本文選題：哈薩克族　切入點(diǎn)：食管鱗癌　出處：《石河子大學(xué)》2017年碩士論文

【摘要】：目的:探討PLCE1基因啟動(dòng)子甲基化程度與PLCE1蛋白表達(dá)的關(guān)系及其與臨床病理特征的關(guān)系;檢測(cè)4株不同食管鱗癌細(xì)胞系Eca109,TE-1,EC9706,Kyse150中PLCE1基因啟動(dòng)子甲基化程度與PLCE1蛋白表達(dá)的關(guān)系并探討甲基轉(zhuǎn)移酶抑制劑5-aza-d C對(duì)細(xì)胞PLCE1甲基化程度與PLCE1蛋白表達(dá)的影響;應(yīng)用PLC抑制劑U73122后對(duì)食管鱗癌細(xì)胞生長(zhǎng)、凋亡及侵襲轉(zhuǎn)移的影響。方法:收集51例新疆哈薩克族食管鱗癌組織及對(duì)應(yīng)的51例癌旁正常組織,用免疫組化Envision法檢測(cè)PLCE1的蛋白表達(dá),并提取這51對(duì)癌與癌旁組織的DNA,應(yīng)用甲基化特異性PCR(MSP)技術(shù)檢測(cè)PLCE1基因啟動(dòng)子甲基化狀態(tài),分析PLCE1基因啟動(dòng)子甲基化程度與PLCE1蛋白表達(dá)的相關(guān)性,及其與患者性別、年齡、腫瘤最大直徑、淋巴結(jié)和遠(yuǎn)處轉(zhuǎn)移、TNM分期及預(yù)后的相關(guān)性;用Western blot檢測(cè)4株食管癌細(xì)胞系在5-aza-d C作用前后的PLCE1蛋白表達(dá),并用MSP檢測(cè)它們的PLCE1基因啟動(dòng)子甲基化狀態(tài);將PLC抑制劑U73122作用于食管癌細(xì)胞系Eca109、EC9706,應(yīng)用Western blot檢測(cè)抑制效率,并在作用48小時(shí)后,用MTT和平板克隆檢測(cè)細(xì)胞生長(zhǎng)情況,流式細(xì)胞術(shù)和TUNEL染色檢測(cè)細(xì)胞凋亡情況,transwell侵襲實(shí)驗(yàn)檢測(cè)細(xì)胞侵襲能力,并用Western blot檢測(cè)凋亡及上皮間葉轉(zhuǎn)化的相關(guān)蛋白表達(dá)情況。結(jié)果:(1)新疆哈族食管鱗癌組織中的PLCE1蛋白表達(dá)明顯高于癌旁正常組織;食管鱗癌組織PLCE1基因啟動(dòng)子甲基化率明顯低于正常組織,兩組比較差異有統(tǒng)計(jì)學(xué)意義(P0.001);新疆哈族食管鱗癌組織中,PLCE1蛋白高表達(dá)的組織其PLCE1基因啟動(dòng)子甲基化水平低于PLCE1蛋白低表達(dá)的組織,兩組比較差異有統(tǒng)計(jì)學(xué)意義(P=0.028)。在這51例食管鱗癌組織中PLCE1基因啟動(dòng)子甲基化程度與患者性別、年齡、腫瘤大小及患者術(shù)后生存時(shí)間無(wú)關(guān),與患者淋巴結(jié)及遠(yuǎn)處轉(zhuǎn)移(χ2=7.242,P=0.027)和TNM分期(χ2=7.883,P=0.019)相關(guān)。(2)MSP檢測(cè)四株食管癌細(xì)胞系Eca109,TE-1,EC9706,Kyse150中PLCE1基因啟動(dòng)子甲基化程度與PLCE1蛋白表達(dá)成反比,甲基轉(zhuǎn)移酶抑制劑5-aza-d C抑制了TE-1和Kyse150的PLCE1甲基化程度、增高了其蛋白表達(dá)水平。(3)U73122在濃度為10u M時(shí)作用于食管鱗癌細(xì)胞48h后對(duì)PLCE1的抑制效果最好,當(dāng)U73122作用后,MTT和平板克隆技術(shù)證明細(xì)胞生長(zhǎng)能力降低,流式細(xì)胞術(shù)和TUNEL法證明細(xì)胞凋亡增多,促凋亡分子Bax、Cleaved-PARP、Caspase3、Caspase7表達(dá)均增高,而抗凋亡分子Bcl-2表達(dá)降低;transwell實(shí)驗(yàn)證明細(xì)胞侵襲能力減弱,上皮標(biāo)記物E-cadherin表達(dá)升高,間葉標(biāo)記物Vimentin降低。結(jié)論:PLCE1的去甲基化導(dǎo)致其在食管鱗癌細(xì)胞中高表達(dá)從而促進(jìn)食管癌細(xì)胞的惡性生物學(xué)行為。
[Abstract]:Objective: To investigate the relationship between methylation of PLCE1 gene promoter and expression of PLCE1 protein and its relationship with clinicopathological features; detection of 4 different strains of esophageal squamous cell carcinoma cell lines Eca109, TE-1, EC9706, Kyse150 in PLCE1 gene promoter methylation and expression of PLCE1 protein and explore the methyltransferase inhibitor 5-aza-d C on cells the degree of PLCE1 methylation and expression of PLCE1 protein on the growth of esophageal squamous cell carcinoma; application of PLC inhibitor U73122, apoptosis and metastasis. Methods: 51 cases were collected in Xinjiang Kazakh esophageal squamous cell carcinoma and 51 cases of cancer adjacent normal tissues, the expression of PLCE1 by immunohistochemical Envision method to detect and extract the protein. 51 of the carcinoma and adjacent tissue DNA by methylation specific PCR (MSP) technique to detect the methylation status of PLCE1 gene promoter and analysis of PLCE1 gene promoter methylation degree and PLCE1 protein The correlation between the expression, and age and gender, and the maximum diameter of tumor, lymph node and distant metastasis, the correlation between TNM staging and prognosis; 4 strains of human esophageal cancer cell line Western to detect the blot expression of 5-aza-d before and after C in the role of the PLCE1 protein, and MSP detection of PLCE1 gene promoter methylation status of their will; PLC inhibitor U73122 on esophageal cancer cell lines Eca109, EC9706, blot application of Western detection and suppression efficiency, and in 48 hours after using MTT and clone detection cell growth, flow cytometry and TUNEL staining were used to detect cell apoptosis, Transwell to detect the invasive ability of cells, and the expression of related protein in leaves the transformation of epithelial apoptosis and Western blot detection between. Results: (1) the expression of Xinjiang Kazakh esophageal squamous cell carcinoma PLCE1 protein was significantly higher than that of adjacent normal tissue; PLCE1 gene in esophageal squamous cell carcinoma. The promoter methylation rate was significantly lower than that in normal tissues, there was significant difference between two groups (P0.001); Xinjiang Kazakh esophageal squamous cell carcinoma, promoter methylation level is lower than the low expression of PLCE1 protein in the tissue of high expression of PLCE1 protein in the tissue of PLCE1 gene, there was significant difference between two groups (P=0.028) in the PLCE1. In 51 cases of esophageal carcinoma gene promoter methylation in patients with gender, age, tumor size and survival time. Patients with lymph node and distant metastasis (2=7.242, P=0.027) and TNM stage (2= 7.883, P=0.019). (2) MSP was detected in four strains of esophageal carcinoma cell line Eca109, TE-1, EC9706, Kyse150 in PLCE1 gene promoter methylation and PLCE1 protein expression is inversely proportional to the methyltransferase inhibitor 5-aza-d inhibited the C methylation level of PLCE1 TE-1 and Kyse150, increased the expression of U73122 (3). At the concentration of 10u M in esophageal squamous cell carcinoma cell line 48h after the best inhibitory effect on PLCE1, when U73122, MTT and plate cloning technology that reduces cell growth ability, that the number of apoptosis cells by flow cytometry and TUNEL assay, apoptosis molecule Bax, Cleaved-PARP, Caspase3, Caspase7 expression was significantly increased, while the anti the decreased expression of apoptosis Bcl-2; Transwell experiments proved that the weakened cell invasion, epithelial marker E-cadherin increased expression of mesenchymal marker Vimentin decreased. Conclusion: PLCE1 demethylation leads to its high expression in esophageal squamous cell carcinoma and promote the malignant biological behavior of esophageal carcinoma cells.

【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.1

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本文編號(hào)：1708439