不同种属小鼠用于淋巴细胞增殖试验的比较研究

目的：对使用不同种属小鼠进行脾淋巴细胞增殖试验的适宜性和可比性进行研究。方法：分别获取BALB/c小鼠、野生型C57BL/6（简称C57）小鼠、Gal抗原缺失（C57背景，GGTA1基因被敲除）小鼠的脾淋巴细胞，在确定脾淋巴细胞数量与CCK8吸收度的线性范围后，设立阳性对照组（刀豆蛋白A，Con A）、不同稀释度的动物原材料匀浆液和脱细胞生物材料匀浆液等试验组，使用CCK8检测淋巴细胞增殖，流式细胞术测定淋巴细胞亚型百分比，以确定BALB/c小鼠、野生型C57小鼠和Gal抗原缺失小鼠在体外淋巴细胞增殖试验的适宜性和可比性。结果：C57小鼠脾淋巴细胞数量和CCK8的吸收度的线性范围要明显大于BALB/c小鼠，而BALB/c小鼠线性曲线的斜率明显大于C57小鼠。从CCK8检测的淋巴细胞增殖结果看，BALB/c小鼠与C57小鼠相比，2.5μg·mL^-1的Con A对2种小鼠脾淋巴细胞增殖均出现明显刺激作用；牛跟腱（含低水平的Gal抗原）匀浆液对2种小鼠脾淋巴细胞增殖均显示刺激作用，并显示出量效关系；不同稀释度的胶原海绵（牛跟腱来源）匀浆液对2种小鼠脾淋巴细胞增殖均未显示刺激作用，表现了一致的结果，说明没有淋巴细胞促增殖作用。另外，来源于Gal抗原缺失小鼠与野生型C57小鼠的脾脏淋巴细胞对Con A、牛心包（含有高水平的Gal抗原）匀浆液均表现出强的增殖反应和相似的反应强度。流式细胞术检测结果显示牛心包匀浆原液组在Gal抗原缺失小鼠仅显示活化B细胞百分比与对照组相比有统计学显著性差异，而在野生型C57小鼠组其活化B细胞、活化T细胞、活化NK细胞、NKT细胞、CD4 T细胞百分比与对照组相比均有统计学显著性差异。结论：CCK8检测的各试验组淋巴细胞增殖率结果在C57小鼠与BALB/c小鼠、Gal抗原缺失小鼠与野生型C57小鼠间具有可比性，表明这些种属的小鼠均可用于体外淋巴细胞增殖试验。  

Objective: To study the applicability and comparability of spleen lymphocytes proliferation test among different species of mice. Methods: The spleen lymphocytes of BALB/c mice, wild-type C57BL/6 (C57)mice and Gal antigen-deficient (with C57 background and GGTA1gene knockout)mice were harvested, respectively. The linear range between the number of spleen lymphocytes and values of absorption (A)detected using CCK8 reagent was primarily determined, and the concanavalin A (Con A)group, the homogenate groups of animal tissue-derived acellular biomaterials and their raw materials with different dilutions were set up. The proliferation of lymphocytes was detected by CCK8 reagent, and the percentage of lymphocyte subtypes was evaluated by flow cytometry, for determining the applicability and comparability of using BALB/c mice, wild-type C57 and Gal antigen-deficient mice in in vitro lymphocyte proliferation test. Results: The linear range of spleen lymphocytes and A value detected by CCK8 in C57 mice was significantly higher than that in BALB/c mice, while the slope of linear curve in BALB/c mice was significantly higher than that in C57 mice. 2.5 μg·mL^-1 of Con A significantly stimulated the proliferation of spleen lymphocytes in both C57 and BALB/c mice, and the homogenate of calcaneal tendon containing lower level of Gal epitopes showed stimulating effect on the proliferation of spleen lymphocytes in both mice with a dosedependent manner. Collagen sponge homogenate which derived from calcaneal tendon with different dilutions did not stimulate the proliferation of spleen lymphocytes in BALB/c and C57 mice consistently, indicating that there was no lymphocyte proliferative effect. In addition, spleen lymphocytes derived from Gal antigen-deficient mice and wild-type C57 mice showed strong proliferative responses and similar reaction intensities to both ConA and bovine pericardium (containing high levels of Gal antigen)homogenates. The results of flow cytometry demonstrated that only the percentage of activated B cells in lymphocytes in bovine pericardial homogenate group from Gal antigen-deficient mice was significantly different from that in the control group, while the percentage of activated B cells, activated T cells, activated NK cells, the percentage of NKT cells and the percentage of CD4 T cells in lymphocytes from wild-type C57 mice were significantly different from those in the control group. Conclusion: The results of lymphocytes proliferation rate detected by CCK8 were comparable between C57 mice and BALB/c mice, Gal antigen deficient mice and wild C57 mice, indicating that these species of mice can be used for lymphocyte proliferation test in vitro. 

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