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 detect and explore the expression of ARD1 and its clinical significance in the nasopharyngeal in-flammatory tissue ,nasopharyngeal carcinoma group and its subgroups .Methods Expression of ARD1 in nasopharyngeal carcinoma (56 cases) and nasopharyngeal inflammatory tissue (20 cases) were detected by immunohistochemical staining SP ,the correlation between the expression of ARD1 and age ,gender ,histological grade ,TNM clinical stage and tumor metastasis were analysed . Results The positive expression rate of ARD1 were 10 .00% (2/20) ,55 .35% (31/56) in the nasopharyngeal inflammatory tissue and nasopharyngeal carcinoma ,respectively .The expression level of ARD1 in nasopharyngeal carcinoma was significantly higher than in the nasopharyngeal inflammatory tissue ,the difference was significant (P< 0 .05) ;expression of ARD1 in the nasopharynge-al carcinoma was correlated with the histological grade of nasopharyngeal carcinoma(P< 0 .05) and the expression was increased in poor differenciation tissue .But there was no statistical difference between the expression of ARD1 and the patient′s age ,gender , TNM clinical stage ,tumor metastasis(P> 0 .05) .Conclusion The expression of ARD1 is high in nasopharyngeal carcinoma ,and has a closely correlation with diffferentiation level of tumor ,which suggested that ARD1 may be involved in the the occurrence and development of nasopharyngeal carcinoma .However ,further research needs to be done for its mechanism in the nasopharyngeal car-cinoma .

Objective To investigate cell proliferation and invasiveness of cervical cancer HeLa229 cell after knockdown of NF-?B signaling pathway by P65 siRNA.Methods RNA interference was employed for specific inhibition of the expression of P65.HeLa229 cell was divided into transfected group and untransfected group.Cell viability was detected by MTT after the HeLa229 cells were transfected with or without P65 siRNA for 24,48,72 h.The sensitivity to 5-Fu of the HeLa229 cell,transfected with or without P65 siRNA,was evaluated also by MTT.Boyden chamber experiment in vitro was used to detect the invasion of HeLa229 cell.Results P65 siRNA inhibited the cell proliferation as compared with the untransfected cells.Proliferations of both cells transfected with and without P65 siRNA were inhibited in a concentration-dependent manner,while at the same concentration of 5-Fu the viability of transfected HeLa229 cells was significantly suppressed(P

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 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.

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.

Objective To obtain Chinese hamsterovary (CHO) cell line expressing human decay accelerating activity (hDAF) stably and to observe the protective effect of hDAF on heterologous cells under the circumstance of complement activation. Methods The eukaryotic expression vector DAF pcDNA3.1 was constructed and then transfected into CHO cells by lipofection. Monoclones of cells expressing hDAF stably were screened by the method of limiting dilution. hDAF expression was detected by flow cytometry. The decay accelerating activity of hDAF was determined by assay of C3 deposition and 51Cr release. Results The expression vector DAF pcDNA3.1 was successfully constructed, and monoclones of cells expressing hDAF were obtained. CHO cells expressing hDAF could decrease C3 deposition and attenuate the killing effect of activation of the complement system. Conclusion We have obtained CHO cell clones expressing hDAF stably, which is helpful for the further studies of the relationship of the structure with the functions of hDAF.

Background and purpose:Perioperative anesthetic management is thought to be critical to the success of free flap breast reconstruction. The purpose of this study was to discuss intraoperative fluid, hemodynamic and temperature management in patients undergoing deep inferior epigastric perforator (DIEP) flap breast reconstruction.Methods:From Jun. 2011 to Dec. 2015, 126 patients underwent DIEP lfap breast reconstruction. Postoperative complications were reviewed. Intraoperative fluid infusion rate was analyzed. Mean arterial blood pressure (MAP) and core temperature were measured before induction (T0), after lfap elevation but before lfap transfer (T1), 15 min after flap revascularization (T2), and at the end of surgery (T3).Results:Nine patients developed flap compromised: 7 were salvaged and 2 failed. The mean intraoperative lfuid infusion rate was (5.44±1.66) (mL?kg-1)/h. MAP at T0, T1, T2 and T3 were (87.45±8.90), (74.19±8.63), (74.60±8.71) and (79.62±7.88) mmHg, respectively. Core temperature at T0, T1, T2 and T3 were (36.69±0.14), (36.36±0.18), (36.27±0.14) and (36.21±0.15)℃, respectively. Conclusion:Standard practice focusing on intraoperative lfuid management, hemodynamic adjustment and temperature control in microsurgical reconstruction of the breast should be established to further improve free lfap outcome.

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 evaluate the efficacy and safety of domestic olmesartan in treatment of mild to moderate essential hypertension in comparison with losartan. Methods Two hundred and thirty-seven patients with mild to moderate essential hypertension were enrolled in a randomized, double-blind, multi-center, paralleded and active-controlled trial, and were divided into olmesartan group (olmesartan 20 mg + losartan 50 mg placebo) and losartan group (losartan 50 mg + olmesartan 20 mg placebo) for a 8-week therapy. Four weeks after treatment, dosages of drugs were doubled in patients with seated diastolic blood pressure ≥90 mmHg (1 mmHg =0.133 kPa). All patients were followed up every two weeks, and the efficacy and adverse effects were observed. Another 32 patients with mild to moderate essential hypertension were enrolled and given olmesartan only, and ambulatory blood pressure monitoring was performed before and 8 weeks after treatment. Results Compared with those before treatment, both systolic blood pressure and diastolic blood pressure significantly decreased in olmesartan group and losartan group 8 weeks after treatment [(15.2 ±13.3) mmHg and (19.5 ±11.8) mmHg, respectively for systolic blood pressure (P <0.001); (15.9 ±7.48) mmHg and (16.2 ± 5.95) mmHg, respectively for diastolic blood pressure (P < 0.01) ], while there was no significant difference between these two groups (P > 0.05). There was no significant difference in total effective rate and incidence of adverse effect between these two groups (86.9% vs 93.7% and 7.63% vs 5.88% , P > 0.05) . Ambulatory blood pressure monitoring demonstrated that trough to peak ratios of systolic blood pressure and diastolic blood pressure were 86% and 71%, respectively. Conclusion Domestic olmesaratan provides an effective, safe and long action in the treatment of mild to moderate essential hypertension.