宫颈上皮内瘤变中E-cadherin,β-catenin的表达与HPV16/18感染的关系
|
摘要
前言
宫颈癌(cervical cancer,CC)是妇女最常见的恶性肿瘤之一,现已确认高危型人乳头瘤病毒(high risk human papillomavirus,HR-HPV)是宫颈癌形成最重要的危险因子,99.7%的浸润性宫颈癌中可以检测到HR-HPV的存在。因此,通过高危HPV的检测来筛查易患人群,追踪HPV感染状态,随访HPV是否被清除,是预防宫颈癌发生的关键。但并非所有感染HPV的患者都会发展为宫颈癌,仅检测到患者感染了HPV而不进一步检查就无法了解宫颈病变的程度和发展趋势。E-cadherin是一类介导细胞-细胞间黏附、具有维持组织结构完整性和极性的钙依赖性跨膜蛋白。E-cad的功能不仅需钙离子存在,还需要与其配体连接素(catenin,cat)结合成复合体才能起作用,β-cat与E-cad直接结合,α-cat介导E-cad/β-cat与细胞骨架的肌动蛋白相连。故对维持上皮细胞的极性及分化起到重要作用,E-cadherin功能下降或缺失可能导致细胞—细胞间黏附作用的降低,从而导致肿瘤的发生,发展.在宫颈上皮内病变(cervical intraepithelial neoplasia,CIN)和宫颈癌中呈低表达,在正常对照组高表达,因此如果通过对宫颈癌病因学因素HPV和生物标志物因素E-cadherin,β-catenin的联合检测,来对宫颈癌高危人群进行筛查,可能具有重要意义。本研究对93例宫颈液基细胞残液标本应用导流杂交HPV-DNA检测技术检测HPV各亚型的表达,并对相应组织组织学标本用免疫组化法检测E-cadherin,β-catenin,以探讨二者在子宫颈癌发生发展中的生物学意义及临床应用价值,试图为提高宫颈癌易患人群检出率,进而为宫颈癌的预警和早期诊断提供更科学有效的方法,达到早诊早治,降低宫颈癌发病率死亡率的目的;并为开发简便易得的分子筛查技术资源提供理论基础。
材料和方法
一、材料
(一)研究对象
600例宫颈液基细胞学残液标本,均经LCT(Liquid-based Cytology Test,LCT系统)诊为宫颈上皮内病变及宫颈癌,来自2007年9月～2008年10月中国医科大学附属第一医院妇科。无宫颈手术史、未孕、无盆腔放射治疗史,年龄26～77岁,平均年龄48.4岁。40例子宫颈癌、40例CIN患者及13例对照组其中子宫颈浸润癌40例、CINⅡ-Ⅲ19例、CINⅠ21例。
(二)主要试剂、仪器和来源
鼠抗人HPV16/18E6抗体和羊抗鼠IgG-HRP抗体,鼠抗人E-cadherin,β-catenin抗体均购自美国Santa Cruz公司。HPV-DNA分型检测试剂盒购自凯普公司。免疫组化S-P试剂盒和DAB显色试剂盒购自福州迈新生物技术有限公司。
二、方法
(一)免疫组织化学染色
用链霉素抗生物素蛋白-过氧化物酶免疫组化法(S-P法)检测E-cadherin,β-catenin的表达。结果判定:E-cadherin,β-catenin蛋白定位于细胞膜,胞膜内见棕黄色颗粒为阳性。由3名有经验的病理医师在双盲法下独立计数。细胞涂片主要计数有病变的细胞,异常宫颈上皮细胞着色大于10%则判定为阳性标本。细胞片和组织片在光镜下根据着色范围分为:阳性细胞数小于10～24%为(+);阳性细胞数25～49%为(++);阳性细胞数50～74%为(+++);阳性细胞数大于75%为(++++)。
(二)原位杂交
检测HPV16/18DNA的表达。细胞核内出现棕黄色颗粒为阳性。阳性细胞数小于25%为(+);阳性细胞数25%～49%为(++);阳性细胞数50%～74%为(+++);阳性细胞数75%～100%为(++++).
(三)导流杂交HPV-DNA分型检测
检测HPV-DNA各亚型的表达.在检测结果模板上相应亚型区域内出现实心圆点即为该亚型阳性表达.
(四)western
HPV蛋白提取:将盛有样本的试管置于碎冰上,用超声破碎机破碎细胞,4℃12 000/g离心,45 min。取上清液,定量、备用。电泳、显色:采用十二烷基硫酸钠变性聚丙烯酰胺凝胶不连续缓冲系统,先后配制15%的分离胶和5%浓缩胶;取蛋白样品,上样20μl(约含蛋白60μg),电泳后将醋酸纤维素膜铺到胶上进行转印,42v,2h.加入一抗(1:200)过夜,加入二抗(1:2000)37℃,DAB显色3～5 min。该膜上有显色带出现为阳性;若无显色,为阴性表现。
(五)统计学分析
对所得结果各组间采用x~2检验及Fisher's确切概率法检验,P＜0.05有统计学意义.
结果
一、免疫组化法检测E-cadherin,β-catenin的表达
E-cadherin,在细胞膜内出现棕黄色颗粒为阳性,β-catenin在细胞膜内出现棕黄色颗粒为阳性,过表达时胞核胞浆均着色,周围正常结缔组织、血管、间质细胞显示弱的着色。
E-cadherin,在正常宫颈脱落细胞及宫颈组织中高表达,在各组宫颈病变中有不同程度的表达:E-cadherin,在正常,CIN,浸润癌中的表达率分别为84.6%(11/13),72.5%(29/40),27.5%(11/40),β-catenin在正常,CIN,浸润癌中的表达率分别为76.9%(10/13),67.5%(27/40),32.5%(13/40)。随着宫颈上皮病变程度的加重呈现减低的趋势
二、原位杂交法检测HPV16/18 DNA的表达
HPV16/18在细胞核内出现棕黄色颗粒为阳性,部分病例可在胞浆中见到棕黄色颗粒。
HPV16/18在正常宫颈脱落细胞中及相应组织中不表达或弱表达,在各组宫颈病变中有不同程度的表达:在正常、CIN和宫颈癌中,HPV16/18的阳性率分别为15.4%(2/13)、50.0%(20/40)和82.5%(33/40),随着宫颈上皮病变程度的加重呈现增高的趋势。
三、导流杂交HPV-DNA分型检测技术检测HPV各亚型的表达
HPV-DNA各亚型在检测结果模板相应亚型区域内出现实心圆点,即为该亚型阳性.
HPV-DNA各亚型在正常宫颈脱落细胞中不表达或弱表达,在各组宫颈病变中有不同程度的表达:600例患者HPV检测阳性共245例,阳性率为40.83%(245/600),其中包括复合感染,具体感染情况见表1.表2.在HPV21种亚型中,除了HPV43,HPV44外有19种亚型被检出,感染率最高的是HPV16(87/245),其它常见型别为HPV58(36/245),由此可见我省妇女感染HPV主要亚型是HPV16,HPV58及HPV6.
四、Western法检测HPV16/18的表达
HPV16/18在正常、CIN和宫颈癌中,阳性率分别为23.1%(3/13),62.5%(25/40),85%(34/40)。随着宫颈上皮病变程度的加重呈现增高的趋势。
结论
1.E-cadherin,β-catenin在宫颈上皮内病变及宫颈癌中,随着病变程度的加重而阳性率减低。
2.HPV16/18的阳性率在宫颈上皮内病变及宫颈癌中,随着病变程度的加重而检出率增高。
3.E-cadherin的表达和HPV16/18的感染在宫颈病变中呈负相关,二者联合检测可提高宫颈病变筛查的准确度。
4.Western-blot检测HPV16/18E6具有较高的灵敏性,尤其是在CIN组,适合于对宫颈疑难病例的检测,作为HPV16/18检测的辅助手段。
5.HPV16/18,在宫颈液基细胞学残液和相应组织中的检测结果都是基本一致的,作为宫颈病变检测的辅助手段。
6.导流杂交HPV-DNA分型检测我省600例妇女感染HPV的阳性率为40.83%(245/600),感染的主要亚型是HPV16,HPV58及HPV6.
Introduction
Cervical cancer(CC) is one of the most frequently founded malignant tumors in women world wide.Current evidences implicated high risk humn papillomavirus(HPV) especially HPV16/18 infection as a causative part of the natural history of cervical carcinogenesis.HPV can be found in 99.7%of cervical cancer patients.It's critical for precaution of cervical cancer to detect HPV.However only a part of the patients who contracted HPV may advance to cervical cancer.E-cadherin is mediated by a class of cell-cell adhesion,with the organizational structure to maintain the integrity and polarity of the calcium-dependent transmembrane protein.E-cad function is not only required the existence of calcium ion,but also the needs of their Ligand catenin (catenin,cat) can be combined into a complex role,β-cat and E-cad direct binding,α-cat-mediated E-cad /β-cat and actin cytoskeleton-linked protein.Therefore,the maintenance of the polarity of epithelial cells and play an important role in differentiation,E-cadherin function may result in decrease or loss of cells-the role of intercellular adhesion reduced,resulting in tumor occurrence and development.In cervical intraepithelial lesions and cervical cancer in expression was low in the high expression of the normal control group,so if the factors on the etiology of cervical cancer and HPV biomarkers factor E-cadherin,β-catenin co-testing,to high-risk groups for cervical cancer screening,may have important significance.In our study,we detect HPV16/18 by the hybridization in situ and E-cadherin,β-catenin by immuohistochemical analysis,to research the value of them in cutting down the mortality of CC,and to supply rationale to exploitation of convenient samples.
Materials and Methods
1.Materials
(1) specimen
600 cervical liquid-based cells remained samples were obtained from the Department of gynecology in the First Affiliated Hospital of China Medical University, during Nov 20067to Nov 2008.All of them were treated as CC or CIN,Among them 13 samples as control gropes.All of them possess corresponding histological samples as controls.The patients with CC were 40,with CINI were 21,with CINⅡ\Ⅲwere 19. The average age is 48.4(range,26～77 years).The standard of cytology grouping based on histodiagnosis.All patients had not the history of the operation on cervix,pregnancy and radiation therapy in the cavity of pelvic.
(2) Main agents,equipment and source
Kit of HPV16/18 in situ hybridization was purchased from MAIXIN biological technology company in Fuzhou,antibody of E-cadherin,β-catenin protein was friendly presented by Jin woo Kim professor in Korea.Sub-type HPV-DNA detection kit purchased from Cape company.S-P Kit and DAB Kit were obtained from MAIXIN biological technology company in Fuzhou.
2.Methods
(1) Immunohistochemistry
Detection of E-cadherin,β-catenin by Immuohistochemical analysis S-P methods. Result determination:The overexpressed staining was in the membrane.The number of staining cells were graded as follows:10-24%(+);25-49%(++);50-74%(+++);≥75% (++++).
(2) In situ hybridization
Detection of HPV16/18 by Hybridization in situ.The masculine staining was in the nucleus.The overexpressed staining was in the cytoplasm.The number of staining cells were graded as follows:10-24%(+);25-49%(++);50-74%(+++);≥75%(++++).
(3) HPV-DNA hybridization diversion type detection
Detection of HPV-DNA expression in the various subtypes.Results in a corresponding subtype of the template on a solid dot the region is the emergence of positive expression of the subtypes
(4)Detection of HPV16/18 by western-blot.
Stamples were incubated on ice for 30 min in an appropriate RIPA buffer(Solarbio) with 1%PMSF(Solarbio).The stamples was centrifuged at 15000g for 45 min at 4℃, and the supematant was fractionated by electrophoresis on a 15%PAGE and transferred to a nitrocellulose membrane.After blocking with 5%skimmed milk,the membrane was incubated with the appropriate primary antibody overnight at 4℃,and with labeled secondary antibodies(1:2000,Chemicon,USA) for 2h at 37℃.Proteins were visualized by DAB(Perking Applygen Co.,Ltd.) according to the manufacturer's protocol.
(5) Statistical analysis
The results of the various groups usingχ2 test and Fisher's exact probability test,P <0.05 statistical significance.
Results
1.Expressions of E-cadherin,β-catenin
Expressions of E-cadherin,β-catenin in cervical exfoliated cells and cervical tissue by Immuohistochemical analysis S-P methods.Membrane E-cadherin,in normal cervical exfoliated cells and high expression in cervical tissue,cervical lesions in each group there are different levels of expression:E-cadherin,in normal,CIN,invasive carcinoma of the expression rates were 84.6%(11/13),72.5%(29/40),27.5%(11/40),β-catenin in normal,CIN,invasive carcinoma of the expression rates were 76.9% (10/13),67.5%(27/40),32.5%(13/40).As the extent of cervical intraepithelial lesions showed a downward trend to increase
2.Expression of HPV16/18 by Hybridization in situ.
HPV16/18 was overexpressed in cervical cancer82.5%(33/40) and CIN 50.0%(20/40) compared with it in normal15.4%(2/13),P<0.05.The expression of HPV16/18 in cervical cancer and precancerous lesion correlated well with cervical neoplasia(CIN).It's consistent both in cervical exfoliated cell and in cervical tissue.
3.HPV-DNA hybridization diversion type detection to detect the expression of HPV subtypes
HPV-DNA test results of the subtypes in the corresponding subtype of the template region appear solid dots,which is positive for the subtype.
HPV-DNA of various subtypes in normal cervical exfoliated cells are not expressed or weakly expressed in cervical lesions in each group there are different levels of expression:600 patients were positive for HPV detection were 245 cases,the positive rate was 40.83%(245/600),including the composite infection,specific infections shown in Table 1.Table 2.HPV21 subtypes,in addition to HPV43,HPV44 There are 19 kinds of subtypes have been detected,the highest rate of infection is HPV16(87/245),other common type for the HPV58(36/245),HPV6(33/2-45), HPV53(28/245),HPV18(20/245),HPV31(19/245),HPV52(19/245),cp8304(16/245) and HPV11(15/245).HPV infection in women can be seen in our province is the main subtype of HPV 16,HPV58 and HPV6.
4.Expression of HPV 16/18 by western
HPV16/18 was overexpressed in cervical cancer85%(34/40) and CIN62.5%(25/40) compared with it in normal23.1%(3/13),P<0.05.The expression of HPV16/18 in cervical cancer and precancerous lesion correlated well with cervical neoplasia(CIN). It's consistent both in cervical exfoliated cell and in cervical tissue.
Conclusions
1 In CIN and CC,The positive rates of E-cadherin,β-catenin decrease with the aggravation of cervix affection.
2 In CIN and CC,The positive rates of HPV16/18 increase with the aggravation of cervix affection.
3 In CIN and CC,the expression rate of E-cadherin,β-catenin and HPV16/18 showed a negative correlation,It will be more accurate for screening CIN and CC to detect them together.
4 Testing HPV16/18 by western has high sensitivity,especially in CIN.It is suitable for questioned case by this way.It helps testing HPV16/18
5 The possible role of HPV16/18 in the remained samples of cervical liquid-based cells is coincident with that in cervical tissues.There is great feasibility in screening cervical affection by utilization of the remained samples of cervical liquid-based cells.
6 HPV-DNA hybridization diversion typing detected 600 eases of women in our province of the positive rate of HPV infection to 40.83%(245/600),infection is the main subtype of HPV16,HPV58 and HPV6.
宫颈癌(cervical cancer,CC)是妇女最常见的恶性肿瘤之一,现已确认高危型人乳头瘤病毒(high risk human papillomavirus,HR-HPV)是宫颈癌形成最重要的危险因子,99.7%的浸润性宫颈癌中可以检测到HR-HPV的存在。因此,通过高危HPV的检测来筛查易患人群,追踪HPV感染状态,随访HPV是否被清除,是预防宫颈癌发生的关键。但并非所有感染HPV的患者都会发展为宫颈癌,仅检测到患者感染了HPV而不进一步检查就无法了解宫颈病变的程度和发展趋势。E-cadherin是一类介导细胞-细胞间黏附、具有维持组织结构完整性和极性的钙依赖性跨膜蛋白。E-cad的功能不仅需钙离子存在,还需要与其配体连接素(catenin,cat)结合成复合体才能起作用,β-cat与E-cad直接结合,α-cat介导E-cad/β-cat与细胞骨架的肌动蛋白相连。故对维持上皮细胞的极性及分化起到重要作用,E-cadherin功能下降或缺失可能导致细胞—细胞间黏附作用的降低,从而导致肿瘤的发生,发展.在宫颈上皮内病变(cervical intraepithelial neoplasia,CIN)和宫颈癌中呈低表达,在正常对照组高表达,因此如果通过对宫颈癌病因学因素HPV和生物标志物因素E-cadherin,β-catenin的联合检测,来对宫颈癌高危人群进行筛查,可能具有重要意义。本研究对93例宫颈液基细胞残液标本应用导流杂交HPV-DNA检测技术检测HPV各亚型的表达,并对相应组织组织学标本用免疫组化法检测E-cadherin,β-catenin,以探讨二者在子宫颈癌发生发展中的生物学意义及临床应用价值,试图为提高宫颈癌易患人群检出率,进而为宫颈癌的预警和早期诊断提供更科学有效的方法,达到早诊早治,降低宫颈癌发病率死亡率的目的;并为开发简便易得的分子筛查技术资源提供理论基础。
材料和方法
一、材料
(一)研究对象
600例宫颈液基细胞学残液标本,均经LCT(Liquid-based Cytology Test,LCT系统)诊为宫颈上皮内病变及宫颈癌,来自2007年9月～2008年10月中国医科大学附属第一医院妇科。无宫颈手术史、未孕、无盆腔放射治疗史,年龄26～77岁,平均年龄48.4岁。40例子宫颈癌、40例CIN患者及13例对照组其中子宫颈浸润癌40例、CINⅡ-Ⅲ19例、CINⅠ21例。
(二)主要试剂、仪器和来源
鼠抗人HPV16/18E6抗体和羊抗鼠IgG-HRP抗体,鼠抗人E-cadherin,β-catenin抗体均购自美国Santa Cruz公司。HPV-DNA分型检测试剂盒购自凯普公司。免疫组化S-P试剂盒和DAB显色试剂盒购自福州迈新生物技术有限公司。
二、方法
(一)免疫组织化学染色
用链霉素抗生物素蛋白-过氧化物酶免疫组化法(S-P法)检测E-cadherin,β-catenin的表达。结果判定:E-cadherin,β-catenin蛋白定位于细胞膜,胞膜内见棕黄色颗粒为阳性。由3名有经验的病理医师在双盲法下独立计数。细胞涂片主要计数有病变的细胞,异常宫颈上皮细胞着色大于10%则判定为阳性标本。细胞片和组织片在光镜下根据着色范围分为:阳性细胞数小于10～24%为(+);阳性细胞数25～49%为(++);阳性细胞数50～74%为(+++);阳性细胞数大于75%为(++++)。
(二)原位杂交
检测HPV16/18DNA的表达。细胞核内出现棕黄色颗粒为阳性。阳性细胞数小于25%为(+);阳性细胞数25%～49%为(++);阳性细胞数50%～74%为(+++);阳性细胞数75%～100%为(++++).
(三)导流杂交HPV-DNA分型检测
检测HPV-DNA各亚型的表达.在检测结果模板上相应亚型区域内出现实心圆点即为该亚型阳性表达.
(四)western
HPV蛋白提取:将盛有样本的试管置于碎冰上,用超声破碎机破碎细胞,4℃12 000/g离心,45 min。取上清液,定量、备用。电泳、显色:采用十二烷基硫酸钠变性聚丙烯酰胺凝胶不连续缓冲系统,先后配制15%的分离胶和5%浓缩胶;取蛋白样品,上样20μl(约含蛋白60μg),电泳后将醋酸纤维素膜铺到胶上进行转印,42v,2h.加入一抗(1:200)过夜,加入二抗(1:2000)37℃,DAB显色3～5 min。该膜上有显色带出现为阳性;若无显色,为阴性表现。
(五)统计学分析
对所得结果各组间采用x~2检验及Fisher's确切概率法检验,P＜0.05有统计学意义.
结果
一、免疫组化法检测E-cadherin,β-catenin的表达
E-cadherin,在细胞膜内出现棕黄色颗粒为阳性,β-catenin在细胞膜内出现棕黄色颗粒为阳性,过表达时胞核胞浆均着色,周围正常结缔组织、血管、间质细胞显示弱的着色。
E-cadherin,在正常宫颈脱落细胞及宫颈组织中高表达,在各组宫颈病变中有不同程度的表达:E-cadherin,在正常,CIN,浸润癌中的表达率分别为84.6%(11/13),72.5%(29/40),27.5%(11/40),β-catenin在正常,CIN,浸润癌中的表达率分别为76.9%(10/13),67.5%(27/40),32.5%(13/40)。随着宫颈上皮病变程度的加重呈现减低的趋势
二、原位杂交法检测HPV16/18 DNA的表达
HPV16/18在细胞核内出现棕黄色颗粒为阳性,部分病例可在胞浆中见到棕黄色颗粒。
HPV16/18在正常宫颈脱落细胞中及相应组织中不表达或弱表达,在各组宫颈病变中有不同程度的表达:在正常、CIN和宫颈癌中,HPV16/18的阳性率分别为15.4%(2/13)、50.0%(20/40)和82.5%(33/40),随着宫颈上皮病变程度的加重呈现增高的趋势。
三、导流杂交HPV-DNA分型检测技术检测HPV各亚型的表达
HPV-DNA各亚型在检测结果模板相应亚型区域内出现实心圆点,即为该亚型阳性.
HPV-DNA各亚型在正常宫颈脱落细胞中不表达或弱表达,在各组宫颈病变中有不同程度的表达:600例患者HPV检测阳性共245例,阳性率为40.83%(245/600),其中包括复合感染,具体感染情况见表1.表2.在HPV21种亚型中,除了HPV43,HPV44外有19种亚型被检出,感染率最高的是HPV16(87/245),其它常见型别为HPV58(36/245),由此可见我省妇女感染HPV主要亚型是HPV16,HPV58及HPV6.
四、Western法检测HPV16/18的表达
HPV16/18在正常、CIN和宫颈癌中,阳性率分别为23.1%(3/13),62.5%(25/40),85%(34/40)。随着宫颈上皮病变程度的加重呈现增高的趋势。
结论
1.E-cadherin,β-catenin在宫颈上皮内病变及宫颈癌中,随着病变程度的加重而阳性率减低。
2.HPV16/18的阳性率在宫颈上皮内病变及宫颈癌中,随着病变程度的加重而检出率增高。
3.E-cadherin的表达和HPV16/18的感染在宫颈病变中呈负相关,二者联合检测可提高宫颈病变筛查的准确度。
4.Western-blot检测HPV16/18E6具有较高的灵敏性,尤其是在CIN组,适合于对宫颈疑难病例的检测,作为HPV16/18检测的辅助手段。
5.HPV16/18,在宫颈液基细胞学残液和相应组织中的检测结果都是基本一致的,作为宫颈病变检测的辅助手段。
6.导流杂交HPV-DNA分型检测我省600例妇女感染HPV的阳性率为40.83%(245/600),感染的主要亚型是HPV16,HPV58及HPV6.
Introduction
Cervical cancer(CC) is one of the most frequently founded malignant tumors in women world wide.Current evidences implicated high risk humn papillomavirus(HPV) especially HPV16/18 infection as a causative part of the natural history of cervical carcinogenesis.HPV can be found in 99.7%of cervical cancer patients.It's critical for precaution of cervical cancer to detect HPV.However only a part of the patients who contracted HPV may advance to cervical cancer.E-cadherin is mediated by a class of cell-cell adhesion,with the organizational structure to maintain the integrity and polarity of the calcium-dependent transmembrane protein.E-cad function is not only required the existence of calcium ion,but also the needs of their Ligand catenin (catenin,cat) can be combined into a complex role,β-cat and E-cad direct binding,α-cat-mediated E-cad /β-cat and actin cytoskeleton-linked protein.Therefore,the maintenance of the polarity of epithelial cells and play an important role in differentiation,E-cadherin function may result in decrease or loss of cells-the role of intercellular adhesion reduced,resulting in tumor occurrence and development.In cervical intraepithelial lesions and cervical cancer in expression was low in the high expression of the normal control group,so if the factors on the etiology of cervical cancer and HPV biomarkers factor E-cadherin,β-catenin co-testing,to high-risk groups for cervical cancer screening,may have important significance.In our study,we detect HPV16/18 by the hybridization in situ and E-cadherin,β-catenin by immuohistochemical analysis,to research the value of them in cutting down the mortality of CC,and to supply rationale to exploitation of convenient samples.
Materials and Methods
1.Materials
(1) specimen
600 cervical liquid-based cells remained samples were obtained from the Department of gynecology in the First Affiliated Hospital of China Medical University, during Nov 20067to Nov 2008.All of them were treated as CC or CIN,Among them 13 samples as control gropes.All of them possess corresponding histological samples as controls.The patients with CC were 40,with CINI were 21,with CINⅡ\Ⅲwere 19. The average age is 48.4(range,26～77 years).The standard of cytology grouping based on histodiagnosis.All patients had not the history of the operation on cervix,pregnancy and radiation therapy in the cavity of pelvic.
(2) Main agents,equipment and source
Kit of HPV16/18 in situ hybridization was purchased from MAIXIN biological technology company in Fuzhou,antibody of E-cadherin,β-catenin protein was friendly presented by Jin woo Kim professor in Korea.Sub-type HPV-DNA detection kit purchased from Cape company.S-P Kit and DAB Kit were obtained from MAIXIN biological technology company in Fuzhou.
2.Methods
(1) Immunohistochemistry
Detection of E-cadherin,β-catenin by Immuohistochemical analysis S-P methods. Result determination:The overexpressed staining was in the membrane.The number of staining cells were graded as follows:10-24%(+);25-49%(++);50-74%(+++);≥75% (++++).
(2) In situ hybridization
Detection of HPV16/18 by Hybridization in situ.The masculine staining was in the nucleus.The overexpressed staining was in the cytoplasm.The number of staining cells were graded as follows:10-24%(+);25-49%(++);50-74%(+++);≥75%(++++).
(3) HPV-DNA hybridization diversion type detection
Detection of HPV-DNA expression in the various subtypes.Results in a corresponding subtype of the template on a solid dot the region is the emergence of positive expression of the subtypes
(4)Detection of HPV16/18 by western-blot.
Stamples were incubated on ice for 30 min in an appropriate RIPA buffer(Solarbio) with 1%PMSF(Solarbio).The stamples was centrifuged at 15000g for 45 min at 4℃, and the supematant was fractionated by electrophoresis on a 15%PAGE and transferred to a nitrocellulose membrane.After blocking with 5%skimmed milk,the membrane was incubated with the appropriate primary antibody overnight at 4℃,and with labeled secondary antibodies(1:2000,Chemicon,USA) for 2h at 37℃.Proteins were visualized by DAB(Perking Applygen Co.,Ltd.) according to the manufacturer's protocol.
(5) Statistical analysis
The results of the various groups usingχ2 test and Fisher's exact probability test,P <0.05 statistical significance.
Results
1.Expressions of E-cadherin,β-catenin
Expressions of E-cadherin,β-catenin in cervical exfoliated cells and cervical tissue by Immuohistochemical analysis S-P methods.Membrane E-cadherin,in normal cervical exfoliated cells and high expression in cervical tissue,cervical lesions in each group there are different levels of expression:E-cadherin,in normal,CIN,invasive carcinoma of the expression rates were 84.6%(11/13),72.5%(29/40),27.5%(11/40),β-catenin in normal,CIN,invasive carcinoma of the expression rates were 76.9% (10/13),67.5%(27/40),32.5%(13/40).As the extent of cervical intraepithelial lesions showed a downward trend to increase
2.Expression of HPV16/18 by Hybridization in situ.
HPV16/18 was overexpressed in cervical cancer82.5%(33/40) and CIN 50.0%(20/40) compared with it in normal15.4%(2/13),P<0.05.The expression of HPV16/18 in cervical cancer and precancerous lesion correlated well with cervical neoplasia(CIN).It's consistent both in cervical exfoliated cell and in cervical tissue.
3.HPV-DNA hybridization diversion type detection to detect the expression of HPV subtypes
HPV-DNA test results of the subtypes in the corresponding subtype of the template region appear solid dots,which is positive for the subtype.
HPV-DNA of various subtypes in normal cervical exfoliated cells are not expressed or weakly expressed in cervical lesions in each group there are different levels of expression:600 patients were positive for HPV detection were 245 cases,the positive rate was 40.83%(245/600),including the composite infection,specific infections shown in Table 1.Table 2.HPV21 subtypes,in addition to HPV43,HPV44 There are 19 kinds of subtypes have been detected,the highest rate of infection is HPV16(87/245),other common type for the HPV58(36/245),HPV6(33/2-45), HPV53(28/245),HPV18(20/245),HPV31(19/245),HPV52(19/245),cp8304(16/245) and HPV11(15/245).HPV infection in women can be seen in our province is the main subtype of HPV 16,HPV58 and HPV6.
4.Expression of HPV 16/18 by western
HPV16/18 was overexpressed in cervical cancer85%(34/40) and CIN62.5%(25/40) compared with it in normal23.1%(3/13),P<0.05.The expression of HPV16/18 in cervical cancer and precancerous lesion correlated well with cervical neoplasia(CIN). It's consistent both in cervical exfoliated cell and in cervical tissue.
Conclusions
1 In CIN and CC,The positive rates of E-cadherin,β-catenin decrease with the aggravation of cervix affection.
2 In CIN and CC,The positive rates of HPV16/18 increase with the aggravation of cervix affection.
3 In CIN and CC,the expression rate of E-cadherin,β-catenin and HPV16/18 showed a negative correlation,It will be more accurate for screening CIN and CC to detect them together.
4 Testing HPV16/18 by western has high sensitivity,especially in CIN.It is suitable for questioned case by this way.It helps testing HPV16/18
5 The possible role of HPV16/18 in the remained samples of cervical liquid-based cells is coincident with that in cervical tissues.There is great feasibility in screening cervical affection by utilization of the remained samples of cervical liquid-based cells.
6 HPV-DNA hybridization diversion typing detected 600 eases of women in our province of the positive rate of HPV infection to 40.83%(245/600),infection is the main subtype of HPV16,HPV58 and HPV6.
引文
1 Walboomers J,Jacobs M,et al.Human papillomavirus is a necessary cause of invasive cervical cancer worldwide[J].Pathol.1999 Sep;189(1):12-19.
2 Lu K P.Pinning down cell signaling,cancer and Alzheimer's disease[J].Trends Biochem Sci,2004,29(4):200- 209.
3 郎景和.迎接子宫颈癌预防的全球挑战与机遇[J].中华妇产科杂志.2002;37(2):129-131.
4 刘泽虎,刘贞富.高危型人类乳头瘤病毒的致瘤分子机制[J].国外医学皮肤性病学分册,2002.28(2):116-118.
5 Helen Trottier,PhD,MSc,et al.Human Papillomavirus and Cervical Cancer:Burden of Illness and Basis for Prevention[J].The American Journal of Managed care,2006;12(17):462-472
6 Regina B,Park D,Elliot J.Androphy.Genetic Analysis of High-Risk E6 in Episomal Maintence of Human Papillomavirus Genomes in Primary Human Kertinocytes[J].Journal of Virology.2002;76(22):11359.
7 Van Tine BA,Knops J,Broker TR,et al.In situ analysis of the transcript-tional activity of integrated viral DNA using tyramide FISH[J].Dev Biol(Basel).2001;106:381.
8 钱德英,岑坚敏,王丁,等.高危型人乳头状瘤病毒DNA检测与细胞学联合检查对子宫颈癌前病变筛查的研究[J].中华妇产科杂志2006;41(1):34-37.
9 孙晓杰,崔满华,左宇,等.原位杂交HPV16/18检测在宫颈癌及癌前病变诊断中的作用[J].中华实验诊断学.2006;10(5):482-484.
10 郎景和.妇科学新进展.子宫颈病变的防治.人类乳头状瘤病毒感染的处理.中华电子音像出版社.2005;3(1):83-89.
11 李洁,刘宝印,zur Hausen H,等.中国妇女宫颈癌组织中人乳头瘤病毒感染及其地理分布的调查[J].中华实验和临床病毒学杂志.1996;10:50-55.
12 Ahn W S,Lee J M,Namkoong S E,et al.Effect of retinoic acid on HPV titration and colposcopic changes in Korean patients with dysplasia of the uterine cervix[J].J Cell Biochem Suppl.1997;28-29;133-139.
13 Matsukura T,Sugase M.Identification of genital human papilloma-viruses in cervical biopsy specimens:Segregation of specific virus types in specific clinicopathologic lesions[J].Int J Cancer.1995;61:13-22.
14 Nindl I,Greinke C,Zahm DM,et al.Human papillomavirus distribution in cervical tissues of different morphology as determined by hybrid capture assay and PCR[J].Int J Gynecol Pathol.1997;15:197-204.
15 Han C,Qiao G,Hubbert NL,et al.Serologic association between human papillomavirus type 16 infection and esophageal cancer in Shanxi Province,China[J].J Natl Cancer Inst.1996;88(20):1467-1471.
16 张儒英,乔惠珍.Snail E-cadhernin与宫颈病变的相关性研究 内蒙古医学杂志 Inner Mongolia Med J 2008年第40卷第8期
1.Nusse R. Wnt signaling in disease and in development. CellRes 2005; 15: 28-32
2.Wehrli M, Dougan ST, Caldwell K, Okeefe L, Schwartz S,Vaizel-Ohayon D, Schejter E,Tomlinson A, Dinardo S. Arrow encodes an LDL-receptor-related protein essential for Wingless signalling. Nature 2000; 407: 527-530
3.Mao J, Wang J, Liu B, Pan W, Farr GH 3rd, Flynn C, Yuan H, Takada S, Kimelman D, Li L,Wu D. Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway. Mol Cell 2001; 7: 801-809
4.Mao B, Wu W, Davidson G, Marhold J, Li M, Mechler BM, Delius H, Hoppe D, Stannek P,Walter C, Glinka A, Niehrs C. Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling. Nature 2002; 417: 664-667
5.Amit S, Hatzubai A,Birman Y,Andersen JS, Ben-Shushan E, Mann M, Ben-Neriah Y, Alkalay I.Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway. Genes Dev 2002;16: 1066-1076
6.Gloy J, Hikasa H, Sokol SY. Frodo interacts with Dishevelled to transduce Wnt signals. Nat Cell Biol 2002; 4: 351-357
7.Heisenberg CP. Wnt signaling: refocusing on Strabismus. Curr Biol 2002; 12: 657-659
8.Kirikoshi H, Sekihara H, Katoh M. Molecular cloning and characterization of human WNT11.Int J Mol Med 2001; 8: 651-656
9.Lustig B ,Behrens J .TheWntsignaling pathway and its role in tumor development. J CancerRes Clin Oncol, 2003,129(4):199-221
10.Rodrguez-Sastre MA,Gonzalez-Maya L,Delgado R,et al.Abnormal distribution of E-cadherin and (3-catenin in different histologictypes of cancer of the uterine cervix. GynecolOncol, 2005,97(2):330-336
11.Pereira-Suarez AL,Meraz MA,Lizano M,et al.Frequent alterations of the beta-catenin potein in cancer of the uterine cervix.Tumour Biol,2002,23(1):45-53
12.Uren A,Fallen S,Yuan H,et al. Activation of the can onicalwnt pathway during genital keratinocyte transformation: A model for cervical cancer progression. CancerRes, 2005, 65(14): 6199-6206
13.Vande Putte G,Kristensen GB,Baekelandt M,et al. E-cadherin and catenins in early squamus cervical carcinoma. GynecolOncol,2004,94(2):521-527
14.Fadare O,Reddy H,Wang i,et al. E-cadherin and P-catenine xpression in early stage cervical carcinoma: a tissue microarray study of 147 cases.World J SurgOncol, 2005,3(1):38-48
15.Khalida MY, Huilgol NG, Hongyo T,et al. Mutations in cervical cancer as predictive factos for radiotherapy. International Congress Series, 2002,1236(7):459-461
16.Shinohara A, Yokoyama Y,Wan X,et al. Cytoplasmic/nuclear expression without mutation of exon3 of the beta-catenin gene is frequent in the development of the neoplasm of the uterine cervix. Gynecol Oncol, 2001,82(3):450-455
17.Ueda M, Gemmill RM, West J,et al. Mutations of the beta- and gamma-catenin genes are uncommon in human lung, breast, kidney, cervical and ovarian carcinomas. Br J Cancer,2001,85(1): 64-68
18.Dong SM, Kim HS, Rha SH, et al. Promoter hypermethylation of multiple genes in carcinoma of the uterinecervix.Clin Cancer Res, 2001,7(7): 1982-1986
19.Zambrano P, Segura-Pacheco B, Perez-Cardenas E,et al. Aphase I study of hydralazine to demethylate and reactivate the expression of tumor suppressor genes. BMC Cancer, 2005,5(1):44-55
20.Kirikoshi H, Katoh M. Expression and regulation of WNT 10B in human cancer: up-regulation of WNT 10B in MCF-7 cells by estradioland down-regulation of WNT 10B in NT2 cells by retinoicacid. Int J MolMed, 2002, 10(4):507-511
21.Okino K, Nagai H,Hatta M,et al.Up-regulation and overproduction of DVL-1, the human counterpart of the Drosophiladishevelledgene, in cervicals quamous cell carcinoma. Oncol Rep,2003, 10(5): 1219-1223
22.Saitoh T,Moriwaki J,Koike J,et al. Molecular cloning and characterization of FRAT2, encoding a positive regulator of the WNT signaling pathway. Biochem BiophysRes Commun,2001,281(3): 815-820
23.Terasaki H, Saitoh T, Shiokawa K,et al. Frizzled-10, up-regulated in primary colorectal cancer, is a positive regulator of the WNT-beta-catenin-TCF signaling pathway. Int J Mol Med,2002,9(2): 107-112
24. Ko J, Ryu KS, Lee YH, et al.Human secreted frizzled-related protein is down-regulated and induces apoptosis in human cervical cancer. Exp CellRes, 2002,280(2):280-287
2 Lu K P.Pinning down cell signaling,cancer and Alzheimer's disease[J].Trends Biochem Sci,2004,29(4):200- 209.
3 郎景和.迎接子宫颈癌预防的全球挑战与机遇[J].中华妇产科杂志.2002;37(2):129-131.
4 刘泽虎,刘贞富.高危型人类乳头瘤病毒的致瘤分子机制[J].国外医学皮肤性病学分册,2002.28(2):116-118.
5 Helen Trottier,PhD,MSc,et al.Human Papillomavirus and Cervical Cancer:Burden of Illness and Basis for Prevention[J].The American Journal of Managed care,2006;12(17):462-472
6 Regina B,Park D,Elliot J.Androphy.Genetic Analysis of High-Risk E6 in Episomal Maintence of Human Papillomavirus Genomes in Primary Human Kertinocytes[J].Journal of Virology.2002;76(22):11359.
7 Van Tine BA,Knops J,Broker TR,et al.In situ analysis of the transcript-tional activity of integrated viral DNA using tyramide FISH[J].Dev Biol(Basel).2001;106:381.
8 钱德英,岑坚敏,王丁,等.高危型人乳头状瘤病毒DNA检测与细胞学联合检查对子宫颈癌前病变筛查的研究[J].中华妇产科杂志2006;41(1):34-37.
9 孙晓杰,崔满华,左宇,等.原位杂交HPV16/18检测在宫颈癌及癌前病变诊断中的作用[J].中华实验诊断学.2006;10(5):482-484.
10 郎景和.妇科学新进展.子宫颈病变的防治.人类乳头状瘤病毒感染的处理.中华电子音像出版社.2005;3(1):83-89.
11 李洁,刘宝印,zur Hausen H,等.中国妇女宫颈癌组织中人乳头瘤病毒感染及其地理分布的调查[J].中华实验和临床病毒学杂志.1996;10:50-55.
12 Ahn W S,Lee J M,Namkoong S E,et al.Effect of retinoic acid on HPV titration and colposcopic changes in Korean patients with dysplasia of the uterine cervix[J].J Cell Biochem Suppl.1997;28-29;133-139.
13 Matsukura T,Sugase M.Identification of genital human papilloma-viruses in cervical biopsy specimens:Segregation of specific virus types in specific clinicopathologic lesions[J].Int J Cancer.1995;61:13-22.
14 Nindl I,Greinke C,Zahm DM,et al.Human papillomavirus distribution in cervical tissues of different morphology as determined by hybrid capture assay and PCR[J].Int J Gynecol Pathol.1997;15:197-204.
15 Han C,Qiao G,Hubbert NL,et al.Serologic association between human papillomavirus type 16 infection and esophageal cancer in Shanxi Province,China[J].J Natl Cancer Inst.1996;88(20):1467-1471.
16 张儒英,乔惠珍.Snail E-cadhernin与宫颈病变的相关性研究 内蒙古医学杂志 Inner Mongolia Med J 2008年第40卷第8期
1.Nusse R. Wnt signaling in disease and in development. CellRes 2005; 15: 28-32
2.Wehrli M, Dougan ST, Caldwell K, Okeefe L, Schwartz S,Vaizel-Ohayon D, Schejter E,Tomlinson A, Dinardo S. Arrow encodes an LDL-receptor-related protein essential for Wingless signalling. Nature 2000; 407: 527-530
3.Mao J, Wang J, Liu B, Pan W, Farr GH 3rd, Flynn C, Yuan H, Takada S, Kimelman D, Li L,Wu D. Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway. Mol Cell 2001; 7: 801-809
4.Mao B, Wu W, Davidson G, Marhold J, Li M, Mechler BM, Delius H, Hoppe D, Stannek P,Walter C, Glinka A, Niehrs C. Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling. Nature 2002; 417: 664-667
5.Amit S, Hatzubai A,Birman Y,Andersen JS, Ben-Shushan E, Mann M, Ben-Neriah Y, Alkalay I.Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway. Genes Dev 2002;16: 1066-1076
6.Gloy J, Hikasa H, Sokol SY. Frodo interacts with Dishevelled to transduce Wnt signals. Nat Cell Biol 2002; 4: 351-357
7.Heisenberg CP. Wnt signaling: refocusing on Strabismus. Curr Biol 2002; 12: 657-659
8.Kirikoshi H, Sekihara H, Katoh M. Molecular cloning and characterization of human WNT11.Int J Mol Med 2001; 8: 651-656
9.Lustig B ,Behrens J .TheWntsignaling pathway and its role in tumor development. J CancerRes Clin Oncol, 2003,129(4):199-221
10.Rodrguez-Sastre MA,Gonzalez-Maya L,Delgado R,et al.Abnormal distribution of E-cadherin and (3-catenin in different histologictypes of cancer of the uterine cervix. GynecolOncol, 2005,97(2):330-336
11.Pereira-Suarez AL,Meraz MA,Lizano M,et al.Frequent alterations of the beta-catenin potein in cancer of the uterine cervix.Tumour Biol,2002,23(1):45-53
12.Uren A,Fallen S,Yuan H,et al. Activation of the can onicalwnt pathway during genital keratinocyte transformation: A model for cervical cancer progression. CancerRes, 2005, 65(14): 6199-6206
13.Vande Putte G,Kristensen GB,Baekelandt M,et al. E-cadherin and catenins in early squamus cervical carcinoma. GynecolOncol,2004,94(2):521-527
14.Fadare O,Reddy H,Wang i,et al. E-cadherin and P-catenine xpression in early stage cervical carcinoma: a tissue microarray study of 147 cases.World J SurgOncol, 2005,3(1):38-48
15.Khalida MY, Huilgol NG, Hongyo T,et al. Mutations in cervical cancer as predictive factos for radiotherapy. International Congress Series, 2002,1236(7):459-461
16.Shinohara A, Yokoyama Y,Wan X,et al. Cytoplasmic/nuclear expression without mutation of exon3 of the beta-catenin gene is frequent in the development of the neoplasm of the uterine cervix. Gynecol Oncol, 2001,82(3):450-455
17.Ueda M, Gemmill RM, West J,et al. Mutations of the beta- and gamma-catenin genes are uncommon in human lung, breast, kidney, cervical and ovarian carcinomas. Br J Cancer,2001,85(1): 64-68
18.Dong SM, Kim HS, Rha SH, et al. Promoter hypermethylation of multiple genes in carcinoma of the uterinecervix.Clin Cancer Res, 2001,7(7): 1982-1986
19.Zambrano P, Segura-Pacheco B, Perez-Cardenas E,et al. Aphase I study of hydralazine to demethylate and reactivate the expression of tumor suppressor genes. BMC Cancer, 2005,5(1):44-55
20.Kirikoshi H, Katoh M. Expression and regulation of WNT 10B in human cancer: up-regulation of WNT 10B in MCF-7 cells by estradioland down-regulation of WNT 10B in NT2 cells by retinoicacid. Int J MolMed, 2002, 10(4):507-511
21.Okino K, Nagai H,Hatta M,et al.Up-regulation and overproduction of DVL-1, the human counterpart of the Drosophiladishevelledgene, in cervicals quamous cell carcinoma. Oncol Rep,2003, 10(5): 1219-1223
22.Saitoh T,Moriwaki J,Koike J,et al. Molecular cloning and characterization of FRAT2, encoding a positive regulator of the WNT signaling pathway. Biochem BiophysRes Commun,2001,281(3): 815-820
23.Terasaki H, Saitoh T, Shiokawa K,et al. Frizzled-10, up-regulated in primary colorectal cancer, is a positive regulator of the WNT-beta-catenin-TCF signaling pathway. Int J Mol Med,2002,9(2): 107-112
24. Ko J, Ryu KS, Lee YH, et al.Human secreted frizzled-related protein is down-regulated and induces apoptosis in human cervical cancer. Exp CellRes, 2002,280(2):280-287