5-羧基荧光素琥珀酰亚胺酯直观检测经交联处理异体组织的交联程度研究

Study on 5-Carboxyfluorescein N-succinimidyl Ester in Detecting the Degree of Cross-linked Allogenic Tissue

利用荧光技术创建一种新的方法评价经交联处理异体组织的交联程度。以新西兰兔胸主动脉作为研究对象,按与其作用的戊二醛(GA)浓度不同分为空白对照组--未与GA作用(A组),GA浓度0.625%组(B组),GA浓度1.25%组(C组),GA浓度2.5%组(D组),各组与GA交联作用后经5-羧基荧光素琥珀酰亚胺酯(5-FAMSE)处理,然后在荧光显微镜下照相,照片进行荧光强度分析(应用专业图像分析软件--Image-Pro Plus 6.0),结果A组>B组>C组>D组,各组之间差异具有显著统计学意义(P<0.01)。各组同时取一定量血管组织经GA交联后提取其组织蛋白,进行传统的聚丙烯酰胺凝胶电泳(PGE)与5-FAMSE法进行对照,结果组织蛋白提取量A组>B组/C组/D组,无法定量比较B、C、D各组间差异。以上可说明5-FAMSE是一种有效的荧光试剂,可以清楚地反映小血管氨基残端的变化,可以更为方便且直观的评价经GA处理的小血管的交联程度。

This study was designed to creat a new method for detecting the degree of cross-linked allogenic tissue based on fluorescent technique.The thoracic aorta of New Zealand rabbits were divided randomly into four groups according to the concentration of Glutaraldehyde（GA）,which were group A（control group-with no GA）,group B（cross-linked with GA of 0.625%）,group C（1.25%）,and group D（2.5%）. Each group was cross-linked with GA and reacted with 5-FAMSE,and then the fluorescence intensity was observed via fluorescence microscopy（analyzed with Image-Pro Plus 6.0,a professional image analysis software）.The differences between groups in order of fluorescence intensity were found to be：group Aroup Bgroup Cgroup D（P0.01）.Meanwhile,the tissue proteins extracted from aorta in each group were submitted to conventional polyacrylamide gel electrophoresis（PGE） after being cross-linked with GA;the result from this method was compared with that from the method of 5-FAMSE.In group A,the tissue proteins extracted from the aorta cross-linked with GA were obviously less than those not cross-linked with GA.However,this phenomenom was not clearly seen among the B,C and D groups.Neverthelss,5-FAMSE can detect the degree of cross-linkage more conveniently and directly.