小鼠腹腔巨噬细胞趋化和吞噬功能中SRC-3的作用研究

目的 探讨类固醇受体辅活化子（SRC）-3蛋白在脂多糖诱导小鼠腹腔巨噬细胞趋化和吞噬功能中的作用.方法 健康SPF级雌性野生型（SRC-3＋/＋）小鼠、SRC-3基因敲除（SRC-3-/-）小鼠各5只,分别分离和原代培养腹腔巨噬细胞,并相应分为SRC-3＋/＋组和SRC-3-/-组.收集并调整细胞浓度为1×106/ml,接种于6孔板,1 ml/孔,给予10 μg/ml LPS刺激,分别在刺激前（0 h）、刺激后4 h和12 h收集腹腔巨噬细胞.通过Western blot测定腹腔巨噬细胞Toll样受体4（TLR4）的表达,并通过transwell趋化实验和中性红吞噬实验,分别测定腹腔巨噬细胞的趋化指数（CI）和吞噬功能.结果 LPS诱导前两组腹腔巨噬细胞TLR4的蛋白表达和CI差别无统计学意义,但SRC-3-/-组的吞噬功能显著低于SRC-3＋/＋组（P＜0.01）;无论是SRC-3＋/＋组,还是SRC-3-/-组,LPS诱导4 h和12 h后,两组腹腔巨噬细胞TLR4的表达和CI均显著增高（P＜0.01）,但在相应时间点,SRC-3-/-组与SRC-3＋/＋组相比差别无统计学意义.LPS刺激4 h后,两组吞噬功能均显著增加（P＜0.01）,但SRC-3-/-组增加的程度显著低于SRC-3＋/＋组（P＜0.01）;相反,LPS刺激12 h后,两组吞噬功能均受到不同程度的抑制（P＜0.05或P＜0.01）,且SRC-3-/-组较SRC-3＋/＋组抑制的程度更低（P＜0.01）.结论 SRC-3调节腹腔巨噬细胞的先天性免疫功能可能与吞噬功能有关,而与其趋化功能无关.

Study on the role of steroid receptor coactivator-3 in chemotactic and phagocytic function of mouse peritoneal macrophages

Objective To investigate the effects of steroid receptor coactivator （SRC） -3 on the chemotactic and phagocytic function of lipopolysaccharide （LPS） induced mouse peritoneal macrophages （PMФ）. Methods Healthy SPF-grade wild female type （SRC-3＋/＋） mice and SRC-3 gene knock-out （SRC-3-/-） mice were used in this study. Their PMФ were isolated and primary cultured, respectively. After the collection, the concentration of PMcb was adjusted to 1 × 106/ml. PMФ were cultured in 6 orifice plates with 1 ml/orifice and stimulated by 10 Ixg/ml LPS. Peritoneal macrophages were collected before the stimulation,4 h, and 12 h after the stimulation. The expressions of Toll-like receptor 4 （ TLR4 ） were determined by Western blot assay. The chemotactic index （CI） and phagocytic function of PMФ were examined through the Transwell chemotaxis assay and the phagocytosis of neutral red assay. Results Before LPS induction,there was no significant difference in the protein expression of TLR4 and CI between the two groups, but the phagocytic function in SRC-3 -/- group was significantly poorer than that in SRC-3 ＋/＋ group （P 〈 0.01 ）. The TLR4 expression level and the CI of PMФin both groups significantly increased 4 and 12 h after the LPS induction（P 〈 0.01 ） , but there was no significant difference between the two groups at the corresponding time points. The phagocytic function of PMФ in both groups increased 4 h after the induction, but the increasing degree in the SRC-3 -/- group was significantly lower than that in SRC-3 ＋/＋ group（P 〈 0.01 ）. On the contrary,the phagocytic function in two groups were both inhibited at different degrees （P 〈 0.05 or P 〈 0.01 ） 12 h after the LPS induction,but the inhibition degree in SRC-3 -/- group was lower than that in SRC-3 ＋/＋ group （P 〈 0.01 ）. Conclusion The innate immunity function of SRC-3 which regulates the peritoneal macrophages may be correlated with the phagocytic function but has no correlation with its chemotactic function.