不同冻存液及降温方式对心脏瓣膜组织学及免疫原性的影响

Effects of different cryoprotectants and cooling methods on the histology and immunogenicity of heart valves

目的探讨液氮冻存心脏瓣膜的合适冻存保护剂及降温方式。方法将健康捐赠者的心脏瓣膜剪切为7份:新鲜对照组1份,冻存液A、B、C 3个组每组2份,分别为-80℃直接降温组(G1组)和程控降温仪缓慢降温组(G2组)。采用苏木精-伊红染色、弹力纤维染色、Masson染色法观察显微结构变化,透射电子显微镜(TEM)观察超微结构变化,采用Western blotting法及免疫组织化学法检测瓣膜表面人类白细胞抗原复合体(HLA)的相对表达量,比较各组瓣膜组织免疫原性。结果低温冻存会造成瓣膜组织结构损伤,使用含DMSO和硫酸软骨素的冻存液B组结构最完整,组织损伤最小,HLA-I蛋白的表达明显下调;G2组与G1组相比,胶原纤维着色偏浅,纤维排列更加紊乱,但HLA-I蛋白的表达相对较低。结论同时含有DMSO和硫酸软骨素对冻存组织保护效果最好,免疫原性最低。-80℃直接降温更有利于胶原纤维的保存,但免疫原性高于程控降温法。

Objective To explore the proper cryoprotectants and cooling methods for storing heart valves in liquid nitrogen. Methods Each heart valve from 6 healthy donors were divided into 7 specimens： 1 was observed and tested directly as control group,and other 6 were divided into 3 cryoprotectant groups（ Group A,B and C）,with 2 specimens in each groups. Two cooling methods were applied,including- 80 ℃ direct cooling（ G1 groups） and programmed cooling（ G2 groups）. The histological changes were observed with hematoxylin-eosin staining,Weigert-Van Gieson（ ET ＋ VG） staining,and Masson Trichrome staining. The ultrastructural changes were observed with transmission electron microscopy（ TEM）. The expression of human leukocyte antigen（ HLA） was detected with Western blotting and immunohistochemistry. The immunogenicity of each group was compared. Results Cryopreservation injured the organizational structure of valves,while valves stored in DMSO and chondroitin sulfate sodium showed the least lesions and obviously down-regulated expression of HLA-I. Collagen fibrils were less stained,while the orientation was more disorderly in the programmed cooling groups than in the- 80 ℃ direct cooling groups,but the expression of HLA-I was lower. Conclusion DMSO in combination with chondroitin sulfate sodium can best preserve the heart valves,with the lowest immunogenicity. The method of- 80 ℃ direct cooling is conducive for the preservation of collagen fibrils,but it produces higher immunogenicity than programmed cooling.