动物源性生物材料体外淋巴细胞增殖试验方法的建立

目的：建立可用于评价动物源性生物材料细胞免疫的体外淋巴细胞增殖试验方法。方法：采用C57小鼠脾脏淋巴细胞，通过淋巴细胞数量、阳性对照剂（刀豆蛋白A，Con A）浓度和培养时间等条件筛选，用CCK8试剂检测细胞增殖率，初步优化体外淋巴细胞增殖试验方法。采用牛心包组织（原材料）的浸提液和匀浆液，作为抗原特异性的阳性对照组考察样品前处理方式对试验体系的影响，并用流式细胞仪分析增殖后的淋巴细胞亚型百分比，考察样品制备方式对淋巴细胞亚型增殖影响的差异。结果：本试验中小鼠脾淋巴细胞体外增殖试验条件确定为每孔接种6×10⁵个淋巴细胞，阳性对照孔Con A浓度为2.5μg·mL^-1，培养时间为3 d。牛心包浸提液和匀浆液的淋巴细胞增殖率均约为细胞对照组的2倍，两者的淋巴细胞亚型百分比变化不同于Con A组，未出现T细胞、CD4 T细胞、CD8 T细胞百分比的增加。浸提液组与匀浆液组的活化NK细胞和NKT细胞百分比与对照组相比均明显升高，但浸提液组的升高幅度数倍高于匀浆液组，且仅浸提液组NK细胞百分比与对照相比出现了明显倍增。结论：本研究建立了以动物组织（原材料）为抗原特异性阳性对照的体外淋巴细胞增殖试验方法。动物组织（原材料）浸提和匀浆样品均可以产生淋巴细胞增殖阳性结果，其促进淋巴细胞增殖的机理（亚型分类）不同于Con A等有丝分裂原，提示使用作为动物组织（原材料）阳性对照更能反映试验体系的敏感性。 

Objective: To establish an in vitro lymphocyte proliferation test method for evaluating cellular immunity of animal tissue-derived biomaterials. Methods: Splenic lymphocytes were obtained from the C57 mice.The experimental conditions, such as the number of lymphocytes, the concentration of Con A and the culture time, were optimized and the lymphocyte proliferation ratio was detected by CCK8 reagent for a preliminary optimizing of an in vitro lymphocyte proliferation test method. The extract and homogenate samples of bovine pericardium raw materials were used as the antigen-specific positive control groups for detecting the effect of sample pretreatment to the test system, and the percentage of lymphocytes subtypes after proliferation was analyzed by flow cytometry to investigate the difference of the proliferation of different lymphocyte subtypes in different preparations. Results: In this proliferation test of mouse spleen lymphocytes in vitro, 6×10⁵ lymphocytes per well, 2.5 μg·mL^-1 Con A as the non-specific positive control, and the 3 day-culture duration were chosen as the optimized experimental conditions. The lymphocyte proliferation rates of bovine pericardium extract group and homogenate group were both about twice as high as that of the cell control group. The percentages of lymphocyte subtypes in bovine pericardial extract and homogenate groups were different from those in the Con A group, where there was no increase in the percentages of T cells, CD4 T cells and CD8 T cells. The percentages of activated NK cells and NKT cells in the extract group and homogenate group increased significantly compared with the control group, and the increase amplitude in the extract group was several times higher than that in the homogenate group, and the percentage of NK cells in the extract group increased significantly compared with the control group. Conclusion: In this study we established an in vitro lymphocyte poliferation assay using animal tissues (raw materials)as antigen specific positive control. Both the extraction and homogenate samples of animal tissues (raw materials)can produce positive results of lymphocyte proliferation, of which mechanism of promoting lymphocyte proliferation (subtype classification)is different from that of Con A and other mitogens, suggesting that using animal tissues (raw materials)as positive control can better reflect the sensitivity of the experimental system. 

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