玻璃体腔重复注射抗血管内皮生长因子单克隆抗体bevacizumab对糖尿病大鼠视网膜的毒性观察

Objective To observe the retinal toxicity of repeated intravitreal injection with bevacizumab(Avastin)in diabetic rats.Methods Forty male Sprague Dawley(SD)rats were randomly divided into normal group(Group A,10 rats)and diabetes mellitus group(30 rats).The rats in diabetes mellitus group were induced with streptozotocin injection for diabetic retinopathy model.And then randomly divided into diabetic retinopathy(DR)group(Group B,10 rats),the rats were not intervened;the left eyes of the other 20 rats were intravitreal injected with bevaeizumab 3 μ1(25 mg/m1)for 3 times as experimental group(Group C);the right eyes of the 20 rats were not intervened as experimental control group(Group D),20 days after last intravitreal injection,retinal function was measured by Flicker Electroretinogram (F-ERG);retinal vascular pattern was determined by fluorescence microscopy of ethidium bromide(EB)stained retinal flat mounts;retinal morphological changes were determined by light microscope on hematoxylin-eosin (HE) stained sections;Thy-1 and VEGF expression was measured by immunohistochemistry staining.Results F-ERG showed that-the differences of a-and b-waves-the b-wave amplitude and the Ops-wave amplitude in the implicit time between group A,B,C and D were significant (F=33.165,36.162,19.955,23.243;P=0.000);the differences of a-wave amplitude between group A,B,C and D was not significant(F=0.097,P=0v961).Retinal blood vessel pattern was normalin Group A;retinal vascular vessels were tortuous and irregularly expanded in Group B:retinal vascular vessels of Group C were regular and thinner than Group A;microaneurysm were showed in Group D.Light microscope displayed that the layers of the rat retina of Group A were regular,the retinal architectures of Group B were irregular,the retinal layers were regular in Group C,the retinal layers were irregular in Group D.Immunohistochemistry staining discovered that Thy-1 and VEGF were mainly expressed in ganglion cell layer(GCL).Conclusion Repeated intravitreal injection of bevacizumab iS toxic tO retina of diabetes mellitus rats.     

玻璃体腔重复注射抗血管内皮生长因子单克隆抗体bevacizumab对糖尿病大鼠视网膜的毒性观察

Objective To observe the retinal toxicity of repeated intravitreal injection with bevacizumab(Avastin)in diabetic rats.Methods Forty male Sprague Dawley(SD)rats were randomly divided into normal group(Group A,10 rats)and diabetes mellitus group(30 rats).The rats in diabetes mellitus group were induced with streptozotocin injection for diabetic retinopathy model.And then randomly divided into diabetic retinopathy(DR)group(Group B,10 rats),the rats were not intervened;the left eyes of the other 20 rats were intravitreal injected with bevaeizumab 3 μ1(25 mg/m1)for 3 times as experimental group(Group C);the right eyes of the 20 rats were not intervened as experimental control group(Group D),20 days after last intravitreal injection,retinal function was measured by Flicker Electroretinogram (F-ERG);retinal vascular pattern was determined by fluorescence microscopy of ethidium bromide(EB)stained retinal flat mounts;retinal morphological changes were determined by light microscope on hematoxylin-eosin (HE) stained sections;Thy-1 and VEGF expression was measured by immunohistochemistry staining.Results F-ERG showed that-the differences of a-and b-waves-the b-wave amplitude and the Ops-wave amplitude in the implicit time between group A,B,C and D were significant (F=33.165,36.162,19.955,23.243;P=0.000);the differences of a-wave amplitude between group A,B,C and D was not significant(F=0.097,P=0v961).Retinal blood vessel pattern was normalin Group A;retinal vascular vessels were tortuous and irregularly expanded in Group B:retinal vascular vessels of Group C were regular and thinner than Group A;microaneurysm were showed in Group D.Light microscope displayed that the layers of the rat retina of Group A were regular,the retinal architectures of Group B were irregular,the retinal layers were regular in Group C,the retinal layers were irregular in Group D.Immunohistochemistry staining discovered that Thy-1 and VEGF were mainly expressed in ganglion cell layer(GCL).Conclusion Repeated intravitreal injection of bevacizumab iS toxic tO retina of diabetes mellitus rats.     

鼠神经生长因子对早期糖尿病大鼠玻璃体中感光细胞间维生素A类结合蛋白含量的影响

Objective To observe the effect of intravitreal injection of mouse nerve growth factor (NGF)on interphotoreceptor retinoid-binding protein(IRBP)in the vitreous of diabetic rats at early stages.Methods Ninety-six male Sprague Dawley(SD)rats were divided into control group(group A,24 rats)and experimental group(72 rats).The rats in experimental group were induced with streptozotocin injection for diabetic retinopathy model,and then randomly divided into positive control group(group B),normal saline group(group C)and NGF group(group D),24 rats in each group.The rats in the group A and B were not intervened.The rats were received intravitreal injection with 4μl normal saline(group C)or 4 μl(0.5 μg/μ1)NGF(group D).At 2,4,6 and 8 weeks after injection,IRBP levels were detected bv enzvmelinked immunosorbent assay(ELISA);hematoxylin-eosin(HE)staining and light microscope were used to observe the morphological changes of the retina;transmission electron microscope was used to observe the retinal uhrastructure.Results At 2 weeks after injection,there was no significant difference in IRBP expression between group A,B,C and D(F=2.833,P=0.052).At 4,6,8 weeks after injection,the differences of IRBP expression between group A,B,C and D were significant(F=22.252,108.459,105.726;P=0.000).At different time points after injection,there was no significant difference in IRBP expression of group A(F=1.462,P=0.241),but there were significant differences in IRBP expression of group B.C and D(F=150.98,63.519,64.604;P=0.000).Light microscope found that the retinal structure was clear in group A and in group B,C,D at 2,4 weeks after injection;the retinal thickness were thinner in group B,C,D at 8 weeks after injection.Transmission electron microscope displayed that the structure of rod outer segments was clear in group A and in group B,C,D at 2 weeks after injection;partly unclear structure of rod outer segments and slightly enlarged gap were observed in group B,C,D at 4,8Weeks after injection.Conclusion Intravitreal injection with NGF can stabilize the IRBP expression in the vitreous of diabetic rats at early stages effectively.     

鼠神经生长因子对早期糖尿病大鼠玻璃体中感光细胞间维生素A类结合蛋白含量的影响

Objective To observe the effect of intravitreal injection of mouse nerve growth factor (NGF)on interphotoreceptor retinoid-binding protein(IRBP)in the vitreous of diabetic rats at early stages.Methods Ninety-six male Sprague Dawley(SD)rats were divided into control group(group A,24 rats)and experimental group(72 rats).The rats in experimental group were induced with streptozotocin injection for diabetic retinopathy model,and then randomly divided into positive control group(group B),normal saline group(group C)and NGF group(group D),24 rats in each group.The rats in the group A and B were not intervened.The rats were received intravitreal injection with 4μl normal saline(group C)or 4 μl(0.5 μg/μ1)NGF(group D).At 2,4,6 and 8 weeks after injection,IRBP levels were detected bv enzvmelinked immunosorbent assay(ELISA);hematoxylin-eosin(HE)staining and light microscope were used to observe the morphological changes of the retina;transmission electron microscope was used to observe the retinal uhrastructure.Results At 2 weeks after injection,there was no significant difference in IRBP expression between group A,B,C and D(F=2.833,P=0.052).At 4,6,8 weeks after injection,the differences of IRBP expression between group A,B,C and D were significant(F=22.252,108.459,105.726;P=0.000).At different time points after injection,there was no significant difference in IRBP expression of group A(F=1.462,P=0.241),but there were significant differences in IRBP expression of group B.C and D(F=150.98,63.519,64.604;P=0.000).Light microscope found that the retinal structure was clear in group A and in group B,C,D at 2,4 weeks after injection;the retinal thickness were thinner in group B,C,D at 8 weeks after injection.Transmission electron microscope displayed that the structure of rod outer segments was clear in group A and in group B,C,D at 2 weeks after injection;partly unclear structure of rod outer segments and slightly enlarged gap were observed in group B,C,D at 4,8Weeks after injection.Conclusion Intravitreal injection with NGF can stabilize the IRBP expression in the vitreous of diabetic rats at early stages effectively.     

鼠神经生长因子对早期糖尿病大鼠玻璃体中感光细胞间维生素A类结合蛋白含量的影响

目的 观察玻璃体腔注射鼠神经生长因子(NGF)对早期糖尿病大鼠玻璃体中感光细胞间维生素A类结合蛋白(IRBP)含量的影响.方法 Sprague-Dawley雄性大鼠96只,随机分为正常对照组(A 组)及实验组,分别为24、72只.实验组采用链脲佐菌霉素(STZ)一次性腹腔注射制作糖尿病动物模型,再随机分为糖尿病阳性对照组(B组)、糖尿病玻璃体腔生理盐水注射组(C组)和糖尿病玻璃体腔鼠NGF注射组(D组),每组24只大鼠.A组及B组建模后不给予任何干预;C组注射生理盐水4μl,1次/周;D组注射0.5μg/μl的鼠NGF 4 μl,1次/周.注射后2、4、6、8周,采用酶联免疫吸附测定(ELISA)法检测大鼠玻璃体中IRBP含量;作石蜡切片行苏木精-伊红(HE)染色,光学显微镜观察视网膜结构;作超微切片,透射电子显微镜观察视网膜超微结构.结果 注射后2周,A、B、C、D组大鼠玻璃体中IRBP含量比较,差异无统计学意义(F=2.833,P=0.052).注射后4、6、8周,A、B、C、D组大鼠玻璃体中IRBP含量比较,差异均有统计学意义(F=22.252,108.459,105.726;P值均=0.000).A组组内注射后不同时间点大鼠玻璃体中IRBP含量比较,差异无统计学意义(F=1.462,P=0.241);B、C、D组组内注射后不同时间点大鼠玻璃体中IRBP含量比较,差异均有统计学意义(F=150.98,63.519,64.604;P值均=0.000).光学显微镜观察发现,A组大鼠视网膜各层组织层次清楚,排列整齐.注射后2、4周,B、C、D组大鼠视网膜结构较A组无明显改变.注射后8周,B、C、D组大鼠视网膜厚度变薄.透射电子显微镜观察发现,A组大鼠视细胞外节膜盘结构清晰,排列规则.注射后2周,B、C、D组大鼠视细胞外节膜盘结构清晰,排列规则整齐.注射后4周,B、C、D组大鼠视细胞外节膜盘局部模糊不清,间隙略有扩大.注射后8周,B、C组大鼠视细胞外节膜盘局部模糊不清,间隙扩大,部分出现溶解、断裂;D组大鼠视细胞外节膜盘局部模糊不清,间隙扩大,未见明显溶解断裂.结论 玻璃体腔注射鼠NGF可以有效稳定糖尿病大鼠早期玻璃体中IRBP的含量.

Abstract：

Objective To observe the effect of intravitreal injection of mouse nerve growth factor (NGF)on interphotoreceptor retinoid-binding protein(IRBP)in the vitreous of diabetic rats at early stages.Methods Ninety-six male Sprague Dawley(SD)rats were divided into control group(group A,24 rats)and experimental group(72 rats).The rats in experimental group were induced with streptozotocin injection for diabetic retinopathy model,and then randomly divided into positive control group(group B),normal saline group(group C)and NGF group(group D),24 rats in each group.The rats in the group A and B were not intervened.The rats were received intravitreal injection with 4μl normal saline(group C)or 4 μl(0.5 μg/μ1)NGF(group D).At 2,4,6 and 8 weeks after injection,IRBP levels were detected bv enzvmelinked immunosorbent assay(ELISA);hematoxylin-eosin(HE)staining and light microscope were used to observe the morphological changes of the retina;transmission electron microscope was used to observe the retinal uhrastructure.Results At 2 weeks after injection,there was no significant difference in IRBP expression between group A,B,C and D(F=2.833,P=0.052).At 4,6,8 weeks after injection,the differences of IRBP expression between group A,B,C and D were significant(F=22.252,108.459,105.726;P=0.000).At different time points after injection,there was no significant difference in IRBP expression of group A(F=1.462,P=0.241),but there were significant differences in IRBP expression of group B.C and D(F=150.98,63.519,64.604;P=0.000).Light microscope found that the retinal structure was clear in group A and in group B,C,D at 2,4 weeks after injection;the retinal thickness were thinner in group B,C,D at 8 weeks after injection.Transmission electron microscope displayed that the structure of rod outer segments was clear in group A and in group B,C,D at 2 weeks after injection;partly unclear structure of rod outer segments and slightly enlarged gap were observed in group B,C,D at 4,8Weeks after injection.Conclusion Intravitreal injection with NGF can stabilize the IRBP expression in the vitreous of diabetic rats at early stages effectively.     

超声微泡造影剂促进重组腺相关病毒载体介导基因转染视网膜神经节细胞的体内实验

Objective To investigate the enhancing effect of ultrasound microbubbles on transfection of recombinant adeno-associated virus (rAAV) mediated green fluorecent protein (EGFP) gene into retinal ganglion cells (RGC) in vivo. Methods A total of 40 adult Sprague-Dawley (SD) rats were divided into four groups randomly (group A, B, C, D) with 10 rats in each. Group A was the normal control, in which the rats underwent intravitreal injection with 5 μl phosphate buffered solution. The rats in group B underwent intravitreal injection with 5 μl recombinant adeno-associated virus encoding EGFP gene (rAAV2-EGFP). The rats in group C underwent ultrasound irradiation on eyes right after intravitreal injection with 5μl rAAV2-EGFP; The ultrasound irradiation was performed on the rats in group D right after intravitreal injection with the mixture solution of microbubbles and rAAV2-EGFP ultrasound. After 21 days, RGC were labeled retogradely with fluogold. Seven days after labeling, the retinal flatmounts and frozen sections were made from five rats in each group. Expression of EGFP reporter gene was observed by laser scanning confocal microscope and evaluated via average optical intensity (AOD) and RGC transfection rate. Labeled RGC were counted to evaluate the adverse effects. Results Green fluorescence can be observed exactly in labeled RGC in B,C, and D groups. The AOD and transfection rate in group D was (95.02 ± 7. 25)% and (20. 10±0. 74)% , respectively; which were higher than those in group B and C (F=25. 970,25. 799;P<0.01). The difference of the number of RGC among the four groups was not significant(F= 0. 877, P>0. 05). Conclusion Under the condition of low frequency and with certain energy, ultrasound-mediated microbubble destruction can effectively and safely enhance rAAV delivery to RGC in rats.     

超声微泡造影剂促进重组腺相关病毒载体介导基因转染视网膜神经节细胞的体内实验

Objective To investigate the enhancing effect of ultrasound microbubbles on transfection of recombinant adeno-associated virus (rAAV) mediated green fluorecent protein (EGFP) gene into retinal ganglion cells (RGC) in vivo. Methods A total of 40 adult Sprague-Dawley (SD) rats were divided into four groups randomly (group A, B, C, D) with 10 rats in each. Group A was the normal control, in which the rats underwent intravitreal injection with 5 μl phosphate buffered solution. The rats in group B underwent intravitreal injection with 5 μl recombinant adeno-associated virus encoding EGFP gene (rAAV2-EGFP). The rats in group C underwent ultrasound irradiation on eyes right after intravitreal injection with 5μl rAAV2-EGFP; The ultrasound irradiation was performed on the rats in group D right after intravitreal injection with the mixture solution of microbubbles and rAAV2-EGFP ultrasound. After 21 days, RGC were labeled retogradely with fluogold. Seven days after labeling, the retinal flatmounts and frozen sections were made from five rats in each group. Expression of EGFP reporter gene was observed by laser scanning confocal microscope and evaluated via average optical intensity (AOD) and RGC transfection rate. Labeled RGC were counted to evaluate the adverse effects. Results Green fluorescence can be observed exactly in labeled RGC in B,C, and D groups. The AOD and transfection rate in group D was (95.02 ± 7. 25)% and (20. 10±0. 74)% , respectively; which were higher than those in group B and C (F=25. 970,25. 799;P<0.01). The difference of the number of RGC among the four groups was not significant(F= 0. 877, P>0. 05). Conclusion Under the condition of low frequency and with certain energy, ultrasound-mediated microbubble destruction can effectively and safely enhance rAAV delivery to RGC in rats.     

色素上皮衍生因子对糖尿病大鼠视网膜谷氨酸代谢的影响

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.     

色素上皮衍生因子对糖尿病大鼠视网膜谷氨酸代谢的影响

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.     

色素上皮衍生因子对糖尿病大鼠视网膜谷氨酸代谢的影响

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.

Abstract：

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.     

A comparative study on the transplantation of different concentrations of human umbilical mesenchymal cells into diabetic rats

AIM: To observe the effects of intravitreal injections of different concentrations of human umbilical mesenchymal stem cells on retinopathy in rats with diabetes mellitus. METHODS: Healthy and adult male Sprague-Dawley (SD) rats were randomly assigned to a normal control group (group A), a diabetic retinopathy (DR) blank control group (group B), a high-concentration transplantation group (group C), a low-concentration transplantation group (group D) and a placebo transplantation group (group E). The expression of nerve growth factor (NGF) protein in the retinal layers was detected by immunohistochemical staining at 2, 4, 6 and 8wk. RESULTS: The expression of NGF was positive in group A and most positive in the retinal ganglion cell layer. In groups B and E, the expression of NGF was positive 2wk after transplantation and showed an increase in all layers. However, the level of expression had decreased in all layers at 4wk and was significantly reduced at 8wk. In groups C and D, the expression of NGF had increased at 2wk and continued to increase up to 8wk. The level of expression in group C was much higher than that in group D. CONCLUSION: DR can be improved by intravitreal injection of human umbilical mesenchymal stem cells. High concentrations of human umbilical mesenchymal stem cells confer a better protective effect on DR than low concentrations.     

Protective effect of LIF-huMSCs on the retina of diabetic model rats

AIM: To investigate the protective effect of human umbilical cord mesenchymal stem cells (hUCMSCs) modified by the LIF gene on the retinal function of diabetic model rats and preliminarily explore the possible mechanism. METHODS: A stably transfected cell line of hUCMSCs overexpressing leukemia inhibitory factor (LIF) was constructed. Overexpression was verified by fluorescent quantitative polymerase chain reaction (qPCR). Forty-eight adult Sprague-Dawley rats were randomly divided into a normal control group (group A), streptozotocin-induced diabetic control group (group B), diabetic rats at 3mo injected with empty vector-transfected hUCMSCs (group C) or injected with LIF-hUCMSCs (group D). Four weeks after the intravitreal injection, analyses in all groups included retinal function using flash electroretinogram (F-ERG), retinal blood vessel examination of retinal flat mounts perfused with fluorescein isothiocyanate-dextran (FITC-dextran), and retinal structure examination of sections using hematoxylin and eosin staining. Expression levels of adiponectin (APN), high-sensitivity C-reactive protein (hs-CRP), and neurotrophin-4 (NT-4) in each group was detected using immunohistochemistry, PCR, Western blotting, and ELISA, respectively. RESULTS: A stable transgenic cell line of LIF-hUCMSCs was constructed. F-ERG and FITC-dextran examinations revealed no abnormalities of retinal structure and function in group A, severe damage of the retinal blood vessels and function in group B, and improved retinal structure and function in group C and especially group D. qPCR, ELISA, and Western blot analyses revealed progressively higher APN and NT-4 expression levels in groups B, C, and D than in group A. hs-CRP expression was significantly higher in group B than in groups A, C, and D, and was significantly higher in group C than in group D (P<0.05). CONCLUSION: LIF-hUCMSCs protect the retina of diabetic rats by upregulating APN and NT-4 expression and downregulating hs-CRP expression in the retina.     

兔眼玻璃体腔注射抗血管内皮生长因子单克隆抗体Bevacizumab的视网膜毒性研究

目的 观察不同剂量抗血管内皮生长因子单克隆抗体Bevacizumab兔眼玻璃体腔注射的视网膜毒性作用。 方法 16只新西兰无色素兔的32只眼随机分为药物注射组和对照组，药物注射组又根据玻璃体腔注射药物剂量不同分为A、B、C组，玻璃体腔注射Bevacizumab剂量分别为0.05、0.10、0.25 ml，分别含Bevacizumab 1.25、2.50、6.25 mg。对照组玻璃体腔注射0.9％生理盐水0.10 ml。注药后1、2、4周行视网膜电图（ERG）检查。另外 ，在兔眼玻璃体腔注射Bevacizumab后1、2、4周，每组各摘除2只兔眼，行视网膜组织形态及超 微结构的光学显微镜和透射电子显微镜观察。 结果 兔眼玻璃体腔注射 Bevacizumab后1、2、4周，兔眼ERG各项反应波形均正常，振幅均未出现异常改变（P＞0.05）。光学显微镜下观察 ，药物注射组和对照组视网膜各层组织形态在各时间点均未见异常。透射电子显微镜观察， A、B组与对照组无明显差异；C组视网膜光感受器细胞出现部分线粒体损伤，发生肿胀和积 水变，4周时病变无缓解。 结论 单次兔眼玻璃体腔注射Bevacizumab 1.25 mg或2.50 mg是安全的。 （中华眼底病杂志，2008，24：193-196）     

P38丝裂素活化蛋白激酶信号通路阻断对糖尿病鼠早期血视网膜屏障和视网膜节细胞的保护作用

目的　观察阻断p38丝裂素活化蛋白激酶(MAPK)信号通路对糖尿病早期Wistar大鼠血视网膜屏障（BRB）和视网膜节细胞（RGC）的保护作用。方法　将60只大鼠分为正常对照组及糖尿病组,每组各30只大鼠。对糖尿病组大鼠进行链脲佐菌素腹腔注射建立糖尿病模型,血糖值＞16.7 mmol/L为模型建立成功的判断标准。正常对照组大鼠腹腔注射等量的枸橼酸钠溶液作为对照。采用IgG渗漏法量化BRB功能破坏和血管渗漏,免疫组织化学法检测视网膜上caspase-3和 血管内皮生长因子（VEGF）的荧光表达。糖尿病鼠建模后2周,对实验组行玻璃体腔注射p38 MAPK抑制剂 SB203580,注射后6周检测caspase-3和 VEGF的荧光表达,caspase-3免疫荧光染色法计数RGC凋亡数量。采用SPSS 13.0应用软件进行统计分析。结果　正常对照组大鼠中,IgG 染色局限于血管腔内,几乎没有渗漏的迹象。糖尿病鼠建模后8周,IgG渗漏明显增加。SB203580璃体腔注射6周后的糖尿病鼠IgG 渗漏减少明显。荧光免疫组织化学及相对定量分析结果显示,SB203580玻璃体腔注射6周后的糖尿病鼠视网膜上VEGF荧光表达呈下降趋势,VEGF相对荧光量从糖尿病鼠8周时较正常对照组高2.9倍减少到仅高于正常对照组1.8倍,两者比较差异有统计学意义（t＝5.203,P＜0.01）。caspase-3免疫荧光染色法RGC凋亡计数结果显示,SB203580玻璃体腔注射6周后,caspase-3阳性节细胞凋亡数与未注射SB203580相比明显减少,两者差异有统计学意义（t＝5.731,P＜0.01）。结论　p38 MAPK抑制剂 SB203580能够减轻糖尿病早期血视网膜屏障的破坏和视网膜节细胞的调亡,提示p38 MAPK信号通路在糖尿病早期对DR的发展起着一定的作用。      

超声微泡造影剂促进重组腺相关病毒载体介导基因转染视网膜神经节细胞的体内实验

目的 观察超声微泡造影剂提高重组腺相关病毒(rAAV2)介导增强型绿色荧光蛋白(EGFP)基因在体内转染视网膜神经节细胞(RGC)的效率.方法 Sprague-Dawley大鼠40只,随机分为A、B、C、D 4组,每组各10只大鼠.A组玻璃体腔注射磷酸盐缓冲液(PBS)5μl;B组玻璃体腔注射rAAV2-EGFP 5 μl;C组玻璃体腔注射rAAV2-EGFP 5 μl后立即用超声波辐照眼球;D组玻璃体腔注射rAAV2-EGFP和微泡造影剂的混悬液5 μl后立即用超声辐照眼球.玻璃体腔注射21 d后,3%荧光金逆行标记RGC.逆行标记7 d后取出眼球,制作视网膜铺片及视网膜冰冻切片,在激光共聚焦显微镜下观察并计算EGFP基因在RGC的转染率及在RGC表达的平均吸光度[A,旧称光密度(OD)]值;用RGC计数判断损伤情况.结果 荧光金标记RGC后,B、C、D 3组均可观察到RGC中有EGFP表达.其中,D组平均A值为95.02±7.25,RGC转染率为(20.10±0.74)%、均明显高于B、C组,差异有统计学意义(F平均A值=25.970,F转染率=25.799;P<0.01);A、B、C、D组RGC计数差异无统计学意义(F=0.877,P>0.05).结论 在低频和一定能量的超声和微泡造影剂作用下,rAAV2介导EGFP基因转染体内RGC的效率能够安全、有效地提高.     

超声微泡造影剂介导脑源性神经营养因子联合转染视网膜和视皮质对视神经损伤后视网膜神经节细胞的保护作用

　　目的　观察超声微泡造影剂介导脑源性神经营养因子（BDNF）联合转染大鼠视网膜和视皮质区细胞对视神经损伤后视网膜神经节细胞（RGC）的保护作用。方法　雄性Sprague-Dawley（SD）大鼠88只随机分为正常组（A组）、假手术组（B组）、空白对照组（C组）、单纯眼转染组（D组）、单纯脑转染组（E组）、联合转染组（F组）;A组8只大鼠,B～F组每组16只大鼠。建立钳夹视神经损伤模型,将B～F组大鼠随机分为视神经损伤1、2周亚组,各亚组8只大鼠。B、C组玻璃体腔和视皮质区分别注射磷酸盐缓冲液（PBS）,D、E组玻璃体腔和视皮质区分别注射BDNF质粒（pBDNF）微泡造影剂悬液,F组玻璃体腔和视皮质区同时注射pBDNF微泡造影剂悬液。D～F组注射pBDNF微泡微泡造影剂悬液后,立即用超声辐照相应转染部位。视神经损伤后1、2周,各组行逆行荧光金标记RGC计数;半胱氨酸蛋白酶-3(caspase-3）蛋白免疫组织化学染色,观察其阳性表达情况;图形视网膜电流图（PERG）检测,记录N95振幅。结果　荧光金标记RGC结果显示,各组均可见金黄色荧光散布于视网膜定向铺片上。A~F组间RGC计数差异有统计学意义（F=256.30,P＜0.01）;B~F组视神经损伤1、2周亚组间RGC计数差异也有统计学意义（F=6518,P＜0.01）。光学显微镜观察发现,A、B组大鼠视网膜均未见caspase-3蛋白阳性表达;C~F组均可见主要位于神经节细胞层的caspase-3蛋白阳性表达。PERG检测发现,A~F组间N₉₅振幅差异有统计学意义（F=121.56,P＜0.01）;B~F组视神经损伤1、2周亚组间N95振幅差异也有统计学意义（F=8238,P＜0.01）。结论　超声微泡造影剂介导BDNF联合转染视网膜和视皮质区细胞能抑制视神经损伤后RGC凋亡,提高RGC存活数,保护其视功能。      

玻璃体腔注射Bevacizumab对PDR患者增殖膜中CTGF及PEDF的影响

目的:研究增殖性糖尿病视网膜病变(PDR)玻璃体腔注射抗-VEGF药物bevacizumab后对增殖膜中结缔组织生长因子(CTGF)及色素上皮衍生因子(PEDF)的影响.方法:回顾2015-01/2016-12入本院行增殖性糖尿病视网膜病变治疗的患者117例126眼,采用病例对照的研究方法,将所选病例随机分为两组,分别为A组60例63眼和B组57例63眼.其中A组单纯进行玻璃体切割手术,B组患者在玻璃体切割术前玻璃体腔注射0.05mL/1.25mg bevacizumab.在手术中剥离取用两组患者的视网膜增殖膜进行染色,然后进行组织病理学观察,观察两组患者视网膜增殖膜中原始细胞和新生血管的变化,以及患者增殖膜中CTGF和PEDF因子的表达.结果:在对两组患者CTGF、PEDF因子表达进行观察发现,两组患者视网膜增殖膜中的CTGF和PEDF都在细胞质内表达.其中A组呈现出38眼阳性表达,阳性表达率为60.3%,相比于A组而言,B组的CTGF的阳性表达率92.1%明显更高,两组差异有统计学意义(P<0.05).而两组的PEDF阳性表达率分别为90.5%和95.2%,差异无统计学意义(P>0.05).结论:PDR患者在玻璃体腔注射抗VEGF药物bevacizumab后,视网膜的新生血管明显减少,有利于玻璃体切割手术的进行.且PDR患者玻璃体腔注射bevacizumab后,CTGF的阳性表达率明显增高,而PEDF因子在前膜上的表达则没有明显的变化.     

不同途径移植人脐带间充质干细胞对糖尿病大鼠视网膜病变的影响

目的 观察人脐带间充质干细胞（hUCMSC）对糖尿病（DM）大鼠血糖的影响及对视网膜病变的治疗作用。方法 健康雄性Sprague-Dawley大鼠45只,随机分为正常对照组（A组）、DM组,分别为10、35只大鼠。DM组经尾静脉注射链脲佐菌素诱导DM模型。10周时,A组、DM组分别随机选取2只大鼠行视网膜血管铺片。DM组剩余的33只大鼠随机分为糖尿病视网膜病变组（B组）、尾静脉注射hUCMSC 组（C组）、玻璃体腔注射hUCMSC 组（D组）,各组均为11只大鼠。A、B组不进行干预。干预后2、4、6、8周为观察处理时间点。各处理时间点前,连续3 d每次随机选取2只大鼠检测随机血糖。DM组大鼠血糖与同期A组大鼠血糖比较,差异均有统计学意义（t=-64.400、-60.601、-44.065、-43.872,P=0.000）。8周时从B、C、D组随机选取2只大鼠行视网膜血管铺片。免疫组织化学法观察视网膜脑源性神经营养因子（BDNF）阳性染色情况;实时荧光定量逆转录聚合酶链反应（RT-PCR）检测视网膜BDNF mRNA相对表达量。结果 干预后,不同处理时间点A、B、C、D组大鼠血糖比较,差异均有统计学意义（F=400.017、404.410、422.043、344.109,P=0.000）;C组大鼠血糖与B、D组大鼠血糖比较,差异有统计学意义（t=4.447、4.990、P＜0.01）。免疫组织化学染色结果显示,A组BDNF表达呈阳性,主要分布在神经节细胞层;B组BDNF表达呈弱阳性;C、D组BDNF表达增多。RT-PCR检测结果显示,4、6、8周时,B、C、D组间视网膜BDNF mRNA相对表达量比较,差异均有统计学意义（F=29.372、188.492、421.537,P=0.000）;C、D组视网膜BDNF mRNA相对表达量与B组比较,差异均有统计学意义（t=66.781、72.401、63.880、88.423、75.120、83.002,P＜0.01）;C组视网膜BDNF mRNA相对表达量与D组比较,仅8周时差异有统计学意义（t=127.321,P=0.005）。结论 尾静脉注射hUCMSC能够显著降低大鼠血糖水平;尾静脉或玻璃体腔注射hUCMSC均可增加BDNF的表达。     

血管内皮生长因子反义寡脱氧核苷酸联合血管生成素-1对糖尿病视网膜病变大鼠视网膜血管渗漏及新生血管生成的影响

目的 观察血管内皮生长因子(vascular endothelial growth factor,VEGF)反义寡脱氧核苷酸(ASODN)联合血管生成素-1(angiogenin-1,Ang-1)对糖尿病视网膜病变(diabetic retinopathy,DR)大鼠视网膜血管渗漏及新生血管生成的影响.方法 选取健康雄性SD大鼠60只,随机分为正常对照组(A组)5只和糖尿病组55只,糖尿病组通过链脲佐菌素腹腔注射诱导糖尿病模型成功后再饲养3个月行眼底荧光血管造影检查,确定DR大鼠模型且视网膜病变程度相仿的大鼠,随机抽取45只分为DR对照组(B组,5只)、PBS缓冲液对照组(C组,10只)、VEGF ASODN干预组(D组,10只)、Ang-1干预组(E组,10只)、联合干预组(F组,10只).C组玻璃体内注射PBS缓冲液5μL;D组玻璃体内注射浓度为100 μmol·L-1 VEGF ASODN 5 μL;E组玻璃体内注射浓度为160 mg·L-1Ang-1 5 μL;F组玻璃体内注射100 μmol·L-1 VEGF ASODN及160 mg·L-1 Ang-1各5μL;3 d后再次行上述操作.各组大鼠行眼底荧光血管造影检查,对比观察不同组别大鼠视网膜血管渗漏情况,病理组织切片光学显微镜下突破内界膜的观察视网膜新生血管芽细胞核数.结果 A、B、C、D、E和F组视网膜新生血管渗漏面积分别为0、( 20.98±1.14) mm2、(21.47±1.65) mm2、(14.60±1.55) mm2、(13.80 ±1.19) mm2、( 10.81±1.35) mm2;D、E、F组新生血管渗漏面积低于B、C组,差异有统计学意义(F=103.99,P ＜0.05);F组渗漏面积低于D、E组,差异有统计学意义(F=190.94,P＜0.05);B组与C组之间差异无统计学意义(t =0.22,P＞0.05).A、B、C、D、E和F组突破内界膜的新生血管芽细胞核数分别为(1.13±0.31)个、(80.31±5.21)个、(81.08 ±2.57)个、(37.37±3.41)个、(41.07±2.09)个、(14.41±1.23)个;D、E、F组突破内界膜的血管芽细胞核数均低于B、C组,差异有统计学意义(F=1339.41,P＜0.05);F组突破内界膜的血管芽细胞核数低于D、E组,差异有统计学意义(F =714.91,P ＜0.05);B组与C组之间差异无统计学意义(t=0.35,P＞0.05).结论 VEGFASODN联合Ang-1玻璃体内注射能明显抑DR大鼠视网膜新生血管的生成,减少视网膜血管渗漏.