重组腺病毒介导反义C-myc基因对人肝癌细胞治疗的有效性及安全性研究
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摘要
肝癌的发病率和死亡率高，是我国第二位常见恶性肿瘤，传统的手
术、化疗及放射治疗效果均不理想，为此，国内外都在寻找和开展肝癌
治疗新方法—基因治疗的研究。对肝癌相关基因的研究，使人们清楚的
认识到肝癌的发生发展是一个多种因素、多种机制、多个阶段的复杂过
程，其中涉及到多种癌基因的激活和抑癌基因的功能丧失。阻断或抑制
癌基因的高表达是肿瘤基因治疗的策略之一，为此反义技术得以发展应
用，并在一些肿瘤的基因治疗中取得良好的效果而具有广泛应用的前景。
c-myc属于细胞原癌基因，其编码的62KD核蛋白通过与核蛋白Max
形成二聚体结合特异的DNA序列（CACTGT）激活许多靶基因的转录，其
中很多基因产物与细胞周期调控密切相关。myc癌基因的异常表达是形
成肿瘤的步骤之一，同时myc基因又与其它癌基因在肿瘤形成的复杂过
程中具有协同作用。从细胞癌变到肿瘤形成再到转移侵润的启动、促癌、
演进及转移各个阶段，c-myc均与其它一些原癌基因被激活参与作用。c-
myc癌基因的高表达参与了肝癌的形成和生长。通过反义RNA抑制c-myc
基因的过表达，使依赖其基因产物的肿瘤细胞生长周期改变、发生凋亡，
有报道在前列腺癌及转移乳腺癌的基因治疗中有效。
随着基因转移技术的不断完善和分子生物学的飞速发展，基因治疗已
从实验室走向临床。理想的基因转移载体是基因治疗能够进入临床的关
键之一。在众多的基因转移方法中，腺病毒载体介导的基因转移，以其
高转染率和良好的靶向性而成为肿瘤基因治疗中广为使用的方法。腺病
毒载体具有很好的嗜肝向性，体内外研究均证明了腺病毒载体介导的基
因在肝脏细胞的高效转移。但是，腺病毒载体介导的肝癌基因治疗的真
正应用于临床，还必需解决临床安全性问题，即其临床使用对机体的毒
副作用。因此临床应用前对其安全性进行评价至关重要。
本研究采用兔疫组化法，以检测C－myc、hcf毛癌基因在肝癌组织中
表达情况，探讨其在肝癌发生发展中的作用及其在判断预后中的价值。
采用构建成功的Ad－ASmyc，利用反义基因在翻译水平上阻断c－p的过
表达，抑制肝癌细胞的生长并促使其凋亡，以寻找肝癌基因治疗新的有
效方法。经Beagle犬肝动脉、门静脉途径注射重组腺病毒介导反义c－D叹c
基因 （Ad－ASm）载体，观测其在体内是否能有效转导入正常肝细胞、持
续时间、体内器官分布及毒副作用，以评价AdAS侧治疗肝癌的临床
前期安全性。
本研究获得以下主要结果：
1．原发性肝癌组织中C侧C蛋白阳性表达率较高，B＊K蛋白阳性表
达率虽然较低，但其阳性表达往往伴随着C 4r1V4r1VC蛋白阳性表达，二者联合
检测有助于判断肝癌的恶性程度及预后。
2．重组腺病毒介导的反义 C－pC基因（A－ASmyC）滴度较高，可达到
5 X 10” PFMl，并可高效转导入斗种人肝癌细胞系①ELj402、QSG－
7701、SMMCJ、HCCq204X人胚肺H倍体细胞ZBS及正常人肝细
胞LOZ细胞。
3．Ad－AS啊c自够引起人肝癌细胞c－p、hcf－2 RNA及 c－myC、hcf－2
蛋白表达下调，抑制肝癌细胞生长和导致肝癌细胞凋亡。
4．c－p c表达水平不同的肝癌细胞系，Ad·ASmyc的作用效果不同，
与细胞c－msc表达水平高低有关，c－msc表达高者治疗效果更明显。说明
反义C－my C基因疗法对不同的细胞类型治疗效果有差异性。
5．通过两种肝癌细胞系①ELJ402、S删CJ）裸鼠皮下移植瘤的
体内实验证实，瘤内直接注射Ad－AS呷治疗裸鼠皮下移植肝癌有效。
6．经犬肝动脉或门静脉途径注射AdASIny均可持续转导至肝细胞
达3周，病毒可播散至脾脏、肾脏、胃、心脏及皮肤，但对组织器官无明
·Vll·
X》
显毒副作用，仅在较大剂量时肝脏组织镜下可见Ad－ASmrc剂量依赖性
的轻微炎症反应。
7．注射Ad－ASmyc后，血清产生针对腺病毒载体的中和抗体，大剂量
Ad叭Smyc犬较小剂量A小ASmyC犬抗体滴度高，但平均抗体滴度皆较
低。临床应用AdASmyc进行肝癌的基因治疗是安全可行的。
本研究结果表明，原癌基因C－IT［yC在肝癌组织及人肝癌细胞系中均为
高表达；AdAS侧对体外培养的人肝癌细胞系及裸鼠体内皮下移植肝癌
治疗有效；经犬肝动脉或门静脉途径注射AdAS啊C对机体无明显毒副作
用，为临床应用Ad－ASmyc基因治疗肝癌提供了有力的实验基础。
Hepatocellular carcinoma ( HCC ) is one the commonest cancer in the
world and one the most difficult tumors to treat. The morbidity and mortality of
this disease is high. The most effective therapy is surgical resection, but still
limited. The results of other approaches, such as chemotherapy or radiotherapy
are also disappointing. Therefore, it is urgent to explore a new and effective
treatment method. Gene therapy, although in its infancy at the present time, may
have a significant role to play in the future management of HCC.
Proto-oncogene c-myc encodes a nuclear 62KD phosphoprotein, which
acts as a key regulator of cell growth and differentiation, when dimerized with
its partner protein Max, c-myc functions as transcription factor, capable of both
activating and repressing transcription. Increased expression of c-myc is seen in
many cancers such as breast, prostate, lung, and others. Importantly, elevated
expression of c-myc combined with other oncogene has a synergistic effect in
the development of tumor. Overexpression of c-myc exists in all stage from the
tumorigenesis to growth of tumor, metastasis in many tumors and so does in the
development of HCC. Thus, using antisense RINA blocks the overexpression of
c-myc gene in HCC cells, the decreased expression of c-myc gene can
contribute to change the cells growth cycle and induce apoptosis. This kind of
antisense gene therapy may be a potential clinical utility for HCC.
With the improvement of gene transferring system and the progression of
molecular biology, gene therapy had performed human clinical trial. First,
efficient gene transfer is essential for successful gene therapy. A number of
gene transfer vectors have been developed. Adenoviral vectors transduce
dividing and non-dividing cells of tissues with efficiency. These vectors have
been used to transduce foreign genes into a variety of tumor cells. Adenoviral
vectors can efficiently infect normal hepatocytes in vitro and in vivo because of
?III
their natural tropism for hepatocytes. Second, the safety required to be
evaluated before adenovirus mediated gene transfer vector really becomes a
feasibility therapy for clinical liver cancer.
In this study, immunohistochemical stainning technique (S-P) was used to
evaluate the effect of the expression of c-myc, Bcl-2 protein in the
carcinogenesis and tumor development of hepatocellular carcinoma and their
clinical values. The observation of morphological changes, growth curve, DNA
ladder, FCM, RT-PCR and Western blot analysis after hepatocellular cells
infected Adenovirus-mediated antisense c-myc gene (Ad-ASmyc) were used in
this study to explore the inhibitory effect of Ad-ASmyc on growth of
hepatocellular cells in vifro and Three kinds dose of Ad-ASmyc, 1O7pfu~ lO2pfii
and ~o~pftj, for treatment of hetero-transplanted tumor in nude mice was studied
in vivo. Ad-ASmyc was infused into the hepatic artery or portal vein in Beagle
dogs to observe its transduction efficiency, duration, distribution in organs,
toxicity and to assess the preclinical safety of Ad-ASmyc.
Conclusion presented in this study can be summarized as follows:
1. The higher positive expression of c-myc was found in HCC tissues. The
positive expression of Bcl-2 protein was lower and associated with the positive
expression of c-myc protein. c-myc and Bcl-2 were associated with the
occurrence and development of I-ICC and both com
术、化疗及放射治疗效果均不理想，为此，国内外都在寻找和开展肝癌
治疗新方法—基因治疗的研究。对肝癌相关基因的研究，使人们清楚的
认识到肝癌的发生发展是一个多种因素、多种机制、多个阶段的复杂过
程，其中涉及到多种癌基因的激活和抑癌基因的功能丧失。阻断或抑制
癌基因的高表达是肿瘤基因治疗的策略之一，为此反义技术得以发展应
用，并在一些肿瘤的基因治疗中取得良好的效果而具有广泛应用的前景。
c-myc属于细胞原癌基因，其编码的62KD核蛋白通过与核蛋白Max
形成二聚体结合特异的DNA序列（CACTGT）激活许多靶基因的转录，其
中很多基因产物与细胞周期调控密切相关。myc癌基因的异常表达是形
成肿瘤的步骤之一，同时myc基因又与其它癌基因在肿瘤形成的复杂过
程中具有协同作用。从细胞癌变到肿瘤形成再到转移侵润的启动、促癌、
演进及转移各个阶段，c-myc均与其它一些原癌基因被激活参与作用。c-
myc癌基因的高表达参与了肝癌的形成和生长。通过反义RNA抑制c-myc
基因的过表达，使依赖其基因产物的肿瘤细胞生长周期改变、发生凋亡，
有报道在前列腺癌及转移乳腺癌的基因治疗中有效。
随着基因转移技术的不断完善和分子生物学的飞速发展，基因治疗已
从实验室走向临床。理想的基因转移载体是基因治疗能够进入临床的关
键之一。在众多的基因转移方法中，腺病毒载体介导的基因转移，以其
高转染率和良好的靶向性而成为肿瘤基因治疗中广为使用的方法。腺病
毒载体具有很好的嗜肝向性，体内外研究均证明了腺病毒载体介导的基
因在肝脏细胞的高效转移。但是，腺病毒载体介导的肝癌基因治疗的真
正应用于临床，还必需解决临床安全性问题，即其临床使用对机体的毒
副作用。因此临床应用前对其安全性进行评价至关重要。
本研究采用兔疫组化法，以检测C－myc、hcf毛癌基因在肝癌组织中
表达情况，探讨其在肝癌发生发展中的作用及其在判断预后中的价值。
采用构建成功的Ad－ASmyc，利用反义基因在翻译水平上阻断c－p的过
表达，抑制肝癌细胞的生长并促使其凋亡，以寻找肝癌基因治疗新的有
效方法。经Beagle犬肝动脉、门静脉途径注射重组腺病毒介导反义c－D叹c
基因 （Ad－ASm）载体，观测其在体内是否能有效转导入正常肝细胞、持
续时间、体内器官分布及毒副作用，以评价AdAS侧治疗肝癌的临床
前期安全性。
本研究获得以下主要结果：
1．原发性肝癌组织中C侧C蛋白阳性表达率较高，B＊K蛋白阳性表
达率虽然较低，但其阳性表达往往伴随着C 4r1V4r1VC蛋白阳性表达，二者联合
检测有助于判断肝癌的恶性程度及预后。
2．重组腺病毒介导的反义 C－pC基因（A－ASmyC）滴度较高，可达到
5 X 10” PFMl，并可高效转导入斗种人肝癌细胞系①ELj402、QSG－
7701、SMMCJ、HCCq204X人胚肺H倍体细胞ZBS及正常人肝细
胞LOZ细胞。
3．Ad－AS啊c自够引起人肝癌细胞c－p、hcf－2 RNA及 c－myC、hcf－2
蛋白表达下调，抑制肝癌细胞生长和导致肝癌细胞凋亡。
4．c－p c表达水平不同的肝癌细胞系，Ad·ASmyc的作用效果不同，
与细胞c－msc表达水平高低有关，c－msc表达高者治疗效果更明显。说明
反义C－my C基因疗法对不同的细胞类型治疗效果有差异性。
5．通过两种肝癌细胞系①ELJ402、S删CJ）裸鼠皮下移植瘤的
体内实验证实，瘤内直接注射Ad－AS呷治疗裸鼠皮下移植肝癌有效。
6．经犬肝动脉或门静脉途径注射AdASIny均可持续转导至肝细胞
达3周，病毒可播散至脾脏、肾脏、胃、心脏及皮肤，但对组织器官无明
·Vll·
X》
显毒副作用，仅在较大剂量时肝脏组织镜下可见Ad－ASmrc剂量依赖性
的轻微炎症反应。
7．注射Ad－ASmyc后，血清产生针对腺病毒载体的中和抗体，大剂量
Ad叭Smyc犬较小剂量A小ASmyC犬抗体滴度高，但平均抗体滴度皆较
低。临床应用AdASmyc进行肝癌的基因治疗是安全可行的。
本研究结果表明，原癌基因C－IT［yC在肝癌组织及人肝癌细胞系中均为
高表达；AdAS侧对体外培养的人肝癌细胞系及裸鼠体内皮下移植肝癌
治疗有效；经犬肝动脉或门静脉途径注射AdAS啊C对机体无明显毒副作
用，为临床应用Ad－ASmyc基因治疗肝癌提供了有力的实验基础。
Hepatocellular carcinoma ( HCC ) is one the commonest cancer in the
world and one the most difficult tumors to treat. The morbidity and mortality of
this disease is high. The most effective therapy is surgical resection, but still
limited. The results of other approaches, such as chemotherapy or radiotherapy
are also disappointing. Therefore, it is urgent to explore a new and effective
treatment method. Gene therapy, although in its infancy at the present time, may
have a significant role to play in the future management of HCC.
Proto-oncogene c-myc encodes a nuclear 62KD phosphoprotein, which
acts as a key regulator of cell growth and differentiation, when dimerized with
its partner protein Max, c-myc functions as transcription factor, capable of both
activating and repressing transcription. Increased expression of c-myc is seen in
many cancers such as breast, prostate, lung, and others. Importantly, elevated
expression of c-myc combined with other oncogene has a synergistic effect in
the development of tumor. Overexpression of c-myc exists in all stage from the
tumorigenesis to growth of tumor, metastasis in many tumors and so does in the
development of HCC. Thus, using antisense RINA blocks the overexpression of
c-myc gene in HCC cells, the decreased expression of c-myc gene can
contribute to change the cells growth cycle and induce apoptosis. This kind of
antisense gene therapy may be a potential clinical utility for HCC.
With the improvement of gene transferring system and the progression of
molecular biology, gene therapy had performed human clinical trial. First,
efficient gene transfer is essential for successful gene therapy. A number of
gene transfer vectors have been developed. Adenoviral vectors transduce
dividing and non-dividing cells of tissues with efficiency. These vectors have
been used to transduce foreign genes into a variety of tumor cells. Adenoviral
vectors can efficiently infect normal hepatocytes in vitro and in vivo because of
?III
their natural tropism for hepatocytes. Second, the safety required to be
evaluated before adenovirus mediated gene transfer vector really becomes a
feasibility therapy for clinical liver cancer.
In this study, immunohistochemical stainning technique (S-P) was used to
evaluate the effect of the expression of c-myc, Bcl-2 protein in the
carcinogenesis and tumor development of hepatocellular carcinoma and their
clinical values. The observation of morphological changes, growth curve, DNA
ladder, FCM, RT-PCR and Western blot analysis after hepatocellular cells
infected Adenovirus-mediated antisense c-myc gene (Ad-ASmyc) were used in
this study to explore the inhibitory effect of Ad-ASmyc on growth of
hepatocellular cells in vifro and Three kinds dose of Ad-ASmyc, 1O7pfu~ lO2pfii
and ~o~pftj, for treatment of hetero-transplanted tumor in nude mice was studied
in vivo. Ad-ASmyc was infused into the hepatic artery or portal vein in Beagle
dogs to observe its transduction efficiency, duration, distribution in organs,
toxicity and to assess the preclinical safety of Ad-ASmyc.
Conclusion presented in this study can be summarized as follows:
1. The higher positive expression of c-myc was found in HCC tissues. The
positive expression of Bcl-2 protein was lower and associated with the positive
expression of c-myc protein. c-myc and Bcl-2 were associated with the
occurrence and development of I-ICC and both com
引文
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23． Ishida S, Takada S, Koike K. Isolation and analysis of cellular DNA fragments directly binding to c-myc protein. Leukemia. 1997 Apr, 11 Suppl 3:399-401．
24． Ljubimova JY, Petrovic LM, Wilson SE, et al. Expression of HGF, its receptor c-met, c-myc, and albumin in cirrhotic and neoplastic human liver tissue. J Histochem Cytochem, 1997 Jan, 45(l):79-87．
25． Liu P, Terradillos 0, Renard CA, et al. Hepatocarcinogenesis in woodchuck hepatitis virus/c-myc mice: sustained cell proliferation and biphasic activation of insulin-like growth factor Ⅱ. Hepatology, 1997 Apr, 25(4) :874-883．
26． Sargent LM, Zhou X, Keck CL, et al. Nonrandom cytogenetic alterations in hepatocellular carcinoma from transgenic mice overexpressing c-myc and transforming growth factor-alpha in the liver. Am J Pathol, 1999 Apr,
154(4):1047-1055.
27.Pollicino T, Terradillos O, Lecoeur H, et al. Pro-apoptotic effect of the hepatitis B virus X gene. Biomed Pharmacother,1998,52(9):363-368.
28．张利宁，刘素侠，孙汶生，等．原发性肝癌中HBs X，S，pre-S，C基因片段整合与ras，myc癌基因表达的关系．山东医科大学学报，1998，36(1)：36-39．
29.Schuster R, Gerlich WH, Schaefer-S. Induction of apoptosis by the transactivating domains of the hepatitis B virus X gene leads to suppression of oncogenic transformation of primary rat embryo fibroblasts. Oncogene, 2000 Feb 24,19(9):1173-1180.
30.Kim H, Lee H, Yun Y. X-gene ptoduct of hepatitis B virus induces apoptosis in liver cells.J Bio Chem, 1998 Jan 2, 273(1):381-385.
31.Hockenbery D, Nunez G, Milliman C, et al. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature,1990,348:334-336.
32.Nakopoulou L, Stefanaki K, Vourlakou C, et al. Bcl-2 ptotein expression in acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. Pathol Res Pract,1999,195(1):19-24.
33.Santoni RE, Jensen MR, Thorgeirsson SS, et al. Disruption of the pRb/E2F pathway and inhibition of apoptosis are major oncogenic events in liver constitutively expressing c-myc and transforming growth factor alpha. Am J Pathol, 1999 Jun, 154(6):1693-1700.
34.Charlotte F, L'Hermine A, Martin-N, et al. Immunohistochemical detection of bcl-2 protein in normal and pathological human liver. Am J Pathol,1994 Mar,144(3): 460-465.
35.Cheng J, Luo J, Zhang X, et al. Inhibition of cell proliferation in HCC-9204 hepatoma cells by a c-myc specific riozyme. Cancer Gene Ther, 2000 Mar,7(3):407-412.
36.Lee GH. Correlation between Bcl-2 expression and histopathology in diethylnitrosamine-induced mouse hepatocellular tumors. Am J Pathol, 1997 Oct, 151 (4):957-961.
37.Ebinuma H, Saito H, Saito Y, et al. Antisense oligodeoxynucleotide against c-myc mRNA induces differentiation of human hepatocellular carcinoma cells. Int J Oncol,1999 Nov, 15(5):991-999.
38.Soini Y, Virkajarvi N, Lehto VP, et al. Hepatocellular carcinomas with a high proliferation index and a liw degree of apoptosis and necrosis are associatted with a shortened survival. Br J Cancer,1996 Mayl,73(9):1025-1030.
39.Frommel TO, Yong S, Zarling EJ. Immunohistochemical evaluation of Bcl-2 gene family expression in liver of hepatitis C and cirrhotic patients: a novel mechanism to explain the high incidence of hepatocarcinoma in cirrhotics. Am J Gastroenterol,1999 Jan, 94(1):178-182.
40.Saito-H, Ebinuma-H, Takahashi-M, et al. Loss of butyrate-induced apoptosis in human hepatoma cell lines HCC-M and HCC-T having substantial Bcl-2 expression. Hepatology,1998 May, 27(5):1233-1240.
41.Fiorentino M, D'Errico A, Altimari A, et al. High levels of BCL-2 messenger RNA detected by in situ hybridization in human hepatocellular and cholangiocellular carcinomas. Diagn Mol Pathol, 1999 Dec,8(4):189-194.
42．郭琳琅，肖莎．肝癌bcl-2基因异常及p53蛋白表达的关系．中华肝胆外科杂志，1998，4(1)：14-16．
43．朱珍道，郭旦敏，胡子元，等．bcl-2，P53和c-erb-B2癌基因在肝癌中的表达．中西医结合肝病杂志，1998，8(1)：12-13．
44.Wang DG, Johnston CF, Marley JJ, et al. Expression of the apoptosis-suppressing gene Bcl-2 in pheochromocytoma is associated with the expression of c-myc. J Clin Endocrinol Metab,1997,82(6):1949-1952.
45． Wang DG, Liu WH, Johnston CF, et al. Bcl-2 and c-myc, not bax and p53, are expressed during human medullary thyroid tumorigenesis. Am J Pathol, 1998,152(6) : 1407-1413．
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22． Shen L, Fang J, Qiu D, et al. Correlation between DNA methylation and pathological changes in human hepatocellular carcinoma. Hepatogastroenterology, 1998 Sep-Oct,45(23) :1753-1759．
23． Ishida S, Takada S, Koike K. Isolation and analysis of cellular DNA fragments directly binding to c-myc protein. Leukemia. 1997 Apr, 11 Suppl 3:399-401．
24． Ljubimova JY, Petrovic LM, Wilson SE, et al. Expression of HGF, its receptor c-met, c-myc, and albumin in cirrhotic and neoplastic human liver tissue. J Histochem Cytochem, 1997 Jan, 45(l):79-87．
25． Liu P, Terradillos 0, Renard CA, et al. Hepatocarcinogenesis in woodchuck hepatitis virus/c-myc mice: sustained cell proliferation and biphasic activation of insulin-like growth factor Ⅱ. Hepatology, 1997 Apr, 25(4) :874-883．
26． Sargent LM, Zhou X, Keck CL, et al. Nonrandom cytogenetic alterations in hepatocellular carcinoma from transgenic mice overexpressing c-myc and transforming growth factor-alpha in the liver. Am J Pathol, 1999 Apr,
154(4):1047-1055.
27.Pollicino T, Terradillos O, Lecoeur H, et al. Pro-apoptotic effect of the hepatitis B virus X gene. Biomed Pharmacother,1998,52(9):363-368.
28．张利宁，刘素侠，孙汶生，等．原发性肝癌中HBs X，S，pre-S，C基因片段整合与ras，myc癌基因表达的关系．山东医科大学学报，1998，36(1)：36-39．
29.Schuster R, Gerlich WH, Schaefer-S. Induction of apoptosis by the transactivating domains of the hepatitis B virus X gene leads to suppression of oncogenic transformation of primary rat embryo fibroblasts. Oncogene, 2000 Feb 24,19(9):1173-1180.
30.Kim H, Lee H, Yun Y. X-gene ptoduct of hepatitis B virus induces apoptosis in liver cells.J Bio Chem, 1998 Jan 2, 273(1):381-385.
31.Hockenbery D, Nunez G, Milliman C, et al. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature,1990,348:334-336.
32.Nakopoulou L, Stefanaki K, Vourlakou C, et al. Bcl-2 ptotein expression in acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. Pathol Res Pract,1999,195(1):19-24.
33.Santoni RE, Jensen MR, Thorgeirsson SS, et al. Disruption of the pRb/E2F pathway and inhibition of apoptosis are major oncogenic events in liver constitutively expressing c-myc and transforming growth factor alpha. Am J Pathol, 1999 Jun, 154(6):1693-1700.
34.Charlotte F, L'Hermine A, Martin-N, et al. Immunohistochemical detection of bcl-2 protein in normal and pathological human liver. Am J Pathol,1994 Mar,144(3): 460-465.
35.Cheng J, Luo J, Zhang X, et al. Inhibition of cell proliferation in HCC-9204 hepatoma cells by a c-myc specific riozyme. Cancer Gene Ther, 2000 Mar,7(3):407-412.
36.Lee GH. Correlation between Bcl-2 expression and histopathology in diethylnitrosamine-induced mouse hepatocellular tumors. Am J Pathol, 1997 Oct, 151 (4):957-961.
37.Ebinuma H, Saito H, Saito Y, et al. Antisense oligodeoxynucleotide against c-myc mRNA induces differentiation of human hepatocellular carcinoma cells. Int J Oncol,1999 Nov, 15(5):991-999.
38.Soini Y, Virkajarvi N, Lehto VP, et al. Hepatocellular carcinomas with a high proliferation index and a liw degree of apoptosis and necrosis are associatted with a shortened survival. Br J Cancer,1996 Mayl,73(9):1025-1030.
39.Frommel TO, Yong S, Zarling EJ. Immunohistochemical evaluation of Bcl-2 gene family expression in liver of hepatitis C and cirrhotic patients: a novel mechanism to explain the high incidence of hepatocarcinoma in cirrhotics. Am J Gastroenterol,1999 Jan, 94(1):178-182.
40.Saito-H, Ebinuma-H, Takahashi-M, et al. Loss of butyrate-induced apoptosis in human hepatoma cell lines HCC-M and HCC-T having substantial Bcl-2 expression. Hepatology,1998 May, 27(5):1233-1240.
41.Fiorentino M, D'Errico A, Altimari A, et al. High levels of BCL-2 messenger RNA detected by in situ hybridization in human hepatocellular and cholangiocellular carcinomas. Diagn Mol Pathol, 1999 Dec,8(4):189-194.
42．郭琳琅，肖莎．肝癌bcl-2基因异常及p53蛋白表达的关系．中华肝胆外科杂志，1998，4(1)：14-16．
43．朱珍道，郭旦敏，胡子元，等．bcl-2，P53和c-erb-B2癌基因在肝癌中的表达．中西医结合肝病杂志，1998，8(1)：12-13．
44.Wang DG, Johnston CF, Marley JJ, et al. Expression of the apoptosis-suppressing gene Bcl-2 in pheochromocytoma is associated with the expression of c-myc. J Clin Endocrinol Metab,1997,82(6):1949-1952.
45． Wang DG, Liu WH, Johnston CF, et al. Bcl-2 and c-myc, not bax and p53, are expressed during human medullary thyroid tumorigenesis. Am J Pathol, 1998,152(6) : 1407-1413．