Effects of static pressure on the expression of ET-1 and NO in cultured retinal microvascular endothelial cells / 眼科研究

ABSTRACT

Background Researches have demonstrated that ocular hypertension induces the ischemia-reperfusion of retina and further leads to the degeneration of retinal ganglion cells,but its mechanism is beyond understanding.Objective The present study aims to observe the effects of static pressure on the morphology,proliferation activity and viability of cultured retinal microvascular endothelial cells (RMECs) and evaluate the expression of ET-1 and NO in these cells under variant static pressure.Methods RMECs were isolated from 30 healthy Wistar rats and cultured using explant culture method by Ⅷ factor antibody and PECAM-1 antibody.The static pressure of 1.33kPa,2.67kPa,5.33kPa and 10.67kPa was used in culture bottle respectively.The RMECs without static pressure were used as normal control group.The morphology of RMECs under the different static pressure was observed by inverted phase contrast microscopy,and the number of RMECs was counted using the counting plate.Cellular viability was studied by trypan blue staining.The changes of ET-1 and NO_2~-/NO_3~-,two metabolic products of NO,in the medium were detected by radioimmunoassay and Griess's nitrate reductase method.The expression of ET-1,eNOS and iNOS mRNA in RMECs was analyzed by semi-quantitative RT-PCR 24 hours after treatment of variant static pressure.Results Cultured RMECs sticked well at 24 hours and reached to confluence at 48 hours and showed the red fluorescence for Ⅷ factor antibody and PECAM-1 antibody.Enlargement of nuclei,extenders of cell bodies and suspension of RMECs in medium were observed.The number of RMECs was gradually increased.The cell viability was reduced with the raise of static pressure among these four groups(F=12.205,P＜0.01;F=11.180,P＜0.01).The static pressure increased the content of ET-1 released by RMECs in 2.67kPa,5.33kPa and 10.67kPa of static pressure groups,and concentrations of NO_2~-/NO_3~- in the medium showed a significant increase in 5.33kPa and 10.67kPa of static pressure groups compared with normal and 1.33kPa of static pressure groups(P＜0.01).The expressions of ET-1 mRNA,eNOS mRNA and iNOS mRNA were considerably enhanced in 5.33kPa and 10.67kPa of static pressure groups compared with normal control group(P＜0.01).Conclusion Raised static pressure causes the alteration of RMGCs structure and morphology.Static pressure could upregulate the expressions of ET-1,eNOS and iNOS mRNA in RMECs and increase the release of ET-1 and NO.This pathway might be one of pathologic mechanisms of retinal injury induced by high intraocular pressure.