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ID:34634607
大小:6.45 MB
页数:49页
时间:2019-03-08
《左卡尼汀对体外高糖培养鼠视网膜神经细胞的保护作用及机制探讨》由会员上传分享,免费在线阅读,更多相关内容在学术论文-天天文库。
1、左卡尼汀对体外高糖培养鼠视网膜神经细胞的保护作用及机制探讨摘要目的探讨左忙尼汀(L—carnitine,LC)对高糖损伤鼠视网膜神经细胞的保护作用及可能的机制。方法选择出生1~3d的SD大鼠,取视网膜,0.125%的胰蛋白酶消化制备单细胞悬液后进行体外培养。倒置相差显微镜观察细胞生长形态,采用Thyl.1单克隆抗体和微管相差蛋白一2多克隆抗体(Map2)对培养4d的细胞进行鉴定。台盼蓝拒染实验观察不同浓度葡萄糖作用‘t-视网膜神经细胞生长状况,并计算细胞存活率。待细胞达80%融合时进行实验分组如卜.:正常细胞培养组(对照组)、单纯LC组
2、、高糖损伤组、LC保护组、N一乙酰半胱氨酸(NAC),MTT法检测各组细胞活力;流式细胞仪测各组细胞内活性氧(ROS)水平;Hoechst33342染色观察各组细胞凋亡率;JC—l染色后采用流式细胞仪检测各组细胞线粒体膜电位的变化。结果体外培养细胞免疫荧光双标鉴定显示大部分为Thyl.1及Map2阳性。通过台盼蓝拒染实验比较不同浓度葡萄糖及不同干预时间视网膜神经细胞存活率,最终选择30mmol/L葡萄糖干预48h建立高糖损伤模型。MTT结果显示高糖损伤组细胞活性显著卜.降(尸<0.01),左}尼汀保护组细胞活力较高糖组显著提高(尸3、01)。高糖损伤组ROS水平高于止常细胞组,差别有统计学意义(尸<0.01),LC保护组ROS水平显著低于高糖损伤组(尸<0.01)。高糖损伤组Hoechst33342染色可见大量细胞出现凋亡的形态学改变(如细胞核致密浓染、核固缩、边缘化等),细胞凋亡率显著高于正常组(P<0.01),LC保护组细胞凋亡率显著低于高糖损伤组(P4、,其保护5、机制可能与其增强细胞的抗氧化能力、抑制细胞凋亡及改善线粒体功能有关。硕士研究生指导教师陈杰(眼科学)孔庆兰教授曹玉副教授关键词:左卡尼汀;视网膜神经细胞;氧化应激;线粒体ThestudyoftheprotectionandmechanismofL-Carnitineonculturedratretinalganglioncellsinhigh—glucoseAbstractObjective:TostudytheprotectionandmechanismofL-carnitine(LC、ontheratretinalganglionc6、ellsinhigh—glucose.Methods:TheretinalofSprague—Dawratsintocellsuspensionwithtrypsindigestionwhichafterbirth1~3daysweredisgested(0.125%)andthenculturedinvitro.Usingthephasecontrastmicroscopeobservedthecellgrowth.Aftercultured4daysretinalganglioncellswereidentifiedwithanti7、.ratThv1.1monoclonalantibodyandanti.ratMap2polyclonalantibodybyimmunofluorescencemethod.ThecellgrowthstatusandsurvivalrateofdifferentconcentrationsofglucosewasobservedbyTrypanbluestainingtest.Theculturedcellsweregroupedasfollows:normalcontrolgroup,simpleLCgroup.high—gluc8、osedamagegroup.LCprotectedgroupandNACgroup.MTTassaysdetectedcellviability.FlowcytometrywasusedtodetectintracellularROS.NuclearstainingaSSaybyusingchromatindyeHoechst3342wasusedtoevaluatedthecellapoptosis.Flowcytometrywasusedtodetectethemitochondrialmembranepotentialbyusi9、ngthefluorescentdyeJC一1.Results:Theresultofcellimmunofluorescenceidentificationshowedthatthemostofcells
3、01)。高糖损伤组ROS水平高于止常细胞组,差别有统计学意义(尸<0.01),LC保护组ROS水平显著低于高糖损伤组(尸<0.01)。高糖损伤组Hoechst33342染色可见大量细胞出现凋亡的形态学改变(如细胞核致密浓染、核固缩、边缘化等),细胞凋亡率显著高于正常组(P<0.01),LC保护组细胞凋亡率显著低于高糖损伤组(P4、,其保护5、机制可能与其增强细胞的抗氧化能力、抑制细胞凋亡及改善线粒体功能有关。硕士研究生指导教师陈杰(眼科学)孔庆兰教授曹玉副教授关键词:左卡尼汀;视网膜神经细胞;氧化应激;线粒体ThestudyoftheprotectionandmechanismofL-Carnitineonculturedratretinalganglioncellsinhigh—glucoseAbstractObjective:TostudytheprotectionandmechanismofL-carnitine(LC、ontheratretinalganglionc6、ellsinhigh—glucose.Methods:TheretinalofSprague—Dawratsintocellsuspensionwithtrypsindigestionwhichafterbirth1~3daysweredisgested(0.125%)andthenculturedinvitro.Usingthephasecontrastmicroscopeobservedthecellgrowth.Aftercultured4daysretinalganglioncellswereidentifiedwithanti7、.ratThv1.1monoclonalantibodyandanti.ratMap2polyclonalantibodybyimmunofluorescencemethod.ThecellgrowthstatusandsurvivalrateofdifferentconcentrationsofglucosewasobservedbyTrypanbluestainingtest.Theculturedcellsweregroupedasfollows:normalcontrolgroup,simpleLCgroup.high—gluc8、osedamagegroup.LCprotectedgroupandNACgroup.MTTassaysdetectedcellviability.FlowcytometrywasusedtodetectintracellularROS.NuclearstainingaSSaybyusingchromatindyeHoechst3342wasusedtoevaluatedthecellapoptosis.Flowcytometrywasusedtodetectethemitochondrialmembranepotentialbyusi9、ngthefluorescentdyeJC一1.Results:Theresultofcellimmunofluorescenceidentificationshowedthatthemostofcells
4、,其保护
5、机制可能与其增强细胞的抗氧化能力、抑制细胞凋亡及改善线粒体功能有关。硕士研究生指导教师陈杰(眼科学)孔庆兰教授曹玉副教授关键词:左卡尼汀;视网膜神经细胞;氧化应激;线粒体ThestudyoftheprotectionandmechanismofL-Carnitineonculturedratretinalganglioncellsinhigh—glucoseAbstractObjective:TostudytheprotectionandmechanismofL-carnitine(LC、ontheratretinalganglionc
6、ellsinhigh—glucose.Methods:TheretinalofSprague—Dawratsintocellsuspensionwithtrypsindigestionwhichafterbirth1~3daysweredisgested(0.125%)andthenculturedinvitro.Usingthephasecontrastmicroscopeobservedthecellgrowth.Aftercultured4daysretinalganglioncellswereidentifiedwithanti
7、.ratThv1.1monoclonalantibodyandanti.ratMap2polyclonalantibodybyimmunofluorescencemethod.ThecellgrowthstatusandsurvivalrateofdifferentconcentrationsofglucosewasobservedbyTrypanbluestainingtest.Theculturedcellsweregroupedasfollows:normalcontrolgroup,simpleLCgroup.high—gluc
8、osedamagegroup.LCprotectedgroupandNACgroup.MTTassaysdetectedcellviability.FlowcytometrywasusedtodetectintracellularROS.NuclearstainingaSSaybyusingchromatindyeHoechst3342wasusedtoevaluatedthecellapoptosis.Flowcytometrywasusedtodetectethemitochondrialmembranepotentialbyusi
9、ngthefluorescentdyeJC一1.Results:Theresultofcellimmunofluorescenceidentificationshowedthatthemostofcells
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