p53、p63、p73基因在舌癌组织中的蛋白表达及意义
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摘要
前言
据病理资料统计分析，口腔颌面部恶性肿瘤约占全身恶性肿瘤的8.2％，它们多数来源于上皮组织，尤以鳞状上皮细胞癌最为常见。
p53基因是一种抑癌基因，在人类肿瘤中p53基因常有突变，在头颈部鳞状细胞癌中(HNSCC)被研究肿瘤的40％-50％已证明与这种基因的改变有关。p53家族的新成员p63与p73基因与p53在结构上具有很高的同源性，所以它们的转录与凋亡功能与p53很相似，被认为可能是候选的肿瘤抑制基因。然而由于p63与p73基因可以形成很多蛋白异构体，在很多肿瘤中过表达及罕见的基因突变，二者也可能是一种癌基因。它们在肿瘤中的作用及与p53的关联有待进一步的探讨。
在我国舌癌占整个HNSCC的40％以上。国内外对p53、p63、p73在舌鳞癌中作用的研究报道极少。本实验拟用免疫组化S-P染色法检测41例舌鳞癌组织中p53、p63、p73的表达情形，探讨其与临床病理特性的关系，为舌癌的辅助诊断及基因治疗提供参考依据。
方法
1．材料：收集中国医科大学第一附属医院口腔颌面外科1995年—2002年舌体鳞癌及舌鳞状上皮非典型性增生存档蜡块标本44例。其中41例为舌体鳞状细胞癌，术前未行化疗及放疗，标本按性别，年龄，UICC标准肿瘤分期(Ⅰ、Ⅱ、Ⅲ、Ⅳ)，肿瘤分化程度(高、中、低)及是否有淋巴结转移等进行分类。男27例，女14例，年龄范围30—68岁，平均50.59岁。Ⅰ—Ⅱ期13例，Ⅲ—Ⅳ期28例。高分化30例，中分化7例，低分化4例。区域性淋巴结转移12例。
对照组17例，其中上皮非典型性增生3例，另外14例源于舌癌癌旁正常粘膜组织经HE染色后确认。
2．实验试剂：一抗p63(4A4+63P02)鼠抗人单克隆抗体、一抗P53(DO—7)单克隆抗体、SP试剂盒、DAB显色剂，均购自福州迈新公司；一抗
夕3(H一刁9)兔抗人多克隆抗体，购自北京中山公司，其稀释度1:100。
3.采用免疫组织化学S一P染色法
(l)主要步骤:蜡块标本制备4卿连续切片，脱蜡水化后，按说明书进
行，必3、p73需经柠檬酸高温高压组织抗原修复(PH二6.0)。一抗户3、两3
保湿盒内37“C下孵育2小时，夕3 4OC过夜。
(2)结果判定:两名病理科医师分别描绘记录结果。户3、两3阳性细胞
为棕黄色，价3染色阳性细胞为棕红色，均定位于细胞核。高倍显微镜下观
察10个视野计数，按阳性细胞所占比例分为:核无染色或阳性细胞数<
10%为(一)，阳性细胞数10%一50%为(+)，51%一75%为(++)，>
75%为(+++)。
(3)统计学分析:在SPSsl 1 .5统计软件中进行，采用XZ检验和四格表
确切概率法，显著性差异P<0.05有意义。
结果
1.正常舌鳞状上皮及不典型增生组织中户3、p63、夕3蛋白分布:p53
蛋白未见阳性染色;两3、夕3蛋白显色较弱，低表达。P63蛋白阳性表达主
要位于上皮基底层并向上到少部分棘细胞层的细胞中，在基底层较为密集，
夕3蛋白阳性表达主要在基底层的细胞中，少部分细胞质同时呈弱棕红色。
2.舌鳞癌中p53、两3、夕3蛋白分布:可见肿瘤细胞阳性反应程度增
加。p53蛋白染色细胞核呈棕黄色，弥漫性分布;两3、p73蛋白大多为弥散
性或局灶性分布，在癌巢中染色细胞核主要分布于癌巢的外周。
3.免疫组化染色统计分析:舌癌中两3、夕3阳性表达率(分别为
90.24%、65.85%)与二者在正常舌鳞状上皮及不典型增生组织中的表达率
(分别为29.41%、17.64%)比较有显著性差异(P<0.05)。户3、P63、夕3
的蛋白表达与舌癌患者的性别、年龄、临床分期、分化程度、颈淋巴结转移等
均无关(P>0.05)。41例舌癌组织中，两3与户3表达一致者23例，P63与
p73表达一致者29例，p53与夕3表达一致者19例，P53、的3、夕3蛋白表达
两两间无明显相关(P>0.05)，P63在舌癌中表达的阳性率与夕3及P53相
比有统计学意义(P<0.05)。
结论
1.两3、价3在舌癌中有较高水平表达。
2.两3、户3在舌癌的发生发展中起着重要的作用。
3.在舌癌中户3、颐3、夕3的表达与舌鳞癌临床病理特性无关。
4.p53、两3、夕3在舌癌中的表达两两间无相关性，三者在舌癌中的作
用是相互独立的。
5.两3在舌癌中起着癌基因的作用，它可以作为一种有用的舌癌检测
的标志物。
Based on the pathological statistics, maxillofacial and oral malignant tumors were almost 8% of the total malignant tumors in our country. Most of them are epidermal origin and squamous cell carcinoma is the most common caner.
p53 is a tumor suppressor gene. It is the most common genetic change found in human tumors. In head and neck squamous cell carcinoma(HNSCC) , 40 -50% of the tumors studied have shown a mutation in this gene. Two new members of the p53 gene family, p63 and p73 share considerable structural ho-mologies with the p53, so their transcriptional and apoptotic functions are similar to those of p53. It has been suggested that they may be manifest tumor suppressor gene. However, in contrast to p53 ,p63 and p73 give rise to multiple functionally distinct protein isoforms as a result of alternative splicing and the use of various promoters , their over expression and exceeding rare mutations in a number of tumors contraindicate this hypothesis of tumor suppressor gene. Their roles and the relationship with p53 in tumors need our next investigation.
This study will perform immunohistochemistry studies for p53, p63 and p73 protein status to determine their differential expression in carcinoma of tongue as well as their clinicopathological significance.
Materials and Methods
1. Tissue specimens: A total of 44 tissue speciments were collected from the
archives of 1995 to 2002 in the Department of oral and maxillofacial surgery of The First Affiliated Hospital China Medical University. Among them,41 cases were squamous cell carcinoma which occurred on the lateral border of anterior 2/3 of the tongue and did not receive any chemotherapy or radiotherapy before . Tumor samples were assorted by sex, age, grade, stage and neck lymph node metastasis. <
The control group included 3 dysplastic lesions and 14 tissues of matches normal epithelium adjacent to the tumor . All of tissues were defined by HE staining.
2. ^Reagent: The primary antibodies p53 ( mouse monoclonal antibody, DO -7) and p63(mouse monoclonal antibody, 4A4 +63P02) ,SP kit and DAB developer were bought from Fuzhou Maixin. The primary antibody p73 ( rabbit polyclonal antibody,H -79. 1 ;100) was bought Peking Zhongshan.
3. Immunohistochemical staining
(1) The important procedure: Immunohistochemical technique was used. For p73 and p53, antigen retrieval was performed by pressure cooking in a PH 6 citrate buffer. p63 was not . The slides were incubated(p73,overnight at 4 ; p53 and p63, 2 hr at room temperature) , according to the manufacturer' s information.
(2) Standard; Two pathologists divided intensity of stainability into . The stained cell nuclei in 10 -50% were + ,51 ~75% were + + , > 75% were
+ + + and below 10% or unstained nuclei were -.
(3 ) Statistical analysis. All data were analyzed using the X2 test and Fisher s Exact Test in SPSS11. 5 . P value <0.05 was required for significance.
Experimental results
1. In normal and hyperplastic epithelia: there were no p53 - positive cells. p63 and p73 staining cells were slight and reduced expression and located on the basal and parabasal layers.
2. In 41 carcinomas, the staining positive cells increased in number and intensity for three markers. The staining was diffuse or localized peripheral cells of
tumor nests for p63 and p73.
3. Statistical analysis of immunohistochemical stainability: The positive expression rate of p63 and p73 protein was significantly higher in carcinomas of tongue(respectively 90.24% and 65. 85% ) than in normal and hyperplastic epi-thelia ( respectively 29.41 % and 17.64% ) ( P < 0.05 ). There was no significant correlation between p63 or p73 protein expression and the different clinico-pathological characteristics ( sex, age, grade stage and neck lymph node matas-tasis). Concordant p63 and p73 expressions were observed in 29 tumors, for p53 and p73 were 19, p53 and p63 were 23. There were no significant correlation each other( P > 0.05). The positive expression rate between p63 and p73 or p53 protein has statistica
据病理资料统计分析，口腔颌面部恶性肿瘤约占全身恶性肿瘤的8.2％，它们多数来源于上皮组织，尤以鳞状上皮细胞癌最为常见。
p53基因是一种抑癌基因，在人类肿瘤中p53基因常有突变，在头颈部鳞状细胞癌中(HNSCC)被研究肿瘤的40％-50％已证明与这种基因的改变有关。p53家族的新成员p63与p73基因与p53在结构上具有很高的同源性，所以它们的转录与凋亡功能与p53很相似，被认为可能是候选的肿瘤抑制基因。然而由于p63与p73基因可以形成很多蛋白异构体，在很多肿瘤中过表达及罕见的基因突变，二者也可能是一种癌基因。它们在肿瘤中的作用及与p53的关联有待进一步的探讨。
在我国舌癌占整个HNSCC的40％以上。国内外对p53、p63、p73在舌鳞癌中作用的研究报道极少。本实验拟用免疫组化S-P染色法检测41例舌鳞癌组织中p53、p63、p73的表达情形，探讨其与临床病理特性的关系，为舌癌的辅助诊断及基因治疗提供参考依据。
方法
1．材料：收集中国医科大学第一附属医院口腔颌面外科1995年—2002年舌体鳞癌及舌鳞状上皮非典型性增生存档蜡块标本44例。其中41例为舌体鳞状细胞癌，术前未行化疗及放疗，标本按性别，年龄，UICC标准肿瘤分期(Ⅰ、Ⅱ、Ⅲ、Ⅳ)，肿瘤分化程度(高、中、低)及是否有淋巴结转移等进行分类。男27例，女14例，年龄范围30—68岁，平均50.59岁。Ⅰ—Ⅱ期13例，Ⅲ—Ⅳ期28例。高分化30例，中分化7例，低分化4例。区域性淋巴结转移12例。
对照组17例，其中上皮非典型性增生3例，另外14例源于舌癌癌旁正常粘膜组织经HE染色后确认。
2．实验试剂：一抗p63(4A4+63P02)鼠抗人单克隆抗体、一抗P53(DO—7)单克隆抗体、SP试剂盒、DAB显色剂，均购自福州迈新公司；一抗
夕3(H一刁9)兔抗人多克隆抗体，购自北京中山公司，其稀释度1:100。
3.采用免疫组织化学S一P染色法
(l)主要步骤:蜡块标本制备4卿连续切片，脱蜡水化后，按说明书进
行，必3、p73需经柠檬酸高温高压组织抗原修复(PH二6.0)。一抗户3、两3
保湿盒内37“C下孵育2小时，夕3 4OC过夜。
(2)结果判定:两名病理科医师分别描绘记录结果。户3、两3阳性细胞
为棕黄色，价3染色阳性细胞为棕红色，均定位于细胞核。高倍显微镜下观
察10个视野计数，按阳性细胞所占比例分为:核无染色或阳性细胞数<
10%为(一)，阳性细胞数10%一50%为(+)，51%一75%为(++)，>
75%为(+++)。
(3)统计学分析:在SPSsl 1 .5统计软件中进行，采用XZ检验和四格表
确切概率法，显著性差异P<0.05有意义。
结果
1.正常舌鳞状上皮及不典型增生组织中户3、p63、夕3蛋白分布:p53
蛋白未见阳性染色;两3、夕3蛋白显色较弱，低表达。P63蛋白阳性表达主
要位于上皮基底层并向上到少部分棘细胞层的细胞中，在基底层较为密集，
夕3蛋白阳性表达主要在基底层的细胞中，少部分细胞质同时呈弱棕红色。
2.舌鳞癌中p53、两3、夕3蛋白分布:可见肿瘤细胞阳性反应程度增
加。p53蛋白染色细胞核呈棕黄色，弥漫性分布;两3、p73蛋白大多为弥散
性或局灶性分布，在癌巢中染色细胞核主要分布于癌巢的外周。
3.免疫组化染色统计分析:舌癌中两3、夕3阳性表达率(分别为
90.24%、65.85%)与二者在正常舌鳞状上皮及不典型增生组织中的表达率
(分别为29.41%、17.64%)比较有显著性差异(P<0.05)。户3、P63、夕3
的蛋白表达与舌癌患者的性别、年龄、临床分期、分化程度、颈淋巴结转移等
均无关(P>0.05)。41例舌癌组织中，两3与户3表达一致者23例，P63与
p73表达一致者29例，p53与夕3表达一致者19例，P53、的3、夕3蛋白表达
两两间无明显相关(P>0.05)，P63在舌癌中表达的阳性率与夕3及P53相
比有统计学意义(P<0.05)。
结论
1.两3、价3在舌癌中有较高水平表达。
2.两3、户3在舌癌的发生发展中起着重要的作用。
3.在舌癌中户3、颐3、夕3的表达与舌鳞癌临床病理特性无关。
4.p53、两3、夕3在舌癌中的表达两两间无相关性，三者在舌癌中的作
用是相互独立的。
5.两3在舌癌中起着癌基因的作用，它可以作为一种有用的舌癌检测
的标志物。
Based on the pathological statistics, maxillofacial and oral malignant tumors were almost 8% of the total malignant tumors in our country. Most of them are epidermal origin and squamous cell carcinoma is the most common caner.
p53 is a tumor suppressor gene. It is the most common genetic change found in human tumors. In head and neck squamous cell carcinoma(HNSCC) , 40 -50% of the tumors studied have shown a mutation in this gene. Two new members of the p53 gene family, p63 and p73 share considerable structural ho-mologies with the p53, so their transcriptional and apoptotic functions are similar to those of p53. It has been suggested that they may be manifest tumor suppressor gene. However, in contrast to p53 ,p63 and p73 give rise to multiple functionally distinct protein isoforms as a result of alternative splicing and the use of various promoters , their over expression and exceeding rare mutations in a number of tumors contraindicate this hypothesis of tumor suppressor gene. Their roles and the relationship with p53 in tumors need our next investigation.
This study will perform immunohistochemistry studies for p53, p63 and p73 protein status to determine their differential expression in carcinoma of tongue as well as their clinicopathological significance.
Materials and Methods
1. Tissue specimens: A total of 44 tissue speciments were collected from the
archives of 1995 to 2002 in the Department of oral and maxillofacial surgery of The First Affiliated Hospital China Medical University. Among them,41 cases were squamous cell carcinoma which occurred on the lateral border of anterior 2/3 of the tongue and did not receive any chemotherapy or radiotherapy before . Tumor samples were assorted by sex, age, grade, stage and neck lymph node metastasis. <
The control group included 3 dysplastic lesions and 14 tissues of matches normal epithelium adjacent to the tumor . All of tissues were defined by HE staining.
2. ^Reagent: The primary antibodies p53 ( mouse monoclonal antibody, DO -7) and p63(mouse monoclonal antibody, 4A4 +63P02) ,SP kit and DAB developer were bought from Fuzhou Maixin. The primary antibody p73 ( rabbit polyclonal antibody,H -79. 1 ;100) was bought Peking Zhongshan.
3. Immunohistochemical staining
(1) The important procedure: Immunohistochemical technique was used. For p73 and p53, antigen retrieval was performed by pressure cooking in a PH 6 citrate buffer. p63 was not . The slides were incubated(p73,overnight at 4 ; p53 and p63, 2 hr at room temperature) , according to the manufacturer' s information.
(2) Standard; Two pathologists divided intensity of stainability into . The stained cell nuclei in 10 -50% were + ,51 ~75% were + + , > 75% were
+ + + and below 10% or unstained nuclei were -.
(3 ) Statistical analysis. All data were analyzed using the X2 test and Fisher s Exact Test in SPSS11. 5 . P value <0.05 was required for significance.
Experimental results
1. In normal and hyperplastic epithelia: there were no p53 - positive cells. p63 and p73 staining cells were slight and reduced expression and located on the basal and parabasal layers.
2. In 41 carcinomas, the staining positive cells increased in number and intensity for three markers. The staining was diffuse or localized peripheral cells of
tumor nests for p63 and p73.
3. Statistical analysis of immunohistochemical stainability: The positive expression rate of p63 and p73 protein was significantly higher in carcinomas of tongue(respectively 90.24% and 65. 85% ) than in normal and hyperplastic epi-thelia ( respectively 29.41 % and 17.64% ) ( P < 0.05 ). There was no significant correlation between p63 or p73 protein expression and the different clinico-pathological characteristics ( sex, age, grade stage and neck lymph node matas-tasis). Concordant p63 and p73 expressions were observed in 29 tumors, for p53 and p73 were 19, p53 and p63 were 23. There were no significant correlation each other( P > 0.05). The positive expression rate between p63 and p73 or p53 protein has statistica
引文
1．邱蔚六，主编．口腔颌面外科学．第四版．北京：人民卫生出版社，2000．212—213．
2. Weber A, Bellmann U, Bootz F, et al. Expression of p53 and its homologues in primary and recurrent squamous cell carcinomas of the head and neck. Int J Cancer, 2002, 99(1): 22-28.
3. Nylander K, Dabelsteen E, Hall AP. The p53 molecule and its prognostic role in squamous cell carcinomas of the head and neck. J Oral Pathol Med, 2000, 29(9): 413-425.
4. Kaghad M, Bonnet H, Yang A, et al. Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell, 1997, 9(4): 809-819.
5. Zhu JH, Jiang JY, Zhou WJ, et al. The potential Tumor suppressor p73 differentially regulates cellular p53 Target Genes. Cancer Res, 1998, 58(15): 5061-5065.
6. Sheikh MS, Fomace AJ Jr. Role of p53 family members in apoptosis. J Cell Physiol, 2000, 182(2): 171-181.
7. Yang A, Kaghad M, Wang Y, et al. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative acticities. Mol Cell, 1998, 2(3): 305-316.
8. Hibi K, Trink B, Patturajan M. et al. AIS is an oncogene amplified in squamous cell carcinoma. Proc Natl Acad Sei USA, 2000, 97(10): 5462-5467.
9. Zaika AI, Kovalev S, Marchenko ND, et al. Overexpression of the wild type p73 gene in breast tissues and new lines. Canoe Res, 1999, 59(13): 3257-3263.
10. Faridoni-Laurens L, Bosq J, Janot F, et al. p73 expression in basal layers of head and need squamous spithelium: a role in differentiation and carcinogenesis in eoneer with p53 and p63? Oneogene, 2001, 20(38): 5302-5312.
11. Chi SG, Chang SG, Lee SJ, et al. Elevated and biallelie expression of p73 is associated with progression of human bladder cancer. Cancer Res, 1999, 59(12): 2791-2793.
12. Ng SW, Yiu GK, Liu Y, et al. Analysis of p73 in human borderline and invasive ovarian tumor. Oneogene, 2000, 19(6): 1885-1890.
13. Yokomizo A, Mai M, Tindall DJ, et al. Overexpression of the wild type p73 gene in. human bladder cancer. Oneogene, 1999, 18(8): 1629-1633.
14. Mai M, Yokomizo A, Qian C, et al. Activation of p73 silent allele in lung cancer. Cancer Res, 1998, 58(11): 2347-2349.
15. Weber A, Langhanki L, Sehutz A, et al. Expression profiles of p53, p63, and p73 in benign salivary gland tumors. Virchows Arch, 2002, 441(5): 428-436.
16. Crook T, Nicholls JM, Brooks L, et al. High level expression of△N-p63: a mechanism for the inactivation of p53 in undifferentiated nasopharyngeal carcinoma. Oneogene, 2000, 19(30): 3439-3444.
17. Hu H, Xia SH, Li AD, et al. Elevated expression of p63 protein in human esophageal squamous cell carcinomas. Int J Cancer, 2002, 102(6): 580-583.
18．邱蔚六．中国口腔颌面肿瘤学进展50年．中华口腔医学杂志，2002，37(3)：161-164．
19．陈竺，主编．医学遗传学．第一版．北京：人民卫生出版社，2001．202-203．
20. Chol HR, John G, Batsakis, et al. Differential expression of p53 gene family Members p63 and p73 in head and neck squamous Tumorigenesis. Hum pathol, 2002, 33(2): 158-164.
21. Yang A, Walker N, Bronson R, et al. p73 -deficient mice have neurological pheromonal and inflammatory defects but lack spontaneous tumours. Nature(Lond), 2000, 404(6773): 99-103.
22. Roth J, Konig C, Wienzek S, et al. Inactivation of p53 but not p73 by abenovirus type 5 E1B 55 -kilodalton and E4 34 -kilodalton oncoproteins. J Virol, 1998, 72(11): 8510-8516.
23. Dobbelstein M, Roth J. The large T antigen of simian virus 40 binds and in-
activates p53 but not p73. J Gen Virol, 1998, 79(12): 3079-3083.
24. Marin MC, Jost C, Irwin M, et al. Viral oncoproteins disvriminate between p53 and the p53 homolog p73. Mol Cell Biol, 1998, 18(10): 6316-6324.
25. E1-Naggar AK, Lai S, Clayman GL, et al. p73 gene alterations and expression in primary oral and Laryneal Squamouscarcinomas. Carcinogenesis, 2001, 22(5): 729-735.
26．张增锋，韦敏怡，浣孝强，等．鼻咽癌组织中p73蛋白表达及其与临床病理特征的关系．实用癌症杂志，2002，17(4)：380-382．
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18．邱蔚六．中国口腔颌面肿瘤学进展50年．中华口腔医学杂志，2002，37(3)：161-164．
19．陈竺，主编．医学遗传学．第一版．北京：人民卫生出版社，2001．202-203．
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26．张增锋，韦敏怡，浣孝强，等．鼻咽癌组织中p73蛋白表达及其与临床病理特征的关系．实用癌症杂志，2002，17(4)：380-382．
27. Chen YK, Huse SS, Lira LM. Differential expression of p53, p63 and p73 proteins in human buccal squamous-cell carcinomas. Clin Otolaryngol. 2003 Oct, 28(5): 451-455.
28. Kaelin WG Jr. The p53 gene family. Oneogene, 1999, 18(53): 7701-7705.
29. Osada M, Ohba M, kauahara C, et al. Cloning and functional analysis of human p51, which structurally and functionally resembles p53. Nat Ned, 1998, 4(7): 839-843
30. Both J, Dobbelstein M. Failure of viral oncoproteins to target the p53 -homologue p51A. J Gen Virol, 1999, 80(12): 3251-3255.
31. Hagiwara K, McMenamin MG, Miura K et al. Mutational analysis of the p63/p73L/p51/p40/CUSP/KET gene in human cancer cell lines using intronic primers. Cancer Res, 1999, 59(17): 4165-4169.
32. Bilal H, Handra-Luca A, Bertrand JC, et al. p63 is expressed in basal and myoepithelial cells of human normal and tumor salivary gland tissues. J Histochem Cytochem, 2003, 51(2): 133-139.
33. Hall PA, Campbell SJ, O'neill M, et al. Expression of the p53 homologue p63α and △Np63α in normal and neoplastic cells. Carcinogenesis, 2000, 21(2): 153-160.
34. Nylander K, Coates PJ, Hall PA. Characterization of the expression pattern of p63α and △Np63α in benign and malignant oral epithelial lesions, Int J Cancer, 2000, 87(3): 368-372.
35. Yang A, Schweitzer R, Sun D, et al. p63 is essential for regenerative prolif-
eration in limb, craniofacial and epithelial developmem. Nature (Lond), 1999, 398(6729): 714 -718.
36. Mills AA, Zheng B, Wang XJ, et al. p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature(Lond), 1999, 398(6729): 708-713.