医学眼科论文

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摘要医学眼科论文rn__氨基胍在兔视神经损伤后对视网膜神经节细__胞的保护性作用研究____AstudyoftheprotectiveeffectofAminogunidineonretinal__ganglioncellsafteropticnerveinjuryinrabbits____目录____中文摘要………………………………………………………………1英文摘要………………………………………………………………4英文缩写………………………………………………………………7研究论文氨基胍在兔视神经损伤后对视网膜神经节细胞的保护__性作用研究__前言……………………………………………………………………8__材料与方法………………………………………………………10__结果………………………………………………………………19__讨论………………………………………………………………28__小结………………………………………………………………36__问题与展望………………………………………………………37__参考文献…………………………………………………………38__附图………………………………………………………………41__综述氨基胍对视神经损伤后视网膜神经节细胞保护性作用……45__参考文献…………………………………………………………50__致谢……………………………………………………………………54个人简历………………………………………………………………55____氨基胍在兔视神经损伤后对视网膜神经节细胞的保护性作用__研究____中文摘要____目的____视神经损伤是眼科常见疾病_多并发于颅脑外伤_预后不良_常致患者失明。由于视神经损伤的发病机制尚未完全明了,所以迄今为止其治疗仍是国内外眼科界的一大难题。本试验通过建立兔眼视神经夹伤的动物模型,及伤后早期应用氨基胍治疗(iNOS抑制剂),动态观察视神经损伤后视网膜病理形态学改变,研究NO、iNOS、MDA、SOD含量变化及耳缘静脉注射氨基胍对其影响,力求探索一条治疗视神经损伤的新思路,为临床治疗提供实验依据,尽最大可能挽救患者视功能。____材料与方法____健康成年大耳白兔66只,3-4月龄,雌雄不限,体重25±02kg_检查双眼屈光间质清_瞳孔等大等圆_对光反射正常_眼底无异常。将实验兔随机分为正常对照组、损伤治疗组、损伤对照组共3组。正常对照组6只,12只眼:从66只兔中随机抽取6只。损伤治疗组:余60只兔用同一反向动脉夹于球后3mm处夹闭视神经20s制成视神经损伤的动物模型_然后随机抽取30只给予2﹪氨基胍(Aminogunidine_AG)80mgkg耳缘静脉注射,每日1次;损伤对照组:另外30只耳缘静脉注射等量生理盐水。按照视神经损伤后1d、3d、7d、14d、21d又随机分为5组,每组兔6只,12只眼.__造模成功后分别于伤后1d、3d、7d、14d、21d处死动物,取出__眼球,其中一部分眼球固定、石蜡包埋常规做视网膜切片,进行HE染色及视网膜凋亡细胞(TUNEL)检测,光镜观察视网膜形态改变。__另一部分眼球于冰盐水上小心剥下视网膜组织,匀浆,分光光度计检测一氧化氮NO、诱导型一氧化氮合成酶iNOS、丙二醛MDA)、超氧化物歧化酶SOD的含量变化,结果进行图像分析,所得数据资料均用spss130软件包进行统计分析。____结果____1视网膜HE染色:正常对照组视网膜内界膜平滑完整,三层细胞分界清楚,节细胞呈单层排列,按胞核大小分为两类:一类大核浅染;另一类小核深染,核内染色质分布均匀。内、外核层细胞均呈多层排列,其厚度、染色均匀,细胞排列整齐紧密。同一时间点损伤治疗组视网膜的病理改变(细胞间隙,组织水肿,细胞减少),均较损伤对照组明显减轻。本实验结果提示视神经损伤后早期耳缘静脉注射AG能够及时挽救未受损的视网膜细胞,对维持视网膜的形态和功能起到了重要作用。__2TUNEL染色凋亡细胞计数和定位:视网膜切片中显示TUNEL__阳性细胞(凋亡细胞)不仅见于视网膜神经节细胞层,还见于内外核层。凋亡细胞胞核染色,呈棕黄色_部分可由于核固缩扭曲破裂而失去正常形态,并可见凋亡小体。正常对照组视网膜切片极少见到凋亡细胞。本实验仅计数节细胞层的凋亡情况进行比较研究。同一时间点损伤对照组和损伤治疗组比较,差异具有统计学意义P005。说明此种治疗方法能够减少视神经损伤后RGCs的凋亡。__3一氧化氮NO含量_诱导型一氧化氮合酶iNOS活力测定:正常视网膜组织中很少表达iNOS,但含有少量NO,在损伤后二者含量逐渐增高,同一时间点损伤对照组和损伤治疗组比较,NO含量和iNOS活性差异有统计学意义P005,提示AG通过抑制iNOS活力减少NO的产生,对视神经损伤后的RGCs起到保护作用。__4丙二醛MDA含量_超氧化物歧化酶SOD活力检测:正常视网膜组织匀浆中含一定量的MDA和SOD。同一时间点损伤对照组和损伤治疗组比较,MDA含量差异有统计学意义P005。同一时间点损伤对照组和损伤治疗组比较,SOD活性差异有统计学意义P005。表明__AG能明显提高超氧化物歧化酶的活性,降低脂质过氧化物的产生,从而降低视网膜受氧自由基损伤的程度。____结论:____1视神经夹伤后,损伤治疗组各时间点视网膜的病理改变,均较损伤对照组明显减轻。提示伤后早期耳缘静脉注射AG进行干预治疗能够及时挽救未受损的视网膜细胞,对维持视网膜的形态和功能起到了重要作用。__2正常视网膜组织中很少表达iNOS,但含有少量NO,在损伤后二者含量逐渐增高,其结果与视神经损伤后RGCs的凋亡趋势相吻合,说__明视神经损伤后NO、iNOS的大量生成是引起RGCs凋亡的一个因素。__3视神经夹伤后视网膜组织中的MDA含量逐渐升高,随着组织损伤的进一步加重,SOD逐渐减少,削弱了其对视神经视网膜的保护作用。结果说明在视神经损伤后视网膜组织内MDA不断产生,SOD不断被消耗_致使自由基不断堆积_导致细胞出现不可逆损伤。__4视神经夹伤后早期耳缘静脉注射AG进行干预治疗能够一定程度减少损伤后RGCs的凋亡。提示AG通过抑制iNOS合成减少NO的产生,同时也抑制自由基的生成,对视神经损伤后的RGCs起到保护作用。__关键词:____氨基胍视神经损伤视网膜神经节细胞凋亡治疗______AstudyoftheprotectiveeffectofAminogunidineonretinal__ganglioncellsafteropticnerveinjuryinrabbits__Abstract__Objective____Opticnerveinjuryisacommoneyedisease_Whichisusuallycoincidencedonskullandbraininjury_Ifthemedicalprognosiswasbleak_itmayleadtopatientblindnessAsthepathogenesisofopticnerveinjuryhasnotyetbeenfullyclear_Sofarthetreatmentisstillahang-upinophthalmologygroupofinternalandexternalTheaimofthestudy_throughestablishingthemodelofopticnerveinjury_andAminogunidineinduciblenitricoxidesynthaseinhibitortreatsearlyitafteropticnerveinjury_toobservethepathomorphologiccharacteristicsofretinaandtheexpressionofNOiNOS、MDASODThroughthisstudy_WhichwillexploreanewmethodoftreatmentingopticnervedamagesandprovidetheexperimentevidencefortheclinicaltreatmentTosavetheVisualfunctionofthepatients____Materialsandmethod____Sixty-sixhealthyadultrabbitsapproximate25kilogramswithouteyediseases_wererandomlydividedintothreegroupsNormalcontrolgroupn=6_injurytreatmentgroupn=30_Injurycontrolgroupn=30Accordingtothesurvivingtimeafterinjury_theinjurygroupweredividedinto5subgroupbysacrificedtime1d_3d_7d_14dand21d_thereare12eyesateachsubgroupTheanimalsoftheinjurytreatmentgroupandinjurycontrolgroupreceivedopticnerveclippinginjurywiththesameartery__clamp15cmH2Oforcepstothenerveat3mmbehindtheglobefor20sTheinjurytreatmentgroupwereinjected2AG80mgKgthroughear-bordervein_onceeverydayTheinjurycontrolgroupwerewereinjectedequalrespectivelyat1d_3d_7d14dand21dafteropticnerveinjury_andtheeyeswereenucleatedHalfofthem_retinalslicestainedHEandTUNELtolocateandcalculateapoptosiscellsHalfofthem_takebiochemistrywaytodeterminetheNitricoxideNO_scontent_induciblenitricoxidesynthaseiNOS_scontent_SuperoxideDismutaseSOD_svitalityandtheMaleicDialdehydeMDA_scontentinretinapasteAllthedatawerestatisticedwiththeSPSS130software____Result____1HEstainofretinaNormalcontrolgrouplayerofretinaissmoothandcomplete,therearethreelayercellsintheretinaThelayerofRGCis__monolayerRGCaredividedintotwotypesaccordingtothesizeofnucleusbignucleusesarelight-coloredandsmallnucleusesaredeep-dyed__IntranuclearcaryotiniswelldistributedThecellsininteriorandexternalplexiformlayerarecompactedandevenatthicknessanddyeingTheexperimentcuedthepathologychangecellspaces_retinaedema_RGC_snumberintreatedgroupofeachtime-pointwerelighterthantheinjurygroupSoAGear-borderveininjectioninearlierperiodhadadefiniteprotectiveeffectonretinaafteropticnerveclippinginjury__2ThelocatioandcalculationofTUNELstainedapoptosiscells:CellswithDNAfragmentationsweredetectedusingTUNELstainingApoptosiscellswerenotonlyseenintheganglioncellslayer_butalsointheinteriorandexternalplexiformlayerApoptosiscellswerestainedatnucleuses_nucleuseswereyellowbrownSomemaybelossofnormalmorphologybecausenucleariscondensationanddistortedandbreakdown_SometimelittleapoptosisbodiescouldbeseenFewTUNEL-positivecellswere09NSthroughear-bordervein_onceeverydayAllanimalswerekilled__detectedatnormalretinaInthisstudy_onlycalculatesapoptosiscellsofretinalganglioncellsThenumberofTUNEL-positiveRGCcellsininjurytreatmentgroupsweresignificantlyreducedP005atthesametimepointsSowecanseethatAGear-borderveininjectioninearlierperiodcandefinitelyreduceapoptosisRGCsafteropticnerveclippinginjury__3ThenitricoxideNO_scontentandtheinduciblenitricoxidesynthaseiNOS_svitalityinretinaiNOSisexpressedrarelyinnormalretinaltissue_butcontainsasmallamountofNOBothcontentincreasedgraduallyafterinjuryTheNO_scontentandiNOS_svitalityinretinaoftheinjurytreatmentgroupsweresignificantlydifferentP005atthesametimepointsTheexperimentshowAGcanreduceNOsynthesisbyinhibitingtheproductionofiNOS_thenprotecttheRGCsafteropticnerveinjury__4TheMaleicDialdehydeMDA_scontentandtheSuperoxideDismutaseSOD_svitalityinretinaInnormalretinapaste_thereweresomeMDAandSODTheMDA_scontentinretinaoftheinjurytreatmentgroupsweresignificantlydifferentP005atthesametimepointsTheSuperoxideDismutaseSOD_svitalityinretinaoftheinjurytreatmentgroupsweresignificantlydifferentP005atvarioustimepointsTheexperimentshowthatAGcoulddefinitelyreducetheMDA_scontent_increasetheSOD_svitality_thenprotecttheretinafromoxygenfreeradical____Conclusion____1Thepathologychangeintreatedgroupofeachtime-pointwerelighterthantheinjurygroupSoAGear-borderveininjectioninearlierperiodhadadefiniteprotectiveeffectonretinaafteropticnerveclippinginjury__2INOSisexpressedrarelyinnormalretinaltissue_butcontainsasmallamountofNOBothcontentincreasedgraduallyafterinjurytheresultsisconsistenttrendsapoptosisofRGCsafteropticnerveinjuryIndicatingthatlargenumberofNOandiNOSisafactor_whichwillleadtoapoptosisofRGCsafteropticnerveinjury__3MDA,scontentincreasedgraduallyintheRetinatissueafteropticnerveinjuryWithtissueinjuryincreasefurther_SOD_svitalitydecreasedgraduallyWeakeningitsprotectiveeffectontheopticnerveandretinaFreeradicalsaccumulationcontinued_leadingtocelloccursdamageirreversible4AGear-borderveininjectioninearlierperiodcanreducethedegreeofapoptosisofRGCsAfteropticnerveinjuryTheexperimentshowAGcanreduceNOsynthesisbyinhibitingtheproductionofiNOS_andinhibitfreeradicalformation_thenprotecttheRGCsafteropticnerveinjury____Keywords____Aminogunidineopticnerveinjuryretinalganglioncellsapoptosistreat____英文缩写注释表____AGAminogunidine氨基胍RGCsretinalganglioncells视网膜神经节细胞TUNELterminaldeoxynucleotidy末端脱氧核苷酸转移酶transferase-mediateddUTPnick介导的生物素化UTPendlabeling缺口末端标记NONitricoxide一氧化氮iNOSinduciblenitricoxidesynthase诱导型一氧化氮合酶MADMalondialdehyde丙二醛SODsuperoxidedismutase超氧化物歧化酶HEhematoxylinandeosinstaining苏木精伊红染色PBSphosphatebufferedsaline磷酸盐缓冲液____氨基胍在兔视神经损伤后对视网膜神经节细胞的保护性作用__研究____前言____高眼压、炎性病变、创伤、缺血及肿瘤压迫等均能严重损伤视神经_造成视力严重丧失。自1928年Cajal断言哺乳动物的中枢神经系统无再生能力以来_人们一直认为视神经损害后无法再生或修复。1985年So和Aguayo[1]进行周围神经视网膜移植成功_彻底改变了视神经损伤后不能再生的观念_预示着不久的将来人类前视路损伤后视觉恢复将成为可能。视神经由RGCs的轴突汇聚而成,事实上_在一定条件下哺乳动物视神经损伤后视网膜节细胞retinalganglioncells_RGCs可免于死亡_损伤的轴突也可再生并恢复功能。对于视神经损伤的治疗_需要3个步骤1使已损伤或已有潜在损伤的RGCs免于死亡2诱导轴突已变性的RGCs延伸出新的轴突_并到达其在中枢神经系统的靶部位3新轴突与靶部位必须形成突触并重建连接。使RGCs继发性损伤降至最低,挽救病人的视功能成为研究的重点。__动物模型研究证实视神经损伤后视功能下降的病理基础是RGCs继发性丧失为主[2]凋亡则是继发性改变的主要原因。目前认为其可能涉及的机制包括:1视神经损伤导致自由基的产生增多,富含不饱和脂肪酸的RGCs轴索及细胞膜受到自由基的攻击而发生脂质过氧化反应,同时自由基可介导Ca2+超载及蛋白质损伤等途径诱导细胞凋亡;2兴奋性氨基酸的毒性作用,视神经损伤后,损伤的RGCs和邻近细胞释放谷氨酸,通过与谷氨酸受体结合,促进细胞外Ca2+内流,Ca2+的增加作为第二信使,激活其他诱发凋亡的物质,导致细胞凋亡;3神经营养因子的剥夺:在RGCs的生命周期中,持续的营养因子是维持其正常功能的保证,视神经损伤时神经营养因子的减少导致RGCs损伤加速;4一氧化氮Nitricoxide,NO的毒性作用:NO作为神经毒素因子,参与神经系统的多种病理损伤过程,可介导神经元及胶质细胞的凋亡;5半胱氨酸蛋白酶Caspase的毒性作用:Caspase是RGCs__

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