本文選題：食管鱗癌 + 橋接整合因子-1　； 參考：《河北醫(yī)科大學》2017年碩士論文

【摘要】：第一部分食管鱗狀細胞癌患者Bin1基因啟動子甲基化狀態(tài)及其意義目的:通過檢測橋接整合因子-1(bridging integrator-1,Bin1)基因在食管鱗癌(esophageal squamous cell cancer,ESCC)患者癌組織和其癌旁組織中表達情況及其啟動子甲基化狀態(tài),分析在ESCC患者中Bin1甲基化狀態(tài)與其表達及臨床病理特征的關系,探討B(tài)in1基因甲基化在ESCC臨床中的意義,為Bin1基因甲基化作為新靶點應用于ESCC綜合治療提供理論依據(jù)。方法:1收集2014年6月至2015年6月期間河北醫(yī)科大學第四醫(yī)院胸外科手術切除的58例ESCC患者的癌組織和癌旁組織,并將每例標本分為3份,其中2份標本取下后迅速置于液氮罐中,后放置于-80℃超低溫冰箱中貯存以備提取RNA、DNA時使用;另1份以4%甲醛溶液固定以備制作蠟塊時使用,用于免疫組織化學。2采用免疫組織化學法(immunohistochemistry,IHC)、甲基化特異性PCR(Methylation-Specific polymerase Chain Reaction,MSP)、實時熒光定量聚合酶鏈式反應(Quantitative Real-time PCR,qRT-PCR)分別檢測58例經病理證實的ESCC患者的癌組織、癌旁組織中Bin1蛋白的表達情況、Bin1基因的表達情況和Bin1基因啟動子甲基化狀態(tài),分析ESCC中Bin1甲基化狀態(tài)與Bin1蛋白、基因表達情況及與患者臨床病理特征之間的關系。結果:1在58例食管鱗癌患者中,ESCC組織中Bin1蛋白低表達發(fā)生率明顯高于相應癌旁組織[37/58(63.79%)vs.13/58(22.41%),P0.01];在58例ESCC患者中,ESCC組織中Bin1 mRNA的表達水平明顯低于癌旁組織,分別為(0.78±0.05)vs.(1.03±0.03)(t=9.643,P0.01);Bin1發(fā)生甲基化的組織中Bin1 mRNA表達水平顯著低于未發(fā)生甲基化的組織,分別為(0.68±0.04)vs.(0.85±0.07)(t=2.476,p0.05),bin1基因呈低表達的患者有35例,是bin1蛋白低表達的患者的94.59%。這一結果提示,在escc組織中bin1在基因水平和蛋白水平的表達一致。2在35例bin1mrna呈低表達的escc組織中有32例(91.43%)bin1基因啟動子發(fā)生甲基化,在37例bin1蛋白呈低表達的escc組織中有33例(89.19%)bin1基因啟動子發(fā)生甲基化;說明在escc組織中bin1啟動子區(qū)的異常甲基化與bin1的低表達相關,且bin1甲基化是bin1在escc組織中低表達的原因之一。3bin1甲基化狀態(tài)與患者tnm分期、腫瘤侵犯深度、分化程度、淋巴結轉移相關(p0.05),與患者年齡、性別比較無統(tǒng)計學意義(p0.05)。結論:食管癌患者腫瘤組織中bin1呈高甲基化狀態(tài),bin1高甲基化狀態(tài)與食管癌患者腫瘤分化程度、浸潤深度、淋巴結轉移、tnm分期等臨床病理特征密切相關。第二部分bin1基因啟動子甲基化狀態(tài)在食管鱗癌發(fā)生發(fā)展中的作用目的:通過檢測escc細胞yes-2、te13、te1、kyse30和ec109中bin1表達情況和甲基化狀態(tài),選擇yes-2和te13細胞進行后續(xù)實驗,應用去甲基化藥物5-氮雜-2’脫氧胞苷(5-aza-2’deoxycytidine,5-aza-dc)作用于yes-2和te13細胞,分析去甲基化前后bin1表達水平、escc細胞增殖、凋亡等惡性生物學行為和emt相關蛋白的變化,探討bin1在escc發(fā)生發(fā)展的可能機制,為bin1基因甲基化作為新靶點應用于escc綜合治療提供實驗依據(jù)。方法:1分別采用qrt-pcr、msp方法檢測bin1在5種人食管鱗癌細胞yes-2、te13、te1、kyse30和ec109中的表達情況及甲基化狀態(tài),選出yes-2和te13進行后續(xù)實驗。使用去甲基化藥物5-aza-dc對兩種escc細胞進行干預,并分別用msp、western-blot對兩種escc細胞進行檢測,分析bin1甲基化狀態(tài)與其表達的變化情況。2采用mtt、克隆形成實驗、流式細胞術檢測yes-2和te13細胞去甲基化前后增殖能力的改變情況,分析bin1甲基化對escc細胞增殖能力的影響。3采用流式細胞術(flowcytometryassay,fcm)檢測yes-2和te13細胞去甲基化前后增殖、凋亡能力的改變情況,分析bin1甲基化對escc細胞增殖、凋亡能力的影響。4采用細胞劃痕實驗、transwell實驗檢測yes-2和te13細胞去甲基化前后遷移、侵襲能力的改變情況,分析bin1甲基化對escc細胞遷移、侵襲能力的影響。5采用westernblot實驗檢測yes-2和te13細胞去甲基化前后上皮間質轉化(epithelial-mesenchymaltransition,emt)相關蛋白e鈣粘蛋白(e-cadherin,e-cad)、n鈣粘蛋白(n-cadherin,n-cad)、基質金屬蛋白酶2(matrixmetalloproteinase-2,mmp-2)、基質金屬蛋白酶9(mmp-9)等表達的改變情況,探討bin1甲基化影響escc細胞的上皮間質轉化的發(fā)生情況。結果:1bin1mrna在te13、te1、kyse30、ec109和yes-2五種escc細胞中均呈低表達狀態(tài)(p0.01);五種escc細胞均為甲基化狀態(tài),其中yes-2和te1兩種細胞為完全甲基化狀態(tài);經5-aza-dc去甲基化處理后,yes-2、te13、te1為非甲基化狀態(tài);篩選出yes-2和te13細胞進行后續(xù)實驗;經5-aza-dc去甲基化處理后yes-2和te13兩種人escc細胞bin1蛋白表達提高(p0.01)。2mtt結果顯示,經不同濃度(30μm、60μm、90μm)5-aza-dc干預yes-2細胞后d2天,細胞的增殖抑制率分別為6.7±0.9%、12.3±1.7%和15.4±2.1%,培養(yǎng)d7天時,細胞的增長抑制率分別為7.3±1.0%、49.3±5.7%和59.6±6.3%,與濃度為0μm的對照組相比,細胞增殖率顯著降低;經不同濃度(30μm、60μm、90μm)的5-aza-dc干預te13細胞后d2天,細胞的增殖抑制率分別為6.4±0.8%、11.9±1.7%和15.1±2.0%,培養(yǎng)d7天時,細胞的增長抑制率分別為7.0±1.1%、47.2±5.6%和57.2±5.9%,與濃度為0μm的對照組相比,細胞增殖率顯著降低(p0.01)�？寺⌒纬蓪嶒灲Y果顯示:經不同濃度(0μm、30μm、60μm、90μm)5-aza-dc干預yes-2細胞后,細胞的克隆數(shù)分別為(233±29)、(189±23)、(105±16)和(71±11),與濃度為0μm的對照組相比,克隆數(shù)明顯降低;經不同濃度(0μm、30μm、60μm、90μm)5-aza-dc干預te13細胞后,細胞的克隆數(shù)分別為(281±33)、(235±28)、(139±19)和(83±12),與濃度為0μm的對照組相比,克隆數(shù)明顯降低。說明yes-2和te13細胞經去甲基化處理后增殖活性均顯著下降(p0.01)。3流式細胞術結果顯示,經不同濃度(0μm、30μm、60μm、90μm)5-aza-dc干預yes-2細胞后,細胞處于g0/g1期的百分比分別為:(37.2±3.1)%、(46.5±3.9)%、(58.2±4.3)%和(70.7±4.9)%,處于s期的百分比分別為:(50.6±4.2)%、(42.7±3.6)%、(26.6±3.1)%和(20.5±2.4)%,與濃度為0μm的對照組相比,細胞處于g0/g1期的百分比明顯升高,處于s期的百分比明顯降低;經不同濃度(0μm、30μm、60μm、90μm)5-aza-dc干預te13細胞后,細胞處于g0/g1期的百分比分別為:(33.7±2.8)%、(39.4±3.6)%、(47.3±3.9)%和(56.3±4.5)%,處于s期的百分比分別為:(70.2±4.5)%、(58.3±4.2)%、(45.2±3.1)%和(32.6±2.8)%,與濃度為0μm的對照組相比,細胞處于g0/g1期的百分比明顯升高,處于s期的百分比明顯降低。說明經去甲基化處理后,escc細胞周期發(fā)生改變,處于s期的細胞顯著下降,阻滯于g0/g1期的細胞顯著上升(p0.01)。流式細胞術結果顯示,經不同濃度(0μm、30μm、60μm、90μm)5-aza-dc干預yes-2細胞后,處于凋亡的細胞百分比為:(4.12±0.89)%、(7.49±1.34)%、(10.03±2.13)%和(13.07±2.47),與濃度為0μm的對照組相比,處于凋亡的細胞百分比明顯提高(p0.01);經不同濃度(0μm、30μm、60μm、90μm)的5-aza-dc干預te13細胞后,處于凋亡的細胞百分比為:(3.57±0.78)%、(8.09±1.43)%和(10.15±2.08)%、(15.64±2.55),與濃度為0μm的對照組相比,處于凋亡的細胞百分比明顯提高(p0.01)。上述實驗結果說明bin1基因去甲基化后可以抑制yes-2和te13細胞的增殖能力和促進其凋亡,對于escc細胞的惡性生物學行為具有抑制作用。4劃痕實驗結果顯示:發(fā)現(xiàn)經5-aza-dc去甲基化處理后,yes-2細胞的48h遷移距離為(57.7±4.5)μm,未經去甲基化處理的48h遷移距離為(122.4±9.4)μm,去甲基化處理后的yes-2細胞遷移距離明顯短于處理前,其結果具有統(tǒng)計學意義(p0.01);te13細胞經5-aza-dc去甲基化處理后的48h遷移距離為(64.2±4.8)μm,未經去甲基化處理的48h遷移距離為(133.4±8.1)μm,去甲基化處理后的te13細胞遷移距離明顯短于處理前,其結果具有統(tǒng)計學意義(P0.01)。YES-2和TE13細胞遷移距離顯著縮短,說明Bin1經去甲基化處理后能夠抑制YES-2和TE13細胞的遷移能力。Transwell實驗結果顯示,經不同濃度(0μM、30μM、60μM、90μM)的5-Aza-d C去甲基化處理后的YES-2細胞,穿過Matrigel濾膜的細胞數(shù)分別為(206±35)、(137±29)、(78±18)和(39±9),與濃度為0μM的對照組相比,穿過Matrigel濾膜細胞術明顯減少(P0.01)。經不同濃度(0μM、30μM、60μM、90μM)的5-Aza-dC去甲基化處理后的TE13細胞,穿過Matrigel濾膜的細胞數(shù)分別為(289±42)、(145±33)、(57±16)和(48±12),與濃度為0μM的對照組相比,穿過Matrigel濾膜細胞術明顯減少(P0.01)。YES-2和TE13細胞穿膜細胞數(shù)顯著減少,說明Bin1經去甲基化處理后能夠抑制兩種細胞的侵襲能力。上述實驗說明說明Bin1經去甲基化后能夠抑制YES-2和TE13細胞的遷移、侵襲能力。5 Western blot檢測結果顯示:ESCC細胞YES-2和TE13經5-Aza-dC處理后,EMT相關蛋白E-cad表達上調(P0.01),N-cad、Snail、MMP-2和MMP-9蛋白的表達下調(P0.01),說明Bin1去甲基化處理后能夠改變YES-2和TE13細胞EMT相關蛋白的表達從而抑制上皮間質轉化的發(fā)生。結論:體外實驗證實Bin1甲基化影響食管癌細胞增殖、凋亡、遷移和侵襲能力,通過抑制食管癌細胞上皮間質轉化過程影響食管癌發(fā)生發(fā)展。
[Abstract]:Methylation status of Bin1 gene promoter in the first part of esophageal squamous cell carcinoma (ESCC) and its significance Objective: to detect the expression of bridging integrator-1 (Bin1) gene in the cancerous tissue and the para cancerous tissue of the esophageal squamous cell carcinoma (esophageal squamous cell cancer, ESCC) and its promoter methylation status, and analyze the status of the methylation status of the promoter of the esophageal squamous cell carcinoma (esophageal squamous cell cancer, ESCC). The relationship between Bin1 methylation status and its expression and clinicopathological features in ESCC patients, to explore the significance of Bin1 gene methylation in the clinical ESCC, and to provide a theoretical basis for Bin1 gene methylation as a new target for ESCC comprehensive treatment. Methods: 1 to collect the Department of thoracic surgery operation in the fourth hospital of Hebei Medical University from June 2014 to June 2015. 58 cases of ESCC patients were removed from the cancer tissue and para cancerous tissue, and each sample was divided into 3 samples, of which 2 specimens were quickly placed in a liquid nitrogen tank and then stored in -80 C ultra low temperature refrigerators for RNA, DNA use; the other 1 were used with 4% Formaldehyde Solution to prepare wax blocks and used immuno histochemical.2 for immunization. Immunohistochemistry (IHC), methylation specific PCR (Methylation-Specific polymerase Chain Reaction, MSP), real-time fluorescent quantitative polymerase chain reaction (Quantitative Real-time PCR, qRT-PCR) were used to detect the expression of the protein in the cancerous tissues of 58 pathologically confirmed patients. The relationship between Bin1 methylation status and Bin1 protein, gene expression and the clinicopathological features of patients in ESCC were analyzed. Results: 1 in 58 cases of esophageal squamous cell carcinoma, the incidence of Bin1 protein low expression in ESCC tissues was significantly higher than that of the corresponding para cancerous tissue [37/58 (63.79%) vs.13/58 (2). 2.41%), P0.01]; in the 58 patients with ESCC, the expression level of Bin1 mRNA in ESCC tissues was significantly lower than that of the paracancerous tissue, respectively (0.78 + 0.05) vs. (1.03 + 0.03) (t=9.643, P0.01), and Bin1 mRNA expression in the Bin1 methylation tissues was significantly lower than that of the non methylation tissues, respectively (0.68 + 0.04) vs. (0.85 + 0.07), respectively. There are 35 patients with low expression of gene, 35 cases of low expression of Bin1 protein, the result of 94.59%. in patients with low expression of protein suggests that the expression of Bin1 at the gene level and protein level in ESCC tissues is consistent with.2 in 35 cases of bin1mrna with low expression of ESCC tissue, 32 cases (91.43%) Bin1 gene promoter methylation, and 37 cases of low expression of Bin1 protein in the ESCC group. 33 (89.19%) Bin1 gene promoter methylation occurred in the fabric, indicating that the abnormal methylation of the Bin1 promoter region in the ESCC tissue is associated with the low expression of Bin1, and Bin1 methylation is one of the reasons for the low expression of Bin1 in the ESCC tissue, which is associated with the patient's TNM staging, the depth of the tumor invasion, the degree of differentiation, and lymph node metastasis (p0.). 05), there was no statistical significance between the age of the patients and the sex (P0.05). Conclusion: the methylation of Bin1 in the tumor tissues of the patients with esophageal cancer is highly methylation, and the hypermethylation of Bin1 is closely related to the clinicopathological features of the tumor differentiation, infiltration depth, lymph node metastasis, and TNM staging. The methylation status of the Bin1 gene promoter in the second part of the esophageal cancer patients. The purpose of the development of esophageal squamous cell carcinoma is to detect the Bin1 expression and methylation status in ESCC cells, yes-2, te13, te1, kyse30 and Ec109, select yes-2 and te13 cells for subsequent experiments, and apply the demethylation drug 5- nitrogen -2 'deoxycytidine (5-Aza-2'). Bin1 expression level, ESCC cell proliferation, apoptosis and other malignant biological behaviors and changes of EMT related proteins before and after methylation, explore the possible mechanism of Bin1 in ESCC development, and provide experimental basis for Bin1 gene methylation as a new target for ESCC comprehensive treatment. Methods: qRT-PCR and MSP methods were used to detect Bin1 in 5 kinds of human esophageal scales at 1. The expression and methylation status of cancer cells yes-2, te13, te1, kyse30 and Ec109 were selected, and yes-2 and te13 were selected for follow-up experiments. Two ESCC cells were intervened with the demethylation drug 5-aza-dC, and the two kinds of ESCC cells were detected by MSP and Western-blot, and the changes of the methylation status and expression were analyzed. Clone formation experiment, flow cytometry to detect the changes of proliferation ability of yes-2 and te13 cells before and after demethylation, and analyze the effect of Bin1 methylation on the proliferation of ESCC cells..3 uses flowcytometryassay (FCM) to detect the proliferation of yes-2 and te13 cells before and after demethylation, the change of apoptosis ability and analysis of Bin1 methyl. Effect of ESCC cell proliferation and apoptosis ability.4 use cell scratch test, Transwell test to detect the migration of yes-2 and te13 cells before and after demethylation, change of invasion ability, analyze the effect of Bin1 methylation on ESCC cell migration and invasion ability,.5 adopts Westernblot test to detect the epithelium of yes-2 and te13 cells before and after demethylation. Epithelial-mesenchymaltransition (EMT) related protein E cadherin (E-cadherin, E-cad), n cadherin (N-cadherin, n-cad), matrix metalloproteinase 2 (matrixmetalloproteinase-2, MMP-2), matrix metalloproteinase 9 (MMP-9), etc. Results: 1bin1mrna showed low expression state (P0.01) in five ESCC cells, te13, te1, kyse30, Ec109 and yes-2, and the five ESCC cells were methylation state, and yes-2 and te1 two cells were completely methylation state. After 5-aza-dC demethylation, the expression of Bin1 protein expression in yes-2 and te13 two kinds of human ESCC cells increased (P0.01).2mtt results showed that the proliferation inhibition rates of cells were 6.7 + 0.9%, 12.3 + 1.7% and 15.4 + 2.1%, respectively, by different concentrations (30 u m, 60 u m, 90 micron M). 7.3 + 1%, 49.3 + 5.7% and 59.6 + 6.3% respectively, compared with the control group with a concentration of 0 m, the cell proliferation rate was significantly reduced, and the proliferation inhibition rate of cell proliferation was 6.4 + 0.8%, 11.9 + and + 6.3% after D2 days after te13 cells with different concentrations (30 mu, 60 u m, 90 mu m), and the growth inhibition rate of cell growth was respectively %, 47.2 + 5.6% and 57.2 + 5.9%, compared with the control group with a concentration of 0 micron m, the cell proliferation rate decreased significantly (P0.01). The clone formation experimental results showed that the number of cell clones was (233 + 29), (189 + 23) and (189 + 0), compared with the control group with concentration of M, after 5-aza-dC intervention in yes-2 cells with different concentrations (0 m, 30, m, 60, m, 90 u m). The number of clones was significantly reduced, and the number of clones was (281 + 33), (235 + 28), (139 + 19) and (83 + 12) after the intervention of te13 cells with different concentrations (0 m, 30 m, 60 m, 90 m). The number of clones decreased significantly compared with the control group with concentration of 0 mu m. The proliferation activity of yes-2 and te13 cells after demethylation was significantly decreased (P0.01).3. The results of flow cytometry showed that the percentage of 5-aza-dC cells in g0/g1 phase was (37.2 + 3.1)%, (46.5 + 3.9)%, (58.2 + 4.3)% and (70.7 + 4.9)%, respectively (37.2 + 3.1)%, (58.2 + 4.3)% and (70.7 + 4.9)%) after yes-2 cells were interfered with yes-2 cells at different concentrations (0 m, 30, m, 90 m). Compared with the control group, the percentage of cells in the g0/g1 stage increased significantly, and the percentage in the S phase decreased obviously, and the percentage of the cells at g0/g1 (33.7 + 2.8)%, (39.4 + 3.6)%, (47.3 + 3.9)% and (56.3 + 4.5)%, respectively (70.2 + 60), respectively (33.7 + 2.8)%, (47.3 + 3.6)%, (47.3 + 3.9)% and 56.3 + 4.5) by different concentrations (0 u m, 30 m, 60 mu m, 90 mu m) were respectively .5)%, (58.3 + 4.2)%, (45.2 + 3.1)% and (32.6 + 2.8)%, compared with the control group with the concentration of 0 m, the percentage of cells in the g0/g1 phase increased obviously, and the percentage in the S phase decreased obviously. It indicated that the ESCC cell cycle changed after demethylation treatment, the cells in the S phase decreased significantly, and the cells blocked in g0/g1 phase increased significantly (P0.01). Flow cytometry showed that the percentages of apoptotic cells were (4.12 + 0.89)%, (7.49 + 1.34)%, (10.03 + 2.13)%, (10.03 + 2.13)% and (13.07 + 2.47) after interfering with yes-2 cells with different concentrations (0 m, 30 m, 60 u m, 90 m), and the percentage of apoptotic cells increased significantly (P0.01), compared with the control group with the concentration of 0 mu m (P0.01). The percentage of apoptotic cells in the apoptotic cells was (3.57 + 0.78)%, (8.09 + 1.43)% and (10.15 + 2.08)%, (15.64 + 2.55), and the percentage of apoptotic cells increased significantly (P0.01). The results indicated that Bin1 gene demethylation could inhibit yes-2 and te13 finer after Bin1 gene demethylation, after 5-aza-dC intervention in te13 cells was (3.57 + 0.78)%, (8.09 + 1.43)% and (10.15 + 2.08)%, (15.64 + 2.55), and (15.64 + 2.55), compared with the control group with a concentration of 0 m. The proliferation ability and apoptosis of ESCC cells and the inhibitory effect of.4 on the malignant biological behavior of ESCC cells showed that after 5-aza-dC demethylation treatment, the migration distance of yes-2 cells was (57.7 + 4.5) mu m, 48h migration distance was (122.4 + 9.4) mu m without demethylation treatment, and yes-2 after demethylation treatment. The migration distance of the cells was significantly shorter than before, and the results were statistically significant (P0.01); the migration distance of 48h after 5-aza-dC demethylation was (64.2 + 4.8) mu m, and the migration distance of 48h without demethylation was (133.4 + 8.1) mu m. The migration distance of te13 cells after demethylation treatment was significantly shorter than that before treatment, and the result was a result. The migration distance of.YES-2 and TE13 cells was significantly shortened, indicating that Bin1 could inhibit the migration ability of YES-2 and TE13 cells after demethylation treatment.Transwell experiment results showed that the cell number of the cells passed through the filter membrane was divided into 0 micron (0 u M, 30 u M, 60 mu M, 90 Mu M). Not (206 + 35), (137 + 29), (78 + 18) and (39 + 9), compared with the control group with a concentration of 0 M, Matrigel filtration membrane cells were significantly reduced (P0.01). The number of TE13 cells after 5-Aza-dC demethylation treated by different concentrations (0 mu M, 30 mu M, 60 mu M, 90 M), and the number of cells passing through Matrigel filter membrane were respectively Compared with the control group with a concentration of 0 M, the number of transmembrane cells in.YES-2 and TE13 cells decreased significantly in.YES-2 and TE13 cells through Matrigel filtration cells, indicating that Bin1 could inhibit the invasive ability of the cells after demethylation. The experiments indicated that Bin1 can inhibit the migration and invasion of YES-2 and TE13 cells after the demethylation of Bin1. The results of.5 Western blot detection showed that the E-cad expression of EMT related protein was up regulation (P0.01), N-cad, Snail, Snail, and protein expression after 5-Aza-dC treatment, indicating that the expression of the proteins could be changed after demethylation to inhibit the occurrence of epithelial mesenchymal transition. In vitro experiments: in vitro experiments have proved that Bin1 methylation affects the proliferation, apoptosis, migration and invasion of esophageal cancer cells, and affects the development of esophageal cancer by inhibiting the process of epithelial mesenchymal transition in esophageal cancer cells.

【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.1

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相關期刊論文 前2條

1 Laura Lattanzio;Cristiana Lo Nigro;;Epigenetics and DNA methylation in cancer[J];World Journal of Translational Medicine;2015年01期

2 Ru Liu;Xiao-Huan Zhang;Kun Zhang;Wei Li;Wen-Jun Wang;Di-Xian Luo;Ling Gao;;5-Aza-2'-deoxycytidine inhibits retinoblastoma cell by reactivating epigenetically silenced RASSF1A gene[J];International Journal of Ophthalmology(English Edition);2014年01期

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本文編號：1864899