Experimental study of TGF-β2 antisense oligodeoxynucleotide as an anti -scarring agent in glaucoma surgery

To investigate the effect of TGF-β2 antisense oligode- oxynucleotide on differentiation, proliferation of subconjunctival fibroblast following glaucoma filtration surgery. · METHODS: Glaucoma filtration surgery was performed on both eyes of 28 rabbits. TGF-β2 antisense oligodeoxynucleotide was subconjunctivally injected in the right eyes (group A),and TGF-β2 missense oligodeoxynucleotide (group B) or PBS (group C) was used at the same method in the left eyes as controls. Rabbits were killed at 4, 7, 14 and 28 days after surgery. Intraocular pressure (IOP), bleb characteristics were recorded at different time point. Subconjunctival fibroblasts were examined by immunohistochemistry and electron microscopy. · RESULTS: The IOP of rabbits in group A was significantly lower at 14 days (6.74±1.18mmHg) and 21 days (8.15±1.97mmHg) after operation than the IOP in group B (8.53±1.04, 9.72±1.09mmHg; P <0.01) and group C (8.79±1.21, 9.43±1.27mmHg; P <0.05). The mean bleb survival time was longer (17.2 days) in group A than that of group B (14.5 days) and group C (13.5 days) (P <0.05). The population of the cells expressing α-smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA) was significantly reduced in group A compared with the group B and C. The ultrastructure of fibroblast was not altered by TGF-β2 antisense oligodeoxynucleotide. · CONCLUSION: TGF-β2 antisense oligodeoxynucleotide can prevent the scar formation after glaucoma surgery by inhibiting the differentiation and proliferation of subconjunctival fibroblast. It could be a potentially useful anti-scarring alternative for the prevention of late surgical failure.     

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.     

Comparative Study of the Disinfection Effects of Three Types of Conjunctiva Sac Irrigations

 PURPOSE:The antiseptic effectiveness of 5% anerdian III, 0.016% gentamicin, and 0.5% tobramycin solutions in pre-surgical irrigation of conjunctival sac were compared.   METHODS:A total of 295 cataract patients (302 eyes) who had undergone phacoemulsification aspiration combined with intraocular lens insertion (IOL) were recruited in this prospective study. Operative eyes were given 0.3% levofloxacin eye drops for 3 days and then were randomized into three treatment groups:, anerdian (A), gentamicin (B) and tobramycin (C). The patients received conjunctival sac irrigation using the respective solutions at 10 minutes preoperatively. Conjunctival sac sampling was performed before and after irrigation and the samples were used for subsequent bacterial culture and swab tests. The positive culture rate was used as the main outcome. RESULTS: The positive rates of bacterial culture before conjunctival sac irrigationwere 17.31% (18 eyes) in group A, 13.86% (14 eyes) in group B and 17.3% (14 eyes) in group C. Post irrigation, the positive rates in the three groups decreased to 5.76% (6 eyes), 5.94% (6 eyes) and 7.22% (7 eyes), respectively. The positive rates among the three groups did not differ significantly. However, the positive rate in group A only significantly differed before and after the irrigation (P &;lt;0.05). No toxic or allergic reactions were observed on the ocular surface of any patient after irrigation.  CONCLUSION:The antiseptic effects of the three types of conjunctival sac irrigations did not differ.     

Y27632体外诱导视网膜色素上皮细胞转分化为神经元样细胞的初步研究

Objective To investigate the feasibility of Y27632 to induce transdifferentiation from human retinal pigment epithelial(hRPE)cells into neuron-like cells in vitro.Methods The third to sixth generation of primary hRPE cells were cultured with 2% fetal bovine serum+Dulbecco's modified eagle medium/F12 culture solution,with(experimental group)or without(control group)10 μmol/L Y27632.At 3,6 hours and 1,3,5,7 days after induction,the morphologic changes of RPE cells were observed by inverted microscope.The expression rate of CK18,Map2,NF200 and Pax6 at 3 days after induction in the experimental and control group were detected by immunofluorescent staining.χ2 test was employed for comparison between the two groups.Results 50.1% cells of the experimental group formed axon-like processes and interconnected each other with typical neuron-like appearance.The expression rates of CK18,Map2,NF200 and Pax6 in the experimental group were 43.88% ,31.90% ,57.45% and 65.79% .while the above indexes in the control group were 93.97% ,4.49% ,22.37% and 8.33% respectively.Compared the expression rate of CK18(χ2=64.763),Map2(χ2=23.634),NF200(χ2=21.261)and Pax6(χ2=25.946)between the two groups,the differences were significant(P＜0.01).Conclusion The hRPE cells can be trans-differentiated into neuron-like cells in vitro bv Y27632.     

Y27632体外诱导视网膜色素上皮细胞转分化为神经元样细胞的初步研究

Objective To investigate the feasibility of Y27632 to induce transdifferentiation from human retinal pigment epithelial(hRPE)cells into neuron-like cells in vitro.Methods The third to sixth generation of primary hRPE cells were cultured with 2% fetal bovine serum+Dulbecco's modified eagle medium/F12 culture solution,with(experimental group)or without(control group)10 μmol/L Y27632.At 3,6 hours and 1,3,5,7 days after induction,the morphologic changes of RPE cells were observed by inverted microscope.The expression rate of CK18,Map2,NF200 and Pax6 at 3 days after induction in the experimental and control group were detected by immunofluorescent staining.χ2 test was employed for comparison between the two groups.Results 50.1% cells of the experimental group formed axon-like processes and interconnected each other with typical neuron-like appearance.The expression rates of CK18,Map2,NF200 and Pax6 in the experimental group were 43.88% ,31.90% ,57.45% and 65.79% .while the above indexes in the control group were 93.97% ,4.49% ,22.37% and 8.33% respectively.Compared the expression rate of CK18(χ2=64.763),Map2(χ2=23.634),NF200(χ2=21.261)and Pax6(χ2=25.946)between the two groups,the differences were significant(P＜0.01).Conclusion The hRPE cells can be trans-differentiated into neuron-like cells in vitro bv Y27632.     

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.     

花色苷对高糖培养的视网膜Müller细胞L-谷氨酸/L-天门冬氨酸转运体表达的影响

目的 观察花色苷对体外高糖培养的视网膜Müller细胞L-谷氨酸/L-天门冬氨酸转运体(GLAST)表达的影响.方法 取出生后10 d的雄性Sprague-Dawley(SD)大鼠视网膜组织,体外原代培养Müller细胞.第2～4代细胞用于实验.将实验分为正常对照组(A组)、高糖对照组(B组)、高糖+30 μmol/L花色苷组(C组)、高糖+60 μmol/L花色苷组(D组)、高糖+100μmol/L花色苷组(E组)进行.采用噻唑蓝比色法(MTT)测定波长570 nm处的吸光度[A,旧称光密度(OD)]值,以各组A值计算细胞相对存活率.采用免疫蛋白印迹法(Western blot)检测各组大鼠视网膜Müller细胞上GLAST的蛋白表达.结果 MTT检测显示,A、B、C、D、E组A值分别为0.450 8±0.020 4、0.270 1±0.031 4、0.332 0±0.023 2、0.428 3±0.017 2、0.361 9±0.027 0,细胞相对存活率分别为100.0%、59.9%、73.6%、95.0%、80.3%.其中,C、D、E组A值均较B组增高,差异有统计学意义(F=32.25,P＜0.05);D组A值较C、E组显著增高,差异也有统计学意义(F=21.07,P＜0.05).Western blot检测显示,B组大鼠视网膜Müller细胞的GLAST蛋白表达较A组降低,差异有统计学意义(t=5.25,P＜0.05);A、C、D、E组间大鼠视网膜Müller细胞的GLAST蛋白表达无明显变化,差异无统计学意义(F=2.979,P＞0.05).结论 花色苷可逆转高糖引起的Müller细胞GLAST蛋白表达下降.

Abstract：

Objective To observe the effect of cyanin on the expression of L-glutamate/ L-aspartate transporter (GLAST) in high glucose cultured retina Müller cells. Methods The retinal tissue of SpragueDawley (SD) rats was collected at postnatal 10 day, and Müller cells were isolated and cultured according to literature. The Müller ceils (2nd-4th generations) were treated with five different medium as normal group (group A), high glucose control group (group B), high glucose+30 μmol/L cyanin group (group C), high glucose+60 μmol/L cyanin group (group D) and high glucose+100 μmol/L cyanin group (group E). Cell relative survival rates (A value) were measured by MTT assay at 570 nm. The GLAST protein expression in M011er cells was observed by Western blot. Results MTT assay showed that the A value of the five group were 0. 450 8±0. 020 4, 0. 270 1±0. 031 4, 0. 332 0±0. 023 2, 0. 428 3±0. 017 2, 0. 361 9±0. 027 0,the cell relative survival rate were 100. 0%, 59. 9%, 73.6%, 95%, 80.3% respectively. The A value of group C, D, E were significantly higher than that of group B (F=32.25, P<0.05), the A value of group D were significantly higher than that of group C and E (F=21.07, P<0. 05). Western blot showed that the GLAST protein expression of group B was lower than that of group A (t=5.25, P<0. 05) ; there was no obvious changes of GLAST protein expression in group A, C, D and E (F= 2. 979, P>0.05).Conclusion Cyanin can rescue high glucose-induced GLAST reduction.     

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.     

分泌人内皮抑素的微囊化基因工程细胞系的构建

目的 建立能稳定分泌人内皮抑素(hES)的基因工程细胞系,观察内皮抑素(ES)蛋白和hES的表达.方法 以hES重组质粒pcDNA3.0(pcDNA3-Endo)为模板,通过聚合酶链反应(PCR)扩增获得hES基因片段,且在基因前加信号肽序列,将其定向插入真核表达载体绿色荧光蛋白(pEGFP-N1)质粒中,获得重组质粒pEGFP-N1-Es;利用阳离子脂质体介导将其转染到人胚胎肾细胞(HeK-293)细胞中,G418筛选后得到阳性克隆hES/293,采用蛋白免疫印迹(Western blot)法检测转染细胞上清中ES蛋白的表达;用海澡酸钠壳聚糖(ACA)微囊包裹hES/293细胞,分别收集培养3、7、21、35 d的ACA微囊化hES/293细胞的培养上清液,Western blot法检测包裹后培养液上清中hES的表达.结果 重组质粒pEGFP-N1-ES经限制性核对内切酶HindⅢ和限制性核酸内切酶BamH Ⅰ双酶切得到4700碱基对(bp)和600 bp 2条带;PCR扩增出600 bp条带;测序结果与NCBI上序列比对软件(BLAST)比对,同源性达到100%.pEGFP-N1-ES转染HeK-293细胞,经G418筛选后获得阳性克隆,选取筛选的10株单克隆细胞培养上清液进行Western blot分析,在相对分子质量为20×103处出现蛋白条带.在ACA微囊内hES/293细胞随着培养时间的延长,细胞团逐渐长大,充满整个囊内空间.培养3、7、21、35 d时,在相对分子质量为20×103处出现蛋白条带.结论 重组pEGFP-N1-ES真核表达载体构建正确,转染HeK-293细胞后可有效的表达hES蛋白,并能分泌到细胞外;微囊化hES/293细胞产生的ES蛋白可以自由扩散出微囊膜外,并呈持续性表达.     

自噬抑制剂3-MA对高糖诱导的人视网膜色素上皮细胞增生的抑制作用

背景糖尿病视网膜病变(DR)是糖尿病常见的并发症之一,视网膜色素上皮(RPE)细胞作为视网膜重要的组成细胞在DR的发生和发展中至关重要. 目的 探讨自噬抑制剂3-MA对高糖培养的人RPE细胞(hRPECs)增生的影响.方法 将体外培养的hRPECs分为对照组、高糖组和3-MA+高糖组,对照组细胞用含5 mmol/L葡萄糖的DMEM/F12培养基进行培养,高糖组采用含30 mmol/L葡萄糖的DMEM/F12培养基进行培养,干预组细胞先在DMEM/F12培养基中添加10 mmol/L的3-MA处理1h后,再添加30 mmol/L葡萄糖溶液进行培养.将培养的细胞以1×105/孔的密度接种于24孔细胞培养板中,待细胞80％融合后用含体积分数0.5％胎牛血清的培养基继续孵育24 h,使细胞周期同步化.倒置光学显微镜和透射电子显微镜下观察各组jRPECs的形态及超微结构;采用CCK-8法检测各组hRPECs的增生率;采用Western blot法检测各组hRPECs中自噬相关基因微管相关蛋白轻链3B(LC3B)蛋白的表达.结果 对照组细胞生长状态良好,细胞排列整齐;高糖组细胞体积稍增大,细胞数量明显多于对照组,而3-MA+高糖组细胞形态不规则,排列紊乱.透射电子显微镜下可见对照组细胞核呈圆形或卵圆形,亚细胞器形态均正常,未发现自噬小体;高糖组细胞中可见自噬溶酶体,而3-MA+高糖组可见自噬小体.对照组、高糖组、3-MA+高糖组细胞的增生率分别为(100.0±2.0)％、(116.9±5.2)%和(103.7±4.7)％,总体比较差异有统计学意义(F=13.526,P=0.006),高糖组的细胞增生率明显高于对照组和3-MA+高糖组,差异均有统计学意义(均P＜0.05),3-MA+高糖组与对照组间细胞增生率的差异无统计学意义(P＞0.05).与对照组比较,高糖组细胞中LC3B-Ⅰ蛋白表达减弱,LC3B-Ⅱ蛋白表达增强,而3-MA+高糖组细胞中LC3B-Ⅰ和LC3B-Ⅱ蛋白的表达强度与对照组接近.对照组、高糖组、3-MA+高糖组细胞中LC3B-Ⅱ/LC3B-Ⅰ值分别为0.131±0.065、2.504±0.097和0.274±0.007,组间总体比较差异有统计学意义(F=1 694.676,P=0.000),高糖组细胞中LC3B-Ⅱ/LC3B-Ⅰ值明显高于对照组和3-MA+高糖组,差异均有统计学意义(均P＜0.05),3-MA+高糖组与对照组间细胞中LC3B-Ⅱ/LC3B-Ⅰ值的差异无统计学意义(P＞0.05).结论 高糖可激活自噬过程并促进hRPECs增生,而自噬抑制剂3-MA在一定程度上抑制自噬进程,从而发挥抑制细胞增生的作用.     

CXCR4抑制剂与抗血管内皮生长因子抗体联合应用对实验性脉络膜新生血管形成的干预作用

目的 观察玻璃体腔联合注射CXCR4抑制剂AMD3100与抗血管内皮生长因子(VEGF)抗体对实验性脉络膜新生血管(CNV)形成的干预作用.方法 选取48只棕色挪威(BN)大鼠随机分为AF564干预实验组(A组)、AMD3100干预实验组(B组)、联合干预实验组(C组)、磷酸盐缓冲液(PBS)对照组(D组),每组均为12只大鼠,左眼为实验眼.采用氪红激光光凝建立CNV模型.激光光凝后即刻玻璃体腔分别注射抗鼠VEGF抗体(AF564)、CXCR4特异性抑制剂AMD3100、抗鼠VEGF抗体及AMD3100、PBS各5μl.激光光凝后14 d行荧光素眼底血管造影(FFA),病理组织切片及脉络膜血管铺片检查.观察不同组别大鼠荧光渗漏程度以及CNV相对厚度和面积的变化.结果 激光光凝后14d,A、B、C、D组荧光渗漏评分分别为2.16±0.91、2.16±0.91、1.92±1.03、1.39±0.93.A、B、C组荧光渗漏较D组荧光渗漏明显受抑制,差异均有统计学意义(F=12.91,P＜0.001);C组荧光渗漏程度低于A、B组,差异有统计学意义(F=9.21,P＜0.05).组织病理学检查显示,激光光凝后14 d,A、B、C、D组CNV相对厚度分别为1.82±0.11、1.90±0.22、1.12±0.12、2.82±0.29.A、B、C组相对CNV厚度与D组CNV相对厚度比较,差异均有统计学意义(F=5.92,P＜0.001);C组CNV相对厚度明显变薄,与A、B组CNV相对厚度比较,差异均有统计学意义(F=5.16,P＜0.05).脉络膜血管铺片结果显示,A、B、C、D组CNV面积分别为(8204±122)、(9332±211)、(6533±101)、(13 644±255)μm2.A、B、C组CNV面积较D组CNV面积明显减少,差异均有统计学意义(F=147.50,P＜0.001);C组CNV面积与A、B组CMV面积比较,差异有统计学意义(F=112.60,P＜0.05).结论 CXCR4抑制剂及抗VEGF抗体联合使用,可显著抑制激光诱导的CNV形成.     

重组干扰素诱导蛋白-10对视网膜血管内皮细胞增生、迁移及管腔形成的影响

目的 观察重组干扰素诱导蛋白-10（IP-10）对人视网膜血管内皮细胞（HREC）和人脐静脉血管内皮细胞（HUVEC）增生、迁移及管腔形成能力的影响。方法 逆转录聚合酶链反应（RT-PCR）法检测HREC和HUVEC中CXC趋化因子受体3（CXCR3） mRNA的表达情况;以细胞计数试剂盒-8（CCK-8)增生分析法比较HREC和HUVEC在不同IP-10浓度下增生能力的差异;采用细胞划痕法测定IP-10对HREC和HUVEC迁移的作用;采用基质体外三维成型法检测IP-10对HREC和HUVEC管腔形成的影响。结果 HREC和HUVEC均在基因水平表达CXCR3。CCK 8增生分析法检测结果显示IP-10抑制HREC增生,并呈剂量依赖性（F=6.202,P＜0.05）,但IP-10对HUVEC增生无明显影响（F=1.183,P＞0.05）。细胞划痕法测定结果显示,HREC和HUVEC在10、100 ng/ml IP-10干预时的迁移距离均小于对照组迁移距离,差异有统计学意义（F=25.373、23.858,P＜0.05）。10、100 ng/ml IP-10对HREC完整管腔形成数量无明显影响,1000 ng/ml IP-10可使HREC完整管腔形成数量明显减少;10、100、1000 ng/ml IP-10干预和未行IP-10干预的HREC完整管腔形成数量比较,差异有统计学意义（F=5.359,P＜0.05）。结论 IP-10对HREC的增生、迁移、管腔形成均有抑制作用,对HUVEC的迁移有抑制作用。     

二十二碳六烯酸对人视网膜色素上皮细胞中血红素氧合酶-1表达的诱导作用

背景 氧化应激是年龄相关性黄斑变性发生的重要机制.研究表明,二十二碳六烯酸(DHA)在视网膜光感受细胞的发育中发挥重要作用,并可上调血红素氧合酶1(HO-1)的表达,从而发挥抗氧化效应,但DHA是否能影响人视网膜色素上皮(RPE)细胞表达HO-1尚未阐明. 目的 观察DHA对体外培养的RPE中HO-1表达的影响及其分子机制. 方法 对人RPE细胞系ARPE-19进行培养,分别用30、50、100和120 μmol/L DHA作用4～24 h,以不含DHA培养的细胞作为对照组.采用乳酸脱氢酶(LDH)法检测DHA对细胞的毒性;分别采用实时荧光定量PCR和Western blot法检测各组细胞中HO-1 mRNA及其蛋白的相对表达;采用比色法分析各组细胞中HO-1酶活性变化情况;采用荧光探针H2DCFDA检测细胞中活性氧簇(ROS)的相对比例,并采用免疫荧光技术检测各组培养细胞中核转录因子-E2相关因子2(Nrf2)的核转位情况;分别采用ROS抑制剂N-乙酰半胱氨酸(NAC)干预法和Nrf2小干扰RNA(siRNA)转染法作用于培养的细胞,采用Western blot法检测细胞中Nrf2蛋白的表达,并观察其对细胞中HO-1蛋白表达量的影响. 结果 0、30、50、100和120 μmol/L DHA作用于ARPE-19细胞后24 h,培养上清液中LDH漏出率的总体比较差异有统计学意义(F=8.14,P＜0.05),其中120 μmol/L DHA作用后细胞中LDH漏出率明显高于0、30、50、100 μmol/L DHA组,差异均有统计学意义(均P＜0.05).不同浓度DHA作用ARPE-19细胞后8h,HO-1mRNA及其蛋白的相对表达量以及HO-1的酶活性总体比较差异均有统计学意义(F=16.24,P＜0.05;F=11.34,P＜0.05;F=11.81,P＜0.05),其中30、50、100 μmol/L DHA组细胞中HO-1 mRNA及其蛋白的相对表达量以及HO-1的酶活性均明显高于0μmol/L DHA组,差异均有统计学意义(均P＜0.05).不同浓度DHA作用ARPE-19细胞后4h细胞内ROS相对荧光强度、细胞核Nrf2阳性细胞比例的总体比较差异均有统计学意义(F=11.08,P＜0.05;F=16.42,P＜0.05),其中30、50和100μmol/L DHA组细胞中ROS相对荧光强度、细胞核Nrf2阳性细胞比例均明显高于0μmol/L DHA组,差异均有统计学意义(均P＜0.05).100 μ.mol/L DHA处理条件下,NAC预处理后细胞中HO-1蛋白的相对表达量和细胞核Nrf2阳性细胞比例均明显低于单纯100μmol/L DHA组,Nrf2 siRNA转染组细胞中HO-1的相对表达量和细胞核Nrf2阳性细胞比例均明显低于空白siRNA转染组,差异均有统计学意义(均P＜0.05). 结论 低于100 μmol/L的DHA可能通过ROS/Nrf2途径诱导RPE细胞表达HO-1,从而发挥对细胞的保护作用.     

吲哚美辛对脉络膜黑色素瘤细胞增生活性的影响及作用机制

目的　观察环氧化酶抑制剂吲哚美辛（IN）对脉络膜黑色素瘤细胞OCM-1增生活性的影响并探讨其作用机制。方法　体外培养OCM-1细胞,采用25、50、100、200、400 μmol/L IN对OCM-1细胞进行干预,应用四氮唑化合物（MTT）法检测不同浓度IN对OCM-1细胞增生活性的影响;侵袭实验检测IN对OCM-1细胞侵袭力的影响;逆转录聚合酶链反应（RT-PCR）检测IN对OCM-1细胞内血管内皮生长因子（VEGF）及凋亡抑制因子（survivin） mRNA表达的调节作用;免疫荧光化学检测OCM-1细胞内VEGF、survivin蛋白的定位及表达,酶联免疫吸附测定（ELISA）及蛋白免疫印迹法（Western blot）检测IN对OCM 1细胞内VEGF、survivin蛋白表达的影响。结果　不同浓度IN对OCM 1细胞的增生及侵袭均有明显的抑制作用,其抑制率呈浓度-时间依赖性（MTT: 24 h：F=19.642,P＜0.01;48 h：F=136.597,P＜0.01;72 h：F=582.543,P＜0.01;侵袭实验：F=54.225,P＜0.01）。免疫荧光化学检测结果显示：VEGF、survivin 2种因子在OCM-1细胞内均呈阳性表达且多数表达于细胞胞浆内;RT-PCR检测结果显示：100、200、400 μmol/L IN可抑制OCM-1细胞内survivin mRNA的表达（F=16.679,P＜0.01）,但对细胞内VEGF mRNA的表达无抑制作用。ELISA法测出未用药组、100、400 μmol/L IN作用OCM-1细胞24 h后,细胞内survivin的浓度分别为（787.33±47.37）、（257.00±26.21）、（123.33±8.02） pg/ml;Western blot进一步验证了IN可抑制OCM-1细胞内survivin蛋白的表达,但对VEGF的表达无抑制作用。结论　IN能够抑制OCM-1细胞的增生及侵袭,其机制可能与其下调OCM-1细胞内survivin因子的表达有关。     

蓝光照射对人视网膜色素上皮细胞钙离子蛋白激酶C信号通路的影响

 目的 观察蓝光照射对人视网膜色素上皮细胞钙离子（Ca2+）蛋白激酶C（PKC）信号通路的影响。方法 体外培养并鉴定人视网膜色素上皮（RPE）细胞,取第4代人RPE细胞随机分组进行实验。采用蛋白免疫印迹法测定培养细胞PKC蛋白表达,检测佛波酯（PMA）和钙磷酸结合蛋白（calphostin C）对PKC活性的影响,确定PMA与calphostin C影响PKC活性的最适宜浓度。采用非放射性核素法测定蓝光照射处理对培养细胞PKC活性的影响。采用20 W,波长450～500 nm医用蓝光灯作为光源,光照强度（2000±500） Lux,照射6 h,24 h后终止培养,以此制造体外培养人RPE细胞光损伤。将培养细胞随机分成5个组,即无光照、单纯光照、光照联合硝苯地平、光照联合calphostin C、光照联合PMA组。其中,无光照组不接受光照;单纯光照组仅接受光照;光照联合硝苯地平组接受光照和0.1 mmol/L的硝苯地平;光照联合calphostin C组接受光照和100.0 nmol/L的calphostin C;光照联合PMA组接受光照和100.0 nmol/L的PMA。用乙酰氧基甲基酯Ca²⁺荧光探针标记各组培养细胞,激光扫描共焦显微镜测定各组细胞内Ca2+浓度。比较各组细胞内Ca2+浓度差异。结果 经鉴定,体外培养人RPE细胞成功。100.0、200.0 nmol/L PMA处理的RPE细胞中PKC蛋白相对表达量高于0.1、1.0、10.0、50.0 nmol/L PMA处理的RPE细胞,差异有统计学意义（F=217.537,P＜0.05）,但100.0、200.0 nmol/L PMA处理组间PKC蛋白相对表达量比较,差异无统计学意义（P=0.072）。100.0、200.0 nmol/L calphostin C处理的RPE细胞中PKC蛋白相对表达量低于5.0、25.0、50.0、75.0 nmol/L calphostin C处理的RPE细胞,差异有统计学意义（F=164.543,P＜0.05）,但100.0、200.0 nmol/L calphostin C处理组间PKC蛋白相对表达量比较,差异无统计学意义（P=0.385）。蓝光照射处理后,RPE细胞的PKC活性显著升高,与未接受蓝光照射处理的RPE细胞的PKC活性比较,差异有统计学意义（t=-9.869,P＜0.05）。单纯光照、光照联合硝苯地平、光照联合Calphostin C、光照联合PMA组的RPE细胞内Ca²⁺浓度均高于无光照组,差异有统计学意义（F=26 764.92,P＜0.05）;光照联合PMA组RPE细胞内Ca²⁺浓度高于单纯光照、光照联合硝苯地平及光照联合calphostin C组（P＜0.05）,单纯光照组高于光照联合硝苯地平和光照联合calphostin C组（P＜0.05）。结论 蓝光照射后人RPE细胞内PKC活性增高,Ca²⁺浓度增高。硝苯地平和calphostin C均能降低蓝光照射后人RPE细胞内Ca²⁺浓度,PMA增加细胞内Ca²⁺浓度。     

抗血管内皮生长因子单克隆抗体bevacizumab对实验性脉络膜黑色素瘤生长的影响

目的 观察并探讨抗血管内皮生长因子（VEGF）单克隆抗体bevacizumab对人眼脉络膜黑色素瘤（CM）细胞系OCM-1细胞裸鼠移植瘤生长的影响及其机制。方法　OCM-1细胞植入18只雌性裸鼠腋周皮下,建立移植肿瘤模型后随机分成空白对照组（A组）、阴性对照组（B组）、注药治疗组（C组）。A组未作处理;B组腹腔注射生理盐水0.2 ml;C组以5 mg/kg剂量腹腔注射生理盐水稀释的bevacizumab溶液0.2 ml。连续用药14 d,期间观察肿瘤生长情况。14 d后处死裸鼠,称量肿瘤重量并计算抑瘤率;免疫组织化学染色检测各组肿瘤组织增生细胞相关核抗原（ki67）、凋亡抑制因子（survivin）表达情况;逆转录聚合酶链反应（RT-PCR）分析各组VEGF和survivin mRNA水平的表达。结果　C组肿瘤体积、重量分别为（598.86±321.81） mm3 、（0.66±0.15）g,A、B组肿瘤体积、重量分别为（1 715.15±278.16）mm3、（1.54±0.39）g和（1 750.23±206.36） mm³、（1.54±0.31） g,C组与A、B组肿瘤体积、重量比较,差异均有统计学意义（F=34.53,8.69;P=0.00,0.01）;C组抑瘤率为57.14%,A、B组抑瘤率分别为5.31%、6.25%。A、B、C组,ki67增生指数分别为（51.85±1.32）%、（46.30±1.39）%、27.90±0.90）%,C组与A、B组比较,ki67增生指数差异有统计学意义（H=15.17,P=0.00）。C组survivin mRNA表达为0.49±0.02,A、B组survivin mRNA表达分别为（0.82±0.05）、（0.61±0.05）,C组与A、B组比较,survivin mRNA表达差异有统计学意义（F=15.17,P＜0.05）。C组VEGF mRNA表达为（0.32±0.08）,A、B组VEGF mRNA表达分别为（0.73±0.07）、（0.80±0.04）,C组与A、B组比较,VEGF mRNA表达差异有统计学意义（F=12.05,P＜0.05）。结论　Bevacizumab能够抑制OCM-1裸鼠移植瘤的生长,其机制可能与抑制VEGF活性,下调survivin表达,抑制细胞增生有关。     

雷帕霉素对人视网膜色素上皮细胞增生和凋亡的影响

背景雷帕霉素（RAPA）是哺乳动物RAPA靶蛋白（mTOR）特异性抑制剂,能有效抑制晶状体上皮细胞（LECs）及多种肿瘤细胞增生并具有诱导肿瘤细胞凋亡的作用,研究其对视网膜色素上皮（RPE）细胞是否具有类似作用对临床上预防和治疗增生性玻璃体视网膜病变（PVR）具有重要意义。目的研究RAPA对体外培养的人RPE细胞增生和凋亡的影响。方法对人RPE细胞（D407细胞株）进行体外传代培养,按照培养基中加入药物的不同将培养的细胞分为9组,空白对照组进行常规培养;二甲基亚砜（DMSO）对照组在培养基中加入质量分数0．1％。DMSO;实验各组将5、10、20、40、80、160、320nmol／LRAPA分别加入培养基中并分别培养12、24、48h。应用噻唑蓝（MTT）法检测各组吸光度（A490）值并计算各组人RPE细胞增生的抑制率,应用Hoechst染色法检测各组人RPE细胞的凋亡率,评价上述各浓度RAPA作用不同时间后对人RPE细胞增生和凋亡的影响。结果不同浓度RAPA组对人RPE细胞的抑制率随浓度的增高而明显升高,差异有统计学意义（F=484．451,P〈0．01）,20～320nmol／LRAPA组在各时间点所测细胞增生抑制率均明显高于DMSO溶剂对照组,差异均有统计学意义（P〈0．01）。RAPA对细胞增生的抑制率随作用时间的延长明显增加,差异有统计学意义（F=232．262,P〈0．01）,各浓度的RAPA作用24h和48h后细胞增生的抑制率均明显高于12h,差异均有统计学意义（P〈0．05）。与空白对照组及溶剂对照组比较,10nmol／LRAPA作用12、24、48h均有诱导细胞凋亡的作用,差异均有统计学意义（P〈0．05）;20～320nmol／LRAPA组细胞凋亡率明显增加,差异有统计学意义（P〈0．叭）。各浓度RAPA组随作用时间的延长人RPE细胞的凋亡率明显增加（F=625．584,P〈0．01）。Hoechst33258染色可见凋亡细胞核碎裂并呈块状致密浓染,染色质固缩。结论RAPA以浓度和时间依赖的方式抑制体外培养的人RPE细胞增生并诱导其凋亡。