体内GMA-DNA加合物的研究

目的　在整体动物水平验证大鼠不同器官是否能形成甲基丙烯酸环氧丙脂(GMA)-DNA加合物。方法　大鼠灌胃给予GMA(31.25～250mg/kg体重),14d后,用32P-后标记法分析肝、肾、外周血白细胞和睾丸中GMA-DNA加合物的形成状况。结果　不同组织器官中形成类型不同、数量不等的GMA-DNA加合物。其中白细胞有4种,肝和肾均有3种,睾丸有1种。当GMA剂量为0～125mg/kg体重时,GMA-DNA加合物的形成量随GMA剂量的增加呈上升趋势,且在所检测组织器官中,其形成量的次序为:肾>肝>白细胞>睾丸。加合物N3-甲基丙烯酸-2羟丙基-脱氧胞嘧啶核苷一磷酸(N3-methacrylate-2-hydroxypropy1-dCMP)存在于肝、肾和白细胞中而不存在于睾丸中。结论　具有亲电子集团的GMA能与DNA带负电荷中心反应形成GMA-DNA加合物。     

Formation of Glycidyl Methacrylate—DNA Adducts in vivo

lNTRoDUCnONPreviousstudiesbyourgnuprevealedthatglycidylmethacrylate(GMA)couldcauseaberraioninsperms'ofndce,andinduceunscheduledDNAsynthesisinratcells,andcausemalignanttransformationinanimalandhumancells.FurtherexperimentsinvitroconfirmedthatGMAcouldbindtoDNAormononucleotideandformspecificGMA-DNAadducts(Fangetal.,l998).ThisleadstothequestionofwhetherspecificGMA-DNAadductscanbeformedaftertheexposureofanimalstoGMA,andwhetherthegenetictoxicityisfinallypro-duced?Ingeneral,chendcalscon…     

Studies of the genotoxicity of glycidyl methacrylate (GMA)

The following experiments were conducted to evaluate the genotoxic effects of GMA (glycidyl methacrylate) on mammalian and human cells. (1) Using the absorption spectrum shift method in vitro, we observed that the maximums of calf thymus DNA and GMA were shifted toward longer wavelengths (a change of more than 15 nm) and the absorbance decreased after incubation at room temperature for 15 min or more. The result indicates that binding of DNA and GMA had occurred. The binding force is strong, not affected by the addition of concentrated sodium chloride solution, and only slightly decreased by the addition of 8 M urea solution. Therefore the bond between DNA and GMA might be covalent. (2) In cell cultures, unscheduled DNA synthesis (UDS) in human and/or rat lymphocyte was induced and DNA semiconservative replication was inhibited by GMA at concentrations of less than 5.2 mM. (3) Sperm abnormality tests and assays of UDS in germ cells of male mice were conducted to study the in vivo genotoxicity of GMA. The results revealed that GMA could damage DNA, increase sperm abnormality frequency, and reduce the number of sperm cells.     

聚合物微球固载N-羟基邻苯二甲酰亚胺催化剂的制备及其催化氧化性能初探

首先采用悬浮聚合法,制备了甲基丙烯酸缩水甘油酯(GMA)和甲基丙烯酸甲酯(MMA)的交联聚合物微球(GMA/MMA),然后经过几步大分子反应在微球表面合成与固载了N-羟基邻苯二甲酰亚胺(NHPI),形成固载有NHPI的功能微球GMA/MMA-NHPI。采用红外光谱(FT-IR)及扫描电子显微镜(SEM)等方法对功能微球进行了表征。结果表明,以含环氧基团的GMA/MMA为载体,通过大分子反应可实现NHPI的合成与固载。GMA/MMA-NHPI与醋酸钴（Co(OAc)2）构成的共催化体系,在分子氧对环己烷和环己醇的氧化过程中,显示出良好的催化活性。     

重铬酸钾和谷胱甘肽作用致小牛胸腺DNA损伤的原子力显微镜研究

目的 研究重铬酸钾和谷胱甘肽对小牛胸腺DNA的损伤。方法 利用原子力显微镜、紫外光谱两种分析方法观察DNA的超微结构变化和吸收光谱的改变。结果 单一重铬酸钾不能诱导DNA断裂,但将重铬酸钾和谷胱甘肽按一定比例和浓度混合同时作用于DNA时,则可以诱导小牛胸腺DNA断裂。紫外-可见吸收光谱分析也得到同样的结果,当重铬酸钾和谷胱甘肽共同作用DNA时,其最大吸收峰呈增色效应。结论 从形态学和光谱学角度佐证了谷胱甘肽在引发铬（Ⅵ）化合物发生还原反应产生多种中间产物并导致人体细胞癌变中有重要作用。     

溴乙锭荧光法测定环境污染物的DNA交联作用

目的：评价溴乙锭荧光法检测食用油烟、香烟烟气、蚊香烟气颗粒提取物、氧化型染发剂、硝基苯类化合物的ＤＮＡ交联作用。方法：ＤＮＡ交联溴乙锭荧光分析法（ＥＦＡ）基于双链间交联所致的ＤＮＡ在变性条件下不解链，与溴乙锭结合可产生较强荧光，而正常ＤＮＡ变性后形成的单链ＤＮＡ无此现象的原理对ＤＮＡ交联进行测定。结果：食用油烟和香烟烟气颗粒提取物、氧化型染发剂具有致小牛胸腺ＤＮＡ交联形成的作用，具有遗传毒性的蚊香烟气颗粒提取物和２，４－二硝基甲苯和间二硝基苯等硝基苯化合物未观察到致ＤＮＡ交联作用。结论：ＤＮＡ交联溴乙锭荧光方法灵敏、快速、简便，适用于对环境污染物ＤＮＡ损伤机制的研究。     

PBT／SBS—g—GMA共混体系的研究

采用溶融共混法制备了ＰＢＴ和甲基丙烯酸缩水甘油酯功能化改性ＳＢＳ的共混物，用ＦＴＩＲ、ＷＡＸＤ，ＤＳＣ，ＭＩ等方法对共混物进行了表征，探讨了熔融共混过程中ＰＢＴ和ＳＢＳ－ｇ－ＧＭＡ的反应机理，研究了ＳＢＳ－ｇ－ＧＭＡ对共混体系相容性，结晶性能，热性能和流变性能的影响。     

用脱脂奶粉代替小牛胸腺DNA进行核酸分子杂交

本文应用脱脂奶粉代替小牛胸腺DNA等大分子物质配制预杂交液和杂交液进行核酸分子杂交,同时用经典的含小牛胸腺DNA等大分子物质的预杂交液和杂交液作为对照。结果表明,用脱脂奶粉可以代替小牛胸腺DNA等大分子物质进行核酸分子杂交,此法经济,配制简便,实际可用。     

pH敏感的GMA修饰葡聚糖水凝胶的制备与体外释药研究

目的:研究用于控释体系中的可生物降解的pH敏感水凝胶的制备方法及其智能释药特性. 方法:过硫酸铵(APS)和四甲基乙二胺(TEMED)作引发体系,甲基丙烯酸缩水甘油酯(GMA)修饰的葡聚糖(GMA-dex)与丙烯酸(AAc)自由基共聚制备水凝胶. GMA-dex中GMA的取代度(DS)用核磁共振测定. 在无酶的人工胃液与人工肠液中测定凝胶的平衡溶涨率,并分别测定载有平阳霉素的水凝胶在两种释放介质中的释药曲线. 结果:GMA的取代度是10.5,凝胶在人工肠液中的溶涨率明显大于人工胃液,释放平阳霉素的速率也明显快于胃液. 24 h后,人工胃液与肠液中的累积药物释放率分别为22%和43%. 结论:聚(GMA-dex/AAc)水凝胶的溶涨与释药具有pH敏感性.     

萘亚胺类化合物对DNA光敏剪切作用的初步研究

研究了Ｎ－（Ｎ,Ｎ－二甲基）－乙胺－１,８－萘硫酰亚胺和Ｎ－（Ｎ,Ｎ－二甲基）－乙胺－１,８－萘酰亚胺对小牛胸腺ＤＮＡ的嵌入结合反应,并求取了嵌入常数,它们可以把质粒ｐＵＣ１９的超螺旋ＤＮＡ剪切为开环ＤＮＡ,在紫外光作用下,这种能力会得到不同程度的提高。     

抗单链DNA单克隆抗体的制备及应用

目的:制备特异性的抗单链单克隆抗体,为建立一种特异、敏感、简便的细胞凋亡检测方法奠定基础。DNA方法:根据烷化剂盐酸氮芥能特异性地修饰小牛胸腺链上的鸟嘌呤而暴露互补链上的胞嘧啶这一特性,将修饰后的DNA(G)(C)小牛胸腺连于甲基化的牛血清白蛋白上制备成完全抗原,免疫小鼠获得特异性的抗单链单克隆抗体,并对DNABALB/cDNA单抗的类别和特异性进行鉴定;用叠氮钠和抗肿瘤药物诱导了细胞的坏死和凋亡,利用所制备的抗单链单克VP-16HL60DNA隆抗体对其进行检测,并与形态学方法、凝胶电泳法和法进行了比较。TUNEL结果:所制备的抗单链单克隆抗体能与羊DNA抗小鼠发生特异性反应,且能与凋亡细胞特异性结合,而不结合坏死细胞。IgM结论:所制备的单抗为型,利用其能特异IgM地结合单链的特性,可用于检测细胞凋亡,简便、有效地区别细胞凋亡和坏死。DNA     

Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine

Accurate DNA quantitation is a prerequisite in many biomedical and pharmaceutical studies. Here we established a new DNA quantitation method by nuclease P1 digestion and UPLC-MS/MS analysis. DNA fragments can be efficiently hydrolyzed to single deoxyribonucleotides by nuclease P1 in a short time. The decent stabilities of all the four deoxyribonucleotides were confirmed under different conditions. Deoxyadenosine monophosphate (dAMP) was selected as the surrogate for DNA quantitation because dAMP showed the highest sensitivity among the four deoxyribonucleotides in the UPLC-MS/MS analysis. The linear range in DNA quantitation by this method is 1.2–5000 ng/mL. In the validation, the inter-day and intra-day accuracies were within 90%–110%, and the inter-day and intra-day precision were acceptable (RSD < 10%). The validated method was successfully applied to quantitate DNA isolated from tumors and organs of a mouse xenograft model. Compared to the quantitation methods using UV absorbance, the reported method provides an enhanced sensitivity, and it allows for the accurate quantitation of isolated DNA with contamination of RNA and ribonucleotide.     

评价小分子嵌插剂的光谱、动力学和电泳模型(英文)

发现新小分子DNA嵌插剂是改进肿瘤化疗的有效途径之一。抗肿瘤活性评价模型和嵌插机制研究模型是发现新小分子DNA嵌插剂的重要基础。本文描述了细胞和动物以外的模型,包括DOCK评分、紫外(UV)、荧光、圆二色散(CD)、相对粘度、熔点曲线、小分子与CT DNA堆叠动力学常数、小分子与CT DNA嵌插动力学常数、小分子嵌插剂和CT DNA复合物的稳定常数和凝胶色谱电泳模型。     

甲基丙烯酸环氧丙酯对质粒表现型的改变

甲基丙烯酸环氧丙醋(glycidyl methacrylate,GMA)在航空工业、高分子合成工业和人们日常生活中有着广泛的用途,其结构式为:系双功能团单体。本研究组前已报告,在Ames试验中,GMA能诱发鼠伤寒沙门氏菌碱基置换型突变,并能诱发小鼠骨髓多染红细胞微核率升高,从而证明了GMA的诱变作用。在此基础上,拟对其诱变机理作深入探讨。本文采用质粒PBR 322作为分子靶,从分子生物学和遗传毒理学角度探讨GMA对靶分子上抗药性基因表现型的改变及此种改变的可遗传性。     

6-O-甲基丙烯酰基-D-吡喃半乳糖的水相原子转移自由聚合

合成了一种含半乳糖基团的水溶性单体6-O-甲基丙烯酰基-D-吡喃半乳糖(GMA),详细考察了其在水相中的原子转移自由聚合（ATRP）,并合成了分子量分布较窄的均聚物和共聚物。用1H-NMR和GPC对聚合物进行了表征。研究表明：当引发剂为2-溴丙酸甲酯（2-MBP）,铜盐配合物为CuBr/2,2′-联吡啶时,聚合可控性较差,单体转化率较低;在聚合体系中加入适量溴化铜并同时采用甲醇和水的混合溶剂,可提高GMA的ATRP可控性;加入四甲基溴化铵,并改用N,N,N′,N″,N″-五甲基二亚乙基三胺(PMDETA)为配体,可有效抑制二价铜的水解,提高单体的转化率;用大分子引发剂和顺序加单体法可直接合成GMA的双亲水性两嵌段共聚物。     

蚂蚁提取液对DNA-蛋白质交联修复能力的研究

目的探讨蚂蚁提取液（antextract,AE）对DNA-蛋白质交联（DPC）的修复能力。方法以甲醛为染毒液,预处理小牛胸腺DNA和卵清蛋白混合液,使之形成DPC,再用不同浓度的AE处理,最后采用SDS—KCl沉淀法来检测不同处理组的DPC修复情况。结果当甲醛染毒浓度为0．544mol／L时,10μg／ml的小牛胸腺DNA和10μg／ml的卵清蛋白DNA-蛋白质交联程度最高;制备2％的AE时,选择pH7．6的磷酸氢二钠-磷酸二氢钠缓冲液作为组织匀浆液,AE具有最强的DPC修复能力,且用pH3．6的柠檬酸一柠檬酸三钠缓冲液制备的AE也具有部分DPC修复能力;0．002％、0．02％、0．2％和2％的AE具有一定的DPC修复能力,且存在明显的浓度-效应关系。结论本研究确定了蚂蚁提取液制备过程中缓冲液的最佳pH值,并证明了蚂蚁提取液具有一定的DPC修复能力。     

Analysis of the phenotype and the restriction enzyme mapping level of mutations induced by the new mutagen glycidyl methacrylate

Glycidyl methacrylate (GMA) is a recently recognized chemical mutagen. In order to explore the mutagenicity and mutagenic process of GMA, plasmid pBR322 was used for in vitro binding, mutant screening, and restriction enzyme mapping. The binding between GMA and DNA in vitro has been verified by means of a spectrophotometric method. When pBR322 and GMA-bound pBR322 were used to transform Escherichia coli HB101, the following results were obtained: (1) The transformation efficiency of GMA-bound pBR322 was much lower than that of pBR322 alone. (2) GMA-bound pBR322 induced phenotype changes in competent cells (i.e., tetracycline-resistance inactivation or ampicillin-resistance inactivation). There were two mutants of pBR322, ApRTCS and ApSTcR, in the transformants and a deductive mutant ApsTcs in the nontransformants. (3) All of the selected mutants were stable and heritable. (4) When restriction enzyme maps were used to analyze the mutant ApRTcS, four of seven maps were changed, some sites were shifted to other resistant gene regions, for example, sites of Bg/I, EcoRI, HindIII, HincII, etc., and there was a new recognition site for HincII (252). We did not observe any DNA fragment insertion or deletion on any maps. Our results suggest that when GMA is covalently linked to the plasmid DNA, it gives rise to a premutagenic lesion of DNA that is converted in vivo into a point mutation.     

磷酸钙沉淀法在HPV-18 DNA转染NIH 3T3细胞中的应用

以 HPV-18 DNA 为供体、小牛胸脉 DNA 为载体,NIH 3T3细胞为受体细胞,应用磷酸钙沉淀法将供体 DNA 引入细胞内,得到了清晰的 NIH 3T3细胞形态学转化灶。     

Experimental Study onMalignant Transformation of Human Bronchial Epithelial CellsInduced by Glycidyl Methacrylate and Analysis on its Methylation

Objective To establish the model of human bronchial epithelial cells(16HBE) malignant transformation induced by glycidyl methacrylate(GMA) and define the different methylation genes at different stages.Methods DNA was extracted at different 16 HBE malignant phases and changes of genes DNA methylation at different stages were detected using Methylation chip of 'NimbleGen HG18 CpG Promoter Microarray Methylation'.Methylation-specific PCR(MSP) was used to observe the methylation status of some genes,and then compared with the control groups.Results The result showed that GMA induced 16 HBE morphorlogical transformation at the dose of8 μg/mL,and cell exposed to GMA had 1 374 genes in protophase,825 genes in metaphase,1 149 genes in anaphase,respectively;30 genes are all methylation in the 3 stages;318 genes in protophase but not in metaphase and anaphase;272 genes in metaphase but not in protophase and anaphase;683 genes in anaphase but not in metaphase and protophase;73 genes in protophase and metaphase but not in anaphase;67 genes in protophase and anaphase but not in metaphase;59 genes in metaphase and anaphase but not in protophase.Conclusion The pattern of DNA methylation could change in the process of 16 HBE induced by GMA.     

新型尾式卟啉的制备、表征及其与DNA相互作用的初步研究

目的合成1个新型尾式卟啉化合物（目标化合物1）,并对其与小牛胸腺DNA（CT-DNA）的相互作用方式进行初步研究。方法以吡咯、对羟基苯甲醛和苯甲醛为原料,制备得到以柔性碳链连接的目标化合物1,采用MS1、HNMR以及UV-vis等对化合物进行表征;并采用紫外光谱、荧光光谱以及黏度实验初步研究目标化合物1与CT-DNA的相互作用。结果目标化合物1的电喷雾质谱（ESI-MS）在997.4出现一个归属于[M＋H]＋的分子离子峰,实验结果与理论计算一致;目标化合物1的电子吸收光谱分别在419 nm出现一个归属于卟啉环的特征Soret吸收,在500~700 nm之间出现归属于卟啉环的特征Q-带吸收。结论目标化合物1能够以插入方式与DNA分子发生结合。