注射用蟾皮总碱抗肿瘤有效性和安全性研究
|
摘要
注射用蟾皮总碱为蟾皮提取液的冻干粉针剂型,是中华大蟾蜍阴干全皮提取精制而成的无菌粉针,具有清热解毒、消肿、止痛、提高机体免疫力等功效。本课题创新性的提出了一种新的吲哚类生物碱提取工艺,使提取物中吲哚类生物碱的含量达到90%以上,并按国家一类新药的标准进行了成药性研究--药效学、一般药理学、毒理学研究。并制定了注射用蟾皮总碱的质量标准,为注射用蟾皮总碱的临床实验建立了理论基础。
实验结果表明:
注射用蟾皮总碱的生产和制备工艺稳定,适合大规模工业化生产。
不同浓度注射用蟾皮总碱对人肿瘤细胞增殖呈现出不同程度的抑制作用,浓度为0.4352 ug/ml的注射用蟾皮总碱对人胃癌细胞SGC-7901、人肝癌细胞SMMC-7721和人宫颈癌细胞Hela的抑制率分别为41.41%、62.41%、80.53%,均高于同剂量的华蟾素注射液组的肿瘤抑制率。
注射用蟾皮总碱急性毒性实验检测不到明显的急性毒性反应。
长期毒性实验结果显示,高剂量、长时间(170 ug/kg、连续三个月)给药后大鼠血液学检查肾脏尿素氮含量为9.94mmol/L,较正常值明显升高,恢复期一个月时尿素氮含量恢复正常。肾脏病理学检查在三个月时表现为肾小管细胞肿胀、变形、境界模糊,胞核消失,细胞坏死。恢复期一个月时肾脏病理学检查已恢复正常。Beagle犬连续高剂量、长时间(52ug/kg、连续三个月)给药后,血液学检查尿素氮含量为5.54mmol/L ,尿常规检查尿液pH值为5.13、尿维生素C含量为0.75mmol/L,尿沉渣镜检(x400)可见7-9个红细胞,与对照组比较,差异明显;病理学检查显示给药三个月时犬肾小管细胞形状不规则,单位面积细胞数量减少,部分胞体皱缩,胞浆透亮度下降,细胞变圆。恢复期1个月后病理检查恢复正常。实验表明注射用蟾皮总碱较大剂量连续用药3个月对大鼠及Beagle犬肾脏功能有影响,恢复期1个月肾脏各项指标恢复正常。
各项实验证明,采用新工艺提取的注射用蟾皮总碱质量工艺稳定,药效显著,无明显及长期毒副作用,可进行临床试用。
Tumor is one kind of familiar disease, and with the speed up of aging and pollution, it becomes the most serious disease that endangers the people’s health. The death rate of tumor sufferers is a little less than that of cardiopathy. This makes it the second killer of human health.
The alkaloids extracted from toad skin are broadly used as one of anti-tumor drugs at present. INj-alkaloids in toad skin is a safe and effective medicine to treat the PLC followed the metastatic tumer of lung .
The research of pharmaceutical included pharmaceutical technology and quality standard. The main purpose of this article mainly works over the new Medicine prescription, manufacture technology, quality standard and pharmacology according to the request of the National New Drug Examination and Approval Method.
Studied the extract process of preparation, developed an optimal process of extraction with traditional medicated process and modern technique, the method is water extraction and ethanol precipitation ,water washing-out supernumerary substance in polyamide column, collect ethanol elution. It is obey the rule of Chinese Medical, extract the effective component and simplify the process at the same time. Above all, the best abstract technology of Vapour distillation was determined, INj-alkaloids in toad skin quality standard was established, INj-alkaloids in toad skin is steady.
On the other hand, according to the guide rules of Chinese medical injection in new medicine evaluation, to make sure this injection is safety, effective and stability, assurance the quality of the injection, and found the base of injection’s fingerprint. The content of one was determined by HPLC. The method is simple, accurate reproducible and can be applied for quality control. Research of the quantity standard has been completed, standard of drug and preparation has been set up, the result show this method is simple and feasible and can guarantee the stability of this preparation.
And anti-tumor efficacy and safety of INj-alkaloids in toad skin by acute-term toxicity test has been researched , in vitro pharmacodynamics test, safety test, pharmacology test and long-term toxicity test and so on.
Through to acute-term toxicity test and LD50 determination, give the result of clinical medication, determiones the dose of medicine. About 2min's the drug poisoning mouse starts to appear the active reduction, peak wool, shuts item, walk haltingly, waggle, jitter. The mouse which are poisoned of large dose have a deep and slow breath, weak and limp. Beyond die the body-righting reflex of dead animals doesn't disappear, the breath from slow to tenuity. Dead mouse have respiration ceases first and cardiac arrest last, which are dissected have no abnormality acquire of organs. The undead ones who are killed 2 weeks later also have no abnormality acquire of organs.
The in vitro experiment of pharmacodynamics shows alkaloids of toad skin's inhibition on tumor cells cultured in vitro using MTT analysis, and tumor control rate via cell appearance observed by inverted microscope. The results indicate that INj-alkaloids in toad skin inhibits evidently the human tumor cells proliferation in vitro and the inhibition depends a certain dose.
Estimate the safety of the medicine, results of hepersensibility test, heamolyticus test and BV stimulus test on INj-alkaloids in toad skin show that INj-alkaloids in toad skin has no allergize on guinea pig, no obviously effect of in vitro heamolyticus and agglomeration in rabbit's red cell, it also has no obviously stimulation on rabbit's BV and can not cause pathological change of tissues of BV surrounding.
The Pharmacology research uses to appraise the INj-alkaloids in toad skin regarding the animal cardiovascular, the respiratory and the nervous system influence. Through pressure transducer, tension transducer, needle-electrode connectting BL-420E biological function experiment system, determine INj-alkaloids in toad skin to take the different time compartment dogs femoral artery blood pressure, breath and the electrocardiogram around separately: observes the medicine with multi-purpose mouse activity recording instrument to the mouse spontaneous activity influence; observes the medicine with Fall-tiltboard method to the mouse coordination exercise influence; recording medicine to mouse pentobarbital sodium hypnosis function influence in subthreshold dose by Righting reflex deprivation. The results show that the anaesthesial dogs' systolic pressure reduce slightly, but there has not seen the remarkable difference about heart rate, diastic and the respiratory system.
Moreover, INj-alkaloids in toad skin which in each dose has no obviously affects to the mouse coordnation exercise, automatic activities and pentobarbital sodium hypnosis function in subthreshold dose. The results indicate that INj-alkaloids in toad skin has certainly reduces the systolic pressure function which depends dose to the dog and it has no obviously affects regarding dogs' respiratory system and mouse's nervous system.
Results of long-term toxicity test indicate that INj-alkaloids in toad skin has no effect on animal general activity, weight increased, organs' coefficient, hemogram and function of liver and kidney; every organ has no change of pathohistology. Stop to using this medicine, organs' coefficient, hemogram and function of liver and kidney of rats also have not been affected, so it is safe on the clinical application.
实验结果表明:
注射用蟾皮总碱的生产和制备工艺稳定,适合大规模工业化生产。
不同浓度注射用蟾皮总碱对人肿瘤细胞增殖呈现出不同程度的抑制作用,浓度为0.4352 ug/ml的注射用蟾皮总碱对人胃癌细胞SGC-7901、人肝癌细胞SMMC-7721和人宫颈癌细胞Hela的抑制率分别为41.41%、62.41%、80.53%,均高于同剂量的华蟾素注射液组的肿瘤抑制率。
注射用蟾皮总碱急性毒性实验检测不到明显的急性毒性反应。
长期毒性实验结果显示,高剂量、长时间(170 ug/kg、连续三个月)给药后大鼠血液学检查肾脏尿素氮含量为9.94mmol/L,较正常值明显升高,恢复期一个月时尿素氮含量恢复正常。肾脏病理学检查在三个月时表现为肾小管细胞肿胀、变形、境界模糊,胞核消失,细胞坏死。恢复期一个月时肾脏病理学检查已恢复正常。Beagle犬连续高剂量、长时间(52ug/kg、连续三个月)给药后,血液学检查尿素氮含量为5.54mmol/L ,尿常规检查尿液pH值为5.13、尿维生素C含量为0.75mmol/L,尿沉渣镜检(x400)可见7-9个红细胞,与对照组比较,差异明显;病理学检查显示给药三个月时犬肾小管细胞形状不规则,单位面积细胞数量减少,部分胞体皱缩,胞浆透亮度下降,细胞变圆。恢复期1个月后病理检查恢复正常。实验表明注射用蟾皮总碱较大剂量连续用药3个月对大鼠及Beagle犬肾脏功能有影响,恢复期1个月肾脏各项指标恢复正常。
各项实验证明,采用新工艺提取的注射用蟾皮总碱质量工艺稳定,药效显著,无明显及长期毒副作用,可进行临床试用。
Tumor is one kind of familiar disease, and with the speed up of aging and pollution, it becomes the most serious disease that endangers the people’s health. The death rate of tumor sufferers is a little less than that of cardiopathy. This makes it the second killer of human health.
The alkaloids extracted from toad skin are broadly used as one of anti-tumor drugs at present. INj-alkaloids in toad skin is a safe and effective medicine to treat the PLC followed the metastatic tumer of lung .
The research of pharmaceutical included pharmaceutical technology and quality standard. The main purpose of this article mainly works over the new Medicine prescription, manufacture technology, quality standard and pharmacology according to the request of the National New Drug Examination and Approval Method.
Studied the extract process of preparation, developed an optimal process of extraction with traditional medicated process and modern technique, the method is water extraction and ethanol precipitation ,water washing-out supernumerary substance in polyamide column, collect ethanol elution. It is obey the rule of Chinese Medical, extract the effective component and simplify the process at the same time. Above all, the best abstract technology of Vapour distillation was determined, INj-alkaloids in toad skin quality standard was established, INj-alkaloids in toad skin is steady.
On the other hand, according to the guide rules of Chinese medical injection in new medicine evaluation, to make sure this injection is safety, effective and stability, assurance the quality of the injection, and found the base of injection’s fingerprint. The content of one was determined by HPLC. The method is simple, accurate reproducible and can be applied for quality control. Research of the quantity standard has been completed, standard of drug and preparation has been set up, the result show this method is simple and feasible and can guarantee the stability of this preparation.
And anti-tumor efficacy and safety of INj-alkaloids in toad skin by acute-term toxicity test has been researched , in vitro pharmacodynamics test, safety test, pharmacology test and long-term toxicity test and so on.
Through to acute-term toxicity test and LD50 determination, give the result of clinical medication, determiones the dose of medicine. About 2min's the drug poisoning mouse starts to appear the active reduction, peak wool, shuts item, walk haltingly, waggle, jitter. The mouse which are poisoned of large dose have a deep and slow breath, weak and limp. Beyond die the body-righting reflex of dead animals doesn't disappear, the breath from slow to tenuity. Dead mouse have respiration ceases first and cardiac arrest last, which are dissected have no abnormality acquire of organs. The undead ones who are killed 2 weeks later also have no abnormality acquire of organs.
The in vitro experiment of pharmacodynamics shows alkaloids of toad skin's inhibition on tumor cells cultured in vitro using MTT analysis, and tumor control rate via cell appearance observed by inverted microscope. The results indicate that INj-alkaloids in toad skin inhibits evidently the human tumor cells proliferation in vitro and the inhibition depends a certain dose.
Estimate the safety of the medicine, results of hepersensibility test, heamolyticus test and BV stimulus test on INj-alkaloids in toad skin show that INj-alkaloids in toad skin has no allergize on guinea pig, no obviously effect of in vitro heamolyticus and agglomeration in rabbit's red cell, it also has no obviously stimulation on rabbit's BV and can not cause pathological change of tissues of BV surrounding.
The Pharmacology research uses to appraise the INj-alkaloids in toad skin regarding the animal cardiovascular, the respiratory and the nervous system influence. Through pressure transducer, tension transducer, needle-electrode connectting BL-420E biological function experiment system, determine INj-alkaloids in toad skin to take the different time compartment dogs femoral artery blood pressure, breath and the electrocardiogram around separately: observes the medicine with multi-purpose mouse activity recording instrument to the mouse spontaneous activity influence; observes the medicine with Fall-tiltboard method to the mouse coordination exercise influence; recording medicine to mouse pentobarbital sodium hypnosis function influence in subthreshold dose by Righting reflex deprivation. The results show that the anaesthesial dogs' systolic pressure reduce slightly, but there has not seen the remarkable difference about heart rate, diastic and the respiratory system.
Moreover, INj-alkaloids in toad skin which in each dose has no obviously affects to the mouse coordnation exercise, automatic activities and pentobarbital sodium hypnosis function in subthreshold dose. The results indicate that INj-alkaloids in toad skin has certainly reduces the systolic pressure function which depends dose to the dog and it has no obviously affects regarding dogs' respiratory system and mouse's nervous system.
Results of long-term toxicity test indicate that INj-alkaloids in toad skin has no effect on animal general activity, weight increased, organs' coefficient, hemogram and function of liver and kidney; every organ has no change of pathohistology. Stop to using this medicine, organs' coefficient, hemogram and function of liver and kidney of rats also have not been affected, so it is safe on the clinical application.
引文
[1]张淑秀.最新药品注册工作指南[M].北京:中国医药科技出版社,2008.
[2]蒋有绪,郭泉水,马娟,等.中国森林部落分类及其群落学特征[M].北京:科学出版社,1998.
[3]施祀.现代中医药应用与研究大系(第十四卷肿瘤科)[M].上海:上海中医药大学出版社,1996.
[4]方喜业。医学实验动物学[M].北京:人民卫生出版社,1995.
[5]翟中和.细胞生物学[M].北京:高等教育出版社,1995.
[6]张友会.现代肿瘤学(基础部分)[M].北京:北京医科大学中国协和医科大学联合出版社,1993.
[7]陈奇.中药药理研究方法学[M].北京:人民卫生出版社,1993.
[8]杨仓良.毒药本草[M].中国中药出版社, 1993.
[9]李仪奎.中药药理实验方法学[M].上海:上海科学出版社,1991.
[10]代丽萍.蟾皮及华蟾素注射液中蟾蜍噻咛含量测定[J].中国中药杂志,2007,32(3):224-226.
[11]李维熙.中华大蟾蜍皮化学成分的研究[J].中草药,2007.38(2):183-185
[12]代丽萍.蟾皮化学成分的分离与结构鉴定[J] .药学学报,2007,42(8):858-861.
[13]张英.中华大蟾蜍的研究进展[J].中草药,2006,37(12):1905-1908.
[14]赵大洲.中华大蟾蜍蟾酥与蟾皮化学成分的比较分析[J].天津药学,2006,18(4):21-24.
[15]王益秀,黄伟,张波.中药华蟾素对晚期肿瘤患者生存质量与镇痛作用的临床观察[J].中国民间疗法,2006,4(14):46.
[16]赵文叨,王严庆,汤为学.亚砷酸钠对人胃癌细胞株SGC-7901作用机制的研究[J].肿瘤研究与临床,2006,18(4):18.
[17]刘勇军,梁爱玲,黄迪南.扇贝提取物诱导Hela细胞凋亡的研究[J].MODERN ONCOLOGY,2006,14(1):4
[18]陈达理,黄涛.散结抗肿瘤方体内和体外抑瘤作用的实验研究[J].南方医科大学学报,2006,26(4):479
[19]李洁,石俊英.噻唑蓝法检测半枝莲对人肺巨细胞癌细胞株PG细胞增殖的抑制作用[J].时珍国医国药,2006,17(4):559
[20]何国群,黄琼,黄俊明.噻唑蓝比色法测定化学物皮肤致敏性的研究[J].中国卫生检验杂志,2006,16(8):1007
[21]彭利.NS-398对肝癌SMMC-7721和BEL-7402细胞凋亡和survivin、PTEN表达调节作用的研究[J].肿瘤,2006,26(7):652
[22]盛新华.MTT、WST-8法测定细胞生长抑制率的结果比较[J].山东医药,2006,46(6):20
[23]韩鸿彬.华蟾素抗肿瘤作用及其机制的研究进展[J].中国肿瘤生物治疗杂志,2005,12(2):160-162.
[24]薛月珍. MTT法检测卵巢癌化疗敏感性[J].上海第二医科大学学报, 2005,25(8):847
[25]朱敏,徐伟荣.山莨菪碱预防华蟾素引起静脉损伤的护理[J].中国实用护理杂志,2005,21(10):10
[26]吕东霞.刺五加皂甙对肝癌SMMC-7721细胞凋亡的影响[J].中国老年学杂志,2005,25(7):822
[27]顿爱社.蟾酥制剂治疗恶性肿瘤的研究进展[J].四川解剖学杂志,2004,12(2):131-132.
[28]左小东.华蟾素抗肿瘤作用的临床研究进展[J].临床肿瘤学杂志,2003,8(3):232-235.
[29]薛志科.蟾蜍制剂诱导细胞凋亡的研究进展[J].广西中医学院学报,2003,6(3):72-73.
[30]乐蓓蓓,夏乐三,袁承泰,等.大剂量华蟾素防治恶性血液病化疗时感染及粒细胞减少[J].中国中西医结合杂志,1992,12(3):145-147.
[31]施俊,许玲,魏品康.华蟾素注射液穴位注射治疗肿瘤疼痛17例临床观察[J].中国中西医结合杂志,2002,22(2):121.
[32]李杭.华蟾素单用及与化疗合用治疗中晚期肺癌的疗效观察[J].河南肿瘤学杂志,2002,15(1):70.
[33]孙中杰,潘承恩,王国俊.华蟾素配合TACE治疗肝癌的临床观察[J].肿瘤防治研究,2002,29(1): 67-68.
[34]王昌俊,陈庆强,邓力等.华蟾素肝动脉灌注治疗晚期原发性肝癌的临床观察[J].中西医结合肝病杂志,2001,11(1): 5-7.
[35]范健,郭金和,何仕成等.华蟾素微球合并多柔比星和顺铂肝动脉化学栓塞治疗肝癌[J].中国新药与临床杂志, 200l,20(1): 30-32.
[36]韩金声,吕春鸾,盛翠平等.放疗结合华蟾素治疗中晚期原发性肝癌12例[J].肿瘤防治杂志,2001,8(5):538.
[37]王玉霞,程志强,贾立群.华蟾素提高晚期恶性肿瘤患者抗感染能力的临床观察[J].中日友好医院学报,2001,15(2):102-103.
[38]邵国荣.华蟾素注射液治疗中晚期肺癌33例临床观察[J].天津中医,2001,18(3):46.
[39]张富同,李娜.华蟾素配合放射治疗食管癌疗效分析[J].现代中西医结合杂志,2001,10(16):1553.
[40]张长武,汪庆华.华蟾素联合化疗治疗晚期胃癌35例[J].安徽中医学院学报,2001,20(4):18.
[41]卢文娜,杨世虞.华蟾素治疗骨转移癌疼痛32例疗效分析[J].贵阳中医学院学报,2001,23(1):17.
[42]张树梅,孟秀华.华蟾素治疗癌性疼痛临床疗效探讨[J].中国中医药信息杂志,2001,8(3):6l-62.
[43]周琴,疏肝健脾消积汤合华蟾素治疗原发性肝癌32例小结[J].甘肃中医,2001,14(6):26-27.
[44]盂勇,吴洁清,杨瑞民等.华蟾素在中晚期肺癌介入治疗中的应用[J].新乡医学院学报,2000,17(4):266-267.
[45]王加真,闰家阁,张军等.华蟾素注射液的镇痛作用及其机制研究[J].临沂医专学报,2000,22(2):81-84.
[46]马国海,杨光.华蟾素注射液治疗晚期恶性肿瘤35例近期疗效观察[J].陕西医学杂志,2000,29(7):431-432.
[47]韩仲明,苏红星,黄晋生等.华蟾素对喉癌细胞凋亡的基础研究[J].中国中西医结合耳鼻咽喉科杂志,2000,8(3):111-114.
[48]苏红星,韩仲明.用分子原位杂交技术及流式细胞仪研究华蟾素对喉癌细胞的作用[J].解剖学杂志,2000,23(S:S):177.
[49]吴洪梅,朱德增,凌昌全等.华蟾素肝动脉插管栓塞化疗治疗肝癌临床观察[J].辽宁中医杂志,2000,27(3):127-128.
[50]李新芳,杨青.静滴华蟾素注射液致药热反应11例报告[J].中国中药杂志,2000,25(9):575-576.
[51]张风瑞,宿桂霞,彭利等.华蟾素注射液治疗原发性肝癌肺转移近期疗效观察[J].现代中西医结合杂志,2000,9(13):1193-1194.
[52]蒋芹,卞保强,张为民等.华蟾素治疗晚期原发性肝癌的临床研究[J].临床肿瘤学杂志,2000,5(4):294-295.
[53]臧凯.华蟾素合并针灸治疗恶性肿瘤所致的顽固性呃逆[J].深圳中西医结合杂志,1999,9(2):39-40.
[54]付伟义,杨春生,祖金池.华蟾素治疗中晚期食管癌、贲门癌疗效观察(附28例报告)[J].Henan Joncol,Aug,1999,12(4):280.
[55]李萍,胡佳娜,谢玉芳等.华蟾素注射液致过敏性休克一例[J].第二军医大学学报,1999,20(5):311.
[56]潘敏求等.安替可胶囊对治疗晚期恶性肿瘤30例临床观察[J].中国中医药科技,1999(1):50-51.
[57]陈玉俊,项进等.干蟾化学成分的研究[J].中国中药杂志,1998 (10): 620-621.
[58]兰胜侪,于瑞花,宋吉法等.华蟾素结合化疗及腔内注射阿霉素治疗肺癌伴恶性胸水的疗效观察[J].齐鲁医学杂志,1998,13(1):57.
[59]陈令松,张胜斌,张志琴等.重组白细胞介素-2与华蟾素联合治疗晚期肝癌获完全缓解1例[J].癌症,1998,17(3):219.
[60]陆红梅,林小东.华蟾素联合化疗治疗中晚期胃癌70例观察[J].实用中西医结合杂志,1998,11(13):1201.
[61]周成英等.大剂量华蟾素治疗中晚期恶性肿瘤疗效观察[J].锦州医学院学报,1998,16(4):28-29.
[62]周祖刚等.导管化疗结合华蟾素治疗肺癌47例[J].河北中西医结合杂志,1998,7(4):510-511.
[63]吴剑峰,肖华.华蟾素致过敏性休克1例[J].内科急危重症杂志,1998,4(4):157.
[64]孙希美.华蟾素致大疱性表皮松解萎缩型药疹l例[J].滨州医学院学报,1998,21(4):347.
[65]龚爱平,刘金秀,倪蓓等.华蟾素的不良反应(附201例临床观察)[J].徐州医学院学报,1998,18(5):418-420.
[66]董岩,王桂英.华蟾素治疗恶性肿瘤并发带状疱疹11例疗效观察[J].河南肿瘤学杂志,1998,ll(2):129.
[67]彭大为.内服中药配合B超引导下肝内注射华蟾素治疗原发性肝癌11例[J].安徽中医临床杂志,1997,9(6):303.
[68]张秀云,尹超.选择性动脉内药物灌注化疗加用华蟾素治疗肺癌42例[J].安徽医科大学学报,1997,32(4):377-379.
[69]裴雄,裴斌.254例华蟾素的药物不良反应分析[J].药物流行病学杂志,1997,6:46-47.
[70]彭大为等.内服中药配合B超引导下肝内注射华蟾素治疗原发性肝癌11例[J].安徽中医临床杂志,1997,9(6):303.
[71]周建芳等.华蟾素加化学药物灌注治疗原发肝癌的临床观察[J].中国中西医结合杂志, 1997,17(11):686-687.
[72]林钧华等.华蟾素与羟基树碱肝动脉灌注治疗原发性肝癌的疗效观察[J].上海中医药杂志,1997(11):18-19.
[73]孙建华等.澳洲蟾酥脂溶性提取物对肿瘤细胞的作用.天津师大学报(自然版), 1997,17(4):34-38.
[74]裴雄.1254例华蟾素的药物不良分析[J].药物流行病学杂志, 1997,6(S):46-47.
[75]卫蓉,王福贵等.华蟾素静脉滴注治疗晚期恶性肿瘤60例临床观察[J].实用中西医结合杂志,1996,9(7):403.
[76]王四旺等.复方蟾皮胶囊对晚期消化道肿瘤近期疗效的观察[J].第四军医大学学报, 1996,17(2):97-100.
[77]范健等.原发性肝癌的肝肿瘤动脉栓塞化疗[J].中国消化杂志, 1996,16(6):339-341.
[78]范健等.华蟾酥精微球肝动脉栓塞的实验研究[J].癌症,1995,14 (6):434-437.
[79]J ing Y et al. Selective inhabitory effect of bufalin on grow th of human tumor in v itro: association w ith the induction of apotosis in leukem ia HL 260 Cell. Jpn. J. Cancer Res. 1994,85: 645.
[80]张风林等.蟾酥水溶液注射液治疗晚期恶性肿瘤疼痛的临床分析[J].实用中医内科杂志,1994,8(4):30.
[81]张明智,吴孔明等.华蟾素治疗中晚期恶性肿瘤疗效观察[J].中国中西医结合杂志,1994,14(7):438-439.
[82]王四旺等.复方蟾皮胶囊治疗219例晚期癌疗效观察及其初步分析[J].中国医药学报,1992(3):30-31.
[83]金向群.中华大蟾蜍皮化学成分研究[J].中草药, 1992(3): 117-119.
[84]张立宏,张豁中等.中华大蟾蜍皮化学成分的研究[J].药学学报, 1992,27(9):679-683.
[85]许长照等.蟾酥水溶液总成分注射液治疗晚期癌症18年追踪观察[J].中药药理与临床,1991,(4):39.
[86]张瑜瑶,许长照等.蟾酥水溶性总成分的实验研究[J].南京中医学院学报,1988(2):33.
[87]蒋庭章,郑孝痛等.蟾酥水溶液总成分注射液治疗晚期癌症218例[J].南京中医学院学报,1988(1):22.
[88]刘嘉湘.蟾酥膏用于恶性肿瘤的临床观察(附332例随机双盲治疗对照观察)[J].中医杂志,1988,29(3):30.
[89]闵锋等.蟾酥水溶液治疗110例食管贲门癌临床观察.南京中医学院学报[J],1987(1):13.
[90]刘国荣.原发性巨块型肝细胞癌肿瘤血管形态分析及其生物学我探讨[J].海南医学,2006,17(7):22-24.
[91]赵文叨.亚砷酸钠对人胃癌细胞株SGC-7901作用机制的研究[J].肿瘤研究与临床,2006,18(4):18.
[92]季宇彬.苏丹红Ⅰ、Ⅲ和Ⅳ对HepG-2细胞和SGC-79012细胞增殖的影响[J].环境科学,2006,6(6):1201.
[93]李玉明.靶向环氧合酶-2的RNA干扰诱导胃癌SGC-7901细胞凋亡[J].中华实验外科杂志,2006,23(6):662.
[94]刘勇军.扇贝提取物诱导Hela细胞凋亡的研究[J].MODERN ONCOLOGY,2006,14(1):4.
[95]陈达理,黄涛.散结抗肿瘤方体内和体外抑瘤作用的实验研究[J].南方医科大学报,2006,26(4):479.
[96]王春芳,李彬,张海燕.三氧化二砷抑制人眼脉络膜黑色素瘤细胞生长的体外研究[J].中华实验外科杂志,2006,41(10):887
[2]蒋有绪,郭泉水,马娟,等.中国森林部落分类及其群落学特征[M].北京:科学出版社,1998.
[3]施祀.现代中医药应用与研究大系(第十四卷肿瘤科)[M].上海:上海中医药大学出版社,1996.
[4]方喜业。医学实验动物学[M].北京:人民卫生出版社,1995.
[5]翟中和.细胞生物学[M].北京:高等教育出版社,1995.
[6]张友会.现代肿瘤学(基础部分)[M].北京:北京医科大学中国协和医科大学联合出版社,1993.
[7]陈奇.中药药理研究方法学[M].北京:人民卫生出版社,1993.
[8]杨仓良.毒药本草[M].中国中药出版社, 1993.
[9]李仪奎.中药药理实验方法学[M].上海:上海科学出版社,1991.
[10]代丽萍.蟾皮及华蟾素注射液中蟾蜍噻咛含量测定[J].中国中药杂志,2007,32(3):224-226.
[11]李维熙.中华大蟾蜍皮化学成分的研究[J].中草药,2007.38(2):183-185
[12]代丽萍.蟾皮化学成分的分离与结构鉴定[J] .药学学报,2007,42(8):858-861.
[13]张英.中华大蟾蜍的研究进展[J].中草药,2006,37(12):1905-1908.
[14]赵大洲.中华大蟾蜍蟾酥与蟾皮化学成分的比较分析[J].天津药学,2006,18(4):21-24.
[15]王益秀,黄伟,张波.中药华蟾素对晚期肿瘤患者生存质量与镇痛作用的临床观察[J].中国民间疗法,2006,4(14):46.
[16]赵文叨,王严庆,汤为学.亚砷酸钠对人胃癌细胞株SGC-7901作用机制的研究[J].肿瘤研究与临床,2006,18(4):18.
[17]刘勇军,梁爱玲,黄迪南.扇贝提取物诱导Hela细胞凋亡的研究[J].MODERN ONCOLOGY,2006,14(1):4
[18]陈达理,黄涛.散结抗肿瘤方体内和体外抑瘤作用的实验研究[J].南方医科大学学报,2006,26(4):479
[19]李洁,石俊英.噻唑蓝法检测半枝莲对人肺巨细胞癌细胞株PG细胞增殖的抑制作用[J].时珍国医国药,2006,17(4):559
[20]何国群,黄琼,黄俊明.噻唑蓝比色法测定化学物皮肤致敏性的研究[J].中国卫生检验杂志,2006,16(8):1007
[21]彭利.NS-398对肝癌SMMC-7721和BEL-7402细胞凋亡和survivin、PTEN表达调节作用的研究[J].肿瘤,2006,26(7):652
[22]盛新华.MTT、WST-8法测定细胞生长抑制率的结果比较[J].山东医药,2006,46(6):20
[23]韩鸿彬.华蟾素抗肿瘤作用及其机制的研究进展[J].中国肿瘤生物治疗杂志,2005,12(2):160-162.
[24]薛月珍. MTT法检测卵巢癌化疗敏感性[J].上海第二医科大学学报, 2005,25(8):847
[25]朱敏,徐伟荣.山莨菪碱预防华蟾素引起静脉损伤的护理[J].中国实用护理杂志,2005,21(10):10
[26]吕东霞.刺五加皂甙对肝癌SMMC-7721细胞凋亡的影响[J].中国老年学杂志,2005,25(7):822
[27]顿爱社.蟾酥制剂治疗恶性肿瘤的研究进展[J].四川解剖学杂志,2004,12(2):131-132.
[28]左小东.华蟾素抗肿瘤作用的临床研究进展[J].临床肿瘤学杂志,2003,8(3):232-235.
[29]薛志科.蟾蜍制剂诱导细胞凋亡的研究进展[J].广西中医学院学报,2003,6(3):72-73.
[30]乐蓓蓓,夏乐三,袁承泰,等.大剂量华蟾素防治恶性血液病化疗时感染及粒细胞减少[J].中国中西医结合杂志,1992,12(3):145-147.
[31]施俊,许玲,魏品康.华蟾素注射液穴位注射治疗肿瘤疼痛17例临床观察[J].中国中西医结合杂志,2002,22(2):121.
[32]李杭.华蟾素单用及与化疗合用治疗中晚期肺癌的疗效观察[J].河南肿瘤学杂志,2002,15(1):70.
[33]孙中杰,潘承恩,王国俊.华蟾素配合TACE治疗肝癌的临床观察[J].肿瘤防治研究,2002,29(1): 67-68.
[34]王昌俊,陈庆强,邓力等.华蟾素肝动脉灌注治疗晚期原发性肝癌的临床观察[J].中西医结合肝病杂志,2001,11(1): 5-7.
[35]范健,郭金和,何仕成等.华蟾素微球合并多柔比星和顺铂肝动脉化学栓塞治疗肝癌[J].中国新药与临床杂志, 200l,20(1): 30-32.
[36]韩金声,吕春鸾,盛翠平等.放疗结合华蟾素治疗中晚期原发性肝癌12例[J].肿瘤防治杂志,2001,8(5):538.
[37]王玉霞,程志强,贾立群.华蟾素提高晚期恶性肿瘤患者抗感染能力的临床观察[J].中日友好医院学报,2001,15(2):102-103.
[38]邵国荣.华蟾素注射液治疗中晚期肺癌33例临床观察[J].天津中医,2001,18(3):46.
[39]张富同,李娜.华蟾素配合放射治疗食管癌疗效分析[J].现代中西医结合杂志,2001,10(16):1553.
[40]张长武,汪庆华.华蟾素联合化疗治疗晚期胃癌35例[J].安徽中医学院学报,2001,20(4):18.
[41]卢文娜,杨世虞.华蟾素治疗骨转移癌疼痛32例疗效分析[J].贵阳中医学院学报,2001,23(1):17.
[42]张树梅,孟秀华.华蟾素治疗癌性疼痛临床疗效探讨[J].中国中医药信息杂志,2001,8(3):6l-62.
[43]周琴,疏肝健脾消积汤合华蟾素治疗原发性肝癌32例小结[J].甘肃中医,2001,14(6):26-27.
[44]盂勇,吴洁清,杨瑞民等.华蟾素在中晚期肺癌介入治疗中的应用[J].新乡医学院学报,2000,17(4):266-267.
[45]王加真,闰家阁,张军等.华蟾素注射液的镇痛作用及其机制研究[J].临沂医专学报,2000,22(2):81-84.
[46]马国海,杨光.华蟾素注射液治疗晚期恶性肿瘤35例近期疗效观察[J].陕西医学杂志,2000,29(7):431-432.
[47]韩仲明,苏红星,黄晋生等.华蟾素对喉癌细胞凋亡的基础研究[J].中国中西医结合耳鼻咽喉科杂志,2000,8(3):111-114.
[48]苏红星,韩仲明.用分子原位杂交技术及流式细胞仪研究华蟾素对喉癌细胞的作用[J].解剖学杂志,2000,23(S:S):177.
[49]吴洪梅,朱德增,凌昌全等.华蟾素肝动脉插管栓塞化疗治疗肝癌临床观察[J].辽宁中医杂志,2000,27(3):127-128.
[50]李新芳,杨青.静滴华蟾素注射液致药热反应11例报告[J].中国中药杂志,2000,25(9):575-576.
[51]张风瑞,宿桂霞,彭利等.华蟾素注射液治疗原发性肝癌肺转移近期疗效观察[J].现代中西医结合杂志,2000,9(13):1193-1194.
[52]蒋芹,卞保强,张为民等.华蟾素治疗晚期原发性肝癌的临床研究[J].临床肿瘤学杂志,2000,5(4):294-295.
[53]臧凯.华蟾素合并针灸治疗恶性肿瘤所致的顽固性呃逆[J].深圳中西医结合杂志,1999,9(2):39-40.
[54]付伟义,杨春生,祖金池.华蟾素治疗中晚期食管癌、贲门癌疗效观察(附28例报告)[J].Henan Joncol,Aug,1999,12(4):280.
[55]李萍,胡佳娜,谢玉芳等.华蟾素注射液致过敏性休克一例[J].第二军医大学学报,1999,20(5):311.
[56]潘敏求等.安替可胶囊对治疗晚期恶性肿瘤30例临床观察[J].中国中医药科技,1999(1):50-51.
[57]陈玉俊,项进等.干蟾化学成分的研究[J].中国中药杂志,1998 (10): 620-621.
[58]兰胜侪,于瑞花,宋吉法等.华蟾素结合化疗及腔内注射阿霉素治疗肺癌伴恶性胸水的疗效观察[J].齐鲁医学杂志,1998,13(1):57.
[59]陈令松,张胜斌,张志琴等.重组白细胞介素-2与华蟾素联合治疗晚期肝癌获完全缓解1例[J].癌症,1998,17(3):219.
[60]陆红梅,林小东.华蟾素联合化疗治疗中晚期胃癌70例观察[J].实用中西医结合杂志,1998,11(13):1201.
[61]周成英等.大剂量华蟾素治疗中晚期恶性肿瘤疗效观察[J].锦州医学院学报,1998,16(4):28-29.
[62]周祖刚等.导管化疗结合华蟾素治疗肺癌47例[J].河北中西医结合杂志,1998,7(4):510-511.
[63]吴剑峰,肖华.华蟾素致过敏性休克1例[J].内科急危重症杂志,1998,4(4):157.
[64]孙希美.华蟾素致大疱性表皮松解萎缩型药疹l例[J].滨州医学院学报,1998,21(4):347.
[65]龚爱平,刘金秀,倪蓓等.华蟾素的不良反应(附201例临床观察)[J].徐州医学院学报,1998,18(5):418-420.
[66]董岩,王桂英.华蟾素治疗恶性肿瘤并发带状疱疹11例疗效观察[J].河南肿瘤学杂志,1998,ll(2):129.
[67]彭大为.内服中药配合B超引导下肝内注射华蟾素治疗原发性肝癌11例[J].安徽中医临床杂志,1997,9(6):303.
[68]张秀云,尹超.选择性动脉内药物灌注化疗加用华蟾素治疗肺癌42例[J].安徽医科大学学报,1997,32(4):377-379.
[69]裴雄,裴斌.254例华蟾素的药物不良反应分析[J].药物流行病学杂志,1997,6:46-47.
[70]彭大为等.内服中药配合B超引导下肝内注射华蟾素治疗原发性肝癌11例[J].安徽中医临床杂志,1997,9(6):303.
[71]周建芳等.华蟾素加化学药物灌注治疗原发肝癌的临床观察[J].中国中西医结合杂志, 1997,17(11):686-687.
[72]林钧华等.华蟾素与羟基树碱肝动脉灌注治疗原发性肝癌的疗效观察[J].上海中医药杂志,1997(11):18-19.
[73]孙建华等.澳洲蟾酥脂溶性提取物对肿瘤细胞的作用.天津师大学报(自然版), 1997,17(4):34-38.
[74]裴雄.1254例华蟾素的药物不良分析[J].药物流行病学杂志, 1997,6(S):46-47.
[75]卫蓉,王福贵等.华蟾素静脉滴注治疗晚期恶性肿瘤60例临床观察[J].实用中西医结合杂志,1996,9(7):403.
[76]王四旺等.复方蟾皮胶囊对晚期消化道肿瘤近期疗效的观察[J].第四军医大学学报, 1996,17(2):97-100.
[77]范健等.原发性肝癌的肝肿瘤动脉栓塞化疗[J].中国消化杂志, 1996,16(6):339-341.
[78]范健等.华蟾酥精微球肝动脉栓塞的实验研究[J].癌症,1995,14 (6):434-437.
[79]J ing Y et al. Selective inhabitory effect of bufalin on grow th of human tumor in v itro: association w ith the induction of apotosis in leukem ia HL 260 Cell. Jpn. J. Cancer Res. 1994,85: 645.
[80]张风林等.蟾酥水溶液注射液治疗晚期恶性肿瘤疼痛的临床分析[J].实用中医内科杂志,1994,8(4):30.
[81]张明智,吴孔明等.华蟾素治疗中晚期恶性肿瘤疗效观察[J].中国中西医结合杂志,1994,14(7):438-439.
[82]王四旺等.复方蟾皮胶囊治疗219例晚期癌疗效观察及其初步分析[J].中国医药学报,1992(3):30-31.
[83]金向群.中华大蟾蜍皮化学成分研究[J].中草药, 1992(3): 117-119.
[84]张立宏,张豁中等.中华大蟾蜍皮化学成分的研究[J].药学学报, 1992,27(9):679-683.
[85]许长照等.蟾酥水溶液总成分注射液治疗晚期癌症18年追踪观察[J].中药药理与临床,1991,(4):39.
[86]张瑜瑶,许长照等.蟾酥水溶性总成分的实验研究[J].南京中医学院学报,1988(2):33.
[87]蒋庭章,郑孝痛等.蟾酥水溶液总成分注射液治疗晚期癌症218例[J].南京中医学院学报,1988(1):22.
[88]刘嘉湘.蟾酥膏用于恶性肿瘤的临床观察(附332例随机双盲治疗对照观察)[J].中医杂志,1988,29(3):30.
[89]闵锋等.蟾酥水溶液治疗110例食管贲门癌临床观察.南京中医学院学报[J],1987(1):13.
[90]刘国荣.原发性巨块型肝细胞癌肿瘤血管形态分析及其生物学我探讨[J].海南医学,2006,17(7):22-24.
[91]赵文叨.亚砷酸钠对人胃癌细胞株SGC-7901作用机制的研究[J].肿瘤研究与临床,2006,18(4):18.
[92]季宇彬.苏丹红Ⅰ、Ⅲ和Ⅳ对HepG-2细胞和SGC-79012细胞增殖的影响[J].环境科学,2006,6(6):1201.
[93]李玉明.靶向环氧合酶-2的RNA干扰诱导胃癌SGC-7901细胞凋亡[J].中华实验外科杂志,2006,23(6):662.
[94]刘勇军.扇贝提取物诱导Hela细胞凋亡的研究[J].MODERN ONCOLOGY,2006,14(1):4.
[95]陈达理,黄涛.散结抗肿瘤方体内和体外抑瘤作用的实验研究[J].南方医科大学报,2006,26(4):479.
[96]王春芳,李彬,张海燕.三氧化二砷抑制人眼脉络膜黑色素瘤细胞生长的体外研究[J].中华实验外科杂志,2006,41(10):887