海洋来源放线菌无活性野生株的链霉素抗性抗肿瘤活性突变株新产代谢产物研究

目的研究阐明无活性放线菌野生株L35-1来源硫酸二乙酯（DES）诱变链霉素抗性抗肿瘤活性突变株D2s4-1新产代谢产物及其抗肿瘤活性。方法组合利用活性跟踪与微量预试先导-放大实验制备的实验模式,通过与原始菌样品直接对照,在快速确定差异活性斑点基础上,组合利用各种色谱技术,分离纯化突变株新产代谢产物。根据理化性质和波谱数据鉴定化合物结构。采用MTT法测定抗肿瘤活性。结果从突变株D2s4-1发酵物中分离鉴定了6个该突变株新产代谢产物,即1,9-二甲酯吩嗪（1）、2-羟基苯乙酰胺（2）、2-吡咯甲酸（3）、大豆黄素（4）、环（4-羟基-脯氨酸-亮氨酸）二肽（5）和尿嘧啶（6）。其中1、3、5和6有一定抗肿瘤活性,浓度为100μg/ml时对K562细胞的抑制率分别为33.3%、16.3%、33.3%和21.1%。结论阐明了抗肿瘤活性突变株D2s4-1的6个新产物,其中4个为活性产物。化合物1为新天然产物,其抗肿瘤活性亦属首次筛选发现。用化学诱变组合抗性筛选技术从无活性放线菌野生株转化获取的活性突变株可供筛选新产活性产物,从而拓展放线菌药源活性新菌株资源。     

无活性放线菌野生株抗肿瘤活性突变株的自发突变抗性筛选和化学诱变抗性筛选

目的由无活性放线菌野生株转化获取活性突变株,为药源活性产物研究拓展新菌株资源。方法以海洋来源无活性放线菌野生株L35-1为出发菌株,通过自发突变抗性筛选以及化学诱变结合抗性筛选,筛选获得抗性突变株。采用MTT法检测突变株发酵样品对K562细胞的抑制活性。结果通过自发突变抗性筛选,得到新霉素抗性突变株114株、链霉素抗性突变株68株,其中7株新霉素抗性突变株和3株链霉素抗性突变株样品对K562细胞有抑制作用,在100μg/ml浓度下抑制率大于20%。通过化学诱变结合抗性筛选,得到硫酸二乙酯诱变链霉素抗性突变株41株、硫酸二乙酯诱变新霉素抗性突变株32株、亚硝基胍诱变新霉素抗性突变株46株,其中1株硫酸二乙酯诱变链霉素抗性突变株和1株亚硝基胍诱变新霉素抗性突变株有抗肿瘤活性,100μg/ml样品对K562细胞的抑制率大于20%。结论上述结果初步表明,无活性放线菌野生株可通过抗性筛选转化为活性突变株,因此有可能成为筛选获取药源活性新菌株的重要原始资源。     

海洋放线菌和真菌野生株及其突变株的分离培养与抗肿瘤活性筛选

目的:从海泥样品中分离培养并筛选获取野生型与突变型药源微生物抗肿瘤活性菌株.方法:通过单菌落挑选与划线培养,分离纯化野生菌株;利用核糖体工程抗性筛选技术.筛选获得抗生素抗性突变株;用K562细胞,采用MTT法测试发酵样品的抗肿瘤活性.结果:从天津塘沽驴驹河渤海湾潮间带海泥样品中分离得到放线菌126株、真菌29株,其中,在100μg/ml样品浓度下抑制率大于20%的放线菌17株、真菌3株,在1000μg/ml样品浓度下抑制率小于5.5%的无活性放线菌19株.以1000μg/ml样品浓度下抑制率为4.8%的无活性放线菌野生株L8-5x为原始茼,经抗性筛选获得链霉素抗性突变株78株,其中在100μg/ml样品浓度下抑制率大于25%的活性突变株2株.结论:所获野生型与突变型抗肿瘤活性菌株为后续新药筛选提供了药源微生物活性菌株.由无活性菌株转化获取活性突变株的研究结果表明,已分离得到的大量无活性菌株也是可转化获取药源活性突变株的很好资源.     

Aurora激酶抑制剂ZM447439的合成及生物活性研究

目的优化Aurora激酶抑制剂ZM447439的合成路线,并对其体外生物活性进行评价。方法以4-羟基-3-甲氧基苯甲酸为起始原料,通过酯化、取代、硝化、还原、关环等反应合成ZM447439,涉及到的中间体及目标产物结构经1H-NMR,MS及IR确证。以MTT法研究其体外抗肿瘤活性(U937,K562,A549,LoVo及HT29细胞株),同时评价其体外AuroraA/B激酶抑制活性。结果合成了Aurora A/B激酶抑制剂ZM447439并进行相关生物活性研究。结论新合成路线总收率约34%,目标化合物具有较好的Aurora A/B激酶活性及肿瘤细胞增殖抑制活性。     

阿德福韦酯治疗失败的慢性乙型肝炎患者HBV反转录酶区新变异rtN236V的表型耐药特点分析

目的 鉴定1例阿德福韦酯(ADV)治疗失败的慢性乙型肝炎患者HBV反转录酶(RT)区的新变异rtN236V,并分析其表型耐药特点.方法 从1例ADV治疗失败患者的血清中提取HBV DNA,采用巢式PCR扩增HBV RT区,将PCR产物克隆到pGEM-Teasy载体,转化JM109细胞.挑选34个阳性克隆进行DNA测序并分析耐药相关突变.构建1.1倍HBV野生株和耐药株重组载体,转染人肝癌细胞系HepG2细胞.转染4h后分别加入不同浓度的拉米夫定(LAM)、ADV、恩替卡韦(ETV)和替诺福韦(TDF),转染后第4天收集细胞培养液,采用实时荧光定量PCR法检测不同药物浓度作用下细胞培养上清中HBV DNA的产量,并分析其表型耐药特点.结果 该例患者在接受ADV治疗15个月后出现病毒学突破和生化学突破,测序结果显示34个克隆中,20个(58.8％)为rtN236V变异株,11个(32.4％)为rtN236T变异株,2个(5.9％)为野生株,1个(2.9％)为rtA181V+N236V变异株.rtN236T、rtN236V和rtA181V+N236V变异株的相对病毒复制力分别为野生株的89.43％、83.60％和75.44％.表型耐药分析显示:rtN236T株、rtN236V株和rtA181V+N236V株对ADV的敏感性分别为野生株的1/4.75、1/3.10和1/5.10,但对LAM、ETV和TDF均敏感.结论 在ADV长期治疗失败的慢性乙型肝炎患者血清病毒池中鉴定了新的变异形式rtN236V,并与rtN236T和rtA181V株伴随存在,该变异降低了病毒的复制力及对ADV的敏感性,可能是一个新的ADV耐药相关变异.     

慢性乙肝患者HBV反转录酶区新变异rtL180M+A181C+M204V的鉴定及表型耐药特点分析

目的鉴定经核苷(酸)类似物序贯治疗的慢性乙肝患者HBV反转录酶(RT)区的新变异rtA181C,并分析其表型耐药特点。方法 2010年6月解放军302医院确诊的慢性乙肝患者1例,提取血清HBV DNA,巢式PCR扩增HBVRT全基因,采用PCR产物直接双向测序结合克隆测序分析HBV RT区12个耐药相关突变位点。构建代表性克隆重组表达载体pTriEx-HBV1.1,瞬时转染人肝癌细胞系HepG2细胞,转染4h后加入不同浓度拉米夫定(LAM,终浓度为0、0.01、0.1、1、10、100μmol/L)、阿德福韦(ADV,终浓度为0、0.033、0.1、0.33、1、3.3μmol/L)、恩替卡韦(ETV,终浓度为0、0.001、0.01、0.1、1、10μmol/L)和替诺福韦(TDF,终浓度为0、0.01、0.1、1、10、100μmol/L),4d后采用实时荧光定量PCR检测不同药物浓度作用下培养细胞上清中HBV DNA的复制水平并分析其表型耐药特点。结果此例慢性乙肝患者序贯接受LAM治疗36个月→ADV治疗14个月→ETV治疗29个月,而后发生病毒学突破,HBV DNA由阴性升高至1.1×106IU/ml,ALT由正常升高至235U/L。直接测序显示HBV RT基因rtL180M+A181V+M204V变异,克隆测序得到18个克隆,其中9个为rtL180M+A181V+M204V变异株,7个为rtL180M+A181C+M204V变异株,1个为rtV173L+L180M+A181V变异株,1个为野生株。病毒复制力检测结果从高到低依次为:野生株>rtV173L+L180M+A181V>rtL180M+A181C+M204V>rtL180M+A181V+M204V。表型耐药分析显示,与野生株比较,rtL180M+A181V+M204V变异株和rtV173L+L180M+A181V变异株对LAM和ADV不敏感,rtL180M+A181C+M204V变异株对LAM和ETV不敏感。结论长期核苷(酸)类似物序贯治疗可引起多重耐药;rt181位点新的变异rtA181C与ETV长期用药有关,可能是新的ETV耐药变异位点。     

弗氏志贺菌ΔlysA突变株的构建及SILAC技术的应用尝试

目的构建弗氏2a志贺菌301株赖氨酸营养缺陷型突变株,为将细胞培养条件下稳定同位素标记（SILAC）技术应用于志贺菌蛋白质组学研究奠定基础。方法采用λ-Red重组系统对弗氏2a志贺菌301株lysA基因进行缺失,对野生株和突变株进行基本的表型测评和毒力评价,并利用双向电泳技术比较二者在全菌蛋白表达谱上的差异,最后将突变株分别在含有12C6-赖氨酸和13C6-赖氨酸的培养基中培养,根据双向电泳胶图取对应蛋白点进行胶内酶切及MALDI-TOF/TOF分析。结果成功构建了301 lysA基因缺失突变株,蛋白鉴定结果显示所取对应蛋白点为同一蛋白,在质谱图上轻重同位素峰位移为6 amu。结论本研究所构建的赖氨酸营养缺陷型突变株适合进行SILAC分析。     

苯甲酰胺组蛋白去乙酰化酶抑制剂的合成及抗肿瘤活性

目的 设计合成苯甲酰胺嘧啶衍生物,并初步评价其抗肿瘤活性.方法 以4-氨甲基苯甲酸为原料经过4步反应,合成了12个苯甲酰胺衍生物,结构经过,HNMR和MS鉴定,采用MTT法筛选了目标化合物的抗肿瘤活性并进行初步评价.结果 合成了12个目标化合物,均未见文献报道.5个化合物显示较好的肿瘤细胞增殖抑制活性.结论 化合物5a...     

Oxidation of cellular thiols by hydroxyethyldisulphide inhibits DNA double-strand-break rejoining in G6PD deficient mammalian cells

Purpose : We investigated the effect of protein- and non proteinthiol oxidation on DNA double-strand-break (DSB) rejoining after irradiation and its relevance in the survival of CHO cells. Materials and methods : We used mutant cells null for glucose 6 phosphate dehydrogenase (G6PD) activity since reducing equivalents, required for reduction of oxidized thiols, are typically generated through G6PD regulated production of NADPH. Cellular thiols were oxidized by pre-incubating the cells with hydroxyethyldisulphide (HEDS), the oxidized form of mercaptoethanol (ME). The concentrations of the intracellular and extracellular non-protein thiols (NPSH), glutathione, cysteine and mercaptoethanol were quantitated by HPLC. Protein thiols (PSH) were estimated using Ellman's reagent. Cell survival was determined by clonogenic assay. The induction and rejoining of DSB in cells was quantitated by Pulse Field Gel Electrophoresis after exposure to ionizing radiation. Results : Much lower bioreduction of HEDS was found in the G6PD deficient mutants (E89) than in the wild-type cells (K1). A 1 h treatment of E89 cells with HEDS produced almost complete depletion of non-protein thiol (NPSH) and a 26% decrease in protein thiols. Only minor changes were found under similar conditions with K1 cells. When exposed to gamma radiation in the presence of HEDS, the G6PD null mutants exhibited a higher cell killing and decreased rate and extent of rejoining of DSB than were observed in K1 cells. Moreover, when the G6PD deficient cells were transfected with the gene encoding wild-type G6PD (A1A), they recovered close to wild-type cellular thiol status, cell survival and DSB rejoining. Conclusions : These results suggest that a functioning oxidative pentose phosphate pathway is required for DSB rejoining in cells exposed to a mild thiol oxidant.     

Oxidation of cellular thiols by hydroxyethyldisulphide inhibits DNA double-strand-break rejoining in G6PD deficient mammalian cells

PURPOSE: We investigated the effect of protein- and non protein-thiol oxidation on DNA double-strand-break (DSB) rejoining after irradiation and its relevance in the survival of CHO cells. MATERIALS AND METHODS: We used mutant cells null for glucose 6 phosphate dehydrogenase (G6PD) activity since reducing equivalents, required for reduction of oxidized thiols, are typically generated through G6PD regulated production of NADPH. Cellular thiols were oxidized by pre-incubating the cells with hydroxyethyldisulphide (HEDS), the oxidized form of mercaptoethanol (ME). The concentrations of the intracellular and extracellular non-protein thiols (NPSH), glutathione, cysteine and mercaptoethanol were quantitated by HPLC. Protein thiols (PSH) were estimated using Ellman's reagent. Cell survival was determined by clonogenic assay. The induction and rejoining of DSB in cells was quantitated by Pulse Field Gel Electrophoresis after exposure to ionizing radiation. RESULTS: Much lower bioreduction of HEDS was found in the G6PD deficient mutants (E89) than in the wild-type cells (K1). A 1 h treatment of E89 cells with HEDS produced almost complete depletion of non-protein thiol (NPSH) and a 26% decrease in protein thiols. Only minor changes were found under similar conditions with K1 cells. When exposed to gamma radiation in the presence of HEDS, the G6PD null mutants exhibited a higher cell killing and decreased rate and extent of rejoining of DSB than were observed in K1 cells. Moreover, when the G6PD deficient cells were transfected with the gene encoding wild-type G6PD (A1A), they recovered close to wild-type cellular thiol status, cell survival and DSB rejoining. CONCLUSIONS: These results suggest that a functioning oxidative pentose phosphate pathway is required for DSB rejoining in cells exposed to a mild thiol oxidant.     

体外筛选抑制K562细胞生长的放线菌代谢物

目的：建立一种适合较大量微生物代谢物抑制K562细胞生长的体外筛选方法，筛选对K562细胞具有抑制作用，而对正常细胞Vero无明显抑制作用的土壤放线菌提取物。方法：放线菌培养液加入乙醇后，置于96孔板中。经过减压除去溶剂后，用MTT法检测各样品对K562及Vero细胞的抑制作用。结果与结论：从446种土壤放线菌提取物中筛选获得5种提取物对K562细胞具有明显的抑制作用，而对Vero细胞生长抑制作用较弱。     

黄芪的含药血清诱导K562细胞表达^Gγ-mRNA和合成HbF

目的研究黄芪对K562细胞的^Gγ-珠蛋白基因表达的作用。方法以K562细胞为体外模型，用联苯胺染色方法来确定黄芪的含药血清诱导K562细胞向红系分化的作用；用RT—PCR方法研究黄芪的含药血清诱导K562细胞^Gγ-珠蛋白mRNA的表达；用Western Blot方法研究黄芪的含药血清诱导K562细胞HbF的合成。结果黄芪的含药血清作用于K562细胞5d后联苯胺染色阳性率达19．8％；同时伴有^Gγ-珠蛋白mRNA的表达的增加（最高可达3．6倍）和HbF的合成的增加，与丁酸钠组比较，黄芪组诱导K562细胞^Gγ-珠蛋白基因mRNA的表达可以持续3～5d，而丁酸钠组只有1d。结论黄芪有望成为一种有效的γ蛋白基因诱导剂。     

N-ras基因在K562细胞中的表达研究

目的探讨N-ras基因突变及表达水平与慢性粒细胞白血病发病的关系。方法以慢粒细胞株K562细胞为研究对象，采用RT-PCR和直接测序的方法对N-ras的表达水平及突变进行检测。结果RT-PCR结果示N-ras基因在K562细胞中表达升高，直接测序结果表明N-ras基因在K562细胞中不存在突变。结论N-ras基因在K562细胞中高表达，而其高表达机制与突变无关。