βG基因及βG前体药自杀基因系统对人膀胱癌细胞作用的实验研究
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摘要
膀胱癌是泌尿系中最常见的肿瘤，在我国发病率居泌尿系肿瘤首位，并且
有恶性度高，易复发的特点。目前治疗以手术、化疗为主，但复发、转移率仍
较高，晚期疗效差，需积极探讨新的治疗方法。
自杀基因疗法是目前有效治疗肿瘤中最有前景的基因治疗策略之一。葡萄
糖醛酸苷酶（β-Glucuronidase，βG）是溶酶体中的酸性水解酶，参与机体的
生理、病理及药物代谢等过程。它能够特异性水解葡萄糖醛酸苷糖甙键，释放
出葡萄糖醛酸和配基。βG在ADEPT（抗体导向酶/前体药疗法）的应用研究中，
已证实其前体药对肿瘤的选择性杀伤作用。将βG及前体药系统与基因治疗相
结合，就有可能产生新的能够定向实施分子化疗的方法，用βG的相应前体药
作用于βG高表达膀胱肿瘤，有可能起到杀伤肿瘤作用。为膀胱癌的治疗寻找
一条新的、有效的途径。本实验进行了以下几方面研究：
1、利用质粒PGEM-4及pcDNA3.1（+）具有相同的EcoRI酶切位点，成功
构建带βG基因的真核表达载体PcDNA3.1（+）-βG。
2、利用脂质体（LipofectAMINE）介导法将含有全长βG cDNA的真核表达
载体pcDNA3.1（+）-βG转染至人膀胱癌细胞T24中，经G418筛选，获得了稳
第四军医大学硕士学位论文
定转染 6 G基因的细胞克隆；通过 RNA打点杂交及原位杂交和免疫荧光、免疫
组化、wes七ern Blot方法分别从帕A及蛋白水平证实p G基因己经稳定整合转
入T24细胞基因组中并获得p G高表达，将稳定转染并高表达p G的转染细胞系
命名为：T24什 G细胞。并通过光镜、电镜、细胞增殖周期检测等方法观察了
p G转染的 T24／fi G细胞的生物学特性。
3、对pG前体药Epi－bUS作用于T24／pG细胞的体外效应进行了一系列研
究，结果显示：稳定转染6 G基因的 T24／e G细胞对 Epi十US的敏感性比亲本
细胞提高了 1000倍以上，IC。／半数抑制剂量）为 0．179／L。浓度为 2．929／L
的Epi－b［JS作用4－5天对转染细胞的生长影响很小，但可将高表达p G的转染
细胞全部杀死；0．2925／L的ESi－bus也能在6－7天内将转染细胞全部杀死，
表现出明显的“自杀效应”；将T24／日G细胞与其末转染的亲本细胞T24以不同
比例混合，观察了 Epi－blls对混合细胞的作用，结果示：当 T24旭 G细胞占川％～
20％的时候，有70％～90％的混合细胞被杀死，说明有较强的旁观者效应。
上述研究表明：pG基因叩G前体药物系统可引入自杀基因治疗系统，脂
质体－真核表达载体介导日G基因转染，结合使用日G前体药物，有望成为治疗
膀脱癌的有效策略，为该系统治疗膀耽癌的临床应用提供了理论和实验依据。
Bladder carcinoma is the most common malignancy in the urinary, accounting
for 20% of the malignancy of all the systems. The managements remain to be
surgery and chemotherapy. High frequent recurrence and bad efficiency in the late
stage call for new therapies for it.
The suicide gene therapy is one of the most promising gene therapies for the
tumor. f3-Glucuronidase is a kind of acid hydrolase in lysosome, involved in the
processes of physiology, pathyology and metabolism of the drugs. It can hydrolysis
glucuronide glycoside bond, releasing the glucuronic acid and ligand. It was well
established that the prodrug of 13G can selectively kill the tumor cells. By combining
the j3G, prodrugs and the gene therapy, a new oriented molecular chemotherapy may
be developed. The bladder tumor cells with high expression of ~G take the prodrug
and will be killed selectively.
The aim of this study is to look for a new and effective therapy for the bladder
carcinoma. The experiment was done in the following ways:
1 .The retrovirus eukaryotic expression vector with the f3G gene was constructed
by the same EcoR I site of the PGEM-4 and pcDNA3.1(+).
2.By the lipofectAMlNE, a retrovirus eukaryotic expression vector
pcDNA3.1 (+) containing the whole length sequence of the f3G cDNA was
transfected into the human bladder tumour cell line T24. After the G41 8 screening, a
clone that get the stable transfection of the BG was obtained. It was confirmed that
the ~G gene had been stably integrated into the genomic DNA of the 124 cell and
got a highly expression by the means of RNA blotting, in situ hybridization,
Immunoflurescence, Jmmunohistochemistry, Western Blot. The stably transfected
cell was named T24/ f3G. The biological characteristics of the fG transfected cell
were studied by the methods of microscopy and electron microscop.
3.The series studies of the in vitro effects of the prodrug Epi-bus on T24/ f3G
showed that the stably transfected cells are more sensitive to the Epi-bus by 1000
fold than the parent, with the IC50 0.1 7g/L. The Epi-bus with a concentration of
2.92g/l sustaining for 4-5 days had little affection on the growth of the transfected
cells, but will kill all the cells with high j3G expression. The concentration of
0.292g11 for 6-7 days will kill the transfected cells, showing the obvious uicide
effects The T24/G cells were mixed with parental cells not transfected with
various proportion and 70%-90% of the mixed cells were killed when the 124/ f3G
cell accounting for 1 0%-20%. It showed the potent stand-by effect.
The study showed that the 13G gene and its prodrug could be introduced into the
suicide gene system and the j3G gene transfection mediated by the LipofectAMlNE-
retrovirus eukariotic expression vector combining the 13G prodrug managment will
be the promising strategy for bladder therapy.
有恶性度高，易复发的特点。目前治疗以手术、化疗为主，但复发、转移率仍
较高，晚期疗效差，需积极探讨新的治疗方法。
自杀基因疗法是目前有效治疗肿瘤中最有前景的基因治疗策略之一。葡萄
糖醛酸苷酶（β-Glucuronidase，βG）是溶酶体中的酸性水解酶，参与机体的
生理、病理及药物代谢等过程。它能够特异性水解葡萄糖醛酸苷糖甙键，释放
出葡萄糖醛酸和配基。βG在ADEPT（抗体导向酶/前体药疗法）的应用研究中，
已证实其前体药对肿瘤的选择性杀伤作用。将βG及前体药系统与基因治疗相
结合，就有可能产生新的能够定向实施分子化疗的方法，用βG的相应前体药
作用于βG高表达膀胱肿瘤，有可能起到杀伤肿瘤作用。为膀胱癌的治疗寻找
一条新的、有效的途径。本实验进行了以下几方面研究：
1、利用质粒PGEM-4及pcDNA3.1（+）具有相同的EcoRI酶切位点，成功
构建带βG基因的真核表达载体PcDNA3.1（+）-βG。
2、利用脂质体（LipofectAMINE）介导法将含有全长βG cDNA的真核表达
载体pcDNA3.1（+）-βG转染至人膀胱癌细胞T24中，经G418筛选，获得了稳
第四军医大学硕士学位论文
定转染 6 G基因的细胞克隆；通过 RNA打点杂交及原位杂交和免疫荧光、免疫
组化、wes七ern Blot方法分别从帕A及蛋白水平证实p G基因己经稳定整合转
入T24细胞基因组中并获得p G高表达，将稳定转染并高表达p G的转染细胞系
命名为：T24什 G细胞。并通过光镜、电镜、细胞增殖周期检测等方法观察了
p G转染的 T24／fi G细胞的生物学特性。
3、对pG前体药Epi－bUS作用于T24／pG细胞的体外效应进行了一系列研
究，结果显示：稳定转染6 G基因的 T24／e G细胞对 Epi十US的敏感性比亲本
细胞提高了 1000倍以上，IC。／半数抑制剂量）为 0．179／L。浓度为 2．929／L
的Epi－b［JS作用4－5天对转染细胞的生长影响很小，但可将高表达p G的转染
细胞全部杀死；0．2925／L的ESi－bus也能在6－7天内将转染细胞全部杀死，
表现出明显的“自杀效应”；将T24／日G细胞与其末转染的亲本细胞T24以不同
比例混合，观察了 Epi－blls对混合细胞的作用，结果示：当 T24旭 G细胞占川％～
20％的时候，有70％～90％的混合细胞被杀死，说明有较强的旁观者效应。
上述研究表明：pG基因叩G前体药物系统可引入自杀基因治疗系统，脂
质体－真核表达载体介导日G基因转染，结合使用日G前体药物，有望成为治疗
膀脱癌的有效策略，为该系统治疗膀耽癌的临床应用提供了理论和实验依据。
Bladder carcinoma is the most common malignancy in the urinary, accounting
for 20% of the malignancy of all the systems. The managements remain to be
surgery and chemotherapy. High frequent recurrence and bad efficiency in the late
stage call for new therapies for it.
The suicide gene therapy is one of the most promising gene therapies for the
tumor. f3-Glucuronidase is a kind of acid hydrolase in lysosome, involved in the
processes of physiology, pathyology and metabolism of the drugs. It can hydrolysis
glucuronide glycoside bond, releasing the glucuronic acid and ligand. It was well
established that the prodrug of 13G can selectively kill the tumor cells. By combining
the j3G, prodrugs and the gene therapy, a new oriented molecular chemotherapy may
be developed. The bladder tumor cells with high expression of ~G take the prodrug
and will be killed selectively.
The aim of this study is to look for a new and effective therapy for the bladder
carcinoma. The experiment was done in the following ways:
1 .The retrovirus eukaryotic expression vector with the f3G gene was constructed
by the same EcoR I site of the PGEM-4 and pcDNA3.1(+).
2.By the lipofectAMlNE, a retrovirus eukaryotic expression vector
pcDNA3.1 (+) containing the whole length sequence of the f3G cDNA was
transfected into the human bladder tumour cell line T24. After the G41 8 screening, a
clone that get the stable transfection of the BG was obtained. It was confirmed that
the ~G gene had been stably integrated into the genomic DNA of the 124 cell and
got a highly expression by the means of RNA blotting, in situ hybridization,
Immunoflurescence, Jmmunohistochemistry, Western Blot. The stably transfected
cell was named T24/ f3G. The biological characteristics of the fG transfected cell
were studied by the methods of microscopy and electron microscop.
3.The series studies of the in vitro effects of the prodrug Epi-bus on T24/ f3G
showed that the stably transfected cells are more sensitive to the Epi-bus by 1000
fold than the parent, with the IC50 0.1 7g/L. The Epi-bus with a concentration of
2.92g/l sustaining for 4-5 days had little affection on the growth of the transfected
cells, but will kill all the cells with high j3G expression. The concentration of
0.292g11 for 6-7 days will kill the transfected cells, showing the obvious uicide
effects The T24/G cells were mixed with parental cells not transfected with
various proportion and 70%-90% of the mixed cells were killed when the 124/ f3G
cell accounting for 1 0%-20%. It showed the potent stand-by effect.
The study showed that the 13G gene and its prodrug could be introduced into the
suicide gene system and the j3G gene transfection mediated by the LipofectAMlNE-
retrovirus eukariotic expression vector combining the 13G prodrug managment will
be the promising strategy for bladder therapy.
引文
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70． Bukhar MA, Evereet JL and Ross WC. Aryl-2-halogenoalkylamines-XXVI Glucuronic, sulphuric and phosphoric esters of p-di-2-chlorethylamino-phenoloric. Biochem Pharmacel. 1972;21:963-7
71． Wang Shing-Ming, Shem Ji-Wang, et al. Specific activation of glucuronide prodrug by antibody-targeted conjugates for cancer therapy. Cancer Res. 1992;52:4484-91
72． Roffler SR, Wang SM, et al. Antineoplastic glucuronide prodrug treatment of human tumor cells targeted with a monoclone antibody-enzyme conjugate. Biochem Pharmacol. 1991;42:2062-5
73． Weenen H, Lankelma J, et al. Pharmacokinetic of 4-epidoxorubicin in man. Invest New Drugs. 1983;1 :59
74． Haisma HJ, Boven E, et al. A monoclonal antibody β-glucuronidase conjugated as activator of the prodrug epirabicin-glucruonide for specific treatment of cancer. Br J Cancer. 1992;66:474-8
75． Smit BJ, Albrechtcf LRW, et al. A phase I trial of hypoxoside as an oral prodrug for cancer therapy: absence of toxicity. S Afr Med J. 1995;85(9) :865-70
76． Stahl PD and Fishman WH. β-D-glucuronidase. Methods in Enzamatic Analysis. 1984;5:246-256
77． Connors TA, Farmer PB, Foster AB, et al. Metabolism of aniline mustard[N, N-di(a-chboroethyl)-aniline] . Biochem Pharmacol. 1973;22:1971-1980
78． Weenen H, Van Maanen JMS, et al. Metabolism of 4-modified analogs of doxorubicin: Unique glucuronidation pathway for 4-epidoxorubidin. Eru J Cancer Clin Oncol. 1984;20:919-926
79． Maftouh M, MONSARRAT Bm Rao RC, et al. Identification of the glucuronide and glutath one conjugates of the antitumor drug N~2-methyl-9-hydro xyellip-ticiniun acetate:comparative disposition of this drug with its olivaciniumisomer in rat bile. Drug Metab Dispos. 1984;12:111-119
80． Houba PH, Boven E, et al. Pronounced antitumor efficacy of doxorubicin when given as the prodrug DOX-GA3 in combination with a monoclonal antibody beta-glucuronidase conjugate, Int J Cancer. 2001 Feb 15;91 (4) :550-4
81． Fonseca MJ, Storm G, et al. Cationic polymeric gene delivery of beta-glucuronidase for doxorubicin prodrug therapy. J Gene Med. 1999 Nov-Dec;l(6) :407-14
82． Houba PH, Boven E, et al. Distribution and pharmacokinetics of the prodrug daunorubicin-GA3 in nude mice bearing human ovarian cancer xenografts. Biochem Pharmacol. 1999 Mar l5;57(6) :673-80
83． Murdter TE, Sperker B, et al. Enhanced uptake of doxorubicin into bronchial carcinoma: beta-Oglucuronidase mediates release of doxorubicin from a glucuronide prodrug(HMR 1826) at the tumor site. Cancer Res. 1997 Jun 15;57(12) :2440-5
84． Cheng TL, Chen BM, et al. Eficient clearance of poly(ethylene glycol)-modified immunoenzyme with anti-PEG monoclonal antibody for prodrug cancer therapy. Bioconjug Chem. 2000 Mar-Apr;11(2) :258-66
85． Leu YL, Roffler SR, Chern JW. Design and synthesis of water-soluble glucuronide derivatives of camptothecin for cancer prodrug monotherapy and antibody-directed enzyme prodrug therapy (ADEPT). J Med Chem. 1999 Sep 9;42(18) :3623-8
86． 高啸波 基因治疗新型病毒载体研究进展 国外医学遗传学分册.1999-22 (1) : 9-14
87． Ralph RW, Kufe D. Gene therapy of cancer. The Lancet. 1997; 349(suppl
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88. Radler JO, Koltover Ⅰ, et al. Structure of DNA-cationic liposome-complexes DNA intercalation in multiamellar membrances in distinct interhelic packing regimes. Science. 1997;257:810
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90. Calvez V, Rixxe O, Wang P, et al. Virus-free transfer of the herpes simplex vires thymidine kinase gene followed by ganciclovir treatment induces tumor death. Clin Cancer Res. 1996;2(1):47-51
91. Yarovoi SV, Mouawad R, Colbere-Garapin F,et al.In vitro sensitization of the B16 murine melanoma cells to ganciclovir by different RNA and plasmid DNA construction encoding HSV-tk.Gene Ther. 1996; 3 (10):913-918
92．张腾飞，钱关祥，陈诗书．人类基因治疗研究的现状．生命的化学．1999；19(1)：11-12
93．朱力．阳离子脂质体及其在体内基因转染中的应用．国外医学分子生物学分册．1999；21(3)：148-151
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97. Bagshow KD antibody directed enzymes revive anti-Cancer prodrugs concept.Br J cancer. 1987,56:531-534
98. Huber BE, Richards CA, Krenitsky TA, et al. Retroviral-mediatcd gene therapy:an innovative approach for cancer therapy. Proc Natl Acad sci USA. 1991,88:8039-8043
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67． Oshima A, Kyle JW, et al. Cloning sequencing and expression of cDNA for human β-glucuronidase. Proc Natl Acad Sci USA. 1987;84:685-689
68． Bagshow KD. Antibody directed enzymes revive anti-cancer prodrugs concept. Br J Cancer. 1987;56:531-4
69． Connors TA, Farmer PB, Foster AB, et al. Metabolism of aniline mustard[N, N-di(a-chboroethyl)-aniline] . Biochem Pharmacol. 1973 ;22:1971-1980
70． Bukhar MA, Evereet JL and Ross WC. Aryl-2-halogenoalkylamines-XXVI Glucuronic, sulphuric and phosphoric esters of p-di-2-chlorethylamino-phenoloric. Biochem Pharmacel. 1972;21:963-7
71． Wang Shing-Ming, Shem Ji-Wang, et al. Specific activation of glucuronide prodrug by antibody-targeted conjugates for cancer therapy. Cancer Res. 1992;52:4484-91
72． Roffler SR, Wang SM, et al. Antineoplastic glucuronide prodrug treatment of human tumor cells targeted with a monoclone antibody-enzyme conjugate. Biochem Pharmacol. 1991;42:2062-5
73． Weenen H, Lankelma J, et al. Pharmacokinetic of 4-epidoxorubicin in man. Invest New Drugs. 1983;1 :59
74． Haisma HJ, Boven E, et al. A monoclonal antibody β-glucuronidase conjugated as activator of the prodrug epirabicin-glucruonide for specific treatment of cancer. Br J Cancer. 1992;66:474-8
75． Smit BJ, Albrechtcf LRW, et al. A phase I trial of hypoxoside as an oral prodrug for cancer therapy: absence of toxicity. S Afr Med J. 1995;85(9) :865-70
76． Stahl PD and Fishman WH. β-D-glucuronidase. Methods in Enzamatic Analysis. 1984;5:246-256
77． Connors TA, Farmer PB, Foster AB, et al. Metabolism of aniline mustard[N, N-di(a-chboroethyl)-aniline] . Biochem Pharmacol. 1973;22:1971-1980
78． Weenen H, Van Maanen JMS, et al. Metabolism of 4-modified analogs of doxorubicin: Unique glucuronidation pathway for 4-epidoxorubidin. Eru J Cancer Clin Oncol. 1984;20:919-926
79． Maftouh M, MONSARRAT Bm Rao RC, et al. Identification of the glucuronide and glutath one conjugates of the antitumor drug N~2-methyl-9-hydro xyellip-ticiniun acetate:comparative disposition of this drug with its olivaciniumisomer in rat bile. Drug Metab Dispos. 1984;12:111-119
80． Houba PH, Boven E, et al. Pronounced antitumor efficacy of doxorubicin when given as the prodrug DOX-GA3 in combination with a monoclonal antibody beta-glucuronidase conjugate, Int J Cancer. 2001 Feb 15;91 (4) :550-4
81． Fonseca MJ, Storm G, et al. Cationic polymeric gene delivery of beta-glucuronidase for doxorubicin prodrug therapy. J Gene Med. 1999 Nov-Dec;l(6) :407-14
82． Houba PH, Boven E, et al. Distribution and pharmacokinetics of the prodrug daunorubicin-GA3 in nude mice bearing human ovarian cancer xenografts. Biochem Pharmacol. 1999 Mar l5;57(6) :673-80
83． Murdter TE, Sperker B, et al. Enhanced uptake of doxorubicin into bronchial carcinoma: beta-Oglucuronidase mediates release of doxorubicin from a glucuronide prodrug(HMR 1826) at the tumor site. Cancer Res. 1997 Jun 15;57(12) :2440-5
84． Cheng TL, Chen BM, et al. Eficient clearance of poly(ethylene glycol)-modified immunoenzyme with anti-PEG monoclonal antibody for prodrug cancer therapy. Bioconjug Chem. 2000 Mar-Apr;11(2) :258-66
85． Leu YL, Roffler SR, Chern JW. Design and synthesis of water-soluble glucuronide derivatives of camptothecin for cancer prodrug monotherapy and antibody-directed enzyme prodrug therapy (ADEPT). J Med Chem. 1999 Sep 9;42(18) :3623-8
86． 高啸波 基因治疗新型病毒载体研究进展 国外医学遗传学分册.1999-22 (1) : 9-14
87． Ralph RW, Kufe D. Gene therapy of cancer. The Lancet. 1997; 349(suppl
Ⅱ):10-12
88. Radler JO, Koltover Ⅰ, et al. Structure of DNA-cationic liposome-complexes DNA intercalation in multiamellar membrances in distinct interhelic packing regimes. Science. 1997;257:810
89. Sugaya S, Fujita K, Kikuchi A, et al. Inhibition of tumor growth by direct intratumoral gene transfer of herpes simplex virus thymidine kinase gene with DNA -liposome complexes. Hum Gene Ther.1996;7:223-230
90. Calvez V, Rixxe O, Wang P, et al. Virus-free transfer of the herpes simplex vires thymidine kinase gene followed by ganciclovir treatment induces tumor death. Clin Cancer Res. 1996;2(1):47-51
91. Yarovoi SV, Mouawad R, Colbere-Garapin F,et al.In vitro sensitization of the B16 murine melanoma cells to ganciclovir by different RNA and plasmid DNA construction encoding HSV-tk.Gene Ther. 1996; 3 (10):913-918
92．张腾飞，钱关祥，陈诗书．人类基因治疗研究的现状．生命的化学．1999；19(1)：11-12
93．朱力．阳离子脂质体及其在体内基因转染中的应用．国外医学分子生物学分册．1999；21(3)：148-151
94．司徒镇强，吴军正．细胞培养．世界图书出版社1994 西安
95. Nishihara E, Nagayama Y, Mawatari F, et al. Retrovirus-mediated herpes simplex virus thymidine kinase gene transduction renders human thyroid carcinoma cell lines sensitive to ganciclovir and rediation in vitro and in vivo. Endocrino.1997; 138:4577-4583
96. Haisma HJ, Boven E, Muigen MV, et al. A monoclonal antibody β -glucuronidase conjugate as activator of the prodrug epiabian-glucuronidase for specific treatment of cancer. Br J cancer. 1992,66:474-478
97. Bagshow KD antibody directed enzymes revive anti-Cancer prodrugs concept.Br J cancer. 1987,56:531-534
98. Huber BE, Richards CA, Krenitsky TA, et al. Retroviral-mediatcd gene therapy:an innovative approach for cancer therapy. Proc Natl Acad sci USA. 1991,88:8039-8043
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