不同剂量硫芥对雄性小鼠生殖细胞的毒性作用

硫芥(sulfur mustard,SM),又名芥子气,是重要的化学战剂,也可用于某些过度增生性疾病的治疗。硫芥中毒可造成全身多器官的损伤,生殖系统对硫芥较为敏感,但关于硫芥生殖毒性的研究较少。本实验以不同剂量的硫芥染毒实验动物,观察实验动物的精子形态,以检测硫芥对受试动物雄性生殖细胞的遗传毒性,为进一步阐明硫芥的遗传毒理学机制提供实验依据。     

硫芥淋巴细胞毒性作用与caspase-3表达

目的 探讨硫芥对淋巴细胞的毒性作用方式和分子机制。方法 利用硫芥诱导的体外培养淋巴细胞，采用荧光分光光度法检测DNA的损伤，运用逆转录．聚合酶链式反应(RBPCR)和Western blot方法检测胱氨酸蛋白酶caspase-3的基因和蛋白表达及活化。结果 硫芥可导致淋巴细胞DNA损伤，Caspase-3在基因水平和蛋白水平上表达增加，同时发生活化作用，具有时间依赖效应。结论 硫芥通过caspase依赖途径诱导淋巴细胞毒作用，Caspase-3的表达与活化参与该过程，影响着淋巴细胞的毒性作用。     

硫芥中毒犬外周血IL-2、IL-6含量变化和淋巴细胞DNA损伤

目的 研究硫芥中毒犬外周血IL-2、IL-6变化和淋巴细胞DNA损伤的规律。方法 重庆家犬硫芥中毒(16mg/kg，sc)。中毒后不同时相点取血，分离血清和淋巴细胞。采用放射免疫方法测定血清中IL-2、IL-6含量，以单细胞凝胶电泳技术测淋巴细胞DNA损伤程度。结果 中毒后4h血清IL-2、IL-6含量开始下降，72h达最低值，120h开始恢复。单细胞电泳检测结果提示，硫芥导致明显的淋巴细胞彗星现象。受损细胞率、淋巴细胞DNA片段迁徙度于中毒4h即开始升高，24～72h持续升高。结论 硫芥中毒导致发生早且持续时间较长的犬外周血IL-2、IL-6含量下降和淋巴细胞DNA损伤。     

硫芥对中国仓鼠肺成纤维细胞DNA的损伤及还原型谷胱甘肽的保护作用

目的 研究硫芥对中国仓鼠肺成纤维细胞（Chinese hamster fibroblast cell line，CHL）DNA的损伤作用及还原型谷胱甘肽（reduced glutathione，GSH）的保护作用。方法 将CHL分为2组，均以10、100、1000μmol／L硫芥染毒，其中1组以10mmol/L的GSH加以保护，分别在不同时间点（即刻，6、24、48、72 h）收集细胞进行单细胞凝胶电泳（single cell gel electrophoresis assay，SCG）检测。结果 硫芥染毒组CHL的DNA迁移率和迁移度在染毒后6、24、48、72h均显著高于相应时刻的生理盐水组和溶剂对照组（P〈0．01）。GSH保护组DNA迁移率和迁移度亦显著增高（P〈0．01），但较之单纯硫芥染毒组DNA迁移率下降了13．0％-52．5％，迁移度亦显著下降。结论 硫芥对CHL的DNA有损伤作用，呈时间、剂量关系。GSH对DNA的损伤有一定保护作用。     

硫芥诱导小鼠染色体畸变和微核形成作用的研究

目的　研究硫芥对中国仓鼠肺成纤维细胞 (chinesehamsterfibroblastcellline ,CHL)染色体的诱变作用及对小鼠骨髓微核率的影响。方法　培养CHL细胞 (±S9) ,以不同浓度硫芥处理 2 4h后常规方法制备染色体 ,观察畸变类型并计算畸变率。KM小白鼠皮下注射硫芥染毒 ,2 4h后处死动物 ,取双侧股骨 ,行骨髓涂片和姬姆萨染色 ,计数含微核嗜多染红细胞(polychromaticerythrocyte ,PCE)数。结果　硫芥处理的CHL细胞染色体畸变率显著增加 ,出现裂隙、断裂、缺失、多倍体等多种类型畸变 ,并呈量效依赖趋势。S9不影响硫芥诱导的CHL细胞染色体畸变形式和畸变率。与对照组比较 ,注射硫芥小鼠骨髓PCE微核率显著增加 (P <0 .0 1) ,硫芥剂量越大 ,微核形成率越高。结论　硫芥能直接诱发细胞染色体损伤。     

硫芥对大鼠脾脏淋巴细胞线粒体的损伤作用

目的观察硫芥对大鼠脾脏淋巴细胞线粒体的损伤作用.方法用密度梯度离心法分离大鼠脾淋巴细胞,与硫芥共同进行体外培养.用琼脂糖DNA凝胶电泳观察细胞凋亡的发生;用Western blot观察Cyt-c释放.用MTT法检测线粒体功能状态,以罗丹明123标记的荧光探针检测线粒体跨膜电位.结果 100 μmol/L硫芥作用4 h线粒体功能就有降低,线粒体跨膜电位降低,二者间呈直线相关.硫芥中毒早期即可引起淋巴细胞线粒体细胞色素C释放和细胞凋亡(DNA ladder).结论硫芥可引起大鼠脾淋巴细胞线粒体明显损伤,线粒体参与了硫芥的细胞毒性作用过程.     

低剂量硫芥诱导大鼠脾脏氧化应激状态的改变

目的探讨低剂量硫芥诱导大鼠脾脏氧化应激状态的改变及其意义.方法皮下注射硫芥,梯度离心法分离大鼠脾线粒体.用紫外分光光度法检测ROS、SOD、MDA及细胞色素含量;荧光分光法检测细胞内GSH含量;HE染色法观察脾脏病理损伤.结果低剂量硫芥作用下,脾脏出现水肿;组织和线粒体内ROS、SOD、MDA、GSH、GSSG含量出现不同程度的改变;线粒体电子传递链成分(细胞色素)含量出现不同程度的丢失.结论氧化应激状态的改变是低剂量硫芥诱导机体免疫器官(脾)损伤的毒作用机制之一.     

氧自由基在大鼠硫芥中毒复合烧伤小肠损伤中的影响

目的 探讨氧自由基在硫芥中毒复合烧伤对大鼠小肠损伤中的作用.方法 SD大鼠随机分为对照组、单纯硫芥中毒组、30%TBSAⅢ度烧伤组、中毒复合烧伤组,观察动物致伤后2、6、12、24、48、72 h血浆二胺氧化酶(DAO)活性、超氧化物歧化酶(superoxide dismutase,SOD)活力、丙二醛(malondialdehyde,MDA)含量、回肠组织病理学的改变情况.结果 单纯及复合伤均导致动物外周血中SOD活力明显下降(P＜0.01),MDA含量、DAO活性显著上升(P＜0.01),复合致伤组与单纯烧伤以及单纯中毒组有明显差异(P＜0.01);致伤后小肠组织结构损伤明显,损伤程度与损伤时间呈正相关,复合伤组的改变较相同时相点单纯损伤组明显.结论 硫芥全身中毒早期,小肠即受到损伤,在复合烧伤的情况下所导致的该损伤更为严重,氧化应激反应也更为剧烈.提示自由基与硫芥中毒复合烧伤对大鼠小肠的损伤关系密切.     

硫芥对大鼠淋巴细胞毒性作用的研究

目的 探讨硫芥中毒对淋巴细胞的作用机制,为寻找硫芥中毒早期改变的敏感指标和提高中毒的防护水平奠定其础。方法 建立硫芥中毒淋巴细胞模型,分别用单细胞凝胶电泳法检测淋巴细胞DNA损伤;用镀铜镉还原法和一氧化氮合成酶试剂盒检测中毒后淋巴细胞培养上清液一氧化氮（NO）和一氧化氮合酶（NOS）的变化;用流式细胞仪和琼脂糖凝胶电泳支检测细胞凋亡。结果 硫芥中毒后4h淋巴细胞DNA明显损伤,72h最明显,5d开始恢复;中毒后NO、NOS的变化相似,4h明显升高,1d达到峰值并于3d开始恢复。NOS的抑制剂（L-NAME）可明显降低NO的含量,但对实验组NOS的活性无明显影响。流式细胞仪检测结果显示4h淋巴细胞凋亡明显,而1、3d几乎无凋亡产生。琼脂糖凝胶电泳结果表明4h出现典型的细胞凋亡的征象,而1、3d则表现为细胞死亡。结论（1）SCG法敏感、经济、简便、可用于硫芥中毒细胞毒性、中毒程度的评价,在早期诊断上有良好的应用前景;（2）中毒后因NOS的活性升高导致NO的升高而产生细胞毒作用可能是硫芥的中毒机制之一;（3）细胞凋亡参与了硫芥的中毒过程,亦可能是其中毒机制之一。     

硫芥诱导L5178Y细胞tk基因突变的研究

目的检测硫芥对小鼠淋巴瘤L5178Y3.7.2c-tk 1-细胞的诱变性.方法采用96孔微孔板接种法检测平板接种效率和突变频率.结果硫芥可诱导L5178Y3.7.2c-tk 1-细胞tk位点的突变,诱发突变是自发突变的2～15倍.在tk位点诱发了大克隆和小克隆两种不同表型的突变集落,以小克隆为主.结论硫芥有明确的致突变性和较强的细胞毒性,产生了大范围的DNA损伤.     

Prophylactic effect of gossypin against percutaneously administered sulfur mustard

Objective To evaluate the protective efficacy of gossypin (3,3',4',5,7,8-hexahydroxyflavone 8-glucoside) by administering it intraperitoneally, for dose, time, and vehicle dependent effects against sulphur mustard (SM), administered through percutaneous route in mice. Methods SM (diluted in PEG-300) was administered percutaneously. The protective efficacy of gossypin was evaluated by administering it intraperitoneally (50, 100, 200, and 400 mg/kg), in various vehicles (water, PEG-300 and DMSO), and time intervals (30 min prior, simultaneous and 2 h post). The time dependent protection of gossypin (200 mg/kg in PEG-300; i.p.) was also evaluated using selected biochemical variables (GSH, GSSG, MDA, total antioxidant status, Hb, WBC count, RBC count, glutathione peroxidase, glutathione reductase, and superoxide dismutase) and liver histology. The protection of gossypin by oral route was also evaluated against percutaneously administered SM. Results The protection against systemic toxicity of SM (LD50 8.1 mg/kg) was better when gossypin was given with PEG-300 (8.0 folds) than DMSO (5.7 folds). No protection was observed when gossypin was administered with water. Good protection (8.0 folds) was observed when gossypin was administered (200 mg/kg in PEG-300; i.p.) at 30 min prior or simultaneous to SM exposure, but no protection was observed when gossypin was administered 2 h post to SM exposure. A significant weight loss was observed 7 days after SM administration (2 LD50), with a significant increase in RBC and Hb. A significant decrease in total antioxidant status of plasma, liver GSH and GSSG levels, and in the activities of glutathione peroxidase, glutathione reductase and superoxide dismutase was also observed 7 days after SM administration. SM treated mouse liver also showed necrosis. A significant protection was observed when gossypin (200 mg/kg in PEG-300; i.p.) was administered either as a pretreatment (30 min before) or simultaneous treatment, and not as a post treatment (2 h). The protective efficacy of gossypin was better through oral route when administered with DMSO (4.8 folds) than with PEG-300 (2.4 folds). No protection was observed when gossypin was administered orally with water. Conclusion Percutaneous administration of SM induces oxidative stress and gossypin can protect it as a prophylactic agent by intraperitoneal or oral routes.     

Protective Efficacy of Calcium Channel Blockers in Sulphur Mustard Poisoning

The present study was designed to ascertain the in vivo protective efficacy of Ca^2 -channel blockers against dermally applied sulphur mustard(SM).Male albino mice were exposed to 1.5LD50 of SM(232mg/kg0percutaneously and the control group received an equal volume of vehicle(polyethylene glycol 300),Prior to SM application,the animals were administered nifedipine and dextrose saline containing antibiotic by intraperitoneal route,The protection assessed by the mean survival time(MST)was detrermined by Dunnett‘s method.The MST was significantly increased in nifedipine treated group.Te characteristic biochemical indices of SM intoxication.i.e.lipid peroxidation and reduced glutathione(GSH)were determined in liver from animals sacrificed at 24,48 and 72 after exposure.Sm application(1 LD50)caused a reduction in GSH level which was restored in nifedipine treated grop.Sm-induced lipid peroxidation was also prevented by nifedipine administration.The protective effect of nifedipine may be related to its capacity of attenuating SM-induced lipid peroxidation and glutathione depletion.     

丹参注射液拮抗庆大霉素耳蜗毒性的实验研究

目的：探讨丹参注射液对庆大霉素（GM）耳蜗毒性的防护作用。方法：选择白色豚鼠40只，随机分成4组（n=10）：正常对照组、GM组、GM＋丹参组和丹参组。GM组腹腔注射硫酸庆大霉素100mg/kg/d，丹参组腹腔注射丹参注射液6g/kg/d，GM＋丹参组同时注射硫酸庆大霉素和丹参注射液，正常对照组注射等量生理盐水，连续用药10d后观察耳蜗的功能与形态。结果：GM＋丹参组豚鼠听功能受损明显轻于GM组，听脑干反应ABR阈值两组比较有显著性差异（P＜0．01），且形态学改变与听阈变化相一致。结论：丹参注射液可有效拮抗GM对豚鼠的耳蜗毒性，这种拮抗作用可能是通过对耳蜗血管纹边缘细胞和毛细胞内线体的保护来实现的。     

Induction of actin disruption and downregulation of P-glycoprotein expression by solamargine in multidrug-resistant K562/A02 cells

Background  Solamargine (SM), a steroidal glycoalkaloid isolated from the Chinese herb Solanum incanum, has been shown to inhibit the growth of some cancer cell lines and induce significant apoptosis. However, the effects of SM on multidrug-resistant (MDR) cells and the molecular mechanisms involved are poorly understood. The purpose of this study was to evaluate the anti-MDR effects of SM and the associated mechanisms in MDR K562/A02 cells.

Methods  The cytotoxicity of SM was measured by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay. The 14′,6-diamidino-2-phenylindole (DAPI) nuclear staining and flow cytometry were used to detect SM-induced apoptosis. The mRNA expression of P-glycoprotein (P-gp) was investigated by real-time PCR (RT-PCR). Western blotting was used to determine the expression of Bcl-2, Bax, and actin. The changes in the morphology of actin were examined with immunofluorescence staining.

Results  MTT results showed that SM effectively killed the MDR sublines K562/A02, KB/VCR, and H460/paclitaxel (Taxol), and their parental cell lines K562, KB, and H460 to an equivalent or more sensitive degree. Based on the results by flow cytometry and immunostaining, the pro-apoptotic effects of SM were observed in MDR K562/A02 cells. Furthermore, the RT-PCR results showed that SM induced the downregulation of MDR1 mRNA. In addition, the expression of P-gp and actin was decreased in the SM-treated cells, as measured by western blotting and immunostaining.

Conclusions  These results demonstrate that SM effectively triggers apoptosis in MDR tumor cells, which is associated with actin disruption and downregulation of MDR1 expression. This compound may merit further investigation as a potential therapeutic agent that bypasses the MDR mechanism for the treatment of MDR tumors. 

     

脂磷壁酸合成相关基因dltD对高致龋力变异链球菌株耐酸能力的影响

目的 通过构建变异链球菌593(Streptococcus mutans 593,SM593)dltD基因缺失株,探究dltD在SM593耐酸能力中的作用和可能机制,为龋病的生态防治提供理论依据.方法 ①同源重组构建SM593 dltD基因缺失株SM593-ΔdltD;②采用全自动生长曲线分析仪绘制在不同pH培养条件下...     

Histomorphological and histochemical alterations following short-term inhalation exposure to sulfur mustard on visceral organs of mice

INTRODUCnONSuifurmustard[SM;bis(2-chloroethyl)sultide],achendcalwaIfreagent,producespainfulblisters0nexp0sedskin(S0maniandBabu,l989;Sasseretat.,l996).Thera-diondmeticproPeniesofSMledtoitsclinicaluseasachemoheraPeuticagentagalnsttumors(WarthinandWelled,l919).SMcancauseseverecutaneousinjurytohumanskin.SMnot0nlyaffectstheskinbutalsootherorganssuchaseyesandrespiratorytract(Papirmeis-teretal.,199l;DraceandGOdman,l996).Inhalati0n0fSMvaP0urrnainlythectsthelaryngealandtracheobronchialmuc0…     

Experimental Study on the Preventive Mechanism of Salviae Miltiorrhizae Against Atherosclerosis in Rabbits Models

Therelatedstudiesapprovedthatthepathogene sisofatherosclerosisandacutecoronarysyndromesin volvesinflammationandapoptosismechanisms.Theevidenceoftheroleofinflammationandapoptosisinatheroscleroticplaquesallowstodeveloptherapeuticstrategiesto preventthedisea…     

IPCL分型联合Cyclin D1、PDGFR对食管早期肿瘤的诊断和预后预测价值

目的探讨食管上皮乳头内毛细血管袢（intrapapillary capillary loop,IPCL）分型联合细胞周期素D1（Cyclin D1）、血小板衍生生长因子受体（platelet derived growth factor,PDGFR）对食管癌的诊断和预后预测价值。 方法收治经NBI-ME检查的食管浅表型病变患者118例（病变118处）,对食管癌组织进行IPCL分型和病理分级,检测食管癌组织Cyclin D1、PDGFR的表达,并于术后随访5年,记录患者生存期。 结果食管癌浅表型病变IPLC分型Ⅲ型、Ⅳ型以食管炎、LGIN为主;V1型、V2型、V3A型以早期食管癌为主,诊断早期食管癌的敏感度、特异度、阳性预测值、阴性预测值分别为85.5%（71/83）、80.0%（28/35）、91.0%（71/78）、83.9%（28/40）;V3B型、VN型以SM1、SM2及以深食管癌为主,诊断SM2及以深食管癌的敏感度、特异度、阳性预测值、阴性预测值分别为70.0%（7/10）、92.6%（100/108）、46.7%（7/15）、90.7%（100/103）。早期食管癌、SM1、SM2及以深食管癌患者Cyclin D1、PDGFR表达程度显著高于食管炎、LGIN患者（P＜0.01）。强阳性表达Cyclin D1（P=0.036）、PDGFR（P=0.034）的早期食管癌患者5年生存率显著低于阳性或弱阳性或阴性表达患者。早期食管癌IPCL分型、Cyclin D1及PDGFR的表达均为早期食管癌预后的独立危险因素。 结论IPCL分型对于食管早期肿瘤诊断具有一定的指导意义,检测食管癌组织Cyclin D1、PDGFR表达有助于早期食管癌的预后判断,IPCL分型、Cyclin D1及PDGFR的表达均为早期食管癌预后的独立危险因素。     

芥子气中毒机制及防护药物研究进展

芥子气(SM)是一种双功能烃化剂,能与蛋白质、DNA、RNA等多种生物大分子发生烃化反应,其中与DNA发生反应是其最重要的毒理机制之一.SM损伤机制主要包括DNA损伤、多聚腺苷二磷酸核糖聚合酶(PARP)激活、氧化应激、炎症及免疫调节反应和蛋白水解酶激活等.目前临床上尚无SM特效药物,主要是对症治疗.目前对症治疗SM使用的主要药物有自由基清除剂及抗氧化剂、PARP抑制剂、抗炎药物及蛋白酶抑制剂等.本文总结了SM的毒理机制及相应的防护药物研究进展.