1、 AnnexinⅡ受体介导细胞凋亡的研究 2、Tmed2促进小鼠前成骨细胞增殖的研究

发布时间：2018-07-07 14:28

本文选题：C5of39 + AXIIR　；参考：《北京协和医学院》2012年博士论文

【摘要】：我的工作主要包含两部分内容：一Annexin Ⅱ受体介导细胞凋亡的研究随着人们对凋亡的认识逐渐深入,对凋亡发生的分子机制的了解也越来越透彻,但同时也发现这一过程远非原来想象的那样简单,而是包含了复杂的调控机制。尽管近几年在凋亡信号转导途径、凋亡的生化反应机制以及凋亡的基因调控等方面的研究都取得了显著的进展,但仍有许多问题迄今尚未阐明。本文的研究对象人类钙磷脂结合蛋白Ⅱ受体human Annexin Ⅱ receptor (简称AXIIR),又称为C5orf39,是一个具有193个氨基酸的蛋白质,其编码基因位于人类染色体5p12区。通过基因序列比对和PCR检测认为AXIIR仅存在于人类第5号染色体上而不存在于其他种属,是人类所特有的基因。AXIIR最先是在2006年,研究者从人类骨髓cDNA文库中寻找Annexin Ⅱ的受体时被克隆出来的,推测为Ⅰ型膜蛋白,研究认为AXIIR表达在骨髓基质细胞的表面,介导Annexin Ⅱ的信号传导而促进成骨细胞形成。而随后对于该基因的功能研究结果也仅仅是认为其作为Annexin Ⅱ的细胞膜受体来介导Annexin Ⅱ的信号传递。该基因的功能研究还处于早期阶段,更没有任何证据证明其与细胞凋亡相关。 Annexin Ⅱ是钙磷脂结合蛋白(Annexin)家族中的一个重要成员,存在于细胞膜、胞质和胞外。Annexin Ⅱ具有广泛的功能,Annexin Ⅱ与一系列细胞外基质成分相互作用,参与许多细胞膜相关事件,如细胞外吐和内吞,细胞黏附,纤溶酶原激活,肿瘤迁移和侵袭。在本研究中,首先通过在人髓性白血病K562细胞系中过表达AXIIR基因,确证该基因高表达时能够诱导细胞凋亡。进一步发现AXIIR能引起其他人类细胞发生不同程度的凋亡,但细胞死亡比例均低于K562细胞,说明AXIIR诱导人类细胞凋亡存在一定的普遍性,但也与特定细胞类型有关。在目前的文献报道中,AXIIR均是作为Annexin Ⅱ的细胞膜受体来介导Annexin Ⅱ的信号传递,所以我们需要了解AXIIR的诱导细胞凋亡功能是否由Annexin Ⅱ信号引起。首先,我们通过免疫荧光染色方法定位AXIIR-myc分布在细胞浆中。另外,蛋白酶体抑制剂MG132处理并未增加AXIIR的蛋白水平,这也排除了胞浆内的AXIIR在被溶酶体和泛素化降解的可能性。Annexin Ⅱ在一个广泛的浓度范围内并不能影响AXIIR诱导的细胞凋亡作用,说明AXIIR并不是作为Annexin Ⅱ的受体来诱导凋亡的。为了了解AXIIR诱导细胞凋亡的机制,我们检测了凋亡通路相关蛋白的表达量和活性,主要包括几种caspase和Bcl-2家族成员Bcl-2和BCL-XL。在AXIIR引起的细胞凋亡过程中,Caspase8,3均被激活。Caspase8抑制剂的使用也证实了Caspase8在AXIIR诱导细胞凋亡功能中发挥的重要作用。而随后的免疫沉淀实验表明AXIIR结合并激活pro-Caspase8,却并不依赖于FADD。此外,下调FADD的表达并不会影响AXIIR激活Caspase8的能力。另外AXIIR截短体表达实验说明Caspase8的羧基端对于Caspase8的激活是必需的,当然氨基端也发挥了一定作用。再者,在死亡受体诱导形成的DISC中并未检查到AXIIR的表达,说明AXIIR没有参与死亡受体诱导的Caspase8激活。在我们的研究中,AXIIR不仅激活Caspase8,也同时激活了Caspase9和下调了BCL-2和BCL-XL的蛋白水平,而Bax水平无变化。BCL-XL高表达阻断线粒体途径对AXIIR诱导的细胞凋亡没有影响。这说明线粒体途径在AXIIR诱导的细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。我们进一步分析了AXIIR在不同细胞类型中的表达水平,发现AXIIR的mRNA很容易被检测到,而蛋白却几乎不能够检测到。提示我们：AXIIR从mRNA到蛋白的翻译过程受到严格调控。研究进一步发现AXIIR翻译抑制是受其5'UTR区的调控。虽然我们尝试用几种死亡受体的配体、抗癌药和紫外线照射的方法来找到可以使AXIIR的翻译被激活的信号,但是并没有获得成功,这仍然需要进一步研究。综上所述,我们发现了AXIIR除了作为细胞表面受体介导信号以外,还分布在细胞浆中,并主要通过激活Caspase8来诱导细胞凋亡。我们的研究揭示了AXIIR的新功能和激活Caspase8的一种新的方式,为对凋亡发生的分子机制的研究提供了新的线索。二Tmed2促进小鼠前成骨细胞增殖功能研究我们实验室通过对随机siRNA文库的筛选得到了一些与小鼠MC3T3-El前成骨细胞增殖相关的基因。我们通过基因上调和下调的方法对Tmed2基因促进MC3T3-E1细胞增殖的功能进行了验证。通过MTS、细胞周期分布检测、相关周期蛋白表达水平检测等方法证明该基因通过上调CyclinA的表达水平,增高MC3T3-E1细胞S期比例,从而促进MC3T3-E1细胞增殖加快。此外,MC3T3-E1细胞在雌激素作用下增殖加快,此时Tmed2表达水平增加,提示该基因可能参与雌激素促进MC3T3-E1细胞增殖的作用。此发现将为细胞增殖机制的研究提供新的资料。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

【共引文献】