【作者】 苏晓强；

【导师】 郝敏；

【作者基本信息】 山西医科大学 ， 妇产科学， 2012， 硕士

【摘要】 目的：1.检测宫颈病变细胞中DNMT1蛋白表达状态和抑癌基因DAPK启动子区的甲基化状态，探讨其在宫颈病变发生发展中的作用；2.探讨高危型HPV的感染、DNMT1表达和DAPK启动子区甲基化状态间的相互关系，可进一步了解宫颈病变的发生机制，对该病早期筛查及病情发展的监测提供帮助。方法：1.研究对象随机选择2011年1月至2011年12月我院妇科门诊患者中均行TCT和阴道镜检查的150例患者，根据阴道镜宫颈活检的病理诊断结果，分为子宫颈上皮内瘤变Ⅰ级（CINⅠ）30例，子宫颈上皮内瘤变Ⅱ级（CINⅡ）30例，子宫颈上皮内瘤变Ⅲ级（CINⅢ）30例，鳞状细胞癌（SCC）30例作为研究组；另取未见恶性细胞和上皮内病变细胞（NILM）30例为对照组。2.检测高危型HPV-DNA感染情况取患者的TCT剩余标本，提取DNA，SPR方法检测高危型HPV，运用生物传感芯片阅读仪读取高危型HPV-DNA感染情况。3.检测DNMT1蛋白表达状态取上述所选患者的TCT剩余标本（保存期不超过3周），经Thinprep2000系统重新制片1张，置于95%酒精中室温固定24小时，空气中晾干，置冰箱4℃储存备用拟行DNMT1蛋白的免疫细胞化学检测。显微镜下观察细胞图片，细胞质及细胞间质出现棕褐色颗粒者为阳性。4．检测抑癌基因DAPK启动子区的甲基化状态取入组患者的TCT剩余标本，采用MSP技术，检测患者宫颈脱落细胞DAPK基因启动子区的甲基化状况。摄像观察8% PAGE上电泳结果：获得甲基化产物，无论是否出现非甲基化产物，均判定为甲基化阳性；只出现非甲基化产物判定为甲基化阴性；甲基化产物与非甲基化产物均未获得判定该标本实验失败。5.统计学分析采用SPSS18.0软件包进行统计分析，检验水准α= 0.05。结果：1. 150例宫颈各病变阶段宫颈脱落细胞中高危型HPV-DNA的检测情况：高危型HPV感染阳性率为80.67%（121∕150），随着病变程度的进展，各组间高危型HPV感染率存在统计学差异（χ2=30.658，P＜0.05），且呈现出增高的趋势（趋势χ2=30.658，P＜0.05）。2.宫颈病变各阶段DNMT1蛋白检测结果：NILM组6.67%；CINⅠ组56.67%；CINⅡ组63.33%；CINⅢ组66.67%；SCC组76.67%。DNMT1蛋白在各宫颈病变阶段组中的阳性表达率差异有显著性（P﹤0.05），且随着病变程度的进展，DNMT1蛋白表达率呈现出增高的趋势（P﹤0.05）。3.本实验中随着病变程度的进展，DAPK基因启动子区CpG岛的甲基化率分别为3.33%、10.00%、13.33%、46.67%、60.00%，各组间有显著性差异（P<0.05），且呈现出增高的趋势（P<0.05）。4.在宫颈病变的过程中，高危型HPV感染、DNMT1蛋白表达与DAPK基因甲基化两两间相关系数分别为0.340、0.214、0.444，互呈正相关（P均< 0.05）。结论：随着宫颈病变级别的升高，高危型HPV持续性感染、DNMT1蛋白异常高表达、DAPK抑癌基因的异常高甲基化，各指标间可能存在相互间的促进关系，可以为宫颈病变的发现和病变程度的监测提供新思路、新方法。更多还原

【Abstract】 Objective：1. To detect the expression of DNMT1 protein and the methylation status of suppressorgene DAPK promoter in cervical lesions, discuss their role in the development of cervical lesions2. To Study the relationship among infection of high-risk type HPV, expression of DNMT1and methylation status of DAPK promoter in cervical lesions, and to further understand thepathogenesis of the disease, to help early screening and monitoring of disease progression.Methods:1.Source of specimen We randomly collected the 150 patients were treated with TCT andcolposcopic examinations from 2011 January to 2011 December. According to the results ofcervical biopsy pathologic diagnosis under colposcopy, divided into cervical intraepithelialneoplasia grade I ( CIN I ) in 30 cases, cervical intraepithelial neoplasia grade II ( CINII )30cases, cervical intraepithelial neoplasia ( CIN III ) in 30 cases, squamous cell carcinoma( SCC ) in 30 cases as study group; the other with negative for intraepithelial lesion ormalignancy ( NILM ) as the control group of 30 cases.2. Detection of high-risk type HPV-DNA infection For patients with TCT residualpreservation solution, DNA was extracted, the SPR method for detection of HPV, HPV-DNAinfection was detected by the biosensor chip.3. Detection of the expression of DNMT1 protein state The selected the TCT residualpreservation solution of 150 patients (preservation period of not more than 3weeks), via theThinprep2000 system to produce 1cell slides. Then put the sliders under 95% alcohol fixation for24 hours at room temperature, then dry them in the air, store them in the refrigerator at 4 degreesbackup to DNMT1 protein was with immunocytochemistry. Cells picture were observed under amicroscope. The case with brown particles in cytoplasm and stromal cells was positive.4. Detection of tumor suppressor gene DAPK promoter methylation status Patients’TCTresidual preservation solution, with MSP technology，were detected the methylation status on thecervical exfoliated cells DAPK gene promoter region. Determination of results PCR productswere walking in 8% PAGE. Get methylated product, whether they appear non-methylatedproduct, both determinated to be methylated positive; only a non-methylated product were foundfor unmethylated; has not been methylated product and non-methylated product is the sign of thefailure of the experiment.5. Statistical analysis Using SPSS18.0 software package for the Statistical analysis,α= 0.05 of inspection standard.Result:1.150cases of cervical lesions in cervical exfoliated cells in each phase of high-risk typeHPV-DNA detection: Total high-risk type HPV positive rate was 80.67% (121/ 150), trend ofχ2= 30.658, P < 0.05,with the extent of the lesion progression, HPV infection rate is alsoshowing a trend of increasing.2. Expression of DNMT1protein in each group: In 6.67% of NILM group; CIN group56.67%;CIN II group 63.33% group66.67%; CIN; SCC76.67%DNMT1 protein expression. DNMT1protein in various types of cervical lesions in the positive expression rates have significantdifference (P < 0.05), and with the extent of the lesion progression, DNMT1 protein expressionrate showed increasing trend (P < 0.05).3. The experiment of promoter region of DAPK gene in CpG island methylation positive ratewas 26.67% (40/ 150), with the extent progression of the lesion, DAPK gene methylation rateswere 3.33%, 10.00%, 13.33%, 46.67%, 60.00%, there are significant differences among thegroups ( P < 0.05), and showed a trend of increasing( P < 0.05).4. In cervical lesions during the development of sick, fection of high-risk type HPV,DNMT1protein expression and DAPK gene methylation every two were positively correlated ( P< 0.05), The correlation coefficient is respectively: 0.340、0.214、0.444.Conclusion：With the rise of the level of cervical lesions, high-risk type HPV persistent infection,abnormal high expression of DNMT1protein and DAPK tumor suppressor gene abnormalhypermethylation, the promotion of relations may exist between between each Indicators. Newideas and new methods were proposed for the discovery and monitoring of the severity ofcervical lesions.更多还原