| Research Purpose:1. To investigate the effects of ligustrazine under hypoxia states vascular and choroidal retinal endothelial cells of protection.2. To investigate the effects of ligustrazine under hypoxia states vascular and choroidal retinal vaso-motor effect.3. Through the experiment, thought and the methods to build up the reasonable application of the traditional Chinese medicine treatment of ischemic disease invigorate the eyes provide theory and experiment basis.Methods:1. Cultivate bovine retinal, people choroidal vascular endothelial cells, Bovine retinal endothelial cell lines (the sixth generation of) is bought in Beijing the sino-japanese friendship hospital cell bank, choroidal vascular endothelial cells is bought in Shanghai biological technology Co. We train the retina, choroidal vascular endothelial cells and we use The Ⅷ factor related antigen by immunohistochemical staining method appraisal endothelial cells.The experimental method:RVEC were cultured in vitro,will ChuanDai cells into three groups:a control group of normal cells; hypoxia model group; anoxia+different concentrations of ligustrazine(content of0.002,0.02,0.2and2μ g/ml ligustrazine medium for culture.) We use inverted phase contrast microscope to watch cell morphology change after24,48hours.By MTT method to detect the medium in conditions of the endothelial cells proliferation training.People choroidal vascular endothelial cells experimental methods are the same as above.RVEC were cultured in vitro,will ChuanDai cells into two groups:hypoxia model group; anoxia+different concentrations of ligustrazine(content of0.02,0.2μ g/ml ligustrazine medium for culture.)We collectt cells24hours after.We use western blotting method to detect effects of ligustrazine on vascular endothelial cells NOS, ET-1the influence of expression. People choroidal vascular endothelial cells experimental methods are the same as above.The results:The cart before the horse is examined under a microscope, the normal cells ChuanDai4h began to stick wall growth,confluent some radiate out in alignment,Living cells and bright,with a spindle shape, each other closely connected. Under hypoxic conditions, cell number significantly less, intercellular increase, visible parts dead cells, Ligustrazine injection effect24h,48h, hypoxic cells by growing the orange into brick red. Cells form some into short spindle shape, dead cell number significantly less.,cells growing number, piece fusion, cells arranged tightly, a typical paving the sample pebble structure.2.Bovine retinal vascular endothelial cellsMTT experiment①Lack of oxygen group and group compared to normal cells, its each time A value significantlyreduced.(P<0.01)②Ligustrazine group0.002u g/ml24h,48h role when the value is A lack of oxygen group had no significant difference contrast (P>0.05)③Ligustrazine group0.02～0.2u g/ml24h,48h role when the A value are higher than to anoxia group, and in certain concentration range with A value of drug concentration increased (P<0.05)④Ligustrazine group2μ g/ml24h,48h role when the value of A group than the other drugs decline. Immune imprinting method (western blot) tests showed that:ET-1protein expression:①Ligustrazine group0.02u g/ml24h role when its ET-1expression is significantly less oxygen control group (P<0.05); ②Ligustrazine group0.2u g/ml24h role when its ET-1express a low concentration of oxygen group, the group was significantly reduced in the control (P <0.05).NOS protein expression:①Ligustrazine group0.02u g/ml24h role the expression of oxygen control group is NOS increased significantly (P<0.05);②Ligustrazine group0.2u g/ml24h role the express a lack of oxygen group, NOS low concentration significantly higher contrast group (P<0.05). People choroidal vascular endothelial cells:MTT experiment①Lack of oxygen group and group compared to normal cells, its each time A value significantly reduced.(P<0.01)②Ligustrazine group0.002u g/ml24h, role when the value is A lack of oxygen group had no significant difference contrast (P>0.05)48h a significant increase in oxygen control group (P<0.05);③Ligustrazine group0.02～0.2u g/ml24h,48h role when the A value are higher than to anoxia group, and in certain concentration range with A value of drug concentration increased (P<0.05)④Ligustrazine group2μ g/ml24h,48h role when the value of A group than the other drugs decline.Immune imprinting method (western blot) tests showed that:ET-1protein expression:①Ligustrazine group0.02u g/ml24h role when its ET-1expression is significantly less oxygen control group (P<0.05);②Ligustrazine group0.2u g/ml24h role when its ET-1express a low concentration of oxygen group, the group was significantly reduced in the control (P <0.05). NOS protein expression:①Ligustrazine group0.02u g/ml24h role the expression of oxygen control group is NOS increased significantly (P<0.05);②Ligustrazine group0.2u g/ml24h role the express a lack of oxygen group(P<0.05).Conclusion:1、Under the anoxia state,it can cause endothelial cell activity reduced, but ligustrazine can restrain this state.Effects of ligustrazine on bovine retina vascular endothelial cells have certain promote reproductive role.,and with the dose dependent.2、Ligustrazine in certain concentration significantly to adjust the retina, choroidal vascular endothelial cells secrete a function and vaso-motor function (ET level down significantly, NO level to rise significantly).This may be protection retinal vascular endothelial cells and functions of one of the mechanism. |
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