自制组织芯片研究p16mRNA、p16~（INK4A）及ZPPS在宫颈鳞癌、CIN中的意义
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摘要
目的:子宫颈癌是危害妇女生命健康的常见恶性肿瘤之一。全球每年约有46.6万例宫颈癌的新发病例,其中80%发生在发展中国家,它已成为发展中国家妇女死亡的主要的原因,死亡率仅次于乳腺癌,居于第二位。因宫颈癌筛查的全面开展,其发病率大大降低,但年轻妇女宫颈癌发病率有所增加。子宫颈癌的发生是涉及多种因素、多个步骤的病理过程。过去几十年里,人们从实验室到临床等不同方面进行了各种研究,但其发病机制尚未完全明了。
p16基因又称多肿瘤抑制基因(multiple tumor suppressor, MTS1)它是人们发现的第一个直接作用于细胞周期,抑制细胞分裂的基因,其在宫颈癌的发生过程中起到何种作用,已成为研究的热点。许多研究证实,在子宫颈癌发生过程中存在p16蛋白表达异常,但其表达是升高,还是降低说法不一。p16基因在子宫颈癌的发生过程中其失活的机制究竟是什么,作为在宫颈癌研究中最受人关注的抑癌基因之一,p16与宫颈癌发生之间的关系还存在许多疑问。
郑氏植物蛋白(Zheng’s plant protein, ZPP)是一种从低等植物中提取的糖蛋白,具有刺激肿瘤细胞增殖的作用,研究表明它是一种与人肿瘤抗原相关的植物蛋白。郑氏植物蛋白片段(Zheng’s plant protein segment, ZPPS)是邹岳奇等人经过进一步对ZPP结构以及相关基因的研究,对其继续提纯而制成。已有研究表明经~(131)I标记的ZPPS在小鼠恶性肿瘤放射性核素显像中的摄取明显高于正常组织,且表达速度及程度均高于~(131)I标记的ZPP。但ZPPS在宫颈癌组织中表达情况,还未见研究报道。
本课题采用组织芯片、原位杂交及免疫组化技术研究抑癌基因p16 mRNA、P16蛋白及ZPPS在宫颈不同程度病变组织中的表达情况。探讨p16 mRNA与宫颈不同程度病变生物学行为的关系,探讨p16 mRNA的异常表达在宫颈癌的发生发展中的作用,探讨p16在蛋白水平和基因水平的表达是否相符及其在正常宫颈组织向CIN和宫颈癌发生、发展中的作用。这对于了解肿瘤的发病机制,提高宫颈肿瘤的诊断率有重要意义。
方法:
1收集研究样本宫颈组织共247例,其中宫颈鳞癌54例, CIN组织154例,正常宫颈组织39例。
2应用自制组织芯片技术将247例供体标本,按阵列设计顺序依次放入受体蜡块中,融合、切片、捞片、烤片制成需要的组织芯片。
3应用免疫组织化学技术(SP法)测定宫颈鳞癌、CIN组织及正常宫颈鳞状上皮组织中ZPPS及P16~(INK4A)的表达。
4采用原位杂交技术检测p16 mRNA在宫颈鳞癌、CIN组织及正常宫颈鳞状上皮组织中的表达情况。
5采用SPSS11.5统计软件进行统计学处理,以α=0.05为检验水准,当P<0.05时具有统计学差异。
结果:
1组织芯片制备
按课题需要制作6×5,7×5芯片共8张,组织点阵排列整齐,无点阵移位现象,有8个位点缺失,39个位点组织无意义,其他位点均可见足够的组织结构。组织位点的形态可观测率在83.3%～93.3%之间。
HE染色均匀,无脱片、异位和皱折。免疫组织化学染色及原位杂交,阳性定位清楚,背景干净,无掉片现象。
2 P16~(INK4A)在子宫颈上皮不同病变中的表达采用免疫组织化学方法检测48例宫颈鳞癌、119例CIN,33例正常宫颈鳞状上皮中P16~(INK4A)的表达。结果显示,正常宫颈鳞状上皮中P16~(INK4A)无表达或仅在基底层部分细胞弱阳性表达。在CIN和宫颈鳞癌病例中,无论是基底层还是其他病变各层均可见细胞强阳性表达,宫颈鳞癌(93.75%)及各级别CIN(CINⅢ81.25%, CINⅡ64.10%, CINⅠ18.75%)均明显高于正常宫颈鳞状上皮(0%)中p16表达,差异具有显著性(p<0.01);P16~(INK4A)过表达在宫颈鳞癌中明显高于CINⅠ和CINⅡ(p<0.01) ;宫颈癌和CINⅢ之间差异无显著性(p>0.05)。其他各级别CIN之间P16~(INK4A)的表达差异均有显著性(p<0.05),且随级别增加表达增强。
3 ZPPS在宫颈上皮不同病变中的表达
采用免疫组织化学方法检测48例宫颈鳞癌、119例宫颈CIN及33例正常宫颈鳞状上皮组织中ZPPS的表达。在正常宫颈鳞状上皮中,ZPPS不表达或少量表达,从正常宫颈鳞状上皮(0%)、CINⅠ(6.25%)、CINⅡ(51.28%)、CINⅢ(58.33%)到宫颈鳞癌(72.92%),ZPPS表达阳性细胞逐渐增多。ZPPS表达在正常宫颈上皮和CINⅠ中差异无显著性(p>0.05);ZPPS在宫颈鳞癌及CINⅡ、CINⅢ中均高于正常宫颈鳞状上皮表达,差异具有显著性(p<0.05);CINⅠ与正常宫颈鳞状上皮中ZPPS表达差异无显著性(p>0.05)。宫颈鳞癌与CINⅠ、CINⅡ均差异有显著性(p<0.05),随级别增加表达增强,但与CINⅢ之间差异无显著性(p>0.05)。
4 p16 mRNA在宫颈上皮不同病变中的表达
采用原位杂交技术检测p16 mRNA在48例宫颈鳞癌、119例CIN(包括48例CINⅢ,39例CINⅡ,32例CINⅠ)及33例正常宫颈鳞状上皮组织中的表达。从正常宫颈鳞状上皮、CINⅠ、CINⅡ、CINⅢ到宫颈鳞癌,p16 mRNA的表达情况依次为18.18% (6/27)、43.75%(14/32)、56.41%(22/39)、75%(36/48)及79.17%(38/48),差异具有显著性(p<0.01)。p16 mRNA阳性率在CINⅠ、CINⅡ、CINⅢ及宫颈鳞癌中明显高于正常宫颈鳞状上皮(p<0.01);宫颈鳞癌与CINⅠ及CINⅡ之间p16 mRNA阳性率差异具有显著性(p<0.01)。但在宫颈鳞癌和CINⅢ之间p16 mRNA表达差异无显著性(p>0.05)。
5子宫颈鳞癌中p16 mRNA表达与ZPPS、P16~(INK4A)表达的相关性分析
在子宫颈鳞癌中p16 mRNA与P16~(INK4A)表达具有显著正相关性(p<0.05,r=0.503);p16 mRNA与ZPPS表达具有显著正相关性(p<0.05,r=0.380);P16~(INK4A)与ZPPS表达具有正相关性(p<0.05,r=0.402)。
6子宫颈鳞癌中p16 mRNA、P16~(INK4A)、ZPPS表达与年龄、肿瘤大小、分化程度、临床分期、淋巴结转移情况及病理分级之间的关系
p16mRNA、P16~(INK4A)与临床分期有显著相关性(p<0.05),与年龄、肿瘤大小、分化程度、淋巴结转移情况及病理分级之间均无明显相关性(p>0.05)。ZPPS表达与年龄、肿瘤大小、分化程度、病理分级之间均无明显相关性(p>0.05),与淋巴结转移之间有显著相关性(p<0.05)。
结论:
1免疫组化方法标记P16~(INK4A)的表达率从正常宫颈鳞状上皮、CIN到子宫颈鳞癌逐渐增高。
2原位杂交检测p16 mRNA在正常宫颈组织中可见表达,并且随CIN级别升高到子宫颈鳞癌,p16 mRNA检出率逐渐增高。
3子宫颈鳞癌中P16~(INK4A)和p16 mRNA有正相关,说明p16基因在蛋白水平和mRNA水平是相符的。
4免疫组化方法标记ZPPS的表达率从正常宫颈鳞状上皮、CIN到子宫颈鳞癌逐渐增高。
5子宫颈鳞癌中P16~(INK4A)过表达与ZPPS异常表达相关,二者呈正相关。提示两种蛋白的异常表达在宫颈癌的诊断过程中有协同作用,在诊断宫颈癌时可以同时进行检测。
6 p16过表达与临床分期有显著相关性,与其他各临床病理因素之间均不存在相关性。ZPPS异常表达与淋巴结转移有显著相关性。p16 mRNA与临床分期有相关性。
7组织芯片手工制做,程序简单、经济方便,是一种简便可行,高效稳定的技术方法,能满足相关科研课题和实际工作的需要。
Objective: Cervical cancer is one of common malignant tumors which influence women’s life and healthy. Worldwide, incidence of almost half a million new cases annually, 80% of them in developing countrys. It have become one of common malignant tumors and main reason of death. Death rate is second only breast cancer. Screening for cervical cancer widely available has reduced its incidence. Pathogenesis of cervical cancer is associated with many factors and phases. In the past decades, laboratory and clinical researches into different aspects of cervical carcinoma had been performed, but the carcinogenesis of cervical carcinoma is still not clear enough.
p16 is a multiple tumor suppressor, it is the first anti-oncogene which direct effect cell cycle to suppression cell division. What kind of effection it is in the cervical cancer occurrence process has become the hot spot of the research. Many researchs have confirmed that the P16 protein expression unusual in the cervix carcinogenesis process, but its expression is elevates, or reduces, the view not one. Actually, What the deactivation mechanism of the p16 gene in the carcinoma of cervix occurrence process, p16 gene becomes one of the most important anti-oncogene in the cervical cancer studies. There are many questions between p16 and occurrence process of the cervical cancer.
ZPP is one kind the glycoprotein which withdraws from the lower forms of plant. ZPP can stimulate the tumor cell multiplication. The research indicated that it is plant-related-person tumor antigen. Yueqi Zou passed through further to this antigen protein structure as well as the correlation gene research, continues to epurate it to made the ZPPS. The research had indicated that ZPPS after ~(131)I mark obviously higher than the normal tissue in the mouse malignant tumor in radionuclide, also the expression speed and the degree are higher than ~(131)I mark ZPP. But the research of the expression of ZPPS has not seen reported in the cervical cancer.
This topic uses the tissue chip, In situ hybridization to research p16 mRNA, and uses the immunohistochemistry to research the expression ZPPS and P16 protein in normal cervical squamous epithelia, CIN and cervical carcinoma. To discusses the relations between p16 mRNA and biology behavior in normal cervical squamous epithelia, CIN and cervical carcinoma. To discuss the function of expression of p16 mRNA in the cervical cancer occurrencing and developing. To discuss the expression of p16 gene in the protein level and the mRNA level whether does tall. To discuss the p16 gene in the protein level and the molecular level in the development from the normal cervical squamous epithelia, CIN to cervical carcinoma.
Methods:
1 39 cases of normal cervical squamous epithelia, 154 cases of CIN and 54 cases of cervical carcinoma were collected.
2 By manual tissue chip technique, this is done by using a needle to biopsy a standard histologic section and placing the core into an array on a recipient paraffin block, then fusing, slicing, gaining and baking.
3 Immunohistochemistry SP method was used to examine for the expression, relation and the effect of p16 and ZPPS in cervical carcinoma, CIN and normal cervical squamous epithelia.
4 In situ hybridization (ISH) was used to detect p16 mRNA in 200 cases of cervical samples, including 48 cases of cervical carcinoma, 119 cases of CIN and 33 cases of normal cervical squamous epithelia.
5 Analysis the data use spss11.5, significance was set at p<0.05.
Results:
1 The result of tissue chip
8 tissue chips (6×5,7×5) were made. Dots of tissue chip lined up in order, uniformed in size and no shifting. 8 tissue dots were absence, 39 tissue no significant tissue structures. The significant tissue structures were watched in the other dots. The rate of complete tissue dots was 83.3%~93.3%.
The haematoxylin-eosin (HE) staining was uniformity and no dropping, moving and wrinkling.
The immunohistochemical and ISH results show that the positive location was accurate and the background was clear.
2 The expression of P16~(INK4A)
In normal cervical epithelia, the expression of P16~(INK4A) is absence. P16~(INK4A) expression is significant differentiated at normal cervical squamous epithelia with CINⅠ, CINⅡ, CINⅢand cervical invasive squamous cell carcinoma (P<0.01). P16~(INK4A) expression at CINⅠ, CINⅡwith cervical invasive squamous cell carcinoma is significant different (p<0.01), the expression at cervical invasive squamous cell carcinoma and CINⅢis no significant different(p>0.05), the expression at CINⅠ, CINⅡand CINⅢis significant different (p<0.05).
3 The expression of ZPPS
In normal cervical epithelia the expression of ZPPS is absence. ZPPS expression is no significant differentiated at normal cervical epithelia with CINⅠ. There is significant differentiated at CINⅠ, CINⅡwith cervical invasive squamous cell carcinoma (p<0.05), but the expression of ZPPS is no significant differentiated at cervical invasive squamous cell carcinoma and CINⅢ(p>0.05).
4 The expression of p16 mRNA
Expression of p16 mRNA in various lesions of cervix, 48 cases of cervical carcinoma, 119 cases of CIN and 33 cases of normal cervical seqamous epithelia were tested. The positive rates of p16 mRNA in normal cervical epithela, CINⅠ, CINⅡ, CINⅢand cervical carcinoma group were 18.18%, 43.75%, 56.41%, 75%, 79.17% respectively. p16 mRNA expression is significant differentiated at normal cervical epithelia with CINⅡ, CINⅢand cervical invasive squamous cell carcinoma (p<0.01). There is significant differentiated at cervical invasive squamous cell carcinoma with CINⅠ, CINⅡ(p<0.01), but the expression at CINⅢand cervical invasive squamous cell carcinoma is no significant different (p>0.05).
5 The relation of p16 mRNA、ZPPS、P16~(INK4A)
There was a positive correlation between expression of P16~(INK4A) and p16 mRNA(p<0.01), expression of p16 mRNA and ZPPS (p<0.05), expression of P16~(INK4A) and ZPPS.
6 The relationship between the expression of p16 mRNA、P16~(INK4A)、ZPPS and clinical pathological features of cervical carcinoma.
No significant relationship were found between the expression of P16~(INK4A) and clinical pathological features, but clinical stage. There was significant relationship between the expression of p16 mRNA and clinical stage. Significant relationship were found between the expression of ZPPS and metastasis.
Conclusions:
1 P16~(INK4A) overexpression rates become higher and higher from normal cervical squamous epithelia, CIN to cervical invasive squamous cell carcinoma.
2 There was expression of p16 mRNA in normal cervical squamous epithelia. The positive rate of p16 mRNA from CINⅠ, CINⅡ, CINⅢto cervical invasive squamous cell carcinoma becomes gradually more higher.
3 There was a positive correlation between overexpression of p16 and p16mRNA in occurrence and development cervical squamous cell carcinomas.
4 ZPPS overexpression rates become higher and higher from normal cervical squamous epithelia, CIN to cervical invasive squamous cell carcinoma.
5 There was a positive correlation between overexpression of P16~(INK4A) and ZPPS. Probably significant correlation of P16 and ZPPS in diagnose cervical squamous cell carcinomas.
6 There was no significant relationship between overexpression of p16 and clinical pathological features, but clinical stage. There was significant relationship between expression of ZPPS and metastasis. Significant relationship were found between the expression of p16 mRNA and clinical stage.
7 Manual tissue chip technique is simple, cost-effective, and reliable. This technique can provide a highly efficient, high-throughput mechanism for some research.
p16基因又称多肿瘤抑制基因(multiple tumor suppressor, MTS1)它是人们发现的第一个直接作用于细胞周期,抑制细胞分裂的基因,其在宫颈癌的发生过程中起到何种作用,已成为研究的热点。许多研究证实,在子宫颈癌发生过程中存在p16蛋白表达异常,但其表达是升高,还是降低说法不一。p16基因在子宫颈癌的发生过程中其失活的机制究竟是什么,作为在宫颈癌研究中最受人关注的抑癌基因之一,p16与宫颈癌发生之间的关系还存在许多疑问。
郑氏植物蛋白(Zheng’s plant protein, ZPP)是一种从低等植物中提取的糖蛋白,具有刺激肿瘤细胞增殖的作用,研究表明它是一种与人肿瘤抗原相关的植物蛋白。郑氏植物蛋白片段(Zheng’s plant protein segment, ZPPS)是邹岳奇等人经过进一步对ZPP结构以及相关基因的研究,对其继续提纯而制成。已有研究表明经~(131)I标记的ZPPS在小鼠恶性肿瘤放射性核素显像中的摄取明显高于正常组织,且表达速度及程度均高于~(131)I标记的ZPP。但ZPPS在宫颈癌组织中表达情况,还未见研究报道。
本课题采用组织芯片、原位杂交及免疫组化技术研究抑癌基因p16 mRNA、P16蛋白及ZPPS在宫颈不同程度病变组织中的表达情况。探讨p16 mRNA与宫颈不同程度病变生物学行为的关系,探讨p16 mRNA的异常表达在宫颈癌的发生发展中的作用,探讨p16在蛋白水平和基因水平的表达是否相符及其在正常宫颈组织向CIN和宫颈癌发生、发展中的作用。这对于了解肿瘤的发病机制,提高宫颈肿瘤的诊断率有重要意义。
方法:
1收集研究样本宫颈组织共247例,其中宫颈鳞癌54例, CIN组织154例,正常宫颈组织39例。
2应用自制组织芯片技术将247例供体标本,按阵列设计顺序依次放入受体蜡块中,融合、切片、捞片、烤片制成需要的组织芯片。
3应用免疫组织化学技术(SP法)测定宫颈鳞癌、CIN组织及正常宫颈鳞状上皮组织中ZPPS及P16~(INK4A)的表达。
4采用原位杂交技术检测p16 mRNA在宫颈鳞癌、CIN组织及正常宫颈鳞状上皮组织中的表达情况。
5采用SPSS11.5统计软件进行统计学处理,以α=0.05为检验水准,当P<0.05时具有统计学差异。
结果:
1组织芯片制备
按课题需要制作6×5,7×5芯片共8张,组织点阵排列整齐,无点阵移位现象,有8个位点缺失,39个位点组织无意义,其他位点均可见足够的组织结构。组织位点的形态可观测率在83.3%～93.3%之间。
HE染色均匀,无脱片、异位和皱折。免疫组织化学染色及原位杂交,阳性定位清楚,背景干净,无掉片现象。
2 P16~(INK4A)在子宫颈上皮不同病变中的表达采用免疫组织化学方法检测48例宫颈鳞癌、119例CIN,33例正常宫颈鳞状上皮中P16~(INK4A)的表达。结果显示,正常宫颈鳞状上皮中P16~(INK4A)无表达或仅在基底层部分细胞弱阳性表达。在CIN和宫颈鳞癌病例中,无论是基底层还是其他病变各层均可见细胞强阳性表达,宫颈鳞癌(93.75%)及各级别CIN(CINⅢ81.25%, CINⅡ64.10%, CINⅠ18.75%)均明显高于正常宫颈鳞状上皮(0%)中p16表达,差异具有显著性(p<0.01);P16~(INK4A)过表达在宫颈鳞癌中明显高于CINⅠ和CINⅡ(p<0.01) ;宫颈癌和CINⅢ之间差异无显著性(p>0.05)。其他各级别CIN之间P16~(INK4A)的表达差异均有显著性(p<0.05),且随级别增加表达增强。
3 ZPPS在宫颈上皮不同病变中的表达
采用免疫组织化学方法检测48例宫颈鳞癌、119例宫颈CIN及33例正常宫颈鳞状上皮组织中ZPPS的表达。在正常宫颈鳞状上皮中,ZPPS不表达或少量表达,从正常宫颈鳞状上皮(0%)、CINⅠ(6.25%)、CINⅡ(51.28%)、CINⅢ(58.33%)到宫颈鳞癌(72.92%),ZPPS表达阳性细胞逐渐增多。ZPPS表达在正常宫颈上皮和CINⅠ中差异无显著性(p>0.05);ZPPS在宫颈鳞癌及CINⅡ、CINⅢ中均高于正常宫颈鳞状上皮表达,差异具有显著性(p<0.05);CINⅠ与正常宫颈鳞状上皮中ZPPS表达差异无显著性(p>0.05)。宫颈鳞癌与CINⅠ、CINⅡ均差异有显著性(p<0.05),随级别增加表达增强,但与CINⅢ之间差异无显著性(p>0.05)。
4 p16 mRNA在宫颈上皮不同病变中的表达
采用原位杂交技术检测p16 mRNA在48例宫颈鳞癌、119例CIN(包括48例CINⅢ,39例CINⅡ,32例CINⅠ)及33例正常宫颈鳞状上皮组织中的表达。从正常宫颈鳞状上皮、CINⅠ、CINⅡ、CINⅢ到宫颈鳞癌,p16 mRNA的表达情况依次为18.18% (6/27)、43.75%(14/32)、56.41%(22/39)、75%(36/48)及79.17%(38/48),差异具有显著性(p<0.01)。p16 mRNA阳性率在CINⅠ、CINⅡ、CINⅢ及宫颈鳞癌中明显高于正常宫颈鳞状上皮(p<0.01);宫颈鳞癌与CINⅠ及CINⅡ之间p16 mRNA阳性率差异具有显著性(p<0.01)。但在宫颈鳞癌和CINⅢ之间p16 mRNA表达差异无显著性(p>0.05)。
5子宫颈鳞癌中p16 mRNA表达与ZPPS、P16~(INK4A)表达的相关性分析
在子宫颈鳞癌中p16 mRNA与P16~(INK4A)表达具有显著正相关性(p<0.05,r=0.503);p16 mRNA与ZPPS表达具有显著正相关性(p<0.05,r=0.380);P16~(INK4A)与ZPPS表达具有正相关性(p<0.05,r=0.402)。
6子宫颈鳞癌中p16 mRNA、P16~(INK4A)、ZPPS表达与年龄、肿瘤大小、分化程度、临床分期、淋巴结转移情况及病理分级之间的关系
p16mRNA、P16~(INK4A)与临床分期有显著相关性(p<0.05),与年龄、肿瘤大小、分化程度、淋巴结转移情况及病理分级之间均无明显相关性(p>0.05)。ZPPS表达与年龄、肿瘤大小、分化程度、病理分级之间均无明显相关性(p>0.05),与淋巴结转移之间有显著相关性(p<0.05)。
结论:
1免疫组化方法标记P16~(INK4A)的表达率从正常宫颈鳞状上皮、CIN到子宫颈鳞癌逐渐增高。
2原位杂交检测p16 mRNA在正常宫颈组织中可见表达,并且随CIN级别升高到子宫颈鳞癌,p16 mRNA检出率逐渐增高。
3子宫颈鳞癌中P16~(INK4A)和p16 mRNA有正相关,说明p16基因在蛋白水平和mRNA水平是相符的。
4免疫组化方法标记ZPPS的表达率从正常宫颈鳞状上皮、CIN到子宫颈鳞癌逐渐增高。
5子宫颈鳞癌中P16~(INK4A)过表达与ZPPS异常表达相关,二者呈正相关。提示两种蛋白的异常表达在宫颈癌的诊断过程中有协同作用,在诊断宫颈癌时可以同时进行检测。
6 p16过表达与临床分期有显著相关性,与其他各临床病理因素之间均不存在相关性。ZPPS异常表达与淋巴结转移有显著相关性。p16 mRNA与临床分期有相关性。
7组织芯片手工制做,程序简单、经济方便,是一种简便可行,高效稳定的技术方法,能满足相关科研课题和实际工作的需要。
Objective: Cervical cancer is one of common malignant tumors which influence women’s life and healthy. Worldwide, incidence of almost half a million new cases annually, 80% of them in developing countrys. It have become one of common malignant tumors and main reason of death. Death rate is second only breast cancer. Screening for cervical cancer widely available has reduced its incidence. Pathogenesis of cervical cancer is associated with many factors and phases. In the past decades, laboratory and clinical researches into different aspects of cervical carcinoma had been performed, but the carcinogenesis of cervical carcinoma is still not clear enough.
p16 is a multiple tumor suppressor, it is the first anti-oncogene which direct effect cell cycle to suppression cell division. What kind of effection it is in the cervical cancer occurrence process has become the hot spot of the research. Many researchs have confirmed that the P16 protein expression unusual in the cervix carcinogenesis process, but its expression is elevates, or reduces, the view not one. Actually, What the deactivation mechanism of the p16 gene in the carcinoma of cervix occurrence process, p16 gene becomes one of the most important anti-oncogene in the cervical cancer studies. There are many questions between p16 and occurrence process of the cervical cancer.
ZPP is one kind the glycoprotein which withdraws from the lower forms of plant. ZPP can stimulate the tumor cell multiplication. The research indicated that it is plant-related-person tumor antigen. Yueqi Zou passed through further to this antigen protein structure as well as the correlation gene research, continues to epurate it to made the ZPPS. The research had indicated that ZPPS after ~(131)I mark obviously higher than the normal tissue in the mouse malignant tumor in radionuclide, also the expression speed and the degree are higher than ~(131)I mark ZPP. But the research of the expression of ZPPS has not seen reported in the cervical cancer.
This topic uses the tissue chip, In situ hybridization to research p16 mRNA, and uses the immunohistochemistry to research the expression ZPPS and P16 protein in normal cervical squamous epithelia, CIN and cervical carcinoma. To discusses the relations between p16 mRNA and biology behavior in normal cervical squamous epithelia, CIN and cervical carcinoma. To discuss the function of expression of p16 mRNA in the cervical cancer occurrencing and developing. To discuss the expression of p16 gene in the protein level and the mRNA level whether does tall. To discuss the p16 gene in the protein level and the molecular level in the development from the normal cervical squamous epithelia, CIN to cervical carcinoma.
Methods:
1 39 cases of normal cervical squamous epithelia, 154 cases of CIN and 54 cases of cervical carcinoma were collected.
2 By manual tissue chip technique, this is done by using a needle to biopsy a standard histologic section and placing the core into an array on a recipient paraffin block, then fusing, slicing, gaining and baking.
3 Immunohistochemistry SP method was used to examine for the expression, relation and the effect of p16 and ZPPS in cervical carcinoma, CIN and normal cervical squamous epithelia.
4 In situ hybridization (ISH) was used to detect p16 mRNA in 200 cases of cervical samples, including 48 cases of cervical carcinoma, 119 cases of CIN and 33 cases of normal cervical squamous epithelia.
5 Analysis the data use spss11.5, significance was set at p<0.05.
Results:
1 The result of tissue chip
8 tissue chips (6×5,7×5) were made. Dots of tissue chip lined up in order, uniformed in size and no shifting. 8 tissue dots were absence, 39 tissue no significant tissue structures. The significant tissue structures were watched in the other dots. The rate of complete tissue dots was 83.3%~93.3%.
The haematoxylin-eosin (HE) staining was uniformity and no dropping, moving and wrinkling.
The immunohistochemical and ISH results show that the positive location was accurate and the background was clear.
2 The expression of P16~(INK4A)
In normal cervical epithelia, the expression of P16~(INK4A) is absence. P16~(INK4A) expression is significant differentiated at normal cervical squamous epithelia with CINⅠ, CINⅡ, CINⅢand cervical invasive squamous cell carcinoma (P<0.01). P16~(INK4A) expression at CINⅠ, CINⅡwith cervical invasive squamous cell carcinoma is significant different (p<0.01), the expression at cervical invasive squamous cell carcinoma and CINⅢis no significant different(p>0.05), the expression at CINⅠ, CINⅡand CINⅢis significant different (p<0.05).
3 The expression of ZPPS
In normal cervical epithelia the expression of ZPPS is absence. ZPPS expression is no significant differentiated at normal cervical epithelia with CINⅠ. There is significant differentiated at CINⅠ, CINⅡwith cervical invasive squamous cell carcinoma (p<0.05), but the expression of ZPPS is no significant differentiated at cervical invasive squamous cell carcinoma and CINⅢ(p>0.05).
4 The expression of p16 mRNA
Expression of p16 mRNA in various lesions of cervix, 48 cases of cervical carcinoma, 119 cases of CIN and 33 cases of normal cervical seqamous epithelia were tested. The positive rates of p16 mRNA in normal cervical epithela, CINⅠ, CINⅡ, CINⅢand cervical carcinoma group were 18.18%, 43.75%, 56.41%, 75%, 79.17% respectively. p16 mRNA expression is significant differentiated at normal cervical epithelia with CINⅡ, CINⅢand cervical invasive squamous cell carcinoma (p<0.01). There is significant differentiated at cervical invasive squamous cell carcinoma with CINⅠ, CINⅡ(p<0.01), but the expression at CINⅢand cervical invasive squamous cell carcinoma is no significant different (p>0.05).
5 The relation of p16 mRNA、ZPPS、P16~(INK4A)
There was a positive correlation between expression of P16~(INK4A) and p16 mRNA(p<0.01), expression of p16 mRNA and ZPPS (p<0.05), expression of P16~(INK4A) and ZPPS.
6 The relationship between the expression of p16 mRNA、P16~(INK4A)、ZPPS and clinical pathological features of cervical carcinoma.
No significant relationship were found between the expression of P16~(INK4A) and clinical pathological features, but clinical stage. There was significant relationship between the expression of p16 mRNA and clinical stage. Significant relationship were found between the expression of ZPPS and metastasis.
Conclusions:
1 P16~(INK4A) overexpression rates become higher and higher from normal cervical squamous epithelia, CIN to cervical invasive squamous cell carcinoma.
2 There was expression of p16 mRNA in normal cervical squamous epithelia. The positive rate of p16 mRNA from CINⅠ, CINⅡ, CINⅢto cervical invasive squamous cell carcinoma becomes gradually more higher.
3 There was a positive correlation between overexpression of p16 and p16mRNA in occurrence and development cervical squamous cell carcinomas.
4 ZPPS overexpression rates become higher and higher from normal cervical squamous epithelia, CIN to cervical invasive squamous cell carcinoma.
5 There was a positive correlation between overexpression of P16~(INK4A) and ZPPS. Probably significant correlation of P16 and ZPPS in diagnose cervical squamous cell carcinomas.
6 There was no significant relationship between overexpression of p16 and clinical pathological features, but clinical stage. There was significant relationship between expression of ZPPS and metastasis. Significant relationship were found between the expression of p16 mRNA and clinical stage.
7 Manual tissue chip technique is simple, cost-effective, and reliable. This technique can provide a highly efficient, high-throughput mechanism for some research.
引文
1 Serrano M, Hannon GJ, Beach D, et al. A new regulatory motifin cell cycle ontrol causing specific inhibition of cy-clinD/CDK4. Nature, 1993, 366(6456): 704~707
2 Kamb A, Cruis NA, Weaverfeldhaus J, et al. A cell cycle regulator potentially involve dingenesis of many tumor types. Science, 1994, 264(3): 436~440
3 Nalabothula N, Lakka SS, Dinh DH, et al. Sense p16 and antisense uPAR bicistronic construct inhibits angiogenesis and induces glioma cell death. Int J Oncol, 2007, 30(3): 669~678
4 Fargnoli MC, Argenziano G, Zalaudek I. High-and low-penetrance cutaneous melanoma susceptibility genes. Expert Rev Anticancer Ther, 2006, 6(5): 657~670
5 Koscielny S, Dahse R, Ernst G, et al. The prognostic relevance of p16 inactivation in head and neck cancer. ORL J Otorhinolaryngol Relat Spec, 2007, 69(1): 30~36
6 Sharma G, Mirza S, Prasad CP, et al. Promoterhypermethylation of p16INK4A, p14ARF, CyclinD2 and Slit2 in serum and tumor DNA from breast cancer patients. Life Sci, 2007, 80(20): 1873~1881
7 Chen R, Aaltonen LM, Vaheri A. Human papillomavirus type
16 in head and neck carcinogenesis. Rev Med Virol, 2005, 15(6): 351~363
8 Sarina S, Julie I, Marian C, et al. Comprehensive Analysis of p16INK4a in Childhood Acute Lymphoblastic Leukemia Reveals Homozygous Deletion, Haploinsufficiency and Acquired Isodisomy at the 9p Locus with Intact p16INK4a.Blood (ASH Annual Meeting Abstracts), 2006, 108: 4332
9 Zhang H, Jin Y, Chen X, et al. Papillomavirus type 16 E6/E7 and human telomerase reverse transcriptase in esophageal cell immortalization and early transformation. Cancer Lett, 2007, 245(1-2): 184~194
10 Hou P, Shen JY, Ji MJ, et al. Microarray-based method for detecting methylation changes of p16(Ink4a) gene 5'-CpG islands in gastric carcinomas. World J Gastroenterol, 2004, 10(24): 3553~3558
11 Cui X, Wakai T, Shirai Y, et al. Arsenic trioxide inhibits DNA methyltransferase and restores methylation-silenced genes in human liver cancer cells. Hum Pathol, 2006, 37(3): 298~311
12 Ulivi P, Zoli W, Calistri D, et al. p16INK4A and CDH13 hypermethylation in tumor and serum of non-small cell lung cancer patients. J Cell Physiol, 2006, 206(3): 611~615
13 Jeong J, Park YN, Park JS, et al.Clinical significance of p16 protein expression loss and aberrant p53 protein expression in pancreatic cancer. Yonsei Med J, 2005, 46(4): 519~525
14 Kong CS, Balzer BL, Troxell ML, et al. p16INK4A immunohistochemistry is superior to HPV in situ hybridization for the detection of high-risk HPV in atypical squamous metaplasia. Am J Surg Pathol. 2007, 31(1): 33~43
15 Zhang Q, Kuhn L, Denn LA y, et al, Impact of utilizing p16INK4A immunohistochemistry on estimated performance of three cervical cancer screening tests.Int J Cancer, 2007, 120(2): 351~356
16 Benevolo M, Mottolese M, Marandino F, et al. Immunohistochemical expression of p16(INK4a) is predictive of HR-HPV infection in cervical low-grade lesions. Mod Pathol, 2006, 19(3): 384~391
17 Nam EJ, Kim JW, Kim SW, et al. The expressions of the Rb pathway in cervical intraepithelial neoplasia; predictive and prognostic significance. Gynecol Oncol, 2007, 104(1): 207~211
18 Murphy N, Ring M, Killalea AG, et al. p16INK4A as a marker for cervical dyskaryosis: CIN and cGIN in cervical biopsies and ThinPrep smears. J Clin Pathol, 2003, 56(1): 56~63
19 Qiao X, Bhuiya TA, Spitzer M. Differentiating high-grade cervical intraepithelial lesion from atrophy in postmenopausal women using Ki-67, cyclin E, and p16immunohistochemical analysis. J Low Genit Tract Dis, 2005, 9(2): 100~107
20 Klaes R, Benner A, Friedrich T, et al. p16INK4a immunohistochemistry improves interobserver agreement in the diagnosis of cervical intraepithelial neoplasia . Am J Surg Pathol, 2002, 26 (11): 1389~1399
21 Queiroz C, Silva TC, Alves VA, et al. P16(INK4a) expression as a potential prognostic marker in cervical pre-neoplastic and neoplastic lesions. Pathol Res Pract, 2006, 202(2): 77~83
22 魏晓丽. p16 基因 mRNA 及其编码蛋白在宫颈癌中的表达. 陕西医学杂志, 2004, 33(6): 543~546
23 Ivanova TA, Golovina DA, Zavalishina LE, et al. Up-regulation of expression and lack of 5' CpG island hypermethylation of p16 INK4a in HPV-positive cervical carcinomas. BMC Cancer, 2007, 7: 47
24 单保恩,郑振海,张子杰,等. 一种植物由来的糖蛋白(郑氏植物蛋白)-肿瘤相同性抗原的研究. 中国免疫学杂志,2003, 19: 328~331
25 丁献义,郑振海,韩冰,等. 131I 标记的一种抗植物蛋白抗体及其片段在荷瘤小鼠体内的分布. 河北医科大学学报, 2003, 24: 321~324
26 Rimm DL, Camp RL, Charette LA, et al. Amplification of tissue by construction of tissue microarrays. Exp Mol Pathol, 2001, 70(3): 255-264
27 Battifora H. The multitumor (sausage) tissue block: novelmethod for immunohistochemical antibody testing. Invest Lab, 1986, 55(2): 244~248
28 Kononen J, Bubendorf L, Kallionien A, et al. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nature Med, 1998, 4(7): 844~847
29 Rosen DG, Huang X, Deavers MT, et al. Validation of tissue microarray technology in ovarian carcinoma. Mod Pathol, 2004, 17(7): 790~797
30 Gomaa W, Ke Y, Fujii H, et al. Tissue microarray of head and neck squamous carcinoma: validation of the methodology for the study of cutaneous fatty acid-binding protein, vascular endothelial growth factor, involucrin and Ki-67. Virchows Arch, 2005, 447(4): 701~709
31 Moch H, Kononen T, Kallioniemi OP, et al. Tissue microarrays: what will they bring to molecular and anatomic pathology? Adv Anat Pathol, 2001, 8(1): 14~20
32 朱明华.组织微阵列及其在肿瘤病理研究中的应用.中华病理学杂志, 2002, 31: 72~73
1 Koscielny S, Dahse R, Ernst G, et al. The prognostic relevance of p16 inactivation in head and neck cancer. ORL J Otorhinolaryngol Relat Spec, 2007, 69(1): 30~36
2 Sulong S, Irving J, Case M, et al. Comprehensive Analysis of p16INK4a in Childhood Acute Lymphoblastic Leukemia Reveals Homozygous Deletion,Haploinsufficiency and Acquired Isodisomy at the 9p Locus with Intact p16INK4a. Blood (ASH Annual Meeting Abstracts), 2006, 108: 4332
3 Cui X, Wakai T, Shirai Y, et al. Arsenic trioxide inhibits DNA methyltransferase and restores methylation-silenced genes in human liver cancer cells. Hum Pathol, 2006, 37(3): 298~311
4 Ulivi P, Zoli W, Calistri D, et al. p16INK4A and CDH13 hypermethylation in tumor and serum of non-small cell lung cancer patients. J Cell Physiol, 2006, 206(3): 611~615
5 Saito M, Nakagawa K, Hamada K, et al. Introduction of p16INK4a inhibits telomerase activity through transcriptional suppression of human telomerase reverse transcriptase expression in human gliomas. Int J Oncol, 2004, 24(5): 1213~1220
6 Liu Y, Lan Q, Siegfried JM, et al. Aberrant promoter methylation of p16 and MGMT genes in lung tumors from smoking and never-smoking lung cancer patients. Neoplasia, 2006, 8(1): 46~51
7 Jicai Z, Zongtao Y, Jun L, et al. Persistent infection of hepatitis B virus is involved in high rate of p16 methylation in hepatocellular carcinoma. Mol Carcinog, 2006, 45(7): 530~536
8 Ojima H, Saito K, Yamauchi H, et al. P16 protein abnormality in Epstein-Barr virus-associated gastric carcinomas. Anticancer Res, 2006, 26(2): 933~937
9 Benevolo M, Mottolese M, Marandino F, et al. Immunohistochemical expression of p16(INK4a) is predictive of HR-HPV infection in cervical low-grade lesions. Mod Pathol, 2006, 19(3):384~391
10 Klaes R, Benner A, Friedrich T, et al. p16INK4a immunohistochemistry improves interobserver agreement in the diagnosis of cervical intraepithelial neoplasia. Am J Surg Pathol, 2002, 26(11): 1389~1399
11 Geza A, Paul J, Cindy M, et al. Hypoxia-Inducible Erythropoietin Signaling in Squamous Dysplasia and Squamous Cell Carcinoma of the Uterine Cervix and Its Potential Role in Cervical Carcinogenesis and Tumor Progression. Am. J. Pathol, 2003, 162(6): 1789~1806
12 Nam EJ, Kim JW, Kim SW, et al. The expressions of the Rb pathway in cervical intraepithelial neoplasia; predictive and prognostic significance. Gynecol Oncol, 2007, 104(1): 207~211
13 Kang S, Kim J, Kim HB, et al. Methylation of p16INK4a is a non-rare event in cervical intraepithelial neoplasia. Diagn Mol Pathol, 2006, 15(2): 74~82
14 Kim NR, Lin Z, Kim KR, et al. Epstein-Barr virus and p16INK4A methylation in squamous cell carcinoma and precancerous lesions of the cervix uteri. J Korean Med Sci, 2005, 20(4): 636~642
15 Queiroz C, Silva TC, Alves VA, et al. P16(INK4a) expression as a potential prognostic marker in cervical pre-neoplastic and neoplastic lesions. Pathol Res Pract, 2006, 202(2):77~83
16 Kanao H, Enomoto T, Ueda Y, et al. Correlation between p14 (ARF)/p16 (INK4A) expression and HPV infection in uterine cervical cancer. Cancer Lett, 2004, 213(1): 31~37
17 Wang HL, Lu DW. Detection of human papillomavirus DNA and expression of p16, Rb, and p53 proteins in small cell carcinomas of the uterine cervix. Am J Surg Pathol, 2004, 28(7): 901~908
18 Ansari-Lari MA, Staebler A, Zaino RJ, et al. Distinction of endocervical and endometrial adenocarcinomas: immunohistochemical p16 expression correlated with human papillomavirus (HPV) DNA detection. Am J Surg Pathol, 2004, 28(2): 160~167
19 Benevolo M, Mottolese M, Marandino F, et al. Immunohistochemical expression of p16(INK4a) is predictive of HR-HPV infection in cervical low-grade lesions. Mod Pathol, 2006, 19(3): 384~391
20 Eleuterio Jr, Giraldo PC, Goncalves AK, et al. Prognostic markers of high-grade squamous intraepithelial lesions: the role of p16INK4a and high-risk human papillomavirus. Acta Obstet Gynecol Scand, 2007, 86(1): 94~98
2 Kamb A, Cruis NA, Weaverfeldhaus J, et al. A cell cycle regulator potentially involve dingenesis of many tumor types. Science, 1994, 264(3): 436~440
3 Nalabothula N, Lakka SS, Dinh DH, et al. Sense p16 and antisense uPAR bicistronic construct inhibits angiogenesis and induces glioma cell death. Int J Oncol, 2007, 30(3): 669~678
4 Fargnoli MC, Argenziano G, Zalaudek I. High-and low-penetrance cutaneous melanoma susceptibility genes. Expert Rev Anticancer Ther, 2006, 6(5): 657~670
5 Koscielny S, Dahse R, Ernst G, et al. The prognostic relevance of p16 inactivation in head and neck cancer. ORL J Otorhinolaryngol Relat Spec, 2007, 69(1): 30~36
6 Sharma G, Mirza S, Prasad CP, et al. Promoterhypermethylation of p16INK4A, p14ARF, CyclinD2 and Slit2 in serum and tumor DNA from breast cancer patients. Life Sci, 2007, 80(20): 1873~1881
7 Chen R, Aaltonen LM, Vaheri A. Human papillomavirus type
16 in head and neck carcinogenesis. Rev Med Virol, 2005, 15(6): 351~363
8 Sarina S, Julie I, Marian C, et al. Comprehensive Analysis of p16INK4a in Childhood Acute Lymphoblastic Leukemia Reveals Homozygous Deletion, Haploinsufficiency and Acquired Isodisomy at the 9p Locus with Intact p16INK4a.Blood (ASH Annual Meeting Abstracts), 2006, 108: 4332
9 Zhang H, Jin Y, Chen X, et al. Papillomavirus type 16 E6/E7 and human telomerase reverse transcriptase in esophageal cell immortalization and early transformation. Cancer Lett, 2007, 245(1-2): 184~194
10 Hou P, Shen JY, Ji MJ, et al. Microarray-based method for detecting methylation changes of p16(Ink4a) gene 5'-CpG islands in gastric carcinomas. World J Gastroenterol, 2004, 10(24): 3553~3558
11 Cui X, Wakai T, Shirai Y, et al. Arsenic trioxide inhibits DNA methyltransferase and restores methylation-silenced genes in human liver cancer cells. Hum Pathol, 2006, 37(3): 298~311
12 Ulivi P, Zoli W, Calistri D, et al. p16INK4A and CDH13 hypermethylation in tumor and serum of non-small cell lung cancer patients. J Cell Physiol, 2006, 206(3): 611~615
13 Jeong J, Park YN, Park JS, et al.Clinical significance of p16 protein expression loss and aberrant p53 protein expression in pancreatic cancer. Yonsei Med J, 2005, 46(4): 519~525
14 Kong CS, Balzer BL, Troxell ML, et al. p16INK4A immunohistochemistry is superior to HPV in situ hybridization for the detection of high-risk HPV in atypical squamous metaplasia. Am J Surg Pathol. 2007, 31(1): 33~43
15 Zhang Q, Kuhn L, Denn LA y, et al, Impact of utilizing p16INK4A immunohistochemistry on estimated performance of three cervical cancer screening tests.Int J Cancer, 2007, 120(2): 351~356
16 Benevolo M, Mottolese M, Marandino F, et al. Immunohistochemical expression of p16(INK4a) is predictive of HR-HPV infection in cervical low-grade lesions. Mod Pathol, 2006, 19(3): 384~391
17 Nam EJ, Kim JW, Kim SW, et al. The expressions of the Rb pathway in cervical intraepithelial neoplasia; predictive and prognostic significance. Gynecol Oncol, 2007, 104(1): 207~211
18 Murphy N, Ring M, Killalea AG, et al. p16INK4A as a marker for cervical dyskaryosis: CIN and cGIN in cervical biopsies and ThinPrep smears. J Clin Pathol, 2003, 56(1): 56~63
19 Qiao X, Bhuiya TA, Spitzer M. Differentiating high-grade cervical intraepithelial lesion from atrophy in postmenopausal women using Ki-67, cyclin E, and p16immunohistochemical analysis. J Low Genit Tract Dis, 2005, 9(2): 100~107
20 Klaes R, Benner A, Friedrich T, et al. p16INK4a immunohistochemistry improves interobserver agreement in the diagnosis of cervical intraepithelial neoplasia . Am J Surg Pathol, 2002, 26 (11): 1389~1399
21 Queiroz C, Silva TC, Alves VA, et al. P16(INK4a) expression as a potential prognostic marker in cervical pre-neoplastic and neoplastic lesions. Pathol Res Pract, 2006, 202(2): 77~83
22 魏晓丽. p16 基因 mRNA 及其编码蛋白在宫颈癌中的表达. 陕西医学杂志, 2004, 33(6): 543~546
23 Ivanova TA, Golovina DA, Zavalishina LE, et al. Up-regulation of expression and lack of 5' CpG island hypermethylation of p16 INK4a in HPV-positive cervical carcinomas. BMC Cancer, 2007, 7: 47
24 单保恩,郑振海,张子杰,等. 一种植物由来的糖蛋白(郑氏植物蛋白)-肿瘤相同性抗原的研究. 中国免疫学杂志,2003, 19: 328~331
25 丁献义,郑振海,韩冰,等. 131I 标记的一种抗植物蛋白抗体及其片段在荷瘤小鼠体内的分布. 河北医科大学学报, 2003, 24: 321~324
26 Rimm DL, Camp RL, Charette LA, et al. Amplification of tissue by construction of tissue microarrays. Exp Mol Pathol, 2001, 70(3): 255-264
27 Battifora H. The multitumor (sausage) tissue block: novelmethod for immunohistochemical antibody testing. Invest Lab, 1986, 55(2): 244~248
28 Kononen J, Bubendorf L, Kallionien A, et al. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nature Med, 1998, 4(7): 844~847
29 Rosen DG, Huang X, Deavers MT, et al. Validation of tissue microarray technology in ovarian carcinoma. Mod Pathol, 2004, 17(7): 790~797
30 Gomaa W, Ke Y, Fujii H, et al. Tissue microarray of head and neck squamous carcinoma: validation of the methodology for the study of cutaneous fatty acid-binding protein, vascular endothelial growth factor, involucrin and Ki-67. Virchows Arch, 2005, 447(4): 701~709
31 Moch H, Kononen T, Kallioniemi OP, et al. Tissue microarrays: what will they bring to molecular and anatomic pathology? Adv Anat Pathol, 2001, 8(1): 14~20
32 朱明华.组织微阵列及其在肿瘤病理研究中的应用.中华病理学杂志, 2002, 31: 72~73
1 Koscielny S, Dahse R, Ernst G, et al. The prognostic relevance of p16 inactivation in head and neck cancer. ORL J Otorhinolaryngol Relat Spec, 2007, 69(1): 30~36
2 Sulong S, Irving J, Case M, et al. Comprehensive Analysis of p16INK4a in Childhood Acute Lymphoblastic Leukemia Reveals Homozygous Deletion,Haploinsufficiency and Acquired Isodisomy at the 9p Locus with Intact p16INK4a. Blood (ASH Annual Meeting Abstracts), 2006, 108: 4332
3 Cui X, Wakai T, Shirai Y, et al. Arsenic trioxide inhibits DNA methyltransferase and restores methylation-silenced genes in human liver cancer cells. Hum Pathol, 2006, 37(3): 298~311
4 Ulivi P, Zoli W, Calistri D, et al. p16INK4A and CDH13 hypermethylation in tumor and serum of non-small cell lung cancer patients. J Cell Physiol, 2006, 206(3): 611~615
5 Saito M, Nakagawa K, Hamada K, et al. Introduction of p16INK4a inhibits telomerase activity through transcriptional suppression of human telomerase reverse transcriptase expression in human gliomas. Int J Oncol, 2004, 24(5): 1213~1220
6 Liu Y, Lan Q, Siegfried JM, et al. Aberrant promoter methylation of p16 and MGMT genes in lung tumors from smoking and never-smoking lung cancer patients. Neoplasia, 2006, 8(1): 46~51
7 Jicai Z, Zongtao Y, Jun L, et al. Persistent infection of hepatitis B virus is involved in high rate of p16 methylation in hepatocellular carcinoma. Mol Carcinog, 2006, 45(7): 530~536
8 Ojima H, Saito K, Yamauchi H, et al. P16 protein abnormality in Epstein-Barr virus-associated gastric carcinomas. Anticancer Res, 2006, 26(2): 933~937
9 Benevolo M, Mottolese M, Marandino F, et al. Immunohistochemical expression of p16(INK4a) is predictive of HR-HPV infection in cervical low-grade lesions. Mod Pathol, 2006, 19(3):384~391
10 Klaes R, Benner A, Friedrich T, et al. p16INK4a immunohistochemistry improves interobserver agreement in the diagnosis of cervical intraepithelial neoplasia. Am J Surg Pathol, 2002, 26(11): 1389~1399
11 Geza A, Paul J, Cindy M, et al. Hypoxia-Inducible Erythropoietin Signaling in Squamous Dysplasia and Squamous Cell Carcinoma of the Uterine Cervix and Its Potential Role in Cervical Carcinogenesis and Tumor Progression. Am. J. Pathol, 2003, 162(6): 1789~1806
12 Nam EJ, Kim JW, Kim SW, et al. The expressions of the Rb pathway in cervical intraepithelial neoplasia; predictive and prognostic significance. Gynecol Oncol, 2007, 104(1): 207~211
13 Kang S, Kim J, Kim HB, et al. Methylation of p16INK4a is a non-rare event in cervical intraepithelial neoplasia. Diagn Mol Pathol, 2006, 15(2): 74~82
14 Kim NR, Lin Z, Kim KR, et al. Epstein-Barr virus and p16INK4A methylation in squamous cell carcinoma and precancerous lesions of the cervix uteri. J Korean Med Sci, 2005, 20(4): 636~642
15 Queiroz C, Silva TC, Alves VA, et al. P16(INK4a) expression as a potential prognostic marker in cervical pre-neoplastic and neoplastic lesions. Pathol Res Pract, 2006, 202(2):77~83
16 Kanao H, Enomoto T, Ueda Y, et al. Correlation between p14 (ARF)/p16 (INK4A) expression and HPV infection in uterine cervical cancer. Cancer Lett, 2004, 213(1): 31~37
17 Wang HL, Lu DW. Detection of human papillomavirus DNA and expression of p16, Rb, and p53 proteins in small cell carcinomas of the uterine cervix. Am J Surg Pathol, 2004, 28(7): 901~908
18 Ansari-Lari MA, Staebler A, Zaino RJ, et al. Distinction of endocervical and endometrial adenocarcinomas: immunohistochemical p16 expression correlated with human papillomavirus (HPV) DNA detection. Am J Surg Pathol, 2004, 28(2): 160~167
19 Benevolo M, Mottolese M, Marandino F, et al. Immunohistochemical expression of p16(INK4a) is predictive of HR-HPV infection in cervical low-grade lesions. Mod Pathol, 2006, 19(3): 384~391
20 Eleuterio Jr, Giraldo PC, Goncalves AK, et al. Prognostic markers of high-grade squamous intraepithelial lesions: the role of p16INK4a and high-risk human papillomavirus. Acta Obstet Gynecol Scand, 2007, 86(1): 94~98