Potential therapeutic effects of pigment epithelium-derived factor for treatment of diabetic retinopathy

Diabetic retinopathy (DR), a major micro-vascular complication of diabetes, has emerged as a leading cause of visual impairment and blindness among working adults in the worldwide. The pathobiology of DR involves multiple molecular pathways and is characterized chronic neurovascular degeneration. Current approaches to prevent or to treat DR are still far from satisfactory. Therefore, it is important to develop new therapeutic strategies for the prevention and treatment to DR. Pigment epithelium-derived factor (PEDF), a 50-kDa secreted glycoprotein, has been described as a multi-functional protein. Some emerging evidences indicate that PEDF are able to target multiple pathways exerting neurotropic, neuroprotective, anti-angiogenic, antivasopermeability, anti-inflammation, anti-thrombogenic and anti-oxidative effects in DR. In this review, we addressed the functions of PEDF in different pathways, which could lead to potential therapeutics on the treatment to DR.

Study of Pigment Epithelium-derived Factor in Pathogenesis of Diabetic Retinopathy

Diabetic retinopathy(DR), a major micro-vascular complication of diabetes, has emerged as a leading cause of visual impairment and blindness among adults worldwide. However,aside from pathological damage, the traditional laser and multi-needle operation treatments required for more advanced disease can cause further damage to the visual field and increase the operation risk. Therefore, the development of new therapeutic strategies for the prevention and treatment of DR is essential..Some emerging evidence now indicates that pigment epithelium-derived factor(PEDF), a multifunctional protein,can target multiple pathways to exert neurotropic,.neuroprotective, anti-angiogenic, anti-vasopermeability, anti-inflammation, anti-thrombogenic, and anti-oxidative effects against DR. This review addresses the functions of PEDF in different pathways that could lead to potential therapeutics for the treatment of DR.

Expression of Pigment Epithelium-derived Factor in Bladder Tumour Is Correlated with Interleukin-8 yet Not with Interleukin-1α

Pigment epithelium-derived factor （PEDF） is an antiangiogenic factor which is effective in tumour inhibition in a variety of tumours and has not yet been studied in bladder tumour before. In this study the expression of PEDF, interleukin-1α （IL-1α） and -8 （IL-8） in bladder tumours was investigated. Immunohistochemistry was performed on 64 bladder tumour and 23 normal uroepithelium samples. Expression change of the factors was compared with clinicopathological parameters. Correlations between PEDF, IL-1α and IL-8 were analyzed. None of the factors was in relation to gender, tumour occurrence, and size or onset pattern. PEDF （P=0.014） and IL-1α （P=0.049） expression was down-regulated with grade progression. PEDF expression was lower in normal uroepithelium than in papillary urothelial neoplasm of low malignant potential （PUNLMP） （P=0.000） and carcinoma （P=0.009） whilst IL-1α （P=0.000 and P=0.000 respectively） and IL-8 （P=0.000 and P=0.023 respectively） expression was higher in the same grouping. PEDF expression had a negative correlation with IL-8 in PUNLMP （P=0.049, r=-0.578） as well as in tumour grouping （P=0.033, r=-0.276）. Deranged expressional change of PEDF, IL-1α and IL-8 could be in relation to loss of differentiation from normal uroepithelium to papillary lesion and eventually to carcinoma.

Pigment Epithelium-derived Factor in Cataractous Aqueous Humor and Lens Epithelial Cells

Purpose: To study the characteristics of PEDF in cataractous aqueous humor and its expression in human lens epithelium. Methods: The PEDF concentration in the aqueous humor was measured by enzyme -linked immunosorbent assay in senile (130cases) and congenital (18cases) cataract patients who underwent cataract phacoemulsification extraction surgery. Anterior lens capsular specimens were obtained from these patients to count lens epithelial cells (LEC) density. The Lens Opacities Classification System Ⅲ was used to classify the senile cataracts as cortical, nuclear, posterior subcapsular and mixed types of opacity, and quantitative analysis of the nuclear opacities was performed by Pentacam Scheimpflug imaging system. Anterior lens capsular specimens from another senile (10cases) and congenital (10cases) cataract were collected for immunofluorescence with polyclonal antibodies specific to human pigment epithelium -derived factor (PEDF). Results:The mean aqueous level of PEDF was(178. 9±87. 5)ng/ml, and there was negative linear correlation of PEDF level and age (r=0. 811, P<0. 001). In senile cases, the aqueous PEDF concentration decreased with increasing nuclear opacities (r=0. 447, P < 0.01) , and the mean PEDF level in nuclear cataract was significantly lower than that in posterior subcapsular opacity (P < 0.01) . PEDF immunostaining was detected in LEC of all capsular specimens. Conclusion : The PEDF level in human aqueous humor is related to age, types of cataracts and lens opacity. PEDF also express in human LEC. The study results suggest PEDF may regulate and/or protect LEC by paracrine and autocrine, and lack of PEDF may play a role in cataractogenesis.

Pigment epithelium-derived factor protects retinal ganglion cells from hypoxia-induced apoptosis by preventing mitochondrial dysfunction

AIM： To investigate the potential of pigment epitheliumderived factor（PEDF） to protect the immortalized rat retinal ganglion cells-5（RGC-5） exposed to Co Cl2-induced chemical hypoxia. METHODS： After being differentiated with staurosporine（SS）, RGC-5 cells were cultured in four conditions： control group cells cultured in Dulbecco ＇s modified eagle medium（DMEM） supplemented with 10% fetal bovine serum, 100 μmol/m L streptomycin and penicillin（named as normal conditions）; hypoxia group cells cultured in DMEM containing 300 μmol/m L Co Cl2; cells in the group protected by PEDF were first pretreated with 100 ng/m L PEDF for 2h and then cultured in the same condition as hypoxia group cells; and PEDF group cells that were cultured in the presence of 100 ng/m L PEDF under normal conditions. The cell viability was assessed by MTT assay, the percentage of apoptotic cells was quantified using Annexin V-FITC apoptosis kit, and intra-cellar reactive oxygen species（ROS） was measured by dichloro-dihydro-fluorescein diacetate（DCFH-DA） probe. The mitochondria-mediated apoptosis was also examined to further study the underlying mechanism of the protective effect of PEDF. The opening of mitochondrial permeability transition pores（m PTPs） and membrane potential（Δψm） were tested as cellular adenosine triphosphate（ATP） level and glutathione（GSH）. Also, the expression and distribution of Cyt C and apoptosis inducing factor（AIF） were observed.RESULTS： SS induced differentiation of RGC-5 cells resulting in elongation of their neurites and establishing contacts between outgrowths. Exposure to 300 μmol/m L Co Cl2 triggered death of 30% of the total cells in cultures within 24 h. At the same time, pretreatment with 100 ng/m L PEDF significantly suppressed the cell death induced by hypoxia（P〈0.05）. The apoptosis induced by treatment of Co Cl2 was that induced cell death accompanied with increasing intracellar ROS and decreasing GSH and ATP level. PEDF pretreatment suppressed these effects（P〈0.05）. Additionally, PEDF treatment inhibited the opening of m PTPs and suppressed decreasing of Δψm in RGC-5 cells, resulting in blocking of the mitochondrial apoptotic pathway.CONCLUSION： Pretreatment of RGC-5 cells with 100 ng/m L PEDF significantly decreases the extent of apoptosis. PEDF inhibits the opening of m PTPs and suppresses decreasing of Δψm. Moreover, PEDF also reduces ROS production and inhibits cellular ATP level＇s reduction. Cyt C and AIF activation in PEDF-pretreated cultures are also reduced. These results demonstrate the potential for PEDF to protect RGCs against hypoxic damage in vitro by preventing mitochondrial dysfunction.

Construction of a plasmid for human brain-derived neurotrophic factor and its effect on retinal pigment epithelial cell viability

Several studies have investigated the protective functions of brain-derived neurotrophic factor（BDNF） in retinitis pigmentosa. However, a BDNF-based therapy for retinitis pigmentosa is not yet available. To develop an efficient treatment for fundus disease, an eukaryotic expression plasmid was generated and used to transfect human 293 T cells to assess the expression and bioactivity of BDNF on acute retinal pigment epithelial-19（ARPE-19） cells, a human retinal epithelial cell line. After 96 hours of co-culture in a Transwell chamber, ARPE-19 cells exposed to BDNF secreted by 293 T cells were more viable than ARPE-19 cells not exposed to secreted BDNF. Western blot assay showed that Bax levels were downregulated and that Bcl-2 levels were upregulated in human ARPE-19 cells exposed to BDNF. Furthermore, 293 T cells transfected with the BDNF gene steadily secreted the protein. The powerful anti-apoptotic function of this BDNF may be useful for the treatment of retinitis pigmentosa and other retinal degenerative diseases.

Pigment epithelium derived factor(PEDF)prevents methyl methacrylate monomer-induced cytotoxicity in H9c2 cells

Acrylic bone cements are currently the most frequently and extensively used materials in orthopedic implant treatment. However, adverse effects have been described of acrylic bone cement on the cardiovascular system. In the present study, we examined the cytotoxicity of bone cement ingredient methyl methacrylate（MMA） to cardiomyocytes and the potential detoxifying effect of pigment epithelium-derived factor（PEDF） in H9c2 cells.We found that high concentration of MM A（〉 120 mmol/L） led to necrotic cell death in H9c2 cells. However, MMA at low concentrations（30-90 mmol/L） caused apoptosis. Pretreatment of PEDF prevented MMA-induced cytotoxicity. In addition, PEDF enhanced total superoxide dismutase activities, and decreased MMA-induced production of malonaldehyde. Furthermore, MMA-induced downregulation of Akt activity was suppressed by PEDF.PEDF also increased the levels of peroxisome proliferator activated receptor gamma（PPARγ）and lysophosphatidic acids（LPA） through PEDF receptor. These results indicated that PEDF inhibited MMA-induced cytotoxicity through attenuating oxidative stress, activating the phosphatidylinositol 3-kinase（PI3K）/Akt pathway and/or PEDF receptorLPA-PPARy pathways in H9c2 cells. PEDF may be explored as a candidate therapeutic agent for alleviating bone cement implantation syndrome during orthopedic surgery.

Ultrasound-mediated microbubble delivery of pigment epithelium-derived factor gene into retina inhibits choroidal neovascularization

Background Many studies have suggested that the imbalance of angiogenic factor and anti-angiogenic factor expression contributes significantly to the development of choroidal neovascularization （CNV）, and ultrasound microbubble combination system can increase the gene transfection efficiency successfully. This study was designed to investigate whether ultrasound-mediated microbubble destruction could effectively deliver therapeutic plasmid into the retina of rat, and whether gene transfer of pigment epithelium-derived factor （PEDF） could inhibit CNV.Methods Human retinal pigment epithelial cells were isolated and treated either with ultrasound or plasmid alone, or with a combination of plasmid, ultrasound and microbubbles to approach feasibility of microbubble-enhanced ultrasound enhance PEDFgene expression; For in vivo animal studies, CNV was induced by argon lasgon laser in rats. These rats were randomly assigned to five groups and were treated by infusing microbubbles attached with the naked plasmid DNA of PEDF into the vitreous of rats followed by immediate ultrasound exposure （intravitreal injection）; infusing liposomes with the naked plasmid DNA of PEDF into the vitreous （lipofectamine ＋ PEDF）; infusing microbubbles attached with PEDF into the orbit of rats with ultrasound irradiation immediately （retrobular injection）; infusing microbubbles attached with PEDF into the femoral vein of rats with exposed to ultrasound immediately （vein injection）. The CNV rats without any treatment served as control. Rats were sacrificed and eyes were enucleated at 7, 14, and 28 days after treatment. Gene and protein expression of PEDF was detected by quantitative real-time RT-PCR, Western blotting and immunofluorescence staining, respectively. The effect of PEDF gene transfer on CNV was examined by fluorescein fundus angiography.Results In vitro cell experiments showed that microbubbles with ultrasound irradiation could significantly enhance PEDF delivery as compared with microbubbles or ultrasound alone. In the rat CNV model, transfection efficiency mediated by ultrasound/microbubbles was significantly higher than that by lipofectamine-mediated gene transfer at 28 days after treatment. The study also showed that with the administration of ultrasound-mediated microbubbles destruction, the CNV of rats was inhibited effectively. Conclusions Ultrasound-microbubble technique could increase PEDF gene transfer into rats＇ retina and chorioid, in association with a significant inhibition of the development of CNV, suggesting that this noninvasive gene transfer method may provide a useful tool for clinical gene therapy.

Anti-inflammatory effects of a synthetic peptide derived from pigment epithelium-derived factor on H2O2-induced corneal injury in vitro

Background The common pathological characteristics of corneal injury include inflammatory factors activation, vascular endothelial cells or inflammatory cells infiltration into lesions, corneal edema, corneal neovascularization （CNV）, and scar formation. PEDF-34 is the functional fragment of pigment epithelium-derived factor （PEDF） that has anti-angiogenic and anti-inflammatory properties and contains an N-terminal 34-amino acid peptide. This study was to investigate the anti- inflammatory effects of PEDF-34 on H202-induced corneal injury in vitro. Methods After cultured in H202 （0.1 mmol/L） for 2 hours, human corneal fibroblasts （HCFs） and human umbilical vein endothelial cells （HUVECs） were treated with PEDF-34-nanoparticles （NPs） at different concentrations （0.1, 0.5, 1.0, 2.0 μg/ml） or 2.0 μg/ml controI-NPs for 24 hours. The viable cells were quantified using the MTT assay. Western blotting or ELISA analysis was performed for measuring the human vascular endothelial growth factor （VEGF） and intercellular adhesion molecule-1 （ICAM-1） expression of both HCFs and HUVECs. VEGF and nuclear factor KB （NF-KB） mRNA levels of HCFs were semi-quantified by RT-PCR. Results The survival rates of HCFs or HUVECs stimulated by H202 did not decrease significantly （P 〉0.05） compared to those in the normal conditions. As compared to controI-NP group, PEDF-34-NPs had dose-dependent inhibitive effect on HUVECs with the MTT assay, but not HCFs. Western blotting analysis showed that the VEGF and ICAM-1 levels in the HCFs and HUVECs stimulated by H202 were significantly higher than those in the normal conditions, which were decreased dramatically in those treated with PEDF-34-NPs. RT-PCR analysis revealed that the VEGF mRNA and NF-KB mRNA levels increased in H202-stimulated HCFs, while both of them decreased in PEDF-34-NP groups dose dependently. Conclusions PEDF-34-NPs may play an important role in regulating the NF-kB pathway, inhibiting inflammatory activity. PEDF-34-NPs may be a potential new drug for treating corneal injury in the future.

Role of pigment epithelium-derived factor on proliferation and migration of choroidal capillary endothelium induced by vascular endothelial growth factor in vitro

Background Pigment epithelium-derived factor （PEDF） is expressed in several normal organs and identified as an inhibitor of neovascularization. In the present study, we investigated the effect of PEDF in an in vitro model of ocular choroidal neovascularization. Methods Microdissection was used to isolate the human choroidal endothelial cells （CECs）, followed by the use of superparamagnetic beads （Dynabeads） coated with the CD31 antibody, which selectively binds to the endothelial cell surface. The mitogenic and motogenic effects of vascular endothelial growth factor （VEGF） on cultured choroidal capillary endothelial cells were examined in the presence or absence of PEDF （1, 10, 100, and 1000 ng/ml） using cell counts and migration assays. Results Cells bound to the beads were isolated using a magnetic particle concentrator and they were successfully cultured and characterized to be endothelial cells that possessed greater than 95% immunoreactivity to von Willebrand factor. PEDF suppressed the proliferation and migration of VEGF-induced choroidal capillary endothelial cells. However, the concentration of PEDF which we used has little effect on normal CECs. Conclusions PEDF played an important role on the growth and migration of VEGF-stimulated choroidal endothelial cell These findings suggest that PEDF may be an effective approach to the treatment of choroidal neovascular disorders.

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.

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.

色素上皮衍生因子在结直肠癌组织中的表达

目的探讨结直肠癌组织中色素上皮衍生因子(PEDF)的表达及其与临床病理参数及预后的关系。方法采用实时荧光定量PCR和免疫组化染色技术检测结直肠癌、淋巴结转移癌和正常肠黏膜组织中PEDF mRNA和蛋白的表达,分析PEDF的表达与临床病理参数及预后的关系。结果结直肠癌组织PEDF mRNA的相对表达量为0.115±0.023,淋巴结转移癌组织为0.201±0.063,正常肠黏膜组织为0.821±0.076(F=9.324,P=0.025),而且结直肠癌组织和淋巴结转移癌组织中PEDF mRNA相对表达量均低于正常肠黏膜组织,结直肠癌组织PEDF mRNA相对表达量低于淋巴结转移癌组织(P均<0.001)。结直肠癌患者中,PEDF蛋白低表达组患者占比为68.33%(82/120),高表达组患者占比为31.67%(38/120)。结直肠癌组织中PEDF蛋白表达与TNM分期、淋巴结转移有关(χ^(2)=57.449,P<0.001;χ^(2)=50.612,P<0.001)。PEDF低表达组患者中位生存期为30.82个月,低于高表达患者的46.89个月(Log-rankχ^(2)=18.140,P<0.001)。结论PEDF表达与结直肠癌患者的TNM分期和淋巴结转移密切相关,结直肠癌组织中PEDF低表达为结直肠癌患者预后差的风险因素。

人参皂苷Rg1对缺氧诱导所致人视网膜色素上皮细胞损伤的抑制作用及机制

目的探究人参皂苷Rg1(GRg1)对缺氧诱导所致人视网膜色素上皮(ARPE)细胞损伤的作用及机制。方法体外培养ARPE-19细胞,将其随机分为对照组、模型组及GRg1高、中、低剂量组与缺氧诱导因子1α(HIF-1α)抑制剂组。除对照组外,其余组采取氯化钴诱导ARPE-19细胞构建缺氧模型,通过CCK-8法、TUNEL法测定各组不同时间点细胞活性及凋亡情况,DCFH-DA荧光探针对各组活性氧(ROS)水平,经由RT-qPCR、Western blotting法测定各组细胞内HIF-1α、BDNF、PACAP38 mRNA与蛋白表达。结果与对照组相比,各组给药0、12、24 h时ARPE-19细胞活性均低、凋亡率高(P均<0.05);GRg1高剂量组给药12、24、48 h时ARPE-19细胞活性均高于模型组、GRg1低剂量组(P均<0.05),细胞凋亡率均低于模型组、GRg1低剂量组(P均<0.05);GRg1高剂量组给药24、48 h时细胞活性均高于GRg1中剂量组(P均<0.05)。与对照组相比,各组给药48 h时ARPE-19细胞ROS、HIF-1αmRNA及蛋白表达均高(P均<0.05);GRg1高剂量组ARPE-19细胞ROSHIF-1αmRNA及蛋白表达均低于模型组、GRg1低剂量组(P均<0.05);GRg1高剂量组ARPE-19细胞BDNF、PACAP38 mRNA及蛋白表达均高于模型组、GRg1低剂量组(P均<0.05);GRg1高剂量组与HIF-1α抑制剂组给药不同时间点细胞活性与凋亡率、细胞ROS、HIF-1α、BDNF、PACAP38表达比较差异均无统计学意义(P均>0.05)。结论GRg1能减轻缺氧诱导所致ARPE-19细胞损伤,其机制可能与抑制细胞ROS、HIF-1α表达,上调BDNF、PACAP38表达有关。

THP1细胞过表达腺病毒介导的PEDF抑制炎症过程中关键基因的鉴别

目的鉴别人单核细胞白血病细胞THP1过表达腺病毒介导的色素上皮衍生因子(PEDF)抑制炎症过程中的关键基因。方法对THP1细胞过表达腺病毒介导的PEDF进行蛋白质组学分析。将THP1细胞分为GFP组和PEDF组,分别用GFP腺病毒和PEDF腺病毒感染细胞;将THP1细胞分为甘露醇组、高糖组、高糖+GFP组和高糖+PEDF组,分别用D-甘露醇、D-无水葡萄糖、GFP腺病毒和PEDF腺病毒培养4、4、3和3 d;将Pedf^(-/-)小鼠采用随机数表法分为Pedf^(-/-)组和Pedf^(-/-)糖尿病组,每组12只,另取10只C57BL/6小鼠为对照组,取小鼠视网膜进行实验。采用实时荧光定量PCR验证差异表达基因(DEGs)的mRNA在视网膜组织和THP1细胞系中的表达水平。与GSE5504数据集进行DEGs交集,使用String数据库构建蛋白-蛋白相互作用(PPI)网络,Cytoscape软件及MCODE应用程序提取PPI网络模块,与Set1数据集取交集并找到关键基因。采用Western blot在THP1细胞和Pedf^(-/-)小鼠中验证关键基因的表达水平。结果通过蛋白质组学和生物信息学分析,筛选出Set1数据集中的105个差异蛋白。实时荧光定量PCR结果显示,PEDF组细胞中ARF5、TCF25和KCTD9 mRNA相对表达量明显高于GFP组,RNPS1、CSF1R、OGA、IBA57和MGST2 mRNA相对表达量明显低于GFP组,差异均有统计学意义(均P<0.001)。对照组、Pedf^(-/-)组和Pedf^(-/-)糖尿病组视网膜组织中表达显著下调的TCF25、KCTD9、ARF5 mRNA和表达显著上调的CSF1R、RNPS1、IBA57 mRNA相对表达量总体比较,差异均有统计学意义(F=64.057、27.561、37.179、65.757、44.024、34.248,均P<0.001);与对照组相比,Pedf^(-/-)组TCF25、KCTD9、ARF5 mRNA相对表达量降低,CSF1R、RNPS1 mRNA相对表达量升高,差异均有统计学意义(均P<0.05);Pedf^(-/-)糖尿病组TCF25、KCTD9、ARF5 mRNA相对表达量降低,CSF1R、RNPS1、IBA57 mRNA相对表达量升高,差异均有统计学意义(均P<0.05)。与Pedf^(-/-)组相比,Pedf^(-/-)糖尿病组TCF25 mRNA相对表达量降低,CSF1R、RNPS1和IBA57 mRNA相对表达量升高,差异均有统计学意义(均P<0.05)。Set1数据集与GSE5504数据集交集后得到20个差异蛋白,主要富集于基因表达的正向调控、ERK1和ERK2级联的正向调节、胰岛素分泌的正向调节参与细胞对葡萄糖刺激的反应和抗原加工与递呈通路上。通过构建PPI网络和Cytoscape软件中MCODE插件筛选出关键基因CSF 1 R。Western blot结果显示,高糖组和高糖+GFP组中细胞CSF1R相对表达量分别为1.961±0.085和1.000±0.069,分别高于甘露醇组的1.000±0.072和高糖+PEDF组的0.469±0.079,差异均有统计学意义(t=14.940、8.765,均P<0.01);Pedf^(-/-)糖尿病组中视网膜CSF1R相对表达量为1.633±0.192,高于Pedf^(-/-)组的1.000±0.050,差异有统计学意义(t=5.537,P<0.01)。结论CSF 1 R可能为THP1细胞过表达腺病毒介导的PEDF抑制炎症过程中的关键基因和治疗靶点。

康柏西普联合全视网膜激光光凝术治疗增殖性糖尿病视网膜病变的疗效及对血清VEGF及PEDF水平的影响

目的研究康柏西普联合全视网膜激光光凝术(PRP)治疗增殖性糖尿病视网膜病变(PDR)的疗效及对患者血清血管内皮生长因子(VEGF)、色素内皮生长因子(PEDF)水平的影响。方法选择2022年2月至2023年12月洛阳市偃师人民医院收治的78例PDR患者作为研究对象,依据随机数表法将患者分为对照组和观察组各39例。对照组患者采用PRP治疗,观察组患者基于对照组治疗基础上联合康柏西普治疗,两组患者均在治疗后随访3个月并回院复查。比较两组患者的临床疗效,以及治疗前和随访3个月后的最佳矫正视力、黄斑中心凹厚度和血清VEGF及PEDF水平;同时比较两组患者的激光治疗情况(光斑数量、激光能量、激光次数)及并发症发生情况。结果观察组患者的治疗总有效率为97.44%,明显高于对照组的82.05%,差异有统计学意义(P<0.05);随访3个月后,两组患者的最佳矫正视力均提高,黄斑中心凹厚度均缩小,且观察组患者的最佳矫正视力为0.46±0.05,明显高于对照组的0.40±0.04,黄斑中心凹厚度为(243.09±30.26)μm,明显低于对照组的(278.93±34.11)μm,差异均有统计学意义(P<0.05);治疗后,两组患者的VEGF水平均降低,PEDF水平均上升,且观察组患者的血清VEGF水平为(124.53±14.93)μg/L,明显低于对照组的(178.06±20.01)μg/L,血清PEDF水平为(83.47±10.55)μg/L,明显高于对照组的(75.64±8.93)μg/L,差异均有统计学意义(P<0.05);治疗后观察组患者的光斑数量、激光能量、激光次数均少于对照组,差异均有统计学意义(P<0.05);观察组患者的并发症发生率为7.69%,略高于对照组的5.13%,但差异无统计学意义(P>0.05)。结论康柏西普联合PRP能够显著改善患者术后视力,缩小黄斑中心凹厚度,降低血清VEGF水平,促进PEDF的表达,并减少PRP治疗所需激光次数和能量,安全性高。

色素上皮衍生因子在糖尿病视网膜病变中的作用机制研究进展

糖尿病视网膜病变(diabetic retinopathy, DR)是糖尿病常见的微血管并发症之一,其发病率逐年升高,1980年到2018年发病率为2.2%~12.7%。我国DR发病率为18.45%,全球DR患病率达22.27%。目前,DR已成为全世界成人失明的主要原因,严重影响了糖尿病患者的视网膜正常功能和生活质量。视网膜色素上皮(retinal pigment epithelium, RPE)是视网膜的最外层,对于维持视觉功能至关重要。RPE细胞可分泌色素上皮衍生因子(pigment epithelium-derived factor, PEDF)。PEDF是一种相对分子质量为50 000的天然糖蛋白,属于丝氨酸蛋白酶超家族的一员。现有研究表明,PEDF具有抑制血管生成、抗氧化、抗炎、营养神经等多种生物学活性,对包括DR在内的多种疾病具有保护作用。DR的发病机制较复杂,与氧化应激、炎症反应、线粒体功能障碍、血管内皮生长因子(vascular endothelial growth factor, VEGF)、小胶质细胞异常活化、晚期糖基化终末产物(advanced glycosylation end-product, AGE)蓄积等有关,其中涉及了Wnt/β-cantenin信号通路、线粒体解偶联蛋白(mitochondrial uncoupling protein,UCP)、核因子κB (nuclear factor-κB, NF-κB)通路、过氧化物酶体增殖物激活受体γ、AGE等。PEDF可以通过这些靶点和通路结合发挥作用,从而阻止DR的发生和进展。本文从PEDF抑制新生血管形成、抗氧化应激、抗炎这3个方面着手,阐述PEDF的不同作用靶点和通路在抑制DR发生、发展中的作用机制,为后期开发可广泛应用于临床的DR治疗新药提供理论依据。

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。

蛴螬提取物对兔脉络膜新生血管中Ang1和PEDF表达的影响

目的:通过建立实验性有色兔脉络膜新生血管(CNV)的动物模型,研究蛴螬提取物对血管生成素1(Ang1)和色素上皮衍生因子(PEDF)表达的影响,从而探讨其对CNV的抑制作用。方法:选取40只有色兔,随机分成5组,A组:健康空白组;B组:模型组;C组:维生素E组;D组:驻景丸加减方组;E组:蛴螬提取物组。每组8只16眼,通过氩激光光凝方式建立CNV模型。激光光凝后24h;7,14,21,28d行眼底彩色照相;7,14,21,28d行荧光素眼底血管造影(FFA);14,28d行光学相干断层扫描(OCT)。然后将每组家兔随机分成2批,分别于14,28d用空气栓塞法处死并摘取眼球后段组织行切片、HE染色,光镜下观察视网膜组织病理形态学改变,并行Ang1和PEDF免疫组织化学染色,以研究蛴螬提取物对CNV的抑制作用。结果:Ang1含量表达测定显示,空白组较实验组低(P<0.05),实验组中驻景丸加减方组和蛴螬提取物组较其它三组有显著性差异(P<0.05),蛴螬提取物组较驻景丸加减方组低,但无显著性差异,蛴螬提取物组28d较14d有显著性差异(P<0.05)。PEDF含量表达测定显示,空白组较实验组高(P<0.05),实验组中驻景丸加减方组和蛴螬提取物组较其它三组有显著性差异(P<0.05),蛴螬提取物组较驻景丸加减方组高,但无显著性差异,蛴螬提取物组28d较14d有显著性差异(P<0.05)。结论:蛴螬提取物对实验性有色兔CNV中Ang1的高表达存在良性影响,能有效干预PEDF的降低,保护视网膜组织,对CNV具有抑制作用。

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。