脐血管去内皮细胞后血管顺应性及抗原表达量变化的实验研究

目的 评价酶原法、低温法去除脐血管内皮细胞的优劣，为研究开发符合生理、无排斥、结构和功能类似自体的血管支架提供理论依据。方法 自愿捐赠的健康、足月顺产、无损伤胎儿新鲜脐动、静脉各60根，每根长10cm，抗生素液37C灭菌24h后，分为正常组、酶原组和冷冻组（n=20）。分组时每根血管取0．5cm细菌培养。酶原组加入0．1％Ⅱ型胶原酶后夹闭两端，37C水浴消化5、10、15、20min，去除内皮细胞；冷冻组置入冷存管中，加入DMEM培养液，逐步冷冻，最后置入液氮中，贮藏24h以上，37C水浴30～60s快速复温，无菌生理盐水灌冲血管腔数次，去除残留血管内皮细胞；正常组4CKerb’s保存液恒温存放。将标本分别行HE、弹力纤维及胶原纤维染色及扫描电镜观察；比较各组血管顺应性差异；采用免疫组织化学染色测定人类白细胞抗原ABC（human leukocyte antigen ABC，HLA-ABC）和HLA-DR，分析抗原表达量的变化。结果 各组标本均无细菌生长。组织学观察：冷冻组，内皮细胞完全去除，脐动脉显示内皮下发达的纵行平滑肌层，脐静脉可见内弹力膜；酶原组，胶原酶作用20min，血管内皮细胞完全去除，脐动脉露出内层纵行平滑肌细胞，脐静脉显示内皮下层。血管顺应性：冷冻组脐血管顺应性大于正常组，正常组大于酶原组，但组间差异均无统计学意义（P〉0．05）；脐静脉的顺应性约为脐动脉的2～3倍。HLA-ABC、HLA-DR免疫组织化学染色：冷冻组、酶原组与正常组比较，脐动、静脉抗原性均明显降低（P〈0．01）；冷冻组与酶原组比较，脐动、静脉HLA-ABC、HLA-DR抗原表达量均降低，且差异有统计学意义（P〈0．01）。结论冷冻法及0．1％Ⅱ型胶原酶作用20min均可完整去除脐血管内皮细胞，低温冷冻法优于酶原法。

EXPERIMENTAL STUDY ON UMBILICAL VASCULAR COMPLIANCE AND EXPRESSION OF ANTIGEN AFTER REMOVING ENDOTHELIAL CELL

OBJECTIVE: To evaluate which is better method zymogen or low temperature frozen in removing vascular endothelial cell so as to lay a foundation for creating a kind of brace which is not to be rejected and the same as own blood vessel. METHODS: Fresh and not damaged umbilical blood vessel was collected from natural labour women, human umbilical blood vessel was remove carefully from normal foetus, then was put into disinfectant at 37 C for 24 hours. They were divided into 3 groups:normal group (NG), zymogen group (ZG) and low temperature frozen group (LG). ZG: 0.1% collagen II enzyme was added in umbilical blood vessel and closed the both sides and the vascular endothelial cell was removed in 37 C water. LG:Umbilical blood vessel was put into liquid nitrogen for 24 hours after frozened step by step, and then it was put into 37 C water for 30-60s and the vascular endothelial cells were washed away by normal saline. NG:Umbilical blood vessel was kept into 4 C Kerb's liquid. The bacteria were cultured in each group. The samples were stained by HE, elastic fiber and collagen fiber were observed by light and scanning electron microscope. The difference of compliance was compared. Human leukocyte antigen ABC (HLA-ABC) and HLA-DR were observed by immunohistochemical method and the expression of antigen of umbilical blood vessel was analysed. Results In LG, umbilical vascular endothelial cells were removed completely; artery showed vertical smooth muscle and vein showed elastic membrane. In ZG, umbilical vascular endothelial cells were removed completely after 20 minutes; artery showed vertical smooth muscle cells and vein showed lower endothelial layer. The vascular compliance in LG was higher than that in NG, and the latter was also higher than that in ZG, but showing no significant differences (P > 0.05). The compliance of umbilical vein was 2-3 times as much as that of umbilical artery. The expression of HLA-ABC and HLA-DR in LG and ZG were lower than that in NG, showing significant differences (P < 0.01). CONCLUSION: Low temperature frozen method and zymogen method (0.1% collagen II enzyme for 20 min) can remove vascular endothelial cells of human umbilical blood vessel completely. Low temperature frozen method was better than zymogen method.