【摘要】：骨組織結(jié)構(gòu)完整性的維持依賴于骨祖細(xì)胞池中成骨細(xì)胞的不斷更新,該生物學(xué)過(guò)程由許多生長(zhǎng)因子進(jìn)行精細(xì)調(diào)控。表皮生長(zhǎng)因子受體(EGFR)配體是目前已知的骨祖細(xì)胞有絲分裂原。但這些配體調(diào)節(jié)骨祖細(xì)胞池的具體機(jī)制沒有得到詳細(xì)闡述,尤其是EGFR信號(hào)通路在調(diào)節(jié)骨祖細(xì)胞凋亡中的作用沒有得到詳細(xì)研究。在本研究中,我們證實(shí)了EGFR信號(hào)通路的激活通過(guò)促進(jìn)骨祖細(xì)胞增殖并抑制骨祖細(xì)胞凋亡,增加骨祖細(xì)胞數(shù)量。該結(jié)論在大鼠顱骨組織器官培養(yǎng)實(shí)驗(yàn)中得到進(jìn)一步證實(shí)。在大鼠顱骨組織器官培養(yǎng)實(shí)驗(yàn)中,表皮生長(zhǎng)因子(EGF)在增加增殖細(xì)胞數(shù)量的同時(shí),降低了凋亡細(xì)胞數(shù)量,從而增加成骨細(xì)胞總數(shù)量。骨祖細(xì)胞系MC3T3細(xì)胞Microarray分析提示,EGFR信號(hào)通路能誘導(dǎo)抗凋亡基因Mcl1以及早期生長(zhǎng)反應(yīng)基因(Egr1, Egr2, Egr3)的表達(dá)。過(guò)表達(dá)Egrs的抑制蛋白Nab2可抑制EGF誘導(dǎo)的骨祖細(xì)胞數(shù)量增加。進(jìn)一步實(shí)驗(yàn)表明,以SiRNA降低Egr2的表達(dá),可抑制EGF誘導(dǎo)的骨祖細(xì)胞數(shù)量增加,而以SiRNA降低Egrl、Egr3的表達(dá),則對(duì)EGF誘導(dǎo)的骨祖細(xì)胞數(shù)量增加沒有影響。進(jìn)一步研究證實(shí)Egr2是EGF促進(jìn)骨祖細(xì)胞增殖并抑制骨祖細(xì)胞凋亡的主要介導(dǎo)基因。采用腺病毒過(guò)表達(dá)、抑制劑以及SiRNA等實(shí)驗(yàn)方法,我們證實(shí)了EGFR信號(hào)通路激活MAPK/Erk通路,進(jìn)而誘導(dǎo)Egr2的表達(dá),從而導(dǎo)致了Mcll的增加,最終抑制細(xì)胞凋亡。同時(shí),另一骨祖細(xì)胞有絲分裂原和存活因子成纖維細(xì)胞生長(zhǎng)因子(FGF)2,也能通過(guò)誘導(dǎo)Egr2的表達(dá),進(jìn)而促進(jìn)骨祖細(xì)胞增殖,抑制骨祖細(xì)胞凋亡。這些結(jié)果表明,Egr2在生長(zhǎng)因子介導(dǎo)的骨祖細(xì)胞數(shù)量增加中起著重要作用。總的來(lái)說(shuō),我們的實(shí)驗(yàn)結(jié)果清楚地說(shuō)明了EGFR信號(hào)傳導(dǎo)通路,通過(guò)誘導(dǎo)Egr2的表達(dá),促進(jìn)骨祖細(xì)胞增殖,抑制骨祖細(xì)胞凋亡,進(jìn)而在調(diào)節(jié)骨祖細(xì)胞庫(kù)的生物學(xué)功能中,起著重要的作用。
[Abstract]:The maintenance of the structural integrity of the bone tissue depends on the continuous update of the osteoblast in the bone progenitor cell, which is finely regulated by a number of growth factors. The epidermal growth factor receptor (EGFR) ligand is currently known as the mitogen of the bone progenitor cells. However, the specific mechanism of these ligands for modulating the pool of bone progenitor cells is not described in detail, in particular the role of the EGFR signaling pathway in the regulation of the apoptosis of the bone progenitor cells. In this study, we confirmed the activation of the EGFR signaling pathway by promoting the proliferation of bone progenitor cells and inhibiting the apoptosis of the bone progenitor cells and increasing the number of bone progenitor cells. The conclusion is further confirmed in the experiment of rat skull tissue organ culture. In the experiment of rat skull tissue organ culture, the epidermal growth factor (EGF) increased the number of proliferating cells and decreased the number of apoptotic cells, thus increasing the total amount of osteoblast. Microarray analysis of the cell line MC3T3 cells suggested that the EGFR signaling pathway could induce the expression of anti-apoptotic gene Mcl1 and early growth response genes (Egr1, Egr2, Egr3). The inhibition protein Nab2 over-expression of Egrs can inhibit the increase in the number of EGF-induced bone progenitor cells. Further experiments show that the expression of Egr2 is reduced by SiRNA, and the increase of the amount of the EGF-induced bone progenitor cells can be suppressed, and the increase of the amount of the EGF-induced bone progenitor cells is not affected by the reduction of the expression of Egrl, Egr3 in the SiRNA. Further studies have shown that Egr2 is a major mediating gene for EGF to promote the proliferation of bone progenitor cells and to inhibit the apoptosis of bone progenitor cells. Using the method of adenovirus overexpression, inhibitor and SiRNA, we confirmed the activation of the MAPK/ Erk pathway in the EGFR signaling pathway, and further induced the expression of Egr2, which resulted in an increase of Mcll, which ultimately inhibited the apoptosis of the cells. At the same time, the other bone progenitor cell mitogen and the survival factor fibroblast growth factor (FGF)2 can also promote the proliferation of the bone progenitor cells and inhibit the apoptosis of the bone progenitor cells by inducing the expression of the Egr2. These results indicate that Egr2 plays an important role in the growth factor-mediated increase in the number of bone progenitor cells. In general, our experimental results clearly illustrate the EGFR signaling pathway and play an important role in the regulation of the biological function of the bone progenitor cell by inducing the expression of Egr2, promoting the proliferation of the bone progenitor cells, and inhibiting the apoptosis of the bone progenitor cells.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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