斑蝥素酸镁与斑蝥酸钠对喉癌Hep-2细胞抑制活性比较的初步研究

目的:探讨自主研发产物--斑蝥素酸镁对人喉癌上皮细胞的增殖抑制活性是否好于临床抗癌药物斑蝥酸钠。方法:采用磺酰罗丹明染色法(SRB法)、细胞集落形成实验分别考察斑蝥素酸镁和斑蝥酸钠对人喉癌上皮细胞Hep-2的影响。结果:与斑蝥酸钠相比,斑蝥素酸镁作用喉癌Hep-2细胞的效果更加显著。其IC50仅为2.19μmol·L-1,远低于斑蝥酸钠的IC50(15.75μmol·L-1)。细胞集落形成实验显示,与斑蝥酸钠相比,斑蝥素酸镁明显阻止细胞集落的形成,最低有效浓度为1.75μmol·L-1,远低于斑蝥酸钠的最低有效浓度(3.50μmol·L-1)。结论:与斑蝥酸钠相比,斑蝥素酸镁对喉癌Hep-2细胞具有更加显著的抑制效果,可进行更加深入的研究。     

三种去甲基斑蝥素衍生物体外对人肝癌HepG-2细胞增殖的抑制作用

目的:探讨去甲基斑蝥素酸钠、去甲基斑蝥素酸钾、去甲基斑蝥素酸钙体外对人肝癌HepG-2细胞增殖的影响。方法:采用磺酰罗丹明(SRB法)、细胞集落形成实验、流式细胞技术检测三种去甲基斑蝥素衍生物对HepG-2细胞增殖和凋亡的影响。qRT-PCR技术考察三种去甲基斑蝥素衍生物对HepG-2细胞Bcl-2、Bax mRNA表达的影响。结果:三种去甲基斑蝥素衍生物能明显抑制HepG-2细胞的增殖,其抑制率呈剂量时效依赖性;与空白对照组相比,三种去甲基斑蝥素衍生物能显著性减少HepG-2细胞集落形成数量,并且可诱导HepG-2细胞的凋亡,凋亡率分别为8±0.56%、6.93±0.91%、6.16±0.37%;qRT-PCR显示,三种去甲基斑蝥素衍生物能抑制HepG-2细胞Bcl-2mRNA的表达,增强Bax mRNA的表达。结论:三种去甲基斑蝥素衍生物可通过下调Bcl-2 mRNA表达,上调Bax mRNA表达,诱导HepG-2细胞的凋亡,从而抑制HepG-2细胞的增殖;三种衍生物中,去甲基斑蝥素酸钠对HepG-2细胞的增殖的抑制作用最强。     

去甲斑蝥酸钠对小鼠卵母细胞第一次减数分裂的影响

体外培养的小鼠卵母细胞在１２ｈ内可完成第一次减数分裂,排出第一极体。将卵母细胞培养在含２５０μｇ／ｍｌ去甲斑蝥酸钠的培养液中,生发泡破裂（ＧＶＢＤ）过程不受影响,但卵母细胞不能完成减数分裂过程,卵母细胞中没有减数分裂器的形成,染色体紧密凝缩在一起;去甲斑蝥酸钠对小鼠卵母细胞减数分裂的影响在６ｈ内具有可逆性：卵母细胞ＧＶ破裂后用去甲斑蝥酸钠处理２ｈ换正常培养液培养,整个减数分裂过程不受影响;ＧＶ期卵母细胞用去甲斑蝥酸钠连续处理６ｈ,洗去药物继续培养,减数分裂可继续进行,但第一极体的排放时间推迟。去甲斑蝥酸钠对分裂期细胞特异性磷蛋白的出现影响不显著,在连续处理的卵母细胞中分裂期细胞特异性磷蛋白仍然存在。     

人胃癌BGC-823细胞中去甲斑蝥素抑癌作用机理的蛋白质组学研究

去甲斑蝥素是我国自行研制的抗肿瘤药物,在临床上主要用于消化道肿瘤的治疗．实验表明,去甲斑蝥素可引起人胃癌BGC-823细胞发生 M期阻滞及细胞凋亡．进一步利用双向电泳和质谱技术,筛选出了去甲斑蝥素抑癌作用相关蛋白．研究显示,线粒体热休克蛋白CH60、线粒体ATP合酶d亚单位、内质网葡萄糖调节蛋白GRP78、线粒体Hsp70的辅助因子GRPE1、SH3L3以及染色质组装因子1小亚基RBBP4参与了去甲斑蝥素的抑癌作用．研究提示,去甲斑蝥素可能通过促进线粒体热休克蛋白及p53的表达进而激活caspase-3依赖的凋亡通路,并且去甲斑蝥素在引发内质网协迫之后,可通过抑制胞外信号调节激酶(extracellular signal regulated kinase, ERK)的活性促进肿瘤细胞的凋亡．进一步分析了去甲斑蝥素与线粒体ATP合酶抑制剂寡霉素A的联合用药对人胃癌细胞生长的影响,结果表明,联合用药的抑瘤效果比单独用药的抑瘤效果显著,提示去甲斑蝥素可能通过抑制线粒体ATP合酶功能抑制BGC-823生长．上述结果为优化去甲斑蝥素的联合用药方案提供了新线索．     

豹蛙核酸酶与斑蝥酸钠对肺腺癌细胞增殖的协同抑制作用

豹蛙核酸酶(onconase,Onc)是从美洲北方豹蛙卵母细胞中提取的一种核糖核酸酶,对许多肿瘤细胞都具有杀伤作用。斑蝥素(cantharidin)是存在于芫青科昆虫斑蝥体内的一种天然防御性毒素,斑蝥酸钠(sodium cantharidate,SCA)是斑蝥素半合成衍生物。鉴于Onc与SCA对非小细胞肺癌都具有杀伤作用,采用MTT法测定Onc与SCA单独与联合作用于两株肺腺癌细胞的IC50值,运用联合作用指数(combination index,CI)和等效线分析评价两者联合作用的效果。结果表明,Onc与SCA联合作用时,CI值均小于0.7,等效线分析图显示,代表Onc与SCA联合作用的点均位于加成线下方,Onc与SCA对肺腺癌SPC-A-1、A549细胞株增殖的抑制作用具有协同效应。用流式细胞仪进行的凋亡细胞检测结果也支持上述"Onc/SCA联合使用具有协同抗癌作用"的结论。     

环境条件对南方大斑蝥成虫取食行为的影响

南方大斑蝥的主要药效成分斑蝥素及其衍生物对肝癌、食道癌等20余种癌症有显著疗效,但斑蝥素不能大规模人工合成,仅依赖于产斑蝥素的昆虫。南方大斑蝥是斑蝥素的重要来源,其野生资源已不能满足市场需求,人工养殖是解决南方大斑蝥资源紧缺的必然选择,而取食是人工养殖中影响其生长繁殖的重要因子之一。本研究在恒温培养箱内对南方大斑蝥成虫的最大取食量、觅食规律、光照、最佳觅食的温度和食物含水量等方面进行了研究,明确了南方大斑蝥成虫的取食条件和规律,为南方大斑蝥成虫的人工养殖提供技术参考。结果显示,南方大斑蝥成虫每天连续取食时,日平均取食量为身体重量的0. 797±0. 062倍,而饥饿24 h后的日平均取食量为体重的1. 712±0. 110倍,两者间取食量差异显著(P 0. 05)。根据其取食日变化规律,上午7∶00为南方大斑蝥成虫的取食高峰期,在不需觅食的基础上有无光照对平均取食量无显著的影响(P 0. 05)。当温度为30℃-33℃、食物含水量40%-60%时南方大斑蝥成虫的平均取食量最大,呈显著水平(P 0. 05)。根据研究结果可知,在南方大斑蝥成虫的人工养殖中,应于每天的上午7∶00前投放食物,食物量为成虫重量的0. 8倍左右,食物含水量控制在40%-60%,环境温度30℃-33℃。     

斑蝥素类衍生物对粘虫胃毒活性及解毒酶系的影响

【目的】筛选斑蝥素衍生物,寻找高效、低毒、低残留的新型化合物研发杀虫剂。【方法】采用生物测定和酶活性测定方法,对100种斑蝥素类衍生物进行筛选,采用叶碟饲喂法测定其对粘虫3龄幼虫的胃毒活性;测定亚致死剂量下试虫体内解毒酶系的活性变化。【结果】通过生物测定发现斑蝥素衍生物Ⅰ-7~#和Ⅳ-13~#的杀虫活性优于去甲斑蝥素,Ⅰ-7~#与斑蝥素对粘虫的胃毒LC_(50)值相近,其LC_(50)分别为49.818mg/m L和43.677mg/m L。酶活性测定结果表明:斑蝥素、斑蝥素衍生物去甲斑蝥素、Ⅰ-7~#和Ⅳ-13~#对粘虫解毒酶系酶比活力均有不同程度的影响,其中对细胞色素P450酶比活力有明显的抑制活性,对羧酸酯酶比活力有不同程度的激活作用,而对谷胱甘肽S-转移酶比活力大体表现出先激活后抑制的活性。【结论】斑蝥素衍生物Ⅰ-7~#和Ⅳ-13~#对鳞翅目害虫有较强杀虫活性,具有作为生物源农药加以开发利用的价值。     

去甲斑蝥素对小菜蛾幼虫的拒食作用及取食行为的影响

去甲斑蝥素(NCTD)是斑蝥素(CTD)的衍生物,可人工合成。去甲斑蝥素对害虫有毒杀作用。为明确它是否有拒食作用及其作用方式,本研究以小菜蛾幼虫为供试昆虫,采用叶碟浸液法对试虫拒食作用及取食量、取食选择、取食次数和持续时间等取食行为进行了生物测定。拒食作用结果显示,2-48 h内NCTD处理组的幼虫取食量明显低于对照组且两者差距随时间逐渐增加,拒食率与剂量呈显著正相关,增加剂量会增加拒食率。行为研究结果显示,小菜蛾处理组试虫的单次平均取食时长明显减少,仅为对照组的70. 6%,而持续取食次数和试探次数均明显增多,分别是对照组的1. 4倍和1. 9倍。结果表明,NCTD对小菜蛾幼虫的拒食作用主要表现在降低试虫的取食量,减少取食时间,增加试探次数。     

芫菁体内结合斑蝥素对胃癌SGC-7901细胞增殖的抑制作用

本文考察了芫菁体内结合斑蝥素和人工合成的斑蝥素盐类衍生物斑蝥素酸镁的体外抗肿瘤活性。采用WST-1法检测两者在体外对人胃腺癌SGC-7901细胞增殖的抑制作用。实验结果显示两者对SGC-7901细胞均表现出明显的抑制效果,且随药物浓度升高其抑制作用增强,呈剂量效应关系;其半数抑制浓度(IC50)分别为10.86和8.65μmol/L。此外,通过流式细胞术检测表明,结合斑蝥素能引起SGC-7901细胞G0～G1期阻滞;斑蝥素酸镁则引起SGC-7901细胞S期阻滞,两者均能通过干预SGC-7901细胞的周期来抑制其增殖。     

去甲斑蝥素对糖尿病大鼠肾小球纤维粘连蛋白及钙调磷酸酶的影响

目的:观察去甲斑蝥索(NCTD)对链脲佐菌素(STZ)诱导的糖尿病(DM)模型大鼠肾小球纤维粘连蛋白以及钙调磷酸酶表达的影响,探讨去甲斑蝥素抗肾小球纤维化的机制.方法:10只大鼠正常饮食为正常对照组(C)组,30只大鼠给予高脂高糖饮食+链脲佐菌素(STZ)制备DM模型,随机分3组:DM模型(D)组10只,小剂量去甲斑蝥素治疗(N1)组10只,大剂量去甲斑蝥素治疗(N2)组10只,给药8周后检测血糖,血肌酐水平变化;用免疫组化法检测肾组织肾小球纤维粘连蛋白(fibronectin,FN)以及钙调磷酸酶(calcineurin,CaN)表达,分别用realtime-PCR以及western blot法检测肾组织FN以及CaN mRNA和蛋白水平的表达.结果:模型(D)组血糖,血肌酐水平上升(P<0.05),同时肾小球区FN,CaN的表达高于正常(C)组(P<0.05).去甲斑蝥素干预(N)组与模型组比较,去甲斑蝥素组肾小球区FN,CaN表达下调(P<0.05).大剂量组效果更显著(P<0.05).去甲斑蝥素(N)治疗组血糖水平,肾功能较(D)组无明显变化.结论:去甲斑蝥素能减少糖尿病大鼠肾小球区FN的表达,其作用机制可能是通过使CaN的表达下调而实现.     

复方斑蝥胶囊联合放疗治疗鼻咽癌的临床疗效分析

摘要目的：观察复方斑蝥胶囊联合放疗治疗鼻咽癌的临床效果及不良反应。方法：将2007年6月至2010年12月来我科就诊的120例Ⅱ、Ⅲ期低分化鳞状细胞鼻咽癌伴有颈淋巴结转移的患者随机分为复方斑蝥胶囊辅助放疗组、甘氨双唑钠辅助放疗组（阳性对照组）和单纯放疗组（阴性对照组）,每组各40例,比较治疗后各组患者的治疗效果和不良反应。结果：各组患者在接受相同放射疗程的情况下,复方盥蝥胶囊辅助放疗组和甘氨双唑钠辅助放疗组的治疗总有效率均明显优于单纯放疗组（P〈0．05）,但复方斑蝥胶囊辅助放疗组与甘氨双唑钠辅助放疗组比较无明显差异（P〉0．05）。复方斑蝥胶囊辅助放疗组皮肤反应的发生率明显低于单纯放疗组（P〈O．05）;皮肤反应、黏膜反应、口腔溃疡和心电图异常的发生率均明显低于甘氨双唑钠辅助放疗组（P〈O．05）,但两组恶心呕吐的发生率比较无明显差异（P〉0．05）。结论：复方斑蝥胶囊辅助治疗可显著提高放疗治疗鼻咽癌的临床疗效,且无明显毒副作用,值得临床推广应用。     

芫菁科不同种类成虫体内斑蝥素的含量

芫菁体内含有斑蝥素,是一种重要的药用昆虫。近年来我国对斑蝥素的临床应用研究表明：斑蝥素及其衍生物对治疗原发性肝癌疗效显著。为了摸清我国芫菁科昆虫的自然资源和虫体内斑蝥素的含量,作者调查了不同地区、不同寄主植物上芫菁科昆虫的种类分布,并利用气相色谱内标法测定了不同性别以及交尾高峰前后的芫菁成虫体内斑蝥素的含量。发现雄性成虫体内斑蝥素的含量均高于雌性成虫。交配高峰后的芫菁雌性成虫体内斑蝥素含量高于交配高峰前的芫菁雌性成虫体内斑蝥素含量。     

Cantharidin poisoning associated with specific binding site in liver

Cantharidin, the potent vesicant and toxicant of blister beetles, was prepared as a radioligand to probe its mechanism of action. [3H]Cantharidin interacts in a saturable and specific manner with a binding site in mouse liver cytosol with apparent Kd and Bmax values of 30 nM and 1.8 pmol/mg protein, respectively. Comparisons of cantharidic acid, the related herbicide endothal, and 20 other oxabicycloheptane-dicarboxylic acids show that their potency as inhibitors of [3H]cantharidin binding is closely correlated with their intraperitoneal toxicity to mice. This binding site is also inhibited in vivo by toxic doses of cantharidin. The [3H]cantharidin binding site in mouse liver cytosol therefore represents, or serves as a model for, the site of toxic action of cantharidin and structurally-related compounds.     

Cantharidin inhibits competitively heme‐Fe(III) binding to the FA1 site of human serum albumin

Cantharidin, a monoterpene isolated from the insect blister beetle, has long been used as a medicinal agent in the traditional Chinese medicine. Cantharidin inhibits a subgroup of serine/threonine phosphatases, thus inducing cell growth inhibition and cytotoxicity. Cantharidin has anticancer activity in vitro, since it is able of inducing p53‐dependent apoptosis and double‐strand breakage of DNA in cancer cells. Although the toxicity of cantharidin to the gastrointestinal and urinary tracts prevents its medical use, it is a promising lead compound for chemical modification to develop new anticancer therapeutics. In fact, cantharidin does not cause myelosuppression and displays anticancer activity against cells with a multidrug resistance phenotype. Here, the competitive inhibitory effect of cantharidin on heme‐Fe(III) binding to the fatty acid site 1 (FA1) of human serum albumin (HSA) is reported. Docking and molecular dynamics simulations support functional data indicating the preferential binding of cantharidin to the FA1 site of HSA. Present results may be relevant in vivo as HSA could transport cantharidin, which in turn could affect heme‐Fe(III) scavenging by HSA.     

Attraction of the fungus gnat Bradysia optata to cantharidin

The toxic terpenoid cantharidin has been reported to attract some specimens of undetermined fungus gnats. In order to investigate this phenomenon, the fungus gnat Bradysia optata Rudzinski, 1994 (Diptera: Sciaridae) was reared and its attraction to cantharidin was registered. Significant attractivity of the substance to males could be confirmed with both sticky baits in a greenhouse and an olfactometer under laboratory conditions. Ingestion of the toxic compound for chemical defence, which has been shown for some so-called canthariphilous insects, was not observed. However, a high toxicity of cantharidin for the attracted species B. optata could be demonstrated. These unusual findings could be the result of the mycetophagous sciarids mistaking the terpenoid for certain structurally related fungal metabolites. These secondary metabolites might indicate a convenient meeting-place for mating and oviposition.     

Enhanced toxicity of Cry2Ab with cantharidin and its analogues on Mythimna separata

The oriental armyworm, Mythimna separata Walker (Lepidoptera: Noctuidae), is a severe pest of graminaceous crops in Asia and Australia. In this study, we investigated the impacts of Cry2Ab, cantharidin and its analogues (cantharidin‐23 and cantharidin‐24) on M. separata growth, hydrolytic enzymes and detoxifying enzymes. Differences in weight gain and enzyme activities among all treatments were observed. Larval and pupal weight gain and hydrolytic enzyme activities declined when larvae were treated with Cry2Ab, cantharidin and its analogues, individually or in combination. The combination of Cry2Ab and cantharidin or cantharidin‐24 had a markedly adverse effect on larval weight gain. Alkaline phosphatase and acid phosphatase were inhibited, whereas glutathione S‐transferase was upregulated in all treatments with sublethal doses. The maximum reduction in alkaline phosphatase activity and increase in glutathione S‐transferase activity occurred after the larvae were treated with a combination of Cry2Ab and cantharidin‐24 over 48 h. The results suggest that the compositions of Cry2Ab, cantharidin and cantharidin‐24 have a potential use in pest management.     

芫菁体内斑蟊素的合成、 转移和生物学功能

斑蟊素是芫菁科昆虫合成的一种防御物质, 已经被证实对多种癌症和其他疾病有着特殊的疗效。芫菁体内存在不同结合态的斑蟊素或斑蟊素衍生物, 包括斑蟊素酸镁、斑蟊素酸钙、羟基斑蟊素、甲基斑蟊胺和脱甲斑蟊素等。不同芫菁种类、不同发育阶段其斑蝥素合成量有显著的差异, 并且有着典型的性二型现象, 性成熟的雄成虫斑蝥素含量最高可达10%。关于斑蝥素的生物合成途径以及斑蝥素在虫体内的分布, 尽管有一些研究, 但仍然没有定论。本文从斑蟊素在芫菁体内的含量、分布、生物合成、代谢及生物学功能等方面对国内外的研究进行概括, 以期为充分发掘芫菁科昆虫资源、指导芫菁的人工养殖、合理的利用资源以及人工合成斑蟊素提供参考。     

Dichloroacetate derivatives. Metabolic effects and pharmacodynamics in normal rats

Dichloroacetate (DCA) reduces blood glucose, lactate and lipids in diabetes or during fasting. Chronic use of DCA, however, is limited by toxicity, probably due in part to its rapid conversion to oxalate in vivo. In theory, therefore, DCA's efficacy may be retained and its toxicity minimized by controlling its rate of metabolism. We attempted to alter DCA pharmacokinetics and bioavailability by synthesizing various derivatives comprising DCA esters with polyols and DCA ionic complexes. Twenty-four hour fasted, nondiabetic rats received single, orogastric doses of saline (control) sodium DCA (100mg/kg) or the following derivatives (D1-4): the esters D1-D3: potassium tetra (dichloroacetyl) glucuronate (D1), inositol-monophosphate-tetradichloroacetate (D2), inositol-hexadichloroacetate (D3) and inositol-hexa [N-methylnicotinate] hexadichloroacetate salt (D4). Each derivative was administered at a dose that would ultimately provide 100 mg/kg DCA as the anion. All derivatives were orally effective in significantly decreasing blood glucose and lactate. D4 exerted the most potent and long-lasting glucose- and lactate-lowering effects, yet increased plasma DCA concentrations less than an equivalent dose of the sodium salt. When administered to reverse light-cycled rats, D4 markedly inhibited the incorporation of tritiated water into cholesterol and triglycerides. We conclude that derivatives of DCA retain the biological activity of the parent compound, but may exhibit different pharmacokinetics. They may eventually prove useful in the treatment of diabetes mellitus, hyperlipidemia and lactic acidosis in man.     

芫菁体内斑蝥素的含量及存在形式

【目的】测定芫菁体内斑蝥素的含量及其存在形式。【方法】利用气相色谱法测定了8种芫菁和作为对照的黑翅红蝉体内总斑蝥素和游离斑蝥素的含量,总斑蝥素含量是通过酸水解法测得的,游离斑蝥素含量是通过直接浸提法测得的,以总斑蝥素与游离斑蝥素含量之差作为结合斑蝥素含量; 并比较了结合斑蝥素与钙、镁含量之间的相关性。【结果】芫菁体内总斑蝥素的含量为游离斑蝥素含量的1～9倍,总斑蝥素的含量一般多于虫体干重的2.0%,而游离斑蝥素的含量均低于虫体干重的1.7%。黑翅红蝉体内不含任何形式的斑蝥素。斑芫菁属和豆芫菁属昆虫中的结合斑蝥素含量与钙元素呈正相关,斑芫菁属中结合斑蝥素含量与镁元素呈正相关,而豆芫菁属中结合斑蝥素含量与镁元素呈负相关。斑芫菁属昆虫体内钙元素的摩尔量要低于结合斑蝥素的摩尔量。【结论】芫菁体内总斑蝥素含量远高于游离斑蝥素的含量;结合斑蝥素可能是以斑蝥素酸钙和斑蝥素酸镁形式存在。