小鼠腹腔巨噬细胞不同分离方法的比较

Comparison of different isolation methods of peritoneal macrophages in mice

目的比较不同方法分离的小鼠腹腔巨噬细胞的生物学特性并找出最优方法。方法采用3种方法分离小鼠腹腔巨噬细胞:5%淀粉肉汤小鼠腹腔注射3 d后灌洗(5%淀粉肉汤组);直接DMEM液腹腔灌洗(无血清DMEM组);含75%血清DMEM液腹腔注射30 min后灌洗(75%血清DMEM组),每组6只小鼠。观察各组巨噬细胞形态、计数细胞数量、流式细胞仪测细胞纯度、台盼蓝染色测细胞存活率、划痕实验测迁移能力及ELISA检测脂多糖(LPS)刺激后巨噬细胞分泌TNFα、IL-6及IL-1β的水平。结果 3组巨噬细胞的形态、纯度、存活率相似,75%血清DMEM组巨噬细胞数量为(80.47±0.82)×105个,明显多于5%淀粉肉汤组(5.64±0.31)×105个及无血清DMEM组(2.63±0.42)×105个(P<0.05);75%血清DMEM组巨噬细胞迁移能力明显高于其他两组(P<0.05);75%血清DMEM组巨噬细胞分泌TNFα(15.51±0.51)ng/m L、IL-6(415.33±1.55)pg/m L、IL-1β(421.01±2.64)pg/m L,均多于5%淀粉肉汤组TNFα(10.50±0.50)ng/m L、IL-6(386.33±1.52)pg/m L、IL-1β(375.33±2.51)pg/m L(P<0.05),也多于无血清DMEM组TNFα(9.56±0.25)ng/m L、IL-6(384.66±1.15)pg/m L、IL-1β(393.34±2.62)pg/m L(P<0.05)。结论 75%血清DMEM液腹腔注射30 min后灌洗是一个比较好的分离小鼠腹腔巨噬细胞的方法。

Objective To compare the characteristics of mouse peritoneal macrophages isolated by different methods and find out the best method. Methods Murine peritoneal macrophages were isolated by three methods： intraperitoneal injection of 5% starch broth for 3 d then lavage （5% starch broth group） ; direct DMEM solution lavage （non serum DMEM group） ; intraperitoneal injection of DMEM solution containing 75% serum for 30 rain then lavage （75% serum DMEM group）. Resuits The morphology, purity and survival rate of macrophages in three groups were similar. , the number of macrophages in 75% serum DMEM group was （80.47 ±0.82）×10^5, significantly more than 5% starch broth group（5.64 ±0.31 ）×10^5 and non serum DMEM group（2.63 ± 0.42）×10^5（P 〈 0.05）, the migration ability of macrophages in 75% serum DMEM group was significantly higher than those in other two groups （P 〈 0.05）. Macrophages in 75% serum DMEM group secreted TNFα（15.51 ±0.51） ng/mL, IL-6（415.33 ± 1.55） pg/mL and IL - 1β（421.01 ±2.64） pg/mL, more than 5% starch broth group TNFα （ 10.50 ± 0.50） ng/mL, IL - 6 （386.33 ± 1.52） pg/mL and IL - 1β （ 375.33± 2.51 ） pg/mL （ P 〈 0.05 ）, also more than non serum DMEM group TNFot （9.56 ± 0.25 ） ng/mL, IL - 6 （ 384.66 ± 1.15 ） pg/mL, IL - 113（393.34 ±2.62）pg/mL （P 〈0.05）. Conclusion Intraperitoneal injection of 75% serum DMEM for 30 min then layage, is a good method for extraction of mouse peritoneal macrophages.