【摘要】：骨骼是一個(gè)不斷更新的具有多種功能的器官,包括調(diào)節(jié)鈣平衡,支撐軟組織提供造血場(chǎng)所等。這些功能是通過(guò)骨組織的不管更新,即重塑而完成的。破骨細(xì)胞是唯一具有骨吸收能力的多核細(xì)胞。破骨細(xì)胞來(lái)源于定向的髓系前體細(xì)胞。多種細(xì)胞及其分泌的因子可以調(diào)節(jié)破骨細(xì)胞的分化及功能,尤其是骨髓基質(zhì)細(xì)胞及其分泌的M-CSF和RANKL。過(guò)度的骨溶解是多種病理性損傷中的一個(gè)重要的臨床問題,如癌癥骨轉(zhuǎn)移,類風(fēng)濕關(guān)節(jié)炎,骨質(zhì)疏松以及Paget's骨病等。因此臨床上需要有效的能抑制骨過(guò)度溶解或促進(jìn)骨生成的治療方法,以預(yù)防或減輕骨質(zhì)疏松的發(fā)生,提高病人的生活質(zhì)量。過(guò)去的十年里,在解析破骨細(xì)胞生成方面有許多重要的突破,破骨細(xì)胞的分化過(guò)程及功能越來(lái)越清晰。許多因子在這過(guò)程中發(fā)揮重要的作用,如M-CSF, RANKL, OPG, PU.1以及MITF等。激活轉(zhuǎn)錄因子4(ATF4)是一種重要的轉(zhuǎn)錄因子,通過(guò)定點(diǎn)突變的方法,ATF4最早被發(fā)現(xiàn)對(duì)于eye lens纖維的形成至關(guān)重要。隨后越來(lái)越多的研究證明其在成骨細(xì)胞的分化及骨形成的過(guò)程中發(fā)揮著重要的作用。有研究證明ATF4可以通過(guò)影響成骨細(xì)胞產(chǎn)生RANKL的量來(lái)間接調(diào)節(jié)破骨細(xì)胞的分化和骨吸收。但是目前尚未見有關(guān)ATF4可以直接調(diào)節(jié)破骨細(xì)胞的分化的研究報(bào)道。我們認(rèn)為ATF4可以直接影響破骨細(xì)胞的分化。 首先,我們通過(guò)蛋白印跡的方法以及免疫組織化學(xué)染色的方法確立了ATF4在破骨細(xì)胞系中的表達(dá),并且通過(guò)磷酸酶處理的方法,發(fā)現(xiàn)了其磷酸化形式的存在。然后我們利用了功能喪失和獲得的方法確立了此因子在破骨細(xì)胞形成中的直接作用。在Atf4-/-小鼠的骨中,Trap陽(yáng)性區(qū)域所占的比例明顯減小,信號(hào)強(qiáng)度也減弱；在體外的分化實(shí)驗(yàn)中,由Atf4-/-BMM細(xì)胞所形成的多核破骨細(xì)胞MNCs(大于等于三個(gè)核)的數(shù)量也顯著減少。而且通過(guò)骨片溶解吸收實(shí)驗(yàn)(Pitassay)可知,在體外由Atf4-/-BMM所形成破骨細(xì)胞的骨吸收凹陷數(shù)量也明顯減少,但是骨吸收凹陷數(shù)目與MNCs數(shù)目的比值沒有明顯的變化,說(shuō)明ATF4敲除后,破骨細(xì)胞的骨吸收能力沒有明顯改變。在由破骨細(xì)胞特異的Trap啟動(dòng)子驅(qū)動(dòng)下,使ATF4在破骨細(xì)胞中轉(zhuǎn)基因過(guò)表達(dá)時(shí),轉(zhuǎn)基因小鼠呈現(xiàn)出明顯的骨減少癥；血清CTX的水平大幅度升高；破骨細(xì)胞的形成在體內(nèi)和體外都有顯著增加,并且與破骨細(xì)胞分化相關(guān)的基因的表達(dá)水平,不論是蛋白水平還是mRNA水平都顯著上調(diào)。 進(jìn)一步研究發(fā)現(xiàn),由Atf4-/-小鼠的骨髓細(xì)胞所形成的GM-CFU的集落數(shù)量也明顯少于野生型對(duì)照組,GM-CFU是具有向破骨細(xì)胞分化能力的最原始的造血前體細(xì)胞。 將Atf4-/-BMM細(xì)胞與野生型的成骨細(xì)胞共培養(yǎng)或用高濃度的RANKL來(lái)刺激其分化,都不能彌補(bǔ)其向破骨細(xì)胞分化的缺陷。RANKL的信號(hào)是由其受體RANK來(lái)傳遞的。我們通過(guò)免疫組織化學(xué)染色的方法以及免疫印跡方法發(fā)現(xiàn),在Atf4-/-BMM細(xì)胞中,RANK的表達(dá)顯著降低,并且其mRNA水平也不能被M-CSF所上調(diào)。此外RANKL對(duì)多條MAPK信號(hào)通路的誘導(dǎo)激活也受ATF4的調(diào)節(jié)。在缺乏ATF4的狀態(tài)下,RANKL對(duì)于三條MAPK的通路的激活能力明顯下降,而NF-kB途徑、PI3K/Akt途徑卻不受影響。但是,ATF4對(duì)于M-CSF的信號(hào)卻沒有明顯的調(diào)節(jié)作用。 到目前為止,NFATcl是破骨細(xì)胞分化的最關(guān)鍵基因。無(wú)論是在體內(nèi)還是在體外,ATF4的缺乏都導(dǎo)致NFATcl的表達(dá)水平明顯下降。用逆轉(zhuǎn)錄病毒為載體,使NFATcl在ATF4WT和KO BMM中過(guò)表達(dá),可以以劑量依賴性的方式使Trap陽(yáng)性的MNCs增多。我們利用腺病毒作為載體,在BMM細(xì)胞中過(guò)表達(dá)ATF4,發(fā)現(xiàn)ATF4可以以劑量依賴性的方式上調(diào)NFATcl蛋白的表達(dá)水平。在體外,ATF4可以與NFATcl的啟動(dòng)子結(jié)合并能劑量依賴性的激活NFATcl (?)勺啟動(dòng)子,另外,通過(guò)ChIP assay的方法可以發(fā)現(xiàn)ATF4可以與NFATcl近啟動(dòng)子端的片段相作用并且該作用可以在RANKL的誘導(dǎo)下增強(qiáng)。在BMM細(xì)胞中,ATF4的蛋白水平受M-CSF以及PI3K/AKT途徑的調(diào)節(jié)。在缺乏M-CSF的情況下,ATF4的蛋白水平以時(shí)間依賴性的方式明顯減少,M-CSF可以阻斷這一進(jìn)程。M-CSF的這一作用可以被PI3K/Akt途徑的抑制劑LY294002阻斷,并且隨著LY2094002的濃度的增高,BMM細(xì)胞向破骨細(xì)胞的分化也相應(yīng)的受抑制。ATF4的缺乏可以使BMM的分化由破骨細(xì)胞系向巨噬細(xì)胞系遷移,導(dǎo)致巨噬細(xì)胞數(shù)量的增多。我們的研究結(jié)果證明ATF4在調(diào)節(jié)破骨細(xì)胞分化方面有著重要的內(nèi)在作用,這也許可以作為治療與破骨細(xì)胞相關(guān)的骨疾病的治療靶點(diǎn)。
[Abstract]:Bone is a constantly updated organ with multiple functions, including regulating calcium balance, supporting the soft tissue to provide hematopoiesis, and so on. These functions are accomplished through the regeneration of bone tissue, that is, remolding. Osteoclasts are the only multinucleated cells with bone absorptive capacity. Osteoclasts are derived from directional myeloid precursor cells. Cells and their secreted factors can regulate the differentiation and function of osteoclasts, especially bone marrow stromal cells and their secreted M-CSF and RANKL. osteolysis, which are important clinical problems in a variety of pathological injuries, such as cancer bone metastases, rheumatoid arthritis, osteoporosis and Paget's bone disease. Effective methods of inhibiting the excessive dissolving of bone or promoting bone formation in order to prevent or reduce the occurrence of osteoporosis and improve the quality of life of the patient. In the past ten years, there are many important breakthroughs in the analysis of osteoclast formation, the differentiation process and function of osteoclast are becoming more and more clear. Many factors play a heavy role in this process. The role of M-CSF, RANKL, OPG, PU.1 and MITF. Activation of transcription factor 4 (ATF4) is an important transcription factor. Through site directed mutagenesis, the earliest discovery of ATF4 is essential to the formation of eye lens fibers. More and more studies have shown that it plays an important role in the process of osteoblast differentiation and bone formation. Some studies have shown that ATF4 can indirectly regulate the differentiation and bone resorption of osteoclasts by affecting the amount of RANKL produced by osteoblasts. However, there has been no study on the differentiation of osteoclasts directly regulated by ATF4. We think that ATF4 can directly affect the differentiation of osteoclast.
First, we established the expression of ATF4 in the osteoclast cell line by the method of Western blot and immunohistochemical staining, and found the form of phosphorylation by the method of phosphatase treatment. Then we used the method of function loss and acquisition to establish the direct of this factor in the formation of osteoclast. In the bone of Atf4-/- mice, the proportion of the Trap positive region was significantly reduced and the signal intensity was weakened; in the differentiation experiment in vitro, the number of MNCs (more than three nuclei) formed by Atf4-/-BMM cells was also significantly reduced. And by the bone dissolving absorption experiment (Pitassay), it is known that Atf4 in vitro The number of bone resorption sags of osteoclasts formed in -/-BMM also decreased significantly, but the ratio of the number of bone resorption depression to the number of MNCs did not change significantly, indicating that the bone resorption capacity of osteoclasts was not significantly changed after ATF4 knockout. Under the drive of osteoclast specific Trap promoter, ATF4 was genetically modified in osteoclasts. At the time of arrival, the transgenic mice showed obvious osteopenia, the level of serum CTX increased significantly, and the formation of osteoclasts increased significantly in both in vivo and in vitro, and the level of gene expression related to osteoclast differentiation, both in protein level and in mRNA level, was significantly up-regulated.
Further studies have found that the number of colonies of GM-CFU formed by bone marrow cells from Atf4-/- mice is also significantly less than that in the wild type control group, and GM-CFU is the most primitive hematopoietic progenitor cells that have the ability to differentiate into osteoclasts.
To co culture Atf4-/-BMM cells with wild type osteoblasts or to stimulate their differentiation with high concentration of RANKL, the signal to the defect to osteoclast differentiation,.RANKL, is transmitted by its receptor RANK. We found that RANK in Atf4-/-BMM cells through immunohistochemical staining and immunological trace methods. The expression was significantly reduced and its mRNA level could not be raised by M-CSF. In addition, the activation of RANKL on multiple MAPK signaling pathways was also regulated by ATF4. In the absence of ATF4, the activation ability of RANKL to the three MAPK pathway was significantly decreased, while the NF-kB pathway was not affected by the PI3K/Akt path. There is no obvious adjustment.
So far, NFATcl is the most critical gene for osteoclast differentiation. The lack of ATF4 in both in vivo and in vitro leads to a significant decline in the expression level of NFATcl. Using retrovirus as the carrier, NFATcl is overexpressed in ATF4WT and KO BMM, and the positive MNCs of Trap can be increased in a dose-dependent manner. We use adenosis. As a carrier, ATF4 is overexpressed in BMM cells, and it is found that ATF4 can increase the expression level of NFATcl protein in a dose dependent manner. In vitro, ATF4 can be combined with NFATcl promoter and can activate the NFATcl (?) spoon promoter in a dose-dependent manner. In addition, ATF4 can be found by ChIP assay method and NFATcl near promoter. In BMM cells, the protein level of ATF4 is regulated by the M-CSF and the PI3K/AKT pathway. In the absence of M-CSF, the protein level of ATF4 is significantly reduced in a time dependent manner, and M-CSF can block this process of.M-CSF by PI3K/Akt, which can be blocked by M-CSF. The inhibitor LY294002 of the diameter is blocked, and with the increase of the concentration of LY2094002, the differentiation of BMM cells to the osteoclast is also corresponding to the lack of.ATF4, which can cause the differentiation of BMM to migrate from the osteoclast line to the macrophage system, which leads to the increase of the number of macrophages. Our research results show that ATF4 is regulating the osteoclast differentiation side. The surface has important internal effects, which may be used as a therapeutic target for osteoclast related bone diseases.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392.1

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