【作者】 赵明芳；

【导师】 刘志；

【作者基本信息】 中国医科大学 ， 急救医学， 2003， 硕士

【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：H₂₂小鼠肝癌细胞株，S₁₈₀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5一6x106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 24.3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;而更多还原

【Abstract】 IntroductionToad 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 componentof 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 methods1. Materials and instruments1.1 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. Methods2. 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 methods2.4.1 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更多还原