Background The suppression of retinal angiogenesis is one of primary treatment targets for retinal vascular diseases,so seeking the intervention targets of retinal neovascularization is a hot research.Studies showed that insulin-like growth factor 1 (IGF-1) can promote the growth and restrain the apoptosis of vascular endothelial cells.However,whether IGF-1 is an intervention target for the treatment of retinal vascular diseases is unelucidated.Objective This study was to address the effects of IGF-1 on the migration,apoptosis and capillary tube formation of human retinal vascular endothelial cells (HRECs) and mechanism.Methods HRECs were cultured in vitro,and the cells in the exponential phase were prepared for subsequent experiments.The expression of IGF-1R mRNA in the cells was examined using reverse transcriptase PCR assay.Different concentrations of IGF-1 were added in the medium based on the difference of tests.The relative free-cell area difference (△S) after test was measured by Photoshop CS4 software and compared among 0,10 and 200 ng/ml IGF-1 groups 12 and 24 hours after cell scratching,respectively.The cell apoptotic rate was assayed by flow cytometry and compared between 0 ng/ml IGF-1 group and 1 000 ng/ml IGF-1 group,and the number of capillary tubes was examined by Matrigel test and assessed among 0,10,100 and 200 ng/ml IGF-1 groups 24 hours after addition of IGF-1.The expressions of platelet derived growth factor (PDGF)-BB mRNA and caspase-3 mRNA in the cells of the 0,500 and 1 000 ng/ml IGF-1 groups were detected by real-time fluorescence quantitative PCR after adding IGF-1 for 6 hours.Results Cultured cells grew well and attached 90％ confluence 2-3 days after incubation,and IGF-1R mRNA was positively expressed in the cells.In 12 and 24 hours after scratching,the relative migrating area of the cells was gradually reduced with the increase of IGF-1 contents.The △S was (4.83 ± 0.61) × 105 μm2 in the 200 ng/ml IGF-1 group,which was significantly larger than (3.28±0.64) ×105 μm2 in the 0 ng/ml IGF-1 group 24 hours after stretching (t=-3.707,P=0.021).The apoptotic rate in the 0 ng/ml IGF-1 group and 1 000 ng/ml IGF-1 group was (18.77±2.37) ％ and (12.05 ±0.88) ％,with a significant difference between them (t =2.869,P =0.046).The number of intact tubes was significantly increased in the 200 ng/ml IGF-1 group compared with the 0 ng/ml IGF-1 group ([20.33±2.83]/well vs.[17.94± 1.96]/well;t =-2.940,P =0.042).Compared with 0 ng/ml IGF-1 group,the relative expression level of PDGF-BB mRNA was elevated and that caspase-3 mRNA was evidently reduced in the 1 000 ng/ml IGF-1 group (t=-3.489,P =0.025;t =7.287,P =0.002).Conclusions IGF-1 can promote the migration and angiogenesis of HRECs and inhibit the apoptosis of HRECs.These effects of IGF-1 probably are associated with the up-regulation of PDGF-BB and down-regulation of caspase-3 in the cells.

Background Corneal neovascularization (CNV) is one of the causes of corneal blindness.Studies showed that ADP-ribosylation factor (ARF) can regulate the growth of tumor cells,and inhibiting ARF will decrease angiogenesis.However,whether ARF antagonist plays an action on CNV is unclear.Objective The aim of this study was to explore the effect and mechanism of ARF inhibitor on alkali-burn induced CNV.Methods Sixty clean male BABL/c mice aged 7-8 weeks were divided into PBS group and ARF antagonist group according to randomized number table.CNV models were induced by NaOH burn method in all the mice.ARF at the concentration of 0.5 g/L(0.5 ml) was intraperitoneally injected 3 times per week for 1 week followed the induction of CNV in the ARF antagonist group,and 0.5 ml PBS was used in the PBS group.CNV was examined 2,4,7,14 days after injection by the slit lamp microscope and the CNV related area in the cornea was calculated.Betore modeling(0 day) and 4,7,14 days after modeling,real-time PCR and Western blot were used to analyze the expressions of ARF mRNA and protein in the corneas.Forteen days after modeling,the expression of the CD31 in the CNV was detected using immnofluorescence of corneal whole mount;the expression of vascular endothelial growth factor(VEGF) in the cornea was assayed by Western blot.Cellular wound scratch test was employed to evaluate the effects of ARF antagonist on proliferation and migration of human retinal vascular endothelial cells (RECs).All animal experiments were done in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and Guideline for the Care and Use of Laboratory Animals on the the Soochow University Animal Care Committee.Results ARF mRNA and protein were expressed in the mice corneas in both the PBS group and the ARF antagonist group at various time points.The expression of ARF mRNA in mice corneas was enhanced with the lapse of the time (Ftime =65.17,P =0.00),but no significant difference was found among the groups (Fsroup =1.98,P=0.18).There was also significant difference in the expression of ARF protein in mice corneas at different time points in the ARF antagonist group (F =10.77,P =0.00).The related CNV area was 0.45±0.05 in the ARF antagonist group,and that in the PBS group was 0.72±0.11,with significant difference between them (t =-3.87,P ＜ 0.05).The green fluorescence area of C D31 expression in the cornea was smaller in the ARF antagonist group than that of the PBS group.Expression level of VEGF in the ARF antagonist group was 1.20±0.21,and that in the PBS group was 2.47±0.33,showing a significant difference (t =-5.62,P ＜ 0.05).As the increase of ARF antagonist concentration,the inhibiting rate of cell proliferation was reinforced among 10,100 and 1 000 μg/L ARF antagonist groups (F=8.47,P =0.02).Twenty-four hours after scratch test,the migrating distance of human RECs was (5.46±1.32) μm and (5.04±1.68) μm in the 100 μg/L and 1 000 μg/L ARF antagonist groups,respectively,which were shorter than (8.49± 1.18) μm of the PBS group (t=-2.94,-2.91,both at P＜0.05).Conclusions ARF inhibitor can reduce CNV by down-regulating the expression of VEGF in alkaline burn cornea and inhibiting the proliferation and migration of vascular endothelial cells.

Background The pathogenesis and mechanism research of corneal neovascularization is of important significance for the prevention and management of corneal neovascularization.Some relative researches are being performed on non-corneal neovascularization-derived vascular endothelial cells, so the results are affected to a certain extent.Objective This study was to isolate and culture vascular endothelial cells from experimental corneal neovascularization tissue and detect the expression of chemokine receptors in vitro.Methods Corneal neovascularization models were established on 10 SPF male BALB/c mice with the age of 7-8 weeks by sticking the filter papers with NaOH on the central corneas, and then the immunofluorescence technique was use to assay the CD31 expression in corneal flatmount 2 weeks after modeling.Corneal pieces were made in 2 weeks after alkali burn and then were digested by collagenase type D.Vascular endothelial cells were isolated from neovascularized tissue by affinity purification using magnetic beads coated with anti-CD31.The cells were cultured on fibronectin-coated walls and then identified by immunocytochemistry.Reverse transcription-PCR was employed to detect the expressions of chemokine receptors in the cells.The use and care of the animals complied with ARVO Statement and this experimental procedure was approved by Soochow University Animal Care Committee.Results Corneal neovascularization occurred at 7 days and peaked at 2 weeks after modeling, and immunofluorescence exhibited the green network-like fluorescence for CD31 antibody in corneas.The cells grew against the wall 2 hours after culture with the polygon shape and large dimension, and the growth obviously quickened after passage.The cultured cells showed the positive response for CD31 antibody, showing the brown dye in cytoplasm,in contrast,the expression of CD31 was absent in corneal stromal cells.Chemokine receptors were positively expressed in the cells with the strongest expression levels in CCR1 ,CCR2,CCR3 and CCR4 mRNA and the weakest expression levels in CCR9,CXCR4 and CXCR5 mRNA,while CXCR3, CCR6, CCR10 and CX3CR1 mRNA were expressed with the moderate intensity.Conclusions Vascular endothelial cells can be obtained from experimental neovascularized corneas by affinity purification and express chemokine receptors,which facilitate the study of their biological properties.

Objective To observe the clinical efficacy of wrist-ankle acupuncture in treating pain due to laparoscopic cholecystectomy.Method Totally 150 patients who were going to receive laparoscopic cholecystectomy were randomized into group A, group B, and group C, 50 cases in each group. Group A was intervened by wrist-ankle acupuncture prior to anesthesia, with the needles retained for 12 h; group B was by subcutaneous needling at the area nearby the points prior to anesthesia, with the needles retained for 12 h; group C didn’t receive any intervention before anesthesia. For the three groups, general inhalational and intravenous anesthesia was adopted for surgery, and patient-controlled intravenous analgesia for post-operation analgesia. The incision pain and visceral pain in the three groups were recorded by using Visual Analogue Scale (VAS) respectively 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h after the operation. The total effective rate, analgesics consumption after operation, and incidence rate of adverse reaction were compared.Result There were significant differences in comparing the VAS scores of incision pain and visceral pain between group A and group C 4 h, 8 h, 12 h, 24 h, and 36 h after the operation (P<0.01,P<0.05). Between group A and group B, there were significant differences in comparing the VAS score of incision pain 8 h, 12 h, 24 h, and 36 h after the operation and the VAS score of visceral pain 12 h, 24 h, and 36 h after the operation (P<0.05). The total effective rate was 96.0% in group A, which was significantly different from 84.0% in group B and 86.0% in group C (P<0.05). The consumption of Fentaneyl citrate injection was (52.4±10.8)μg in group A, which was significantly different from (92.2±11.0)μg in group B and (107.2±11.5)μg in group C (P<0.05,P<0.01). The incidence rate of adverse reactions was 12.0% in group A, which was significantly different from 58.0% in group B and 66.0% in group C (P<0.01).Conclusion Wrist-ankle acupuncture plus patient-controlled intravenous analgesia can mitigate pain after laparoscopic cholecystectomy, and thus it can be taken as one of the post-operational analgesic approaches.

The fine ultrastructure and localization of acid phosphatase in cell ultrastructuralevel of a HGPRT-human promyelocytic leukemia cell mutant(HL-60-AR)arestudied by scanning electron microscopy,transmission electron microscopy,freezeetching,and electron microscopic cytochemistry techniques.The results of theobservation show that the ultrastructural characteristics of HL-60-AR cells aresimilar to that of HL-60 cells.There are microvilli and ridges over cell surface.Thecells have large nucleus with prominent nucleoli,and numerous nuclear pores.Thereare less developed Golgi complex,expanded rough endoplasmic reticulum andabundant polyribosomes.After treatment with retinoic acid(RA)at 10~(-6) mol/L for 5days,HL-60-AR cells differentiate along myeloid pathway and have a decreasednucleocytoplasmic ratio accompanied with nuclear condensation and segmention.A (?)ignificant increase of specific granules is demonstrated.Microvilli of the cellsdisappear,surface features of the treated cells become more irregular and largeprotrusion and blunt pseudopodia appear.Increase of acid phosphatase content localizedon azurophilic granules(lysosomes)and Golgi complex is showed.The application offour kinds of electron microscopic techniques might provide the best way foridentifying cell ultrastructure.

Background Intracorneal macrophages play a critical role in corneal neovascularization (CNV)by secreting relative chemokines.But macrophages are characteristic by heterogeneity which has different biologic functions under different induction or stimulation from microenvironment.Objective This study was to detect the effects of chemokine (C-X3-C motif) ligand 1 (CX3CL1) and chemokine (C-C motif) ligand 2 (CCL2) on macrophages in vitro.Methods CNV was induced by corneal alkali burn in the left eyes of 20 male BALB/c mice aged 7-8 weeks.The CNV was evaluated under the slit lamp microscope 4 days after alkali burn,and then the corneal sections were prepared after mice were sacrificed.The expressions of CCR2 and CX3CR1 in the corneal specimens were detected by histo-fluorescence staining.Human peripheral blood mononuclear cells were separated using density gradient centrifugation and incubated in RPMI-1640 medium containing 10％ fetal bovine seruml(FBS) with 30 μg/L granulocyte-macrophage colony-stimulating factor (GM-CSF).The cells were divided into CD68 +CCR2 group and CD68+CX3CR1 group,and the percentage of the CX3CR1 and CCR2 expressions in the infiltrated macrophages of corneal specimens and human monocyte-derived macrophages was assayed by flow cytometry.The cultured cells were stimulated using human recombinant CX3CL1 and CCL2 proteins,and real-time PCR was used to detect the relative expressions of angiogenesis-related factors in macrophages.Results CNV was found in corneas 4 days after alkali burn and the CNV onsets from corneal limbus to central zone observed by a slit lamp.CCR2 and CX3CR1 were expressed in the F4/80-positive macrophages in alikali burned corneas.The macrophages grew for two weeks and appeared more dead cells in without GM-CSF group,but in GM-CSF induced group,the number of macrophages was increased.The percentage of CX3CR1-positive cells was 75％ and that of CCR2-positive cells was 45％.Real-time PCR showed that expression level of vascular endothelial growth factor (VEGF) mRNA increased and that ADAMTS-1 mRNA or TSP-1 mRNA decreased on macrophages after CCL2 stimulation,with significant differences in the 150 mg/L CCL2 group compared with the control group (t =-5.09,P =0.03 ; t =3.01,P =0.04 ; t =4.27,P =0.02).However,the VEGF mRNA expression decreased and ADAMTS-1 mRNA and TSP-1 mRNA increased after CX3CL1 stimulation,showing significant differences between the 150 mg/L CX3CL1 group and the control group (t=6.35,P=0.O2;t=-2.92,P=0.04; t=-3.81,P=0.03).Conclusions These results suggest that the macrophages have high heterogeneity.CCL2-and CX3CL1-expressing macrophages can regulate the expressions of angiogenesis-related factors.Macrophage chemokine signal may be a good target for treatment of neovascular ocular disease.

Background Corneal neovascularization (CNV) is one of the causes of corneal blindness.Studies showed that zonula occludens-1 (ZO-1) can inhibit pathological angiogenesis through physical barrier formed by tight junction structure.However,whether ZO-1 plays a role in CNV is unclear.Objective The aim of this study was to explore the effect of ZO-1,a tight junction protein on experimental CNV.Methods The CNV models were established in the left eyes of 24 clear male BALB/c mice aged 7-8 weeks by putting NaOH filter paper in the center of corneas for 15 seconds (15 s group) or 40 seconds (40 s group).CNV was examined and evaluated under the slit lamp microscope,and the expression of ZO-1 mRNA in the corneas were detected and compared by reverse transcription PCR (RT-PCR) between the two groups 2 weeks after modeling.In addition,54 models created by the same method were assigned to 3 groups according to randomized number table,0.2％ hyaluronic acid (HA),antiZO-1 neutralizing antibody (10 mg/L) +0.2％ HA and mouse hypoxia inducible factor-1α (HIF-1α) recombinant protein (5 mg/L)+0.2％ HA were topically administrated in the mice three times a day for 1 week after modeling respectively.The corneas were extracted 2 weeks after application of the drugs.Expression of CD31 in the CNV was assayed to calculate the number and the area of CNV by immunohistochemistry.The expression of VEGF mRNA in the corneas was detected by RT-PCR.The percentages of macrophage-specific F4/80 positive cells and neutrophilsspecific Ly-6G positive cells were calculated to evaluate the infiltrations of inflammatory cells in the corneas by flow cytometry.Results In 2 weeks after alkali burn of corneas,the number of severe CNV was more in the 40 s group than that in the 15 s group (x2 =6.032,P=0.049),and the expression level of ZO-1 mRNA was lower in the 40 s group than that in the 15 s group (1.15±0.08 versus 1.53±0.04) (t=4.157,P=0.014).CD31 positive cell number was more and the staining area was larger in the ZO-1 antibody group and HIF-1α positive control group than those in the 0.2％ HA group (cells:t=-129.590,-226.820,both at P=0.000;area:t =-5.310,-8.840,both at P=0.000).The relative expressions level of vascular endothelial growth factor (VEGF) mRNA was 1.33±0.10 and 1.46±0.11 in the ZO-1 antibody group and HIF-1 α positive control group respectively,which were significantly higher than 0.93±0.06 of the 0.2％ HA group (t =-5.820,-7.284,both at P =0.000).The percentages of positive cells in the ZO-1 antibody group and HIF-1α positive control group were significantly increased in comparison with the 0.2％ HA group for F4/80 (t =-16.750,-17.480,both at P =0.000) and for Ly-6G (t =-21.450,-27.680,both at P=0.000).Conclusions Alkali burn induced CNV downregulates the expression of ZO-1 mRNA.Administration of ZO-1 antibody causes the rise of VEGF mRNA in CNV and the infiltration inflammation cells,which suggests that the influence of ZO-1 on CNV is associated with the expression of VEGF.

Background Researches showed that ADP-ribosylation factor (ARF) promotes intracorneal secretion of multiple angiogenesis-related factors,such as vascular endothelial growth factor (VEGF) and nitricoxide synthase (NOS) etc., and therefore results in corneal neovascularization.However, whether ARF affects the tube formation of human retinal endothelial cells(HRECs) is unelucidated.Understanding the effect of ARF tube formation of HRECs is important for the target treatment of retinal vascular diseases.Objective The aim of this study was to explore the effect and mechanism of ARF inhibitor on tube formation of HRECs in vitro.Methods HRECs (HREC line) were cultured and passaged.The growth-well cells were harvested and divided into two groups.The cells were regularly cultured in the control group,and ARF antagonist (lml) was added in the culture medium in the ARF antagonist group.The expression levels of ARFmRNA and protein in the cells were examined by reverse transcription (RT)-PCR and Western blot.The morphology and number of HREC tube formation were detected by using three-dimensional Matrigel assay.The relative expression levels of VEGF, NOS, focal adhesion kinase (FAK) and heat shock protein 90 (HSP90) at gene level and protein level were examined by RT-PCR and Western blot in vitro.Results The relative expression levels of ARFmRNA in the cells were 0.65 ±0.14 and 0.32±0.10, and those of ARF protein were 0.85±0.15 and 0.24±0.17 in the control group and ARF antagonist group,showing significant differnces between the two groups (t =7.32, P =0.00;t =5.15, P =0.00).The number of HREC tube formation was (34.66±8.57)/field in the ARF antagonist group, which was significantly lower than (51.46±7.12)/field in the control group (t=2.99 ,P=0.04).The relative expression levels of VEGF mRNA, NOSmRNA and their proteins in the cells were significantly lower than those of the control group (t =3.02, P =0.04;t =3.68, P =0.02;t =3.33,P=0.03;t=2.89 ,P=0.04).The relative expression levels of FAKmRNA and HSP90mRNA in the ARF antagonist group were 0.65±0.18 and 0.28±0.05 ,which were significantly lower than 0.76±0.25 and 0.46±0.09 in the control group (all at P＜0.05).Conclusions ARF antagonist appears to have an inhibitory effect on the tube formation ability of HRECs propably by down-regulating the expressions of VEGF, NOS and the downstream signal transduction factors FAK and HSP90 in HRECs in vitro.

Objective To explore the role of viral infection in the development of drug eruption in patients with HIV infection, and to evaluate the efficacy of antiviral treatment. Methods This study enrolled 87 HIV-positive patients, including 11 with and 76 without drug eruption, all of whom received highly active antiretroviral therapy(HAART). Clinical data on, baseline CD4+ and CD8+ T cell counts and CD4/CD8 ratio in these subjects were retrospectively analyzed. Results The severity of drug eruption was mild in the 11 HIV-positive patients, with a mean latency period of (14.00 ± 8.10)(range, 8 - 34)days. Of the 11 patients with drug eruption, 7 had liver function impairment, which was not in accordance with the severity of skin lesions. Drug eruption was controlled in all the 11 patients after anti-anaphylactic treatment without withdrawal of antiviral drugs. Compared with 75 HIV-positive patients without drug eruption, the 11 HIV-positive patients with drug eruption showed significantly increased baseline CD4 + T cell counts (493.00 ± 245.68 (range, 42 - 810)/μl vs. 347.81 ± 167.00 (range, 11 - 814)/μl, t = 647.50, P < 0.05), but decreased proportion of patients with baseline CD4+ T cell counts below the lower limit of normal(3/11 vs. 48/75(64.00%), X2 = 3.95, P < 0.05). There were no significant differences between 10 patients with drug eruption and 69 patients without drug eruption in the baseline CD8+ T cell count(1472.30 ± 858.55/μl vs. 1356.59 ± 684.06/μl, P > 0.05), CD4/CD8 ratio(0.40 ± 0.27 vs. 0.29 ± 0.16, P > 0.05), or percentage of patients with a CD4/CD8 ratio below the lower limit of normal (9/10 vs. 68/69 (98.55%), P >0.05). Conclusions The latency period of drug eruption seems to be long in HIV-positive patients receiving HAART, and mild drug eruption can be complicated by liver function impairment in the patients. Relatively high CD4 + counts may be a risk factor for the development and aggravation of drug eruption in HIV-positive patients.

Objective To observe the clinical characteristics and treatment of cytomegalovirus retinitis (CMVR) in leukemia patients.Methods This is a retrospective analysis. Seven leukemia patients (13 eyes) with CMVR were studied. All patients underwent examinations of visual acuity, slit lamp microscope, ophthalmoscope, color fundus photography, peripheral blood CD4+T cell count and serum/aqueous CMV-DNA test. All patients were treated with ganciclovir or zoledronic acid combined with intravitreal injection of ganciclovir. The follow-up period was 3-14 months.Results Six patients were treated with hematopoietic stem cell transplantation and 1 patient was with chronic leukemia. All patients were CMV-DNA positive for serum, and 18.5％ (2/7) for aqueous humor. CMVR in leukemia patients showed mild anterior segment inflammation, ocular fundus with irregular yellowish-white retinal necrosis and radial hemorrhage (7 eyes). Some (2 eyes) also shoed gray and white granular retinal infiltrates. Intravenous ganciclovir/zoledronic acid combined with intravitreal injection of high concentration ganciclovir was an effective treatment, while systemic corticosteroids were effective in reducing vitreous opacity.Conclusions CMVR is characterized by progressive necrotic retinitis with hemorrhage and vasculitis. Intravenous ganciclovir/zoledronic acid combined with intravitreal injection of ganciclovir is effective in the treatment of CMVR with leukemia.