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背景:低温冻存可以降低组织和器官的抗原性,而对细胞的影响,存在较大争论。目的:探讨液氮冷冻对成骨细胞免疫原性的影响。设计:随机对照,配对设计实验。单位:实验于2003-07/2004-03在山东大学附属齐鲁医院实验中心完成。材料:4只新西兰大白兔,雌雄不限。3H-TdR由山东大学核医学教研室提供。方法:于兔胫骨骨膜取材,体外原代培养成骨细胞,传代鉴定后液氮冻存3个月,复苏。设新鲜培养细胞为未冻存组,冻存3个月后复苏细胞为冻存组。利用流式细胞仪,通过间接免疫荧光技术测定冻存前后成骨细胞主要组织相容性复合体(major histocompatibility complex-I,MHC-I)抗原阳性率。同时建立成骨细胞、淋巴细胞混合培养模型,利用β液闪计数仪测定每分钟闪烁数,计算淋巴细胞刺激指数。主要观察指标:冻存前后成骨细胞MHC-I抗原阳性率及淋巴细胞刺激指数。结果:未冻存组成骨细胞MHC-I抗原阳性率和刺激指数明显高于冻存组,差异有显著性意义(P<0.01)。结论:液氮冻存后成骨细胞免疫原性显著降低,低温液氮冷冻是一种理想的降低成骨细胞免疫原性的方法。
BACKGROUND: Cryopreservation can reduce the antigenicity of tissues and organs, and the effect on cells is much controversial. Objective: To investigate the effect of liquid nitrogen freezing on the immunogenicity of osteoblasts. Design: Randomized control, matching design experiment. SETTING: The experiment was performed at the Experimental Center of Qilu Hospital affiliated to Shandong University from July 2003 to March 2004. Material: 4 New Zealand white rabbits, male or female. 3H-TdR by the Department of Nuclear Medicine, Shandong University. Methods: Osteoblasts were harvested from the tibia of rabbits. Primary osteoblasts were cultured in vitro. After passage identification, liquid nitrogen was frozen for 3 months and resuscitated. The freshly cultured cells were set as non-cryopreservation group, and the resuscitation cells were cryopreserved after 3 months of cryopreservation. The positive rate of major histocompatibility complex-I (MHC-I) antigen in osteoblast before and after cryopreservation was determined by indirect immunofluorescence assay using flow cytometry. At the same time, osteoblast and lymphocyte mixed culture model was established, and the number of scintillation per minute was determined by β liquid scintillation counter to calculate the lymphocyte stimulating index. MAIN OUTCOME MEASURES: The positive rate of MHC-I antigen and lymphocyte stimulating index of osteoblasts before and after cryopreservation. Results: The positive rate and stimulation index of MHC-I antigen in non-cryopreserved osteoblasts were significantly higher than those in cryoprotectant group (P <0.01). CONCLUSION: The immunogenicity of osteoblasts decreased significantly after cryopreservation of liquid nitrogen. Cryogenic liquid nitrogen freezing is an ideal method to reduce the immunogenicity of osteoblasts.