【作者】 胡滨；

【导师】 崔金全；

【作者基本信息】 郑州大学 ， 妇产科学， 2007， 硕士

【摘要】 宫颈癌是最常见的妇科恶性肿瘤之一，性传播疾病和HPV感染使其发病年龄逐渐趋向于年轻化，由于广泛开展宫颈癌预防普查，子宫颈癌的早期诊断率大大提高，子宫颈癌患者生存率得到提高。文献报告宫颈癌中HPV的检出率可以达到90％以上。HPV是一种环状双链结构的DNA病毒，由8000左右碱基对组成。高危型HPV E6、E7基因编码的原癌蛋白是导致宫颈癌的重要原因。它们均为小分子蛋白，分别含有158个和98个氨基酸，具有锌指结构。这种锌指结构与细胞的恶性转化密切相关。表达了E6、E7原癌蛋白的体外培养细胞能继续增殖到永生化，这种永生化可能是肿瘤发生的前奏。转基因研究还发现高危型HPV E6、E7能直接导致肿瘤形成。目前已经有学者研制成功了HPV疫苗，宫颈癌的发病率可能将会明显下降。基因治疗是以后肿瘤治疗的一个趋势，但有关宫颈癌特异性基因的研究的报道很少，影响了宫颈癌基因治疗进展。WAPL基因是在果蝇体内发现的一个基因，在人体内的同源序列称为HWAPL基因，其具体作用可能与染色体的稳定有关。Oikawa等发现宫颈癌相对于卵巢癌、乳腺癌、肺癌、肾癌、结肠癌及其相应的正常组织，HWAPL基因的mRNA表达升高更明显，相对于其他细胞系，宫颈癌细胞系中HWAPL有更高程度的表达。初步推测HWAPL基因可能是一种宫颈癌特异性高表达基因，推动了宫颈癌基因治疗的研究进展。本试验研究目的在于检测出宫颈癌石蜡标本中HPV并进行分型，为用石蜡标本对HPV研究提供理论依据；同时结合HWAPL蛋白免疫组化的结果，从临床角度找出高危型HPV与HWAPL基因之间的关系，为以后宫颈癌的预防和基因治疗的研究提供理论依据。材料和方法收集2004年2月到2006年2月郑州大学第二附属医院75例宫颈石蜡标本。30例宫颈鳞癌，患者术前均未进行放疗或化疗，年龄29～57岁，平均年龄43岁。45例宫颈上皮内瘤变(CIN)，包括CINⅠ级15例、CINⅡ级15例、CINⅢ级15例，年龄24～50岁，平均年龄37岁。采用普通PCR技术和基因芯片检测技术，检测出标本中的HPV并对之进行分型。结合HPV分型结果和HWAPL蛋白免疫组化研究结果，找出HPV分型与HWAPL基因之间的关系。用SPSS10.0软件系统对数据进行分析，以α=0.05作为判断显著差异的标准。结果(1)30例宫颈鳞癌石蜡标本中28例(93.3％)普通PCR扩增后显示HPVDNA阳性，基因芯片检测显示27例(90％)显示HPV16/18阳性，1(3.3％)例显示HPV6阳性；15例CINⅢ石蜡标本中14例(93.3％)普通PCR扩增后显示HPVDNA阳性，基因芯片检测显示10例(66.7％)HPV16/18阳性，1例(6.7％)58型阳性，3(20％)例HPV6/11阳性；15例CINⅡ石蜡标本中14例(93.3％)普通PCR扩增后显示HPV DNA阳性，基因芯片检测显示11(73.3％)例HPV 6/11阳性，3例(20％)HPV16/18阳性；15例CINⅠ石蜡标本中12例(80％)普通PCR扩增后显示HPV DNA阳性，基因芯片检测显示10例(66.7％)HPV6/11阳性，2例(13.3％)HPV16/18阳性。与CINⅠ、CINⅡ比较，宫颈癌和CINⅢ中高危HPV感染所占比率较大，差异有统计学意义(χ~2=32.248，P=0.001)；(2)30例宫颈癌标本中HWAPL蛋白表达强度5例(16.7％)为弱阳性(+)，8例(26.7％)为阳性(++)，17例(56.7％)为强阳性(+++)；15例CINⅢ标本中HWAPL蛋白表达强度4例(26.7％)为+，4例(26.7％)为++，7例(46.7％)为+++；15例CINⅡ标本中6例(40％)为+，5例(33.3％)为++，4例(26.7％)为+++；15例CINⅠ标本中HWAPL蛋白表达强度7例(46.7％)为+，6例(40％)为++，2例(13.3％)为+++。宫颈癌和CINⅢ中HWAPL蛋白表达强度远远高于CINⅠ和CINⅡ中，差异有统计学意义(χ~2=8.928，P=0.012)；(3)HPV阴性的7例标本中4例(57.1％)HWAPL蛋白表达为+，2例(28.6％)为++，1例(14.3％)为+++；低危HPV感染的26例标本中12例(46.2％)HWAPL蛋白表达为+，9例(34.6％)为H，5例(19.2％)为+++；检测出高危HPV的42例标本中6例(14.3％)HWAPL蛋白表达为弱阳性+，12例(28.6％)为++，24例(57.1％)为强阳性+++。高危HPV感染的标本与低危HPV感染的标本及HPV阴性标本相比较HWAPL蛋白表达强度差异有统计学意义(χ~2=14.844，P=0.005)。Spearmen相关性分析显示高危HPV感染与HWAPL基因表呈正相关(r=0.440，P=0.000)。结论(1)宫颈癌石蜡标本中可以检测出93.3％HPV DNA,可用作宫颈癌HPV感染的研究手段；(2)基因芯片技术有自动化、通量大、准确率高、特异性强等优点可以作为临床HPV检测和分型的常用手段；(3)宫颈癌中HWAPL基因呈高表达，高危HPV感染可增强HWAPL基因的复制表达，HWAPL基因异常表达可能是HPV致宫颈癌发病机制的重要环节。更多还原

【Abstract】 Background and objectiveCervical cancer was one maligant tumor of female. For sexual transmitted diseases and HPV infection, the patients with cervical cancer were becoming younger and younger. For the wide-ranging general survey used in cervical cancer, the rate of earlier diagnose of cervical cancer was improved. And the patients’ survival rate have been increased.As the literature reported the detection rate of HPV in cervical cancer was over 90 percentage. HPV is a double-stranded circular structure DNA virus and it is made up of about 8000 base pairs. The major cause of cervical cancer was the original cancer protein encoded by E6 gene and E7 gene of high risk HPV. They are small protein molecules, which contain 158 and 98 amino acids. They have zinc finger structure, which related to the malignant transformation of cells closely. Cells in vitro, which expressed original cancer protein of E6 gene and E7 gene, would reach eternal life. And the eternal life would be a prelude of tumor. Transgenic study also founded that in high-risk HPV E6 gene and E7 gene could directly lead to tumor. For the reasons above, some scholars have already successfully developed the HPV vaccine. With clinical application of HPV vaccine, the incidence of cervical cancer would be significantly decreased. Gene therapy would be a trend in clinical in the future. For the research about specific gene of cervical cancer was few, the progress of gene therapy in cervical cancer was inhibited. WAPL gene was found in Drosophila. And in human the homology sequence was HWAPL gene. The specific role of HWAPL gene may be keeping the stability of chromosome. Oikawa and so on found that compared to ovarian cancer, breast cancer, lung cancer, kidney cancer, colon cancer and the corresponding normal tissues the expression of HWAPL mRNA in cervical cancer increased more obviously. They also found that compared to other cell lines, HWAPL gene in cervical cancer cell lines expressed a higher level. So they guessed that HWAPL gene may be a cervical cancer-specific gene and this promoted the research progress of gene therapy in cervical cancer.In this test, HPV would be detected and typed from paraffin-embedded specimens of cervical cancer, then provided a theoretical basis for studying HPV. At the same time, immunohistochemical results of HWAPL protein would be compined to find out the relationship between HPV and HWAPL gene expression. This study will provide a theoretical basis for the prevention and gene therapy of cervical cancer in the future.Materials and methods75 cases of cervical paraffin-embedded specimens were collected in Zhengzhou University 2nd affiliated hospital from February 2004 to February 2006. The age of 30 cases of cervical squamous cell carcinoma was from 29 years old to 57 years old and the average age was 43 years old. In 45 cases of cervical intraepthelial neoplasia , there were 15 cases of CIN I,15 cases of CIN II and 15 cases of CINIII. The age of 45 cases of CIN was from 24 years old to 50 years old and the average age was 37 years old. All women hadn’t been treated with chemotherapy or radiotherapy.PCR and gene chip technology were used to detect and type HPV from paraffin-embedded cervical specimens. According to typing results and immunohistochemical results of HWAPL protein, we would find the relationship between HPV with HWAPL gene expression. SPSS10.0 was used to analyze the datum.Results (1) After amplifying with PCR, the results showed that there were 28 cases of HPV DNA positive in 30 cases of cervical cancer and the positive rate was 93.3%. In 15 cases of CIN III, there were 14 cases of HPV DNA positive and the positive rate was 93.3%. In 15 cases of CIN II, there were 14 cases of HPV DNA positive and the positive rate was 93.3%. In 15 cases of CIN I , there were 12 cases of HPV DNA positive and the positive rate was 80%. Detected by gene chip, the results showed that in 30 cases of cervical cancer there were 27 cases of HPV16/18 positive and 1 case of HPV6 positive. The positive rate was 90% and 6.7% respectively. In 15 cases of CIN III, there were 10 cases of HPV16 /18 positive, 1 case of HPV58 positive and 3 cases of HPV6/11 positive. The positive rate was 66.7%, 6.7% and 20% respectively. In 15 cases of CIN II, there were 11 cases of HPV6/11 positive and 3 cases of HPV16/18 positive. The positive rate was 73.3% and 20% respectively. In 15 cases of CIN I , there were 10 cases of HPV6/11 positive and 2 cases of HPV 16/18 positive. The positive rate was 66.7% and 13.3% respectively. In cervical cancer and CIN III the detection rate of high-risk HPV was significant higher than in CIN II and CIN I (x~2 = 32.248, P=0.000).(2) The immunohistochemical results of HWAPL protein showed that in 30 cases of cervical cancer there were 5 cases with weak positive staining intensity (+), 8 cases with middle positive staining intensity (++) and 17 cases with strong positive staining intensity(+++). In 15 cases of CIN III, there were 4 cases with +, 4 cases with ++ and 7 cases with +++. In 15 cases of CIN II, there were 6 cases with +, 5 cases with ++ and 4 cases with +++. In 15 cases of CIN I , there were 7 cases with +, 6 cases with ++ and 2 cases with +++. There were significant difference between the expression of HWAPL protein in CIN III and cervical with the expression of HWAPL protein in CIN II and CIN I (x~2 = 8.928, P=0.012).(3) There were 7 cases of HPV negative specimens, in which 4 cases with HWAPL protein +, 2 cases with ++ and 1 case with +++. There were 26 cases of low risk HPV positive specimens, in which 12 cases with HWAPL protein +, 5 cases with ++ and 9 cases with +++. There were 42 cases of high risk HPV positive specimens, in which 6 cases with HWAPL protein +, 12 cases with ++ and 24 cases with +++. Specimens with high-risk HPV positive expressed HWAPL protein significant higher than specimens without high risk HPV positive (x~2=14.844, P=0.005). Correlation analysis showed that high risk HPV and the expression of HWAPL gene correlated (r =0.440, P= 0.000).Conclusions(1) 90% HPV DNA could be detected from Paraffin-embedded specimens of cervical cancer. And these specimens could be used as cervical HPV infection research.(2) For automation throughput high accuracy and specificity advantages, gene chip technology could be used as an important method for clinical HPV testing and classification.(3) In cervical cancer HWAPL gene expression was high. High risk HPV infection may increased the replication and expression of HWAPL gene. The abnormal expression of HWAPL gene may play an important role in the pathogenesis of cervical cancer.更多还原