人外周血淋巴细胞增殖试验的优化及其应用

目的：优化人外周血淋巴细胞增殖试验条件；考察动物源材料的Gal抗原含量与淋巴细胞增殖效应的相关性；并比较人外周血淋巴细胞与小鼠脾脏淋巴细胞增殖试验对动物源材料的敏感性差异。方法：从细胞接种量，阳性对照（刀豆球蛋白A，Con A）的工作浓度与细胞培养时间，优化人外周血淋巴细胞增殖试验的最佳条件；利用优化的试验方法，通过人外周血淋巴细胞与动物源性材料的（牛跟腱或由牛跟腱纯化的胶原蛋白海绵）匀浆液或浸提液共培养，考察材料的不同前处理方式对人外周血淋巴细胞增殖的影响；参考标准YY/T 1561-2017，检测材料匀浆液或浸提液的Gal抗原含量，分析其抗原含量与淋巴细胞增殖效应的相关性；同时参考标准YY/T 1465.1-2016，考察材料匀浆液与浸提液对小鼠脾脏淋巴细胞增殖效应的影响，并对比分析对动物源材料的敏感性差异。结果：人外周血淋巴细胞增殖试验最佳反应条件经优化确定为：细胞接种量为1×10⁵，阳性对照（Con A）的终浓度选择5~10μg·mL^-1，培养时间为4 d。人外周血淋巴细胞与胶原蛋白海绵（匀浆组与浸提液组）共培养后，样品组与正常细胞对照组相比不存在明显差异；与牛跟腱匀浆液共培养后，与正常细胞对照组相比不存在显著性差异。胶原蛋白海绵（匀浆组与浸提液组）对小鼠脾脏淋巴细胞均无明显增殖效应。牛跟腱匀浆液在5倍稀释与50倍稀释后，对小鼠脾脏淋巴细胞均有明显增殖效应（增殖率为143.20%和128.71%）。牛跟腱匀浆液的Gal抗原含量为（3.98±1.06）×10¹³个·mg^-1、胶原蛋白海绵匀浆液为（2.24±0.60）×10¹³个·mg^-1、胶原蛋白海绵浸提液为（1.89±0.64）×10¹³个·mg^-1。结论：淋巴细胞增殖效应与动物源性生物材料中残留Gal抗原含量的正向相关性不强。与小鼠脾脏淋巴细胞增殖试验相比，采用人外周血淋巴细胞增殖试验评价含Gal抗原的动物源性生物材料细胞免疫的敏感性未见优势。淋巴细胞增殖试验是评价动物源生物材料细胞免疫的可用方法之一。  

Objective: To optimize the experimental conditions of human peripheral blood lymphocyte proliferation test, and investigate the correlation between Gal antigen content of animal tissue-derived biomaterials and the proliferation effect of human peripheral blood lymphocytes, and to compare the difference in the sensitivity of proliferation effect between human peripheral blood lymphocytes and mouse spleen lymphocytes for animal tissuederived biomaterials. Methods: The optimized experimental conditions of human peripheral blood lymphocyte proliferation test, includes cell seeding quantity, working concentration of positive control (Concanavalin A, Con A)and cell culture period were investigated. The refined human peripheral blood lymphocyte proliferation test was applied to investigate the cellular immunity of animal tissue derived biomaterials by co-culture of human peripheral blood lymphocytes with homogenate or extraction of animal derived materials (bovine achilles tendon or collagen sponge made from bovine achilles tendon). The Gal antigen content contained in the homogenate or extraction of materials was determined in according to the standard YY/T 1561-2017. The effects of homogenate and extraction on the proliferation of mouse spleen lymphocytes were investigated and the sensitivity for animalderived materials was compared with human peripheral blood lymphocyte proliferation test in according to the standard YY/T 1465.1-2016. Results: The optimized experimental conditions for the proliferation test of human peripheral blood lymphocytes were as follows:the cell seeding quantity was 1×10⁵ cells per well, the final concentration of the positive control (Con A)was 5-10 μg·mL^-1, and the culture time was 4 days. After a coculture of human peripheral blood lymphocytes with collagen sponge (including homogenate group and extraction group), there was no significant proliferation effect in the test group compared with the control group; and after a co-culture with bovine achilles tendon homogenate, no significant difference was compared to the control group. Both homogenate and extraction of collagen sponge had no obvious proliferation effect on mouse spleen lymphocytes. A and the significant proliferation effects on mouse spleen lymphocytes were observed in bovine achilles tendon homogenate group (143.20% and 128.71%). The Gal antigen content of bovine achilles tendon homogenate was (3.98±1.06)×10¹³ number·mg^-1, collagen sponge homogenate was (2.24±0.60)×10¹³ number·mg^-1 and extract was (1.89±0.64)×10¹³ number·mg^-1. Conclusion: There was no strong positive correlation between the lymphocytes proliferation and the content of residual Gal antigens contained in biomaterials. Compared with mouse spleen lymphocytes, the sensitivity of using human peripheral blood lymphocytes to evaluate cellular immunity of Gal antigen contained animal-derived biomaterials did not show more benefit. Lymphocyte proliferation test was one of the available methods for evaluating cellular immunity of animal-derived biomaterials. 

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