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目的制备去细胞人羊膜基质(AHAM)并分析其移植至小鼠体内后的免疫反应,评价去细胞人羊膜基质作为细胞移植支架的免疫原性。方法利用胰蛋白酶-EDTA消化人羊膜(HAM),去除人羊膜上皮细胞,制备新鲜去细胞人羊膜基质;新鲜去细胞人羊膜基质置于含硫酸软骨素及甘油的冷冻保护液中,-80℃保存;使用前经PBS复水处理后即冷冻去细胞人羊膜基质;免疫细胞化学分析去细胞人羊膜基质中人类白细胞抗原的表达情况;将冷冻的去细胞人羊膜基质移植至BALB/c小鼠肝脏内4周后,分析脾脏组织中CD4~+、CD8~+T淋巴细胞比例的变化。结果新鲜及冷冻保存后的去细胞人羊膜基质均不表达人类白细胞抗原;冷冻的去细胞人羊膜基质移植至小鼠体内后,脾脏组织中CD4~+、CD8~+T淋巴细胞无明显变化。结论去细胞人羊膜基质的免疫原性较低,移植后不会引起由T细胞介导的特异性排斥反应,可以作为免疫安全性的生物支架用于疾病的细胞移植治疗。
Objective To prepare acellular human amniotic membrane matrix (AHAM) and analyze its immune response after transplanted into mice to evaluate the immunogenicity of acellular human amniotic membrane matrix as a cell transplantation scaffold. Methods Human amniotic membrane (HAM) was digested with trypsin-EDTA to remove human amniotic epithelial cells and prepare fresh acellular human amniotic membrane matrix. The fresh acellular human amniotic membrane matrix was placed in a cryoprotectant solution containing chondroitin sulfate and glycerol at -80 ℃ The frozen-thawed human amniotic membrane (amniotic membrane) matrix was frozen after reconstitution with PBS prior to use. Immunocytochemistry was used to analyze the expression of human leukocyte antigens in acellular amniotic membrane matrix. The frozen acellular human amniotic membrane matrix was transplanted into BALB / c mice After 4 weeks in the liver, the changes of the proportion of CD4 ~ + and CD8 ~ + T lymphocytes in spleen were analyzed. Results No human leukocyte antigen was expressed in fresh and cryopreserved amnion. No significant changes in CD4 ~ + and CD8 ~ + T lymphocytes were observed in spleen after transplantation of frozen acellular human amniotic membrane matrix into mice. Conclusion The immunogenicity of amniotic membrane-derived human amniotic membrane matrix is low, and it does not cause specific rejection mediated by T cells after transplantation. It can be used as a biological scaffold for immune safety for cell transplantation of diseases.