STUDY ON GMA-DNA ADDUCTS

Objective. DNA modification fixed as mutations in the cells may be an essential factor in the initiation step of chemical ca~inogenesis. In order to explore the mechanism of gene mutation and cell transformation induced by glyaldyl methacrylate (GMA), the current test studied the characteristics of GMA-DNA adducts formation in vitro. Methods. in vitro test, dAMP, dCMP, dGMP, dTMP and calf thymus DNA were allowed to react with GMA (Glycldyl Methacrylate). After the reaction, the mixtures were detected by UV and subjected to reversed-phase HPLC on ultrasphere ODS reversed-phase column, the reaction products were eluted with a 1inear gradients of methanol (solvent A) and 10mmol/L ammonium formate, pH5.0 (solvent B).The synthesized adducts were then characterized by UV spectroscopy in acid (pH1. 0), neutral (pH7.2),alkaline (pH11.0) and by mass speetroacopy. Results. The results showed that GMA could bind with dAMP, dCMP, dGMP and calf thynms DNA by covalent bond, and the binding sites were specific (N6 of adenine, Na of cytosine). Meanwhile, a main GMA-DNA adduct in the reaction of GMA with calf thymus DNA was confirmed as N3 methaerylate-2-hydroxypropyl-dCMP. Conclusions. GMA can react with DNA and /or deoxynucleotide monophosphate and generate some adducts such as N6-methaerylate-2-hydroxypropyl-dAMP and N3 methacrylate-2 hydroxypropyl-dCMP,ets. Formation of GMA-DNA adducts is an important molecular event in gene mutation and cell transformation induced by GMA.     

姜黄素类似物对正常动物细胞和肿瘤细胞间通讯传递的影响

采用正常中国仓鼠肺细胞（Ｖ７９）、Ｂａｌｂ／ｃ小鼠成纤维细胞（Ｂａｌｂ／ｃ－３Ｔ３）、大鼠肝上皮细胞（ＷＢ）、人胚肺细胞（２８Ｓ）观察了间隙连接介导的细胞间通讯功能（ＧＪＩＣ）以及促癌剂ＴＰＡ对ＧＪＩＣ的抑制作用和药物对ＴＰＡ的拮抗作用。结果表明：Ｖ７９、ＷＢ以及３Ｔ３、２ＢＳ细胞均具有中等强度的细胞间隙通讯传导功能，ＴＰＡ对其均呈一定的抑制作用，姜黄素类似物９１０２２、９１０２２－Ｓ对ＴＰＡ具有一定的拮抗作用，即可增强上述４种细胞的通讯传导功能，同时观察到人肺腺癌Ａ５４９和ＧＬＣ无明显间隙连接通讯。９１０２２之所以增强Ａ５４９的传导功能，可能与其具有抗肿瘤作用有关。     

MOLECULAR ANALYSIS OF RADIATION-INDUCED MUTATION IN EXON 7/8 OF RAT HPRT GENE

Objective To investigate the relationship between the radiation dose and the HPRT gene lo-cus mutation in rat smooth muscle cells, and provide the molecular basis for prevention of restenosis after percutaneous transluminal coronary angioplasty (PTC4). Methods The smooth muscle cells cultured in vitro were irradiated by radionuclide 188Re in different doses. HPRT gene mutation colonies were selected and isolatedby 6-thioguanine. Analysis of mutation in exon 7/8 of HPRT gene were accomplished by polymerase chain reaction and single-strand conformation polymorphism. Results The HPRT gene mutation frequency of rat smooth muscle cells that were irradiated by radionuclide 188Re ranged from 5.5× 10-6 to 13 ×10-6. Of 91 HPRT gene mutation colonies, 13 (14.3%) contained exon 7/8 deletion and 15(16.5%) had point mutation.The exon 7/8 mutation frequency was 30.8% . There were significant relationships between radiation dose and mutation frequency of HPRT gene and exon 7/8 . Conclusion The DNA damage and gene mu     

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.     

白藜芦醇抑制HepG2细胞生长和对细胞间隙连接通讯的影响

目的研究白藜芦醇对人肝癌细胞HepG2的生长抑制作用以及对细胞间隙连接通讯（GJIC）的影响。方法利用MTT法观察白藜芦醇对HepG2细胞的生长抑制作用。流式细胞仪检测细胞周期的时相分布，使用荧光探针5＇-CFDA／AM标记细胞．采用荧光漂白后重恢复技术（FRAP）和共聚焦显微镜技术观察白藜芦醇对HepG2细胞间隙连接通讯的影响。结果不同浓度的白藜芦醇处理细胞不同时间后，HepG2细胞的生长增殖明显受到抑制，抑制率最高达92．1％，各处理组间比较均有显著性差异（F=18．532，P〈0．05）；细胞周期分析显示，白藜芦醇能诱导HepG2细胞在S期停滞．抑制细胞DNA的合成并可诱导细胞凋亡：另外，白藜芦醇有恢复HepG2细胞间隙连接通讯的功能，且随浓度增加而增加。结论白藜芦醇可抑制HepG2细胞增殖，使细胞停滞于S期，并能诱导细胞凋亡；白藜芦醇还有恢复HepG2细胞间隙连接通讯功能的作用，提示此作用可能是白藜芦醇抑制HepG2细胞增殖和抗肿瘤作用的机制之一。     

微囊藻毒素、佛波酯和苯巴比妥钠对细胞间隙通讯和细胞内Ca2+浓度的影响

目的:探讨细胞间隙通讯和细胞钙离子信号传导系统在非遗传毒性致癌物的遗传外致癌过程中的作用.方法:应用划痕染料示踪技术(SLDT)和fluo-3荧光标记法,测定微囊藻毒素、佛波酯(TPA)、苯巴比妥钠3种非遗传毒性致癌物对BALB/c 3T3细胞间隙通讯功能和细胞内游离Ca2+离子浓度的影响.结果:3种非遗传毒性致癌物均可不同程度抑制细胞间隙通讯功能,并呈良好的剂量效应关系.TPA和微囊藻毒素可明显诱导细胞内游离钙离子浓度升高,但在试验剂量下苯巴比妥钠未见引起细胞内Ca2+离子浓度的明显改变.结论:细胞间隙通讯系统可能是3种非遗传毒性致癌物的共同作用位点,对细胞Ca2+系统的影响以TPA类非遗传毒性致癌物为主.     

Aminoguanidine delays the replicative senescence of human diploid fibroblasts

Background The accumulation of free radicals and advanced glycation end products (AGEs) in cell plays a very important role in replicative senescence. Aminoguanidine (AG) has potential antioxidant effects and decreases AGE levels. This study aimed to investigate its effect on replicative senescence in vitro. Methods The effects of aminoguanidine on morphology, replicative lifespan, cell growth and proliferation, AGEs, DNA damage, DNA repair ability and telomere length were observed in human fetal lung diploid fibroblasts (2BS). Results Aminoguanidine maintained the non-senescent phenotype of 2BS cells even at late population doubling (PD) and increased cumulative population doublings by at least 17-21 PDs. Aminoguanidine also improved the potentials of growth and proliferation of 2BS cells as detected by the MTT assay. The AGE levels of late PD cells grown from early PD in DMEM containing aminiguanidine decreased significantly compared with those of late PD control cells and were similar to those of young control cells. In addition, the cells pretreated with aminoguanidine had a significant reduction in DNA strand breaks when they were exposed to 200 μmol/L H(2)O(2) for 5 minutes which indicated that the compound had a strong potential to protect genomic DNA against oxidative stress. And most of the cells exposed to 100 μmol/L H(2)O(2) had much shorter comet tails and smaller tail areas after incubation with aminoguanidine-supplemented DMEM, which indicated that the compound strongly improved the DNA repair abilities of 2BS cells. Moreover, PD55 cells grown from PD28 in 2 mmol/L or 4 mmol/L aminoguanidine retain telomere lengths of 7.94 kb or 8.12 kb, which was 0.83 kb or 1.11 kb longer than that of the control cells.Conclusion Aminoguanidine delays replicative senescence of 2BS cells and the senescence-delaying effect of aminoguanidine appear to be due to its many biological properties including its potential for proliferation improvement, its inhibitory effect of AGE formation, antioxidant effect, improvement of DNA repair ability and the slowdown of telomere shortening.     

纳米SiO2颗粒对HL-7702细胞的毒性作用和缝隙连接通讯的影响

目的：探讨纳米二氧化硅(SiO2)颗粒对HL-7702细胞的毒性作用和细胞缝隙连接通讯（GJIC)的影响,为纳米SiO2体内外毒性的预测和安全性应用提供实验依据。方法：透射电镜(TEM)观察2种SiO2颗粒的粒径、分散性和形状;动态光散射法(DLS)检测SiO2颗粒在高纯水和培养液中的粒度分布;MTT法检测细胞存活率;乳酸脱氢酶（LDH）活力实验检测细胞膜完整性;划痕染料示踪技术检测GJIC。结果：透射电镜,2种SiO2颗粒呈圆形,大小均一,分散性良好,2种颗粒的粒径分别为（447.60±20.78）和（67.42±5.69） nm,分别为亚微米级和纳米级颗粒。动态光散射法,2种颗粒在高纯水和RPMI-1640培养液中的水合粒径分别为（684.37±18.76）、（697.02±19.57） nm　和（128.31±7.64）、（133.74±8.97） nm,颗粒均未发生聚集,分散性良好。MTT法,2种SiO2颗粒作用细胞24 h后,同一粒径的颗粒,随着浓度的增加,细胞存活率下降;同一浓度下,纳米颗粒比亚微米颗粒的毒性大。LDH活力实验,当作用细胞24 h后,2种SiO2颗粒均能够损伤细胞膜,同一浓度下,纳米SiO2颗粒比亚微米颗粒对细胞膜的损伤能力大;同一粒径的颗粒,随着作用浓度的增加,细胞膜的损伤程度加重。划痕染料示踪实验,纳米SiO2颗粒可抑制GJIC,并且随着作用浓度的增加,抑制作用增强;而在相同浓度下,纳米SiO2颗粒比亚微米颗粒对GJIC的抑制作用更明显。结论：纳米SiO2颗粒能够对HL-7702细胞产生毒性作用,且抑制GJIC。     

纳米SiO_2颗粒对HL-7702细胞的毒性作用和缝隙连接通讯的影响

目的:探讨纳米二氧化硅(SiO2)颗粒对HL-7702细胞的毒性作用和细胞缝隙连接通讯(GJIC)的影响,为纳米SiO2体内外毒性的预测和安全性应用提供实验依据。方法:透射电镜(TEM)观察2种SiO2颗粒的粒径、分散性和形状;动态光散射法(DLS)检测SiO2颗粒在高纯水和培养液中的粒度分布;MTT法检测细胞存活率;乳酸脱氢酶(LDH)活力实验检测细胞膜完整性;划痕染料示踪技术检测GJIC。结果:透射电镜,2种SiO2颗粒呈圆形,大小均一,分散性良好,2种颗粒的粒径分别为(447.60±20.78)和(67.42±5.69)nm,分别为亚微米级和纳米级颗粒。动态光散射法,2种颗粒在高纯水和RPMI-1640培养液中的水合粒径分别为(684.37±18.76)、(697.02±19.57)nm和(128.31±7.64)、(133.74±8.97)nm,颗粒均未发生聚集,分散性良好。MTT法,2种SiO2颗粒作用细胞24h后,同一粒径的颗粒,随着浓度的增加,细胞存活率下降;同一浓度下,纳米颗粒比亚微米颗粒的毒性大。LDH活力实验,当作用细胞24h后,2种SiO2颗粒均能够损伤细胞膜,同一浓度下,纳米SiO2颗粒比亚微米颗粒对细胞膜的损伤能力大;同一粒径的颗粒,随着作用浓度的增加,细胞膜的损伤程度加重。划痕染料示踪实验,纳米SiO2颗粒可抑制GJIC,并且随着作用浓度的增加,抑制作用增强;而在相同浓度下,纳米SiO2颗粒比亚微米颗粒对GJIC的抑制作用更明显。结论:纳米SiO2颗粒能够对HL-7702细胞产生毒性作用,且抑制GJIC。     

基于Cre重组酶体系的小鼠HPRT定点整合打靶载体的构建及其应用

目的：构建带有Cre重组酶识别位点变异体lox66的针对小鼠HPRT基因的基因打靶载体,并用于小鼠胚胎干细胞基因打靶研究。方法：利用含HPRT基因组DNA片段的质粒pMP8和人工合成含lox66序列的寡核苷酸,构建含Cre重组酶识别位点变异体lox66的置换型打靶载体,经过限制性内切酶酶切鉴定其结构正确后,用电穿孔法对小鼠胚胎干细胞R1株进行基因转染,经G418／6－TG（6－thioguanine)分组药物筛选,进行pSP-HPRT-lox66-Neo载体的应用研究。结果：得到了与设计一臻的置换型打靶载体pSP-HPRT-lox66-Neo;将线性化的打靶载体导入ES细胞后,能与靶位点有效地发生同源重组,获得了20个靶基因被灭活了的ES细胞克隆。结论：此研究成功构建了含Cre重组酶识别位点变异位lox66针对小鼠HPRT基因位点的置换型打靶载体,在胚胎干细胞基因打勒中得到有效的应用。     

不同频率电磁场对胆囊癌细胞增殖及DNA损伤作用

目的 探讨不同频率的低功率电磁场对胆囊癌细胞恶性增殖及DNA损伤的作用及其意义。方法 采用绘制细胞生长曲线及单细胞凝胶电泳技术分别检测经不同频率电磁场作用后,胆囊癌细胞的增殖及DNA损伤情况。结果 经不同频率（0．1～40MHZ）电磁场作用后,各实验组总体上均可见细胞增殖受到抑制,DNA链产生断裂损伤,统计学检验较正常对照有显著意义。结论 0．1-40MHZ范围低功率电磁场对胆囊癌细胞增殖及DNA损伤产生明显影响,主要表现为使DNA链产生断裂损伤,进一步抑制细胞增殖,且这种作用和频率有关,但并不是“线性”关系。     

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.     

康莱特诱导人胰腺癌细胞凋亡的实验研究

目的 探讨康莱特(KLT)对人胰腺癌细胞凋亡的影响及其作用机制。方法 采用MTT法观察KLT对人胰腺癌细胞株8988细胞增殖的抑制作用。采用TUNEL染色、DNA梯度电泳法和流式细胞术检测细胞凋亡改变，并以RT-PCR和Western blot检测凋亡调节基因p53和bcl-2的表达。结果 KLT能明显抑制人胰腺癌8988细胞的生长，且呈时间和浓度依赖性。KLT作用后8988细胞呈现凋亡特征，TUNEL染色可见发黄绿色荧光的凋亡细胞，DNA电泳可见典型梯形条带，流式细胞术显示凋亡细胞比例升高。RT-PCR和Western blot检测可见p53基因表达显著增加，而bcl-2基因表达减少。结论 KLT能诱导人胰腺癌细胞凋亡，其作用可能与凋亡调节基因p53的上调和bcl-2的下调有关。     

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     

紫杉醇对人食管癌Eca109细胞株生长的影响

目的：观察不同浓度的紫杉醇对食管癌细胞Eca109的影响。方法：应用四甲基偶氮唑蓝（MTT）实验,细胞形态学观察及流式细胞仪等方法对人食管癌细胞Eca109进行检测和观察。结果：MTT实验显示紫杉醇可抑制食管癌细胞增殖;光镜及电镜可发现药物作用组细胞核固缩、解聚以及凋亡小体,流式细胞仪检测显示G1峰前有明显的凋亡峰。细胞周期分析提示紫杉醇可将Eca细胞阻滞于G2－M期,且与浓度相关。结论：紫杉醇对人食管癌细胞系Eca109细胞的生长有明显的抑制作用,使细胞分裂阻滞于G2－M期,并诱导肿瘤细胞凋亡。     

反义阻断REV3基因表达对细胞自发突变和MNNG诱导突变的影响

目的 :通过反义核酸技术研究REV3基因对次黄嘌呤鸟嘌呤磷酸核糖基转移酶基因HPRT的自发突变频率及烷化剂甲基硝基亚硝基胍 (methy1-nitro -nitrosoguanidine ,MNNG)的诱发突变频率的影响。方法 :用REV3基因反义核酸表达质粒pMAM -REV3-转染人羊膜上皮细胞 (FL) ,建立了由地塞米松诱导下的REV3基因表达可被反义阻断的细胞系 (FL-REV3-) ,并应用经典的筛选HPRT基因位点的突变子的实验进行REV3基因对HPRT基因位点的自发与诱发突变影响的检测研究。结果 :在自发突变率的检测中 ,FL细胞系的自发突变率比它的诱发突变率低 3 0 1倍 (P <0 0 5 ) ,而在FL -REV3-细胞系中始终未能筛选到自发突变子 ;用 0 .4 μmol/LMNNG分别处理FL -REV3-和FL细胞系 ,前者诱发突变率比后者低 6 0 2倍 (P <0 0 5 )。结论 :REV3基因不仅参与细胞自发突变 ,而且参与细胞的诱发突变 ,与DNA的跨损伤修复有关     

人衰老相关DNA片段的筛选及特征分析

目的:探索衰老相关新基因.方法:采用RAPD法筛选衰老相关的DNA片段,采用Southern blot 分析基因状态,Northern blot 分析mRNA水平.结果:筛选出衰老特异的DNA片段,长894 bp,命名为sad.将sad片段克隆、测序,查询GenBank,确认为一种新的衰老相关序列,基因登录号为AQ324104.Southern blot分析表明,sad基因可能是一种单拷贝基因;sad在衰老2BS细胞中的Southern blot图谱不同于年轻细胞,而与人胃癌细胞系BGC-823类似.Northern blot分析显示,sad基因具有表达活性,其RNA长约0.5 kb,相对水平在各代龄的细胞间差异无显著性.结论:sad是一种新的与衰老相关的DNA片段,sad基因可能为单拷贝基因,具有表达活性.     

CD44表达减弱致人鼻咽癌细胞CNE-2L2体外生长的抑制

目的探讨CD44表达抑制对人鼻咽癌细胞株CNE-2L2生长的影响。方法采用RNA干扰技术抑制细胞CD44表达,基因组PCR检测siRNA整合,Western blot检测CD44表达,CellTiter96 AQueous One Solution Cell Proliferation Assay kit检测细胞生长,PI染色流式细胞仪检测细胞DNA含量。结果藉逆转录病毒转导入CNE-2L2细胞的siRNA均整合入了细胞基因组DNA。与整合了siegfp的对照细胞相比,整合了siCD44的细胞CD44表达均明显抑制,以整合了siCD44-1或siCD44-2的细胞中抑制最强。整合了siCD44-1或siCD44-2的细胞在培养中的生长明显受抑。细胞DNA含量分析显示野生型细胞、整合了siegfp的细胞、整合了siCD44-1的细胞及整合了siCD44-2的细胞处于G0/G1期的比例分别为44.4%、45.5%、53.9%及53.3%,处于S期的比例分别为39.3%、40.0%、27.1%及28.2%,处于G2/M期的比例分别为16.3%、14.5%、19.0%及18.5%。结论CD44表达减弱抑制了CNE-2L2细胞在培养中的生长,抑制细胞从G0/G1期进入S期,但略促进细胞从S期进入G2/M期。     

烹调油烟冷凝物诱发支气管上皮细胞HPRT 基因位点突变的研究

目的 研究烹调油烟冷凝物致人支气管上皮细胞HPRT 基因位点突变的影响及二甲基亚砜的拮抗作用,以探讨烹调油烟的致突变性及其防护.方法 采用体外多核细胞法研究烹调油烟冷凝物对人支气管上皮细胞HPRT 基因位点的影响及二甲基亚砜的防护作用.结果 烹调油烟冷凝物诱发人支气管上皮细胞HPRT 基因位点突变, 且突变频率随着染毒浓度的增加而升高, 突变频率与烹调油烟冷凝物浓度之间存在一定的剂量- 效应关系,二甲基亚砜对HPRT 基因位点突变有显著的拮抗作用.结论 烹调油烟冷凝物能诱发人支气管上皮细胞HPRT 基因位点突变,二甲基亚砜能抑制烹调油烟冷凝物的诱变作用.HPRT 基因突变频率可望成为接触环境致突变物人群检测的敏感指标.