早中期食管鳞状细胞癌中P16、PCNA表达研究
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摘要
目的：食管癌是世界第三大恶性肿瘤,占中国恶性肿瘤死亡的第二位，严重影响人类健康，但其癌变机制仍不很清楚[1]。食管癌的发生有家族聚集性，提示遗传因素在食管癌的发生中起一定作用。近年来的分子生物学进展已发现了多个可能与食管癌的发生或演进有关的相关基因或相关的基因片段，而且，对细胞周期调控的异常在肿瘤发生中的作用有了更为深刻的认识，已经发现在食管癌中有多种G1／S期调控元件的表达异常，和食管癌的发生、发展、临床病理特征有密切关系。其中，抑癌基因P16与和食管癌的淋巴结转移及病理分级关系密切，p16位于染色体9p21，cDNA长960bp, 亦称为多肿瘤抑制因子(MTS1)，含有4个ankyrin结构，能结合并阻断CDK4和CDK6，它是CDK4/CDK6特异的抑制剂。p16全长8.5Kb，包括E1，E2，E3三个外显子，长分别为E1 1266bp、E2 307bp、E3 1166bp，共同编码一个相对分子量16KDa的蛋白质，即P16蛋白。野生型P16蛋白含有一个活性部位，由4个回钩状重叠的空间构型组成，是作为细胞生长调控的结构基因。P16的主要作用在于抑制CDK4/CDK6介导的RB基因蛋白产物的磷酸化，阻止细胞从G1期进入S期。P16与CDK、cyclin、Rb形成一个反馈调节环路。当P16蛋白基因缺失、突变、启动子CPG岛异常甲基化使其表达异常时，同样导致细胞生长失控、细胞转化和癌变。CDK4或CDK6与CyclinD1结合之后能使细胞周
期进入G1期，而P16蛋白氨基末端具有与CyclinD1竞争CDK4／CDK6的部位，抑制其激酶活性，并使其激活功能丧失，从而使细胞生长停滞。细胞增殖核抗原(proliferation cell nuclear antigen PCNA)是细胞核内的一种酸性蛋白质，分子量为36000D，是DNA聚合酶的辅助因子，参与DNA合成并在细胞周期中起着重要作用［13］。PCNA在DNA合成的G1期开始增加，S起达高峰，G2、M期迅速下降。因此PCNA可作为评价细胞增殖状态的标志物，用以鉴别癌细胞和正常细胞。PCNA和食管癌的淋巴结转移及病理分级关系也十分密切。本实验拟通过对食管鳞状细胞癌组织中P16与PCNA的蛋白表达的研究，探讨二者与食管鳞状细胞癌生物学行为的关系，以期对早期，中期食管鳞状细胞癌的诊断与治疗提供佐证。
方法：试验所用49例标本系河北医科大学附属第二医院手术切除的食管癌组织蜡块，均为术后病理证实食管鳞状细胞癌(年龄30--78岁,平均56.9岁)。临床病理学分级按WHO1990年标准，Ⅰ级16例，Ⅱ级16例，Ⅲ级17例。临床病理分期(PTNM)按UICC标准，0期2例，Ⅰ期1例，ⅡA期22例，ⅡB期24例。淋巴结转移状况:有淋巴结转移者24例，无淋巴结转移者25例。抽取石蜡标本HE染色玻璃切片，经两位病理科主任确定食管鳞状细胞癌的病理分级状况与淋巴结转移情况。(1) 按是否有淋巴结转移分为两组：淋巴结转移组与淋巴结非转移组，其中，淋巴结转移组24例，淋巴结非转移组25例。(2) 按病理分级分组，共分三组：第一组Ⅰ级16例，第二组Ⅱ级16例，第三组Ⅲ级17例，所有标本经采用免疫组织化学方法对标本进行半定量分析。
分别检测P16与PCNA在淋巴结转移组和无淋巴结转移组中的表达量，检测P16与PCNA在不同的病理分级中的表达量，各指标的表达用阳性细胞面积比率，阳性强度均值表示。统计学方法使用SPSS10.0软件，其中，淋巴结转移组与无淋巴结转移组在方差齐性分析基础上进行两样本均数比较，采用t检验(T--test)；按病理分级所分的三组在方差齐性分析基础上采用单因素方差分析(One--way ANOVA)。
结果：(1) P16蛋白的阳性细胞面积比率及阳性强度均值在淋巴结非转移组(71.92%，8.375)明显高于淋巴结转移组(60.0417%，6.64)，两组之间比较，具有显著性差异P<0.05。(2) PCNA蛋白的阳性细胞面积比率及阳性强度均值在淋巴结非转移组(42.7666%，11.625)，明显低于淋巴结转移组(74.6667%，15.08)，两组之间比较，具有显著性差异P<0.01。(3) P16蛋白阳性细胞面积比率及阳性强度均值随病理分级的升高而降低(Ⅰ79.6875%，10.9375；Ⅱ57.5%，9.125；Ⅲ45.2941%，6.8824)，三组之间，两两比较，均有显著性差异p<0.01。(4) PCNA蛋白阳性细胞面积比率及阳性强度均值随病理分级的升高而升高(Ⅰ38.5%，10.4118；Ⅱ63.625%，13.375；Ⅲ78%，16.125), 三组之间，两两比较，均有显著性差异p<0.01。
结论：P16、PCNA与食管鳞状细胞癌关系密切，本实验结果证实在食管鳞状细胞癌，P16作为一种多肿瘤抑制基因，其蛋白表达与食管鳞状细胞癌的病理分级、淋巴结浸润、侵袭呈负相关，有淋巴结转移的P16的蛋白表达量明显低于无淋巴结转移的，P16随病理分级的升高，其表达量逐渐减少，而PCNA作为一种细胞增殖活性的标记物，恰恰相反，
其蛋白表达与食管鳞状细胞癌的病理分级、淋巴结浸润、侵袭呈正相关，其蛋白表达量在有淋巴结转移的明显高于无淋巴结转移的。PCNA则随食管癌细胞恶性程度的增高，其表达量逐渐增多，因此，测定P16及PCNA可能对鉴别ⅡA与ⅡB期食管鳞状细胞癌有指导作用,对早中期食管鳞状细胞癌的诊断，淋巴结浸润、侵袭及转移，恶性度的判定及治疗治疗有重要作用。
Objective: esophageal carcinoma is in the third of malignant tumor in the world, accounting for the second dead causes of malignant tumor in China, but the mechanism of cell malignant alteration is not clear yet. There are a family character in the ESCC's occuring, which shows that the heredity factor play a role in the occuring of ESCC. Recently, a few correlated gene or gene pitch have been found in the molecule biology, which may be correlated to the occuring or progression of ESCC. Moreover, we have had more knowledge about the effect of abnormal cell cycle regulatory in malignant tumor progression. It has been found that there are a variety of abnormal expression of regulatory factors in G1/S phrase, which is related to lymph node metastasize and clinicopathological feature of Esophageal Squamous-cell carcinoma (ESCC), in particularly, p16 has more correlation of lymph node metastasis and pathological grade in ESCC. P16 is located chromosome of 9p21, whose cDNA length is 960bp. P16 is also named more tumor suppressor, including four ankyrin configuration. Being a differential suppressor, it can combine with and stop CDK4 and CDK6 activity. The whole lengths of p16 are 8.5kb, including
three exon named E1、E2、E3 respectively, their length are E1 266bp、E2 307bp、E3 1166bp respectively, coding a protein together, whose comparative molecule quantum is 16 KD, that is p16 protein. Wild p16 protein has an active part, composed of four hook wrap space fabricate. It is viewed as frame gene controlling cell growth. P16 main function is suppressing the phosphation of Rb protein transmited by CDK4/CDK6, stopping cell entering into S phase from G1 phase. p16、CDK、cyclin and Rb are formed a feedback control circle line. When the gene deletion、mutation、and promoter CPG island aberrant methylation of p16 protein make p16 abnormal expression, it would lead the growing cell lost of control, cell transmission and carcinoalteration. CDK4 or CDK6 could make cell enter into G1 phase after combining with cyclinD1, while p16 protein amino-tail end have a place, which can compete with cyclinD1 for CDK4/CDK6, suppressing cyclinD1 enzyme's activity and making cyclinD1 lose its activity function and stop the growing cell. Proliferation cell nuclear antigen is a kind of acidity protein in cell nucleus, its molecule quality is 36000D. It is an assistant factor of DNA poly enzyme, participating in DNA synthesize and playing a role in cell cycle period. In the possess of DNA synthesize , PCNA expression start increasing in G1、reaching apex in S、rapidly decreasing in G2 and M phase. Therefore, PCNA is taken as a symbol of evaluating the cell proliferation activity, used as distinguishing carcinoma cell from normal cell. There is also a closed correlation between PCNA and lymph
node metastasis and pathological grades of ESCC. In this study, we explored the relationship of both p16 and PCNA with ESCC biological behavior by studying the expression of p16 and PCNA in protein lever in ESCC tissues. Exploring the relationship between both p16 and PCNA and esophageal carcinoma biology behavior, aiming at giving evidence in the diagnosing and treating of earlier stage and metaphase's esophageal carcinoma.
Methods: Immunohistochemistry analysis of p16 and PCNA proteins was carried out in paraffin-embedded section from 49 surgically resected ESCC’s in the affiliated second hospital, HeBei medical university, all specimen were divided into two groups: lymph node metastasis group(24) and non-lymph node metastasis group(25),in addition, all specimens were divided into three groups according to pathological grade: Ⅰ16, Ⅱ16, Ⅲ17. Macroscopically, all specimens are ESCC tissues (aged from30-78 years old, mean 56.9 years old). The data were expressed with positive cell labeled area ratio (percentage,%) and positive degree means. The results of correlating with p16 and PCNA are analyzed by using T test and one-way ANOVA in SPSS10.0.
Results: (1) The positive cell area ratio and positive degree means of p16 were significantly highe
期进入G1期，而P16蛋白氨基末端具有与CyclinD1竞争CDK4／CDK6的部位，抑制其激酶活性，并使其激活功能丧失，从而使细胞生长停滞。细胞增殖核抗原(proliferation cell nuclear antigen PCNA)是细胞核内的一种酸性蛋白质，分子量为36000D，是DNA聚合酶的辅助因子，参与DNA合成并在细胞周期中起着重要作用［13］。PCNA在DNA合成的G1期开始增加，S起达高峰，G2、M期迅速下降。因此PCNA可作为评价细胞增殖状态的标志物，用以鉴别癌细胞和正常细胞。PCNA和食管癌的淋巴结转移及病理分级关系也十分密切。本实验拟通过对食管鳞状细胞癌组织中P16与PCNA的蛋白表达的研究，探讨二者与食管鳞状细胞癌生物学行为的关系，以期对早期，中期食管鳞状细胞癌的诊断与治疗提供佐证。
方法：试验所用49例标本系河北医科大学附属第二医院手术切除的食管癌组织蜡块，均为术后病理证实食管鳞状细胞癌(年龄30--78岁,平均56.9岁)。临床病理学分级按WHO1990年标准，Ⅰ级16例，Ⅱ级16例，Ⅲ级17例。临床病理分期(PTNM)按UICC标准，0期2例，Ⅰ期1例，ⅡA期22例，ⅡB期24例。淋巴结转移状况:有淋巴结转移者24例，无淋巴结转移者25例。抽取石蜡标本HE染色玻璃切片，经两位病理科主任确定食管鳞状细胞癌的病理分级状况与淋巴结转移情况。(1) 按是否有淋巴结转移分为两组：淋巴结转移组与淋巴结非转移组，其中，淋巴结转移组24例，淋巴结非转移组25例。(2) 按病理分级分组，共分三组：第一组Ⅰ级16例，第二组Ⅱ级16例，第三组Ⅲ级17例，所有标本经采用免疫组织化学方法对标本进行半定量分析。
分别检测P16与PCNA在淋巴结转移组和无淋巴结转移组中的表达量，检测P16与PCNA在不同的病理分级中的表达量，各指标的表达用阳性细胞面积比率，阳性强度均值表示。统计学方法使用SPSS10.0软件，其中，淋巴结转移组与无淋巴结转移组在方差齐性分析基础上进行两样本均数比较，采用t检验(T--test)；按病理分级所分的三组在方差齐性分析基础上采用单因素方差分析(One--way ANOVA)。
结果：(1) P16蛋白的阳性细胞面积比率及阳性强度均值在淋巴结非转移组(71.92%，8.375)明显高于淋巴结转移组(60.0417%，6.64)，两组之间比较，具有显著性差异P<0.05。(2) PCNA蛋白的阳性细胞面积比率及阳性强度均值在淋巴结非转移组(42.7666%，11.625)，明显低于淋巴结转移组(74.6667%，15.08)，两组之间比较，具有显著性差异P<0.01。(3) P16蛋白阳性细胞面积比率及阳性强度均值随病理分级的升高而降低(Ⅰ79.6875%，10.9375；Ⅱ57.5%，9.125；Ⅲ45.2941%，6.8824)，三组之间，两两比较，均有显著性差异p<0.01。(4) PCNA蛋白阳性细胞面积比率及阳性强度均值随病理分级的升高而升高(Ⅰ38.5%，10.4118；Ⅱ63.625%，13.375；Ⅲ78%，16.125), 三组之间，两两比较，均有显著性差异p<0.01。
结论：P16、PCNA与食管鳞状细胞癌关系密切，本实验结果证实在食管鳞状细胞癌，P16作为一种多肿瘤抑制基因，其蛋白表达与食管鳞状细胞癌的病理分级、淋巴结浸润、侵袭呈负相关，有淋巴结转移的P16的蛋白表达量明显低于无淋巴结转移的，P16随病理分级的升高，其表达量逐渐减少，而PCNA作为一种细胞增殖活性的标记物，恰恰相反，
其蛋白表达与食管鳞状细胞癌的病理分级、淋巴结浸润、侵袭呈正相关，其蛋白表达量在有淋巴结转移的明显高于无淋巴结转移的。PCNA则随食管癌细胞恶性程度的增高，其表达量逐渐增多，因此，测定P16及PCNA可能对鉴别ⅡA与ⅡB期食管鳞状细胞癌有指导作用,对早中期食管鳞状细胞癌的诊断，淋巴结浸润、侵袭及转移，恶性度的判定及治疗治疗有重要作用。
Objective: esophageal carcinoma is in the third of malignant tumor in the world, accounting for the second dead causes of malignant tumor in China, but the mechanism of cell malignant alteration is not clear yet. There are a family character in the ESCC's occuring, which shows that the heredity factor play a role in the occuring of ESCC. Recently, a few correlated gene or gene pitch have been found in the molecule biology, which may be correlated to the occuring or progression of ESCC. Moreover, we have had more knowledge about the effect of abnormal cell cycle regulatory in malignant tumor progression. It has been found that there are a variety of abnormal expression of regulatory factors in G1/S phrase, which is related to lymph node metastasize and clinicopathological feature of Esophageal Squamous-cell carcinoma (ESCC), in particularly, p16 has more correlation of lymph node metastasis and pathological grade in ESCC. P16 is located chromosome of 9p21, whose cDNA length is 960bp. P16 is also named more tumor suppressor, including four ankyrin configuration. Being a differential suppressor, it can combine with and stop CDK4 and CDK6 activity. The whole lengths of p16 are 8.5kb, including
three exon named E1、E2、E3 respectively, their length are E1 266bp、E2 307bp、E3 1166bp respectively, coding a protein together, whose comparative molecule quantum is 16 KD, that is p16 protein. Wild p16 protein has an active part, composed of four hook wrap space fabricate. It is viewed as frame gene controlling cell growth. P16 main function is suppressing the phosphation of Rb protein transmited by CDK4/CDK6, stopping cell entering into S phase from G1 phase. p16、CDK、cyclin and Rb are formed a feedback control circle line. When the gene deletion、mutation、and promoter CPG island aberrant methylation of p16 protein make p16 abnormal expression, it would lead the growing cell lost of control, cell transmission and carcinoalteration. CDK4 or CDK6 could make cell enter into G1 phase after combining with cyclinD1, while p16 protein amino-tail end have a place, which can compete with cyclinD1 for CDK4/CDK6, suppressing cyclinD1 enzyme's activity and making cyclinD1 lose its activity function and stop the growing cell. Proliferation cell nuclear antigen is a kind of acidity protein in cell nucleus, its molecule quality is 36000D. It is an assistant factor of DNA poly enzyme, participating in DNA synthesize and playing a role in cell cycle period. In the possess of DNA synthesize , PCNA expression start increasing in G1、reaching apex in S、rapidly decreasing in G2 and M phase. Therefore, PCNA is taken as a symbol of evaluating the cell proliferation activity, used as distinguishing carcinoma cell from normal cell. There is also a closed correlation between PCNA and lymph
node metastasis and pathological grades of ESCC. In this study, we explored the relationship of both p16 and PCNA with ESCC biological behavior by studying the expression of p16 and PCNA in protein lever in ESCC tissues. Exploring the relationship between both p16 and PCNA and esophageal carcinoma biology behavior, aiming at giving evidence in the diagnosing and treating of earlier stage and metaphase's esophageal carcinoma.
Methods: Immunohistochemistry analysis of p16 and PCNA proteins was carried out in paraffin-embedded section from 49 surgically resected ESCC’s in the affiliated second hospital, HeBei medical university, all specimen were divided into two groups: lymph node metastasis group(24) and non-lymph node metastasis group(25),in addition, all specimens were divided into three groups according to pathological grade: Ⅰ16, Ⅱ16, Ⅲ17. Macroscopically, all specimens are ESCC tissues (aged from30-78 years old, mean 56.9 years old). The data were expressed with positive cell labeled area ratio (percentage,%) and positive degree means. The results of correlating with p16 and PCNA are analyzed by using T test and one-way ANOVA in SPSS10.0.
Results: (1) The positive cell area ratio and positive degree means of p16 were significantly highe
引文
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26 Mori M, Kakeji Y, Adachi Y, et al. The prognostic significance of proliferating cell nuclear antigen in clinical gastric cancer. Surgery, 1993, 113(6): 683~90.
27 Mori M, Mimori K, Sadanaga N, et al. Polypoid carcinoma of the esophagus. Jpn J Cancer Res, 1994, 85(11): 1131~1136.
28 Dabrowski A, Szumilo J, Brajerski G, et al. Proliferating nuclear antigen(PCNA) as a prognostic factor of squamous cell carcinoma of the oesophagus. Ann Univ Mariae Curie Sklodowska[Med]2001, 56, 59~67.
29 Kinugasa S, Tachibana M, Hishikawa Y, et al. Prognosis significance of proliferating cell nuclear antigen (PCNA) in squmous cell carcinoma of the esophgus. Jpn J clin Oncol, 1996, 26(5):405~410.
30 Mouisaki Y, Shima S, Yoshizumi Y, et al. PCNA immunostaining combined with AgNOR staining in esophageal. squamous cell carcinoma to identify patients with a poor prognosis. Surg Today, 1995, 25(5): 389~395.