蟾蜍毒素和华蟾素对H_(22)和S_(180)荷瘤小鼠的疗效及毒副作用的实验研究
摘要
前言
蟾蜍毒素是传统中药蟾酥的主要有效成分，主要以蟾酥的脂溶性成分为主，是一些具有相同骨架的甾族化合物，总称蟾蜍配基(Bufodienolides)。近年来的研究发现，蟾蜍毒素在诱导细胞分化、促进细胞凋亡、抑制血管生成、改变肿瘤细胞相关基因的表达等方面发挥抗肿瘤的作用。本实验室的前期工作发现，部分分离纯化的蟾酥的脂溶性成分(蟾蜍毒素混合物)可以明显抑制人类白血病HL60，K562，NB4细胞的增殖，增强ATRA对NB4及APL原代培养细胞的诱导分化作用，诱导HL60，K562，NB4，OVCAR3细胞的凋亡，诱导K562细胞发生G2/M期阻滞，OVCAR3细胞发生M期阻滞，下调HL60细胞BCL-2蛋白、K562细胞WT1mRNA及蛋白的表达。但其主要的抗肿瘤成分及作用机制目前尚不十分清楚。
本实验的目的是用乙醇提取蟾蜍耳后腺及皮肤腺分泌物原液，获得部分分离纯化的醇溶性的蟾蜍毒素混合物(以下简称蟾蜍毒素)，并探讨蟾蜍毒素是否可抑制荷瘤小鼠体内肿瘤细胞的生长，和在有效治疗剂量范围内对各内脏器官的毒副作用，来进一步证实蟾蜍毒素的抗肿瘤作用。
实验材料与方法
1．实验材料及仪器
1．1 中华大蟾蜍的耳后腺及皮脂腺分泌物原浆。华蟾素注射液(安徽金蟾生化股份有限公司产品)。
1．2 细胞株：H_(22)小鼠肝癌细胞株，S_(180)小鼠肉瘤细胞株，由中
国药科大学提供，用昆明种小鼠传代保种。
1 .3试剂:无水乙醇，蒸馏水，二甲基亚矾，RPMI一1640培养
基，10%胎牛血清，庆大霉素，肝素钠，硫化钠，H一E染色材料等。
1.4主要仪器:SIGMA 3K30低温离心机，减压蒸馏装置，
cH甩sT一Loc一IM真空冷冻干燥机，光学显微镜，医用净化工作
台，37度水浴箱，VITROS250全自动干化学分析仪，SYSMXKx-
21全自动血球分析仪等。
1 .5动物:昆明种小鼠由中国医科大学动物部提供。
2实验方法
2 .1蟾蛛毒素的制备:采用特殊工艺提取蟾赊耳后腺及皮脂
腺分泌物，用双蒸馏水充分搅拌溶解后离心，沉淀物用无水乙醇溶
解后离心取上清，减压蒸馏，制成干粉称重，计算提纯百分率。
2 .2肿瘤细胞的准备:取液氮保存的H22、5150细胞复苏后，加
人含10%胎牛血清的RPMI一1 640培养液，调整细胞浓度为5一6
x106个/d，再加人肝素钠抗凝，庆大霉素预防感染。小鼠下腹
部碘伏消毒后，腹腔注射0.2血细胞悬液，10天后用细针头消毒
后取腹水，呈乳白色，用生理盐水稀释，调整细胞浓度5一6 x106
个/血，加人肝素钠和庆大霉素，准备接种昆明种小鼠。
2.3荷瘤小鼠的制备:
2.3.1腹水瘤小鼠的制备:同上面细胞的准备，将调整好浓
度的细胞悬液0.2撇接种于小鼠的腹腔。
2.3.2实体瘤小鼠的制备:取小鼠，用脱毛剂褪净小鼠右下
肢外侧绒毛，用1而注射器皮下注射0.2血计数好的细胞悬液。
2 .4实验方法:
2.4.1蟾赊毒素的亚急性毒性实验:取昆明种小鼠80只，18
一22克，分成7组(取蟾赊毒素干粉用DMSO溶解后，用双蒸馏水
配制成不同浓度的蟾蛛毒素溶液，保证等容量注人)，按lm岁kg，
sm扩kg，IOm扩kg，20m扩kg，40m扩kg，80m扩kg，16Om岁kg给
药，连续给药9天，次日处死，取内脏，作石蜡切片，H一E染色。
，2 .4.2腹水瘤小鼠实验方法:每次取昆明种小鼠80只，18-
22克，腹腔接种S;so小鼠肉瘤细胞，次日依体重分成7组，实验组
分别给予sm岁kg，0 .sm扩kg，0.lmg/kg的蟾赊毒素，阳性对照组
分别给予17Om群kg，17m扩kg，3 .4m岁kg的华蟾素，正常对照组给
予0 .5%的二甲基亚矾(DMSO)。连续给药9天后停药，观察小鼠
的生存时间，计算生命延长率。H22接种方法同上，给药剂量为:蟾
赊毒素组sm扩kg，华蟾素组为170m岁kg，正常对照组给以0 .5%
DMSO
2
4.3实体瘤小鼠的实验方法:每次取昆明种小鼠80只，18
一22克。皮下接种5180小鼠肉瘤细胞，次日依体重分成7组，给药
剂量及给药时间同5180腹水瘤组，次日处死取血、瘤体及内脏，检
测血常规，肝肾功能和心肌酶谱，瘤体和内脏作石蜡H一E染色切
片，胸骨切片检测骨髓造血系统功能。H22实体瘤组方法同518。组，
给药剂量同H22腹水瘤组。
3.统计学处理:所有数据用均数士标准差表示，显著性检验用
q检验和t检验，P<0.05为差异显著。
结果
1.用无水乙醇分离提取的蟾赊毒素占蟾蛛分泌物原浆的百
分比为10.55%。
2.1一smg/kg的蟾蛛毒素对小鼠无明显影响，10m留kg以上
的蟾蛛毒素可以引起小鼠的毒性反应，40m扩kg的蟾蛛毒素可以
引起小鼠的抽搐，loomg/kg的蟾蛛毒素可引起小鼠全部抽搐死
亡。病理切片显示对心脏、肝脏的损伤较重，对肾脏、胃肠道的毒
性较小。
3.0.5一sm岁kg的蟾赊毒素有延长荷518。腹水瘤组小鼠的生
存时间的趋势(P>0.05)。sm扩kg蟾赊毒素可以明显抑制5150实
体瘤组织的生长(P<0.05);5 mg/kg蟾蛛毒素可以明显延长荷
H22腹水瘤小鼠的生存时间(P<0.05)，抑制H22实体瘤组织的生
长(P<0.05)。H一E石蜡切片可以见到肿瘤细胞局灶性或大片
状坏死区，肿瘤细胞失去正常形态，细胞核固缩、碎裂或崩解;而
Introduction
Toad venom is a major component derived from Chan Su,a kind of traditional Chinese medicine. It is generally called bufodienoHdes based on the same steroid nucleus. In previously study , toad venom may play a role of anti - tumor by inducing differentiation , improving apoptosis , inhibiting angiogenesis and altering gene expression correlated with tumor cell. An extract of partial purification was prepared from Chinese toad secrection ( Toad venom mixture , TVM ) , which could inhibit the cell proliferation of the human leukemic cell line HL60,K562,NB4, improve the ATRA action of inducing differentiation to the leukemia cell line NB4 and the primary culture cell of APL, induce apoptosis of HL60,K562,NB4,OVCAR3, downregulate Bcl - 2 protain expression and WT1 gene expression during human leukemic cell differentiation and apoptosis. But the mainly component
of anti - tumor and the anti - tumor mechanisms is still unclear.
The purpose of this study was to determine the efficacy of the eth-anolic extract ( Toad venom mixture , TVM ) from Chinese toad secretion in treating kun - ming mice loaded H22 and S180 tumor cell.
Materials and methods
1. Materials and instruments
1.1 Chinese toad secretion, parenteral solution of hua chan su (made in anhui jinchan corporation).
1.2 Cell line: H22 cell line of mice hepatic tumor and S180 cell line of mice sarcom was provided by China Drug University.
1. 3 Reagent; anhydrous alcohol, distilled water, Dimethyl Sulfoxide (DMSO) ,RPMI - 1640 culture medium, 10% fetal bovine serum, gentamycin, heparin sodium, sodium sulfide, H - E staining materials ect.
1.4 Major Instruments; sigma 3k30 centrifuger, decompression stilling assembly,light microscope,depuration table,etc.
1. 5 Animals: kun - ming mice was provided by China Medical University .
2. Methods
2. 1 Preparation of toad venom: We extract the secretion of toad sebaceous gland by special technique and dissolve them in double -stilling water, then centrifugate them. Put the depositor in anhydrous alcohol,then centrifugate them and decompress stilling,made them into shaf - powder at last, calculate the percentage of purification
. 2. 2 Preparation of tumor cell; Putting the H22,S180 cell into RPMI 1640 supplemented with 10% fetal bovine serum after they were resuscitated. We took 0. 2ml cell suspension and injected them into intraperitoneal cavity. We drew ascites after 10 days , calculated and adjusted cell concentrition to 5 - 6 106 /ml with containing 0. 3 l/ml gentamycine and 3.2 J/ml heparin sodium.
2.3 Preparation of mice loaded with tumor cell;
2.3. 1 Preparation of mice loaded with ascites tumor cell: see 2. 2. Inoculating 0.2ml cell suspension(5 -6 106 /ml) into mice's intraperitoneal cavity.
2.3.2 Preparation of mice loaded entity tumor: We cleared the villi with depilator containing sodium sulfide and then injected 0. 2ml cell suspension to subcutaneous tissue of right lower extremity.
2.4 methods
2.4.1 Subacute toxicity expriment of toad venom: All 80 mouse were divided 7 groups depending on their weight. TVM were administrated to mouse ranging from Img/kg to 160mg/kg for 9 days. The next day, we kill them and take out of bowel to embed in paraffin wax, make sections and stain with H - E staining.
2.4.2 Experiment of mice loaded with H22 or S180 ascites tumor cell;We inoculated S180 tumor cells by intraperitoneal injections and the next day divided them into 7 groups. Experiment groups (3groups) were administrated TVM at 5mg/kg, 0.5mg/kg,0. Img/kg. Huachan-su groups were administrated 170mg/kg, 17mg/kg, 3. 4mg/kg used as positive groups and negitive group were given 0. 5% DMSO for 9 days. After stopping administration, we observed the life span and calculated extend rate of life span of load - tumor mouse. The mouse loaded H22 tumor cell were divided 3 groups and the dose is 5mg/kg of toad venom, 170mg/kg of huachansu and 0. 5% DMSO by sequence.
2.4.3 Experiment of mice loaded with H22 or S180 entity tumor cell:We inoculated
蟾蜍毒素是传统中药蟾酥的主要有效成分，主要以蟾酥的脂溶性成分为主，是一些具有相同骨架的甾族化合物，总称蟾蜍配基(Bufodienolides)。近年来的研究发现，蟾蜍毒素在诱导细胞分化、促进细胞凋亡、抑制血管生成、改变肿瘤细胞相关基因的表达等方面发挥抗肿瘤的作用。本实验室的前期工作发现，部分分离纯化的蟾酥的脂溶性成分(蟾蜍毒素混合物)可以明显抑制人类白血病HL60，K562，NB4细胞的增殖，增强ATRA对NB4及APL原代培养细胞的诱导分化作用，诱导HL60，K562，NB4，OVCAR3细胞的凋亡，诱导K562细胞发生G2/M期阻滞，OVCAR3细胞发生M期阻滞，下调HL60细胞BCL-2蛋白、K562细胞WT1mRNA及蛋白的表达。但其主要的抗肿瘤成分及作用机制目前尚不十分清楚。
本实验的目的是用乙醇提取蟾蜍耳后腺及皮肤腺分泌物原液，获得部分分离纯化的醇溶性的蟾蜍毒素混合物(以下简称蟾蜍毒素)，并探讨蟾蜍毒素是否可抑制荷瘤小鼠体内肿瘤细胞的生长，和在有效治疗剂量范围内对各内脏器官的毒副作用，来进一步证实蟾蜍毒素的抗肿瘤作用。
实验材料与方法
1．实验材料及仪器
1．1 中华大蟾蜍的耳后腺及皮脂腺分泌物原浆。华蟾素注射液(安徽金蟾生化股份有限公司产品)。
1．2 细胞株：H_(22)小鼠肝癌细胞株，S_(180)小鼠肉瘤细胞株，由中
国药科大学提供，用昆明种小鼠传代保种。
1 .3试剂:无水乙醇，蒸馏水，二甲基亚矾，RPMI一1640培养
基，10%胎牛血清，庆大霉素，肝素钠，硫化钠，H一E染色材料等。
1.4主要仪器:SIGMA 3K30低温离心机，减压蒸馏装置，
cH甩sT一Loc一IM真空冷冻干燥机，光学显微镜，医用净化工作
台，37度水浴箱，VITROS250全自动干化学分析仪，SYSMXKx-
21全自动血球分析仪等。
1 .5动物:昆明种小鼠由中国医科大学动物部提供。
2实验方法
2 .1蟾蛛毒素的制备:采用特殊工艺提取蟾赊耳后腺及皮脂
腺分泌物，用双蒸馏水充分搅拌溶解后离心，沉淀物用无水乙醇溶
解后离心取上清，减压蒸馏，制成干粉称重，计算提纯百分率。
2 .2肿瘤细胞的准备:取液氮保存的H22、5150细胞复苏后，加
人含10%胎牛血清的RPMI一1 640培养液，调整细胞浓度为5一6
x106个/d，再加人肝素钠抗凝，庆大霉素预防感染。小鼠下腹
部碘伏消毒后，腹腔注射0.2血细胞悬液，10天后用细针头消毒
后取腹水，呈乳白色，用生理盐水稀释，调整细胞浓度5一6 x106
个/血，加人肝素钠和庆大霉素，准备接种昆明种小鼠。
2.3荷瘤小鼠的制备:
2.3.1腹水瘤小鼠的制备:同上面细胞的准备，将调整好浓
度的细胞悬液0.2撇接种于小鼠的腹腔。
2.3.2实体瘤小鼠的制备:取小鼠，用脱毛剂褪净小鼠右下
肢外侧绒毛，用1而注射器皮下注射0.2血计数好的细胞悬液。
2 .4实验方法:
2.4.1蟾赊毒素的亚急性毒性实验:取昆明种小鼠80只，18
一22克，分成7组(取蟾赊毒素干粉用DMSO溶解后，用双蒸馏水
配制成不同浓度的蟾蛛毒素溶液，保证等容量注人)，按lm岁kg，
sm扩kg，IOm扩kg，20m扩kg，40m扩kg，80m扩kg，16Om岁kg给
药，连续给药9天，次日处死，取内脏，作石蜡切片，H一E染色。
，2 .4.2腹水瘤小鼠实验方法:每次取昆明种小鼠80只，18-
22克，腹腔接种S;so小鼠肉瘤细胞，次日依体重分成7组，实验组
分别给予sm岁kg，0 .sm扩kg，0.lmg/kg的蟾赊毒素，阳性对照组
分别给予17Om群kg，17m扩kg，3 .4m岁kg的华蟾素，正常对照组给
予0 .5%的二甲基亚矾(DMSO)。连续给药9天后停药，观察小鼠
的生存时间，计算生命延长率。H22接种方法同上，给药剂量为:蟾
赊毒素组sm扩kg，华蟾素组为170m岁kg，正常对照组给以0 .5%
DMSO
2
4.3实体瘤小鼠的实验方法:每次取昆明种小鼠80只，18
一22克。皮下接种5180小鼠肉瘤细胞，次日依体重分成7组，给药
剂量及给药时间同5180腹水瘤组，次日处死取血、瘤体及内脏，检
测血常规，肝肾功能和心肌酶谱，瘤体和内脏作石蜡H一E染色切
片，胸骨切片检测骨髓造血系统功能。H22实体瘤组方法同518。组，
给药剂量同H22腹水瘤组。
3.统计学处理:所有数据用均数士标准差表示，显著性检验用
q检验和t检验，P<0.05为差异显著。
结果
1.用无水乙醇分离提取的蟾赊毒素占蟾蛛分泌物原浆的百
分比为10.55%。
2.1一smg/kg的蟾蛛毒素对小鼠无明显影响，10m留kg以上
的蟾蛛毒素可以引起小鼠的毒性反应，40m扩kg的蟾蛛毒素可以
引起小鼠的抽搐，loomg/kg的蟾蛛毒素可引起小鼠全部抽搐死
亡。病理切片显示对心脏、肝脏的损伤较重，对肾脏、胃肠道的毒
性较小。
3.0.5一sm岁kg的蟾赊毒素有延长荷518。腹水瘤组小鼠的生
存时间的趋势(P>0.05)。sm扩kg蟾赊毒素可以明显抑制5150实
体瘤组织的生长(P<0.05);5 mg/kg蟾蛛毒素可以明显延长荷
H22腹水瘤小鼠的生存时间(P<0.05)，抑制H22实体瘤组织的生
长(P<0.05)。H一E石蜡切片可以见到肿瘤细胞局灶性或大片
状坏死区，肿瘤细胞失去正常形态，细胞核固缩、碎裂或崩解;而
Introduction
Toad venom is a major component derived from Chan Su,a kind of traditional Chinese medicine. It is generally called bufodienoHdes based on the same steroid nucleus. In previously study , toad venom may play a role of anti - tumor by inducing differentiation , improving apoptosis , inhibiting angiogenesis and altering gene expression correlated with tumor cell. An extract of partial purification was prepared from Chinese toad secrection ( Toad venom mixture , TVM ) , which could inhibit the cell proliferation of the human leukemic cell line HL60,K562,NB4, improve the ATRA action of inducing differentiation to the leukemia cell line NB4 and the primary culture cell of APL, induce apoptosis of HL60,K562,NB4,OVCAR3, downregulate Bcl - 2 protain expression and WT1 gene expression during human leukemic cell differentiation and apoptosis. But the mainly component
of anti - tumor and the anti - tumor mechanisms is still unclear.
The purpose of this study was to determine the efficacy of the eth-anolic extract ( Toad venom mixture , TVM ) from Chinese toad secretion in treating kun - ming mice loaded H22 and S180 tumor cell.
Materials and methods
1. Materials and instruments
1.1 Chinese toad secretion, parenteral solution of hua chan su (made in anhui jinchan corporation).
1.2 Cell line: H22 cell line of mice hepatic tumor and S180 cell line of mice sarcom was provided by China Drug University.
1. 3 Reagent; anhydrous alcohol, distilled water, Dimethyl Sulfoxide (DMSO) ,RPMI - 1640 culture medium, 10% fetal bovine serum, gentamycin, heparin sodium, sodium sulfide, H - E staining materials ect.
1.4 Major Instruments; sigma 3k30 centrifuger, decompression stilling assembly,light microscope,depuration table,etc.
1. 5 Animals: kun - ming mice was provided by China Medical University .
2. Methods
2. 1 Preparation of toad venom: We extract the secretion of toad sebaceous gland by special technique and dissolve them in double -stilling water, then centrifugate them. Put the depositor in anhydrous alcohol,then centrifugate them and decompress stilling,made them into shaf - powder at last, calculate the percentage of purification
. 2. 2 Preparation of tumor cell; Putting the H22,S180 cell into RPMI 1640 supplemented with 10% fetal bovine serum after they were resuscitated. We took 0. 2ml cell suspension and injected them into intraperitoneal cavity. We drew ascites after 10 days , calculated and adjusted cell concentrition to 5 - 6 106 /ml with containing 0. 3 l/ml gentamycine and 3.2 J/ml heparin sodium.
2.3 Preparation of mice loaded with tumor cell;
2.3. 1 Preparation of mice loaded with ascites tumor cell: see 2. 2. Inoculating 0.2ml cell suspension(5 -6 106 /ml) into mice's intraperitoneal cavity.
2.3.2 Preparation of mice loaded entity tumor: We cleared the villi with depilator containing sodium sulfide and then injected 0. 2ml cell suspension to subcutaneous tissue of right lower extremity.
2.4 methods
2.4.1 Subacute toxicity expriment of toad venom: All 80 mouse were divided 7 groups depending on their weight. TVM were administrated to mouse ranging from Img/kg to 160mg/kg for 9 days. The next day, we kill them and take out of bowel to embed in paraffin wax, make sections and stain with H - E staining.
2.4.2 Experiment of mice loaded with H22 or S180 ascites tumor cell;We inoculated S180 tumor cells by intraperitoneal injections and the next day divided them into 7 groups. Experiment groups (3groups) were administrated TVM at 5mg/kg, 0.5mg/kg,0. Img/kg. Huachan-su groups were administrated 170mg/kg, 17mg/kg, 3. 4mg/kg used as positive groups and negitive group were given 0. 5% DMSO for 9 days. After stopping administration, we observed the life span and calculated extend rate of life span of load - tumor mouse. The mouse loaded H22 tumor cell were divided 3 groups and the dose is 5mg/kg of toad venom, 170mg/kg of huachansu and 0. 5% DMSO by sequence.
2.4.3 Experiment of mice loaded with H22 or S180 entity tumor cell:We inoculated
引文
1．刘云鹏，等．蟾蜍灵诱导K562细胞分化和凋亡过程中WT1表达的下调中华血液学杂志，2002 Jul；23(7)：356-9
2．平井康晴，庄司政满，等．含蟾酥制剂“救心”的抗肿瘤作用，Natural medicines，1995；49(3)279-83
3．李风云，陈浩然，张喜兰，等．中药制剂抗肿瘤作用实验研究，中医药学报，1999；27(封三)
4．王德昌，范贤俊，李润田，王永泉．蟾酥某些药理作用的实验研究药学通报1980；5：426
5．王浴生，等．中药药理与应用第一版人民卫生出版社，1983；1258-1261
6．徐向田，等．华蟾素治疗慢性已肝病毒携带者疗效观察临床医学，1990，10(4)：169
7．刘莉，蒋亚生，张士华，等．抗癌中药制剂局部注射对裸鼠人肝癌细胞核DNA含量的影响中国肿瘤临床，1993；20(20)：140-42
8．蒋庭章，等．蟾酥水溶性总成分注射液治疗晚期癌症218例 南京中医学院学报1988；1：22
9．葛勤等．蟾酥的化学及制剂质量研究近况 中成药，1998；20：38-40
10. Numazawa S, Shinoki MA, Ito H. Involvement of Na~+, K~+ - ATPase inhibition in K562 cell differentiation induced by bufalin. J Cell Physiol 1994 Jul;160(1):113 -20
11. Zhang LS, Nakaya K,Yoshida T, et al。Bufalin as a potent inducer of differentiation of human myeloid leukemia cells. Biolchem Biophys Res Commun 1991 Jul 31;178(2):686-93
12. Jing Y,Watabe M, Hashimoto S,et al. Cell cycle arrestand protein kinase modulating effect of bufalin on human leukemia ML1 cells. Anticancer Res 1994 May - Jun; 14(3A):1193 - 8
13. Pastor N, Dominguez I, Mateos S, Cortes F. A comparative study of genotoxic effects of anti-Topoisomerase Ⅱ drugs ICRF-193 and bufalin in Chinese hamster ovary cells. Mutat Res 2002 Mar 25;515(1-2):171-80
14. Yamada K, Hino K, Tomoyasu S, et al. Enhancement by bufalin of retinoic acid - induced differentiation of acute promyelocytic leukemia cells in primary culture. Leuk Res 1998 Jul;22(7):589 - 95
15. Akiyama M., Ogura M, Iwai M, et al. Effect of bufalin on growth and differentiation of human skin carcinoma cells in vitro. Hum Cell 1999 Dec;12(4):205 -9
16. Numazawa S, Inoue N, Nakura H, et al. A cardiotonic steroid bufalin - induced differentiation of THP - 1 cells. Involvement of Na~+, K~+ -ATPase inhibition in the early changes in proto-oncogene expression. Biochem Pharmacol, 1996 Jul 26;52(2):321 - 9
17. Efferth T, Davey M, etc. Activity of drugs from traditional Chinese medicine toward sensitive and MDR1 or MRP1 -overexpressing multidrug-resistant human CCRF-CEM leukemia cells Blood Cells Mol Dis 2002 Mar - apr;28(2): 160 - 8)
18．陈小义，买霞，徐瑞成，等．Bufalin对MGC-803细胞生长、分化的影响．武警医学院学报2000；9(3)：155-6
19．陈小义，徐瑞成，陈莉，等．蟾蜍灵对HL60细胞的生长抑制及凋亡诱导作用．中华血液杂志2000；21(7)：359-361
20. Masuda Y, Kawazoe N, Nakajo S, et al. Bufalin induces apoptosis and influences the expression of apoptosis - related genes in hu-
man leukemia cells. Leuk Res 1995 Aug;19(8):549-56
21. Watabe M, Masuda Y, Nakajo S, et al. The cooperative interaction of two different signaling pathways in response to bufalin induces apoptosis in human leukemia UA937 Cells. J Biol Chem. 1996; 271(24):14067-72
22. Kurosawa M, Tani Y, Nishimura S, et al. Distinct PKC isozymes regulate bufalin-induced differenation and apoptosis in human monocytic cells. Am J Physiol Cell Physiol 2001 Mar; 280(3):CA59-64
23. Yeh JY, Huang WJ, Kan SF, Wang PS. Effect of bufalin and cinobufagin on the proliferation of androgen dependent and independent prostate cancer cells. Prostate 2003 Feb 1;54(2):112-24
24．赵建斌，崔勤等．华蟾毒精抗癌作用的体外研究第四军医大学学报2001，22(16)．-1504-1507
25．韩仲明，苏红星，黄普生，等．华蟾素对喉癌细胞凋亡的基础研究．中国中西医结合耳鼻咽喉科杂志，2000；8(3)：111-14
26. Lee DY, Yasuda M, Yammoto T, et al. Bufalin inhibits endothelial cell proliferation and angiogenesis in vitro. Life Sci 1997;60(2):127-34
27．孙关林，等．中西医结合杂志，1984；4(5)：297
28．张静．华蟾素联合化疗治疗晚期恶性肿瘤的疗效分析．肿瘤；2000；20(5)：379-8129．王忠良，曲华，李健丽．华蟾素联合平消胶囊治疗中晚期肝癌．河北中西医结合杂志，1995；4(3)：63
30. Brubacher JR, Ravikumar PR, Bania T, Treatment of toad venom poisoning with digoxin - specific Fab fragments. Chest 1996 Nov; 110(5):1282-8
31. Brubacher JR, Lachmanen D, Ravikumar PR Efficacy of digoxin specific Fab fragments (Digibind) in the treatment of toad venom
poisoning. Toxicon 1999 Jun; 37 (6) : 931-42
32． Bick RJ,Poindexter BJ,Sweney RR,Dasgupta A. Effects of Chan Su, a traditional Chinese medicine, on the calcium transients of isolated cardiomyocytes: cardiotoxity due to more then Na, K-AT-Pase blocking. Life Sci 2002 Dec 27;72(6) :699-709
2．平井康晴，庄司政满，等．含蟾酥制剂“救心”的抗肿瘤作用，Natural medicines，1995；49(3)279-83
3．李风云，陈浩然，张喜兰，等．中药制剂抗肿瘤作用实验研究，中医药学报，1999；27(封三)
4．王德昌，范贤俊，李润田，王永泉．蟾酥某些药理作用的实验研究药学通报1980；5：426
5．王浴生，等．中药药理与应用第一版人民卫生出版社，1983；1258-1261
6．徐向田，等．华蟾素治疗慢性已肝病毒携带者疗效观察临床医学，1990，10(4)：169
7．刘莉，蒋亚生，张士华，等．抗癌中药制剂局部注射对裸鼠人肝癌细胞核DNA含量的影响中国肿瘤临床，1993；20(20)：140-42
8．蒋庭章，等．蟾酥水溶性总成分注射液治疗晚期癌症218例 南京中医学院学报1988；1：22
9．葛勤等．蟾酥的化学及制剂质量研究近况 中成药，1998；20：38-40
10. Numazawa S, Shinoki MA, Ito H. Involvement of Na~+, K~+ - ATPase inhibition in K562 cell differentiation induced by bufalin. J Cell Physiol 1994 Jul;160(1):113 -20
11. Zhang LS, Nakaya K,Yoshida T, et al。Bufalin as a potent inducer of differentiation of human myeloid leukemia cells. Biolchem Biophys Res Commun 1991 Jul 31;178(2):686-93
12. Jing Y,Watabe M, Hashimoto S,et al. Cell cycle arrestand protein kinase modulating effect of bufalin on human leukemia ML1 cells. Anticancer Res 1994 May - Jun; 14(3A):1193 - 8
13. Pastor N, Dominguez I, Mateos S, Cortes F. A comparative study of genotoxic effects of anti-Topoisomerase Ⅱ drugs ICRF-193 and bufalin in Chinese hamster ovary cells. Mutat Res 2002 Mar 25;515(1-2):171-80
14. Yamada K, Hino K, Tomoyasu S, et al. Enhancement by bufalin of retinoic acid - induced differentiation of acute promyelocytic leukemia cells in primary culture. Leuk Res 1998 Jul;22(7):589 - 95
15. Akiyama M., Ogura M, Iwai M, et al. Effect of bufalin on growth and differentiation of human skin carcinoma cells in vitro. Hum Cell 1999 Dec;12(4):205 -9
16. Numazawa S, Inoue N, Nakura H, et al. A cardiotonic steroid bufalin - induced differentiation of THP - 1 cells. Involvement of Na~+, K~+ -ATPase inhibition in the early changes in proto-oncogene expression. Biochem Pharmacol, 1996 Jul 26;52(2):321 - 9
17. Efferth T, Davey M, etc. Activity of drugs from traditional Chinese medicine toward sensitive and MDR1 or MRP1 -overexpressing multidrug-resistant human CCRF-CEM leukemia cells Blood Cells Mol Dis 2002 Mar - apr;28(2): 160 - 8)
18．陈小义，买霞，徐瑞成，等．Bufalin对MGC-803细胞生长、分化的影响．武警医学院学报2000；9(3)：155-6
19．陈小义，徐瑞成，陈莉，等．蟾蜍灵对HL60细胞的生长抑制及凋亡诱导作用．中华血液杂志2000；21(7)：359-361
20. Masuda Y, Kawazoe N, Nakajo S, et al. Bufalin induces apoptosis and influences the expression of apoptosis - related genes in hu-
man leukemia cells. Leuk Res 1995 Aug;19(8):549-56
21. Watabe M, Masuda Y, Nakajo S, et al. The cooperative interaction of two different signaling pathways in response to bufalin induces apoptosis in human leukemia UA937 Cells. J Biol Chem. 1996; 271(24):14067-72
22. Kurosawa M, Tani Y, Nishimura S, et al. Distinct PKC isozymes regulate bufalin-induced differenation and apoptosis in human monocytic cells. Am J Physiol Cell Physiol 2001 Mar; 280(3):CA59-64
23. Yeh JY, Huang WJ, Kan SF, Wang PS. Effect of bufalin and cinobufagin on the proliferation of androgen dependent and independent prostate cancer cells. Prostate 2003 Feb 1;54(2):112-24
24．赵建斌，崔勤等．华蟾毒精抗癌作用的体外研究第四军医大学学报2001，22(16)．-1504-1507
25．韩仲明，苏红星，黄普生，等．华蟾素对喉癌细胞凋亡的基础研究．中国中西医结合耳鼻咽喉科杂志，2000；8(3)：111-14
26. Lee DY, Yasuda M, Yammoto T, et al. Bufalin inhibits endothelial cell proliferation and angiogenesis in vitro. Life Sci 1997;60(2):127-34
27．孙关林，等．中西医结合杂志，1984；4(5)：297
28．张静．华蟾素联合化疗治疗晚期恶性肿瘤的疗效分析．肿瘤；2000；20(5)：379-8129．王忠良，曲华，李健丽．华蟾素联合平消胶囊治疗中晚期肝癌．河北中西医结合杂志，1995；4(3)：63
30. Brubacher JR, Ravikumar PR, Bania T, Treatment of toad venom poisoning with digoxin - specific Fab fragments. Chest 1996 Nov; 110(5):1282-8
31. Brubacher JR, Lachmanen D, Ravikumar PR Efficacy of digoxin specific Fab fragments (Digibind) in the treatment of toad venom
poisoning. Toxicon 1999 Jun; 37 (6) : 931-42
32． Bick RJ,Poindexter BJ,Sweney RR,Dasgupta A. Effects of Chan Su, a traditional Chinese medicine, on the calcium transients of isolated cardiomyocytes: cardiotoxity due to more then Na, K-AT-Pase blocking. Life Sci 2002 Dec 27;72(6) :699-709
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