Objective To determine whether kringle 4-5 could inhibit choroidal neovascularization (CNV) in mice induced by argon laser photocoagulation. Methods Fundus laser photocoagulation was performed on C57BL/6J mice to induce CNV. In treatment group, 20 ?g (low dosage group) and 50 ?g (high dosage group) kringle 4-5 were injected retrobulbarly after photocoagulation. In control group, equilibrium liquid was injected retrobulbarly. Choroidal neovascularization was evaluated on the 7th and 14th day after photocoagulation by fundus fluorescein angiography. The mice were killed on the 14th day after photocoagulation, the lesions were evaluated histologically and immunohistochemically, and the expression of CD105 was detected. The Expression of VEGF and bFGF was detected by immunohistochemistry on the 4th day after photocoagulation. Results The incidence of CNV was 64.3% in control group, 51.2%(P

Objective: To evaluate the frequency of MSI in epithelial ovarian tumors and its relationship with clinicopathologic features. Methods: Ninety fresh specimens of epithelial ovarian tumors, including 74 primary and 16 secondary tumors, were collected. Microsatellite analysis was carried out using 5 mono- and dinucleotide markers from the National Cancer Institute Consensus Panel by fluorescence-labeled polymerase chain reaction. Results: Of 90 epithelial ovarian tumors analyzed, 18 demonstrated a high level of microsatellite instability (MSI-H), 30 demonstrated a low level of microsatellite instability (MSI-L), and the remaining 42 exhibited microsatellite stability (MSS). Frequency of microsatellite instability (MSI) at loci BAT-25 was higher than that at any other loci. No correlation was found between MSI level and patient age, tumor type, tumor differentiation (P>0.05). But the microsatellite instability-high phenotype correlates with clinical stage.It tended to occur more frequently in early-stage tumors (P=0.03). Conclusion: The frequent MSI in epithelial ovarian tumors suggests that it is an early event to involve in the development of epithelial ovarian tumors.

Aim To produce poly (lactic-co-glycolic acid) (PLGA) microspheres, containing a staphylokinase variant (K35R, DGR) with reduced immunogenecity and antiplatclet aggregation activities,which allowed the preservation of protein stability during both particle processing and drug release.Methods DGR-loaded microspheres were fabricated using a double emulsion-solvent evaporation technique. The effects of preparative parameters, such as stirring rate, polymer concentration, and the excipients of both internal and external aqueous phase (W2 ), on DGR encapsulation efficiency and microsphere characteristics were investigated. In vitro and in vivo release of DGR were conducted and the cause for instability of DGR during release was also investigated. Results Moderate ultrasonic treatment of aqueous DGR/dichloromethane mixtures caused approximately. Eighty four per cent DGR denaturation.However, the activity recovery of DGR almost amounted to 100% when 2% polyvinyl alcohol (PVA) was added into the aqueous phase. It was found that NaCl in the external water phase significantly increased DGR encapsulation efficiency. Furthermore, NaCl in the external water phase played a role in determining size and surface morphology of microsphere. In vitro release test showed a burst release of DGR from microspheres, followed by sustained release of 50% total activity over 15 days. In vivo experiments showed that DGR released from microspheres sustained 5 days. Denaturation of DGR within microspheres might be resulted from acidic microclimate. Conclusion The stability of DGR was effectively protected during microencapsulation and a relatively high encapsulation efficiency of DGR was obtained. PLGA microspheres could be an effective carrier for DGR.

Purpose To purify recombinant angiogenesis inhibitor r-K4K5 and investigate its inhibitoryeffects on bovine capillary endothelial (BCE) cell proliferation, chick embryo chorioallantoic membrane(CAM) angiogenesis and growth of experimental human non-small cell lung cancer (adeno). Methodsr-K4K5 was obtained by salting out and gel filtration with the purity of 95% determined by SDS-PAGE.BCE cells were cultured with DMEM media containing r-K4K5. The cells were counted in 24,48,72 hrespectively. r-K4K5 was injected daily into all 7-day chick embryo CAMs and CAM angiogenesis wasobserved at 72 h after incubation. The Balb/c (nu/nu) mice implanted with human SPC-Al tumor pieceswere grown for 10 days and then randomly divided into three groups. One group was treated with PBS, theother two groups were treated with local subcutaneous injection of purified r-K4K5 at 8 μg and 80 μg lpermouse every other day. They were daily observed and sacrificed in 14 days. Each tumor was weighed.Results The number of BCE cells, blood vessels diameter less than 50 μn of chick embryo CAM and theaverage weight of experimental tumor were decreased markedly in all the groups treated with r-K4K5.Conclusions r-K4K5 inhibits proliferation of BCE cells, angiogenesis of chick embryo CAMs and thegrowth of experimental human SPC-A1 non small lung cancer (adeno).

Objective To master the technique of mouse embryonic stem (ES) cells differentiate into endothelial cells,which would be a new therapeutic approach for cardiovascular disease.Methods Expression of selfrenewal marker genes in E 14 cells was assessed.Expression of vascular endothelial growth factor receptor 2 (Flk 1) in monolayer differentiation on day 4 and vascular endothelial cadherin (VE-cadherin) on day 8 were detected.On day 8,differentiation cells were also observed under phase contrast microscopy (PCM) and transmission electron microscope (TEM).ES cells and endothelial-specific molecular markers were assessed by RT-PCR at different time-points.Results As self-renewal marker genes were expressed in E14 cells,E14 cells was identified to maintain their selfrenewal pluripotency.The marker gene of letarl,Flk1 was expressed on differentiation day 4.On differentiation day 8 the marker gene VE-cadherin was expressed and as observed under PCM endothelial cells with spindle shape and TEM with Weibel-Palade body,thus were the major populations generated after VEGF induction,and E14 cells were confirmed differentiated into mature endothelial cells.The expressions of genes octamer binding transcription factor 4 (Oct4),Flk1 and VE-cadherin were detected on differentiation day 2,4,6,8 and 10.Conclusions As VE-cadherin gene was expressed in monolayer on differentiation day 8,E14 cells were confirmed differentiated into endothelial cells,which would be a new therapeutic approach for cardiovascular disease.

Background The vitreoretinal traction plays a critical role in the formation of macular hole and cystoid macular edema.Enzymatic vitreolysis has potential in relieving vitreoretinal traction as a simple and less invasive method in comparison with pars plane vitrectomy.ObjectiveThis study is to investigate the effects of plasmin mutant with kringle domains deficiency(Plm△K)on vitreoretinal interface in new Zealand white rabbits.Methods Plm△K was prepared through activating plasminogen mutant with Kringle domains deficiency (Plg△K) by tissue plasminogen activator (tPA).100μL of Plm△K at the dose of 0.5,1.0 and 1.5μmol/min was injected respectively into the vitreous of 48 New Zealand white rabbits and 16 eyes for each dose.B-scan and optical coherence tomography (OCT) were performed to detect the structure variety at the vitreoretinal interface in 1 day and 7 days after injection.The gross anatomy analysis with triamcinolone acetonide fine particle suspension,as well as histopathological examinations by scanning electron microscopy,was performed in the different time points mentioned above.Results Two peptide chains were determined with the relative molecular weight about 26000 and 5000 by the gel imaging analysis of reduced SDS-PAGE.Separation of the posterior vitreous cortex from retina was found after intravitreous injection under the B-scan and OCT.The ultrastructure change of vitreoretinal interface as well as the examination of fine particle suspension by triamcinolone acetonide demonstrated the same outcome.The degree of remnants of vitreous cortex showed the negative correlation with the dosage of Plm△K (r=-0.9516,P=0.048).No significant correlation was found between the degree of remnants of vitreous cortex and the action time(r=-0.720,P=0.470).There was no obvious morphological difference in outer layer of retina between control eyes and Plm△K-treated eyes.No drug-related adverse event was found after intravitreous injection of Plm△K.Conclusion Intravitreous injection of Plm△K alone can induce complete separation of vitreous from retina.This procedure is safe and effective.

A sandwich ELISA for the measurement of urokinase(UK)antigen was developed based on anti—UK monoclonal antibodies(McAbs)against two non—overlapping epitopes.The lower limit of sensitivity of the assay was 0.15ng/ml.Coefficients of variation of the assay at physilogi-cal levels of UK were 4.3 percent within assaysw and 8.7 percent between assays.The recovery of added UK was about 98 percent.Culture media of some human malignant cell lines contains more UK than that of normal cell lines measured by our assay.The ELISA was used to measurethe concentration of UK in plasma from 82 healthy donors.The mean value for the healthy donors was 1.31?60.6ng/ml of UK in plasma.

Objective To study the effect of retinal dehydrogenase type 2 inhibitor (4-diethylaminobenzaldehyde,DEAB) on embryonic CSrdiac develclpment of zebrafish model with retinoic acid(RA)deficiency. Methods Zebrafish embryos were treated with DEAB at various concentrations including 1×10~(-6),5×10~(-6),10×10~(-6),25×10~(-6)mol/L at 5,8 and 10.3 hours post fertilization,respectively.The effects of DEAB on the embryonic development were assessed under microscope.1×10~(-9)mol/L exogenous RA was then added to detect the antagonistic effect against DEAB.The abnormal cardiac phenotype,heart rate and ventricular systolic fraction were observed and analyzed between wild type and DEAB treated groups.The expression of specific cardiac gene, natriuretic peptide precursor A,was monitored by whole-mount in situ hybridization to demonstrate the effect of RA signaling on early cardiac development. Results The survival rate of zebrafish embryos declined with the increase of DEAB concentration at different developmental stage.The percentage of abnormal embryos reached 100% when DEAB over 5×10~(-6)mol/L.1×10~(-9) mol/L exogenous RA could eliminate the teratogenic effect of DEAB(≥5×10~(-6)mol/L).DEAB treated embryos presented abnormal cardiac phenotype,including tubular heart,incomplete D-loop,abnormal atrioventricular development,regurgitation,slow blood flow and weak heart beat.The difference of heart rate and ventrieular systolic fraction between wild type and RA deficiency embryos was of statistical significance(P<0.05).The natriuretic peptide precursor A expression remained in the ventricle,but reduced obviously in the atrium with RA signaling deficiency. Conclusions The effects of DEAB on the embryonic development are dose-dependent and time-dependent,and could be rescued by exogenous RA.RA signaling plays a critical role in several key stages of early cardiac development and natriuretie peptide precursor A expression.

The primary neural stem cells were isolated from SD rat and formed the neuropheres, the neuropheres were passaged and planted on the dish coated with 0.1% gelatin, the colony was picked up under the microscope, then dispersed and cultured, to obtain the clone proliferated from one cell, passaging and picking up the cells 5～6 times at least. The NSC and its differentiated cells were identified with the marker genes respectively. The results showed that the neural stem cells were isolated from the SD rat embryos and the real clone were obtained by picking up the cells again and again, and then cultured in the form of monolayer. The marker genes of the neural stem cells and its differentiated cells could be detected at last. It will provide the rat model the resource of the cells for the treatment and the basic research for the morphology standard.

Objective To study whether the expression Smad 7 protein by the recombinant adenovirus with Egr-1 promoter and Smad 7 cDNA in fibroblast cell can block the signal transduction pathway of transforming growth factor-beta1 (TGF-?1) under irradiation thereby inhibiting collagen synthesis in vitro. Methods The location of endogenous Smad 7 and exogenous Smad 7 protein in recombinant adenovirus infected fibroblast cells(3T6) were determined by immunocytochemical method. The infected 3T6 cells were irradiated and then cultured with TGF-?1 4 hours after irradiation. The activity of preventing radiation-induced fibrosis by expression Smad 7 protein was evaluated by the amount of collagen synthesis and proliferation of 3T6 cells. The amount of collagen synthesis was shown by the coruscant per minute (cmp) through the 3?H-Proline incorporation technique. Results The endogenous Smad 7 and exogenous Smad 7 protein both were located in the cytoplasm. When cultured with TGF-?1 4 hours after irradiation, the amount of collagen synthesis in the 3T6 cells infected with the recombinant adenovirus was significantly less than that in the cells without infecting adenovirus after irradiation(P=0.001), But, there was no difference in the proliferation of 3T6 cells between those with and without adenovirus infection (P= 0.312 ). Conclusions The Egr-1 promoter in the recombinant adenovirus can regulate the expression of downstream Smad 7 cDNA in 3T6 cells. The expression Smad 7 protein could block the TGF-?1 signal transduction pathway thereby inhibiting the collagen synthesis. The mechanism of inhibiting the collagen synthesis may be accomplished at the transcription level.