Research On Degree Of Cross Linking Of Cross-linked Allogenic Tissue By 5-FAMSE Fluorescence Intensity Analysis

Artificial small vessel grafts used for bypass grafting surgeries have been limited because of many unsolved problems. Instead of autologous vein grafts,cross-linked allogenic small vessel grafts are the most prospective grafting materials and leading the researches focus.Glutaraldehyde is a cross linking agent in common use and the cross linking mechanisms are explicit.Cross linking happens at amino terminus and the effect is confirmed. However,glutaraldehyde cross-linked vessels have a series of adverse reactions,such as cytotoxicity,tissue calcification and so on.A new method of cross linking by dye-mediated photooxidation has no adverse reactions of glutaraldehyde,but the mechanisms are still ambiguous.In this research we discover and use a new method—fluorescence intensity analysis to detect the degree of cross linking.Fluorescence material-5-Carboxyfluorescein N-succinimidyl ester can be specifically bound with amino terminus of protein and show brightly green fluorescence excited by 488 nm wavelength light. According to the character mentioned above,it can be used to detect the degree of cross linking of small vessels treated with glutaraldehyde and dye-mediated photooxidation and explore the cross linking mechanism of dye-mediated photooxidation initially. Part one Research on Detection of Degree of Cross Linking of Small Vessels Treated with GlutaraldehydeOBJECTIVE:To detect the degree of cross linking of small vessels treated with glutaraldehyde in different concentrations,based on the method of fluorescence intensity analysis and polyacrylamide gel electrophoresis.5-Carboxyfluorescein N-succinimidyl ester can be specifically bound with amino terminus of protein and show brightly green fluorescence excited by 488 nm wavelength light.METHODS:Decellularized thoracic aorta of New Zealand Rabbits were divided randomly into four groups,which were group A(control group—untreated with glutaraldehyde),group B(cross-linked with glutaraldehyde in concentration of 0.625%),group C(cross-linked with glutaraldehyde in concentration of 1.25%),group D(cross-linked with glutaraldehyde in concentration of 2.5%).The cross linking time in all groups of thoracic aorta specimens were 30 minutes.After eluting the remnant glutaraldehyde,the aorta were made into frozen section.Then the frozen section reacted with 5-Carboxyfluorescein N-succinimidyl ester for 2 hours.After eluting the remnant 5-Carboxyfluorescein N-succinimidyl ester,fluorescence intensity was observed through fluorescence microscope.Meanwhile,the tissue proteins extracted from thoracic aorta were used for polyacrylamide gel electrophoresis.The two ways were used to detect the degree of cross linking of small vessels treated with glutaraldehyde.RESULTS(1) Fluorescence intensity was different significantly,group A＞group B＞group C＞group D.There was a significant statistically difference(P＜0.01).(2) The tissue proteins extracted from thoracic aorta treated with glutaraldehyde were smaller than untreated with glutaraldehyde. However,the tissue proteins extracted from thoracic aorta treated with glutaraldehyde in different concentration were no significant differences.CONCLUSIONS5-Carboxyfluorescein N-succinimidyl ester can detect the degree of cross linking of small vessels treated with glutaraldehyde more conveniently and directly;in a certain phase,the degree of cross linking was increased with the increase of glutaraldehyde concentration.Part two Research on Mechanism and Degree of Cross Linking of Dye-mediated Photooxidation Cross-linked Small Vessels InitiallyOBJECTIVE:To research the mechanism and degree of cross linking of dye-mediated photooxidation cross-linked small vessels initially by evaluating the changes of amino terminus,according to the character of 5-Carboxyfluorescein N-succinimidyl ester which can be specifically bound with amino terminus of protein and show brightly green fluorescence excited by 488 nm wavelength light.METHODS:Decellularized thoracic aorta of New Zealand Rabbits were divided randomly into three groups,which were group A(control group—uncross linked by dye-mediated photooxidation),group B(cross linked by dye-mediated photooxidation for 2 hours),group C(cross linked by dye-mediated photooxidation for 4 hours),After eluting the remnant dye,the aorta were made into frozen section.Then the frozen section reacted with 5-Carboxyfluorescein N-succinimidyl ester for 2 hours.After eluting the remnant 5-Carboxyfluorescein N-succinimidyl ester,fluorescence intensity was observed through fluorescence microscope.Therefore the changes of amino terminus can be evaluated,the mechanism of cross linking of dye-mediated photooxidation can be explained initially and the degree of cross linking can be detected.RESULTS:Fluorescence intensity was different significantly, group A＞group B＞group C.There was a significant statistically difference(P＜0.01).CONCLUSIONS:At least partly the small vessels were cross linked at amino terminus by dye-mediated photooxidation because of the reduction of amino terminus;in a certain phase,the degree of cross linking was increased as time of light exposure prolonged.