脱细胞绵羊带瓣静脉支架材料的制备及其免疫原性评价

Immunogenic evaluation of decellularized ovine vein with valve

目的：制备脱细胞绵羊带瓣静脉支架材料,通过探讨其对BALB/c小鼠体液免疫和细胞免疫的影响,以此评价该脱细胞基质的免疫原性,进而为脱细胞组织工程支架的安全性提供实验依据.方法：正常成年绵羊带瓣股静脉2cm长的片段,联合使用弱碱、非离子去垢剂及核酶进行脱细胞处理.H-E染色观察细胞组分残留及细胞外基质完整性.免疫组织化学检测其HLA-1的表达.将脱细胞基质植入BALB/c小鼠背部皮下,ELISA法检测植入前、植入后24h内、1周、3周、9周小鼠血清IgG.此外,采用刀豆素A诱导的脾体外淋巴细胞增殖实验检测脱细胞支架材料浸提液对BALB/c小鼠淋巴细胞增殖的影响.结果：经弱碱、非离子去垢剂及核酶处理后的绵羊带瓣静脉,未见细胞组织,纤维未见断裂;HLA-1未见阳性表达.植入脱细胞基质后,小鼠体内IgG水平与植入前相比,各时相点差异均无统计学意义.施加脱细胞基质浸提液后,BALB/c小鼠淋巴细胞还原率与阴性对照相比,差异无统计学意义.而与施加刀豆素A相比,差异有统计学意义.结论：联合弱碱、非离子去垢剂及核酶处理可制备良好的脱细胞绵羊带瓣静脉支架材料;该支架对BALB/c小鼠体液免疫和细胞免疫无明显影响.

Objective： To explore the effect of decellularized vein with valve of sheep on the humoral immunity and cellular immunity of BALB/c, therefore to provide experimental basis for clinical transplantation of decelluarized scaffold. Methods： 2 cm long femoral veins with valve from healthy adult sheep were decellularized with Triton X-100, ammonium hydroxide, RNase and DNase. After histological examination and immunohistochemical detection against HLA-1, the decellularized matrix was implanted subcutaneously into the back of BAB/c mice, and the IgG level in serum was detected using ELISA before implantation, within 24h after implantation, as well as at 1 week, 3 weeks and 9 weeks after implantation. In addition, the leaching liquor of decellularized matrix was validated its effect on proliferation of lymphocytes derived from spleen of BALB/ c mice in vitro, whereas the concanavalin A and culture media were used as positive and negative stimulator respectively. Results： After decellularization, the cellular components were not observed in the ovine vein with valve. The decellularized matrix possessed complete collagen fibers and negative reactivity against HLA-1. Implantation of decellularized matrix caused insignificant changes in serum IgG of mice. Compared with lymphocytes stimulated by concanavalin A, addition of leaching liquor of the decellularized matrix resulted in a statistical inhibition on lymphocyte proliferation, which had no significant difference to those cultured in media only. Conclusion： Treatment with Triton X-100, ammonium hydroxide and nu- cleotidase can create a reliable decellurized ovine vein with valve that has no nonadvantageous effect on both humoral immunity and cellular immunity of BALB/c mice.