本文選題：PTEN + 宮頸癌��； 參考：《青島大學》2014年碩士論文

【摘要】：PTEN(磷酸酶和張力蛋白同源物)是一種具有雙蛋白質(zhì)和脂質(zhì)磷酸酶活性的腫瘤抑制基因。CpG島甲基化是導致抑癌基因失活的一種機制,近年來許多學者從PTEN基因啟動子區(qū)甲基化方向著手研究PTEN基因在腫瘤組織中下調(diào)的機制。然而關于宮頸癌組織中PTEN基因啟動子區(qū)的甲基化狀態(tài)存在爭議,因此宮頸癌組織中PTEN基因啟動子區(qū)甲基化狀態(tài)及甲基化對PTEN基因表達的影響有待進一步研究。 目的 探討宮頸癌細胞系及宮頸組織中PTEN基因啟動子甲基化狀態(tài)以及對其表達的影響 方法 選取體外培養(yǎng)的4種宮頸癌細胞系HeLa, Caski, C-33A, HT-3以及18例宮頸癌組織和8例宮頸正常組織作為研究對象,采用BSP聯(lián)合TA克隆測序檢測PTEN基因啟動子甲基化狀態(tài)；采用去甲基化藥物5-氮雜-2′-脫氧胞苷(5-Aza-dC)處理體外培養(yǎng)的4種宮頸癌細胞系,應用實時熒光定量PCR檢測藥物處理前后PTEN基因表達差異。 結(jié)果 1.4種宮頸癌細胞系PTEN基因啟動子區(qū)呈低甲基化狀態(tài),HPV陽性細胞系(HeLa, Caski)與HPV陰性細胞系(C-33A,HT-3) PTEN基因啟動子甲基化率無顯著性差異(CpGl:t=2.83, P=0.11; CpG2:t=1.00, P=0.42); 2.宮頸癌組織以及正常宮頸組織中PTEN基因啟動子區(qū)均呈低甲基化狀態(tài),且兩者間PTEN基因啟動子甲基化水平無明顯差異(CpGl:T=83.50,P=0.15; CpG2;T=34.5,P=0.720)； 3. Real-time qPCR顯示,4種宮頸癌細胞系5-Aza-dC處理前后PTEN基因mRNA表達無明顯差異(HeLa:t=-1.18, P=0.36; Caski:t=2.07,P=0.18;C-33A:t=-0.07, P=0.95; HT-3:t=-0.53, P=0.65)。 結(jié)論 1.BSP聯(lián)合TA克隆測序結(jié)果分析結(jié)果顯示宮頸癌細胞系中HPV感染與PTIN基因啟動子區(qū)甲基化程度無明顯相關性； 2.BSP聯(lián)合TA克隆測序結(jié)果比較及統(tǒng)計學分析提示宮頸癌組織中PTEN基因啟動子區(qū)甲基化不是導致該基因表達下調(diào)的主要原因； 3.抑癌基因啟動子高度甲基化被認為是除突變和缺失以外抑癌基因功能失活的關鍵機制,但僅應用MSP定性分析抑癌基因的甲基化水平具有一定的局限性,抑癌基因啟動子區(qū)域甲基化研究中同時采用BSP聯(lián)合TA克隆測序可更為客觀的反映其甲基化狀態(tài)。
[Abstract]:PTEN (phosphatase and tension protein congener) is a tumor suppressor gene with double protein and lipid phosphatase activity. CpG island methylation is a mechanism leading to the inactivation of tumor suppressor gene. In recent years, many researchers have started to study the down-regulation of PTEN gene in tumor tissues from the direction of promoter methylation of PTEN gene. However, the methylation status of PTEN promoter region in cervical cancer tissues is controversial. Therefore, the methylation status of PTEN promoter region and the effect of methylation on the expression of PTEN gene in cervical cancer tissues need to be further studied. Purpose Study on methylation status and expression of PTEN gene promoter in cervical cancer cell line and cervical tissue Method Four cervical cancer cell lines, HeLa, Caski, C-33A, HT-3, 18 cervical cancer tissues and 8 normal cervix tissues were selected to detect the methylation status of PTEN gene promoter by BSP combined with TA clone sequencing. Four cervical cancer cell lines cultured in vitro were treated with demethylated drug 5-aza-dC. The difference of PTEN gene expression before and after drug treatment was detected by real-time fluorescence quantitative PCR. Result 1.4 there was no significant difference in the methylation rate of PTEN promoter between the HPV-positive cell line (HeLa, Caski) and the HPV negative cell line (CpGl2: t1. 83, P0. 11, CpG2: t1. 00, P0. 42), and the HPV negative cell line (CpG2: t1. 00, P0. 42, P0. 42, CpG2: t1. 00, P0. 42), CpG2: t1. 00, P0. 42, CpG2: 1. 2. The promoter region of PTEN gene in cervical cancer and normal cervix tissues was hypomethylated, and there was no significant difference in methylation level of PTEN gene promoter between them. 3. Real-time qPCR showed that there was no significant difference in mRNA expression of PTEN gene in four cervical cancer cell lines before and after 5-Aza-dC treatment. The expression of PTEN gene was 0.36; Caski: t0. 07; C33: T0. 07, P0. 05; HT-3: t0. 53, p0. 65. Conclusion The results of 1.BSP combined with TA clone sequencing showed that there was no significant correlation between HPV infection and methylation degree of PTIN gene promoter in cervical cancer cell line. The results of 2.BSP combined with TA clone sequencing and statistical analysis showed that methylation of the promoter region of PTEN gene was not the main cause of down-regulation of PTEN gene expression in cervical cancer tissues. 3. Hypermethylation of tumor suppressor gene promoter is considered to be the key mechanism for the functional inactivation of tumor suppressor gene except mutation and deletion. However, only qualitative analysis of the methylation level of tumor suppressor gene by MSP has some limitations. In the study of methylation of tumor suppressor gene promoter region, BSP combined with TA clone sequencing can more objectively reflect the methylation status of tumor suppressor gene.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.33

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本文編號：1912683