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目的:揭示不同的炎症因子在体外培养的条件下,对小胶质细胞系N9不同极化方向的影响。方法:将小鼠小胶质细胞系N9细胞分为四组:空白对照组(正常培养基)、LPS组、LPS+INF-γ组、IL-4组。各组细胞在上述刺激条件下培养24 h,以及撤去上述刺激条件,继续培养24 h后,分别提取各组细胞总RNA,利用荧光实时定量PCR的方法检测各组N9细胞表达的细胞因子在mRNA水平的变化;利用Western blot检测各组N9细胞极化相关蛋白的变化。结果:在LPS以及LPS+INF-γ刺激条件下培养24 h,M1型巨噬细胞特异性标记物iNOS和CD86的mRNA及蛋白表达水平在N9细胞中明显升高,并且LPS+INF-γ刺激组升高更为明显;而撤除LPS+INF-γ刺激因子后继续培养24 h,N9细胞表达的iNOS和CD86 mRNA的水平均有下调,但仍明显高于对照组。在IL-4刺激条件下培养24 h,M2型巨噬细胞特异性标记物Arg1和CD206的mRNA及蛋白表达水平均在N9细胞中明显上调;同样撤除IL-4刺激因子后,继续培养24 h,N9细胞表达的ArgI和CD206的mRNA水平均有明显下调,但仍然高于对照组。此时ArgI的蛋白表达变化也与其mRNA水平的变化相符。结论:小胶质细胞的分型分化具有明显的细胞因子依赖性,LPS和LPS+INF-γ可使N9小胶质细胞向M1型巨噬细胞方向极化;IL-4可促使N9小胶质细胞向M2型巨噬细胞方向极化;LPS与IFN-γ二者在极化N9小胶质细胞中具有协同作用。极化后的N9细胞在去除刺激因子后,仍可在一定程度上维持其极化状态。
Objective: To reveal the effect of different inflammatory factors on the polarization direction of microglia cell line N9 in vitro. Methods: The mouse microglial cell line N9 was divided into four groups: blank control group (normal medium), LPS group, LPS + INF-γ group and IL-4 group. The cells in each group were cultured under the above stimulus conditions for 24 h, and the above stimulation conditions were removed. After 24 hours of continuous culture, total RNA was extracted from each group. Fluorescence quantitative real-time PCR was used to detect the expression of cytokines in N9 cells The changes of polarization-related proteins in N9 cells of each group were detected by Western blot. Results: The mRNA and protein expressions of M1 macrophage-specific markers iNOS and CD86 were significantly increased in N9 cells after LPS and LPS + INF-γ stimulation for 24 h, and LPS + INF-γ stimulation Group increased more obviously. However, the levels of iNOS and CD86 mRNA expression in N9 cells were all decreased after 24 h of LPS + INF-γ stimulation, but still significantly higher than that of control group. The mRNA and protein expression levels of M2-specific macrophage markers Arg1 and CD206 were significantly up-regulated in N9 cells after IL-4 stimulation for 24 h. After IL-4-stimulated factors were similarly removed, the cells were cultured for 24 h , N9 cells expressed ArgI and CD206 mRNA levels were significantly down, but still higher than the control group. At this point ArgI protein expression changes consistent with changes in its mRNA levels. Conclusion: The differentiation of microglial cells is obviously cytokine dependent. LPS and LPS + INF-γ can polarize N9 microglial cells towards M1 macrophages. IL-4 can promote the formation of N9 microgunds The stromal cells are polarized towards M2 macrophages; both LPS and IFN-γ have a synergistic effect in polarized N9 microglia. Polarized N9 cells can still maintain their polarized state to a certain extent after removing stimuli.