本文選題：C5of39 + AXIIR��； 參考：《北京協(xié)和醫(yī)學(xué)院》2012年博士論文

【摘要】：我的工作主要包含兩部分內(nèi)容： 一Annexin Ⅱ受體介導(dǎo)細(xì)胞凋亡的研究 隨著人們對(duì)凋亡的認(rèn)識(shí)逐漸深入,對(duì)凋亡發(fā)生的分子機(jī)制的了解也越來(lái)越透徹,但同時(shí)也發(fā)現(xiàn)這一過(guò)程遠(yuǎn)非原來(lái)想象的那樣簡(jiǎn)單,而是包含了復(fù)雜的調(diào)控機(jī)制。盡管近幾年在凋亡信號(hào)轉(zhuǎn)導(dǎo)途徑、凋亡的生化反應(yīng)機(jī)制以及凋亡的基因調(diào)控等方面的研究都取得了顯著的進(jìn)展,但仍有許多問(wèn)題迄今尚未闡明。 本文的研究對(duì)象人類鈣磷脂結(jié)合蛋白Ⅱ受體human Annexin Ⅱ receptor (簡(jiǎn)稱AXIIR),又稱為C5orf39,是一個(gè)具有193個(gè)氨基酸的蛋白質(zhì),其編碼基因位于人類染色體5p12區(qū)。通過(guò)基因序列比對(duì)和PCR檢測(cè)認(rèn)為AXIIR僅存在于人類第5號(hào)染色體上而不存在于其他種屬,是人類所特有的基因。AXIIR最先是在2006年,研究者從人類骨髓cDNA文庫(kù)中尋找Annexin Ⅱ的受體時(shí)被克隆出來(lái)的,推測(cè)為Ⅰ型膜蛋白,研究認(rèn)為AXIIR表達(dá)在骨髓基質(zhì)細(xì)胞的表面,介導(dǎo)Annexin Ⅱ的信號(hào)傳導(dǎo)而促進(jìn)成骨細(xì)胞形成。而隨后對(duì)于該基因的功能研究結(jié)果也僅僅是認(rèn)為其作為Annexin Ⅱ的細(xì)胞膜受體來(lái)介導(dǎo)Annexin Ⅱ的信號(hào)傳遞。該基因的功能研究還處于早期階段,更沒(méi)有任何證據(jù)證明其與細(xì)胞凋亡相關(guān)。 Annexin Ⅱ是鈣磷脂結(jié)合蛋白(Annexin)家族中的一個(gè)重要成員,存在于細(xì)胞膜、胞質(zhì)和胞外。Annexin Ⅱ具有廣泛的功能,Annexin Ⅱ與一系列細(xì)胞外基質(zhì)成分相互作用,參與許多細(xì)胞膜相關(guān)事件,如細(xì)胞外吐和內(nèi)吞,細(xì)胞黏附,纖溶酶原激活,腫瘤遷移和侵襲。 在本研究中,首先通過(guò)在人髓性白血病K562細(xì)胞系中過(guò)表達(dá)AXIIR基因,確證該基因高表達(dá)時(shí)能夠誘導(dǎo)細(xì)胞凋亡。進(jìn)一步發(fā)現(xiàn)AXIIR能引起其他人類細(xì)胞發(fā)生不同程度的凋亡,但細(xì)胞死亡比例均低于K562細(xì)胞,說(shuō)明AXIIR誘導(dǎo)人類細(xì)胞凋亡存在一定的普遍性,但也與特定細(xì)胞類型有關(guān)。 在目前的文獻(xiàn)報(bào)道中,AXIIR均是作為Annexin Ⅱ的細(xì)胞膜受體來(lái)介導(dǎo)Annexin Ⅱ的信號(hào)傳遞,所以我們需要了解AXIIR的誘導(dǎo)細(xì)胞凋亡功能是否由Annexin Ⅱ信號(hào)引起。首先,我們通過(guò)免疫熒光染色方法定位AXIIR-myc分布在細(xì)胞漿中。另外,蛋白酶體抑制劑MG132處理并未增加AXIIR的蛋白水平,這也排除了胞漿內(nèi)的AXIIR在被溶酶體和泛素化降解的可能性。Annexin Ⅱ在一個(gè)廣泛的濃度范圍內(nèi)并不能影響AXIIR誘導(dǎo)的細(xì)胞凋亡作用,說(shuō)明AXIIR并不是作為Annexin Ⅱ的受體來(lái)誘導(dǎo)凋亡的。 為了了解AXIIR誘導(dǎo)細(xì)胞凋亡的機(jī)制,我們檢測(cè)了凋亡通路相關(guān)蛋白的表達(dá)量和活性,主要包括幾種caspase和Bcl-2家族成員Bcl-2和BCL-XL。在AXIIR引起的細(xì)胞凋亡過(guò)程中,Caspase8,3均被激活。Caspase8抑制劑的使用也證實(shí)了Caspase8在AXIIR誘導(dǎo)細(xì)胞凋亡功能中發(fā)揮的重要作用。而隨后的免疫沉淀實(shí)驗(yàn)表明AXIIR結(jié)合并激活pro-Caspase8,卻并不依賴于FADD。此外,下調(diào)FADD的表達(dá)并不會(huì)影響AXIIR激活Caspase8的能力。另外AXIIR截短體表達(dá)實(shí)驗(yàn)說(shuō)明Caspase8的羧基端對(duì)于Caspase8的激活是必需的,當(dāng)然氨基端也發(fā)揮了一定作用。再者,在死亡受體誘導(dǎo)形成的DISC中并未檢查到AXIIR的表達(dá),說(shuō)明AXIIR沒(méi)有參與死亡受體誘導(dǎo)的Caspase8激活。 在我們的研究中,AXIIR不僅激活Caspase8,也同時(shí)激活了Caspase9和下調(diào)了BCL-2和BCL-XL的蛋白水平,而B(niǎo)ax水平無(wú)變化。BCL-XL高表達(dá)阻斷線粒體途徑對(duì)AXIIR誘導(dǎo)的細(xì)胞凋亡沒(méi)有影響。這說(shuō)明線粒體途徑在AXIIR誘導(dǎo)的細(xì)胞凋亡中并不是必需的,激活的Caspase8足以直接激活Caspase3引起凋亡。 我們進(jìn)一步分析了AXIIR在不同細(xì)胞類型中的表達(dá)水平,發(fā)現(xiàn)AXIIR的mRNA很容易被檢測(cè)到,而蛋白卻幾乎不能夠檢測(cè)到。提示我們：AXIIR從mRNA到蛋白的翻譯過(guò)程受到嚴(yán)格調(diào)控。研究進(jìn)一步發(fā)現(xiàn)AXIIR翻譯抑制是受其5'UTR區(qū)的調(diào)控。雖然我們嘗試用幾種死亡受體的配體、抗癌藥和紫外線照射的方法來(lái)找到可以使AXIIR的翻譯被激活的信號(hào),但是并沒(méi)有獲得成功,這仍然需要進(jìn)一步研究。 綜上所述,我們發(fā)現(xiàn)了AXIIR除了作為細(xì)胞表面受體介導(dǎo)信號(hào)以外,還分布在細(xì)胞漿中,并主要通過(guò)激活Caspase8來(lái)誘導(dǎo)細(xì)胞凋亡。我們的研究揭示了AXIIR的新功能和激活Caspase8的一種新的方式,為對(duì)凋亡發(fā)生的分子機(jī)制的研究提供了新的線索。 二Tmed2促進(jìn)小鼠前成骨細(xì)胞增殖功能研究 我們實(shí)驗(yàn)室通過(guò)對(duì)隨機(jī)siRNA文庫(kù)的篩選得到了一些與小鼠MC3T3-El前成骨細(xì)胞增殖相關(guān)的基因。我們通過(guò)基因上調(diào)和下調(diào)的方法對(duì)Tmed2基因促進(jìn)MC3T3-E1細(xì)胞增殖的功能進(jìn)行了驗(yàn)證。通過(guò)MTS、細(xì)胞周期分布檢測(cè)、相關(guān)周期蛋白表達(dá)水平檢測(cè)等方法證明該基因通過(guò)上調(diào)CyclinA的表達(dá)水平,增高M(jìn)C3T3-E1細(xì)胞S期比例,從而促進(jìn)MC3T3-E1細(xì)胞增殖加快。此外,MC3T3-E1細(xì)胞在雌激素作用下增殖加快,此時(shí)Tmed2表達(dá)水平增加,提示該基因可能參與雌激素促進(jìn)MC3T3-E1細(xì)胞增殖的作用。 此發(fā)現(xiàn)將為細(xì)胞增殖機(jī)制的研究提供新的資料。
[Abstract]:My work consists mainly of two parts:
A study of Annexin II receptor mediated apoptosis
With the understanding of apoptosis, the understanding of the molecular mechanism of apoptosis is becoming more and more thorough, but it is also found that this process is far from the original imagination, but contains complex regulatory mechanisms. Although in recent years, the biochemical mechanism of apoptosis and the gene regulation of apoptosis in the signal transduction pathway of apoptosis Significant progress has been made in other aspects, but there are still many problems that have not yet been elucidated.
The study of the human Calc phospholipid binding protein II receptor human Annexin II receptor (AXIIR), also known as C5orf39, is a protein with 193 amino acids, its encoding gene is located in the human chromosome 5p12 region. By gene sequence alignment and PCR detection, AXIIR only exists on the human chromosome fifth and does not exist. In other species, the gene.AXIIR specific to human is the first in 2006. Researchers were cloned when looking for Annexin II receptors from the human bone marrow cDNA library. It is presumed to be type I membrane protein. It is considered that AXIIR is expressed on the surface of bone marrow stromal cells and mediates the signal conduction of Annexin II to promote osteoblast formation. The functional study of the gene was only considered as a cell membrane receptor of Annexin II to mediate the signal transmission of Annexin II. The function of the gene is still in the early stage, and there is no evidence that it is associated with cell apoptosis.
Annexin II is an important member of the calcium phosphatide binding protein (Annexin) family. It exists in the cell membrane, cytoplasm and extracellular.Annexin II has extensive functions. Annexin II interacts with a series of extracellular matrix components, and participates in many cell membrane related events, such as exocytosis and endocytosis, cell adhesion, plasminogen activation, and cancer. Migration and invasion.
In this study, the expression of AXIIR gene was first expressed in the K562 cell line of human myeloid leukemia, and it was confirmed that the gene could induce apoptosis when the gene was highly expressed. It was found that AXIIR could induce apoptosis of other human cells in varying degrees, but the proportion of cell death was lower than that of K562 cells, indicating that AXIIR induced apoptosis in human cells. The universality is determined, but it is also related to a specific cell type.
In the current literature, AXIIR is used as a cell membrane receptor for Annexin II to mediate the signal transmission of Annexin II. So we need to know whether the apoptosis function of AXIIR is caused by the Annexin II signal. First, we locate AXIIR-myc in the cytoplasm by immunofluorescence staining. In addition, the proteasome The inhibitor MG132 treatment did not increase the protein level of AXIIR, which also excluded the possibility of AXIIR in the cytoplasm of the lysosome and the ubiquitination of.Annexin II in a wide range of concentrations and did not affect the apoptosis induced by AXIIR, indicating that AXIIR did not induce apoptosis as a receptor for Annexin II.
In order to understand the mechanism of AXIIR induced apoptosis, we detected the expression and activity of apoptosis pathway related proteins, mainly including several caspase and Bcl-2 family members Bcl-2 and BCL-XL. in the process of apoptosis induced by AXIIR, Caspase8,3 was activated by.Caspase8 inhibitors and also confirmed Caspase8 in AXIIR induced cell withering. The subsequent immunoprecipitation experiments showed that AXIIR binding and activating pro-Caspase8 did not depend on FADD., and the expression of FADD did not affect the ability of AXIIR to activate Caspase8. In addition, the AXIIR truncate expression experiment indicated that the carboxyl terminal of Caspase8 was necessary for the activation of Caspase8, of course ammonia The base end also played a role. Furthermore, the expression of AXIIR was not detected in the DISC induced by death receptor, indicating that AXIIR did not participate in the Caspase8 activation induced by death receptor.
In our study, AXIIR not only activates Caspase8, but also activates Caspase9 and down down the protein level of BCL-2 and BCL-XL, while Bax level does not change the.BCL-XL high expression, blocking mitochondrial pathway has no effect on AXIIR induced apoptosis. This suggests that mitochondrial pathway is not essential in AXIIR induced apoptosis. Caspase8 is sufficient to directly activate Caspase3 to induce apoptosis.
We further analyzed the expression level of AXIIR in different cell types, and found that the mRNA of AXIIR was easily detected and the protein was almost impossible to detect. It was suggested that the translation process of AXIIR from mRNA to protein was strictly regulated. The study further found that AXIIR translation inhibition was regulated by the 5'UTR region. Although we tried it, we tried. The use of several death receptor ligands, anticancer drugs and ultraviolet radiation to find signals that can enable the translation of AXIIR to be activated is not successful, which still needs further study.
In summary, we found that AXIIR, in addition to being a cell surface receptor mediated signal, is also distributed in the cytoplasm and mainly by activating Caspase8 to induce apoptosis. Our study revealed a new function of AXIIR and a new way to activate Caspase8, providing a new line for the study of the molecular mechanism of the occurrence of withering. Cable.
Two Tmed2 promotes the proliferation of mouse pre osteoblasts
In our laboratory, we screened some genes related to the proliferation of MC3T3-El pre osteoblast in mice by screening the random siRNA library. We validated the function of Tmed2 gene to promote the proliferation of MC3T3-E1 cells by gene regulation and down regulation. The detection of cell cycle distribution by MTS, detection of cell cycle distribution, and detection of related cyclin protein expression level In addition, the proliferation of MC3T3-E1 cells is accelerated by increasing the expression level of CyclinA and increasing the S phase ratio of MC3T3-E1 cells. Furthermore, the proliferation of MC3T3-E1 cells is accelerated under the action of estrogen, and the expression level of Tmed2 is increased at this time, suggesting that the gene may be involved in the role of estrogen in promoting the proliferation of MC3T3-E1 cells.
This finding will provide new information for the study of cell proliferation mechanism.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

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7 歐珍艷;NiTi合金表面制備HA/BMP、HA/COL/BMP復(fù)合涂層[D];天津大學(xué);2008年

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9 李aU，

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