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目的利用羧基荧光素琥珀酰亚胺酯(CFSE)能与氨基基团发生特异性不可逆结合,且在波长为488 nm的激发光下发出绿色荧光的特性,检测人工心脏瓣膜的交联程度。方法将去细胞的牛心包浸泡于0.5%的戊二醛溶液中进行交联处理,根据浸泡时间不同分为空白对照组、10 min组、30 min组和6 h组。各组分别制成冰冻切片,经CFSE溶液处理后,通过荧光显微镜观察每组切片的荧光强度,采用图像分析软件计算每组图像的平均荧光强度。同时,从戊二醛处理过的牛心包中提取蛋白质,用聚丙烯酰胺凝胶电泳法(SDS-PAGE)检测各组牛心包的剩余蛋白含量,与氨基免疫荧光探针法进行比较。结果未交联的氨基数量随交联时间增加逐渐减少,荧光强度空白对照组>10 min组>30 min组>6 h组,各组间差异有统计学意义(P<0.01)。聚丙烯酰胺凝胶电泳法检测结果显示,从空白对照组到6 h组可提取的蛋白越来越少。结论与蛋白电泳法检测生物瓣膜交联度相比,CFSE荧光免疫法更为直观灵敏,且能反映瓣膜组织任何部位、层厚的交联效果,为人工生物瓣膜交联度的检测提供新的理论依据和思路。
Objective To detect the degree of cross-linking of prosthetic heart valves by using carboxyfluorescein succinimidyl ester (CFSE) to bind with amino groups irreversibly and emit green fluorescence under 488 nm excitation light. Methods Acellular bovine pericardium was immersed in 0.5% glutaraldehyde solution for crosslinking. According to different immersion time, the rats were divided into blank control group, 10 min group, 30 min group and 6 h group. Frozen sections were made in each group. After treatment with CFSE solution, the fluorescence intensity of each group was observed by fluorescence microscopy. The average fluorescence intensity of each group of images was calculated by image analysis software. At the same time, protein was extracted from glutaraldehyde-treated bovine pericardium, and the remaining protein content of bovine pericardium in each group was detected by polyacrylamide gel electrophoresis (SDS-PAGE), and compared with amino-immunofluorescent probe method. Results The number of unlinked amino groups decreased gradually with the increase of crosslinking time. The fluorescence intensity of control group> 10 min group> 30 min group> 6 h group, the difference was statistically significant (P <0.01). Polyacrylamide gel electrophoresis test results show that from the blank control group to 6 h group extractable protein less and less. Conclusions CFSE fluorescence immunoassay is more intuitive and sensitive than protein electrophoresis to detect the degree of cross-linking of biological valves. It can reflect the cross-linking effect of any part of the valve tissue and layer thickness, and provide a new method for the detection of the cross-linking degree of artificial biological valves Theoretical basis and ideas.