【作者】 贾英；

【导师】 唐良萏；

【作者基本信息】 重庆医科大学 ， 妇产科， 2012， 博士

【摘要】 目的：1、本文通过检测宫颈病变患者宫颈上皮脱落细胞中人端粒酶mRNA (human telomerase mRNA component, hTERC)基因和c-myc基因的表达以及人乳头瘤病毒(human papillomavirus, HPV)的感染情况和液基细胞学检测的分级,以临床病理结果为金标准,分析比较液基细胞学、HPV检测、hTERC和c-myc基因扩增检测对宫颈病变的预测价值。2、将c-myc基因、hTERC基因、HPV亚型及薄层液基细胞学(thinPrep cytologic test,TCT)的检查结果进行比较及相关性的分析,拟寻求宫颈病变筛查更准确的联合检测方法。3、对行宫颈锥切和子宫切除治疗的CIN患者进行随访,检测hTERC、c-myc基因、HPV亚型及TCT,探讨hTERC、c-myc基因检测及其联合HPV、TCT检测在CIN手术治疗后随访中的价值。方法：选择2011年1月～2011年9月就诊于重庆医科大学附属第一医院妇科的1000例已婚或有性生活史,年龄25～64岁,符合入选要求的妇女。所有入选患者均刷取宫颈脱落细胞行液基细胞学检查,其残留液同时用作荧光原位杂交(fluorescence in situ hxbridization, FISH)技术检测c-myc基因、hTERC基因的表达,表面等离子共振(surface plasmon resonance,SPR)技术检测HPV亚型。液基细胞学、HPV亚型、hTERC基因、c-myc基因任一指标阳性的患者均行阴道镜及病理活组织检查。以病理学结果为金标准,分析液基细胞学、HPV检测、hTERC基因和c-myc基因检测单独筛查对于子宫颈癌筛查的灵敏度和特异性,评价与临床病理结果的相关性。经筛选有117例患者需行阴道镜检查及活检,但有2例主动放弃。所以有115例患者有病理学结果。对病检正常/炎症和CINI者直接随访而不治疗；CINII、CINIII、SCC均采取手术治疗为主,术后6月随访。随访中,26例失访,其中以炎症、全子宫切除患者为主；有89例复诊,并于6个月时对其再次刷取宫颈脱落细胞行TCT、HPV、c-myc、 hTERC检测,有异常时行阴道镜下宫颈活检及组织病理学检查。结果：1、CINI、CINII、CINIII、SCC患者中TCT检测结果的阳性率分别为87.5%、96.4%、100%、100%。随着病理级别的增高,TCT检查结果的阳性率增加。但对不同等级CIN的筛查,没有明显的优越差异(P=0.061)。2、随着病变组织病理学程度的增加,c-myc基因阳性扩增患者病例数增加,c-myc基因阳性扩增率增加,在正常组、CINI组、CINII组、CINIII组、SCC组中c-myc基因扩增阳性患者比率分别为：41.67%,18.75%,39.29%,54.90%,87.50%。结果显示：不同病理级别之间c-myc基因扩增的阳性率差异有统计学意义(P<0.05)。3、病理检测级别为正常、CINI、CINI、CINII、SCC的患者中hTERC的阳性率分别是：33.33%、25%、50%、68.63%、87.50%。随着病变严重程度增加,hTERC基因扩增的阳性率增加。4、随着病理学级别的升高,HPV基因扩增的阳性表达率在正常、CINI、CINII、CINIII、SCC的患者HPV的阳性率分别是：75.00%、68.75%、100.00%、88.24%、87.50%。结果表明：HPV阳性表达在不同病理学级别组中,差异有统计学意义。5、TCT、HPV、c-myc基因、hTERC基因单独检测对宫颈癌及CIN检测的灵敏度分别为86.21%、92.00%、52.9%、64.40%,特异度分别为70.00%、26.70%、70.00%、70.00%。TCT+c-myc、TCT+hTERC、 HPV+c-myc、HPV+hTERC、c-myc+hTERC检测的灵敏度分别为93.50%、95.09%、96.23%、97.15%、83.28%,特异度分别为91.00%、91.00%、78.01%、78.01%、91.00%;TCT+HPV+c-myc、 TCT+HPV+hTERC、TCT+HPV+c-myc+hTERC的灵敏度分别为：99.48%、99.61%、99.81%,特异度分别为：93.40%、93.40%、98.02%。hTERC、c-myc基因单独检测的灵敏度和特异度均不高,但分别与常用检测方法HPV+TCT三联合,其特异度和灵敏度均有较大幅度的增高,较c-myc基因,hTERC基因单独检测及传统的TCT+HPV联合检测在灵敏度方面显示出更大的优越性；四联合的灵敏度和特异度分别高达99.81%和98.02%,但同时检测两个基因加重了患者的经济负担。6、CIN患者中69例行锥切手术,其中3例失访,66例6个月复诊。66例CIN治疗前、后TCT、HPV、c-myc基因、hTERC基因的检测结果,进行对比分析,得出CIN治疗后TCT、HPV、c-myc基因、hTERC基因的阳性率明显降低,与对应的治疗前比较,差异均有具有统计学意义。结论：1、C-myc、hTERC基因扩增可能是宫颈癌形成的早期事件,在宫颈癌变的过程中c-myc、hTERC基因可能起着重要的作用,其可作为宫颈癌筛查的分子标志。二者的扩增情况对预测CIN复发或进展有重要参考价值,其可作为CIN治疗后的一种随访手段。2、HPV感染与宫颈上皮内瘤变密切相关。尤其是HPV高危型患者CIN发病率较高,HPV高危型检测在宫颈病变的检查中有重要意义。由于HPV感染率高,特异性太低,与宫颈细胞学检查(TCT)相结合可明显提高宫颈病变的检出率,为目前主要的联合筛查CIN的方法。3、HPV感染与c-my、hTERC基因扩增率成正相关,在宫颈癌前病变中,c-myc、hTERC基因扩增与HPV感染在宫颈癌前病变及宫颈癌的发生发展中起着重要的作用。二者在宫颈病变中的异常表达存在相关性。c-myc、hTERC基因检测与常用检测TCT+HPV联合灵敏度、特异度显示更高,所以认为TCT+HPV+hTERC或TCT+HPV+c-myc为最佳组合。4、FISH技术具有客观、可重复、无创等优点,并且利用宫颈脱落细胞残液对宫颈病变进行筛查,检测c-myc、hTERC基因,可弥补TCT灵敏度差、HPV特异性差的缺点,可作为目前“三阶梯”及HPV检测以外的有力检测方法。5、本研究采用表面等离子共振技术(SPR)对不同宫颈病变的脱落细胞HPV DNA进行检测。HPV阳性表达在不同病理学级别组中,差异有统计学意义。但是,与组织病理学级别没有正相关关系(与文献报道有差异)。这可能与病例数太少或者检测方法有关,有待进一步用SPR技术检测研究。更多还原

【Abstract】 Purposes:1Taking clinical and pathological findings as a gold standard, the thesis analyses and compares the prediction values of Thinprep Cytology, HPV infection, hTERC, and c-myc gene amplification in cervical lesions through the test of hTERC gene, HPV subtypes, c-myc gene and classification of Thinprep Cytology in exfoliated cells of cervical epithelium.2The thesis is designed to seek a more accurate test method to diagnose and predict cervical disease through the analysis and comparison of examination results of the c-myc gene, hTERC gene, HPV subtypes and ThinPrep Cytological Test.3The thesis discusses the values of hTERC, c-myc gene, HPV subtypes as well as TCT in the follow-up after operation in CIN patients who had experienced conization or hysterectomy treatment.Methods We chose1000women, aged25to64years old, who have been married and have sexual life history from those who doctored in department of gynecology of the First Affiliated Hospital of Chongqing Medical University in January to September2011. The selected patients were brushed the cervical exfoliated cell for liquid-based cytology, the residual liquid at the same time as to detect c-myc and hTERC gene expression by fluorescence in situ hybridization, by surface plasmon resonance to detect HPV types, positive patients with any of the indicators as liquid-based cytology, HPV subtypes, hTERC gene and c-myc gene underwent colposcopy and biopsy.After screening, at last117patients required colposcopy and biopsy, but2of them gave up. Therefore, only115patients had pathological results. The patients with Normal/inflammation and CINI directly followed up without treatment; The patients with CINII, CINIII, SCC were taken to surgery and follow-up after6month. During the process of follow-up,26who are mainly inflammation and hysterectomy patients missed the follow-up. There were89taking referral. They were made c-myc, hTERC gene, HPV subtype and TCT test of exfoliated cervical cells in6months. When necessary, they were taken colpscope and biopsy.Results:1The positive rates of CINI, CINII, CINIII, SCC patients in TCT test are respectively87.5%,96.4%,100%,100%. With the hypertension of pathological level, the TCT positive rates are increasing. But for different levels of CIN screening, there is no obvious superiority (P=0.061).2with the increasing severity of the lesion histopathology, c-myc gene amplification rates are increasing, and c-myc gene positive amplification rate are also increasing. In the inflammatory/normal, CINI, CINII, CINIII, SCC group, the positive rates of c-myc gene amplification are as follows:41.67%,18.75%,39.29%,54.90%,87.50%. It shows that the statistics of the c-myc gene amplification positive rate of different pathological levels is significant(P<0.05).3The levels of pathological detection are inflammation\normal, CINI, CINII, CINIII, SCC. The positive rates are respectively:33.33%,25%,50%,68.63%,87.50%. As the lesion severity increases, the positive rate of hTERC gene amplification is also increasing.4As the pathology level increases, the positive expression rates of HPV gene amplification of inflammation, CINI, CINII, CINIII, SCC patients are respectively:75%,68.75%,100%,88.24%,87.50%. The results show that the positive expression of HPV in different pathological levels, the difference was statistically significant.5the test sensitivities of TCT, HPV, c-myc gene, hTERC gene in cervical leision are respectively86.21%,92%,52.9%,64.40%; specificities are70%,26.70%,70.00%,70.00%. The sensitivities of TCT+c-myc, TCT+hTERC, HPV+c-myc, HPV+hTERC, c-myc+hTERC are93.50%, 95.09%,96.23%,97.15%,83.28%; specificities:91.00%,91.00%,78.01%,78.01%,91.00%. The sensitivities of TCT+HPV+c-myc, TCT+HPV+hTERC, TCT+HPV+c-myc+hTERC are99.48%,99.61%,99.81%; specificities:93.40%,93.40%,98.20%. The HTERC, c-myc gene test sensitivity and specificity are not high, but the specificity and sensitivity increase to a certain extent when combining HPV and TCT. Besides, Compared with c-myc gene, hTERC gene are independently tested and TCT+HPV detection in sensitivity and specificity to show greater advantages; four joint sensitivity and specificity were as high as99.81%and98.02%, but at the same time detection of two genes increased the economic burden of patients.6Of the69CIN patients with conization,3failed in follow-up, and66took referral in6months. All the66patients’ positive rate of TCT, HPV, c-myc gene, hTERC gene reduced obviously comparing the results of before and after treatment.Conclusions:1The amplification of c-myc gene and hTERc gene may be the early event during the formation of cervical cancer. C-myc gene and hTERc gene, which play a very important role in the process of cervical cancer, can be considered as the molecular marker of CIN screening. The amplification of two genes has an important reference value on predicting CIN recurrence or progression. They also can be used as a follow-up method after CIN treatment.2HPV infection is closely related to the cervical intraepithelial neoplasia. Especially, HPV high-risk patients have a higher rate CIN incidence. The detection of high-risk HPV has a significant meaning in the examination of cervical lesions. Because of the higher rate of HPV infection and the lower specificity, the combination between the detection of high-risk HPV and the cervical cytology test can obliviously increase the detection rate of cervical lesions. The method is also considered as the major joint screening of CIN.3The HPV infection and the rate of amplification of C-myc gene and hTERc gene are positively correlated in the beginning of cervical lesions, the amplification of C-myc gene and hTERc gene and the HPV infection play a very important role in the process of cervical lesions and cervical carcinoma. Their abnormal expressions in cervical lesions are associated. The combination between the detection of C-myc gene, hTERc gene and the commonly used detection of TCT+HPV has a higher sensitivity and specificity. Thus TCT+HPV+hTERC or TCT+HPV+c-myc are thought as the best combinations.4FISH technology has the advantages of being objective, repeatable and producing no injuries. Moreover, it screens cervix lesions by taking advantage of exfoliated cervical cells, detects the c-myc and hTERC gene. It makes up the low sensibility of TCT and low specificity of HPV and can be taken as an effective detection method of the "three-step".5This research utilizes SPR to make HPV DNA detection in exfoliated cervical cells. Distinctions in HPV positive expressions in different levels of pathology have statistics significance, but they are not positively correlated with levels of histopathology.lt may be related to insufficient clinical cases and detection methods which needs further research by the means of SPR technology.更多还原