發(fā)布時(shí)間：2018-04-27 01:30

  本文選題：干擾素 + 誘導(dǎo)�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2017年碩士論文

【摘要】：腫瘤細(xì)胞與免疫系統(tǒng)的相互作用被歸納為3E理論(Dunn et al.,2004;Schreiber et al.,2011),包括 Eradication(消滅),Equilibrium(平衡),Evasion(逃逸)。在機(jī)體中,腫瘤細(xì)胞在免疫因素的作用下會(huì)出現(xiàn)以上三種生物現(xiàn)象:腫瘤細(xì)胞被免疫系統(tǒng)消滅清除;在免疫系統(tǒng)的監(jiān)視下腫瘤細(xì)胞與機(jī)體達(dá)到均勢;腫瘤細(xì)胞逃脫免疫系統(tǒng)的控制進(jìn)而侵害全身(Romero et al.,2014)。免疫誘導(dǎo)的腫瘤休眠(tumordormancy),是指腫瘤細(xì)胞在機(jī)體免疫系統(tǒng)作用下,出現(xiàn)細(xì)胞周期阻滯、增殖相關(guān)基因表達(dá)下調(diào)、代謝減慢等現(xiàn)象。此種現(xiàn)象是腫瘤與機(jī)體免疫系統(tǒng)相互作用的過程中進(jìn)入平衡穩(wěn)定狀態(tài)的集中體現(xiàn)(Sosa et al.,2014),與免疫監(jiān)視這一假說相呼應(yīng)。目前的科學(xué)研究由于缺少有效公認(rèn)的腫瘤休眠的體內(nèi)外模型,免疫誘導(dǎo)的腫瘤休眠缺乏有效的手段研究其深入的機(jī)制。在對(duì)于腫瘤細(xì)胞休眠這一領(lǐng)域的科學(xué)研究中,目前的研究成果僅限于通過運(yùn)用中和抗體和轉(zhuǎn)基因動(dòng)物探究該現(xiàn)象的存在,及確定與之相關(guān)的免疫分子如Interferonγ(伽馬干擾素,IFNγ)。Interferonγ作為免疫效應(yīng)分子,在腫瘤免疫中,IFNγ主要由腫瘤微環(huán)境中的殺傷性T細(xì)胞(CTLs)所分泌,對(duì)控制腫瘤的生長具有重要的作用(Akira etal.,2006;Dunn etal.,2006),可能會(huì)引起腫瘤細(xì)胞的休眠。但I(xiàn)FNγ如何作用于腫瘤細(xì)胞休眠,其內(nèi)在的作用機(jī)制尚未闡明。腫瘤細(xì)胞具有異質(zhì)性,不同的亞群具有不同的特性。其中,腫瘤干細(xì)胞即腫瘤再生細(xì)胞(tumorigenic cells,TRCs)是一群具有很強(qiáng)成瘤能力和增殖分裂能力的細(xì)胞,且表現(xiàn)為較強(qiáng)的耐藥特性,與腫瘤的復(fù)發(fā)轉(zhuǎn)移具有緊密關(guān)系(Enver et al.,2009;Visvader and Lindeman,2008)。目前尚未有研究證明腫瘤干細(xì)胞是否存在腫瘤休眠這一現(xiàn)象,且其內(nèi)在的機(jī)制也不清楚。本課題研究旨在利用本課題組已經(jīng)建立起來的3D(軟三維膠體外擴(kuò)增培養(yǎng)腫瘤再生細(xì)胞)技術(shù),來探究以下三個(gè)問題:IFNγ能否誘導(dǎo)腫瘤的休眠;IFNγ可具體誘導(dǎo)哪群腫瘤細(xì)胞休眠;IFNγ誘導(dǎo)腫瘤細(xì)胞休眠的機(jī)制。本論文分為三部分:1.IFNγ體外誘導(dǎo)腫瘤干細(xì)胞的休眠的探究;2.IFNγ誘導(dǎo)腫瘤干細(xì)胞休眠的機(jī)制探究;3.運(yùn)用相關(guān)分子的抑制劑逆轉(zhuǎn)腫瘤休眠對(duì)腫瘤細(xì)胞影響的探究第一部分:IFNγ體外誘導(dǎo)腫瘤干細(xì)胞的休眠的研究目的:研究IFNγ是否可誘導(dǎo)腫瘤干細(xì)胞休眠,并區(qū)別于傳統(tǒng)2D培養(yǎng)腫瘤細(xì)胞;且探究IFNγ誘導(dǎo)的腫瘤干細(xì)胞是否存在對(duì)化療藥物甲氨蝶呤(Methotrexate)和紫杉醇(Paclitaxel)的耐藥性。方法:該研究選用本實(shí)驗(yàn)組之前使用過的小鼠黑色素瘤細(xì)胞系B16及三文魚來源的纖維蛋白原建立的3D腫瘤干細(xì)胞培養(yǎng)方法(Liu et al.,2012),來建立體外模型。1.IFNγ處理小鼠來源的B16黑色素瘤細(xì)胞、H22肝癌細(xì)胞系及CT26結(jié)腸癌細(xì)胞系:1)1OOng/ml IFNγ分別作用于傳統(tǒng)2D培養(yǎng)的B16細(xì)胞、H22細(xì)胞及CT26細(xì)胞72h;2)100ng/ml IFNγ作用于3D培養(yǎng)的B16細(xì)胞、H22細(xì)胞及CT26細(xì)胞72h,檢測其對(duì)兩種不同培養(yǎng)模式的三種腫瘤細(xì)胞增殖及凋亡代謝情況。2.化療藥物甲氨蝶呤(MTX)和紫杉醇(Paclitaxel)分別處理3D培養(yǎng)的B16細(xì)胞:1)細(xì)胞對(duì)照組:2D培養(yǎng)B16細(xì)胞+PBS;2D培養(yǎng)B16細(xì)胞+IFNγ;2D培養(yǎng)B16細(xì)胞+MTX;2D培養(yǎng)B16細(xì)胞+IFNγ+MTX;2)實(shí)驗(yàn)細(xì)胞組:3D培養(yǎng)B16細(xì)胞+ MTX;3D培養(yǎng)B16細(xì)胞+IFNγ+MTX;IFNγ預(yù)處理72h的3D培養(yǎng)B16細(xì)胞+MTX+ IFNγ;IFNγ預(yù)處理72h后3D培養(yǎng)B16細(xì)胞+MTX。紫杉醇分組同甲氨蝶呤。結(jié)果:1.用不同濃度的IFNγ處理傳統(tǒng)2D培養(yǎng)皿和3D培養(yǎng)的B16細(xì)胞、H22細(xì)胞和CT26細(xì)胞96h后,2D腫瘤細(xì)胞的凋亡明顯,3D細(xì)胞未見明顯凋亡。3D細(xì)胞主要表現(xiàn)為克隆變小,細(xì)胞周期變慢,葡萄糖消耗量下降,及增殖相關(guān)基因下調(diào)。2.本研究選用常規(guī)化療藥物甲氨蝶呤和紫杉醇處理IFNγ誘導(dǎo)的休眠的腫瘤細(xì)胞和正常培養(yǎng)的2D細(xì)胞和3D細(xì)胞,發(fā)現(xiàn)休眠的腫瘤細(xì)胞有更強(qiáng)的耐藥性。第二部分:IFNγ在誘導(dǎo)腫瘤細(xì)胞休眠的機(jī)制目的:在已經(jīng)建立的IFNγ體外誘導(dǎo)腫瘤休眠模型的條件下探究其內(nèi)在機(jī)制方法:在已經(jīng)建立IFNγ體外誘導(dǎo)腫瘤休眠的模型中,我們運(yùn)用PCR,Western-blot,質(zhì)粒構(gòu)建,病毒包裝,CRISPR-Cas9,免疫熒光,流式分析、分選等分子生物學(xué)、細(xì)胞生物學(xué)的手段來鑒定并驗(yàn)證相關(guān)信號(hào)通路及機(jī)制。結(jié)果:1.根據(jù)文獻(xiàn)報(bào)道及PCR驗(yàn)證,我們確定了吲哚胺2,3雙加氧酶(indoleamine 2,3-dioxygenase,IDO)在被IFNγ處理過的B16腫瘤細(xì)胞中發(fā)生了很大的改變(Takikawa et al.,1990),并且TRCs被IFNγ處理后IDO上調(diào)相較于普通2D細(xì)胞更為明顯,說明IDO在TRCs的休眠的過程中具有重要作用;2.我們將過表達(dá)IDO的TRCs進(jìn)行分析,發(fā)現(xiàn)其細(xì)胞克隆大小及細(xì)胞周期均發(fā)生較明顯的變化,出現(xiàn)了克隆變小及周期變慢的現(xiàn)象;3.IDO是一種催化酶,主要催化色氨酸(Tryptophan,Trp)的代謝,使其代謝成為犬尿氨酸(kynurenine,Kyn),我們用200uM到500uM濃度的Kyn處理TRCs也得到了克隆變小及周期減慢的結(jié)果,說明Kyn在TRCs的休眠中扮演著重要的角色;4.我們從文獻(xiàn)中得知,Kyn為多環(huán)芳香烴受體(Aryl hydrocarbon receptor,Ahr)的配體,可以導(dǎo)致Ahr誘導(dǎo)下游基因的表達(dá)(Opitz et al.,2011),其中可能包括細(xì)胞周期抑制相關(guān)的蛋白,我們通過免疫熒光、western-blot及熒光素酶實(shí)驗(yàn)確定了 Ahr在TRCs中得到激活;5.同時(shí)我們用western-blot篩選相關(guān)周期蛋白,發(fā)現(xiàn)在IFNγ處理后,Cdknlb(cyclin-dependent kinase inhibitor 1B,P27kip)出現(xiàn)了 很明顯的變化,而其他周期蛋白沒有較為明顯的變化,說明P27可能在休眠中扮演重要的角色;6.我們用Kyn處理TRCs72小時(shí)后用western-blot檢測P27的表達(dá)水平,發(fā)現(xiàn)其出現(xiàn)了明顯上調(diào),并同時(shí)調(diào)取P27基因的啟動(dòng)子,構(gòu)建熒光素報(bào)告質(zhì)粒,并同時(shí)聯(lián)合AHR基因行瞬時(shí)轉(zhuǎn)染后,用Kyn處理后檢測,發(fā)現(xiàn)熒光素信號(hào)在AHR、Kyn及IFNγ處理后有明顯增強(qiáng),說明IFNγ對(duì)P27的調(diào)控主要是通過IDO-Kyn-AHR這一信號(hào)通路;7.經(jīng)典的 IFNγ 信號(hào)主要通過 STAT1(signal transducer and activator of transcription 1)發(fā)揮作用,并且主要對(duì)腫瘤細(xì)胞引起凋亡的作用,我們通過western-blot及免疫熒光及流式證實(shí)了這一信號(hào)通路在普通培養(yǎng)的B16中存在,但在休眠的細(xì)胞中,這一通路被明顯抑制,而且我們通過Ip-western發(fā)現(xiàn)IFNy-Stat1這一信號(hào)通路與IFNγ-IDO-AHR-P27這一信號(hào)通路存在交互作用,即P27結(jié)合p-STAT1蛋白阻止其入核;8.我們也在后來建立的IDO、AHR、P27敲低敲除細(xì)胞系中進(jìn)一步驗(yàn)證了這一結(jié)論。第三部分:在體外實(shí)驗(yàn)中運(yùn)用相關(guān)分子的抑制劑逆轉(zhuǎn)腫瘤休眠目的:通過運(yùn)用相關(guān)分子的抑制劑聯(lián)合IFNy看能否打破腫瘤細(xì)胞的休眠,并對(duì)腫瘤治療提出新的想法方法:運(yùn)用3D培養(yǎng)、western-blot及流式檢測等相關(guān)手段檢測相關(guān)分子抑制劑聯(lián)合IFNγ處理后的腫瘤細(xì)胞的增殖凋亡情況。結(jié)果:1.我們聯(lián)合 IDO 抑制劑 1-MT(1-Methyl-L-tryptophan)及 IFNγ 處理3D培養(yǎng)的B16細(xì)胞后,其出現(xiàn)克隆數(shù)減少,克隆大小變小的表現(xiàn),并在整體細(xì)胞水平及分子水平出現(xiàn)了凋亡的增加,說明IFNγ聯(lián)合IDO抑制劑可以有效的打破休眠,殺傷腫瘤;2.我們聯(lián)合AHR抑制劑DMF(3',4'-Dimethoxyflavone)及IFNγ處理3D培養(yǎng)的B16細(xì)胞后,得到了與IDO抑制劑相同的結(jié)果,即休眠打破凋亡增加。說明這兩個(gè)抑制劑在今后的免疫治療中可能有較好的前景。總結(jié):腫瘤休眠這一現(xiàn)象在腫瘤的發(fā)生、發(fā)展、轉(zhuǎn)移及治療預(yù)后中均有重要的意義,免疫所引起的休眠,更是這一現(xiàn)象的很好的體現(xiàn),我們通過運(yùn)用3D培養(yǎng)腫瘤再生細(xì)胞這一方法,很好的在體外模擬了腫瘤休眠這一過程,即用IFNγ在體外處理3D培養(yǎng)的細(xì)胞72小時(shí),就會(huì)誘導(dǎo)其進(jìn)入休眠,并在進(jìn)一步的研究中我們發(fā)現(xiàn),在誘導(dǎo)腫瘤體外休眠的過程中,吲哚胺2,3雙加氧酶(indoleamine 2,3-dioxygenase,IDO),犬尿氨酸(kynurenine,kyn),芳香烴受體(aryl hydrocarbon receptor,AhR)即IDO-kyn-AhR這一代謝調(diào)控的轉(zhuǎn)錄因子信號(hào)通路發(fā)揮著重要的作用。IFNγ作用于腫瘤再生細(xì)胞后,可誘導(dǎo)其進(jìn)入休眠,具體表現(xiàn)為IFNγ誘導(dǎo)腫瘤再生細(xì)胞內(nèi)IDO與AHR的表達(dá)上調(diào),IDO的表達(dá)上調(diào)繼而產(chǎn)生大量的內(nèi)源性的Kyn,Kyn與AhR結(jié)合并激活A(yù)hR,AhR進(jìn)入細(xì)胞核并結(jié)合到相應(yīng)基因的啟動(dòng)子上,導(dǎo)致相關(guān)周期阻滯基因的表達(dá),從而產(chǎn)生細(xì)胞周期的阻滯。我們通過進(jìn)一步的篩選,發(fā)現(xiàn)在這一現(xiàn)象休眠之中,細(xì)胞周期阻滯蛋白P27出現(xiàn)了很明顯的變化,并且受到AhR基因的轉(zhuǎn)錄調(diào)控。IFNγ-IDO-AhR-P27這一信號(hào)通路對(duì)腫瘤再生細(xì)胞的休眠具有巨大的作用,我們同時(shí)通過運(yùn)用相關(guān)抑制劑聯(lián)合IFNγ,發(fā)現(xiàn)腫瘤的休眠可以被打破,并取得了較好的治療效果,為今后臨床上對(duì)腫瘤的治療提出了新的思路。
[Abstract]:The interaction of tumor cells with the immune system is summed up as 3E theory (Dunn et al., 2004; Schreiber et al., 2011), including Eradication (elimination), Equilibrium (balance), Evasion (escape). In the body, the tumor cells will present more than three biological phenomena under the action of immune factors: tumor cells are eliminated by the immune system; in exemption Under the surveillance of the epidemic system, the tumor cells and the body reach the balance; the tumor cells escape the immune system and then infringe the whole body (Romero et al., 2014). The immune induced tumor dormancy (tumordormancy) refers to the cell cycle arrest, the downregulation of the related gene expression and the metabolic slowing down of the tumor cells under the action of the immune system of the body. This phenomenon is the concentration of equilibrium and stability in the process of interaction between the tumor and the immune system of the body (Sosa et al., 2014), which corresponds to the hypothesis of immune surveillance. The current scientific research is lacking effective means of immune induced dormancy due to the lack of an effectively recognized model of tumor dormancy in vivo and in vivo. In a scientific study of the field of tumor cell dormancy, the current research results are limited to the use of neutralizing antibodies and genetically modified animals to explore the presence of the phenomenon and to determine the immune molecules such as Interferon gamma (gamma interferon, IFN gamma).Interferon gamma as an immune response molecule. In the epidemic, IFN gamma is secreted mainly by the killer T cells (CTLs) in the tumor microenvironment. It plays an important role in controlling the growth of tumor (Akira etal., 2006; Dunn etal., 2006), which may cause the dormancy of tumor cells. However, the internal mechanism of the tumor cells has not been elucidated. The tumor cells have heterogeneity, Different subgroups have different characteristics. Among them, tumor stem cells (tumorigenic cells, TRCs) are a group of cells with strong tumor forming ability and proliferation and division, and exhibit strong resistance characteristics, closely related to tumor recurrence and metastasis (Enver et al., 2009; Visvader and Lindeman, 2008). There have been no studies to prove whether tumor stem cells exist in tumor dormancy, and its intrinsic mechanism is not clear. This study aims to explore the following three questions: whether IFN gamma can induce the dormancy of tumor by using the 3D (soft three-dimensional colloid amplification and proliferation of tumor regenerative cells) established by our group: IFN gamma can induce the tumor's dormancy; IFN gamma can be used Which group of tumor cells to induce dormancy and IFN gamma induced dormancy of tumor cells. This paper is divided into three parts: 1.IFN gamma induced dormancy of tumor stem cells in vitro; mechanism of 2.IFN gamma induced dormancy of tumor stem cells; 3. the first part of the exploration of the effect of tumor dormancy on tumor cells using inhibitors of related molecules: the first part: IFN gamma The purpose of in vitro induction of tumor stem cell dormancy is to investigate whether IFN gamma can induce tumor stem cells dormancy and differentiate between traditional 2D and tumor cells; and explore whether IFN gamma induced cancer stem cells have resistance to chemotherapy drugs methotrexate (Methotrexate) and paclitaxel (Paclitaxel). The mouse melanoma cell line B16 used before and the 3D tumor stem cell culture method (Liu et al., 2012) derived from salmon derived fibrinogen to establish B16 melanoma cells derived from.1.IFN gamma treatment mice in vitro model, H22 hepatoma cell line and CT26 colon cancer cell line: 1) 1OOng/ml IFN gamma action on traditional 2D The cultured B16 cells, H22 cells and CT26 cells 72h; 2) 100ng/ml IFN gamma acts on B16 cells cultured in 3D, H22 cells and CT26 cells 72h. The proliferation and apoptosis metabolism of three tumor cells in two different culture modes were detected by.2. chemotherapeutic drugs methotrexate and taxol, respectively, 1) cells The control group: 2D culture B16 cells +PBS; 2D culture B16 cells +IFN gamma; 2D culture B16 cells +MTX; 2D culture B16 cells +IFN gamma; 2) experimental cell group. Results: 1. the apoptosis of 2D tumor cells was obvious after the treatment of traditional 2D culture dish and 3D cultured B16 cells, H22 cells and CT26 cells 96h, and the.3D cells of 3D cells did not have obvious apoptosis, and the main manifestations of the.3D cells were the small clone, the slow cell cycle, the decrease of glucose consumption, and the.2. of proliferation related genes. The treatment of methotrexate and paclitaxel treated IFN gamma induced dormant tumor cells and normal 2D and 3D cells, and found that the dormant tumor cells have stronger resistance. The second part: the mechanism of IFN gamma in inducing tumor cells dormancy: explore the inside of the tumor dormancy model under the established IFN gamma body. In the mechanism method: in the model of IFN gamma induced tumor dormancy in vitro, we use PCR, Western-blot, plasmid construction, virus packaging, CRISPR-Cas9, immunofluorescence, flow analysis, sorting and other molecular biology, cell biology methods to identify and verify the related signaling pathways and mechanisms. Results: 1. according to the literature and PCR test It was confirmed that the indolamine 2,3 dioxygenase (indoleamine 2,3-dioxygenase, IDO) had a great change in the B16 tumor cells treated by IFN gamma (Takikawa et al., 1990), and TRCs was more obvious after TRCs was treated by IFN gamma than that of ordinary B16 cells, indicating that it plays an important role in the dormancy process; 2. We analyzed the TRCs of the expression of IDO, and found that the cell clone size and cell cycle were obviously changed, the clone became smaller and the cycle slowed down; 3.IDO was a kind of catalytic enzyme, which catalyzed the metabolism of Tryptophan, Trp, so that the metabolism became kynurenine, Kyn, and we used 200uM to 500uM concentration. The degree of Kyn treatment TRCs also obtained the result of cloning and periodic slowing down, indicating that Kyn plays an important role in the dormancy of TRCs; 4. we have learned from the literature that Kyn is a ligand of polycyclic aromatic hydrocarbon receptor (Aryl hydrocarbon receptor, Ahr), which can lead to the expression of the Ahr induced gene (Opitz et, 2011), which may include finer The proteins associated with cell cycle inhibition, we determined that Ahr was activated in TRCs by immunofluorescence, Western-blot and luciferase experiment; 5. at the same time, we screened the related cyclin with Western-blot, and found that after IFN gamma treatment, Cdknlb (cyclin-dependent kinase inhibitor 1B, P27kip) had obvious changes, and other cycles. There is no obvious change in protein, indicating that P27 may play an important role in dormancy; 6. we detected the expression level of P27 with Western-blot after TRCs72 hours of Kyn treatment, and found that it was obviously up-regulated, and the promoter of P27 gene was transferred simultaneously, the fluorescein plasmid was constructed, and the AHR gene was simultaneously transfected with the AHR gene. After Kyn treatment, it was found that the fluorescein signal was obviously enhanced after AHR, Kyn and IFN gamma treatment, indicating that the regulation of IFN gamma to P27 was mainly through the IDO-Kyn-AHR signal pathway; 7. the classic IFN gamma signal was mainly played by STAT1 (signal transducer and), and it was mainly caused by the tumor cells. The role of apoptosis was confirmed by Western-blot and immunofluorescence and flow cytometry in the common culture of B16, but in dormant cells, this pathway was obviously suppressed, and we found that the signal pathway of IFNy-Stat1 was interacted with IFN gamma -IDO-AHR-P27 signaling pathway through Ip-western, that is, P2 7 combined with p-STAT1 protein to prevent its nucleation; 8. we also further verified this conclusion in the later IDO, AHR, and P27 knockout cell lines. The third part: using inhibitors of relevant molecules to reverse the tumor dormancy in vitro experiments: the possibility of breaking the dormancy of the tumor cells by using the inhibitors of the relevant molecules combined with IFNy And a new way of thinking for tumor therapy is put forward: using 3D culture, Western-blot and flow detection to detect the proliferation and apoptosis of tumor cells associated with IFN gamma treatment. Results: 1. we combine the IDO inhibitor 1-MT (1-Methyl-L-tryptophan) and IFN gamma treatment of 3D cultured B16 cells, and they appear. The number of clones decreased, the size of clones became smaller, and the apoptosis increased at the whole cell level and molecular level. It showed that IFN gamma combined with IDO inhibitor could effectively break the dormancy and kill the tumor; 2. we are the same as IDO inhibitors with the AHR inhibitor DMF (3', 4'-Dimethoxyflavone) and IFN gamma treatment of 3D cultured B16 cells. The results indicate that dormancy breaks the increase of apoptosis. It shows that these two inhibitors may have good prospects in the future immunotherapy. Conclusion: the phenomenon of tumor dormancy is of great significance in the occurrence, development, metastasis and treatment of tumor, and the dormancy caused by immunization is a good manifestation of this phenomenon, and we use 3D The culture of tumor regenerative cells is a good method to simulate the process of tumor dormancy in vitro, that is, IFN gamma is used to treat 3D cells for 72 hours in vitro, and it will induce its entry into dormancy. In further study, we found that in the process of inducing the tumor to sleep in vitro, the indolamine 2,3 dioxygenase (indoleamine 2,3-dioxygena) Se, IDO), kynurenine (KYN), aromatic hydrocarbon receptor (aryl hydrocarbon receptor, AhR), a metabolic transcriptional factor signaling pathway, plays an important role in the action of.IFN gamma on tumor regenerative cells and can induce its entry into dormancy. The expression of IDO is up and then produces a large number of endogenous Kyn. Kyn combined with AhR and activates AhR, AhR enters the nucleus and binds to the promoter of the corresponding gene, resulting in the expression of the related cycle block gene, resulting in cell cycle arrest. The protein P27 changes obviously, and the signaling pathway of the AhR gene transcription regulation.IFN gamma -IDO-AhR-P27 has a great effect on the dormancy of the tumor regenerated cells. We also find that the dormancy of the tumor can be broken through the use of the associated inhibitor combined with IFN gamma, and we have obtained a better therapeutic effect for the future clinical practice. A new way of thinking for the treatment of tumor is put forward.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730.5
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本文編號(hào)：1808596