Leech Extract On Cultured Rhesus Retinal Endothelial Cell Growth

OBJECTIVETo study the action of leech on retinal vascular epithelium cell in vitro,probe the action and mechanism of leech on anti-Vascularization,and give the prove for exploitation and study on new anti-Vascularization medicine.METHODS1.Cultured RF/6A in vitro,and identified cell by immunohistochemical method;2.Cultured RF/6A in vitro with different final concentration of Extract of leech (0mg/ml,8mg/ml,16mg/ml,32mg/ml,64mg/ml,128mg/ml),used Colorimetry MTT to detect viability of RF/6A at 24 hour,and calculated the 50%inhibitory concentration(IC₅₀) for choosing the right concentration of Exact of leech at 24 hour;3.Cultured RF/6A in vitro with different final concentration of thrombin(0u/ml,2.5u/ml,5u/ml,10u/ml,20u/ml),and used Colorimetry MTT to detect viability of RF/6A at 24 hour for choosing the right concentration of thrombin;4.Used flow cytometry to research the influence of Group Extract of leech,Group thrombin,Group mixing and the control on cell cycle of RF/6A cultured in vitro;5.Used euzymelinked immunosorbent assay to study the influence of every group on MMP-2 of RF/6A cultured in vitro;6.Used radio-immunity to study the influence of every group onâ…£-C of RF/6A cultured in vitro.RESULTS1.RF/6A had the feature of vascular epithelium cells,had positive antigen presentation of factorâ…§,could be usede to study the ophthalmic diseases;2.Exponential phase of growth of RF/6A cultured in vitro was the 48-72 hour;3.High concentration of Extract of leech had inhibition on proliferation of retinal vascular endothelial cell cultured in vitro,low concentration had enhancement on proliferation,the 50%inhibitory concentration(IC₅₀) at 24 hour was 97mg/ml,Extract of leech of concentration 64mg/ml was chosen to be the right concentration for this experiment;4.Thrombin of concentration 20u/ml had enhancement on proliferation of retinal vascular endothelial cell cultured in vitro,and was chosen to be the right concentration for this experiment;5.Most cells cultured in vitro were in the phrase G1 with Extract of leech,in the Group thrombin most were in the phrase S,Extract of leech had inhibition on the action of thrombin on generation cycle of retinal vascular endothelial cell cultured in vitro;6.The level of MMP-2 was decreased in Group Extract of leech,and it was increased in Group thrombin in vitro;7.The synthesis ofâ…£-C was increased in Group Extract of leech in vitro,and it was lightly increased in Group thrombin,while controlled with the normal,it was lower in Group thrombin,thrombin could enhance the degradation of ECM,which could be weekened by leech at 2h.CONCLUSION1.Extract of leech of concentration 64mg/ml has the inhibition on proliferation of retinal vascular endothelial cell cultured in vitro;2.Extract of leech of concentration 64mg/ml can change the growth cycle of retinal vascular endothelial cell cultured in vitro and inhibit the cell proliferation induced by thrombin;3.Extract of leech of concentration 64mg/ml can increase the synthesis ofâ…£-C, decrease the secretion of MMP-2 in the cell microenvironment cultured in vitro induced by thrombin,and change the microenvironment,maybe Extract of leech has the effect on anti-Vascularization.