Effects of travoprost on actin cytoskeleton and β-catenin in the human trabecular meshwork cells treated with Dexamethasone

Objective To investigate the effect of travoprost on changes of actin cytoskeletal and β-catenin protein in the cultured human trabecular meshwork (HTM) cells treated with dexamethasone (DEX). Methods It was a control experiment study. The HTM cells were cultured in vitro and divided into control group, DEX (1 × 10-6mol/L) group, travoprost (1 × 10-6mol/L) group, and DEX (1 ×10-6mol/L) plus travoprost (1 × 10-6mol/L) group. F-actin in the HTM cells was detected by FITC-phallodin and observed under a fluorescence microscope. The expression of β-catenin was determined by immunofluorescence and western-blot. Statistical analysis was performed using SPSS13.0 software. The difference of β-catenin expression among groups was analyzed through variance analysis and, further by q test. Results The cultured HTM cells were identified by immunohistochemistry. A reorganization of actin cytoskeletal and a formation of cross linked actin networks (CLANs) were seen in the HTM cells treated with DEX, which were partially reversed by the treatment with DEX plus travoprost. An increase of the expression of β-catenin was discovered in the HTM cells treated with DEX, which was also partially reversed by the treatment with DEX plus travoprost. The amount of β-catenin protein in untreated control group, DEX group, DEX plus travoprost group and travoprost group were 0. 84 ± 0. 03,1.65 ± 0. 05, 1.21 ± 0. 05, and 0. 65 ±0. 04, respectively. Expression of β-catenin was significantly ( F = 143.07, P ＜ 0. 05 ) different when compared untreated control group with DEX group ( q = 15. 32 ,P ＜0. 05 ), untreated control group with DEX plus travoprost group (q = 11.40,P＜0. 05), DEX group with DEX plus travoprost group (q =9. 38,P ＜ 0. 05 ), DEX group with travoprost group ( q = 16. 55, P ＜ 0. 05 ), and DEX plus travoprost group with travoprost group (q = 14. 31 ,P ＜ 0. 05 ). No difference was found in untreated control group and travoprost group(q = 2. 84, P ＞ 0. 05 ). Conclusions Our results suggest that reversion of the changes of actin organization and β-catenin by travoparost in the HTM cells treated with DEX may partially elucidate the mechanism of action of increasing outflow by which travoprost reduces intraocular pressure.     

Effects of travoprost on actin cytoskeleton and β-catenin in the human trabecular meshwork cells treated with Dexamethasone

Objective To investigate the effect of travoprost on changes of actin cytoskeletal and β-catenin protein in the cultured human trabecular meshwork (HTM) cells treated with dexamethasone (DEX). Methods It was a control experiment study. The HTM cells were cultured in vitro and divided into control group, DEX (1 × 10-6mol/L) group, travoprost (1 × 10-6mol/L) group, and DEX (1 ×10-6mol/L) plus travoprost (1 × 10-6mol/L) group. F-actin in the HTM cells was detected by FITC-phallodin and observed under a fluorescence microscope. The expression of β-catenin was determined by immunofluorescence and western-blot. Statistical analysis was performed using SPSS13.0 software. The difference of β-catenin expression among groups was analyzed through variance analysis and, further by q test. Results The cultured HTM cells were identified by immunohistochemistry. A reorganization of actin cytoskeletal and a formation of cross linked actin networks (CLANs) were seen in the HTM cells treated with DEX, which were partially reversed by the treatment with DEX plus travoprost. An increase of the expression of β-catenin was discovered in the HTM cells treated with DEX, which was also partially reversed by the treatment with DEX plus travoprost. The amount of β-catenin protein in untreated control group, DEX group, DEX plus travoprost group and travoprost group were 0. 84 ± 0. 03,1.65 ± 0. 05, 1.21 ± 0. 05, and 0. 65 ±0. 04, respectively. Expression of β-catenin was significantly ( F = 143.07, P ＜ 0. 05 ) different when compared untreated control group with DEX group ( q = 15. 32 ,P ＜0. 05 ), untreated control group with DEX plus travoprost group (q = 11.40,P＜0. 05), DEX group with DEX plus travoprost group (q =9. 38,P ＜ 0. 05 ), DEX group with travoprost group ( q = 16. 55, P ＜ 0. 05 ), and DEX plus travoprost group with travoprost group (q = 14. 31 ,P ＜ 0. 05 ). No difference was found in untreated control group and travoprost group(q = 2. 84, P ＞ 0. 05 ). Conclusions Our results suggest that reversion of the changes of actin organization and β-catenin by travoparost in the HTM cells treated with DEX may partially elucidate the mechanism of action of increasing outflow by which travoprost reduces intraocular pressure.     

Effects of dexamethasone and HA1077 on actin cytoskeleton and β-catenin in cultured human trabecular meshwork cells

AIM: To investigate the effects of dexamethasone (DEX) and 1-(5-isoquinolinesulfonyl)-homopiperazine (HA1077) on actin cytoskeleton and β-catenin in cultured human trabecular meshwork (HTM) cells. METHODS: The HTM cells were separated from human eyeball and cultured in vitro. They were divided into control group, DEX (1×10-6 mol/L) group, HA1077 (3×10-5 mol/L) group, and DEX (1×10-6 mol/L) and HA1077 (3×10-5 mol/L) group. Actin cytoskeleton and β-catenin in HTM cells of the four groups were examined by immunofluorescence and Western blot analyses. RESULTS: In DEX group, there were reorganization of actin cytoskeleton and formation of cross linked actin networks (CLANs), which were partially reversed in DEX and HA1077 group. DEX treatment also induced an increased expression of β-catenin, which was obviously reduced in DEX and HA1077 group. Meanwhile, the cultured HTM cells in HA1077 group had lower expression of β-catenin than that in the control group. CONCLUSION: Our results show that HA1077 can reverse the changes of actin organization and expression of β-catenin induced by DEX in cultured HTM cells, suggesting that HA1077 may play an important role in increasing outflow and reducing intraocular pressure.     

Human β-NGF gene transferred to cat corneal endothelial cells

AIM: To transfect the cat corneal endothelial cells (CECs) with recombinant human β-nerve growth factor gene adeno-associated virus (AAV-β-NGF) and to observe the effect of the expressed β-NGF protein on the proliferation activity of cat CECs. METHODS: The endothelium of cat cornea was torn under the microscope and rapidly cultivated in Dulbecco’s modified Eagle''s medium (DMEM) to form single layer CECs and the passage 2 endothelial cells were used in this experiment. The recombinant human AAV-β-NGF was constructed. The recombinant human AAV-β-NGF was transferred into cat CECs directly. Three groups were as following: normal CEC control group, CEC-AAV control group and recombinant CEC-AAV-β-NGF group. Forty-eight hours after transfection, the total RNA was extracted from the CEC by Trizol. The expression of the β-NGF target gene detected by fluorescence quantitative polymerase chain reaction; proliferation activity of the transfected CEC detected at 48h by MTT assay; the percentage of G1 cells among CECs after transfect was detected by flow cytometry method (FCM); cell morphology was observed under inverted phase contrast microscope. RESULTS: The torn endothelium culture technique rapidly cultivated single layer cat corneal endothelial cells. The self-designed primers for the target gene and reference gene were efficient and special confirmed through electrophoresis analysis and DNA sequencing. Forty-eight hours after transfect, the human β-NGF gene mRNA detected by fluorescence quantitative polymerase chain reaction showed that there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05); there was significant difference between two control groups and recombinant CEC-AAV-β-NGF group (P<0.05). MTT assay showed that transfect of recombinant AAV-β-NGF promoted the proliferation activity of cat CEC, while there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05). FCM result showed that the percentage of G1cells in CEC-AAV-NGF group was 76.8% while that in normal CEC control group and CEC-AAV control group was 46.6% and 49.8%. CONCLUSION: Recombinant AAV-β-NGF promotes proliferation in cat CECs by expressing bioactive β-NGF protein in high efficiency and suggests that its modulation can be used to treat vision loss secondary to corneal endothelial dysfunction.     

TGF-β1 in retinal ganglion cells in rats with chronic ocular hypertension: its expression and anti-apoptotic effect

AIM: To investigate the anti-apoptotic effect of transforming growth factor beta-1 (TGF-β1) on chronic ocular hypertension. METHODS: The expression of TGF-β1 in retinal ganglion cells (RCGs) was measured using the immunohistochemiscal S-P method and real-time PCR in the normally control group, the ocular hypertension group (experimental group A), the ocular hypertension plus antibody intervention group (experimental group B) and the ocular hypertension plus antigen intervention group (experimental group C) at 1, 2, 3 and 4 weeks postoperatively. The count of apoptotic RCGs was measured using the TUNEL method. RESULTS: The expression of TGF-β1 was significantly higher in experimental group C than that in other three groups (P＜0.05). The expression was the lowest in experimental group B (4.17%). A statistically significant difference was noted between the four groups (P<0.01). The count of apoptotic RCGs was statistically significantly lower in experimental group C than that in the experimental groups A and B (P<0.01). A statistically significant difference was noted in the count of apoptotic RCGs between these three experimental groups (P<0.01). CONCLUSION: TGF-β1 can inhibit the apoptosis of RCGs in rats with chronic ocular hypertension.     

β-catenin expression in rat neovascularized cornea after alkali burn

AIM: To investigate the expression of β-catenin in cornea after alkali burn and explore its role in cornea neovascularization (CNV). METHODS: CNV model was established by putting filter paper with the size of 3.0mm in diameter immersed in 1mol/L NaOH solution on the left cornea of rat for 20 seconds. Twenty-five Sprague Dawley rats were randomly divided into 5 groups: post-operation 1-, 4-, 7-, 14- and 21-day groups while the right eyes as normal control group. The expression level of β-catenin protein, mRNA and VEGF were determined at the 1st, 4th, 7th, 14th and 21st day following the establishment of model by RT-PCR and immunohistochemical technique. RESULTS: No expression of β-catenin immunoreactivity was detected in normal cornea. The expressions of β-catenin and VEGF both reached the peak at the 4th and 7th day and gradually decreased to near baseline 21 days later. Alteration of β-catenin and VEGF levels showed a significant positive correlation (r=0.855, P<0.05). CONCLUSION: The levels of β-catenin are markedly related to inflammatory CNV in rat cornea after alkali burn.     

人角膜内皮细胞压力调节培养的初步研究

Objective To investigate the effect of pressure bionic culture on the morphology and function of rabbit corneal endothelial cells. Methods Corneal endothelial cells were separated and purified by tearing apart the descemet and digesting with trypsin and EDTA, then cultured in the plate. The cells were divided into two groups: group A were cultured under atmosphere; cells exposed to 2 kPa( 14. 66 mm Hg) pressure in vitro was group B; the morphology and growth pattern of cells were observed by inverted microscope; cells origin were identified by neuron-specific enolase immunoassay. Cellular changes in the structure were observed by HE staining and scanning and transmission electron microscopy (SEM and TEM) analysis. Cells activity was detected by flow cytometry. Results NSE antibody of the primary corneal endothelial cells was positive without corneal epithelial cells and corneal stroma cells. Two groups of cells were cultured for 120-144 h respectively, the morphology was flat, polygon, most of cells were hexagon and abundant cytoplasms in group B (pressure bionic culture), but in group A, the cells size was not uniform and there were much granules in the cytoplasm. There was no difference in the time of formation of monolayer in two groups. SEM showed that cells exposed to pressure connected tightly and the surface was rich in microvilli, extended foot processes and attached to the substrate tightly, while cells cultured under atmosphere with more off-chip. In group B, Annexiv-FITC/PI detection of apoptosis showed cell survival rate was 98.2%, early apoptosis rate was 0.7%, late apoptosis rate was 1.0%, death rate was 0. 1%; the corresponding data were 92.2%, 5.2%, 2.3%, and 0.3% in group A, respectively; There was statistically significant difference between the two groups (x2 =594. 0,P ＜0. 01 ). After cultured for 96 h,the expression of ZO-1 protein in cells exposed to pressure was higher than those in control. Conclusions The biological activity of endothelial cells is regulated positively by bionic pressure. The establishment of a new biomimetic pressure model will help to investigate the physiological function and injury repair of corneal endothelial cells in vitro.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

肾素(原)受体小干扰RNA对小鼠视网膜新生血管抑制作用的研究

目的 观察肾素(原)受体(PRR)小干扰RNA(siRNA)对小鼠视网膜新生血管的抑制作用及机制.方法 实验研究.将小鼠分为6组:A～E组建立高氧诱导视网膜新生血管动物模型,其中A～D组分别给予PRRsiRNA质粒、对照质粒、PRRsiRNA质粒联合氯沙坦治疗和氯沙坦治疗,E组不予任何治疗,F组为空白对照组.各组分别于P17时处死小鼠,用视网膜铺片、HE染色方法观察视网膜新生血管生长情况.用免疫印迹法检测各组PRR及ERK1/2的表达情况.用实时PCR方法检测A、B、E、F组中TGF-β1的表达.采用单因素方差分析(LSD法)比较各组视网膜新生血管及PRR、ERK1/2和TGF-β1表达的差异.结果 视网膜灌注铺片显示A、C和D组视网膜新生血管较E组和B组明显减少(F=17.218,P=0.000).A、C和D组突破内界膜的血管内皮细胞数分别为4.47±1.96、4.49±2.53和5.94±2.54,明显少于E组(32.73±6.38)和B组(21.04±5.39).免疫印迹法检测结果显示E组17 d时PRR表达明显高于F组;A组和C组PRR水平明显低于E组,而B组和D组PRR水平与E组相比差异无统计学意义(F=14.584,P=0.000).E组P17时活化的ERKI/2水平明显高于F组;A、C和D组ERK1/2的活化率均明显低于E组;其中A组和C组降低更明显,与D组相比差异有统计学意义(F=11.177,P=0.000).实时PCR结果显示E组小鼠视网膜TGF-β1 mRNA表达水平明显高于F组,A组表达明显降低,而B组与E组无差异(F=12.468,P=0.002).结论 PRR与肾素(原)结合后可独立激活ERK1/2通路,促进视网膜新生血管的生长.PRRsiRNA可明显降低PRR的表达量,减少ERK1/2通路的激活,达到治疗新生血管的目的.

Abstract：

Objective To observe the inhibit effect of (pro)renin receptor siRNA on mice retinal neovascularization, and investigate its possible mechanism. Methods Experimental study. 72 new born C57BL/6J mouse were randomly divided into six groups: group A to group E were exposed to hyperoxia, and then returned to normoxia to induce retinal neovascularization. Group A were treated with PRR siRNA plasmid, group B with control plasmid, group C with PRR siRNA and Losartan, and group D with Losartan.Group E were not treated. Group F was control group. Mouse were sacrificed at postnatal day 17, retinal perfusion stretched preparation and HE dyeing method were used to observe the status of retinal neovascularization. PRR expression and the activation of extracellular regulated protein kinases 1/2(ERK1/2) were detected by Western Blot. And Real Time PCR was used to detect the expression of transforming growth factor-β1 (TGF-β1) in group A, B, E and F. Analysis of one way variance (LSD) was used and statistical difference was considered significant at a P value less than 0. 05. Results The mouse treated with PRR siRNA, Losartan and combind therapy could significantly reduce retina neovascularization and vessel leakage compared with oxygen-induced retinopathy group and control plasmid group. Average counts of vascular endothelial cells which break through the inner limiting membrane performed at postnatal day 17 in PRR siRNA group (4. 47 ± 1.96), Losartan group (5.94 ± 2. 54) and combind therapy group(4. 49 ± 2. 53)were significantly lower than oxygen-induced retinopathy group (32. 73 ± 6. 38) (P＜0. 05) and control plasmid group(21.04 ±5. 39). Western Blot showed that PRR protein express in hyperxia induced group was significantly higher than normal (P=0. 007). After treated with PRR siRNA or combind therapy, the expression of PRR protein was significantly lower than hyperxia induced group (P＜0. 05). There are no significantly differences between control plasmid group, Losartan group and hyperxia induced group (P＞0. 05). The activated ERK1/2 lever in hyperxia group was significantly higher than normal (P=0. 003).After treated with PRRsiRNA, Losartan or combind therapy, activated ERK1/2 lever was significantly lower than hyperxia induced group (P＜0. 05). And the effect of PRR siRNA group and combind therapy group seems more obviously, compared with Losartan group, the difference was significantly (P＜0. 05). Real Time PCR showed that the lever of TGF-β1 in hyperxia group was significantly higher than normal (P=0. 001). After treated with PRR siRNA, the TGF-β1 was significantly reduced (P=0. 004), and there was no significantly difference between control plasmia group and hyperxia induced group (P=0. 222) .Conclusions PRR combined with prorenin or renin could activate ERK1/2 signal transduction passageway,and promote cell proliferation, differentiation and migration, thus promote retinal neovascularization. PRR siRNA could obviously reduce PRR expression, inhibit ERK1/2 signal transduction passageway activation,and diminish retianl neovascularizatior.     

大鼠结膜囊成纤维细胞TGFβ2特异性siRNA的筛选及其载体构建的研究

Objective As the expression of transfornfing growth factor β （TGF2 β） of tenon＇ s fibroblasts is important for filtering bleb scarring formation after glaucoma surgery, it is benefit for preventing filtering bleb scarring through inhibiting its expression. This paper was to screen the effects of different TGF2β siRNAs on the expression of TGF1β mRNA and to construct the specific TGF2β siRNAs plasmid vector of rat Tenon＇ s fibroblasts. Methods Four TGF1β siRNAs （ L1 , L2 , L3 , L4 ） were transcribed in vitro and then transferred to cultured Tenon＇ s capsule fibroblasts in 30 SD rats. RT-PCR was performed to evaluate the levels of TGF1β mRNA in both transferred cells, and untransferred cells were as the controls. The specific TGF2 β siRNAs plasmid vector was constructed according to the screened TGF1β siRNAs. Results The expression of TGF1β mRNA of fibroblasts transferred with only L4 （0. 163 ± 0. 009 ） was reduced significantly in comparison with the control group（0. 433 ± 0. 011 ）, hut the interference function of others siRNAs seemed to be inactive. The plasmid vector of siRNA specific for TGFβ2 was proved to be constructed successfully by detecting its sequence. Conclusion Different siRNAs is designed to target the relevant TGFβ2 mRNA of rats Tenon＇ s fibroblast. The vector of siRNA specific for TGFβ2 is of potent interference ability for target tissue.     

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.     

Modulation of TGFβ2 and dopamine by PKC in retinal Müller cells of guinea pig myopic eye

AIM: To investigate the effect of protein kinase C (PKC) on transforming growth factor-β2 (TGFβ2) and dopamine in retinal Müller cells of guinea pig myopic eye. METHODS: Myopia was induced by translucent goggles in guinea pig, whose retinal Müller cells were cultured using the enzyme-digesting method. Retinal Müller cells were divided into 5 groups: normal control, myopia, myopia plus GF109203X, myopia plus PMA, myopia plus DMSO. PKC activities were detected by the non-radioactive methods. TGFβ2 and tyrosine hydroxylase (TH) proteins were analyzed by Western Blotting in retinal Müller cells. Dopamine was determined by the high-performance liquid chromatography-electrochemical detection in suspensions. RESULTS: After 14 days deprived, the occluded eyes became myopic with ocular axle elongating. Müller cells of guinea pigs were obtained using enzyme digestion. Compared with normal control group, the increase in PKC activity and the up-regulation in TGFβ2 expression were found in retinal Müller cells of myopic eyes, with the decrease of TH and dopamine content (P<0.05). After PKC activated by PMA, TGFβ2 and TH content were up-regulated with the increase of dopamine content (P<0.05). While the PKC activities was inhibited by GF109203X, proteins of TGFβ2 and TH were down-regulated in the myopic eyes, with the decrease of dopamine content (P<0.05). CONCLUSION: TGFβ2 and dopamine are modulated by PKC in Müller cells of the myopic eyes in guinea pig.     

Clinical significance of serum biochemistry changes in mice with targeted disruption of βB2-crystallin gene

AIM: To explore the pathogenesis, influencing factors, ways of medical intervention and evaluation indicators of cataract by observing changes in serum biochemical indices in mice with targeted disruption of βB2-crystallin. METHODS: Nine 6-week-old male mice with targeted knockout of βB2-crystallin were used as the study group, and nine age- and sex-matched normal wild-type mice as the control group. The genetype of the modeled mice was identified by PCR technique. Tropicamide and phenylephrine eye drops were used as the cycloplegic agents to observe changes in lens opacity with a slit-lamp. The lens was then removed and blood was collected for biochemical evaluation in the serum. RESULTS: Two genotypes were successfully identified by PCR technique. Slit-lamp observation showed that the lens cortex was opaque and GSH level in the lens cortex was remarkably decreased in mice with βB2-crystallin deficiency compared with the control group (P<0.01). Serum Na+, Cl-, Ca2+, Mg2+ and Fe2+ levels, ALT and AST activities, and TP, ALP, Cr, TC, GLU content were decreased significantly compared with the control group (P<0.05). There was no difference in LDH, P, Cu2+, K+ levels between the two groups (P>0.05). CONCLUSION: Compared with the wild-type mice, serum biochemical indices underwent significant changes in mice with targeted disruption of βB2-crystallin gene, especially with abnormal distribution of Na+&Ca2+, which induced the formation of cataract.     

【In Press】 The relationship between insulin resistance/β-cell dysfunction and diabetic retinopathy in Chinese patients with type 2 diabetes mellitus: the Desheng Diabetic Eye Study

AIM: To investigate the relationship between insulin resistance (IR)/β-cell dysfunction and diabetic retinopathy (DR) in Chinese patients with type 2 diabetes mellitus (T2DM), and to explore further whether there were differences in the relationship among diabetic patients with higher and lower body mass index (BMI). METHODS: Cross-sectional study. A total of 1466 subjects with T2DM were recruited in a local Desheng Community of urban Beijing from November 2009 to June 2012 for the cohort of Beijing Desheng Diabetic Eye Study. Standardized evaluation was carried out for each participant, including questionnaire, ocular and anthropometric examinations, and laboratory tests. Seven fields 30° color fundus photographs were used for DR grading according to the Early Treatment Diabetic Retinopathy Study protocols. Homeostatis Model Assessment (HOMA) method was employed for IR and β-cell function assessment. RESULTS: After excluding those participants who were treated with insulin (n=352) or had missing data of fasting insulin (n=96), and further excluding those with poor quality of retinal photographs (n=10), a total of 1008 subjects were included for the final analysis, 406 (40.3%) were men and 602 (59.7%) were women, age ranging from 34 to 86 (64.87±8.28) y. Any DR (levels 14 and above) was present in 278 (27.6%) subjects. After adjusting for possible covariates, the presence of any DR did not correlate with HOMA IR (OR 1.51, 95%CI: 0.87-2.61, P=0.14) or HOMA β-cell (OR 0.71, 95%CI 0.40-1.26, P=0.25). After stratification by BMI, the presence of any DR was associated positively with HOMA IR (OR 2.46, 95%CI: 1.18-5.12, P=0.016), and negatively with HOMA β-cell (OR 0.40, 95%CI: 0.19-0.87, P=0.021) in the group of patients with higher BMI (≥25 kg/m2). In the group of patients with lower BMI (<25 kg/m2), the presence of any DR was not associated with HOMA IR (OR 1.00, 95%CI: 0.43-2.33, P=1.00) or HOMA β-cell (OR 1.41, 95%CI: 0.60-3.32, P=0.43). CONCLUSION: The data suggest that higher IR and lower β-cell function are associated with the presence of DR in the subgroup of diabetic patients with higher BMI. However, this association is not statistically significant in diabetic patients with lower BMI.     

The relationship between insulin resistance/β-cell dysfunction and diabetic retinopathy in Chinese patients with type 2 diabetes mellitus: the Desheng Diabetic Eye Study

AIM: To investigate the relationship between insulin resistance (IR)/β-cell dysfunction and diabetic retinopathy (DR) in Chinese patients with type 2 diabetes mellitus (T2DM), and to explore further whether there were differences in the relationship among diabetic patients with higher and lower body mass index (BMI). METHODS: Cross-sectional study. A total of 1466 subjects with T2DM were recruited in a local Desheng Community of urban Beijing from November 2009 to June 2012 for the cohort of Beijing Desheng Diabetic Eye Study. Standardized evaluation was carried out for each participant, including questionnaire, ocular and anthropometric examinations, and laboratory tests. Seven fields 30° color fundus photographs were used for DR grading according to the Early Treatment Diabetic Retinopathy Study protocols. Homeostatis Model Assessment (HOMA) method was employed for IR and β-cell function assessment. RESULTS: After excluding those participants who were treated with insulin (n=352) or had missing data of fasting insulin (n=96), and further excluding those with poor quality of retinal photographs (n=10), a total of 1008 subjects were included for the final analysis, 406 (40.3%) were men and 602 (59.7%) were women, age ranging from 34 to 86 (64.87±8.28)y. Any DR (levels 14 and above) was present in 278 (27.6%) subjects. After adjusting for possible covariates, the presence of any DR did not correlate with HOMA IR [odds ratio (OR) 1.51, 95% confidence interval (CI) 0.87-2.61, P=0.14] or HOMA β-cell (OR 0.71, 95%CI 0.40-1.26, P=0.25). After stratification by BMI, the presence of any DR was associated positively with HOMA IR (OR 2.46, 95%CI: 1.18-5.12, P=0.016), and negatively with HOMA β-cell (OR 0.40, 95%CI: 0.19-0.87, P=0.021) in the group of patients with higher BMI (≥25 kg/m2). In the group of patients with lower BMI (<25 kg/m2), the presence of any DR was not associated with HOMA IR (OR 1.00, 95%CI: 0.43-2.33, P=1.00) or HOMA β-cell (OR 1.41, 95%CI: 0.60-3.32, P=0.43). CONCLUSION: The data suggest that higher IR and lower β-cell function are associated with the presence of DR in the subgroup of diabetic patients with higher BMI. However, this association is not statistically significant in diabetic patients with lower BMI.     

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.