Effect Of3-aminobenzamide On Proliferation Of Bovine Retinal Capillary Endothelial Cells Cultured In High Concentration Of Glucose

[Background&Objective] Diabetic retinopathy, one of the serious complicationsof diabetes, causes serious vision loss. It mechanisms has already attracted wideattention. With BREC extracted and cultured in vitro, the research is to investigate theeffects of high glucose on bovine retinal capillary endothelial cells, and to explore theeffect of3-AB on proliferation and apoptosis of BREC cultured in high concentration ofglucose. The purpose of the study is to find the theoretical basis and therapeutic methodto treat diabetic retinopathy.[Methods]①With the isolation of active retinal blood vessels, BREC were culturedin Endothelial Cell Medium(ECM). BREC were identified by immunohistochemicalmethod of Von Willebrand factor and then draw the growth curve.②After passage, thecells were set to different concentration of glucose, mannitol group and control group.MTT assay was used to describe the cells proliferation in different groups. Flowcytometry was used to observe cell apoptosis during the culture.③BREC were culturedin Endothelial Cell Medium(ECM) containing30mmol/L of glucose and0.05,0.1,0.25,0.5,0.75,1mmol/L of3-AB were added in above medium respectively. MTTassay were used to observe the effect of3-AB on the cell proliferation. Flow cytometrywas used to analyze cell apoptosis during the culture, and Annexin-V marked the earlierapoptosis cells, while PI marked the dead cells.[Results]①The selectively cultured BREC can be reproduced. The expression ofVon Willebrand factor was positive. The growth curve showed that the cells in thepassage after the first3to4days proliferated by log, and10to12days into the platform period.②While the concentration of glucose between10mmol/L and25mmol/L, MTT showed the proliferation of the cells promoted. While the concentration of glucoseover30mmol/L, MTT showed that the proliferation of the cells inhibited, and flowcytometry showed the apoptotic peak in the cell cycle increased.③The OD value ofcultured cells was obviously increased after treated with3-AB in0.25mmol/L ofconcentration groups in comparison with that of the control group(P<0.01), showing thesurvival rate of BREC was higher after treated with3-AB. Flow cytometry showed thatthe endothelial cells cultured in high concentration of glucose with0.25mmol/L3-ABhad lower cell apoptosis rate compared with those cultured in other groups.[Conclusion]①The high purity BREC could be obtained though the selectivelycultured method which is simple and repeatable without any other procedures.②Theproliferation of BREC were promoted in a certain concentration of glucose, whileinhibited in the high concentration of glucose.③3-AB can improve the survival rate ofBREC cultured in high concentration of glucose, the mechanism may be the certainconcentration of3-AB can reduce these cell apoptosis.