肝癌 Open Access
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2004-04-15; 12(4): 782-784
在线出版日期: 2004-04-15. doi: 10.11569/wcjd.v12.i4.782
肝癌组织HSP70和caspase 3的表达意义
彭绍华, 邓虹, 冯德云, 郑晖
彭绍华, 邓虹, 湖南师范大学医学院病理学教研室 湖南省长沙市 410006
冯德云, 郑晖, 中南大学基础医学院病理学系 湖南省长沙市 410078
彭绍华, 男, 1963-10-14生, 湖南省南县人, 汉族, 副教授, 医学硕士. 主要从事肿瘤病理的研究.
通讯作者: 彭绍华, 410006, 湖南省长沙市咸嘉湖, 湖南师范大学医学院病理学教研室. pengshaohua2004@sina.com
电话: 0731-8630309
收稿日期: 2003-08-28
修回日期: 2004-01-01
接受日期: 2004-02-01
在线出版日期: 2004-04-15

目的: 研究热休克蛋白70和caspase 3在肝细胞癌及癌旁肝组织中的表达及其临床意义.

方法: 利用免疫组织化学检测70例肝细胞癌及其癌旁肝组织中HSP70和caspase 3蛋白的表达.

结果: HCC中HSP70的阳性率和阳性强度明显高于癌旁肝组织(68.6% vs 31.4%, P<0.01), 而caspase3蛋白的阳性率和阳性强度明显低于癌旁肝组织(17.1% vs 35.7%, P<0.01). 在HCC中HSP70和caspase3蛋白的表达强度与HCC的分化程度和肿瘤的大小明显相关, 分化愈差和肿瘤愈大HSP70表达愈强(分别为: F = 5.219和5.421, P均<0.01)而caspase3蛋白表达愈弱(分别为F = 5.944和4.571, P均<0.01). 统计学分析显示在HCC及其癌旁肝组织中HSP70和caspase 3蛋白的表达呈明显负相关(分别为: r = -0.4 126和 -0.5237, P均<0.01).

结论: 本文结果提示在HCC发生过程中HSP70的表达可能通过促进细胞的转化和增生及抑制细胞的凋亡, 使HCC呈现无限制的生长及恶性程度不断增高; 检测HSP70有望能成为判断HCC预后的重要指标之一.

关键词: N/A
引文著录: 彭绍华, 邓虹, 冯德云, 郑晖. 肝癌组织HSP70和caspase 3的表达意义. 世界华人消化杂志 2004; 12(4): 782-784
Expression of HSP 70 and caspase 3 and their significance in hepatocellular carcinoma tissues
Shao-Hua Peng, Hong Deng, De-Yun Feng, Hui Zheng
Shao-Hua Peng, Hong Deng, Department of Pathology, Hunan Normal University Medical College, Changsha 410006, Hunan Province, China
De-Yun Feng, Hui Zheng, Department of Pathology, Xiangya School of Medicine, Central South University, Changsha 410078, Hunan Province, China
Correspondence to: Shao-Hua Peng, Department of Pathology, Hunan Normal University Medical College, Changsha 410006, Hunan Province, China. pengshaohua2004@sina.com
Received: August 28, 2003
Revised: January 1, 2004
Accepted: February 1, 2004
Published online: April 15, 2004

AIM: To investigate the expression of heat shock protein 70 (HSP70) and caspase 3 protein and their clinical significance in hepatocellular carcinomas and surrounding liver tissues.

METHODS: The expression of HSP70 and caspase 3 protein were detected by immunohistochemistry in hepatocellular carcinomas (HCC) and their surrounding liver tissues.

RESULTS: The positive rate and intensity of HSP70 in HCCs were significantly higher than those in pericarcinomatous liver tissues (68.6% vs 31.4%, P < 0.01), and these of caspase protein were significantly lower (17.1% vs 35.7%, P < 0.01). The expression level of HSP70 and caspase protein in HCCs was remarkably related to differentiation degree and tumor size of HCCs, and the poorer differentiation, the stronger the expression of HSP70 (F = 5.219 and 5.421 respectively, P < 0.01), the weaker the expression of caspase 3 protein (F = 5.944 and 4.571 respectively, P < 0.01). The correlation analysis indicated that there was a negative relationship between expression of HSP70 and caspase protein in HCC and their surrounding liver tissues (r = 0.4 126 and 0.5 237 respectively, P < 0.01).

CONCLUSION: The expression of HSP70 may make uncontrolled growth and unceasingly increased malignant degree of HCC by accelerating cell transformation and proliferation and inhibiting apoptosis. HSP70 may be an important marker for evaluation of prognosis in patients with HCC.

Key Words: N/A
0 引言

在我国及日本肝细胞癌(HCC)是一种死亡率非常高的高度恶性肿瘤[1-10], 其发生与肝炎病毒感染密切相关[11-14], 并呈多步骤多阶段的特性[15-16]. 尽管HCC的研究和治疗在近年取得了很大的进展[17-26], 但其发生的分子机制及比较特异的诊断标记物和治疗靶分子均未得到明确的鉴定. 最近Chuma et al[27]利用基因芯片技术研究发现热休克蛋白70(HSP70)可能是一种鉴别早期HCC的有效分子标记物. HSP70是一组重要应激蛋白. 在肿瘤细胞中表达异常, 能与癌基因、抑癌基因产生结合, 并与肿瘤细胞的细胞周期调控、增生、凋亡、分化、多药耐药、肿瘤免疫以及肿瘤细胞的发生发展密切相关[28], caspase 3是凋亡信号转导通路中的重要执行分子, 其激活后导致细胞的不可逆性凋亡[29]. 我们利用组织芯片技术和免疫组织化学方法检测HSP70和caspase 3在HCC及其癌旁肝组织中的表达, 并探讨二者的相关性及其临床意义.

1 材料和方法
1.1 材料

1996-01/2003-02湘雅医院肝胆外科手术切除的HCC标本70例(均附癌旁肝组织)及胆囊炎患者切除的正常肝组织10例. 所有标本经40 g/L甲醛固定, 常规石蜡包埋、切片、病理学确诊. 在70例HCC中男53例, 女17例, 平均年龄53.4 (27-68岁); 肿瘤直径小于或等于5 cm者25例; 5.1-10 cm者29例; 大于10 cm者16例. 其中肝内有多个肿瘤结节者19例; 患者术前均未做过任何放疗和化疗. HCC组织根据Edmondson标准分级, 其中Ⅰ级11例, Ⅱ级31例, Ⅲ级23例, Ⅳ级5例. 所有癌旁肝组织均有不同程度的肝纤维化和肝硬化, 其中有非典型增生者22例. 抗HSP70、抗磷酸化Caspase3蛋白抗体及二步法Elivision试剂盒均购自福州迈新生物技术开发公司.

1.2 方法

利用自行设计制作的模具制备含96孔直径2 mm的蜡胚; 在显微镜下HE切片, 选取要检测的区域, 在石蜡包埋块上用孔径1.8 mm的骨髓穿刺针穿下对应区域的组织, 灌入制备好的蜡胚的小孔中, 并使各组织柱表面平整, 在切片机上切取4 m的连续切片, 60 ℃烤片备用(制作组织芯片). 切片脱蜡至水; 30 mL/L H2O2阻断内源性过氧化物酶30 min; 高火档微波修抗原5 min; 然后按免疫组织化学二步法操作. 用PBS代替一抗作阴性对照. 参照Bresalier半定量公式判断染色结果: 在每张切片中随机选取10个视野, 根据细胞染色强度分为4级, 并分别计分: 阴性, 细胞无着色(0); 弱阳性, 细胞着色为浅黄色(1); 中度阳性, 细胞着色为棕黄色(2); 强阳性, 细胞着色为棕褐色(3). 计数每一强度的视野数, 根据下列计算公式计算每张切片的平均染色强度: IS(intensity score) = ∑{(0×F0)+(1×F1)+(2×F2)+(3×F3)}, F =%×10视野.

统计学处理 用SPSS10.0统计软件进行χ2检验及相关性分析.

2 结果
2.1 HSP70和caspase3蛋白的表达

HSP70和caspase3蛋白的阳性信号主要位于细胞质, 少数细胞核阳性(图1, 2), 在HCC及癌旁肝组织中HSP70蛋白的阳性率分别为68.6%和31.4%, 正常肝组织10例中仅2例呈弱阳性, HCC中的阳性表达率明显高于癌旁肝组织(P<0.01). caspase3蛋白在HCC和癌旁肝组织中的阳性率分别为17.1%和35.7%, 正常肝组织的阳性率为80%, HCC中的阳性表达率明显低于癌旁肝组织和正常肝组织(P<0.01).

图1
图1 HCC胞质中HSP70阳性. SP×400.
图2
图2 HCC胞质中Caspase阳性, 部分胞核阳性. SP×400.
2.2 HSP70和caspase3表达与HCC

在HCC中HSP70和caspase3蛋白的表达强度与HCC的分化程度明显相关, 分化愈差HSP70表达愈强, 而caspase3蛋白表达愈弱, 反之亦然(P<0.01, 表1); HCC肿块愈大HSP70蛋白表达强, 而caspase3蛋白表达愈弱, 反之亦然(P<0.01, 表1).在HCC和癌旁肝组织中HSP70和caspase3蛋白的表达强度呈明显负相关(分别为r = -0.4 126和-0.5 237, P<0.01, 表1).

表1 HSP70和caspase3表达与HCC病理关系.
HCCnHSP70蛋白表达(IS)caspase3蛋白表达(IS)
分级
Ⅰ110.78±0.431.49±0.65
Ⅱ311.64±1.05a1.52±0.95b
Ⅲ231.81±1.10a0.80±0.57b
Ⅳ52.01±0.84a0.46±0.24b
肿块直径
≤5 cm251.11±0.881.42±0.87
5.1-10 cm291.88±1.07c1.01±0.99d
>10 cm162.02±0.77c0.63±0.44d
总计701.59±1.211.07±0.98
癌旁肝组织701.07±0.981.87±1.10
aP<0.01,
bP<0.01 vs I级,
cP<0.01,
dP<0.01 vs ≤5 cm.
3 讨论

既往报道在多种肿瘤和转化的培养细胞中HSP70呈过表达, 对维持肿瘤细胞的增生非常重要[30-32]. 本结果显示HSP70在HCC中的表达率和表达强度均显著高于癌旁肝组织, 并与HCC的分化程度和肿瘤的大小明显相关, 分化愈差表达愈强, 肿瘤愈大表达愈强, 与尹燕明et al的结果基本一致. Chuma et al[27]利用基因芯片技术研究热休克蛋白70在HCC中的表达, 并认为检测HSP70蛋白的表达可作为早期肝细胞癌的重要诊断指标. 根据我们的资料HSP70有望能成为判断HCC预后的重要指标之一.

在HCC的发生中常存在细胞增生和细胞凋亡的失衡, caspase 3是细胞凋亡通路中重要的执行分子, 其以磷酸化方式激活后导致细胞发生不可逆性凋亡[29,33], 利用免疫组织化学检测磷酸化caspase 3蛋白的表达, 能基本反映细胞凋亡的状况. 本资料显示HCC中磷酸化caspase3蛋白的表达率和表达强度均显著低于癌旁肝组织, 亦与HCC的分化程度和肿瘤大小明显相关, 分化愈差和瘤块愈大其表达愈弱. 提示在HCC的发生发展过程中可能存在癌细胞的低凋亡.

HSP70是否具有直接调节细胞凋亡的作用, 目前尚无定论, 尹燕明 et al研究结果显示HCC中HSP70的表达与细胞凋亡指数呈明显正相关; Chen et al发现表达HSP70的HepG2细胞能显著抑制UVC诱导的凋亡; 赵霞 et al利用反义寡核苷酸阻断HSP70的表达可诱导卵巢癌细胞的凋亡; 本文结果显示在HCC和癌旁肝组织中HSP70和caspase3蛋白的表达强度呈明显负相关. 推测在HCC发生过程中HSP70的表达可能通过促进细胞的转化和增生及抑制细胞的凋亡, 使HCC呈现无限制的生长及恶性程度不断增高.

编辑: N/A

1.  Cui J, Zhou XD, Liu YK, Tang ZY, Zile MH. Abnormal beta-catenin gene expression with invasiveness of primary hepatocellular carcinoma in China. World J Gastroenterol. 2001;7:542-546.  [PubMed]  [DOI]
2.  Zhao WH, Ma ZM, Zhou XR, Feng YZ, Fang BS. Prediction of recurrence and prognosis in patients with hepatocellular carcinoma after resection by use of CLIP score. World J Gastroenterol. 2002;8:237-242.  [PubMed]  [DOI]
3.  Li Y, Tang ZY, Ye SL, Liu YK, Chen J, Xue Q, Chen J, Gao DM, Bao WH. Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97. World J Gastroenterol. 2001;7:630-636.  [PubMed]  [DOI]
4.  Li G, Ma HH, Lau GK, Leung YK, Yao CL, Chong YT, Tang WH, Yao JL. Prevalence of hepatitis G virus infection and homology of different viral strains in Southern China. World J Gastroenterol. 2002;8:1081-1087.  [PubMed]  [DOI]
5.  Rabe C, Pilz T, Klostermann C, Berna M, Schild HH, Sauerbruch T, Caselmann WH. Clinical characteristics and outcome of a cohort of 101 patients with hepatocellular carcinoma. World J Gastroenterol. 2001;7:208-215.  [PubMed]  [DOI]
6.  Tang YC, Li Y, Qian GX. Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy. World J Gastroenterol. 2001;7:22-27.  [PubMed]  [DOI]
7.  Song ZQ, Hao F, Min F, Ma QY, Liu GD. Hepatitis C virus infection of human hepatoma cell line 7721 in vitro. World J Gastroenterol. 2001;7:685-689.  [PubMed]  [DOI]
8.  Fan J, Wu ZQ, Tang ZY, Zhou J, Qiu SJ, Ma ZC, Zhou XD, Ye SL. Multimodality treatment in hepatocellular carcinoma patients with tumor thrombi in portal vein. World J Gastroenterol. 2001;7:28-32.  [PubMed]  [DOI]
9.  Tang ZY, Sun FX, Tian J, Ye SL, Liu YK, Liu KD, Xue Q, Chen J, Xia JL, Qin LX. Metastatic human hepatocellular carcinoma models in nude mice and cell line with metastatic potential. World J Gastroenterol. 2001;7:597-601.  [PubMed]  [DOI]
10.  Meier V, Mihm S, Braun Wietzke P, Ramadori G. HCV-RNA positivity in peripheral blood mononuclear cells of patients with chronic HCV infection: does it really mean viral replication? World J Gastroenterol. 2001;7:228-234.  [PubMed]  [DOI]
11.  Huo TI, Wang XW, Forgues M, Wu CG, Spillare EA, Giannini C, Brechot C, Harris CC. Hepatitis B virus X mutants derived from human hepatocellular carcinoma retain the ability to abrogate p53-induced apoptosis. Oncogene. 2001;20:3620-3628.  [PubMed]  [DOI]
12.  Roussos A, Goritsas C, Pappas T, Spanaki M, Papadaki P, Ferti A. Prevalence of hepatitis B and C markers among refugees in Athens. World J Gastroenterol. 2003;9:993-995.  [PubMed]  [DOI]
13.  Wang Y, Liu H, Zhou Q, Li X. Analysis of point mutation in site 1896 of HBV precore and its detection in the tissues and serum of HCC patients. World J Gastroenterol. 2000;6:395-397.  [PubMed]  [DOI]
14.  Li K, Wang L, Cheng J, Lu YY, Zhang LX, Mu JS, Hong Y, Liu Y, Duan HJ, Wang G. Interaction between hepatitis C virus core protein and translin protein--a possible molecular mechanism for hepatocellular carcinoma and lymphoma caused by hepatitis C virus. World J Gastroenterol. 2003;9:300-303.  [PubMed]  [DOI]
15.  Kato T, Miyamoto M, Furusaka A, Date T, Yasui K, Kato J, Matsushima S, Komatsu T, Wakita T. Processing of hepatitis C virus core protein is regulated by its C-terminal sequence. J Med Virol. 2003;69:357-366.  [PubMed]  [DOI]
16.  Tang ZY. Hepatocellular carcinoma--cause, treatment and metastasis. World J Gastroenterol. 2001;7:445-454.  [PubMed]  [DOI]
17.  Zhang J, Zhang JK, Zhuo SH, Chen HB. Effect of a cancer vaccine prepared by fusions of hepatocarcinoma cells with dendritic cells. World J Gastroenterol. 2001;7:690-694.  [PubMed]  [DOI]
18.  Kalogeraki A, Garbagnati F, Santinami M, Zoras O. DNA fragmentation and cell proliferation correlated with tumor grade in patients with hepatocellular carcinoma. Cancer. 2002;96:301-305.  [PubMed]  [DOI]
19.  Wang FS, Liu MX, Zhang B, Shi M, Lei ZY, Sun WB, Du QY, Chen JM. Antitumor activities of human autologous cytokine-induced killer (CIK) cells against hepatocellular carcinoma cells in vitro and in vivo. World J Gastroenterol. 2002;8:464-468.  [PubMed]  [DOI]
20.  Wei CY, Li T, Zhang CM, Yang NW, Tan YC. Treating primary liver cancer with S-TIL combining Chinese traditional medicine. Shijie Huaren Xiaohua Zazhi. 2000;8:478.  [PubMed]  [DOI]
21.  Röcken C, Carl-McGrath S. Pathology and pathogenesis of hepatocellular carcinoma. Dig Dis. 2001;19:269-278.  [PubMed]  [DOI]
22.  Zheng N, Ye SL, Sun RX, Zhao Y, Tang ZY. Effects of cryopreservation and phenylacetate on biological characters of adherent LAK cells from patients with hepatocellular carcinoma. World J Gastroenterol. 2002;8:233-236.  [PubMed]  [DOI]
23.  Guo XZ, Shao XD, Liu MP, Xu JH, Ren LN, Zhao JJ, Li HY, Wang D. Effect of bax, bcl-2 and bcl-xL on regulating apoptosis in tissues of normal liver and hepatocellular carcinoma. World J Gastroenterol. 2002;8:1059-1062.  [PubMed]  [DOI]
24.  Ikeguchi M, Hirooka Y, Kaibara N. Quantitative analysis of apoptosis-related gene expression in hepatocellular carcinoma. Cancer. 2002;95:1938-1945.  [PubMed]  [DOI]
25.  Guan XJ, Guan XJ, Wu YZ, Jia ZC, Shi TD, Tang Y. Construction and characterization of an experimental ISCOMS-based hepatitis B polypeptide vaccine. World J Gastroenterol. 2002;8:294-297.  [PubMed]  [DOI]
26.  Du QY, Wang FS, Xu DP, Liu H, Lei ZY, Liu MX, Wang YD, Chen JM, Wu ZZ. Cytotoxic effects of CIK against hepatocellular carcinoma cells in vitro. Shijie Huaren Xiaohua Zazhi. 2000;8:863-866.  [PubMed]  [DOI]
27.  Chuma M, Sakamoto M, Yamazaki K, Ohta T, Ohki M, Asaka M, Hirohashi S. Expression profiling in multistage hepatocarcinogenesis: identification of HSP70 as a molecular marker of early hepatocellular carcinoma. Hepatology. 2003;37:198-207.  [PubMed]  [DOI]
28.  Schueller G, Paolini P, Friedl J, Stift A, Dubsky P, Bachleitner-Hofmann T, Jakesz R, Gnant M. Heat treatment of hepatocellular carcinoma cells: increased levels of heat shock proteins 70 and 90 correlate with cellular necrosis. Anticancer Res. 2001;21:295-300.  [PubMed]  [DOI]
29.  Sun BH, Zhang J, Wang BJ, Zhao XP, Wang YK, Yu ZQ, Yang DL, Hao LJ. Analysis of in vivo patterns of caspase 3 gene expression in primary hepatocellular carcinoma and its relationship to p21(WAF1) expression and hepatic apoptosis. World J Gastroenterol. 2000;6:356-360.  [PubMed]  [DOI]
30.  Kawanishi K, Shiozaki H, Doki Y, Sakita I, Inoue M, Yano M, Tsujinaka T, Shamma A, Monden M. Prognostic significance of heat shock proteins 27 and 70 in patients with squamous cell carcinoma of the esophagus. Cancer. 1999;85:1649-1657.  [PubMed]  [DOI]
31.  Maehara Y, Oki E, Abe T, Tokunaga E, Shibahara K, Kakeji Y, Sugimachi K. Overexpression of the heat shock protein HSP70 family and p53 protein and prognosis for patients with gastric cancer. Oncology. 2000;58:144-151.  [PubMed]  [DOI]
32.  Vanaja DK, Grossmann ME, Celis E, Young CY. Tumor prevention and antitumor immunity with heat shock protein 70 induced by 15-deoxy-delta12,14-prostaglandin J2 in transgenic adenocarcinoma of mouse prostate cells. Cancer Res. 2000;60:4714-4718.  [PubMed]  [DOI]
33.  Li HL, Chen DD, Li XH, Zhang HW, Lu YQ, Ye CL, Ren XD. Changes of NF-kB, p53, Bcl-2 and caspase in apoptosis induced by JTE-522 in human gastric adenocarcinoma cell line AGS cells: role of reactive oxygen species. World J Gastroenterol. 2002;8:431-435.  [PubMed]  [DOI]