbx3 interfered with the transcriptional activity of OPN promoter mediated by Runx2, thus eliminating the activity of Runx2. Immunofluorescence results showed that Runx2 appeared simultaneously in nuclear and cytoplasm in overexpressed Tbx3 cells. These results indicate that Tbx3 eliminates the activity of Runx2 and increases the level of Tbx3, which leads to the cytoplasmic localization of some Runx2. HDACs is a large family, which is recruited by many transcription factors that are important in cellular processes. We studied whether HDAC1 was involved in the inhibition of Runx2 activity by Tbx3. We studied the expression of HDAC1 and its effect on Tbx3 by RT-PCR, free imprinting and ALP staining. Under normal culture conditions, both Tbx3 and Runx2 overexpression in C3H10T1 / 2 cells increased the mRNA level of HDAC1 twice as compared with that of C3H10T1/2 cells, but the mRNA level of HDAC1 increased threefold in differentiation culture. The protein level of HDAC1 was also significantly increased in these two cell cultures. Under the treatment of TSA (HDAC inhibitor, ALP positive staining was found in the cells with (ALP) activity of alkaline phosphatase mediated by Tbx3, which were inhibited by Tbx3. All the results showed that Tbx3 could effectively inhibit the function and function of Runx2. Tbx3 could effectively inhibit Runx2 and its nuclear localization. Tbx3 upregulated HDAC1, thus affecting the activity and function of Runx2, and inhibiting HDAC1 could alleviate the inhibitory effect of Tbx3 on Runx2.
【学位授予单位】：东北师范大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363
本文编号：2224656
[Abstract]:Bone is an important component of the human body, with a certain shape, structure and hardness. Bone is composed of bone, which is a complex evolution of growth and remodeling in the life of organism. There are two types of osteogenesis: intramembranous ossification (direct mesenchymal cell osteogenesis) and endochondral ossification (cartilage formation followed by osteogenesis). These two processes determine the survival of organisms and are strictly regulated at the transcriptional level. Many transcription factors and proteins play an important role in this process. For example, Runx2 is one of the important transcription factors in osteoblast differentiation. Recently, Tbx3 (Tbox inclusion factor) has been reported to negatively regulate Runx2 and bone formation during osteogenesis induced by growth hormone. This dissertation focuses on elucidating the molecular mechanism of Tbx3 inhibiting Runx2 and bone formation. Runx2 is the main regulatory factor in osteoblasts. We overexpressed Runx2 in C3H10T1/2 cells to study its function and the factors affecting Runx2 function. Many reports suggest that Runx2 recruits co-activators (such as HATs) or co-suppressors (such as HDACs) to regulate its target genes. We selected HDAC1, 5 and P300 (HAT) to analyze the nucleotide localization of acetylase and deacetylase in the presence or absence of Runx2. The results of immunofluorescence showed that the three proteins were located in the nucleus and no nuclear and cytoplasmic shuttle occurred in the presence or absence of Runx2. We studied the regulation of Tbx3 on Runx2 mediated osteoblast differentiation. Tbx3 and Runx2. were transfected into C3H10T1/2 cells. The results of double report analysis showed that Tbx3 interfered with the transcriptional activity of OPN promoter mediated by Runx2, thus eliminating the activity of Runx2. Immunofluorescence results showed that Runx2 appeared simultaneously in nuclear and cytoplasm in overexpressed Tbx3 cells. These results indicate that Tbx3 eliminates the activity of Runx2 and increases the level of Tbx3, which leads to the cytoplasmic localization of some Runx2. HDACs is a large family, which is recruited by many transcription factors that are important in cellular processes. We studied whether HDAC1 was involved in the inhibition of Runx2 activity by Tbx3. We studied the expression of HDAC1 and its effect on Tbx3 by RT-PCR, free imprinting and ALP staining. Under normal culture conditions, both Tbx3 and Runx2 overexpression in C3H10T1 / 2 cells increased the mRNA level of HDAC1 twice as compared with that of C3H10T1/2 cells, but the mRNA level of HDAC1 increased threefold in differentiation culture. The protein level of HDAC1 was also significantly increased in these two cell cultures. Under the treatment of TSA (HDAC inhibitor, ALP positive staining was found in the cells with (ALP) activity of alkaline phosphatase mediated by Tbx3, which were inhibited by Tbx3. All the results showed that Tbx3 could effectively inhibit the function and function of Runx2. Tbx3 could effectively inhibit Runx2 and its nuclear localization. Tbx3 upregulated HDAC1, thus affecting the activity and function of Runx2, and inhibiting HDAC1 could alleviate the inhibitory effect of Tbx3 on Runx2.
【学位授予单位】：东北师范大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363
【参考文献】
相关期刊论文 前1条
1 陈忠华;吕光明;季天海;;TBX3基因mRNA在乳腺癌中表达的检测及其意义[J];南方医科大学学报;2009年01期
，本文编号：2224656
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