抑癌基因MMAC1在胆管癌中的表达及临床意义的研究
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摘要
胆管癌是常见的恶性肿瘤之一，近几年来其病例数有逐渐升高的趋势。随着现代影像学和外科技术的发展，对胆管癌的早期诊断、手术切除率、术后生存率均有所提高。但1、3、5年生存率分别为67％、22％、11％仍不十分理想，其原因除了与患者病情多属较晚期外，主要与胆管癌的生物学特性，特别是浸润和转移有关，高浸润和转移是胆管癌最重要的生物学特征，它除直接浸润和淋巴转移外，血管和神经周围间隙均是重要的转移途径。因此仅依靠单纯的手术切除、术后化疗等治疗方法很难达到理想的效果。近年来人们一直在寻求更为有效的治疗方法，基因治疗是最具希望，并且成为目前肿瘤治疗的研究热点之一，其前景广阔。多发性进展期癌突变基因(Mutated in multple advanced cancerl，MMAC1)又名第10号染色体同源丢失性磷酸酶—张力蛋白基因(phoshatase and tensin homology
deleted on chromosome ten，Pten)是一种多功能蛋白质。最近在人肿瘤易被更改的10q23染色体区被鉴定出来，因具有磷酸酶活性，它不仅可使酪氨酸、丝氨酸或苏氨酸的蛋白脱去磷酸，而且同时还能使磷脂酰肌醇通道的磷酸脂脱去磷酸。是现今发现的第一个具有磷酸酶活性的抑癌基因。该基因的肿瘤抑制作用是：可使酪氨酸、丝氨酸或苏氨酸的蛋白脱去磷酸，同时还能使磷脂酰肌醇通道的磷酸脂脱去磷酸；其磷酸酶蛋白活性能够抑制Ras／Mek／Erk通道以及FAK(灶性粘连激酶)通道，从而对侵袭机制中起重要作用的细胞与细胞外基质的相互作用产生影响；其脂质磷酸酶活性可阻断PI3K／Akt通道。引起细胞周期G1的停止并增加对细胞凋亡的敏感性；影响肿瘤细胞的形态和增殖。资料显示，MMAC1／Pten基因在各种不同的
第三军医大学硕士研究生论文
恶性肿瘤中的表达有突变和缺失，MMACI护ten基因表达异常见于胶质细
胞瘤、前列腺癌、乳癌、内膜样癌、恶性黑色素瘤、消化道肿瘤和肺癌等。
本实验研究应用免疫组织化学、原位杂交技术检测犯例胆管癌患者的胆管
癌石蜡标本、5例胆囊腺瘤标本、5例正常的胆管组织和QBC939胆管癌
细胞株，分别检测其MMACI肤en蛋白和mRNA的表达情况，旨在观察
MMAcl爪en基因的表达水平与肿瘤的病理类型、分化程度、肿瘤浸润转
移与临床、预后的关系，为胆管癌患者术后的预测和开展基因治疗提供依
据。
结果:32例胆管癌组织中，MMACI爪en蛋白阳性率64.0%，阳性反
应分布在胞浆中。QBC939胆管癌细胞株中MMACI八、en蛋白无表达。胆
管癌组织中M州[A Cl月吮en功丑NA阳性表达率73.6%，阳性信号位于胞浆中。
胆管癌细胞株(QBC939)中mRNA无表达。MMACI爪en蛋白及
MMACI用ten mRNA在胆管癌中的表达水平与患者性别、年龄、手术方式
以及肿瘤部位无明显相关性，但与组织分化程度、有无浸润转移、术后生
存时间明显相关，分化程度愈低，恶性程度高的其表达水平低、术后生存
时间短。有肿瘤转移的病例中其表达水平比无转移的病例表达水平低。
结论:胆管癌组织中存在着较高比例的MMACI爪en蛋白和
MMACI爪en mRNA阴性表达，说明在胆管癌的发生发展中，
MM人Cl爪en基因失活起着重要作用，两者的阳性表达均有一定的预后意
义，。对判断病人的预后有重要意义，同时为胆管癌的基因治疗提供依据。
Objective: The MMAC1/MMAC1/Pten gene is the first tumor suppressor encoding a phosphatase identified by far, whose mutation is found in multiple rumors. However, the clinical significance of this gene in cholangiocarcinoma remains unclear. So the purpose of our research aims to investigate the expression of MMAC1/ Pten gene in cholangiocarcinoma and its clinical significance.
Methods: Tissue samples were taken from 32 patients with cholangiocartinorna and 5 adenoma of gallbladder , fixed in formaldehyde and embedded with paraffin . 5 samples from normal bile duct tissues were also withdrawn. Protein and gene expression of MMAC1/ Pten were analyzed by irnmunohistochemistry and in situ hybridzation . Cholangiocarcinoma cell line Q939 was determined for the expression of MMAC 1/ Pten as well.
Results: MMAC1/ Pten protein and mRNA expression were positive not only in all 5 samples of normal bile duct tissues (5/5, 100% ), but also in all 5 cases of adenoma of gallbladder (5/5,100%).
Of the 32 cases of cholangiocarcinoma, MMAC1/ Pten protein expression were positive in 20 cases (64.00%), the positive staining was seen in the cytoplasm; there was no positive staining in cholangiocarcinoma cell line Q939.
73.6% of 32 cases of cholangiocarcinoma were MMAC1/ Pten mRNA positive, and MMAC1/ Pten mRNA was clearly observed in the cytoplasm of cholangiocarcinoma cells. No expression of MMAC1/ Pten mRNA was found
in cholangiocarcinoma cell line Q939 either.
Compared with normal bile duct tissue, the negative expression rates of MMAC1/ Pten mRNA and protein in cholangiocarcinoma were higher.
There was no correlation between the protein and mRNA expression of MMAC1/ Pten and such factors as sex, age in cholangiocarcinoma , but a significant correlation between the expression of MMAC1/ Pten and the differentiation, metastasis and postoperative prognosis were found. The less the MMAC1/ Pten protein and mRNA were, the poorer the differentiation and prognosis were. The positive rates of MMAC1/ Pten protein and mRNA expression in well differentiated adenocarcinoma were significantly higher than those in poorly differentiated adenocarcinoma (P<0.01), 40% vs50% and 72.73% vs 86.36% respectively. The levels of MMAC1/ Pten protein and mRNA in patients with metastasis were significantly higher than in those without metastasis.
Conclusions: Inactivation of MMAC1/ Pten gene may play an important role in the pathogenesis of cholangiocarcinoma. The positive expression of both protein and mRNA of MMAC1/ Pten may be related with favorable prognosis of cholangiocarcinoma.
deleted on chromosome ten，Pten)是一种多功能蛋白质。最近在人肿瘤易被更改的10q23染色体区被鉴定出来，因具有磷酸酶活性，它不仅可使酪氨酸、丝氨酸或苏氨酸的蛋白脱去磷酸，而且同时还能使磷脂酰肌醇通道的磷酸脂脱去磷酸。是现今发现的第一个具有磷酸酶活性的抑癌基因。该基因的肿瘤抑制作用是：可使酪氨酸、丝氨酸或苏氨酸的蛋白脱去磷酸，同时还能使磷脂酰肌醇通道的磷酸脂脱去磷酸；其磷酸酶蛋白活性能够抑制Ras／Mek／Erk通道以及FAK(灶性粘连激酶)通道，从而对侵袭机制中起重要作用的细胞与细胞外基质的相互作用产生影响；其脂质磷酸酶活性可阻断PI3K／Akt通道。引起细胞周期G1的停止并增加对细胞凋亡的敏感性；影响肿瘤细胞的形态和增殖。资料显示，MMAC1／Pten基因在各种不同的
第三军医大学硕士研究生论文
恶性肿瘤中的表达有突变和缺失，MMACI护ten基因表达异常见于胶质细
胞瘤、前列腺癌、乳癌、内膜样癌、恶性黑色素瘤、消化道肿瘤和肺癌等。
本实验研究应用免疫组织化学、原位杂交技术检测犯例胆管癌患者的胆管
癌石蜡标本、5例胆囊腺瘤标本、5例正常的胆管组织和QBC939胆管癌
细胞株，分别检测其MMACI肤en蛋白和mRNA的表达情况，旨在观察
MMAcl爪en基因的表达水平与肿瘤的病理类型、分化程度、肿瘤浸润转
移与临床、预后的关系，为胆管癌患者术后的预测和开展基因治疗提供依
据。
结果:32例胆管癌组织中，MMACI爪en蛋白阳性率64.0%，阳性反
应分布在胞浆中。QBC939胆管癌细胞株中MMACI八、en蛋白无表达。胆
管癌组织中M州[A Cl月吮en功丑NA阳性表达率73.6%，阳性信号位于胞浆中。
胆管癌细胞株(QBC939)中mRNA无表达。MMACI爪en蛋白及
MMACI用ten mRNA在胆管癌中的表达水平与患者性别、年龄、手术方式
以及肿瘤部位无明显相关性，但与组织分化程度、有无浸润转移、术后生
存时间明显相关，分化程度愈低，恶性程度高的其表达水平低、术后生存
时间短。有肿瘤转移的病例中其表达水平比无转移的病例表达水平低。
结论:胆管癌组织中存在着较高比例的MMACI爪en蛋白和
MMACI爪en mRNA阴性表达，说明在胆管癌的发生发展中，
MM人Cl爪en基因失活起着重要作用，两者的阳性表达均有一定的预后意
义，。对判断病人的预后有重要意义，同时为胆管癌的基因治疗提供依据。
Objective: The MMAC1/MMAC1/Pten gene is the first tumor suppressor encoding a phosphatase identified by far, whose mutation is found in multiple rumors. However, the clinical significance of this gene in cholangiocarcinoma remains unclear. So the purpose of our research aims to investigate the expression of MMAC1/ Pten gene in cholangiocarcinoma and its clinical significance.
Methods: Tissue samples were taken from 32 patients with cholangiocartinorna and 5 adenoma of gallbladder , fixed in formaldehyde and embedded with paraffin . 5 samples from normal bile duct tissues were also withdrawn. Protein and gene expression of MMAC1/ Pten were analyzed by irnmunohistochemistry and in situ hybridzation . Cholangiocarcinoma cell line Q939 was determined for the expression of MMAC 1/ Pten as well.
Results: MMAC1/ Pten protein and mRNA expression were positive not only in all 5 samples of normal bile duct tissues (5/5, 100% ), but also in all 5 cases of adenoma of gallbladder (5/5,100%).
Of the 32 cases of cholangiocarcinoma, MMAC1/ Pten protein expression were positive in 20 cases (64.00%), the positive staining was seen in the cytoplasm; there was no positive staining in cholangiocarcinoma cell line Q939.
73.6% of 32 cases of cholangiocarcinoma were MMAC1/ Pten mRNA positive, and MMAC1/ Pten mRNA was clearly observed in the cytoplasm of cholangiocarcinoma cells. No expression of MMAC1/ Pten mRNA was found
in cholangiocarcinoma cell line Q939 either.
Compared with normal bile duct tissue, the negative expression rates of MMAC1/ Pten mRNA and protein in cholangiocarcinoma were higher.
There was no correlation between the protein and mRNA expression of MMAC1/ Pten and such factors as sex, age in cholangiocarcinoma , but a significant correlation between the expression of MMAC1/ Pten and the differentiation, metastasis and postoperative prognosis were found. The less the MMAC1/ Pten protein and mRNA were, the poorer the differentiation and prognosis were. The positive rates of MMAC1/ Pten protein and mRNA expression in well differentiated adenocarcinoma were significantly higher than those in poorly differentiated adenocarcinoma (P<0.01), 40% vs50% and 72.73% vs 86.36% respectively. The levels of MMAC1/ Pten protein and mRNA in patients with metastasis were significantly higher than in those without metastasis.
Conclusions: Inactivation of MMAC1/ Pten gene may play an important role in the pathogenesis of cholangiocarcinoma. The positive expression of both protein and mRNA of MMAC1/ Pten may be related with favorable prognosis of cholangiocarcinoma.
引文
1 黄志强，肝胆外科的发展．中国实用外科杂志，2002，22(1)：1
2 Launonis B, Terblanche J, Lakehal M, et al. proximal bile duct cancer: high respectability rote and 5-year survival. Ann Surg, 1999, 230(2): 266
3 王曙光，韩本立，段恒春，等．肝外QBC939细胞系的建立。中华实验外科杂志。1997，17：245-246
4 Lerseh C, Classen M, palliative therapy of carcinomas of the biliary system.Med Clin, 1997, 92(7): 401
5 Launois B, Terblanche J, Lakehal M, et al. proximal bile duct cancer: high respectability rate and 5-year survival. Ann surg, 1999, 230(2): 266
6 Gerhards MF, van Gulid TM, de Wit LT, et al. Evaluation of morbidity and mortality after resection for hilar cholangiocarcinoma-a single center experience.Surgery, 2000, 127(4): 395
7 Steck PA, pershouse MA, Jasser SA, et al. Identification of candidate tumour suppressor gene; MMAC1. at chromosome 10q23. 3 that is mutated advanced cancers. Nat Genet: 1997; 15: 356-362
8 Lie J, Yen C, Law D, et al. PTEN, a putative protein tyrosine phosphatese gene mutated in human brain, breast, and prostate cancer. Science, 1997,275: 1943-1945
9 Lie DM, Sun H. TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatese regulated by transforming growth factor β. Cancer Res, 1997, 57: 2124-2126
10 Tsou HC, Ping XL, Xie XX, et al. The genetic basis of Cowden's syndrome;
three novel mutation in PTEN/MMAC1/TEP1. Human Genet, 1998, 102: 467-468
11 张晋夏，吕怀盛，张雪．胃癌组织中PTEN基因和蛋白表达的研究．宁夏医学杂志，1999，21：451-453
12 张尚福，苏学英等．肺癌中PTEN蛋白的表达．中华病理学杂志，2000， 29：220-221
13 张雷，刘鸿瑞等．PTEN／MMAC1／TEP1在肺癌中的丢失和失活．中华病理学杂志，2000，29：85-88
14 张利能，俞强，贺建宇等．人肝细胞癌中PTEN基因的DNA杂交及突变分析．上海医科大学学报，2000，27(1)：1—3
15 肖绍文，谢小薰，陈维平等．脑肿瘤中PTEN／MMAC1／TEP1．蛋白表达，中国肿瘤，2000，9：323-324
16 谢小薰，肖绍文．肿瘤抑制基因PTEN／MMAC1．实用癌症杂志，1999，3：237-238。
17 Davies MPA, Gibbs FEM, Halliwell N, et al. Mutation in the PTEN/MMAC1 gene in archial low grade and high grade gliomas, Brithsh J Cancer, 1999, 79(9/10): 1532-1533.
18 Gonxalez-Aulaeta M, et al. Methylation of the 5, CPG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing, Cancer Res, 1995, 55: 4531-4535
2 Launonis B, Terblanche J, Lakehal M, et al. proximal bile duct cancer: high respectability rote and 5-year survival. Ann Surg, 1999, 230(2): 266
3 王曙光，韩本立，段恒春，等．肝外QBC939细胞系的建立。中华实验外科杂志。1997，17：245-246
4 Lerseh C, Classen M, palliative therapy of carcinomas of the biliary system.Med Clin, 1997, 92(7): 401
5 Launois B, Terblanche J, Lakehal M, et al. proximal bile duct cancer: high respectability rate and 5-year survival. Ann surg, 1999, 230(2): 266
6 Gerhards MF, van Gulid TM, de Wit LT, et al. Evaluation of morbidity and mortality after resection for hilar cholangiocarcinoma-a single center experience.Surgery, 2000, 127(4): 395
7 Steck PA, pershouse MA, Jasser SA, et al. Identification of candidate tumour suppressor gene; MMAC1. at chromosome 10q23. 3 that is mutated advanced cancers. Nat Genet: 1997; 15: 356-362
8 Lie J, Yen C, Law D, et al. PTEN, a putative protein tyrosine phosphatese gene mutated in human brain, breast, and prostate cancer. Science, 1997,275: 1943-1945
9 Lie DM, Sun H. TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatese regulated by transforming growth factor β. Cancer Res, 1997, 57: 2124-2126
10 Tsou HC, Ping XL, Xie XX, et al. The genetic basis of Cowden's syndrome;
three novel mutation in PTEN/MMAC1/TEP1. Human Genet, 1998, 102: 467-468
11 张晋夏，吕怀盛，张雪．胃癌组织中PTEN基因和蛋白表达的研究．宁夏医学杂志，1999，21：451-453
12 张尚福，苏学英等．肺癌中PTEN蛋白的表达．中华病理学杂志，2000， 29：220-221
13 张雷，刘鸿瑞等．PTEN／MMAC1／TEP1在肺癌中的丢失和失活．中华病理学杂志，2000，29：85-88
14 张利能，俞强，贺建宇等．人肝细胞癌中PTEN基因的DNA杂交及突变分析．上海医科大学学报，2000，27(1)：1—3
15 肖绍文，谢小薰，陈维平等．脑肿瘤中PTEN／MMAC1／TEP1．蛋白表达，中国肿瘤，2000，9：323-324
16 谢小薰，肖绍文．肿瘤抑制基因PTEN／MMAC1．实用癌症杂志，1999，3：237-238。
17 Davies MPA, Gibbs FEM, Halliwell N, et al. Mutation in the PTEN/MMAC1 gene in archial low grade and high grade gliomas, Brithsh J Cancer, 1999, 79(9/10): 1532-1533.
18 Gonxalez-Aulaeta M, et al. Methylation of the 5, CPG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing, Cancer Res, 1995, 55: 4531-4535