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.     

Protective effect of pomegranate juice on retinal oxidative stress in streptozotocin-induced diabetic rats

AIM: To investigate the effect of pomegranate juice (PJ) intake on overall oxidation status in retinas of diabetic rats. METHODS: Twenty-seven rats were divided into four groups as control (CO), diabetic (DM), control treated with PJ (CO-PJ), and diabetic treated with PJ (DM-PJ).The retina tissues were used to determine 8-hydroxy-2’-deoxyguanosine (8OHdG), malondialdehyde (MDA), reduced glutathione (GSH) levels, and the enzyme activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). RESULTS: The levels of 8OHdG and MDA were significantly increased in the retina of DM group compared to CO group (P=0.001, P<0.001 respectively). Both 8OHdG and MDA levels were decreased in PJ-DM group compared to DM group (P=0.004, P<0.001 respectively). The activities of antioxidant enzymes GSH, SOD, and GDH-Px were significantly decreased in the retina of DM group compared to CO group (P≤0.01). GSH and GSH-Px activities were higher in PJ-DM group compared with DM group (P=0.010, P=0.042, respectively) but SOD activity was not statistically different (P=0.938). CONCLUSION: PJ intake is found to be effective in decreasing oxidative end products, and in increasing the activities of antioxidant enzymes in diabetic retinas of rats, which suggests it may be effective against oxidative stress in diabetic retinas.     

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.     

【In Press】 Protective effect of pomegranate juice on retinal oxidative stress in streptozotocin-induced diabetic rats

AIM: To investigate the effect of pomegranate juice (PJ) intake on overall oxidation status in retinas of diabetic rats. METHODS: Twenty-seven rats were divided into four groups as control (CO), diabetic (DM), control treated with PC (CO-PJ), and diabetic treated with PJ (DM-PJ).The retina tissues were used to determine 8-Hydroxy-2’-deoxyguanosine (8OHdG), malondialdehyde (MDA), reduced glutathione (GSH) levels, and the enzyme activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). RESULTS: The levels of 8OHdG and MDA were significantly increased in the retina of DM group compared to CO group (p=0.001, p<0.001 respectively). Both 8OHdG and MDA levels were decreased in PJ-DM group compared to DM group (p=0.004, p<0.001 respectively). The activities of antioxidant enzymes GSH, SOD, and GDH-Px were significantly decreased in the retina of DM group compared to CO group (p≤0.01). GSH and GSH-Px activities were higher in PJ-DM group compared with DM group (p=0.010, p=0.042, respectively) but SOD activity was not statistically different (p=0.938). CONCLUSION: PJ intake was found to be effective in decreasing oxidative end products, and in increasing the activities of antioxidant enzymes in diabetic retinas of rats, which suggests it may be effective against oxidative stress in diabetic retinas.     

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

目的 观察玻璃体腔重复注射抗血管内皮生长因子(VEGF)单克隆抗体bevacizumab(商品名Avastin)对糖尿病大鼠视网膜的毒性作用.方法 40只健康成年雄性Sprague-Dawley大鼠随机分为正常组(A组)和糖尿病组,分别为10、30只.糖尿病组采用链脲佐菌霉素(STZ)尾静脉注射方法制作糖尿病动物模型.随机选取10只作为糖尿病视网膜病变(DR)组(B组),不作任何处理;其余20只大鼠左眼为实验组(C组),玻璃体腔注射25 mg/ml的bevacizumab 3 μ1,共注射3次,每次间隔10 d;右眼为实验对照组(D组),不给予任何处理.末次注射后20 d,采用闪光视网膜电图(F-ERG)对各组大鼠行视网膜功能检测;溴化乙锭(EB)染色视网膜铺片,荧光显微镜观察各组大鼠视网膜血管变化情况;苏木精-伊红(HE)染色,光学显微镜观察大鼠视网膜形态学变化;免疫组织化学染色法观察大鼠视网膜各层Thy-1及VEGF阳性表达情况.结果 F-ERG检测显示,A、B、C、D 4组暗适应a、b波潜伏期,暗适应b波振幅及振荡电位(Ops)总振幅比较,差异均有统计学意义(F=33.165,36.162,19.955,23.243;P值均=0.000);A、B、C、D4组暗适应a波振幅比较,差异无统计学意义(F=0.097,P=0.961).荧光显微镜观察发现,A组大鼠视网膜血管走行良好,B组大鼠视网膜血管走行纡曲、扩张,C组大鼠视网膜血管走形规则、变细,D组大鼠视网膜可见微血管瘤.光学显微镜观察发现,A组大鼠视网膜层次结构整齐分明,B组大鼠视网膜各层细胞结构排列紊乱,C组大鼠视网膜各层细胞排列整齐,D组大鼠视网膜各层细胞排列不整齐.免疫组织化学染色发现,Thy-1阳性表达主要位于神经节细胞层(GCL),极少数位于内丛状层、内核层.VEGF阳性表达主要定位于GCL,少量位于神经纤维层、内核层及视网膜色素上皮层.结论 玻璃体腔重复注射bevacizumab对糖尿病大鼠视网膜有一定毒性作用.

Abstract：

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.     

Effects of estrogen replacement therapy on apoptosis and vascular endothelial growth factor expression in ocular surface epithelial cells: An experimental study

AIM: To investigate the effects of estrogen replacement therapy (ERT) on apoptosis and vascular endothelial growth factor (VEGF) expression in ocular surface in an experimental rat model. METHODS: Forty female, Wistar rats were randomized in 4 groups in the study. Subcutaneous ERT (17β-estradiol, 10μg/kg/day) was administered to the first group without ovariectomy and to the second group with ovariectomy for three months. Third group had only ovariectomy and fourth group had sham operation. All rats were sacrificed in estrous cycles determined by vaginal smear test and their right eyes were enucleated at the end of the third month. Enucleated eyes were analyzed by immunohistochemical method for expressions of caspase-3, bcl-2, VEGF and TUNEL assay. RESULTS: Caspase-3 expression of conjunctival epithelium was significantly higher in group 3 than group 1 (P=0.005), and group 2 (P=0.007). TUNEL score of conjunctival epithelium was significantly higher in group 3 than group1 (P=0.006). TUNEL score of corneal epithelium was significantly higher in group 3 than group 2 (P=0.012), and group 4 (P=0.002). There was no significant difference between groups in that bcl-2 and VEGF expressions. CONCLUSION: We determined increased apoptosis in ocular surface epithelial cells in ovariectomized rats. ERT and endogen estrogen decreased the apoptosis, and did not result in difference in VEGF expression between the groups. Estrogen may be beneficial for the treatment of apoptosis-mediated ocular surface disorders such as dry eye. Further studies are needed on this subject for a better understanding of the role of estrogen and to provide a new insight for treatment and prevention of apoptosis-mediated ocular surface disorders.     

川芎嗪对氧诱导视网膜病变中视网膜神经细胞凋亡的抑制作用

目的 观察川芎嗪(TMP)对氧诱导视网膜病变(OIR)中视网膜神经细胞凋亡的抑制作用.方法 48只C57BL/6新生幼鼠随机分为正常对照组、OIR对照组及OIR药物组,分别为18、18、12只.正常对照组在正常空气环境中饲养.OIR对照组、OIR药物组幼鼠于出生后7 d置于含氧体积分数(75±3)%的氧气箱中连续生活5 d;出生后12 d,幼鼠回到正常空气环境中饲养,建立OIR模型.出生后12～16d,OIR药物组按200 mg/kg的剂量腹腔注射TMP,1次/d;正常对照组、OIR对照组同时注射相同体积的含0.1%二甲基亚砜(DMSO)的生理盐水.出生后12、14、17 d,摘除幼鼠眼球作石蜡切片并行苏木精-伊红(HE)和脱氧核苷酸末端转移酶介导缺口末端标记(TUNEL)染色.观察视网膜无灌注区形态学变化,检测视网膜神经细胞凋亡数量.结果 正常对照组幼鼠视网膜各层结构清晰.出生后12 d,OIR对照组幼鼠视网膜内核层(INL)可见少量染色质凝聚的细胞核.出生后14 d,OIR对照组幼鼠视网膜INL可见大量染色质凝聚的细胞核,OIR药物组幼鼠视网膜INL均可见少量染色质凝聚的细胞核.出生后17 d,OIR对照组幼鼠视网膜INL、内丛状层(IPL)和外丛状层(OPL)厚度变薄;OIR药物组幼鼠视网膜中周部INL、IPL和OPL厚度较正常对照组薄,较OIR对照组厚.出生后12 d,OIR对照组幼鼠视网膜TUNEL阳性细胞数量为正常对照组的6倍,差异有统计学意义(t=9.432,P＜0.001).出生后14 d,3组幼鼠视网膜TUNEL阳性细胞数量比较,差异有统计学意义(F=587.217,P＜0.001);OIR对照组幼鼠视网膜TUNEL阳性细胞数量为正常对照组的28倍,差异有统计学意义(t=49.813,P＜0.001);OIR药物组幼鼠视网膜TUNEL阳性细胞数量较OIR对照组减少了82.3%,差异有统计学意义(t=42.434,P＜0.001).出生后17 d,3组幼鼠视网膜TUNEL阳性细胞数量比较,差异无统计学意义(F=587.217,P>0.05).结论 TMP能明显抑制OIR中视网膜神经细胞凋亡.

Abstract：

Objective To observe the inhibitory effect of tetramethylpyrazine(TMP)on apoptosis of retinal neurons in oxygen-induced retinopathy(OIR).Methods 48 C57BL/6 mice were randomly divided into normal control group(n=18),OIR control group(n=18)and OIR TMP group(n=12).The mice of normal control group were raised in room air.From the postnatal day 7(P7),mice of the other two groups mice of OIR TMP group received intraperitoneal injection of TMP(200 mg/kg)once a day from P12 to P16.meanwhile the mice of normal control group and OIR control group were iniected with the same volume of normal saline containing of 0.1% DMSO.At P12,P14 and P17,the morphologic changes in retinal avascular zone and the number of retinal apoptotic cell were observed by HE staining and TUNEL assay.Results At P1 2.there were a few of chromatin condensation and pycnic nuclei in the inner nuclear layer (INL)of OIR control group.At P14,a great quantity(OIR control group)or some(OID TMP treated group)chromatin condensation and pycnic nuclei in the central INL were observed.At P17,the thickness of INL,inner plexiform layer(IPL)and outer plexiform layer(OPL)in the OIR control group were reduced;the thickness of INL,IPL and OPL in the OIR TMP group weas thinner than those in the normal control group and thicker than those in the OIR control group.At P12,the TUNEL-positive cells in the OIR control group was 6 times of the normal eontrol group(F=587.217,P＜0.001).At P14,the difference of TUNEL-positive cells in three groups was significant(F=587.217,P＜0.001);the TUNEL-positive cells in the OIR control group was 28 times of the normal control group(t=49.813,P＜0.001);the TUNEL-positive cells in the OIR TMP group has reduced 50% compared with the OIR controI group(t=42.434,P＜0.00 1).At P17,there was no significant difference in TUNEL-positive cells among the three groups (F=587.217,P>0.05).Conclusions TMP can inhibit apoptosis of retinal cells in OIR significantly.     

Sensitized heat shock protein 27 induces retinal ganglion cells apoptosis in rat glaucoma model

AIM: To investigate the relationships between the changes of heat shock protein 27 antibody (anti-HSP27) in serum/cerebrospinal fluid (CSF), intraocular pressure (IOP), retinal ganglion cell (RGC) apoptosis in a rat glaucoma model and disclose the underlying pathogenesis of glaucoma. METHODS: A total of 115 Wistar rats were randomly divided into 4 groups. Group 1 was the ocular hypertension group by condensing 3 episcleral & limbal veins or episcleral area of right eye (HP group, n=25) and sham operation group with conjunctiva incision without coagulation (n=25). Group 2: HSP27 or dose-matched PBS was injected into the vitreous (V-HSP27 group, n=15; V-PBS group, n=15). Group 3: HSP27 and complete Freund’s adjuvant or dose-matched PBS was injected subcutaneously into the hind limb accompanied intraperitoneal injection of pertussis toxin [sensitized group (I-HSP27 group), n=15; I-PBS group, n=15)]. Group 4 was normal group without any treatment (n=5). IOPs of the rats were measured before, day 3, weeks 1, 2, 4, 6, and 8 after treatment. Paraffin-embedded sections were prepared for HE staining and RGCs apoptosis were detected by TUNEL. Anti-HSP27 level in serum and CSF were examined by ELISA. RESULTS: IOPs were elevated significantly in HP and V-HSP27, V-PBS groups (P<0.01) and positively related to anti-HSP27 levels in serum and CSFs. Anti-HSP27 levels in serum and CSF were elevated significantly in I-HSP27 group compared to other groups (P<0.05). However, the IOPs did not show any relationship with the high-level anti-HSP27 in serum and CSFs. RGC apoptosis were all elevated significantly in the HP, V-HSP27, V-PBS and I-HSP27 groups and also positively relative with anti-HSP27 level in serum and CSFs except that high-level of anti-HSP27 in the serum of I-HSP group. CONCLUSION: The increases of anti-HSP27 levels in serum and CSFs both promote IOP escalation and the increase of RGC apoptosis in retina when anti-HSP27 is at low level. The case of high-level anti-HSP27 is opposite and shows protective function in preventing IOP increase and RGC apoptosis.     

Physical activity and risk of age-related cataract

AIM: To investigate the relationships between the changes of heat shock protein 27 antibody (anti-HSP27) in serum/cerebrospinal fluid (CSF), intraocular pressure (IOP), retinal ganglion cell (RGC) apoptosis in a rat glaucoma model and disclose the underlying pathogenesis of glaucoma. METHODS：A total of 115 Wistar rats were randomly divided into 4 groups. Group 1 was the ocular hypertension group by condensing 3 episcleral & limbal veins or episcleral area of right eye (HP group, n=25) and sham operation group with conjunctiva incision without coagulation (n=25). Group 2 was injected with HSP27 or dose-matched PBS into vitreous cavity (V-HSP27 group, n=15; V-PBS group, n=15). Group 3 was injected subcutaneously into the hind limb with HSP27 and complete Freund''s adjuvant or dose-matched PBS accompanied to intaperitoneal injection of pertussis toxin [sensitized group (I-HSP27 group), n=15; I-PBS group, n=15)]. Group 4 was normal group without any treatment (n=5). IOPs of the rats were measured before, day 3, weeks 1, 2, 4, 6, and 8 after treatment. Paraffin-embedded sections were prepared for HE staining and RGCs apoptosis were detected by TUNEL. Anti-HSP27 level in serum and CSF were examined by ELISA. RESULTS: IOPs were elevated significantly in HP and V-HSP27, V-PBS groups (P<0.01) and positively related to anti-HSP27 levels in serum and CSFs. Anti-HSP27 levels in serum and CSF were elevated significantly in I-HSP27 group compared to other groups (P<0.05). However, the IOPs did not show any relationship with the high-level anti-HSP27 in serum and CSFs. RGC apoptosis were all elevated significantly in the HP, V-HSP27, V-PBS and I-HSP27 groups and also positively relative with anti-HSP27 level in serum and CSFs except that high-level of anti-HSP27 in the serum of I-HSP group. CONCLUSION: The increases of anti-HSP27 levels in serum and CSFs both promote IOP escalation and the increase of RGC apoptosis in retina when anti-HSP27 was at low level. The case of high-level anti-HSP27 is opposite and shows protective function in preventing IOP increase and RGC apoptosis.     

胰岛素对链脲佐菌素诱导的糖尿病大鼠视网膜血管内皮生长因子表达的影响

Objective To investigate if insulin can affect the expression of vascular endothelial growth factor (VEGF) in the retina of streptozotocin-induced diabetic rats. Methods A total of 60 male Sprague-Dawley rats were randomly divided into sodium citrate buffer control group (CIT-CON, n= 30) and STZ-induced diabetic group (STZ-DM, n=30). At the 16th week, 24 rats from CIT-CON group at random were randomly divided to group A (sodium citrate buffer control group, n = 12) and group B (sodium citrate buffer plus insulin group, n= 12). The remaining 6 rats from as CIT-CON group served as negative control. At the same time, 24 rats from STZ-DM group at random were randomly divided to group C (STZ-induced diabetic group, n= 12) and group D (STZ-induced diabetic plus insulin group, n= 12). The remaining 6 rats from STZ-DM group also served as negative control. 4 IU of insulin was injected subcutaneously to rats of group B and D. Immunohistochemistry, Western blot and Real-time polymerase chain reaction (RT-PCR) were used to measure the expression level of VEGF protein and mRNA respectively. RESULTS Insulin significantly increased the VEGF mRNA (7.71 ± 0.25 vs 5.36 ±0. 37, t test P< 0. 05) and protein expression (0. 4925 ± 0. 0122 vs 0. 4272 ± 0. 0110, t test P< 0. 05) in the retina of CIT-CON rats.However, in retina of STZ-DM rats, insulin had no effect on VEGF mRNA (8. 92±0. 27 vs 9. 05±0. 28, t test, P>0. 05) and protein expression (0. 5152±0. 0109 vs 0. 5099±0. 0100, t test P>0.05). Conclusions Insulin had no effect on VEGF expression in the retina of STZ-DM rats.     

双靶点干预对糖尿病大鼠视网膜中血管内皮生长因子和结缔组织生长因子基因表达的影响

目的 观察糖尿病大鼠视网膜中以及双靶点干预后血管内皮生长因子（VEGF）和结缔组织生长因子（CTGF）mRNA表达变化。方法 Sprague-Dawley大鼠48只,随机分为正常对照组（CON1组）和糖尿病（DM）组。经鼠尾静脉注射链脲佐菌素制作糖尿病大鼠模型。建模后8、10、12周取大鼠视网膜标本行逆转录实时定量聚合酶链反应（RT-PCR）检测VEGF、CTGF mRNA表达变化。根据上述结果,选取相同条件的大鼠60只,随机选择50只大鼠依照上述方法建立糖尿病大鼠模型;10只大鼠为正常对照组（CON2组）。建模后第10周,将糖尿病大鼠随机分为双靶点干预组、ranibizumab单靶点干预组、CTGF小发夹RNA（shRNA)单靶点干预组、DM未干预组。干预1周后取各组大鼠视网膜标本,行实时定量RT-PCR检测VEGF、CTGF mRNA表达变化。结果 建模后第8周,DM组大鼠视网膜CTGF mRNA水平显著增高且一直持续到第12周,与CON1组比较,差异均有统计学意义（t=-2.49、-2.67、-2.42,P＜0.05）;第8周时DM组大鼠视网膜VEGF mRNA水平与CON1组比较,差异无统计学意义（t=-0.443,P=0.669）;第10周时显著上调且一直维持到第12周,与CON1组比较,差异有统计学意义（t=-2.35、-2.57,,P＜0.05）。 Ranibizumab单靶点干预组大鼠视网膜VEGF mRNA表达水平较DM未干预组显著降低,差异有统计学意义（t=-3.44,P＜0.05）,与CON2组比较,差异无统计学意义(t=-1.37,P＞0.05);CTGF mRNA表达显著高于DM未干预组,差异有统计学意义（t=2.48,P＜0.05）。CTGF shRNA单靶点干预组大鼠视网膜CTGF、VEGF mRNA表达均较DM未干预组显著降低,差异有统计学意义（t=0.23、-2.92,,P＜0.05）。双靶点干预组大鼠视网膜VEGF、CTGF mRNA表达均下调,与DM未干预组比较,差异均有统计学意义（t=-6.09、-5.11,,P＜0.001）;与CON2组比较,差异无统计学差异（t=-1.16、1.139,,P＞0.05）。结论 早期DM大鼠视网膜内VEGF、CTGF基因表达即升高,且CTGF升高较VEGF早。Ranibizumab结合CTGF shRNA双靶点干预能同时降低VEGF、CTGF基因在DM大鼠视网膜中的表达。     

色素上皮衍生因子的表达与早期糖尿病大鼠视网膜血管形态学的改变

目的探讨色素上皮衍生因子(pigment epithelium derived factor,PEDF)mRNA在不同病程糖尿病大鼠模型视网膜中的表达水平及其与早期糖尿病大鼠视网膜血管病变之间的关系。方法 48只健康Wistar大鼠随机分成糖尿病1个月组(DM1)、3个月组(DM3)和6个月组(DM6)与正常对照1个月组(CON1)、3个月组(CON3)、6个月组(CON6),每组各8只。链脲佐菌素诱导糖尿病大鼠动物模型,分别于造模后1个月、3个月、6个月行视网膜血管铺片和PEDFmRNA的原位杂交,观察大鼠视网膜微血管的改变和PEDFmRNA表达情况。结果 (1)DM3和DM6组可见毛细血管迂曲,走形不规则,管腔粗细不均,以DM6组病变最重,可见无细胞毛细血管。(2)周细胞数量:DM1组与CON1组相比差异无统计学意义(CON1组6.45±0.64,DM1组6.52±0.75,P>0.05),DM3及DM6组均较CON3组、CON6组明显减少(CON3组6.51±0.70、DM3组6.06±0.65,CON6组6.47±0.60、DM6组5.45±0.80),差异均有统计学意义(均为P<0.05)。(3)CON1和DM1组PEDFmRNA在节细胞层、内核层及RPE层均有阳性细胞表达,CON1组64±10、DM1组53±8,差异无统计学意义(P>0.05);CON3组和DM3组PDEFmRNA在节细胞层、内核层、RPE层阳性反应细胞表达减少,CON3组68±11、DM3组49±12,差异有统计学意义(P<0.05);CON6组和DM6组PEDFmRNA仅在内核层和节细胞层表达,RPE层只见极少阳性细胞,CON6组58±8、DM6组34±14,差异有显著统计学意义(P<0.01)。结论糖尿病视网膜病变的发生、发展与周细胞数量减少、PEDFmRNA表达降低密切相关,两者的表达同糖尿病视网膜病变的发生、发展存在时效和空间关系。     

曲安奈德对糖尿病鼠血-视网膜屏障破坏的治疗作用

目的探讨曲安奈德(TA)玻璃体注射在糖尿病Wistar鼠血-视网膜屏障(BRB)破坏方面的保护作用。方法选择健康成年Wistar鼠,链脲佐菌素(STZ)诱导大鼠糖尿病。分为正常对照组,糖尿病4个月(DM4)组和6个月(DM6)组,每组各分为形态学观察组和BRB测定组,BRB测定组再分为未行TA治疗(NT)组、TA治疗1周组和2周组。行消化铺片HE染色观察视网膜血管形态,利用视网膜标准化Evansblue(EB)含量测定大鼠BRB的变化。结果正常对照组各组间EB含量差异无统计学意义(P>0.05)。同正常对照组相比,DM4组大鼠出现毛细血管管径粗细不一,血管闭锁,内皮细胞增生,周细胞丢失等形态学改变;DM6组上述病变进一步加重,出现无细胞性毛细血管,糖尿病NT组EB含量明显升高(P>0.01),且6个月组高于4个月组,差异有统计学意义(P>0.01)。同糖尿病NT组相比,各糖尿病治疗组EB含量均显著降低(P>0.01),但除4个月治疗2周组EB含量基本恢复正常(P>0.05)外,余治疗组均仍高于正常对照组(P<0.05)。各糖尿病治疗组间EB含量差异无统计学意义(P>0.05)。结论糖尿病大鼠视网膜的形态学变化与BRB的破坏有关,TA对糖尿病大鼠BRB的破坏具有保护作用。     

CTGFsiRNA ameliorates retinal cells apoptosis in streptozotocin-induced diabetic rats

AIM: To detect the effect of connective tissue growth factor (CTGF) on the apoptosis in the diabetic retina with small interfering RNAs (siRNA) targeting CTGF. METHODS: A total of 60 rats were divided into 6 groups including control group, diabetic 4, 8, 12, 16 weeks groups, and interference group. Diabetic rats were induced by intraperitoneal streptozotocin (STZ). Retinas were obtained from control, diabetic rats and diabetic rats of interference group treated by intravitreal injection of CTGFsiRNA to suppress the expression of CTGF mRNA. Retinal cells apoptosis was detected by Tunnel staining and mRNA expression of CTGF was analyzed by RT-PCR. RESULTS: The levels of CTGF and the apoptosis in the retinas of diabetic rats were significantly higher than those in the controls. Apoptosis occurred at 4 weeks after a diabetic model being set up, became serious with the diabetes developing, while CTGF elevated at 8 weeks. The apoptosis cell counts increased to 25.8cells/mm2 at 24weeks of diabetes. SiRNA-mediated inhibition of CTGF mRNA resulted in a significant decrease in apoptosis. Significant correlations were found between CTGF and apoptosis in the retina. CONCLUSION: It was suggested that CTGF might be involved in retinal cells apoptosis which is a characteristic of early diabetic retina. SiRNA targeting CTGF seems to have the advantage of ameliorating retinal cells apoptosis.     

CTGFsiRNA改善STZ诱导糖尿病大鼠视网膜细胞凋亡

目的:探讨CTGF对糖尿病大鼠视网膜内细胞凋亡的影响。方法:将60大鼠分为对照组,糖尿病4,8,12,16wk组和干预组。糖尿病大鼠应用STZ腹腔注射诱导成模。干预组大鼠于糖尿病成模后16wk玻璃体腔注射CTGFsiRNA来造成CTGF基因的沉默。取各组视网膜组织,应用RT-PCR检测视网膜内的CTGF基因表达,Tunnel法检测视网膜凋亡细胞的情况。结果:糖尿病组视网膜内CTGF表达和凋亡细胞数较正常组明显增加。凋亡发生在建模后4wk,并随着时间的延长而加重,在24wk时,凋亡细胞数为25.8个/mm2。CTGF表达于8wk时出现升高,到16wk时升高更明显。CTGFsiRNA处理后凋亡细胞数明显降低。视网膜内CTGF和细胞凋亡之间具有明显的相关性。结论:CTGF参与了糖尿病视网膜早期病变的细胞凋亡机制,CTGFsiRNA有利于改善糖尿病早期视网膜由于凋亡所致的细胞丢失。     

糖尿病大鼠视网膜毛细血管细胞凋亡及其凋亡相关基因的表达

目的 确定早期糖尿病大鼠视网膜毛细血管细胞凋亡并观测凋亡相关基因（Bax、bcl-2）在早期糖尿病大鼠视网膜的表达。方法 选择健康成年Wistar大鼠40只,随机分成正常对照组（contrast group,CON）和糖尿病（diabetes mellitus)1个月（DM1）、3个月（DM3）及6个月（DM6）组。腹腔内注射链脲佐菌素（streptozotocin,STZ)诱发大鼠糖尿病。制备大鼠视网膜血管辅片及切片,分别行TUNEL法和免疫组化ABC法染色,并对ABC法染色结果进行计算机图像分析。结果 TUNEL法标记的阳性周细胞核出现于DM3和DM6组,而内皮细胞见于DM6组,CON和DM1组未见阳性反应。TUNEL阳性反应细胞核染色质分布不均,表现为环形核、新月形核等典型凋亡细胞特征。ABC法染色：于CON及DM各组,Bax和bcl-2二基因的蛋白均在视网膜血管表达。随病程进展,其阳性反应强度逐渐增加。此外,bcl-2阳性反应在DM3组于色素上皮细胞出现。在DM6组进一步扩大到视杆内节和节细胞。而Bax在DM6组亦出现于细胞。结论 早期糖尿病大鼠视网膜毛细血管周细胞丧失的性质为凋亡,内皮细胞亦存在凋亡;Bax和bcl-2在早期糖尿病大鼠视网膜的表达增加。     

玻璃体腔注射抗氧化剂NAC对早期糖尿病大鼠视网膜的保护作用

目的:探讨抗氧化剂N-乙酰半胱氨酸(NAC)对早期糖尿病大鼠视网膜保护作用的机制。方法:健康成年雄性SD大鼠30只,按照随机分配的原则分为正常对照组(CON组,n=10),糖尿病组(DM组,n=20)。DM组禁食12h后,按照60mg/kg体质量剂量一次性左下腹腔注射1%链脲佐菌素(STZ)溶液,CON组注入等量的柠檬酸缓冲溶液。用药72h后,鼠尾静脉取血检测血糖,≥16.7mmol/L者定为糖尿病模型动物。成模大鼠随机分为糖尿病对照组(D组)和NAC治疗组(N组)。模型建立后,N组大鼠每周通过玻璃体腔注射4μL 1.6μg/μL的NAC,CON组、D组大鼠则给予玻璃体腔注射4μL 0.01mmol/L的磷酸缓冲盐溶液(pbs)不限饮食水,分组喂养。每周记录各组大鼠体质量及血糖。糖尿病成模后2mo,处死实验动物,通过HE染色法,检测各组大鼠视网膜内核层厚度;通过免疫荧光法,检测各组大鼠视网膜神经节细胞数量及视网膜中色素上皮衍生因子(PEDF)水平。结果:N组视网膜内核层厚度较D组厚度增加(P<0.01),与CON组厚度无差异(P>0.05)。与CON组相比,D组视网膜神经节细胞数量减少(P<0.01),N组视网膜神经节细胞略减少(P>0.05)。D组视网膜神经节细胞较N组减少(P<0.01)。与CON组相比,D组PEDF表达下降(P<0.01),N组PEDF表达略下降(P>0.05)。D组PEDF表达较N组下降(P<0.01)。结论:抗氧化剂NAC对早期糖尿病大鼠视网膜组织有保护作用的机制可能是通过上调视网膜中PEDF水平。     

血管内皮生长因子在糖尿病大鼠视网膜中的表达

目的 :观察血管内皮生长因子 (VEGF)蛋白在链脲佐菌素 (STZ)诱导的糖尿病大鼠视网膜中的表达情况 ,以探讨其在早期糖尿病视网膜病变 (DR)中的作用。方法 :选择健康成年Wistar大鼠 4 0只 ,随机分成正常对照组 (CON)、糖尿病 1个月组 (DM1)、糖尿病 3个月组 (DM3)及糖尿病 6个月 (DM6 )组 ,每组 10只。大鼠腹腔内注射STZ诱发大鼠糖尿病。制备大鼠视网膜石蜡切片行免疫组化ABC法染色 ,光镜观察。结果 :VEGF于CON和DM1组只表达于内核层及节细胞层部分细胞 ;病程 3个月时 ,尚可见视网膜血管的阳性反应 ;6个月时 ,阳性反应范围又扩大至视杆内节和色素上皮细胞。结论 :随病程进展 ,VEGF在视网膜中的表达呈逐渐增强的趋势 ,提示它在早期DR的发生、发展中起重要作用     

糖尿病大鼠早期视网膜神经节细胞凋亡机制的探讨

目的：探讨糖尿病大鼠模型早期视网膜神经节细胞(retinal ganglion cells,RGCs)凋亡的机制。

方法：SD大鼠60只,随机分为对照组(CON)及糖尿病组(DM),糖尿病组一次性腹腔注射1% STZ诱发糖尿病鼠模型,两组于第4,8,12wk分别行HE、透射电镜及TUNEL法检测RGCs的凋亡情况,并应用激光共聚焦显微镜检测RGCs内钙离子浓度的变化。

结果：糖尿病组第8wk开始出现RGCs数量减少、细胞排列紊乱的病理改变,第12wk更为明显。糖尿病组透射电镜下可见第4wk时RGCs出现线粒体肿胀; 第8wk时RGCs内肿胀的线粒体更为明显、数目增多,染色质边集于核膜周边,部分细胞体积缩小、细胞器减少; 第12wk时出现RGCs体积变小,甚至出现细胞核断裂。TUNEL阳性RGCs最早于糖尿病组第4wk时出现,随病程延长凋亡的阳性细胞逐渐增多,凋亡指数与同期对照组相比,差异有统计学意义(P<0.01)。糖尿病组第8,12wk时RGCs内钙离子浓度明显高于同期对照组,差异显著(P<0.01); 糖尿病组第8wk与第12wk比较,升高差异亦有统计学意义(P<0.05)。

结论：糖尿病早期出现了视网膜神经节细胞的凋亡,其机制可能与细胞内钙离子浓度升高有关。     

糖皮质激素对糖尿病大鼠视网膜病变的影响

目的：探讨糖皮质激素对糖尿病( diabetes mellitu,DM)大鼠视网膜神经组织的损伤作用及机制。
　　方法：利用链脲佐菌素诱导大鼠DM模型,玻璃体内注射腺病毒( DM组)或腺病毒包装的糖皮质激素受体反义寡核苷酸( siGR组)、阴性核苷酸序列( scRNA组)。另取正常SD 大鼠,玻璃体腔注射腺病毒为对照组( CON 组)。12wk后,HE染色检测视网膜神经节细胞( retinal ganglion cell,RGC)密度,测量大鼠视网膜厚度,免疫组织化学和Western-blot检测ROCK表达变化。
　　结果：与CON组相比,DM组、siGR组及scRNA组糖皮质激素浓度均明显升高(均 P<0.01)。 HE 染色显示,与CON组相比,DM组及scRNA组RGC密度明显降低,视网膜厚度下降,ROCK的蛋白表达增加(均P<0.01),而siGR组无明显变化(均P>0.05)。
　　结论：抑制糖皮质激素能下调糖尿病大鼠视网膜ROCK表达,恢复视网膜RGC密度降低及视网膜厚度。