Inhibition of EGFR attenuates EGF-induced activation of retinal pigment epithelium cell via EGFR/AKT signaling pathway

AIM:To explore the effect of epidermal growth factor receptor(EGFR)inhibition by erlotinib and EGFR siRNA on epidermal growth factor(EGF)-induced activation of retinal pigment epithelium(RPE)cells.METHODS:Human RPE cell line(ARPE-19 cells)was activated by 100 ng/mL EGF.Erlotinib and EGFR siRNA were used to intervene EGF treatment.Cellular viability,proliferation,and migration were detected by methyl thiazolyl tetrazolium(MTT)assay,bromodeoxyuridine(BrdU)staining assay and wound healing assay,respectively.EGFR/protein kinase B(AKT)pathway proteins and N-cadherin,α-smooth muscle actin(α-SMA),and vimentin were tested by Western blot assay.EGFR was also determined by immunofluorescence staining.RESULTS:EGF treatment for 24h induced a significant increase of ARPE-19 cells’viability,proliferation and migration,phosphorylation of EGFR/AKT proteins,and decreased total EGFR expression.Erlotinib suppressed ARPE-19 cells’viability,proliferation and migration through down regulating total EGFR and AKT protein expressions.Erlotinib also inhibited EGF-induced an increase of proliferative and migrative ability in ARPE-19 cells and clearly suppressed EGF-induced EGFR/AKT proteins phosphorylation and decreased expression of N-cadherin,α-SMA,and vimentin proteins.Similarly,EGFR inhibition by EGFR siRNA significantly affected EGF-induced an increase of cell proliferation,viability,and migration,phosphorylation of EGFR/AKT proteins,and up-regulation of N-cadherin,α-SMA,and vimentin proteins.CONCLUSION:Erlotinib and EGFR-knockdown suppress EGF-induced cell viability,proliferation,and migration via EGFR/AKT pathway in RPE cells.EGFR inhibition may be a possible therapeutic approach for proliferative vitreoretinopathy(PVR).

Effect of acetyl L-carnitine on human retinal pigment epithelium-19 cells in hypoxic conditions

AIM:To investigate the effect of acetyl-L-carnitine(ALCAR)on cell viability,morphological integrity,and vascular endothelial growth factor(VEGF)expression in human retinal pigment epithelium(ARPE-19)cells using a hypoxic model.METHODS:In the first set of experiments,the optimal CoCl_(2) dose was determined by exposing ARPE-19 cell cultures to different concentrations.To evaluate the effect of ALCAR on cell viability,five groups of ARPE-19 cell culture were established that included a control group,a sham group(200μM CoCl_(2)),and groups that received 1,10 and 100 mM doses of ALCAR combined with 200μM CoCl_(2),respectively.The cell viability was measured by MTT assay.The morphological characteristics of cells were observed by an inverted phase contrast microscope.The levels of VEGF and HIF-1α secretion by ARPE-19 cells were detected by enzyme linked immunosorbent assay(ELISA)assay.RESULTS:ARPE-19 cells were exposed to different doses of CoCl_(2) in order to create a hypoxia model.Nevertheless,when exposed to a concentration of 200μM CoCl_(2),a notable decrease in viability to 83% was noted.ALCAR was found to increase the cell viability at 1 mM and 10 mM concentrations,while the highest concentration(100 mM)did not have an added effect.The cell viability was found to be significantly higher in the groups treated with a concentration of 1 mM and 10 mM ALCAR compared to the Sham group(P=0.041,P=0.019,respectively).The cell viability and morphology remained unaffected by the greatest dose of ALCAR(100 mM).The administration of 10 mM ALCAR demonstrated a statistically significant reduction in the levels of VEGF and HIF-1α compared with the Sham group(P=0.013,P=0.033,respectively).CONCLUSION:The findings from the current study indicate that ALCAR could represent a viable therapeutic option with the potential to open up novel treatment pathways for retinal diseases,particular relevance for age-related macular degeneration(AMD).However,to fully elucidate ALCAR’s application potential in retinal diseases,additional investigation is necessary to clearly define the exact mechanisms involved.

New antioxidant SkQ1 is an effective protector of rat eye retinal pigment epithelium and choroid under conditions of long-term organotypic cultivation

Cells have intrinsic mechanisms for cleaning harmful oxidants represented mainly by reactive oxygen species (ROS). Despite the antioxidant defense, ROS can cause serious damage to the retina that with age leads to various eye diseases and even blindness. Among numerous cell sites of ROS generation, mitochondrial electron transport is of crucial importance. Recently, for the purpose of cleaning ROS in the mitochondrial matrix, powerful mitochondria- targeted antioxidant “SkQ1” has been invented. We studied SkQ1 effects upon tissues of rat posterior eye cup that consisted: retinal pigment epithelium (RPE) ? choroidal coat ? scleral coat. The eye cups were isolated from the eyes of adult albino rats and cultivated in rotary tissue culture system in the presence of 20 nM SkQ1 or without this compound. After 7 days - 1 month in vitro eye cup samples were studied by immunohistochemistry, routine histology, morphometry, and digital image analysis. We have found that under chosen, “in vitro like in vivo” conditions 20 nM SkQ1 effectively reduced cell death in RPE and choroid, protected RPE from disintegration caused by cell phenotypic transformation and withdrawal from the layer, suppressed transmigration of choroidal coat cells. In the ex vivo model we used degenerative processes were more pronounced in the eye cup center where SkQ1 effect was most vivid. All this give us hopes for effectiveness of SkQ1 treatment of retinal central part that is very susceptible to light-induced over-oxidation injury and mostly suffering in many age-related diseases, AMD, in particular.

Effects of Nerve Growth Factor on Proliferation and DNA Synthesis of Cultured Human Fetal Retinal Pigment Epithelium Cells

Objective: To investigate the effects of nerve growth factor(NGF)on proliferation and DNAthesis of cultured human fetal retinal pigment epithelium (RPE)cells in vitro.Methods: Primary culture and subculture of human fetal retinal pigment epithelium cellswere established in vitro first. Cultured RPE cells were treated with NGF by variousconcentrations 0μg/L, 50μg/L, 100μg/L, 200μg/L and 300μg/L(final concentration)for 48 hs.After 48 hs, cells proliferation was measured with methyl thiazolyl tetrazolium(MTT)assay method and the amount of DNA was determined by the absorbance at 280nm of nucleic acid & protein analysis.Results: The A values of 100 μg/L, 200 μg/L, 300 μg/L NGF was(0. 213 7 ± 0. 23 3),(0. 218 8 ±0. 018 1), (0. 232 2 ±0. 016 4) as compared with(0. 189 7 ±0. 015 2) of Avalue of 0 μg/L NGF respectively, q value was 3.63,4.40, 6. 42 and P value was0. 015, 0. 000, 0. 000(q-test). The DNA concentrations of 100 μg/L, 200 μg/L, 300μg/L and 400 μg/L NGF was (981. 220 4 ± 123.535 7), (1 375. 848 4 ±244. 471 8),(1 658.707 1 ± 176. 938 1), (2 353.086 3 ±609. 906 4) μg/ml as compared with(666. 818 8 ± 141. 330 2) μg/ml of DNA concentration of 0 μg/L NGF respectively, qvalue was 3.63,8.20,11.47,19.46, P value was 0. 024,0. 000,0. 000,0. 000 (q-test).Conclusion: The data suggested that NGF could stimulate the proliferation and DNAsynthesis of cultured of hRPE cells in vitro in a dose-dependent manner.

Autophagy: a new mechanism for regulating VEGF and PEDF expression in retinal pigment epithelium cells

AIM: To investigate the regulation of vascular endothelial growth factors(VEGF) and pigment epithelium-derived factor(PEDF) expression by autophagy in retinal pigment epithelium(RPE) cells on exposure to hypoxia. METHODS: ARPE-19, an RPE cell line, was treated as following: the control group was kept in a normoxic incubator; the hypoxia group was incubated in a hypoxic incubator with 1% O_2/5% CO_2/94% N_2 for 24 h; the hypoxia + 3-methyladenine(3-MA) group was pretreated with 10 mmol/L 3-MA for 1 h and then in the hypoxic incubator for 24 h; and the hypoxia + chloroquine(CQ) group was pretreated with 50 μmol/L CQ for 1 h and then in the hypoxic incubator for 24 h. The morphology and ultrastructure of the cells was observed by an inverted microscope or a transmission electronic microscope(TEM). Western blot was performed to assay the expression of autophagy-associated markers, including microtubule associated protein 1 light chain 3 B(LC3 B), Beclin-1, Atg5 and p62. The concentration of VEGF and PEDF in the culture supernatant was determined by ELISA, and the ratio of VEGF/PEDF was calculated. RESULTS: There were no obvious differences in cell morphology among different groups and autolysosomes could be observed in the cytoplasm in all groups. Compared to the control cells, the LC3 B-II/I ratio and levels of Beclin-1 and Atg5 were significantly increased and p62 level was significantly decreased in the hypoxia group. With the increase of VEGF and decrease of PEDF concentration, the VEGF/PEDF ratio was significantly increased in the hypoxia group compared to the control cells. The LC3 B-II/I ratio was significantly reduced by 3-MA treatment and increased by CQ treatment. The expressions of Beclin-1 and Atg5 were significantly reduced by 3-MA or CQ treatment, while expression of p62 was increased in the 3-MA or CQ treated cells. The concentration of VEGF was significantly decreased and PEDF increased, thereby the VEGF/PEDF ratio was decreased in the hypoxia + 3-MA group and hypoxia + CQ group compared with that in the hypoxia group. CONCLUSION: Hypoxia leads to elevated autophagy in RPE cells, and expression of VEGF and PEDF might be regulated by autophagy on exposure to hypoxia to further participate in regulating the formation of retinal neovascularization.

The effects of anti-vascular endothelial growth factor agents on human retinal pigment epithelial cells under high glucose conditions

AIM： To investigate the effects of high glucose levels and anti-vascular endothelial growth factor（VEGF） agents（bevacizumab,ranibizumab and aflibercept） on retinal pigment epithelium（RPE） cells.METHODS： ARPE-19 cells were cultured at different glucose levels（5.5 mmol/L,25 mmol/L,and 75 mmol/L）.Cell viability was evaluated by MTT assay at 3d after treatment with D-glucose.Cell migration ability was measured by wound healing assay at 3d.A cell death detection kit was used to assess apoptosis at 3 and 14 d.Cell proliferation was assessed by EdU assay at 3d.The culture medium was treated with anti-VEGF agents at clinically relevant concentrations.The experiment was then repeated at a different glucose level.RESULTS： The viability and migration of ARPE-19 cells were significantly decreased in the presence of 75 mmol/L as compared to 5.5 mmol/L glucose.The percentage of TUNEL-positive cells was significantly increased and the proliferative potential was decreased with 75 mmol/L compared to 5.5 mmol/L glucose.There were no significant differences in the results between 25 mmol/L and 5.5 mmol/L glucose.In the presence of 75 mmol/L glucose,the groups treated with anti-VEGF showed decreased cell viability and proliferation and increased apoptosis.However,there were no significant differences between the anti-VEGF groups.CONCLUSION： High glucose level decreases the viability,wound healing ability,and proliferation of RPE cells,while increasing apoptosis.Furthermore,anti-VEGF agents interfered with the physiological functions of RPE cells under high-glucose conditions,accompanied by decreases in cell viability and proliferation.

Small Hairpin Loop RNA Targeting HIF-1α Down-regulates VEGF and Up-regulates PEDF in Human Retinal Pigment Epithelial Cells under Hypoxic Condition

The aim of this study was to explore the effect of small hairpin loop RNA （shRNA） silencing hypoxia-induced factor 1α （HIF-1α） gene on the expression of vascular endothelial growth factor （VEGF） and pigment epithelium derived factor （PEDF） in human retinal pigment epithelium （RPE） cells under hypoxic condition. Two target sites of HIF-1α mRNA were chosen and two kinds of shRNA were designed and synthesized against the target sites. Then the two kinds of shRNA were transfected into human RPE cells in vitro, respectively. These cells were cultured under hypoxic condition that was simulated by using 150 μmol/L CoCl2. The mRNA expressions of HIF-1α, VEGF and PEDF were tested by semi-quantitative reverse transcription PCR （RT-PCR）. The protein levels of HIF-1α, VEGF and PEDF were analyzed by Western blotting. After the two kinds of HIF-1α-specific shRNA were transfected into RPE cells respectively, the expression of HIF-1α mRNA and the levels of HIF-1α protein were decreased significantly in RPE cells under hypoxic condition. The expression of VEGF mRNA and the levels of protein significantly were also decreased. However, the levels of PEDF protein was significantly increased, but the expression of PEDF mRNA showed no significant changes. In conclusion, HIF-1α-specific shRNA can effectively silence the HIF-1α gene, and consequently down-regulate VEGF and up-regulate PEDF expression against hypoxia. These results reveal that HIF-1 is associated with posttranslational mechanism for down-regulating PEDF under hypoxia and provide an explanation for hypoxia-provoked increases in VEGF/PEDF ratios. These results also suggest that HIF-1 is one of the key cytokines to retinal neovascularization.

Inhibitory Effect of Dexamethasone on TGF-β1 Expression of Rabbit Ciliary Pigment Epithelia Cultured in Vitro

In order to elucidate the effect of dexamethasone on the expression of transforming growth factor-betal （TGF-β1） in ciliary pigment epithelial （CPE） cells cultured in vitro, rabbit CPE cells were cultured in vitro, treated with DMEM medium containing 0, 1 × 10^-8 , 5 × 10^-8 , 10 × 10^-8 and 50 × 10^-8 mol/L dexamethasone respectively for 5 days. The TGF-131 expression was detected by immunohistochemistry Supervision methods and analyzed semi-quantitatively by HMIAS-2000 image system. As opposed to in vivo, rabbit CPE cells expressed TGF-131 under cul- tured circumstance in vitro. The gray scales of the positive yellow staining in the groups of 1 × 10^-8 , 5 × 10^-8 , 10 × 10^-8 and 50 × 10^-8 mol/L dexamethasone were 136.57 ± 4.43, 140.20 ± 6. 10, 142. 98± 2. 99, 146.80±1.68 and 150.05 × 1.94 respectively. When the concentrations of dexamethasone were equal to or higher than 5 × 10^-8mol/L and, the expression of TGF-β1 was inhibited. 10^-7 mol/L dexamethasone showed a significant inhibition. It was suggested that CPE cells possess the potential ability of synthesizing and expressing TGF-β1. The inhibition of TGF-β1 expression by dexamethasone may be beneficial to the treatment of proliferative vitroretinopathy, also exert some influence on the secretion of aqueous humor and ciliary inflammation.

The Effect of Zinc on the Apoptosis of Cultured Human Retinal Pigment Epithelial Cells

To clarify the effects of zinc on the proliferation and apoptosis of cultured human retinal pigment epithelia (RPE) and the expression of caspase 3 in RPE cells. The effect of Zinc on theproliferation of RPE were examined with MTT method. TUNEL method was used to detect the apoptosis of RPE cells. Caspase 3 was detected by immunohistochemistry. A concentration of zinc higher than 0.001 μM could inhibit the proliferation of RPE. And the relationship between concentration of zinc higher than 10 μM and growth prohibition rate of RPE cells was dose dependent. All concentrations of zinc including 0.001 μM enhanced the expression of caspase 3 of RPE. But only the concentration of zinc higher than 0.01 μM could induce apoptosis of RPE. It is concluded that zinc could enhance the expression of caspase 3 of RPE cells and induce apoptosis of RPE cells. Caution should be taken when using zinc supplements for the treatment of ARMD patients without deficiency of zinc.

红景天苷预处理对高糖诱导的ARPE-19细胞上皮-间充质转化的作用与机制研究

目的探讨红景天苷(SAL)对高糖诱导的ARPE-19细胞的上皮-间充质转化的作用及机制。方法采用MTT法筛选刺激ARPE-19细胞增殖的合适的葡萄糖浓度,确定为50 mmol·L^(-1)。将ARPE-19细胞分为正常对照组(Control组)(5 mmol·L^(-1)的D-葡萄糖和45 mmol·L^(-1)甘露醇培养)、高糖组(HG组)(50 mmol·L^(-1)葡萄糖培养)、HG+低SAL组、HG+中SAL组、HG+高SAL组,低、中、高SAL浓度分别为20μmol·L^(-1)、80μmol·L^(-1)、320μmol·L^(-1),SAL预处理4 h后,加50 mmol·L^(-1)葡萄糖,作用48 h。采用MTT法观察细胞增殖率;划痕实验观察细胞迁移活性;免疫荧光检测细胞α平滑肌肌动蛋白(α-SMA)、波形蛋白(Vimentin)的表达;Western blot法检测细胞中α-SMA、Vimentin、纤维粘连素(FN)、Ⅰ型胶原蛋白(Col I)、钙黏附蛋白E(E-Cadherin)、转化生长因子-β1(TGF-β1)、p-Smad2及p-Smad3蛋白的表达水平。结果与Control组相比,HG组ARPE-19细胞增殖率与伤口愈合百分比均增加,细胞内a-SMA、Vimentin、FN、Col I蛋白相对表达量均增加,E-cadherin蛋白相对表达量降低,细胞内TGF-β1、p-Smad2及p-Smad3蛋白相对表达量均增加,差异均有统计学意义(均为P<0.01);与HG组相比,HG+低SAL组、HG+中SAL组及HG+高SAL组ARPE-19细胞增殖率与伤口愈合百分比均降低,细胞内a-SMA、Vimentin、FN、Col I、TGF-β1、p-Smad2及p-Smad3蛋白相对表达量均呈浓度依赖性降低,E-cadherin蛋白相对表达量呈浓度依赖性增加,差异均有统计学意义(均为P<0.05)。结论SAL能够降低高糖诱导的ARPE-19细胞的增殖、迁移能力,以及干预其上皮-间充质转化进程,这可能与SAL抑制了ARPE-19细胞的TGF-β/Smads信号通路有关。

视网膜下抽液联合玻璃体内注射康柏西普和气体治疗息肉样脉络膜血管病变并发浆液性视网膜色素上皮脱离的疗效和安全性

目的探讨视网膜下抽液联合玻璃体内注射康柏西普和气体治疗息肉样脉络膜血管病变(PCV)并发浆液性视网膜色素上皮脱离(sPED)的疗效和安全性。方法选取2019年7月至2021年2月于潍坊眼科医院行视网膜下抽液联合玻璃体内注射康柏西普和气体治疗的PCV并发sPED患者共13例(13眼),所有患眼术前接受过至少3次(每月1次)的玻璃体内抗血管内皮生长因子(VEGF)(雷珠单抗)注射治疗,且治疗无效。检测术前和术后1周、1个月、3个月、6个月患者的最佳矫正视力(BCVA)、中央视网膜厚度(CRT)、黄斑中心凹PED高度及宽度变化,记录术中及术后并发症发生情况。结果术后1周患眼BCVA较术前好转,差异有统计学意义(Z=-3.237,P=0.001);术后1周、1个月、3个月、6个月患眼CRT均较术前变薄,差异均有统计学意义(Z=-3.180、-3.180、-3.110、-3.180,P=0.001、0.001、0.002、0.001);术后1周、1个月、3个月、6个月患眼PED高度和宽度均较术前下降,差异均有统计学意义(均为P<0.05)。术前13眼平均接受过(4.15±1.40)次玻璃体内注药(雷珠单抗)治疗,治疗时间为(5.92±3.95)个月(相当于每6周注射1次)。术后随访6个月内13眼平均接受了(2.31±1.97)次玻璃体内重复注射(康柏西普)治疗(相当于每10周注射1次)。偏相关分析结果显示,术后6个月时患眼BCVA提高量与CRT的降低量呈弱正相关(r=0.416,P=0.203);术后6个月时患眼BCVA提高量与PED高度和宽度的变化量无明显相关性(r=0.218、0.209,P=0.520、0.538)。术后1个月时,9眼出现PED复发或不同程度的视网膜神经上皮下积液,后期配合玻璃体内重复注射康柏西普治疗,PED均改善。术后6个月时,3眼黄斑中心凹下PED完全消失,视网膜解剖完全复位,1眼黄斑区视网膜仍存在活动性渗出。13眼术后随访期间均未出现全身及严重眼部并发症。结论视网膜下抽液联合玻璃体内注射康柏西普和气体治疗PCV并发sPED能够安全有效地降低患者CRT和改善PED,减少PED长期存在对视网膜的损害,但对术后6个月时患者BCVA的提高无明显帮助。

Predictive Value of Serum Vascular Endothelial Growth Factor and Pigment Epithelium-derived Factor in Ovarian Hyperstimulation Syndrome

Objective To explore whether the serum concentrations of vascular endothelial growth factor （VEGF） and pigment epithelium-derived factor （PEDF） could serve as the predictors of ovarian hyperstimulation syndrome （OHSS） in patients undergoing in vitro fertilization-embryo transfer （IVF-ET）. Methods Enzyme-linked immunoadsordent assay （ELISA） was employed to measure the serum concentrations of VEGF and PEDF on the day of hCG administration, oocyte retrieval and embryo transfer, respectively. Based on OHSS classification of the criteria of Golan, 85 patients were divided into three groups. Patients in group A （n=10） showed symptoms of severe OHSS and patients in group B （n=13） suffered from moderate OHSS. The control group （group C, n=62） contained patients without symptoms of OHSS as well as patients with mild OHSS.Results In groups A, B and C, serum concentrations of PEDF on the day of hCG administration （h-PEDF）（166.54 ± 102.81 pg/ml, 159.45 ±136. 77 pg/ml, 172.05±170.95 pg/ml, P=0.48）, oocyte retrieval （o-PEDF）（176.91 ± 103.37 pg/ml, 122.52± 92.54 pg/ml, 179.82±177.47 pg/ml, P=0.27） and embryo transfer （e-PEDF）（169.02± 240.08 pg/ml, 136.80 ±139.21pg/ml, 157.38 ±222.54 pg/ml, P=0.95）, h-VEGF （175.55 ± 103.54 pg/ml, 218.84 ±179.70pg/ml, 153.39±145.06 pg/ml, P=0.36） and o- VEGF （171.93 ± 128.55 pg/ml, 220.36±149.82 pg/ml, 138. 74 ±% 139.30 pg/ml, P=0. 15） showed no significant differences. There was a statistical difference in serum concentration of e-VEGF between group A （197.04±156.63 pg/ml） and group C （110.69±49.55 pg/ml）（P=0.008）. The serum level of estradiol showed a positive correlation with the count of large follicles （r=0. 744）. The ratios of h-VEGF/h-PEDF, o-VEGF/o-PEDF and e-VEGF/e-PEDF were calculated and showed a clear difference among groups A, B and C （4.04±3.39, 2.10±2.14, 1.05± 4.80, P〈0.001; 4.54 5.69, 2.29 ±1.67, 0.94 ±0.59, P〈0.001; 5.43±6.16, 1.81±1.36, 2.42±2.60, P=0.04）. Conclusion While neither serum concentrations of VEGF nor PEDF can be used as an OHSS predictor, the ratios of h-VEGF/h-PEDF, o-VEGF/o-PEDF and e-VEGF/e-PEDF may have great predictive value.

血管内皮生长因子与色素上皮衍生因子在糖尿病猕猴视网膜病变早变早期的表达

目的检测糖尿病视网膜病变早期猕猴视网膜中血管内皮生长因子(VEGF)及色素上皮衍生因子(PEDF)的表达,探讨其意义。方法通过空腹血糖值糖化血红蛋白水平、眼底彩照情况及糖尿病病程,确定入选3只利用高脂饲料诱导成功且处于糖尿病视网膜病变早期的猕猴及3只年龄相当的健康猕猴。行实时荧光定量PCR及免疫组织化学检测,观察猕猴视网膜内VEGF和PEDF表达情况。结果处于糖尿病视网膜病变早期的猕猴视网膜内VEGF在mRNA表达水平(P<0.05)及蛋白表达水平(P<0.05)均明显高于对照组猕猴,糖尿病组猕猴视网膜内PEDF mRNA表达量相比对照组显著减少(P<0.01),PEDF蛋白表达与其mRNA表达趋势一致(P<0.05)。结论猕猴糖尿病视网膜早期,出现VEGF的上调和PEDF的下调,对糖尿病视网膜病变的发生具有一定的提示意义,可为早期糖尿病视网膜病变提供辅助诊断。

血清Cys C、PEDF、VEGF检测在糖尿病肾病诊断及治疗中的意义

目的了解糖尿病肾病(DN)患者血清胱抑素C(Cys C)、色素上皮衍生因子(PEDF)、血管内皮生长因子(VEGF)水平及缬沙坦联合阿魏酸钠干预治疗的效果。方法选择153例糖尿病患者作为糖尿病组,根据尿清蛋白与肌酐的比率(ACR)将其分为正常蛋白尿组、微量蛋白尿组和临床蛋白尿组,并选取30例同期门诊健康体检者作为对照组。微量蛋白尿组及临床蛋白尿组患者分别随机分为缬沙坦治疗组和缬沙坦联合阿魏酸钠治疗组,收集受试对象治疗前、后的血清及尿液标本。采用酶联免疫吸附测定(ELISA)法检测血清PEDF、VEGF水平,同时检测空腹血糖、糖化血红蛋白(GHbA1c)、血肌酐、尿素、Cys C。结果与健康对照组相比,正常蛋白尿组、微量蛋白尿组和临床蛋白尿组患者血清Cys C、PEDF、VEGF水平显著升高(P<0.05),并随DN的进展而逐渐升高。缬沙坦联合阿魏酸钠治疗可显著减少蛋白尿,并降低患者血清Cys C、PEDF、VEGF水平(P<0.05),提示缬沙坦联合阿魏酸钠可能通过改善糖尿病肾脏异常血管新生发挥肾脏保护效应,且联合用药效果优于缬沙坦单药治疗。结论 DN患者血清Cys C、PEDF、VEGF高表达,缬沙坦联合阿魏酸钠治疗效果显著。

笃斯越橘花色苷提取物对光损伤人视网膜色素上皮细胞的保护作用

目的:通过建立人视网膜色素上皮细胞(hRPE)光损伤模型评价笃斯越橘花色苷对眼睛的保护作用及机理。方法:光损伤设置光强、光照时间、后续培养时间3个影响因素,以细胞存活率和乳酸脱氢酶活性评价模型建立情况;根据越橘花色苷提取物的干预方式分为预防组、应激组和修复组,每组设高、中、低3个浓度,检测细胞存活率和血管内皮生长因子(VEGF)水平评价保护效果。结果:选择光照度2500lx,光照24h后培养24h作为最佳造模条件,细胞损伤率达20%;预防组和应激组能有效保护细胞活力(P<0.01),修复组没有效果,各组都能抑制由光损伤诱导hRPE细胞VEGF的过表达,但是不同浓度之间抑制能力有差异。结论:hRPE细胞光损伤程度呈光照强度和光照时间依赖性;越橘花色苷能通过保护hRPE的细胞活力和VEGF的过表达,实现保护眼科健康、预防眼科疾病的作用。

Avastin玻璃体腔注射后糖尿病视网膜病变增生膜中细胞因子的变化

背景增生型糖尿病视网膜病变（PDR）导致患者视力下降的主要原因是纤维血管膜的收缩、牵拉引起的玻璃体积血及视网膜脱离，因此研究视网膜增生膜形成的病理机制、抑制新生血管和纤维血管膜的形成及其进展是治疗PDR的重要研究方向之一。目的研究玻璃体腔注射avastin后PDR增生膜中血管内皮生长因子（VEGF）、结缔组织生长因子（CTGF）及色素上皮衍生因子（PEDF）表达的变化。方法采用前瞻性随机对照研究设计。选择2008年1—6月在天津医科大学眼科医院拟行手术治疗的PDR患者24例26眼，用随机数字表法将患者随机分为单纯玻璃体切割术组和avastin注射＋玻璃体切割术组，每组12例13眼，两组间性别、年龄及病程等基线资料均匹配。Avastin注射＋玻璃体切割术组患者玻璃体腔注射avastin 1．25mg（0．05m1），10d后行玻璃体切割术；单纯玻璃体切割术组患者仅行玻璃体切割术。术中收集术眼视网膜增生膜，采用免疫组织化学法检测增生膜中VEGF、CTGF和PEDF的表达。结果2个组患眼视网膜增生膜中VEGF、CTGF和PEDF的表达均位于细胞质。与单纯玻璃体切割术组比较，avastin注射＋玻璃体切割术组患者视网膜增生膜中VEGF阳性表达率明显下降（30．77％VS．100．00％），CTGF阳性表达率上升（92．31％VS．62．54％），差异均有统计学意义（U=4．000，P〈0．01；U=7．500，P=0．048），而avastin注射＋玻璃体切割术组与单纯玻璃体切割术组间PEDF的阳性表达率差异无统计学意义（100．00％VS．92．31％，U=65．500，P=0．299）。结论PDR患者玻璃体腔注射avastin后视网膜增生膜中VEGF的表达率明显降低，CTGF的表达率明显升高，而PEDF的表达率无明显变化。

金雀异黄素对氯化钴诱导的兔视网膜色素上皮细胞血管内皮生长因子表达的影响

目的:研究氯化钴对体外培养的兔视网膜色素上皮(RPE)细胞血管内皮生长因子(VEGF)表达的诱导作用,以及金雀异黄素(genistein,Gen)对其表达的影响。方法:采用免疫荧光组织化学技术和激光共聚焦显微镜,以荧光比值来表示VEGF的表达,半定量分析氯化钴处理不同时间对体外培养的RPE细胞VEGF蛋白表达的诱导作用,以及Gen对低氧或氯化钴诱导的VEGF表达的影响。结果:对照组有低水平VEGF蛋白的表达,随着低氧或氯化钴处理时间的延长,荧光比值的变化表现为时间依赖性,在6 h点达到高峰。Gen(50、100、200μmol/L组)与二甲基亚砜(DMSO)对照组相比,均可明显抑制氯化钴诱导的VEGF的表达,且此种抑制作用呈浓度依赖性。结论:Gen可抑制氯化钴诱导的RPE细胞VEGF表达,提示Gen对视网膜新生血管的形成具有潜在的预防及治疗价值。

PKC信号传导通路对缺氧人视网膜色素上皮细胞表达VEGF的作用

目的 :探讨蛋白激酶C(PKC)信号传导通路对缺氧状态下培养的视网膜色素上皮 (retinalpigmentepithelium ,RPE)细胞表达VEGF的作用 .方法 :在缺氧状态下分别培养人RPE细胞 6 ,1 2和 2 4h ;将不同浓度的PKC激动剂佛波醇 1 2 豆蔻酰 1 3乙酸 (phorbol 1 2 myristate 1 3 acetate,PMA)及PKC抑制剂Chelerythrine分别加入人RPE细胞培养液 ,在缺氧状态下培养 2 4h .用免疫组化方法检测各组RPE细胞中VEGF的表达 ,经计算机图像处理 ,定量分析 .结果 :在缺氧状态下 ,RPE细胞对VEGF的表达较非缺氧组RPE细胞显著增加 (P <0 .0 1 ) ;较对照组 ,PMA可增强缺氧状态下RPE细胞VEGF的表达 (P <0 .0 1 ) ,Chelerythrine则减弱VEGF的表达 (P <0 .0 1 ) .结论 :缺氧诱导VEGF在RPE细胞中的表达 。

糖尿病大鼠视网膜中VEGF、PEDF的表达与血-视网膜屏障损伤

目的观察糖尿病(diabetes mellitus,DM)大鼠视网膜中血管内皮生长因子(vascular endothelial growth factor,VEGF)和色素上皮源性因子(pigment epithelium-derived factor,PEDF)的表达及早期血-视网膜屏障的改变,探讨二者之间的关系。方法选取87只健康SD大鼠,应用链-脲佐菌素建立DM大鼠模型,随机分为正常对照组(N)、DM0.5个月组、DM1个月组、DM3个月组及DM5个月组。分别行RT-PCR及Western blot检测大鼠视网膜VEGF、PEDF的mENA及蛋白表达,应用伊文思蓝法及透射电镜法观察血-视网膜屏障损伤情况。结果正常大鼠视网膜有VEGF的mRNA及蛋白表达,DM0.5个月组表达开始增高,光密度值分别为0.5700±0.1580和0.5770±0.0279(均为P<0.05),随病程延长,表达量逐渐增高,至DM5个月组VEGF表达量约为正常对照组的2倍,分别为0.9560±0.0560和0.9790±0.0790(均为P<0.05)。PEDF mRNA表达于正常大鼠视网膜,DM1个月组表达开始减少,光密度值为0.3440±0.0437,与正常对照组比较差异有统计学意义(P<0.05),至DM5个月组视网膜表达量减少至0.2360±0.0455,约为正常对照组的1/2(P<0.05)。PEDF蛋白在各组大鼠视网膜的表达与其mRNA表达趋势一致。伊文思蓝法结果显示DM大鼠视网膜血管渗漏较正常大鼠增强,DM0.5个月组鼠即有增高,达(2.6310±0.2725)μL·g-1·h-1(P<0.05),随病程延长,渗漏逐渐增强,至DM5个月组,伊文思蓝渗漏量达(10.0260±1.1121)μL·g-1·h-1,约为正常对照组的6倍(P<0.05)。同时DM1个月时血-视网膜屏障超微结构发生改变,且呈逐渐加重趋势。结论早期DM大鼠血-视网膜屏障损伤,VEGF的正性表达增多与PEDF的负性表达减少可能对此病理改变起一定的促进作用。

体内实验观察bFGF对视网膜色素上皮细胞再生的促进作用

目的 观察重组人碱性成纤维细胞生长因子 ( basic fibroblast growth factor,b FGF)在体内对兔视网膜色素上皮 ( retinal pigm ent epithelium,RPE)细胞再生的作用。方法 应用氩绿激光照射 ,造成兔 RPE损伤模型 ,照射后应用 b FGF 3 0 0 0 U /5 0μL玻璃体腔内注射 ,眼底荧光素血管造影检查 ,测定照射后 1～ 4d透见荧光面积和荧光素渗漏面积 ,并与对照眼进行比较。结果 照射后第 1天和第 2天 ,2组眼的透见荧光面积和荧光素渗漏面积无显著差异 ( P=0 .5 34,0 .2 83) ,第 3天和第 4天 ,2组眼的透见荧光面积和荧光素渗漏面积有显著差异 ( P=0 .0 180 ,0 .0 0 0 7)。结论 体内外源性 b FGF能促进 RPE的再生和修复 ,b FGF不具有种属特异性。