Tbx3對(duì)Runx2活性與功能的影響的研究
發(fā)布時(shí)間：2018-09-05 15:13
【摘要】：骨骼是人體內(nèi)的重要組成成分,具有一定的形狀,結(jié)構(gòu)和硬度。骨骼由骨組成,在生物體的生命過程中不斷的進(jìn)行著生長(zhǎng)和改建的復(fù)雜演變。骨的發(fā)生方式有兩種,膜內(nèi)骨化成骨(間充質(zhì)細(xì)胞直接成骨)和軟骨內(nèi)骨化成骨(先形成軟骨后再成骨)。這兩個(gè)過程決定著生物體的存亡,并在轉(zhuǎn)錄水平上被嚴(yán)格的調(diào)控。在這個(gè)過程中有很多轉(zhuǎn)錄因子和蛋白質(zhì)起著重要作用。例如,Runx2是成骨細(xì)胞分化的重要的轉(zhuǎn)錄因子之一;最近Tbx3(Tbox包含因子)被報(bào)道,在生長(zhǎng)激素的誘導(dǎo)下可以成骨過程中,負(fù)調(diào)控Runx2和骨形成。本學(xué)位論文的研究工作主要集中在闡明Tbx3抑制Runx2和骨形成的分子機(jī)制。 成骨細(xì)胞中Runx2是主要的調(diào)控因子。我們?cè)贑3H10T1/2細(xì)胞中過表達(dá)Runx2來(lái)研究其功能以及影響Runx2功能的因子。很多報(bào)告顯示Runx2招募共激活因子(如HATs)或共抑制因子(如HDACs)來(lái)調(diào)控它的靶基因。我們選擇了HDAC1, 5和P300(HAT)來(lái)分析存在或不存在Runx2的情況下乙酰化酶和去乙�；傅暮硕ㄎ弧Ｃ庖邿晒饨Y(jié)果顯示,在存在和不存在Runx2的情況下這三個(gè)蛋白都在核內(nèi)定位,不發(fā)生核質(zhì)穿梭。 我們研究了Tbx3調(diào)控Runx2介導(dǎo)的成骨細(xì)胞分化。在C3H10T1/2細(xì)胞中共轉(zhuǎn)染了Tbx3和Runx2。雙報(bào)告分析結(jié)果顯示,Tbx3干涉了Runx2介導(dǎo)的OPN啟動(dòng)子的轉(zhuǎn)錄活性,從而消除了Runx2的活性。免疫熒光結(jié)果顯示,在過表達(dá)Tbx3細(xì)胞中Runx2在核質(zhì)中同時(shí)出現(xiàn)。這些結(jié)果證實(shí),Tbx3消除了Runx2的活性,提高Tbx3水平可以導(dǎo)致部分Runx2的細(xì)胞質(zhì)定位。 HDACs是一個(gè)大家族,它們被很多的細(xì)胞過程重要的轉(zhuǎn)錄因子所招募。我們研究了HDAC1是否參與Tbx3抑制Runx2的活性。我們用RT-PCR,免于印記和ALP染色等方法研究了HDAC1的表達(dá)和對(duì)Tbx3影響。在正常培養(yǎng)條件下,C3H10T1/2細(xì)胞同時(shí)過表達(dá)Tbx3和Runx2與C3H10T1/2細(xì)胞過表達(dá)Runx2相比,其HDAC1的mRNA水平增加兩倍;而在分化培養(yǎng)條件下其HDAC1的mRNA水平增加三倍。這兩個(gè)細(xì)胞培養(yǎng)條件中HDAC1的蛋白水平也顯著增加。在TSA(HDAC抑制劑)處理情況下,原本被Tbx3抑制的、Runx2介導(dǎo)的堿性磷酸酶活性(ALP)陽(yáng)性的細(xì)胞又出現(xiàn)ALP陽(yáng)性染色結(jié)果。 全部結(jié)果顯示了Tbx3影響Runx2的功能和作用。Tbx3有效的抑制Runx2及其核定位。Tbx3上調(diào)了HDAC1從而對(duì)Runx2活性和功能產(chǎn)生影響,抑制HDAC1可以緩解Tbx3對(duì)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.
【學(xué)位授予單位】：東北師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363
本文編號(hào)：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.
【學(xué)位授予單位】：東北師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 陳忠華;呂光明;季天海;;TBX3基因mRNA在乳腺癌中表達(dá)的檢測(cè)及其意義[J];南方醫(yī)科大學(xué)學(xué)報(bào);2009年01期
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