CAJ | 학술논문

目的观察色素上皮衍生因子(PEDF)对糖尿病大鼠视网膜谷氨酸代谢的影响.方法 Sprague_Dawley大鼠78只,分为模型组、模型对照组、PEDF干预组(干预组)、干预对照组,实验结束时去除血糖恢复鼠和实验期间死亡鼠,每组以12只大鼠作为统计样本.模型组、干预组、干预对照组大鼠采用链脲佐菌素诱导糖尿病大鼠模型.模型组大鼠不作任何干预,模型对照组为相同月龄的正常大鼠.干预组大鼠左眼玻璃体腔注射0.1 μg/μl的PEDF 5.0μl,干预对照组大鼠左眼玻璃体腔注射相同容积的磷酸盐缓冲液.采用蛋白免疫印迹法(Western bolt)和实时荧光聚合酶链式反应(PCR)法检测视网膜L-谷氨酸-L-门冬氨酸转运体(GLAST)表达的变化,高压液相色谱法(HPLC)观察视网膜谷氨酸的含量变化.将体外培养的大鼠视网膜Müller细胞随机分为对照组、实验组、PEDF干预组(干预组)和干预对照组,荧光免疫法和实时荧光PCR法检测Müller细胞GLAST表达的改变,根据[3H]标记的D,L-谷氨酸摄入量判断Müller细胞的摄取功能.结果实时荧光PCR法和Western bolt检测结果显示,相对于模型对照组大鼠,模型组大鼠视网膜GLAST表达降低(实时荧光PCR法:t=8.86,P＜0.01;Western blot:t=3.42,P＜0.05),视网膜谷氦酸含量升高(t=4.01,P＜0.05);干预组大鼠视网膜GLAST表达与干预对照组视网膜GLAST表达比较,干预组大鼠视网膜GLAST的表达升高(实时荧光PCR法:t=3.56,P＜0.05;Western blot:t=3.52,P＜O.05);视网膜谷氨酸含量下调(t=4.36,P＜0.05).实时荧光PCR法和荧光免疫法检测结果显示,高糖可以降低视网膜Müller细胞GLAST的表达(实时荧光PCR法:t=3.48,P＜O.05;荧光免疫法:t=4.72,P＜O.05);[3H]标记的D,L-谷氨酸摄人量结果显示,高糖可以下调视网膜Mü1ler细胞GIAST的功能(t=3.81,P＜0.05);经PEDF处理后,可以明显改善高糖状态下视网膜Müller细胞GLAST的表达(实时荧光PCR法:t=6.82,P＜O.01;荧光免疫法:t=3.72,P＜0.05)和对谷氨酸的摄取功能(t=4.14,P＜0.05).结论 PEDF可通过改善糖尿病大鼠视网膜Müller细胞中GLAST功能从而改善谷氨酸循环,抑制神经节细胞的死亡. 目的觀察色素上皮衍生因子(PEDF)對糖尿病大鼠視網膜穀氨痠代謝的影響.方法 Sprague_Dawley大鼠78隻,分為模型組、模型對照組、PEDF榦預組(榦預組)、榦預對照組,實驗結束時去除血糖恢複鼠和實驗期間死亡鼠,每組以12隻大鼠作為統計樣本.模型組、榦預組、榦預對照組大鼠採用鏈脲佐菌素誘導糖尿病大鼠模型.模型組大鼠不作任何榦預,模型對照組為相同月齡的正常大鼠.榦預組大鼠左眼玻璃體腔註射0.1 μg/μl的PEDF 5.0μl,榦預對照組大鼠左眼玻璃體腔註射相同容積的燐痠鹽緩遲液.採用蛋白免疫印跡法(Western bolt)和實時熒光聚閤酶鏈式反應(PCR)法檢測視網膜L-穀氨痠-L-門鼕氨痠轉運體(GLAST)錶達的變化,高壓液相色譜法(HPLC)觀察視網膜穀氨痠的含量變化.將體外培養的大鼠視網膜Müller細胞隨機分為對照組、實驗組、PEDF榦預組(榦預組)和榦預對照組,熒光免疫法和實時熒光PCR法檢測Müller細胞GLAST錶達的改變,根據[3H]標記的D,L-穀氨痠攝入量判斷Müller細胞的攝取功能.結果實時熒光PCR法和Western bolt檢測結果顯示,相對于模型對照組大鼠,模型組大鼠視網膜GLAST錶達降低(實時熒光PCR法:t=8.86,P＜0.01;Western blot:t=3.42,P＜0.05),視網膜穀氦痠含量升高(t=4.01,P＜0.05);榦預組大鼠視網膜GLAST錶達與榦預對照組視網膜GLAST錶達比較,榦預組大鼠視網膜GLAST的錶達升高(實時熒光PCR法:t=3.56,P＜0.05;Western blot:t=3.52,P＜O.05);視網膜穀氨痠含量下調(t=4.36,P＜0.05).實時熒光PCR法和熒光免疫法檢測結果顯示,高糖可以降低視網膜Müller細胞GLAST的錶達(實時熒光PCR法:t=3.48,P＜O.05;熒光免疫法:t=4.72,P＜O.05);[3H]標記的D,L-穀氨痠攝人量結果顯示,高糖可以下調視網膜Mü1ler細胞GIAST的功能(t=3.81,P＜0.05);經PEDF處理後,可以明顯改善高糖狀態下視網膜Müller細胞GLAST的錶達(實時熒光PCR法:t=6.82,P＜O.01;熒光免疫法:t=3.72,P＜0.05)和對穀氨痠的攝取功能(t=4.14,P＜0.05).結論 PEDF可通過改善糖尿病大鼠視網膜Müller細胞中GLAST功能從而改善穀氨痠循環,抑製神經節細胞的死亡.
목적 관찰색소상피연생인자(PEDF)대당뇨병대서시망막곡안산대사적영향.방법 Sprague_Dawley대서78지,분위모형조、모형대조조、PEDF간예조(간예조)、간예대조조,실험결속시거제혈당회복서화실험기간사망서,매조이12지대서작위통계양본.모형조、간예조、간예대조조대서채용련뇨좌균소유도당뇨병대서모형.모형조대서불작임하간예,모형대조조위상동월령적정상대서.간예조대서좌안파리체강주사0.1 μg/μl적PEDF 5.0μl,간예대조조대서좌안파리체강주사상동용적적린산염완충액.채용단백면역인적법(Western bolt)화실시형광취합매련식반응(PCR)법검측시망막L-곡안산-L-문동안산전운체(GLAST)표체적변화,고압액상색보법(HPLC)관찰시망막곡안산적함량변화.장체외배양적대서시망막Müller세포수궤분위대조조、실험조、PEDF간예조(간예조)화간예대조조,형광면역법화실시형광PCR법검측Müller세포GLAST표체적개변,근거[3H]표기적D,L-곡안산섭입량판단Müller세포적섭취공능.결과 실시형광PCR법화Western bolt검측결과현시,상대우모형대조조대서,모형조대서시망막GLAST표체강저(실시형광PCR법:t=8.86,P＜0.01;Western blot:t=3.42,P＜0.05),시망막곡양산함량승고(t=4.01,P＜0.05);간예조대서시망막GLAST표체여간예대조조시망막GLAST표체비교,간예조대서시망막GLAST적표체승고(실시형광PCR법:t=3.56,P＜0.05;Western blot:t=3.52,P＜O.05);시망막곡안산함량하조(t=4.36,P＜0.05).실시형광PCR법화형광면역법검측결과현시,고당가이강저시망막Müller세포GLAST적표체(실시형광PCR법:t=3.48,P＜O.05;형광면역법:t=4.72,P＜O.05);[3H]표기적D,L-곡안산섭인량결과현시,고당가이하조시망막Mü1ler세포GIAST적공능(t=3.81,P＜0.05);경PEDF처리후,가이명현개선고당상태하시망막Müller세포GLAST적표체(실시형광PCR법:t=6.82,P＜O.01;형광면역법:t=3.72,P＜0.05)화대곡안산적섭취공능(t=4.14,P＜0.05).결론 PEDF가통과개선당뇨병대서시망막Müller세포중GLAST공능종이개선곡안산순배,억제신경절세포적사망.
Objective To observe the effect of pigment epithelium-derived factor(PEDF)on glutamate metabolism in diabetic rat retina.Methods 78 Sprague-Dawley rats were randomly divided into the model group,model control group,PEDF intervention group and intervention control group.There were some dead and euglycemia rats at the end of experiment,so only 12 rats in each group were included in the statistical analysis.The diabetic retinopathy rat model of the model,PEDF intervention and intervention control group were induced with streptozotocin injection.The rats in the model group were not intervened.The monthly-age matched normal rats of model group were in the model control group.The left eyes of rats were received intravitreal injection with 5μl(0.1μg/μl)PEDF(PEDF intervention group)or 5μlphosphate buffer solution(intervention control group).The expressions of L-glutamate/L-aspartate transporter in retina were analyzed by western blot and real time RT-PCR techniques and glutamate content in retina was analyzed by high-pressure liquid chromatography(HPLC).Cultured rat Mailer cells were divided into the control,experimental,PEDF intervention and intervention control group,GLAST expressions were detected by fluorescence immunofluorescence and real-time RT-PCR techniques.The glutamate up-take activity of Müller cells was determined by intracellular[3H] labeled D,L-glutamate concentration with scintillation counting.Results Western blot and real-time RT-PCR showed that GLAST expression decreased(real-time RT-PCR:t=8.86,P＜0.01;Western blot:t=3.42,P＜0.05).glutamate content increased(t=4.01,P＜0.05)in model group compared with the model control group:GLAST expression increased(real-time RT-PCR:t=3.56,P＜0.05;Western blot:t=3.52,P＜0.05),glutamate content decreased(y=4.36,P＜0.05)in the PEDF intervention group compared with the intervention control group.Real-time RT-PCR and fluorescence immunofluorescenee showed that high glucose down-regulate GLAST expressions in Müller cells(real-time RT-PCR:t=3.48,P＜0.05;fluorescence immunofluoreseence:t=4.72,P＜0.05)and impair glutamate up-take activity of Müller cells(t=3.81,P＜0.05).Under high glucose conditions,PEDF up-regulated GLAST expression significantly(real-time RT-PCR:t=6.82,P＜0.01;fluorescence immunofluorescence:t=3.72,P＜0.05)and ameliorated the glutamate up-take activitv of Mailer cells(t=4.14,P＜0.05).Conclusions In diabetic rats,PEDF may improve the activitv of GLAST in Müller cells,thus ameliorate retinal glutamate metabolism and inhibit death of retinal ganglion cells.