【摘要】：肝癌(Hepatocellular carcinoma,HCC)在中國是死亡率高居第三的惡性腫瘤。盡管,當前我們已經(jīng)把手術切除確立為了治療早期肝癌的一線治療手段,并且輔助結合其他治療方式幫助患者。但是肝癌患者們不僅飽受肝癌耐藥之苦,同時還承擔著較高的術后復發(fā)風險,以至于最終大多都以差預后和短生存期而結束。然而根據(jù)最新研究,人們把癌癥的這種高復發(fā)率歸咎于一群叫做腫瘤干細胞(cancer stem cells,CSCs)的罪魁禍首。由于這群細胞不僅擁有腫瘤形成的始動能力和類似于胚胎干細胞的自我更新特性,還擁有高度的分化潛能,使得它們在腫瘤的耐藥已經(jīng)放療耐受中發(fā)揮重要作用。先前的研究已表明:腫瘤干細胞與腫瘤的形成、轉移、耐藥以及術后復發(fā)密切相關。同時,伴隨著關于腫瘤干細胞的研究越來越多,更多揭示了它的作用機制,一方面可以加深我們對于惡性腫瘤的研究和了解,另一方面也為我們戰(zhàn)勝癌癥提供了新的希望。我們課題組先前的研究已經(jīng)證明了,轉錄因子Nanog是肝癌干細胞的一個干性指標,它可以在體內或者體外條件下,通過IGF-1信號通路來調控肝癌干細胞的自我更新能力。同時我們也發(fā)現(xiàn)這群Nanog陽性的肝癌干細胞是肝癌患者不良預后的重要病因,它們不僅能夠維持肝癌的自我更新,還在肝癌形成以及分化中發(fā)揮重要作用。然而實際上,我們無法闡明肝癌干細胞在肝癌的發(fā)生發(fā)展以及復發(fā)中作用的詳細機制。去乙�；竤irtuin家族是一群高度依賴于煙酰胺腺嘌呤二核苷酸(NAD+)的酶。該家族包含SIRT1-7 一共七個成員,已有研究表明它們參與調控了各種細胞生物學功能,例如:細胞周期、細胞代謝、細胞增殖、細胞分化、基因組穩(wěn)定性以及腫瘤形成。同時,SIRT1也被證實在肝癌的維持以及發(fā)展中發(fā)揮關鍵作用。盡管人們發(fā)現(xiàn)在肝癌中microRNA (miRNA)-34a、miRNA-29c和c-MYC的表達能夠有效調控致癌基因SIRT1的功能,但是SIRT1如何通過肝癌干細胞在肝癌中發(fā)揮作用,這個具體的調控機制我們依舊不得而知。MEK1和MEK2屬于絲裂原活化蛋白激酶激酶(MAPKKs)家族的成員,這種激酶能夠特異性的磷酸化蘇氨酸和酪氨酸殘基進而激活絲裂原活化蛋白激酶(MAPK)基質。MEK1異常調控會引發(fā)很多疾病包括腫瘤。而且,研究發(fā)現(xiàn)MEK1在很多腫瘤形成過程中會呈現(xiàn)上調趨勢。另一方面,MEK1信號通路通常被認為是肝癌維持和發(fā)展的一個重要因素�？墒�,關于MEK1信號通路如何調控肝癌干細胞自我更新的具體機制還有待于進一步探索。我們的此次研究便揭示了一個MEK1失活時在體內外條件下,能通過促進SIRT1泛素化來加速其蛋白降解,進而抑制肝癌干細胞腫瘤形成能力的重要作用機制。為了達到此項研究目的,我們將這個課題分為四步來完成。首先,我們證明了 MEK1信號通路在肝癌干細胞自我更新和增殖中發(fā)揮重要作用:然后,我們又發(fā)現(xiàn)了在肝癌患者中MEK1的表達水平與SIRT1正相關,同時我們在肝癌干細胞中抑制或者敲除MEK1之后會導致SIRT1的明顯下調;接下來我們又闡明了 MEK1對SIRT1的調控是通過維持其蛋白水平降解而達到的,也就證明了 MEK1信號通路是通過維持SIRT1蛋白水平來調控肝癌干細胞的自我更新的;最后,我們還驚喜的發(fā)現(xiàn)了 MEK1和SIRT1蛋白的共表達水平與患者的預后密切相關。本篇文章的主要結果及結論如下所示:1.在肝癌干細胞中抑制MEK1的活性可以有效地減慢其細胞增殖能力(1)我們將Huh7-Nanogpos和PLC/PRF/5-Nanogpos的肝癌干細胞以1×103每孔的密度鋪在96孔板中,并用不同濃度的U0126進行持續(xù)處理6天,然后進行CCk-8檢測。我們發(fā)現(xiàn)U0126能夠顯著抑制肝癌干細胞的增殖,而且這種抑制效果表現(xiàn)出濃度依賴的模式。同時,細胞生存曲線排除了這些抑制濃度對于腫瘤干細胞的藥物毒性殺傷作用。(2)我們將 Huh7-Nanogpos 和 PLC/PRF/5-Nanogpos 肝癌干細胞與 U0126 或者二甲基亞砜(DMSO)共培養(yǎng)48小時之后,免疫熒光(Immunofluorescence, IF)實驗表明比起對照組U0126處理后的肝癌干細胞Ki-67表達水平顯著下降。(3)將丁酸鈉同步化處理后的Huh7-和PLC/PRF/5-Nanogpos肝癌干細胞用U0126或者二甲基亞砜(陰性對照)處理之后,細胞周期實驗發(fā)現(xiàn)U0126處理,誘發(fā)了大量肝癌干細胞將細胞周期停滯在G0/G1期,同時還伴隨著S期和G2/M期細胞的顯著減少。2. MEK1抑制劑U0126能夠明顯地降低肝癌干細胞的自我更新能力(1)我們發(fā)現(xiàn)在肝癌干細胞中抑制MEK1活性后能夠顯著下調其克隆形成及成球能力,且此結論已在兩個細胞系中得到重復驗證。而與肝癌干細胞相反的是,低濃度的U0126處理并不能降低非肝癌干細胞的克隆形成及成球能力。(2)免疫印跡實驗表明在肝癌干細胞中抑制MEK1活性后能夠顯著抑制干性指標例如SOX2、OCT4以及Nanog的表達。(3)當我們把U0126處理后的肝癌干細胞以不同濃度梯度1×102、1×103,、1×104皮下注入NOD-SCID小鼠后,發(fā)現(xiàn)相對于對照組其腫瘤形成及生長能力被顯著抑制。3.敲除MEK1能顯著抑制肝癌干細胞的自我更新和增殖能力(1)蛋白水平檢測發(fā)現(xiàn)在肝癌干細胞中敲除MEK1后能顯著下調MEK1及磷酸化ERK1/2的蛋白表達水平,但是ERK1/2的表達水平?jīng)]有明顯差異。(2)在肝癌干細胞中用兩種不同的MEK1干擾慢病毒敲除MEK1后,發(fā)現(xiàn)其增殖能力被明顯抑制。(3)在肝癌干細胞中敲除MEK1后能顯著抑制肝癌干細胞的成球和克隆形成能力。(4)免疫印跡實驗表明在肝癌干細胞中敲除MEK1后干性相關蛋白的表達例如Nanog、OCT4、c-Myc和SOX2都被明顯抑制。4.抑制或敲除MEK1能顯著降低SIRT1的表達(1)通過檢測我們發(fā)現(xiàn)sirtuin家族中,只有SIRT1和SIRT7在肝癌干細胞中的表達水平高于非肝癌干細胞,然而用U0126抑制MEK1活性之后只有SIRT1的表達水平被降低了。(2)正如免疫印跡實驗結果所示:U0126對于肝癌干細胞中SIRT1表達水平的下調作用不僅與藥物作用時間正相關,還與藥物作用濃度正相關。(3)另一種MEK1抑制劑PD98059同樣證實了在肝癌干細胞中抑制MEK1活性能有效地降低SIRT1蛋白表達。(4)在肝癌干細胞中敲除MEK1后能有效地降低SIRT1蛋白表達水平。5. MEK1信號通路通過調控組蛋白去乙�；窼IRT1來促進肝癌干細胞的自我更新和腫瘤形成能力(1)在非肝癌干細胞中過表達SIRT1后能顯著地提高其克隆形成及成球能力,但是通過抑制MEK1信號通路的活性能夠有效地逆轉這種激活效果。(2)在肝癌干細胞中抑制MEK1活性后,這群細胞的克隆形成及成球能力被顯著抑制,然后通過過表達SIRT1蛋白水平能夠恢復它們的克隆形成和成球能力。(3)在體內腫瘤形成模型中,注射了 MEK1活性抑制的肝癌干細胞的NOD-SCID小鼠其腫瘤形成能力比起陽性對照組差很多。同時,我們還發(fā)現(xiàn)通過過表達SIRT1水平能夠部分恢復肝癌干細胞在NOD-SCID小鼠體內的腫瘤形成能力。6. MEK1信號通路是通過抑制SIRT1的泛素化水平來抑制蛋白酶體的降解作用,從而維持SIRT1蛋白的表達水平(1)我們將肝癌干細胞分為四組:一組為陰性對照即與二甲基亞砜共培養(yǎng),一組為陽性對照即與蛋白酶體抑制劑MG-132共培養(yǎng),一組與MEK1抑制劑U0126共培養(yǎng),一組為MEK1敲除的肝癌干細胞。這四組細胞培養(yǎng)48小時后,同時加入放線菌酮(CHX)處理不同時間,最后檢測SIRT1蛋白水平發(fā)現(xiàn):相對于陰性對照,MEK1抑制或者敲除后,肝癌干細胞中SIRT1蛋白的降解速度明顯加快,而抑制蛋白酶體后SIRT1蛋白的降解速度被顯著減慢。(2)在肝癌干細胞中抑制或者敲除MEK1后,會激活蛋白酶體活性從而有效地促進SIRT1蛋白的降解。(3)免疫共沉淀實驗(Co-IP)結果顯示:在肝癌干細胞中抑制或者敲除MEK1后,SIRT1的泛素化狀態(tài)被極大地提高,導致了SIRT1蛋白的降解。7.在肝癌患者中MEK1/SIRT1的高水平表達與其不良預后呈現(xiàn)顯著的正相關關系(1)我們在148例肝癌患者的組織標本中,通過免疫組化(Immunohistochemistry,IHC)檢測了 p-MEK1、SIRT1和Nanog蛋白的表達水平。其中同時高水平表達p-MEK1/SIRT1的患者有48例,而同時低水平表達p-MEK1/SIRT1的患者有39例。通過卡方檢驗(Person chi-square test)統(tǒng)計分析發(fā)現(xiàn)P值為0.035,也就是說在肝癌患者中MEK1與SIRT1的表達水平是相關的。同樣的方法也證實了 Nanog的表達水平與MEK1/SIRT1的表達在肝癌患者中是相關的,其中兩個P值分別是0.013和0.038。(2)我們將回訪搜集到的148例肝癌患者的臨床病理信息進行統(tǒng)計和分析發(fā)現(xiàn):MEK1/SIRT1的共表達高低狀態(tài)與肝癌患者的年齡、性別、血清甲胎蛋白(AFP)水平、腫瘤間質增生、腫瘤壞死及腫瘤復發(fā)沒有統(tǒng)計學關聯(lián);但是MEK1/SIRT1同時表達水平的高低與肝癌患者的腫瘤大小(p=0.012)、血管癌栓(p0.001)、包膜浸潤(p=p=0.048)和臨床腫瘤分期(p0.001)都具有顯著的統(tǒng)計學關系。(3)我們將患者生存周期與預后聯(lián)合MEK1和SIRT1的表達水平進行分析發(fā)現(xiàn)兩者存在顯著關聯(lián),同時高水平表達MEK1和SIRT1的肝癌患者比起低水平的患者往往預后更差。總而言之,我們的研究結果表明:1.在肝癌患者中MEK1表達水平與SIRT1表達水平顯著相關,同時高水平表達MEK1/SIRT1的肝癌患者提示預后不佳和較短的生存期。2.激活MEK1信號通路能促進肝癌干細胞增殖、自我更新和腫瘤形成特征,敲除或者抑制MEK1能顯著降低干性標記物的表達水平。3. MEK1信號通路是通過SIRT1蛋白表達來調控肝癌干細胞的增殖、腫瘤形成和自我更新。4. MEK1通過泛素化SIRT1控制蛋白酶體降解來維持SIRT1蛋白的表達水平。5.可以將MEK1/SIRT1作為判斷肝癌患者情況的指標,同時MEK1信號通路可以作為一個治療肝癌的潛在靶標。綜上所述,本研究證明了無論在體內還是體外環(huán)境下,抑制或者干擾MEK1能夠有效地減緩肝癌干細胞的腫瘤形成速度。同時,我們還證實了 MEK1信號通路是通過維持SIRT1蛋白水平來促進肝癌干細胞的自我更新和腫瘤形成。從機制上闡明了MEK1信號通路通過抑制SIRT1的泛素化水平來抑制蛋白酶體的降解,從而維持較高的SIRT1蛋白表達水平。肝癌患者臨床預后數(shù)據(jù)分析表明:高表達MEK1和SIRT1的肝癌患者與預后不良相關。我們的研究提示MEK1-SIRT1可以作為一個重要的診斷指標,同時抑制MEK1可能是靶向肝癌干細胞治療的一個潛在作用靶點。
[Abstract]:Hepatocellular carcinoma (HCC) is the third highest death rate in China. Although we have now established surgical excision to treat early liver cancer as a first-line treatment and assist patients with other treatment methods, patients with liver cancer are not only suffering from the resistance of liver cancer, but also in the same way. The high risk of postoperative recurrence ends up mostly in poor prognosis and short life. However, according to the latest research, the high recurrence rate of cancer is blamed for a group of cancer stem cells, CSCs, because the cells do not only have the ability to start the tumor and similar to the embryo. The self renewal characteristics of stem cells also have high differentiation potential that make them play an important role in the tolerance of cancer to radiotherapy. Previous studies have shown that cancer stem cells are closely related to tumor formation, metastasis, drug resistance, and postoperative recurrence, and more and more research on cancer stem cells is accompanied by more and more research. On the one hand, it can deepen our research and understanding of malignant tumors, and on the other hand, it provides new hope for us to overcome cancer. Our previous research group has shown that the transcription factor Nanog is a dry indicator of liver cancer stem cells, which can be used in vivo or in vitro. IGF-1 signaling pathway is used to regulate the self-renewal capacity of liver cancer stem cells. We also found that this group of Nanog positive liver cancer stem cells is an important cause of poor prognosis in the patients with liver cancer. They not only maintain the self renewal of liver cancer, but also play an important role in the formation and differentiation of liver cancer. However, we can not explain the fact. A detailed mechanism for the role of liver cancer stem cells in the development and recurrence of liver cancer. The deacetylase sirtuin family is a group of enzymes highly dependent on nicotinamide adenine dinucleotide (NAD+). The family contains seven members of the SIRT1-7, which has been studied to show that they are involved in the regulation of various cellular biological functions, such as cell cycle. Cell metabolism, cell proliferation, cell differentiation, genomic stability and tumor formation, and SIRT1 also proved to play a key role in the maintenance and development of liver cancer. Although it is found that the expression of microRNA (miRNA) -34a, miRNA-29c and c-MYC can effectively regulate the function of the oncogene SIRT1 in liver cancer, but how SIRT1 passes through it Liver cancer stem cells play a role in liver cancer. This specific regulatory mechanism remains unknown that.MEK1 and MEK2 belong to the members of the mitogen activated protein kinase kinase (MAPKKs) family. This kinase can specifically phosphorylate threonine and tyrosine residues and then activate the abnormality of the matrix.MEK1 of the mitogen activated protein kinase (MAPK) matrix. In addition, the MEK1 signaling pathway is often considered to be an important factor in the maintenance and development of liver cancer. However, the specific mechanism of how the MEK1 signaling pathway regulates the self-renewal of liver cancer stem cells remains to be found in the MEK1 signaling pathway. Further exploration. Our present study reveals the important mechanism of a MEK1 inactivation in vivo and in vitro, which can accelerate its protein degradation by promoting SIRT1 ubiquitination, and then inhibit the ability of liver cancer stem cell tumor formation. In order to achieve this goal, we divide the subject into four steps. First, we The MEK1 signaling pathway plays an important role in the self renewal and proliferation of liver cancer stem cells. Then, we also found that the expression level of MEK1 in liver cancer patients is positively related to SIRT1, and our inhibition or knockout of MEK1 in liver cancer stem cells will lead to the obvious downregulation of SIRT1; then we also elucidate MEK1 to SIRT1. The regulation is achieved by maintaining protein level degradation, and it is also proved that the MEK1 signaling pathway regulates the self renewal of liver cancer stem cells by maintaining the SIRT1 protein level. Finally, we also surprised to find that the co expression level of MEK1 and SIRT1 proteins is closely related to the patient's preconditioning. The main results of this article are as follows The conclusions are as follows: 1. inhibition of MEK1 activity in liver cancer stem cells can effectively slow down its cell proliferation ability (1) we spread the Huh7-Nanogpos and PLC/PRF/5-Nanogpos liver cancer stem cells in 96 orifice with the density of 1 x 103 per pore, and carry on the continuous treatment for 6 days with different concentrations of U0126, and then carry out CCk-8 detection. We found U0126 It can significantly inhibit the proliferation of liver cancer stem cells, and this inhibitory effect shows a concentration dependent pattern. At the same time, the cell survival curve excludes the toxic effects of these inhibitory concentrations on tumor stem cells. (2) we put Huh7-Nanogpos and PLC/PRF/5-Nanogpos liver cancer stem cells with U0126 or two methyl sulfoxide (DMS). O) after 48 hours co culture, the immunofluorescence (Immunofluorescence, IF) experiment showed that the Ki-67 expression level of liver cancer stem cells decreased significantly compared with the control group after U0126 treatment. (3) Huh7- and PLC/PRF/5-Nanogpos liver cancer stem cells treated with sodium butyrate were treated with U0126 or two methyl sulfoxide (negative control), and the cell cycle was true. It was found that U0126 treatment induced a large number of liver cancer stem cells to stagnate the cell cycle in the G0/G1 phase, and also accompanied by the significant reduction of.2. MEK1 inhibitor U0126 in S and G2/M phase cells (1) the self-renewal capacity of liver cancer stem cells was significantly reduced (1) we found that after the inhibition of MEK1 activity in the stem cells of the liver cancer, the clones could be significantly reduced. The formation and formation of ball forming ability, and this conclusion has been repeated in two cell lines. Contrary to liver cancer stem cells, low concentration of U0126 treatment does not reduce the cloning and formation of non hepatoma stem cells. (2) immunoblotting experiments showed that the inhibition of MEK1 activity in liver cancer stem cells could significantly inhibit the dry index. Such as the expression of SOX2, OCT4 and Nanog. (3) when U0126 treated liver cancer stem cells were injected with different concentrations of 1 x 102,1 x 103 and 1 x 104 subcutaneous injected into NOD-SCID mice, it was found that the tumor formation and growth ability of the cancer stem cells compared with the control group was significantly inhibited by.3. knockout MEK1 can significantly inhibit the self renewal and proliferation of liver cancer stem cells (1) Protein level detection showed that after knockout MEK1 in liver cancer stem cells, the protein expression level of MEK1 and phosphorylated ERK1/2 was significantly reduced, but the expression level of ERK1/2 was not significantly different. (2) the proliferation ability was obviously suppressed after two different MEK1 interfering lentivirus knockout MEK1 in liver cancer stem cells. (3) in liver cancer stem cells Knockout MEK1 significantly inhibited the formation of ball and clonogenic ability of liver cancer stem cells. (4) immunoblotting experiments showed that the expression of dry related proteins after knockout MEK1 in liver cancer stem cells such as Nanog, OCT4, c-Myc and SOX2 were significantly inhibited by.4. inhibition or knockout MEK1 can significantly reduce the expression of SIRT1 (1) through detection we found sirtuin home. The expression level of only SIRT1 and SIRT7 in HCC stem cells was higher than that of non hepatoma stem cells. However, only SIRT1 expression level was reduced after U0126 inhibition of MEK1 activity. (2) the downregulation of U0126 to the level of SIRT1 expression in liver cancer stem cells is not only positively related to the time of drug action. There is also a positive correlation with drug concentration. (3) another MEK1 inhibitor PD98059 also confirms that the inhibition of MEK1 activity in liver cancer stem cells can effectively reduce the expression of SIRT1 protein. (4) after the knockout of MEK1 in the liver cancer stem cells, the SIRT1 protein expression level is effectively reduced by the.5. MEK1 signaling pathway by regulating the histone deacetylase SIRT1 Promoting self renewal and tumorigenesis of liver cancer stem cells (1) can significantly increase their cloning and formation after overexpressing SIRT1 in non hepatoma stem cells, but it can effectively reverse the activation effect by inhibiting the activity of MEK1 signaling pathway. (2) cloning of this group of cells after the inhibition of MEK1 activity in liver cancer stem cells The ability to form and form the ball was significantly suppressed, and then their cloning and forming ability could be restored through the expression of SIRT1 protein. (3) in the tumor formation model of the body, the NOD-SCID mice injected with MEK1 active liver cancer stem cells were much less capable of tumorigenesis than the positive control group. Overexpression of SIRT1 level can partially restore the tumor formation ability of liver cancer stem cells in NOD-SCID mice. The.6. MEK1 signaling pathway inhibits the degradation of proteasome by inhibiting the ubiquitination level of SIRT1, thus maintaining the expression level of SIRT1 protein (1) we divide the liver cancer stem cells into four groups: a group of negative controls Two methyl sulfoxide co culture, a group of positive control and proteasome inhibitor MG-132 co culture, a group of MEK1 inhibitor U0126 co culture, a group of MEK1 knockout liver cancer stem cells. These four groups of cell culture 48 hours after the addition of actinomycone (CHX) treatment at the same time, the final detection of SIRT1 protein levels: relative to negative pairs After MEK1 inhibition or knockout, the degradation rate of SIRT1 protein in liver cancer stem cells accelerated significantly, and the degradation rate of SIRT1 protein was significantly slowed down after the inhibition of proteasome. (2) proteasome activity could be activated to effectively promote the degradation of SIRT1 protein in the stem cells of liver cancer. (3) immunoprecipitation experiment (Co) -IP) the results showed that after the suppression or knockout of MEK1 in the liver cancer stem cells, the ubiquitination of SIRT1 was greatly improved, resulting in the degradation of SIRT1 protein in the liver cancer patients, the high expression of MEK1/SIRT1 was positively correlated with the poor prognosis (1) we used immunohistochemistry in the tissue specimens of 148 patients with liver cancer. (Immunohistochemistry, IHC) detected the expression level of p-MEK1, SIRT1 and Nanog protein. At the same time, there were 48 patients with high level of p-MEK1/SIRT1 and 39 patients with low-level expression of p-MEK1/SIRT1. The statistical analysis of Person chi-square test (Person chi-square test) found that P was 0.035, that is to say, MEK1 and in the patients with liver cancer. The expression level of SIRT1 is related. The same method also confirms that the expression level of Nanog is associated with the expression of MEK1/SIRT1 in the patients with liver cancer, of which two P values are 0.013 and 0.038. (2). We will return the clinicopathological information of the 148 patients with liver cancer and find that the co expression of MEK1/SIRT1 is high and low. Status was not associated with age, sex, serum alpha fetoprotein (AFP) level, tumor interstitial hyperplasia, tumor necrosis and tumor recurrence, but the level of MEK1/SIRT1 simultaneous expression was associated with tumor size (p=0.012), vascular tumor thrombus (p0.001), membrane infiltration (p=p=0.048) and clinical tumor staging (p0.001) in patients with liver cancer. There was a significant statistical relationship. (3) we found that there was a significant association between the MEK1 and SIRT1 expression levels associated with the patient's survival cycle and prognosis, and the high level of MEK1 and SIRT1 patients with liver cancer tended to have worse prognosis than those with lower levels. All in all, our results showed that 1. in patients with liver cancer, MEK1 The expression level is significantly related to the level of SIRT1 expression, while patients with high level of MEK1/SIRT1 expression of liver cancer suggest that poor prognosis and shorter survival time.2. activation MEK1 signaling pathway can promote the proliferation of liver cancer stem cells, self renewal and tumor formation characteristics, knockout or inhibition of MEK1 can significantly reduce the expression level of.3. MEK1 letters of dry markers. The signal transduction pathway regulates the proliferation of liver cancer stem cells through the expression of SIRT1 protein. Tumor formation and self-renewal.4. MEK1 control the proteasome degradation through ubiquitination of SIRT1 to maintain the expression level of SIRT1 protein,.5. can be used as an indicator to determine the condition of liver cancer patients, and MEK1 signaling pathway can be used as a treatment for liver cancer. In summary, this study demonstrates that inhibition or interference with MEK1 can effectively slow down both in vivo and in vitro
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.7

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本文編號：2160204