CAJ | 학술논문

目的探讨不同冷冻温度、时间对异种移植骨免疫原性的影响. 方法将猪松质骨粒分为5组:新鲜骨组(A组),-80℃冷冻组(B组),液氮冷冻组(C组),-80℃冷冻加酶处理组(D组),液氮冷冻加酶处理组(E组),其中B、C、D、E组按照不同冷冻时间,分别于冷冻1周、2周、3周、1个月、2个月、3个月制备骨材料.采用酶联免疫吸附实验及淋巴细胞刺激实验筛选出较优冷冻时间的异种骨代表该组.应用组织病理学观察及免疫组织化学染色等检测筛选出各组异种骨免疫原性的大小. 结果 B、C、D、E组异种骨的α-半乳糖(α-Gal)抗原OD值和淋巴细胞刺激指数大小:冷冻1周＞2周＞3周＞1个月＞2个月(P＜0.05),但是冷冻2个月与3个月之间差异无统计学意义(P＞0.05),故冷冻2个月为异种骨较理想的冷冻时间.B、C、D、E组不同冷冻时间异种骨的α-Gal抗原OD值和淋巴细胞刺激指数均明显低于A组,且D、E组明显低于B、C组,差异均有统计学意义(P＜0.05).组织病理学及免疫组织化学检测结果显示:B、C、D、E组冷冻2个月时异种骨的α-Gal、主要组织相容性复合体(MHC)-Ⅰ、MHC-Ⅱ抗原含量均较A组显著降低,D、E组明显低于B、C组.结论-80 ℃冷冻2个月为异种骨较理想的冷冻温度和时间,该冷冻条件加酶处理可以有效去除异种骨的主要抗原,降低其免疫原性.
목적 탐토불동냉동온도、시간대이충이식골면역원성적영향. 방법 장저송질골립분위5조:신선골조(A조),-80℃냉동조(B조),액담냉동조(C조),-80℃냉동가매처리조(D조),액담냉동가매처리조(E조),기중B、C、D、E조안조불동냉동시간,분별우냉동1주、2주、3주、1개월、2개월、3개월제비골재료.채용매련면역흡부실험급림파세포자격실험사선출교우냉동시간적이충골대표해조.응용조직병이학관찰급면역조직화학염색등검측사선출각조이충골면역원성적대소. 결과 B、C、D、E조이충골적α-반유당(α-Gal)항원OD치화림파세포자격지수대소:냉동1주＞2주＞3주＞1개월＞2개월(P＜0.05),단시냉동2개월여3개월지간차이무통계학의의(P＞0.05),고냉동2개월위이충골교이상적냉동시간.B、C、D、E조불동냉동시간이충골적α-Gal항원OD치화림파세포자격지수균명현저우A조,차D、E조명현저우B、C조,차이균유통계학의의(P＜0.05).조직병이학급면역조직화학검측결과현시:B、C、D、E조냉동2개월시이충골적α-Gal、주요조직상용성복합체(MHC)-Ⅰ、MHC-Ⅱ항원함량균교A조현저강저,D、E조명현저우B、C조.결론-80 ℃냉동2개월위이충골교이상적냉동온도화시간,해냉동조건가매처리가이유효거제이충골적주요항원,강저기면역원성.
Objective To investigate the effects of different cryopreservation conditions on the immunogenicity of bone xenografts.Methods Porcine cancellous bone granules were divided into 5 groups according to preservation conditions of bone:fresh bone (A),bone frozen at-80 ℃ (B); bone preserved in liquid nitrogen (C),bone frozen at-80 ℃ after enzyme digestion (D),and bone preserved in liquid nitrogen after enzyme digestion (E).Bone xenografts were prepared using bone granules from groups B,C,D and E at 1,2 and 3 weeks and 1,2 and 3 months.Enzyme-linked immunosorbent assay (ELISA) and lymphocyte stimulation test were conducted to screen the best representative bone xenograft of each group.Immunogenicity of the representative bone xenografts was detected and subsequently compared using ELISA,lymphocyte stimulation test,morphological observation,pathological detection and immunohistochemical staining.Results The optical density (OD) of α-Gal antigen and lymphocytes' stimulation index of the bone xenografts in groups B,C,D and E declined significantly from one week throughout 2 months after freezing (P ＜ 0.05) but showed no significant difference between 2 and 3 months (P ＞ 0.05),indicating 2 months was the ideal time of freezing.At all time points,the OD of o-Gal antigen and lymphocytes' stimulation index of the bone xenografts were significantly lower in groups B,C,D and E than in group A and significantly lower in groups D and E than in groups B and C (P ＜ 0.05).Histopathological and immunohistochemical tests showed that the α-Gal,MHC (major histocompatibility complex class)-Ⅰ and MHC-Ⅱ at 2 months were significantly lower in groups B,C,D and E than in group A and significantly lower in groups D and E than in groups B and C.Conclusion Freezing at-80 ℃for 2 months may be ideal for preparation of xenogeneic bone,because this condition plus enzyme digestion can effectively remove the main antigen in the xenogeneic bone to lower its immunogenicity.