本文關(guān)鍵詞：WWP1在胃癌發(fā)生發(fā)展中的作用及其初步的分子調(diào)控機(jī)制　出處：《鄭州大學(xué)》2015年博士論文　論文類型：學(xué)位論文

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【摘要】：背景和目的胃癌(gastric cancer, GC)是最常見(jiàn)的消化道惡性腫瘤,在腫瘤相關(guān)性死亡中居第二位。盡管胃癌的診斷策略和分子標(biāo)記的研究取得了很大的進(jìn)展,但是晚期胃癌的預(yù)后和生存率仍不理想,胃癌病人的中位存活時(shí)間僅為6-10個(gè)月。因此,尋找和鑒定新的診斷、預(yù)防或治療胃癌病人的分子標(biāo)記具有十分重要的意義。含有WW結(jié)構(gòu)域的E3泛素蛋白連接酶1 (WW domain containing E3 ubiquitin protein ligasel, WWP1)定位于人染色體8q21.3區(qū)域。人WWP1包含922個(gè)氨基酸殘基,分子量大約為110 kDa,由1個(gè)C2結(jié)構(gòu)域,4個(gè)WW結(jié)構(gòu)域和1個(gè)HECT結(jié)構(gòu)域組成。WWPl能與多種不同的信號(hào)途徑相互作用,如Notch信號(hào)途徑、轉(zhuǎn)化生長(zhǎng)因子β(transforming growth factor β, TGFβ)信號(hào)途徑、絲裂原活化蛋白激酶(mitogen-activated protein kinases, MAPKs)信號(hào)途徑和表皮生長(zhǎng)因子(Epithelial growth factor, EGF)信號(hào)途徑等,提示其功能上的多樣性和復(fù)雜性。越來(lái)越多的研究表明,WWP1牽涉到多種不同疾病的發(fā)生、發(fā)展和進(jìn)展,如感染性疾病、神經(jīng)性疾病、衰老,特別是腫瘤的發(fā)生發(fā)展過(guò)程。在前列腺癌和乳腺癌中,WWP1 mRNA和蛋白表達(dá)上調(diào),在口腔癌和肝癌中表達(dá)也顯著增加。這些研究表明WWP1可能在這些腫瘤的發(fā)生發(fā)展及進(jìn)展中發(fā)揮重要的作用。然而,迄今為止,國(guó)內(nèi)外尚未見(jiàn)WWP1在胃癌發(fā)生、發(fā)展中的作用的研究報(bào)道。本研究采用原位雜交、免疫組織化學(xué)、實(shí)時(shí)熒光定量PCR (Real-time qPCR)以及Western blotting等技術(shù)檢測(cè)胃癌組織和正常胃粘膜組織中WWP1蛋白和mRNA的表達(dá),探討胃癌臨床病理學(xué)參數(shù)與其蛋白和mRNA的表達(dá)之間的關(guān)系,并通過(guò)病人資料隨訪,采用Kaplan-Meier存活曲線研究WWP1的表達(dá)與胃癌病人預(yù)后之間的關(guān)系,進(jìn)一步采用Cox比例風(fēng)險(xiǎn)模型分析胃癌病人獨(dú)立的預(yù)后因子；采用Real-time qPCR和Western blotting技術(shù)檢測(cè)多種胃癌細(xì)胞系中WWP1mRNA和蛋白的表達(dá)；采用RNA干擾技術(shù)研究WWP1表達(dá)下調(diào)對(duì)胃癌細(xì)胞增殖、細(xì)胞周期和凋亡的影響,并應(yīng)用生物信息學(xué)手段分析WWP1可能相互作用的蛋白,利用Western blotting技術(shù)驗(yàn)證其可能的相互調(diào)控作用；最后建立胃癌的裸鼠移植瘤模型,并采用該模型研究WWP1表達(dá)下調(diào)對(duì)胃癌裸鼠移植瘤生長(zhǎng)的影響,并通過(guò)Kaplan-Meier存活曲線分析其與裸鼠生存率之間的關(guān)系。第1章 WWP1在胃癌組織中的表達(dá)及其與胃癌病人預(yù)后之間的關(guān)系目的分析WWP1在胃癌組織中的表達(dá),初步探討其在胃癌發(fā)生發(fā)展中的可能作用。方法(1)采用原位雜交和免疫組化技術(shù)分別檢測(cè)131例胃癌組織和相應(yīng)的正常胃黏膜組織中WWP1 mRNA和蛋白的表達(dá)：采用Real-time qPCR和Western blotting技術(shù)檢測(cè)胃癌組織和相對(duì)應(yīng)的正常胃粘膜組織中WWP1 mRNA和蛋白的表達(dá)。(2)采用Kaplan-Meier存活曲線分析WWP1 mRNA和蛋白表達(dá)與胃癌病人預(yù)后之間的關(guān)系,并采用Cox比例風(fēng)險(xiǎn)模型分析胃癌病人獨(dú)立的預(yù)后因子。(3)統(tǒng)計(jì)學(xué)分析：采用統(tǒng)計(jì)學(xué)軟件SPSS17.0對(duì)數(shù)據(jù)進(jìn)行分析處理,數(shù)據(jù)表示為x±s,原位雜交和免疫組化的結(jié)果采用X2進(jìn)行評(píng)估,Real-time qPCR和Western blotting結(jié)果采用單因素方差分析(One-way ANOVA), WWP1 mRNA和蛋白的表達(dá)與胃癌病人預(yù)后之間的關(guān)系采用Kaplan-Meier存活曲線分析,Cox比例風(fēng)險(xiǎn)模型用來(lái)分析胃癌病人的獨(dú)立預(yù)后因子,P＜0.05表示差異具有統(tǒng)計(jì)學(xué)意義。結(jié)果(1)在131例胃癌組織中,WWP1 mRNA和蛋白的陽(yáng)性表達(dá)率分別為66.41%和61.83%,而在131例正常胃黏膜組織中,WWP1 mRNA和蛋白的陽(yáng)性表達(dá)率分別為12.21%和9.92%。WWP1 mRNA和蛋白在胃癌組織和正常胃黏膜組織中的表達(dá)水平均具有統(tǒng)計(jì)學(xué)差異(X2=80.646和76.715,均P=0.000)。(2) Real-time qPCR和Western blotting結(jié)果表明,隨機(jī)選擇的10例胃癌組織中WWP1 mRNA和蛋白的相對(duì)表達(dá)水平均顯著高于正常胃黏膜組織,且差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。(3) WWP1 mRNA和蛋白表達(dá)均與胃癌病人腫瘤分化程度、TNM分期、浸潤(rùn)深度和淋巴結(jié)轉(zhuǎn)移密切相關(guān)(所有P0.05),而與胃癌病人的年齡、性別和腫瘤大小無(wú)關(guān)(所有P0.05)。(4) Log-rank檢驗(yàn)(Mantel-Cox)結(jié)果表明,WWP1 mRNA和蛋白低表達(dá)的胃癌患者的存活時(shí)間均顯著長(zhǎng)于WWP1 mRNA和蛋白高表達(dá)的胃癌患者,且差異具有統(tǒng)計(jì)學(xué)意義(P分別為0.0134和0.0098)。(5)Cox比例風(fēng)險(xiǎn)模型結(jié)果表明,TNM分期、淋巴結(jié)轉(zhuǎn)移、WWP1 mRNA和蛋白均可作為胃癌病人獨(dú)立的預(yù)后因子。第2章 WWP1表達(dá)下調(diào)對(duì)胃癌細(xì)胞增殖、細(xì)胞周期和細(xì)胞凋亡的影響及可能的分子調(diào)控機(jī)制目的探討WWP1表達(dá)下調(diào)對(duì)胃癌細(xì)胞增殖、凋亡和細(xì)胞周期的影響及其調(diào)控機(jī)制。方法(1)采用Real-time qPCR和Western blotting技術(shù)檢測(cè)不同胃癌細(xì)胞系和正常胃黏膜上皮細(xì)胞GES-1中WWP1 mRNA和蛋白的表達(dá)。(2)利用脂質(zhì)體2000將WWP1 siRNA和對(duì)照siRNA分別轉(zhuǎn)染胃癌MKN-45細(xì)胞和胃癌AGS細(xì)胞。(3)采用Western blotting技術(shù)檢測(cè)轉(zhuǎn)染前后胃癌細(xì)胞中WWP1、cyclin D1、 CDK4、Bcl-2和Bax蛋白的表達(dá)以及PTEN-Akt信號(hào)途徑中關(guān)鍵蛋白PTEN、p-Akt和總Akt蛋白表達(dá)的變化；采用Caspase-3比色分析試劑盒檢測(cè)不同方法處理的胃癌細(xì)胞中Caspase-3活性的變化。(4)采用CCK-8增殖實(shí)驗(yàn)檢測(cè)WWP1表達(dá)下調(diào)對(duì)胃癌MKN-45和AGS細(xì)胞增殖的影響；采用流式細(xì)胞術(shù)檢測(cè)WWP1表達(dá)下調(diào)對(duì)胃癌MKN-45和AGS細(xì)胞周期和凋亡的影響。(5)采用STRING 9.1在線軟件對(duì)WWP1的結(jié)構(gòu)及其相互作用的蛋白進(jìn)行預(yù)測(cè)分析。(6)統(tǒng)計(jì)學(xué)處理：采用統(tǒng)計(jì)學(xué)軟件SPSS17.0分析所有的實(shí)驗(yàn)數(shù)據(jù),數(shù)據(jù)表示為x±S,采用單因素方差分析(One way ANOVA)比較多個(gè)樣本的均數(shù),以P0.05表示具有統(tǒng)計(jì)學(xué)差異。結(jié)果(1)所檢測(cè)的胃癌細(xì)胞中WWP1 mRNA和蛋白的相對(duì)表達(dá)水平均顯著高于正常胃粘膜上皮細(xì)胞GES-1中WWP1 mRNA和蛋白的相對(duì)表達(dá)水平,且差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。此外,KN-45和AGS細(xì)胞中WWP1 mRNA和蛋白的相對(duì)表達(dá)水平顯著高于其它各個(gè)胃癌細(xì)胞,且差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。(2) WWP1 siRNA組中WWP1蛋白的表達(dá)水平均顯著低于未處理的MKN-45和AGS以及對(duì)照siRNA,且差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。(3)與未處理組和對(duì)照siRNA組相比,轉(zhuǎn)染W(wǎng)WP1 siRNA組中胃癌MKN-45和AGS細(xì)胞的增殖均顯著受到抑制,且差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。(4) WWP1 siRNA組中MKN-45和AGS細(xì)胞在G0/G1期的比率(64.87±1.48%和64.88±1.34%)分別顯著高于未處理組(50.19±1.46%和50.25±3.02%)和對(duì)照siRNA組(50.93±1.39%和51.60±2.09%),且差異具有統(tǒng)計(jì)學(xué)意義(F=98.515和38.528,均P=0.000),而未處理組和對(duì)照siRNA組中MKN-45和AGS細(xì)胞在G0/G1期的比率無(wú)差異(P＞0.05)。此外,WWP1 siRNA組中MKN-45和AGS細(xì)胞在S期的比率(20.32±1.25%和23.47±2.26%)分別顯著低于未處理組(29.57±1.25%和30.30±0.96%)和對(duì)照siRNA組(30.36±1.17%和30.23±1.34%),差異具有統(tǒng)計(jì)學(xué)意義(F=62.386和17.692,P=0.000和0.003),而未處理組和對(duì)照siRNA組中MKN-45和AGS細(xì)胞在S期的比率無(wú)差異(P＞0.05)。(5)流式細(xì)胞術(shù)結(jié)果表明,WWP1 siRNA組中MKN-45和AGS的早期凋亡率分別為20.56±0.92%和28.99±1.71%,顯著高于未處理組(6.95±0.69%和9.5±1.18%)和對(duì)照siRNA組(8.35±1.87%和10.0±1.46%),且差異具有統(tǒng)計(jì)學(xué)意義(F=104.561和171.636,均P=0.000),而未處理組和對(duì)照siRNA組之間MKN-45和AGS細(xì)胞的早期凋亡率無(wú)差異(P0.05)。WWP1 siRNA組中MKN-45和AGS的總凋亡率分別為22.95±1.92%和36.2±4.36%,顯著高于未處理組(8.38±1.03%和11.49±0.48%)和對(duì)照siRNA組(9.9±1.29%和11.48±0.88%),且差異具有統(tǒng)計(jì)學(xué)意義(F=90.441和91.844,均P=0.000),而未處理組和對(duì)照siRNA組之間MKN-45和AGS細(xì)胞的總凋亡率無(wú)差異(P＞0.05)。(6) WWP1 siRNA組中MKN-45和AGS的CDK4、cyclin D1和Bcl-2蛋白表達(dá)水平顯著低于對(duì)照siRNA組和未處理組,而B(niǎo)ax蛋白的表達(dá)和Caspase-3的活性均顯著高于對(duì)照siRNA組和未處理組,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。(7) STRING 9.1軟件預(yù)測(cè)結(jié)果表明,WWP1與很多蛋白可能具有潛在的相互作用,如PTE、TGFβ1、STAT3和SMAD7等。(8)與未處理組和對(duì)照siRNA組相比,WWP1 siRNA組中MKN-45和AGS細(xì)胞中PTEN蛋白的表達(dá)顯著上調(diào),而p-Akt蛋白表達(dá)顯著下調(diào),但不改變總Akt蛋白表達(dá)。第3章 WWP1在胃癌MKN-45和AGS細(xì)胞裸鼠移植瘤中的作用目的分析WWP1下調(diào)在胃癌裸鼠移植瘤中的作用。方法(1)實(shí)驗(yàn)動(dòng)物分組：42只裸鼠隨機(jī)分為MKN-45細(xì)胞移植組和AGS細(xì)胞移植組,每組21只,進(jìn)行致瘤實(shí)驗(yàn)。每組的裸鼠均隨機(jī)分為三小組,每組7只,分為未處理組、對(duì)照siRNA組、WWP1 siRNA組。(2)MKN-45細(xì)胞和AGS細(xì)胞的皮下接種及治療：在裸鼠右側(cè)背部皮下分別接種2×106 MKN-45細(xì)胞/只和3×106 AGS細(xì)胞/只。當(dāng)腫瘤體積生長(zhǎng)至55-80mm3時(shí),未處理組不進(jìn)行任何處理,其余兩小組,每3天分別注射一次WWP1siRNA和對(duì)照siRNA(100ng/只)。每隔3天采用游標(biāo)卡尺測(cè)量腫瘤的體積,當(dāng)測(cè)量全部結(jié)束后,剝離腫塊,稱量瘤體重量,并采用Kaplan-Meier存活曲線分析WWP1表達(dá)與裸鼠生存率之間的關(guān)系。(3)采用Real-time qPCR和Western blotting分別檢測(cè)胃癌細(xì)胞MKN-45和AGS裸鼠移植瘤中WWP1 mRNA和蛋白的表達(dá)。(4)統(tǒng)計(jì)學(xué)處理：采用SPSS17.0統(tǒng)計(jì)學(xué)軟件進(jìn)行所有實(shí)驗(yàn)數(shù)據(jù)的處理,數(shù)據(jù)表示為x±s,多個(gè)樣本的均數(shù)比較采用單因素方差分析(One way ANOVA), WWP1表達(dá)與裸鼠存活之間的關(guān)系采用Kaplan-Meier存活曲線進(jìn)行分析,P0.05表示差異具有統(tǒng)計(jì)學(xué)意義。結(jié)果(1) Real-time qPCR和Western blotting結(jié)果表明,與未處理組(MKN-45或AGS)和對(duì)照siRNA組相比,WWP1 siRNA組中的MKN-45或AGS裸鼠移植瘤中WWP1 mRNA和蛋白的表達(dá)均顯著降低,且差異具有統(tǒng)計(jì)學(xué)意義(P0.05),而未處理組和對(duì)照siRNA組中WWP1 mRNA和蛋白的相對(duì)表達(dá)水平無(wú)差異(P＞0.05)。(2)與未處理的MKN-45或AGS和對(duì)照siRNA組相比,WWP1 siRNA組中MKN-45或AGS接種的裸鼠腫瘤的生長(zhǎng)顯著被抑制,且差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。(3) WWP1 siRNA組中MKN-45和AGS裸鼠移植瘤的平均重量分別為0.467±0.111和0.457±0.118,顯著低于未處理組(1.823±0.102和1.838±0.095)和對(duì)照siRNA組(1.714±0.119和1.950±0.153),且差異具有統(tǒng)計(jì)學(xué)意義(F=323.932和312.691,均P=0.000),而未處理組和對(duì)照siRNA組的MKN-45和AGS的裸鼠移植瘤的重量無(wú)差異(P0.05)。(4) Kaplan-Meier存活曲線分析結(jié)果顯示,與未處理的MKN-45和對(duì)照siRNA組相比,WWP1 siRNA組中裸鼠的生存時(shí)間顯著延長(zhǎng)(P0.05)。同樣的結(jié)果也在AGS裸鼠移植瘤中發(fā)現(xiàn)。結(jié)論(1)WWP1在胃癌組織中的高表達(dá)與胃癌病人腫瘤分化程度、TNM分期、浸潤(rùn)深度、淋巴結(jié)轉(zhuǎn)移以及預(yù)后關(guān)系密切,并且WWP1可作為胃癌病人獨(dú)立的預(yù)后因子,提示W(wǎng)WP1在胃癌發(fā)生發(fā)展中具有十分重要的作用。(2)WWP1表達(dá)下調(diào)誘導(dǎo)的胃癌細(xì)胞增殖抑制、細(xì)胞周期分布改變和細(xì)胞凋亡,這可能與cyclin D1、CDK4和Bcl-2表達(dá)下調(diào)以及Bax表達(dá)上調(diào)和Caspase-3活性的升高密切相關(guān)。(3)WWP1表達(dá)下調(diào)介導(dǎo)的胃癌生物學(xué)行為的變化可能通過(guò)調(diào)控PTEN-Akt信號(hào)途徑發(fā)揮作用,因而聯(lián)合靶向二者可能成為胃癌治療的新途徑。(4)WWP1表達(dá)下調(diào)在胃癌裸鼠移植瘤中的抑制作用可能為開(kāi)辟胃癌治療新靶點(diǎn)提供實(shí)驗(yàn)依據(jù)。
[Abstract]:Background and objective gastric cancer (GC) is the most common malignant tumor of the digestive tract and is the second largest in tumor related death. Although great progress has been made in the research of gastric cancer diagnosis strategy and molecular marker, the prognosis and survival rate of advanced gastric cancer is still not ideal. The median survival time of gastric cancer patients is only 6-10 months. Therefore, it is of great significance to find and identify the new molecular markers for the diagnosis, prevention, or treatment of gastric cancer patients. The E3 ubiquitin protein ligase 1 (WW domain containing E3 ubiquitin protein ligasel, WWP1), which contains the domain of the WW, is located in the 8q21.3 region of the human chromosome. Human WWP1 contains 922 amino acid residues, with a molecular weight of about 110 kDa, consisting of 1 C2 domains, 4 WW domains and 1 HECT domains. WWPl signaling pathway and various interactions, such as the Notch signaling pathway, transforming growth factor beta (transforming beta growth factor, TGF) signal pathway, mitogen activated protein kinase (mitogen-activated protein, kinases, MAPKs) signal pathway and epidermal growth factor (Epithelial growth, factor, EGF) signal pathway, suggesting its function the diversity and complexity of. More and more studies show that WWP1 is involved in the occurrence, development and progression of various diseases, such as infectious diseases, neurologic diseases and senescence, especially the occurrence and development of tumors. In prostate and breast cancer, the expression of WWP1 mRNA and protein is up-regulated, and the expression in oral and hepatocellular carcinoma is also significantly increased. These studies suggest that WWP1 may play an important role in the development and progression of these tumors. However, up to now, there has been no research report on the role of WWP1 in the occurrence and development of gastric cancer at home and abroad. This research adopts immunohistochemistry in situ hybridization, chemical, real time fluorescence quantitative PCR (Real-time qPCR) expression of WWP1 and mRNA and Western blotting detection of gastric cancer and normal gastric mucosa tissues, to explore the relationship between clinicopathological parameters and the expression of protein and mRNA, and the relationship between the survival of patients followed up. Study on WWP1 curve expression and prognosis of gastric cancer patients by Kaplan-Meier, further using Cox proportional hazards model analysis of prognostic factors in patients with gastric cancer independently; the expression of WWP1mRNA and protein detection in gastric cancer cell lines by Real-time qPCR and Western blotting using RNA WWP1 technology; research on jamming effect of down-regulation on cell cycle and apoptosis of gastric cancer cell proliferation the analysis of WWP1 protein interaction and possible application of bioinformatics method, using Western blot Ting technology to verify the possible effect of mutual regulation; finally to establish nude mouse model of gastric cancer, and using the model to study the effect of down-regulation of WWP1 expression on the growth of gastric cancer xenografts in nude mice, and through its relationship with the survival of Kaplan-Meier nude mice survival curve analysis. The first chapter is the expression of WWP1 in gastric cancer and its relationship with the prognosis of gastric cancer. Objective to analyze the expression of WWP1 in gastric cancer and explore its possible role in the development of gastric cancer. Methods (1) by in situ hybridization and immunohistochemistry techniques were used to detect the expression of WWP1 and mRNA protein in 131 cases of gastric cancer tissues and corresponding normal gastric mucosa tissues: the expression of WWP1 and mRNA protein in gastric cancer was detected by Real-time qPCR and Western Blotting Technology and the corresponding normal gastric tissues. (2) Kaplan-Meier survival curve was used to analyze the relationship between WWP1 mRNA and protein expression and prognosis of gastric cancer patients. Cox independent risk factor analysis was used to analyze the independent prognostic factors of gastric cancer patients. (3) statistical analysis: using statistical software SPSS17.0 to analyze the data, the data were expressed as x + s, in situ hybridization and immunohistochemical results were evaluated by X2, Real-time qPCR and Western blotting results using single factor analysis of variance (One-way ANOVA), the relationship between the expression and prognosis of gastric cancer patients of WWP1 mRNA and protein the Kaplan-Meier survival curve analysis, independent prognostic factor in Cox proportional hazards model for analysis of gastric cancer patients, P < 0.05 said the difference was statistically significant. Results (1) in 131 gastric cancer tissues, the positive expression rates of WWP1 mRNA and protein were 66.41% and 61.83%, respectively. In 131 normal gastric mucosa tissues, the positive expression rates of WWP1 mRNA and protein were 12.21% and 9.92%, respectively. The expression levels of WWP1 mRNA and protein in gastric cancer tissues and normal gastric mucosa were statistically different (X2=80.646 and 76.715, all P=0.000). (2) Real-time qPCR and Western blotting results showed that the relative expression levels of WWP1 mRNA and protein in 10 gastric cancer tissues were significantly higher than those in normal gastric mucosa tissues, and the difference was statistically significant (P0.05). (3) WWP1 mRNA and protein expression were closely related to tumor differentiation, TNM stage, depth of invasion and lymph node metastasis in all gastric cancer patients (all P0.05), but not related to age, sex and tumor size of gastric cancer patients (all P0.05). (4) Log-rank test (Mantel-Cox) showed that the survival time of gastric cancer patients with low expression of WWP1 mRNA and protein was significantly longer than those of patients with high expression of WWP1 mRNA and protein, and the difference was statistically significant (P 0.0134 and 0.0098 respectively). (5) the Cox proportional risk model showed that TNM staging, lymph node metastasis, WWP1 mRNA and protein could be an independent prognostic factor for gastric cancer patients. The second chapter is about the effect of downregulation of WWP1 expression on gastric cancer cell proliferation, cell cycle and apoptosis, and the possible molecular regulation mechanism. Objective to investigate the effect of downregulation of WWP1 expression on gastric cancer cell proliferation, apoptosis and cell cycle and its regulatory mechanism. Methods (1) Real-time qPCR and Western blotting were used to detect WWP1 mRNA and eggs in GES-1 of different gastric cancer cell lines and normal gastric mucosa epithelial cells.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.2

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1 張麗;WWP1在胃癌發(fā)生發(fā)展中的作用及其初步的分子調(diào)控機(jī)制[D];鄭州大學(xué);2015年

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