CCK—8法在淋巴细胞增殖检测中最佳实验条件的筛选

王瑾 马肖容 张王刚

[摘要] 目的 筛选CCK-8法在淋巴细胞增殖检测中的最佳实验条件。 方法 采用正交实验设计，对初始细胞浓度、培养时间、LPS浓度、显色时间这4个主要因素各水平对人外周血单个核细胞（PBMC）和小鼠脾细胞增殖的影响进行试验研究，对各实验组合测得的刺激指数进行方差分析。 结果 CCK-8检测人PBMC增殖试验的最佳条件：初始细胞浓度为2.5×106/mL，培养时间为48 h，LPS浓度为1 μg/mL，加入CCK-8后孵育4.5 h；检测小鼠脾细胞增殖试验的最佳条件：初始细胞浓度为5.0×106/mL，培养时间为48 h，LPS浓度为1 μg/mL，加入CCK-8后孵育4.5 h。 结论 CCK-8法便捷、灵敏、重复性好，可作为检测淋巴细胞增殖的稳定方法。本研究建立的CCK-8最佳实验条件可为免疫调节作用的药物体外筛选和免疫药理学作用的研究提供依据。

[关键词] CCK-8；PBMC；淋巴细胞增殖；正交实验

[中图分类号] R392.33 [文献标识码] A [文章编号] 1673-7210（2018）08（b）-0013-04

[Abstract] Objective To optimize the experimental conditions of CCK-8 in lymphocyte proliferation assays. Methods An orthogonal test was designed to investigate the influence of four major factors （cell density， culture period， concentration of LPS and duration of incubation with CCK-8） on cell proliferation of human PBMC and mouse splenocyte. ANOVA was carried out to analyze the stimulation indices of all experimental condition combinations. Results The optimal conditions for CCK-8 was as follows： for PBMC， cell density was 2.5×106/mL， culture period was 48 h， concentration of LPS was 1 μg/mL， and duration of incubation with CCK-8 was 4.5 h； and for splenocyte， cell density was 5.0×106/mL， culture period was 48 h， concentration of LPS was 1 μg/mL， and duration of incubation with CCK-8 was 4.5 h. Conclusion The optimized CCK-8 protocol is a sensitive， convenient and stable quantitative method to evaluate lymphocyte proliferation. This result can provide evidence in screening of immunomodulating drugs and investigation of their immunopharmacology.

[Key words] CCK-8； PBMC； Lymphocyte proliferation； Orthogonal test

檢测淋巴细胞增殖的方法主要有形态学检查法、放射性核素标记法和四氮唑盐比色法等。形态学检查法和放射性核素标记法因多方面原因应用有限。MTT比色法以其快速简便、灵敏度高、无放射性污染等特点，已经成为细胞生物学及相关领域分析细胞生长、活性的基本方法[1]。但其形成的甲臜结晶（Formazan）不溶于水，需加有机溶剂溶解，重复性不佳。近年来国外开发了多种水溶性的四氮唑盐类，如MTS[2]、XTT和WST-8[3-4]等。CCK-8试剂中含有的WST-8可被细胞线粒体中的脱氢酶还原为具有高度水溶性的黄色甲臢，减少了实验步骤，提高了实验结果的准确性[5]。生成的甲臢数量与活细胞的数量成正比。……