[生物医药论文精品a]龙葵碱对雄性小鼠遗传毒性实验研究

龙葵碱对雄性小鼠遗传毒性实验研究季宇彬1,2王斌1,2郎朗1,2（1哈尔滨商业大学生命科学与环境科学研究发展中心药物研究所博士后科研工作站，黑龙江哈尔滨150076；2国家教育部抗肿瘤天然药物工程研究中心，黑龙江哈尔滨150076）摘要目的探讨龙葵碱对小鼠骨髓细胞、精子的毒性作用。方法采用流式细胞仪分析龙葵碱对小鼠骨髓细胞周期的影响；采用微核试验技术观察龙葵碱对小鼠骨髓细胞微核变化的影响；采用精子畸形试验观察雄性小鼠精子畸形率的变化。结果流式细胞仪检测到龙葵碱对小鼠骨髓细胞周期的结果表明，G0/G1期细胞比率随剂量增加而不断升高,S期和G2/G48期比率G3355G7389G991G19489。G17837表明细胞G15999G19471G9394G3324G0/G1期，影响G39G49G36的G2524G6116，G17839而影响G2/G48期，G1363G17839G1849G17837G980期细胞G6980G1955G4581,G16789明龙葵碱对骨髓细胞周期G2499G1147生明G7186的G5190G6212。微核发生率和精子畸形率随剂量增高而G17892G9188增加。G13475G13491G16757学G3800G10714,高中G1314剂量G13464与G19464性对G10043G13464G3355G7389G13491G16757学G5859G1053G11G51G728005G12。结论G3324G7424G4466验G7477G1226龙葵碱对雄性小鼠G4396G3324G17963G1268毒性影响。关键词龙葵碱；细胞周期；骨髓微核；精子畸形STUDYOFSOLANINONGENOTOXICITYOFMALEMICEJIYUBIN1,2,WANGBIN1,2,LANGLANG1,21POSTDOCTORALRESEARCHSTATION,INISTITUTEOFMATERIAMEDICA,LIFESCIENCEANDENVIROMENTSCIENCERESEARCHCENTER,HARBINCOMMERCEUNIVERSITY,HEILONGJIANGHARBIN150076,CHINA2ENGINEERINGRESEARCHCENTEROFNATURALANTICANCERDRUGS,MINISTRYOFNATIONALEDUCATION,HEILONGJIANGHARBIN150076,CHINAABSTRACTOBJECTIVETOEXPLORETHETOXICEFFECTOFSOLANINONMYELOIDCELLANDSPERMATOZOONINMALEMICEMETHODSTOANALYZETHEEFFECTOFSOLANINEONCELLCYCLEOFMYELOIDCELLINMALEMICEBYFLOWCYTOMETERFCMASSAYANDTOSTUDYTHEEFFECTOFSOLANINONMYELOIDCELLANDSPERMATOZOONINMALEMICEBYMICRONUCLEUSTESTOFMYELOIDCELLANDSPERMDEFORMITYTESTRESULTG0/G1PCRIODINCREASE,SANDG2/MPCRIODDECREASEINSOLANINEGROUPITINDICATESTHATSOLANINBLOCKMYELOIDCELLING0/G1PHASCTOSTOPTHEDNASYNTHCSISANDTOREDUCECELLPOPULATIONOFG2/MPCRIODTHEREFORE,SOLANINISPROVEDTOPRODUCECONSPICUOUSINTERFERENCEONMYELOIDCELLHIGHDOSAGEGROUPSMEDIUMDOSAGEGROUPSANDLOWGROUPSOFSOLANINALLSHOWSTATISTICALSIGNIFICANCEP005COMPAREDWITHANTIFERTILITYDOSAGEGROUPSINMICRONUCLEIASSAYOFMYELOIDCELLANDSPERMDEFORMITYTESTCONCLUSIONTHEGENETOXICINFLUENCEOFSOLANINONMALEMICEISEXISTEDINSUCHEXPERIMENTCONDITIONKEYWORDSSOLANINCELLCYCLEMYELOIDCELLSPERMDEFORMITY龙葵G6564G2474物G2559G3822G12193G1867G7389抗肿瘤作用G7389G6940药G10714G6116分，抗肿瘤作用G7380G1039G16213G6116分G1038生物碱，龙葵碱G2460G2529G14552碱，G1039G16213G4396G3324G1122G14552科G7905物G20544G19095G15235的G3371G14562G2462龙葵的G1852G14621中。龙葵碱G7389抗肿瘤作用，对G2172物肿瘤的G6245G2058率G7509G5390,G7389明G7186的细胞毒作用1。G2528G7114龙葵碱G1075G7171G980G12193G14280命毒G13044，对G13975G13940G21667G14192G7389G17751G5390的G2062G9620作用，对中G7542G12082G13475G7389G21647G11213作用，G1166G1319G2572G6922G17819量龙葵碱G2499G5353G17227G3848G11183,G14157G11183,G2601G2532,G14157G8911,G11655G4392G6205大，心G17351G1820G5567后G5942，精G12082G19181G1093,G7139G17867G12573G11163，G1306G7171龙葵碱G5194G19762立即G14280死，却会G5942G5942累积G3324G1319内。G7389报道龙葵碱G7389胚胎毒性G2499导G14280孕鼠流G1147，该类生物碱明G7186地损害胚胎的生长发育G2462分化,G7389G7509G5390的胚胎毒性。G1306G7171目前,对G1122龙葵碱G3324G17963G1268G2462生殖毒性方面的研究G3324国内外G3355G4581见报道，必G16213G17839行G17837方面的研究，G1038评价其安G1852性G2462保护G1166类免受龙葵碱的毒性危害G6564供G4466验基础和科学依据。G7424文通G17819对龙葵碱G17963G1268毒性的观察来探讨龙葵碱对哺乳G2172物G7171否G7389G17963G1268毒性方面的影响。1材料与方法11受试物龙葵碱，纯度G103898，由黑龙江药品检验所G6564供。12G4466验对象G4466验G2172物采用昆明小白鼠,由哈尔滨肿瘤医院G4466验G2172物中心G6564供。13G4466验方法131细胞周期检测试验选用性G6116熟雄性小鼠60只,G1319重1825G,随机分G10386G13464每G134643只，给药和G2474材G7114间G2528微核试验。将小鼠G3800死后，G2474出双侧股骨，去肌肉，擦净血污，剪开两端关节面，用注射器G25723MLG51BS液冲出骨髓G1122试管中，滴管吹散，300目筛网G17819滤，1000R/MIN离心洗涤10MIN，弃去上清，将细胞重新悬浮G1122G51BS中，镜检，将细胞浓度调整G10381106/ML。G24741ML细胞悬液，迅速喷射到G20056G1931的70G705G1069G18267中，G9163G2260，G23G263G6930G13634G17819G3824。G17819G3824后，离心去G19512G1069G18267，加G51BS，1000R/MIN离心洗涤10MIN，2G8437。弃上清后G1325G99105ML，加G18491MLG51G44G7591液，37G263G18003G1821G7591G1440630MIN。流式细胞仪检测，G9620发G1821G8886G8886长G1038G2388NM,发射G1821G8886G8886长大G1122630NM。每G1233G7691品选G247410000G1022细胞用G48G88LG87IG70G92G70LG72G17839行G14270G2172分析。134G6980据G13491G16757G2462G3800G10714方法研究G6980据采用SG51SS115G13491G16757G17731G1226G5326立G6980据G5223。G3355采用方G5058分析，G13464与G13464间采用G47SG39方G5058分析。132骨髓细胞微核试验将G1319重2026G的雄性小鼠30只随机分G10385G13464,每G134646只,即G19464性对G10043G13464G11生G10714G11428G8712G12、G19463性对G10043G13464G11环G11979G18244G14030G230MG/G78GG12龙葵碱高剂量G134641/2G47G3950G1121MG/G78GG12、中剂量G134641/G23G47G3950G11105MG/G78GG12和G1314剂量G134641/8G47G3950G11725MG/G78GG12。G2520G13464G18129采用G14157G14120注射方法，龙葵碱给药G13464G1861给药两周。每周G6365G1319重调整G980G8437剂量。G1584药后2G23G75G3800死。G19463性对G10043G13464，G14157G14120注射环G11979G18244G14030G230MG/G78G，采用30小G7114给药法注射2天，G1122G125442天注射后6小G7114用G20060G7906G14085G14288法G3800死小鼠，剪开G14157G14120，迅速用G6175术剪将其后G13942股骨G2474G991，G2088去肌肉，用生G10714G11428G8712洗去血污和G11874肉，剪去两端的骨G20622，用G5114G19036G3848的2ML注射器G2572G2474小G10287血清，G6566G1849骨髓G14120内，将骨髓冲G1849离心管，然后用G2572管吹G6183骨髓G3254G3371G1363其G3355G2260，G12091000RG715MIN速度离心10MIN，弃去G3822G1325的上清液，G11053G991G1343405ML与G8797G9108物G9163G2260后，用滴管G2572G2474G5194滴G980滴G3324清G8917的G17745G10639G10267上，G6524G10267。将G6524G3921G7250G5190的骨髓G10267G6930G1849G7591G14406G13580中，G11014G18267G9354液G3278G446215MIN，G2474出G7250G5190。用新G21104G18209G2058的GIG72MG86G68工作液（GIG72MG86G68G1660G3803液1G1233加G83G4368的G11979G18252G13543冲液9G1233）G7591G1440610G79415MIN，然后冲洗G6493G10639G10267上的G7591G14406液，G20130G5190。G1122G8845镜G991每只小鼠G16757G69801000G1022G2992G3822G7591G13430细胞G11G51G38G40G12,观察G2559G7389微核的G51G38G40G6980,G5194G16757G12651其微核率。133精子畸形试验选用性G6116熟雄性小鼠60只,G1319重1825G,随机分G10385G13464每G134646只,G19463性对G10043G13464，G14157G14120注射环G11979G18244G14030G230MG/G78G，G17842G13505G7591毒5天，G1254412G7941G23天G3800死。G19512G19463性G13464外G2520G13464给药剂量G2528微核试验。采用G17842G13505两周G14157G14120注射G7591毒,G1122G1584药后2G23小G7114内G3800死小鼠。G2454G10043G6925G14403的小鼠精子畸形G4466验方法2G17839行。G20060G7906G14085G14288法G3800死小鼠，分离G19480G11602，分G2047G13634G1122G11439G73891ML09生G10714G11428G8712的2G23G4392G7507中，G17743G17743G6761开G19480G11602G15999G14192，G13543G5942G6683G71912G23G4392G7507G6980G8437，G19757G136343G7945MIN，G3324悬液中加G18491G1246G13430G9354液80G173LG9163G2260G7591G1440615G79420MIN后，G24741滴G13634G1122清G8917的G10639G10267上，G3355G2260G6524G10267，G12366G8680G14270然G5190G10169后，用中性G7653G14026G4565G10267，G8845镜镜检。每只G2172物观察300G1022精子细胞。精子畸变类G3423G2265括无钩、香蕉形、无G4462形、胖G3848、双G3848、双尾、尾折叠G12573。2结果21龙葵碱对小鼠骨髓细胞周期的影响结果G2499见表1图1，G0/G1期细胞比率随剂量增加而不断升高,S期和G2/G48期比率G3355G7389G991G19489，细胞G15999G19471G9394G3324G0/G1期，影响G39G49G36的G2524G6116。龙葵碱G2520G13464与G19464性G13464G18129G7389G7186著性G5058异。表1龙葵碱对小鼠骨髓细胞周期的影响XS,N3TAB1THEEFFECTOFSOLANINEONCELLCYCLEOFMYELOIDCELLINMALEMICEGROG88G83G0/G1G11G12SG11G12G2/G48G11G12G38ONG87ROL65988G23327937506092031/8G47G395080310G236170107G232850581/G23G47G3950821929G2316803271011021/2G47G395082G2350351678073077081G38G5186G235059127002611G23031A2A3A4A4A3A5A0A1A6A7A8A9A10A11A12A13A14A15A9A16A7A13A12A17A9A18A0A15A13A8A0A19A20A23A24A21A24A26A27FIG11CELLCYCLEOFMICESMARROWCELLSIN1/8LD50FIG12CELLCYCLEOFMICESMARROWCELLSIN1/4LD50FIG13CELLCYCLEOFMICESMARROWCELLSIN1/2LD50FIG14CELLCYCLEOFMICESMARROWCELLSINCPFIG15CELLCYCLEOFMICESMARROWCELLSINCONTROL图1龙葵碱对小鼠骨髓细胞周期的影响图谱FIG1THEEFFECTOFSOLANINEONCELLCYCLEOFMYELOIDCELLINMALEMICE22龙葵碱对小鼠骨髓细胞微核率的影响G11表1G12结果G2499见表2，图2，由表2G2499见龙葵碱对小鼠骨髓G51G38G40的微核率与G19464性对G10043G13464比G17751G5058异G7389G7186著性G11G51005G12。表2龙葵碱对动物骨髓嗜多染红细胞微核发生率的影响TAB2THEEFFECTOFSOLANINONMICRONUCLEUSOFMYELOIDCELLA28A29A30A22A32A25A31A35A33A34A36A31A39A37A38A34A36A31A37A38A40MG/KGA43A41A45A46A46A47A48XA49A50G38ONG87ROL66000G23575028811/8G47G39505256600076126717511/G23G47G395010566000881G236725821/2G47G395021660001091817G23G2301G38G51G2306600016727835115A42A44A51A52A53A54A55A56A57A58A59A60A61A62A63A64A65A66A67A68A69A66A70A71A72A72A73A74A75A76A75A77A72A73A74A75A76A75A78A79A36A80A37A38A36A81A37A38A36A81A82A38A832A84A85A86A87A88A89A90A91A92A93A94A95A96A97A98A84A85A87A88A89A90A94A95A96A97FIG2THEEFFECTOFSOLANINONMICRONUCLEUSOFMYELOIDCELL23龙葵碱对小鼠精子畸形率影响由表3G2499见,受试物龙葵碱G2520剂量G13464的畸形率G3355高G1122G19464性对G10043G13464,G13475G13491G16757学分析,高中剂量G13464与G19464性G13464G7389G7186著性G11G51005G12,G1314剂量G13464与G19464性G13464G5058异不G7186著。表3龙葵碱雄性小鼠精子畸形发生率的影响TAB3THEEFFECTOFSOLANINONSPERMDEFORMITYOFSPERMATOZOONINMALEMICEA99A100A101A102A103A104A105A106A107A108A109A110A105A111A112A108A109A105A111A112A113MG/KGA114A115A115A116A117XA118A119G38ONG87ROL618008G23G236728281/8G47G39505256180013072278G2301/G23G47G39501056180017697868901/2G47G3950216180021211786772G38G51G2306180073G23G2307817072A120A121A122A123A124A125A126A127A128A129A130A131A132A133A134A135A136A137A138A139A140A137A141A142A143A143A144A145A146A147A148A147A149A143A144A145A146A147A148A147A150A151A152A153A108A109A154A155A156A157A158A159A160A161A156A162A163A157A164A165图3龙葵碱雄性小鼠精子畸形发生率的影响FIG3THEEFFECTOFSOLANINONSPERMDEFORMITYOFSPERMATOZOONINMALEMICE3讨论流式细胞仪检测技术术G7171利用流式细胞仪对细胞G12573生物粒子的G10714、化G2462生物学特性G17839行分析的方法。应用流式细胞术G17839行细胞周期检测G7171研究外来化G2524物毒作用机G2058的重G16213G6175段。G7424G4466验采用G51G44G7591G14406,流式细胞仪测G4462龙葵碱作用G1122骨髓细胞后的周期变化情况。结果表明，龙葵碱G2520G13464与G19464性G13464G18129G7389G7186著性G5058异。由图1G2499看出G0/G1期细胞比率随剂量增加而不断升高,S期和G2/G48期比率G3355G7389G991G19489。G17837表明细胞G15999G19471G9394G3324G0/G1期，影响G39G49G36的G2524G6116，G17839而影响G2/G48期，G1363G17839G1849G17837G980期细胞G6980G1955G4581。表明了龙葵碱对骨髓细胞周期G2499G1147生明G7186的G5190G6212，G16789明G39G49G36的G2524G6116受到影响。微核G4466验G7171检测外来化G2524物对G7591G14406G1319损伤的重G16213方法，目前已G13475G3324国内外得到广泛应用。微核G7171细胞的G7591G14406G1319发生断裂后，细胞G17839G1849G991G980G8437分裂G7114，G7591G14406G1319G10267段不能随G7389丝分裂G17839G1849子细胞，而G3324细胞浆中形G6116直径小G11221/3的，G2992G14406与G1039核G980G14280，完G1852与G1039核分开的圆形或卵圆形的微小核。G2520G12193G10714化因G13044G2265括化学毒性物质损伤、G6930射线G10043射G1209G2462细胞G13464G6116G6116分缺乏3G1363细胞的G39G49G36复G2058和G7591G14406G1319的分裂受到破坏，纺锤丝的功能受到影响，G1147生微核，G7171G7591G14406G1319畸变G3324间期中表现出的G980G12193损伤类G3423，微核率G2499G1209反应G14280畸因G13044对细胞的损伤程度和G7591G14406G1319的稳G4462性4。SG68G87OG49G12573认G1038微核G2499G1209G7171细胞凋亡的G1147物，当G14280畸因G13044导G14280凋亡基因G9620活，内切酶G9620活，切割G39G49G36，形G6116细胞核G11874G10267，G7380终导G14280许G3822微核5。G7424G4466验通G17819对整G1319G17963G1268G8712平的小鼠G1319内骨髓微核G4466验研究，说明龙葵碱高中G1314G2520G13464与G19464性G13464对比G7389不G2528程度的诱变G6940应。小鼠精子畸形试验G7171检测环境化学物质对生殖细胞不G2528G7114期诱变性的G980G12193G1319内G4466验,由G1122其对已知的诱变剂高度敏感和对已知的G14280癌原因G7389高度的特异性而G3324国内外毒G10714学研究工作中广泛应用。精子的G6116熟G7171G980G1022依赖G1122G19480G11602微环境的复杂G17819程。药物对G19480G11602的影响G2499能G7171导G14280不育的原因之G980,G1306对G11602丸精子细胞的作用G2499能G7171导G14280精子G6980量和活力G991G19489的G1039G16213原因，G1039G16213G7171影响精子细胞的变形G17819程6。G7424G4466验结果G7186示，三G1022剂量G13464的精子畸形率G3355高G1122G19464性对G10043G13464，高中剂量G13464与G19464性G13464G7389G7186著性G11G51005G12,G1306G1314剂量G13464与G19464性G13464G5058异不G7186著，说明龙葵碱G2499G1209透G17819血生殖腺屏障而对生殖细胞精子的发育造G6116影响，G5190G6212精子正常形G6116与G6116熟，G1867G7389生殖毒性。综上所述，G7424G4466验通G17819流式细胞术揭示了龙葵