本文選題：宮頸癌 + 表皮生長因子受體��； 參考：《第四軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：宮頸癌是一種嚴重威脅著女性生命和健康、由HPV感染導(dǎo)致的惡性腫瘤，發(fā)病率在全世界女性腫瘤中位居第二，死亡率位居第一，且目前發(fā)病率與死亡率仍呈上升趨勢。多數(shù)患者在確診時已達中晚期，因此大多要在手術(shù)治療后接受放化療。目前的化療藥物對晚期宮頸癌的治療有一定效果，但仍存在嚴重的耐藥性，導(dǎo)致預(yù)后差、生存期短。在大部分宮頸癌患者標本中，可檢出表皮生長因子受體（epidermalgrowth factor receptor, EGFR）高水平表達。近年，以表皮生長因子受體酪氨酸激酶抑制劑(EGFR-TKI)為基礎(chǔ)的分子靶向藥物成為宮頸癌個體化用藥治療的研究熱點，但臨床試驗卻并未獲得預(yù)期療效。研究發(fā)現(xiàn)，在部分宮頸癌患者中，EGFR基因存在甲基化修飾，可能與EGFR靶向治療藥物效果較差有關(guān)。因此，對EGFR基因啟動子甲基化狀態(tài)的檢測，有利于指導(dǎo)宮頸癌EGFR-TKI個體化治療和去DNA甲基化臨床用藥的實施。 熒光偏振技術(shù)（fluorescence polarization, FP）是一種通過檢測熒光標記分子的熒光偏振值變化判斷分子量改變的技術(shù)。偏振熒光的強度與熒光標記分子分子量的大小呈正相關(guān)，因此通過檢測熒光偏振強度值的變化即可判斷待測熒光標記分子是否參與了反應(yīng)。本研究利用此技術(shù)在液相環(huán)境中均相檢測的能力和效率的優(yōu)勢，對宮頸癌患者EGFR啟動子甲基化狀態(tài)進行檢測研究。 目的：建立基于熒光偏振技術(shù)的EGFR啟動子甲基化標準檢測方法與體系，檢測并分析宮頸癌患者組織標本中EGFR基因啟動子甲基化狀態(tài)，并初步探討其與肺癌相關(guān)EGFR-TKI敏感突變、宮頸癌病因HPV感染之間的聯(lián)系，為指導(dǎo)宮頸癌的個體化治療提供新思路和分子診斷技術(shù)方法。 方法：首先建立熒光偏振檢測標準體系。提取健康志愿者血液淋巴細胞DNA，對DNA進行亞硫酸氫鹽修飾，一半經(jīng)SssI甲基轉(zhuǎn)移酶處理。設(shè)計EGFR啟動子序列通用引物，擴增序列后測序，同時構(gòu)建甲基化與非甲基化序列標準質(zhì)粒載體。設(shè)計EGFR啟動子甲基化特異性探針，利用標準品作為模板建立熒光偏振檢測標準體系，對體系各項反應(yīng)條件與敏感性、穩(wěn)定性、特異性等進行研究和優(yōu)化。隨后利用已建立的檢測方法對宮頸癌組織標本進行EGFR啟動子甲基化狀態(tài)檢測，并與測序法結(jié)果進行比較。同時，檢測標本中肺癌相關(guān)EGFR-TKI敏感突變以及HPV感染型別。利用SPSS13.0軟件進行統(tǒng)計學(xué)分析，分析EGFR啟動子甲基化與肺癌相關(guān)EGFR-TKI敏感突變、HPV感染等因素之間的關(guān)系。 結(jié)果：本研究成功構(gòu)建了基于熒光偏振技術(shù)的EGFR啟動子甲基化狀態(tài)的標準檢測體系，對此標準檢測體系的反應(yīng)條件、靈敏度、準確度和穩(wěn)定性均進行了研究和優(yōu)化。本方法最低檢測濃度為50拷貝/μl，，最低檢測含量可達10%，敏感度高。利用該體系成功檢測了宮頸癌標本組織中EGFR啟動子甲基化狀態(tài)，本方法檢測EGFR啟動子甲基化狀態(tài)結(jié)果與直接測序法結(jié)果無統(tǒng)計學(xué)差異。99例宮頸癌標本中EGFR啟動子甲基化陽性37例，陽性率為37.4%。未檢測出EGFR基因18、19、21外顯子EGFR-TKI敏感突變。HPV感染型別中HPV16陽性29例，HPV18陽性12例，HPV58陽性4例，HPV52陽性4例。HPV16感染陽性中EGFR啟動子甲基化18例，占62.1%。HPV16陽性樣本中EGFR啟動子甲基化陽性比率最高。 結(jié)論：本研究方法檢測EGFR啟動子甲基化操作簡單高效，靈敏度與準確度較高，為臨床宮頸癌個體化治療相關(guān)檢測提供了新技術(shù)。本研究中，宮頸癌標本中EGFR啟動子甲基化陽性率為37.4%，未發(fā)現(xiàn)肺癌相關(guān)EGFR-TKI敏感基因突變， HPV16感染的宮頸癌標本中EGFR啟動子甲基化陽性率較其他HPV型顯著增高，提示HPV16感染與EGFR啟動子甲基化可能有一定相關(guān)性。
[Abstract]:Cervical cancer is a malignant tumor that seriously threatens the life and health of women and is caused by HPV infection. The incidence of the disease is second in the world's female tumor. The mortality rate is the first, and the current incidence and mortality are still on the rise. Most patients have reached the middle late stage at the time of diagnosis, so most of them should receive chemotherapy after surgical treatment. The current chemotherapy drugs have some effect on the treatment of advanced cervical cancer, but there are still serious drug resistance, which results in poor prognosis and short survival time. In most of the cervical cancer patients, the epidermalgrowth factor receptor (EGFR) Gao Shui flat expression can be detected. In recent years, the epidermal growth factor receptor tyrosine kinase is used. Inhibitor (EGFR-TKI) based molecular targeting drugs have become a hot spot in the study of individualized drug therapy for cervical cancer, but clinical trials have not been expected. The study found that in some patients with cervical cancer, the EGFR gene methylation modification may be related to the poor effect of EGFR targeting therapy. Therefore, the promoter of the EGFR gene can be used as a promoter. The detection of basal status is helpful to guide the individualized treatment of cervical cancer EGFR-TKI and the implementation of clinical medication for removing DNA methylation.
Fluorescence polarization (FP) is a technique to determine the change of molecular weight by detecting the change of fluorescence polarization value of the fluorescent labeling molecule. The intensity of polarization fluorescence is positively correlated with the molecular weight of the fluorescent labeling molecule, so the fluorescence labeling can be judged by detecting the change of the fluorescence polarization intensity value. This study used this technique to detect the methylation status of EGFR promoter in cervical cancer patients by using the advantage of this technique to detect the ability and efficiency of homogeneous detection in the liquid environment.
Objective: to establish a standard detection method and system of EGFR promoter methylation based on fluorescence polarization technology, to detect and analyze the methylation status of EGFR gene promoter in the tissue specimens of cervical cancer patients, and to explore the relationship between the EGFR-TKI sensitive mutation associated with lung cancer and the HPV infection of the cause of cervical cancer in order to guide the individualized treatment of cervical cancer. The treatment provides new ideas and molecular diagnostic techniques.
Methods: first, a standard system of fluorescence polarization detection was established. DNA of blood lymphocyte in healthy volunteers was extracted, DNA was modified by hydrogen sulfite and half of SssI methyltransferase was treated. The universal primers for EGFR promoter sequence were designed, and the sequence was sequenced, and the standard plasmid vector of methylation and non methylation sequence was constructed. The EGFR initiation was designed. The methylation specific probe was used to establish a standard system for fluorescence polarization detection using standard products as a template. The reaction conditions and sensitivity, stability and specificity of the system were studied and optimized. Then the methylation status of EGFR promoter in cervical cancer tissues was detected by the established detection method, and the sequencing method was combined with the sequencing method. The results were compared. At the same time, the lung cancer related EGFR-TKI sensitive mutations and HPV infection types were detected. The SPSS13.0 software was used for statistical analysis to analyze the relationship between EGFR promoter methylation and EGFR-TKI sensitive mutations in lung cancer, HPV infection and other factors.
Results: the standard detection system for the methylation status of EGFR promoter based on fluorescence polarization technology was successfully constructed. The reaction conditions, sensitivity, accuracy and stability of the standard detection system were studied and optimized. The minimum detection concentration of this method was 50 copies / mu L, the minimum detection content was up to 10%, and the sensitivity was high. The system successfully detected the methylation status of EGFR promoter in cervical carcinoma tissue. This method was used to detect the methylation status of EGFR promoter and the results of direct sequencing, 37 cases of EGFR promoter methylation positive in.99 cases of cervical cancer, the positive rate was 37.4%. not detected the EGFR-TKI sensitivity of EGFR gene 18,19,21 exon There were 29 cases of HPV16 positive in the mutant.HPV infection, 12 cases of HPV18 positive, 4 cases of HPV58 positive, and 18 cases of EGFR promoter methylation in 4 HPV52 positive positive cases, which accounted for the highest percentage of EGFR promoter methylation positive in 62.1%.HPV16 positive samples.
Conclusion: the method of EGFR promoter methylation is simple and efficient, with high sensitivity and accuracy, which provides a new technique for the detection of clinical cervical cancer. In this study, the positive rate of EGFR promoter Zi Jiaji was 37.4% in cervical cancer specimens, and no EGFR-TKI sensitive gene mutation was found in lung cancer, and HPV16 infection was not found in this study. The positive rate of methylation of EGFR promoter in cervical cancer samples was significantly higher than that in other HPV types, suggesting that HPV16 infection may be related to methylation of EGFR promoter.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.33

【參考文獻】

相關(guān)期刊論文 前9條

1 陳楠;江靜;李小平;屠錚;魏麗惠;;宮頸癌分子靶向治療藥物研究進展[J];中國婦產(chǎn)科臨床雜志;2010年01期

2 倪原;張菊;陳國紹;陳智利;楊利紅;;基于熒光偏振技術(shù)的基因檢測分析系統(tǒng)[J];生命科學(xué)儀器;2008年09期

3 趙晨;張亮;倪原;;熒光偏振技術(shù)在生命科學(xué)中的研究進展[J];現(xiàn)代生物醫(yī)學(xué)進展;2010年16期

4 陳蘇瓊;賀修勝;;人乳頭瘤病毒感染與宮頸癌的研究進展[J];現(xiàn)代生物醫(yī)學(xué)進展;2010年24期

5 強少盈;張菊;姜英浩;劉文超;;肺癌患者EGFR基因啟動子甲基化狀態(tài)研究[J];現(xiàn)代腫瘤醫(yī)學(xué);2012年10期

6 彭正良,曹仁賢;甲狀腺腫瘤相關(guān)基因甲基化研究進展[J];國外醫(yī)學(xué)(生理、病理科學(xué)與臨床分冊);2005年02期

7 黨艷麗,馬曉旗,畢紅梅,鄭維國,辛?xí)匝?5-氮胞苷對宮頸癌細胞系DAPK1異常甲基化的影響[J];西安交通大學(xué)學(xué)報(醫(yī)學(xué)版);2005年01期

8 徐成波;沈建箴;沈松菲;傅海英;吳雪梅;吳淡森;朱藝芳;陳璐;;惡性血液病細胞系分泌型卷曲相關(guān)蛋白基因啟動子CpG島甲基化狀態(tài)的檢測及意義[J];中國實驗血液學(xué)雜志;2009年06期

9 郝瑞鳳;王培玉;;宮頸癌流行病學(xué)及高危因素探討[J];醫(yī)學(xué)綜述;2009年10期

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