Study On Vascular Endothelial Cell Injury Induced By High-concentration Glucose And Photosensitive Reactive Oxygen Free Radicals And Determination Of Endothelin

In this thesis,the function,injury and injury markers of endothelial cells（ECs）and the damage of reactive oxygen species to organism were introduced.The electrochemical biosensors and research prospects of electrochemical biosensors were described and briefly summarized.ECs were selected as the research object.The influences of high-concentration glucose and photosensitive active oxygen free radical on cells activity and the protective effect of a small molecule reductant,berberine,on cell injury were investigated.An electrochemical method for the detection of endothelin,a damage marker,was developed by using nanocomposites based on Fenton reaction.The main contents of the research work are listed as follows:1.The cell morphology of human umbilical vein endothelial cells（HUVECs）in the growth medium containing glucose with different concentration（11 m M,20 m M,50 m M and 100 m M）was observed by using the microscope.The effect of glucose concentration on the activity of HUVECs was studied by MTT colorimetry and electrochemical analysis.It is found that the environment containing high-concentration glucose could lead to the obvious change of cell morphology and the decrease of cell activity.The degree of cell injury increased with the enhancement of glucose concentration in the growth medium.0.01μM and 0.1μM berberine played an obvious protective role in HUVECs that suffered from injury in the presence of 50 m M glucose,but the function of 1μM berberine was not obvious due to its own cytotoxicity.The cell morphology and MTT measurement results show that the oxidant H₂O₂could cause oxidative injury of HUVECs and the cellular damage was inhibited by 0.1μM berberine.However,the co-action of H₂O₂and high-concentration glucose resulted in severe cell injury and the protective effect of berberine was insignificant.2.Thechemicalreactionsofphotoilluminated aminophylline-riboflavin（Am-Rf）system were investigated by using absorption spectrometry.The results of cell morphology observation and MTT colorimetric analysis show that ¹O₂and ³O₂produced by photoilluminated Rf and hydroxyl radical（·OH）derived from photoilluminated Am-Rf system led to HUVECs injury,and the cellular damage induced by·OH was more serious.The activity of HUVECs that suffered from injury by photoilluminated Rf and Am-Rf systems was found to increase by 5%in the presence of berberine.It indicates that berberine exerted a certain repairing effect on ECs injury caused by photosensitive reactive oxygen species.In the growth medium containing high-concentration glucose,photoilluminated Rf and Am-Rf systems generated more serious damage of HUVECs,which indicates that these vascular endothelial cells were more susceptible to oxidative damage under the condition of high-concentration glucose.The activity of cells increased less than 2%in the presence of berberine,suggesting that the injury mechanism of HUVECs was complicated and the protective function of small molecules with reducibility were limited in that case.3.A glassy carbon electrode（GCE）modified with poly（thiamine）（PTH）was prepared based on cyclic voltammetry（CV）and endothelin（ET）,antigen（Ag）,was immobilized on the electrode surface by using glutaraldehyde.ET antibody-gold nanoparticles-Cu（II）composites（Ab-GNPs-Cu（II））with enzyme-like function were synthetized and they were characterized by TEM,UV-vis and IR.The Fenton type reaction between Cu（II）and H₂O₂produced?OH and the latter could effectively oxidizePTH.Hence,themodifiedelectrode,GCE/PTH/Ag/Ab-GNPs-Cu（II）,displayed a significant reduction peak during the CV scan process in 0.1 M p H 6.5 PBS containing H₂O₂.The above modified electrode was assembled in the presence of the ET-composites mixture.The strong steric resistance effect on the electrode surface after the assembly of ET decreased the amount of immobilized Ab-GNPs-Cu（II）and covered up the catalytic sites of Cu（II）to a certain extent.As a result,the Fenton type reaction was inhibited and the reduction peak current on the CV curve declined.Therefore,a sensitive electrochemical method for the detection of ET was developed.Under the optimal conditions,a good linear relationship existed between the current signal change derived from the differential pulse voltammetry measurement and the ET concentration in the range of 0.5-500 ng m L^-1.Analysis of the growth media that were used for the culture of HUVECs and contained glucose with different concentration was performed.The results show that the higher the glucose concentration was,the greater the cell damage degree was,and the more ET the cells released.