Objective To investigate whether MEG3 involved in the development of retinoblastoma by down-regulating the expression of P53 protein.Methods The MEG3 expression of retinoblastoma tissues and corresponding non-tumor tissues were detected by quantitative real-time PCR (qRT-PCR).Retinoblastoma cell lines SO-RB50 or HXO-RB44 were transfected with pcDNA-MEG3 or siRNA-MEG3,after which cell apoptosis was tested by flow cytometry and P53 protein expression was tested by Western blot.Results MEG3 expression of retinoblastoma tissues was significantly reduced compared with corresponding non-tumor tissues(P =0.014).MEG3 level was significantly increased in pcDNA-MEG3 transfected SO-RB50 cells (P =0.002) and significantly decreased in siRNA-MEG3 transfected HXO-RB44 cells (P =0.004).Flow cytometry showed that the SO-RB50 cells apoptosis was significantly increased with the MEG3 over-expression(P ＜ 0.05),as well as the HXO-RB44 cells apoptosis was significantly decreased with the MEG3 knockdown(P ＜ 0.05),compared with the control group,respectively.Furthermore,Western blot showed that P53 protein level was significantly increased after SO-RB50 transfected with pcDNA-MEG3 (P ＜ 0.05),while significantly decreased after HXO-RB44 transfected with siRNA-MEG3 (P ＜ 0.05),compared with the control group,respectively.Conclusion MEG3 is down-regulated in retinoblastoma,affect the development of retinoblastoma,and may induce the retinoblastoma cell apoptosis by promoting the expression of P53 protein.

Based on the requirements of Good Preparation Practice in Medical Institutions(GPP) and considering the actual conditions of the hospital,we decided upon a right way in the reconstruction of the hospital's preparation room and amelioration of the equipment and apparatus that constituted 30% of the investment.The results were reduced investment,higher efficiency,with perfect conformity to the GPP requirements.

BACKGROUND:Al ogeneic penetrating keratoplasty is the most effective method for treating corneal blindness. However, the incidence of rejections is high after keratoplasty, so it is urgent to develop an immunosuppressive drug with high efficacy and low toxicity. OBJECTIVE:To establish al ogeneic penetrating keratoplasty models and monitor the expression of tumor necrosis factor-αin blank control group and after transforming growth factor-β1 eyedrop during acute rejection period of corneal grafts. METHODS:Al ogeneic penetrating keratoplasty models were established and were randomly divided into blank control group, ciclosporin A group (1%ciclosporin A), and transforming growth factor-β1 group (1μg/ml transforming growth factor-β1 eyedrop). The medications from each group commenced at 1 day after surgery, one eyedrop once, three eyedrops per day. Al the operated eyes were given 0.3%ofloxacin ophthalmic solutions and 0.5%tropicaide ophthalmic solution, three times per day, for 12 days. The corneal grafts were harvested for hematoxylin-eosin staining and immunihistochemical staining (SABC method), to detect tumor necrosis factor-αexpression in corneal grafts. RESULTS AND CONCLUSION:Hematoxylin-eosin staining showed that, corneal grafts were significantly thickened, a large number of histoleucocytes and lymphocytes infiltrated in the blank control group;corneal grafts showed normal thickness and no inflammatory cel s infiltrated in the transforming growth factor-β1 group. Immunohistochemical staining showed that, there were less cel s positive for tumor necrosis factor-αin the transforming growth factor-β1 group compared with the blank control group (P<0.05). Transforming growth factor-β1 eyedrops can reduce the expression of tumor necrosis factor-αin the corneal grafts during acute rejection period, and reduce the inflammatory cel s infiltration in the corneal grafts, which is probably the mechanism of transforming growth factor-β1 to prevent and treat corneal al ograft rejection.

Objective: To study the effect and mechanism of Rhodobryum giganteum (Schwaegr.) Par. 's anti-atherosclerotic effect. Methods:Vascular endothelial ceils were cultivated and the H2O2 were used to induce the oxidative stress injury of human umbilical vascular endothelial cells (HUVEC). Water extract of Rhodobryum giganteum (Schwaegr.) Par. Was added to cultivated HUVEC, and the activity of the cells was carefully determined by OD value with MTF method. The Griess Reagent was used to detect the NO concentration of different groups. At the same time, the NO fluorescence analysis probe, DAF-FM DA. (3-amino, 4-aminomethyl-2', 7'-difluorescein, diacetate), was used to determine the activity of NO synthase. Results:The most suitable stimulating concentration of H2O2 on HUVEC is 12.5 mmol· L-1. Water extract of Rhodobryum giganteum (Schwaegr.) Par. Was co-cultured with HUVEC damaged byH2O2 .The OD values indicate that 3.33,2.50 mg· mL-1 water extract groups increase activity of the cells significantly compared with the model group (P <0.05) ,and 3.33 mg·mL-1 is much better than 2.50 mg·mL-1 dose group. To determine the content of NO and NOS, the 2.50 mg·mL-1 dose has significant effect (P < 0.05). Conclusions: The H2O2 concentration of 12.5 mmol· L-1 could be used to establish the injured model of endothelial cell successfully. 2.50 and 3.33 mg·mL-1 water extract of Rhodobryum giganteum (Schwaegr.) Par. Have protective effect on ECV304 injured by H2O2. 2.50 mg· mL-1 water extract of Rhodobryurn giganteum (Schw aegr.) Par could increase the activity of NOS and promote the synthesis and secretion of NO.

BACKGROUND: Nuclear factor-kappa B (NF-κB) plays a key role in regulating expressions of cytokines and adhesion factors during transcription and in central adjustment during corneal graft rejection reaction.OBJECTIVE: To study the dynamic expressions of NF-κB, intercellular adhesion molecular 1 (ICAM-1) and vascular endothelial growth factor (VEGF) in corneal graft and to investigate the interventional effect of cyclosporin A (CsA). DESIGN, TIME AND SETTING: A randomized controlled animal study was performed in the Department of Ophthalmology, Zhujiang Hospital, the First Military Medical University of Chinese PLA between January and July 2005.MATERIALS: 40 SD rats and 50 Wistar rats were included and randomly divided into three groups, syngenic transplantation (10 Wistar rats used as donor and another 20 Wistar rats used as acceptor), allogeneic transplantation (10 Wistar rats used as donor, and 20 SD rats used as acceptor), and allogeneic transplantation+CsA treatment (10 Wistar rats used as donor, and 20 SD rats used as acceptor).METHODS: Comeal transplantation models were established. Gentamicin (2 000 U) was subconjunctivally injected into the experimental eyes of all ecceptors every other day for three times in total; chloroptic (2.5 g/L) was then used two droplets every time, twice a day, and 18 successive days in total; additionally, tropicamide (5 g/L) was also used two droplets every time, once a day, and 7 successive days in total. One day after corneal transplantation, CsA eye droplet (10 g/L) was used in the allogeneic transplantation+CsA treatment group two droplets every time, three times a day, and 18 successive days in total.MAIN OUTCOME MEASURES: Scores of corneal graft rejection reaction index were measured at day 3, 7, 12, and 18 after corneal transplantation; pathological changes of corneal graft were observed at the same time points to detect expressions of NF-κB, ICAM-1, and VEGF.RESULTS: Rejection reaction was not observed in the syngenic transplantation group at 18 days; however, indexes of rejection reaction in the allogeneic transplantation group were higher than those in the syngenic transplantation group at four time points (P<0.05), but indexes in the allogeneic transplantation+CsA treatment group were lower than those in the allogeneic transplantation group (P<0.05). Immunohistochemical examination indicated that NF-κB, ICAM-1, and VEGF were located at corneal epithelial lamina and substantia propria layer and in newborn vascular endothelial cells. At each time point, expressions of NF-κB, ICAM-1, and VEGF in the allogeneic transplantation group were higher than those in the syngenic transplantation group (P<0.05) but lower than those in the allogeneic transplantation+CsA treatment group (P<0.05).CONCLUSION: CsA can weaken nuclear translocation and activity of NF-κB to inhibit expressions of cytokines, adhesion molecules, and other relative factors so as to inhibit ccurrence and development of corneal graft rejection reaction.

BACKGROUND:Corneal neovascularization not only seriously affects vision, it is also a high-risk factor for the rejection after allogeneic ceratoplasty, thus it is always a hot issue and in ophthalmology to investigate the pathogenesis of corneal neovascularization and the inhibitors for blocking its formation.OBJECTIVE: To induce model of corneal neovascularization in rats using nylon suture, and investigate the mechanism of nuclear factor-кB (NF-кB) in the occurrence and development of corneal neovascularization using dexamethasone as the glucocorticoid inhibitor for corneal neovascularization.DESIGN: A randomized controlled trial.SETTING: Department of Ophthalmology, Zhujiang Hospital of Southern Medical University.MATERIALS: The experiments were carried out in the Zhujiang Hospital of Southern Medical University from January to April 2005. Fifty-five healthy male Wistar rats of clean degree were used. Rabbit-anti-rat NF-кB P65 monoclonal antibody, rabbit-anti-rat vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1)monoclonal antibodies, and 3,3-diaminobenzidine (DAB) kit were purchased from Wuhan Boster Biological Technology Co., Ltd.METHODS: ① Interventions: The rats were randomly divided in the saline control group (n =25), dexamethasone group(n =25) and normal comea group (n =5). Corneal neovascularization using nylon suture was induced in rats in the saline control group and dexamethasone group, and then saline and dexamethasone was dropped to the right eye of the rats respectively, 2 drops for each time, 3 times a day for 18 days. Not any treatment was given to the rats in the normal cornea group. ② Evaluations: The score of corneal neovascularization was evaluated in the saline control group and dexamethasone group at 1, 3, 7, 12 and 18 days postoperatively. Corneal sections were prepared to observe the histological changes of cornea under light microscope; The expressions of NF-кB, VEGF and ICAM-1 were detected with immunohistochemical staining.MAIN OUTCOME MEASURES: ① Score of corneal neovascularization; ② Histological changes of cornea; ③ Expressions of NF-кB, VEGF and ICAM-1 in cornea.RESULTS: ① Score of corneal neovascularization: The corneal neovascularization was obviously inhibited, and scores of corneal neovascularization at different time points were all significantly lower than those in the saline control group (P ＜0.05-0.01). ② Histological changes of cornea: In the dexamethasone group, corneal neuvascularization and the infiltration of inflammatory cells after suture were obviously alleviated as compared with those in the control group, and the corneal structures in each layer were relatively complete. ③ Expressions of NF-кB, VEGF and ICAM-1 in cornea: In the dexamethasone group, the expressions of NF-кB, VEGF and ICAM-1 at each time points were all lower than those in the saline control group (P ＜ 0.05). The intensity of the expression of NF-кB was positively correlated with the score of corneal neovascularization and the expressions of ICAM-1 and VEGF (r =0.961, 0.922, 0.958, P ＜ 0.01).CONCLUSION: NF-кB is involved in the formation of corneal neovascularization possibly through upregulating the expressions of its downstream genes (VEGF, ICAM-1). Dexamethasone can inhibit the expressions of many factors related to corneal neovascularization regulated by NF-кB, including cytokines and adhesion molecules, through reducing the activity of NF-кB, and then suppresses the occurrence and development of corneal neovascularization.

Objective To evaluate the benefit of three dimensional (3D) reconstruction images with rotational digital subtraction technique for the clinical applications Methods Conventional two dimensional digital substraction angiography ( 2D DSA ) was obtained on A P and lateral view. Three dimensional digital subtraction angiography ( 3D DSA ) images were obtained by reconstruction of a rotational acquisition on a C arm (LCV+, GE Medical Systems) spinning at 40 degrees per second 53 cases of cerebral angiographies were performed (32 men and 21 women; the age ranged from 19 to 72 years, mean 46 3 years) Results In this series of 53 cases of cerebral angiographies, 5 cases of arteriovenous malformation were all correctly diagnosed by 3D DSA and 2D DSA . Seven cases were misdiagnosed as intracranial aneurysms at conventional 2D DSA but confirmed to be kinking of the vessel by 3D DSA . 41 cases were confirmed to be intracranial aneurysms Of the 41 cases, 5 cases were diagnosed as normal at 2D DSA but confirmed to be intracranial aneurysms at 3D DSA . The total consistency rate of 3D DSA and 2D DSA for the diagnosis of intracranial aneurysm is 77 4% (41/53) The consistent test shows that there was consistency between the two modalities (chi square test, ? 2=5 267, P

Background Primary open angle glaucoma (POAG) is one of the frequent glaucomatous types,and genetic factor participates in pathogenesis and development of the disease.Recently,MYOC mutation was found to be associated with POAG.Objective This study was to describe the clinical and genetic findings in a POAG family from Luoyang,China.Methods This study protocol was approved by Ethic Committee of Affiliated First Hospital of Henan University of Science and Technology.The study adhered to Declaration of Helsinki.A POAG family with 29 members of 5 generations was surveyed and followed-up for 5-year duration.The mode of inheritance was determined by the pedigree analysis.The periphery blood sample was collected form 12 families and 100 health controls for the extraction of genomic DNA under the informed consent.The third exon and its flanking introns of MYOC were amplified,and quantitative real time PCR products were sequenced,and the structure and function of mutated gene were examined by restriction fragment length polymorphism analysis.The predicted effects of the detected variants on the secondary structure of MYOC protein were evaluated using Garnier-Osguthorpe-Robson (GOR) method,and homology analysis of protein was carried out by Blast software provided by National Center for Biotechnology Information (NCBI).Results This POAG family included 29 members of 5 generations,and the clinical data were not clear in 11 family members.Three individuals from 3 generations were determined POAG,another one was ocular hypertension,and 2 were carriers.Pedigree analysis appeared an autosomal dominant inheritance.In 12 subjects included 6 members genetically affected and 6 members with normal phenotype,the heterozygous mutation was found in the third exon of MYOC gene in 6 genetically affected members,which revealed a T→C transition at position 1021 (p.S341P),resulting in a switch of serine (Ser) to proline (Pro).It was a missense mutation abolished a CviKI-1 restriction site that segregated with the affected members.Secondary structure prediction of p.S341P suggested that myocilin protein was misfolded.Analysis of protein homology and switched Ser was conservative amine acid at position 1021 (p.S341P).No similar change was found in the 6 normal families and the normal controls.Conclusions Ser341Pro MYOC mutation is disease-causing factor in the POAG family of Luoyang.The clinical and genetic features of this mutation warrant further investigation.The mutation spectrum of MYOC is expanded to offer a better diagnosis and treatment for POAG patients.

BACKGROUND:The preparation of recombinant human epidermal growth factor (rhEGF) and basic fibroblast growth factor (bFGF) has been clinical y used in the repair of ocular surface trauma. However, the concentration of these growth factors that achieve the maximal healing effect and the comparison of two kinds of growth factors on promoting wound healing are stil controversial.
OBJECTIVE:To investigate the effect of rhEGF and bFGF on the cloning of human corneal epithelial cells.
METHODS:The human corneal epithelial cells cultured in vitro were interfered with rhEGF and bFGF under different concentrations. The proliferation of human corneal epithelial cells was detected using MTT assay after 3, 5, 7 days of growth factors treatment. Plate clone formation assay was applied to observe the morphology of cellclone and analyze clone formation rate of human corneal epithelial cells.
RESULTS and CONCLUSION:MTT value shows that 10μg/L rhEGF and 10μg/L bFGF on day 5 were the most powerful concentration. The clone formation rate of human corneal epithelial cells after treated with 10μg/L rhEGF was higher than that with 10μg/L bFGF (P=0.02). The results confirmed that both rhEGF and bFGF could promote the proliferation and increase clone formation ability of human corneal epithelial cells. 10μg/L rhEGF for 5 days achieves the best effect on promoting clone formation of human corneal epithelium cells.

BACKGROUND: Chinese medicines have better effects on preventing and treating diabetic nephropathy at early period, and the effects are caused by the diversity of the effective components of Chinese medicines which acts on the different targets of diabetic nephropathy.OBJECTIVE: To observe the effect of Xiaokening on the proliferation of mesangial cells under high glucose, and investigate the possible mechanism. DESIGN: A controlled observation.SETTING: Department of Endocrinology, the First Affiliated Hospital of Nanjing Medical University.MATERIALS: The experiments were completed in the Research Room of Cell Culture, Research Center of Endocrine Metabolism, the First Affiliated Hospital of Nanjing Medical University from July 2003 to May 2004. The rat mesangial cell lines HBZY-1 were bought from Chinese Center for Typical Culture Collection.METHODS: The mesangial cells were passaged and cultured according to the instructions. ① Grouping according to different concentration of stimulation: In the high glucose+Xiaokening group, Xiaokening of 6 concentrations were used, I.e., 20.0, 40.0, 60.0, 80.0, 120.0, 200.0 g/L. Meanwhile,normal glucose control group and high glucose control group were also set,the glucose concentration in the medium was 5.6 and 30 mmol/L respectively. The proliferations were observed after 72 hours. Grouping according to different time points of stimulation: There were normal glucose control group, high glucose control group and high glucose+Xiaokening group, the glucose concentration in the medium was 5.6, 30 and 30 mmol/L respectively, and the Xiaokening concentration was 60 g/L. The proliferations were observed at 24, 48 and 72 hours respectively in the three groups.② The dispensed cell suspension was placed into the 96-well plate, and 200 μL suspension for each well. The culture plate was placed in the inoculation box containing CO2 (0.05 in volume fraction) at 37 ℃ for 24 hours, then the supernatant was deserted, cell solutions containing different drugs of different concentrations were added in each group, 200 μL for each well. The 96-well plate was then placed in the culture box containing CO2 (0.05 in volume fraction) and 100% humidity at 37 ℃ for inoculation.In the groups of different concentrations, 20 μL MTT (5 g/L) was added into the wells after 72 hours. In the groups of different time points, 20 μL MTT (5 g/L) was added into the wells at 24, 48 and 72 hours. Then the plates were inoculated at 37 ℃ for 4 hours. Under microscope, it was observed that MTT get into the cells, the supernatant was sucked away, then 150 μL dimathyl sulfoxide was added to dissolve methyl alcohol, and shaken to even with plate rocking bed for 30 s, and the absorbance (A) values were recorded with the microplate reader at the wavelength of 492 nm.MAIN OUTCOME MEASURES: The proliferations of mesangial cells (A value) were observed at different time points and in Xiaokening of different concentrations.RESULTS: ①Proliferation of mesangial cells at different time points: The A values in the high glucose control group at 24, 48 and 72 hours (0.685 ±0.010, 0.750±0.087, 0.659±0.018) were higher than those in the normal glucose control group (0.586±0.054, 0.598±0.040, 0.527±0.047, P ＜ 0.05). In both the high and normal glucose control groups, the A value at 48 hours was higher than that at 24 hours (P ＜ 0.05), but the A value at 72 hours was lower than that at 48 hours (P ＜ 0.05). The A value in the high glucose+Xiaokening group at 72 hours was 0.538±0.023, and it was lower than that in the high glucose control group (P ＜ 0.05). ② Proliferation of mesangial cells in Xiaokening of different concentrations: Xiaokening high er than 60.0 g/L could obviously restrain the excessive stimulation of high glucose to the mesangial cells, and the effect was in a concentration-de pendent manner, but too high concentration (120.0 g/L) would result in the abscission and death of cells, whereas no survived cells could be observed in extremely high concentration (200.0 g/L). CONCLUSION: Xiaokening could inhibit the proliferation of mesan gial cells stimulated by high glucose after 72 hours in a concentration dependent manner, but too high concentration would cause strong cytotoxicity.