小鼠ANA-1巨噬细胞不同极化状态下补体组分mRNA动态分析

摘要

Objective:To reveal the dynamic characteristics of the intracellular complement mRNA from mouse macrophage ANA-1 treated with LPS or IL-4. Methods:The polarization models of macrophage ANA-1 were established by treating with LPS(1μg/ml) and IL-4(20 ng/ml),respectively. After treating at 3,8,12 and 24 h,the total RNA were abstracted by Trizol lysis methods . The macrophage polarization were estimated by the expression of IL-1β, CCL2 and Arg-1 mRNA detected by Real-time fluorescent quantitative PCR. The intracellular complement C1q, C3, CfB and CRIg mRNA were quantitatively analyzed. Results: The mouse macrophage ANA-1 cells treated with LPS was polarized to M1 since the levels of IL-1β and CCL2 mRNA were up-regulated significantly,in which their 2-△△Ct value were up to 297. 0±31. 0 and 19. 9±3. 3 respectively at 12 h. On the other hand,the ANA-1 cells treated with IL-4 was polarized to M2 because the level of Arg-1 mRNA was obviously higher( the 2-△△Ct value of Arg-1 mRNA was up to 27.3±9.1 at 24 h)(P<0.05).The intracellular complement C1q,C3,CfB and CRIg mRNAs all were up-regulated in different polarized macrophages. The intracellular C1q and C3 mRNA in polarized M2 were significantly higher,in which the peak value of C1q and C3 were to 94. 9±12. 9 and 11. 3±2. 4 at 12 h,respectively(P<0. 05). Reversely,the CfB mRNA in polarized M1 increased obviously,in which its 2-△△Ct was to 61. 4±6. 2 at 12 h. In addition,the CRIg mRNA in both groups was only up-regulated at 24 h,in which the 2-△△Ct value was 6. 5±1. 8 in M1 and 10. 8±3. 2 in M2(P<0. 05). Conclusion: The macrophage ANA-1 cell polarization models were successfully established by treated with LPS or IL-4. The intracellular complement C1q,C3 and CRIg mRNA in polarized M2 were transcripted more than in M1. But the intracellular CfB mRNA in polarized M1 was up-regulated significantly. These results suggested that the dynamic characteristic of complement components in different polarized macrophage would be correlated with its fun-tions.%目的：观察小鼠ANA-1巨噬细胞不同极化状态下补体相关组分的mRNA动态变化。方法：分别以LPS （1μg/ml)、IL-4（20 ng/ml)体外刺激小鼠ANA-1细胞，于刺激后3、8、12及24 h时间点收获细胞，Trizol法提取细胞总RNA，采用实时荧光定量PCR法检测IL-1β、CCL2、Arg-1的表达，判断细胞极化情况，检测胞内C3、CfB、CRIg、C1q补体分子mRNA的动态表达。结果：细胞极化情况：刺激3 h后LPS组IL-1β、CCL2的mRNA表达上调，12 h达高峰（2－△△Ct分别为297．0±31．0和19．9±3．3)；在IL-4组中以Arg-1mRNA表达上调显著，于24 h达高峰（2－△△Ct：27．3±9．1)，提示LPS组细胞向M1方向极化， IL-4组向M2方向极化。细胞内补体mRNA的动态：两极化组的相应补体组分均表现为不同程度的上调，其中：C1q、C3在M2极化组刺激8 h后显著上调，刺激12 h时达高峰（2－△△Ct分别为94．9±12．9和11．3±2．4)；CfB因子在M1极化组中显著上调，以刺激12 h表达上调达高峰（2－△△Ct为61．4±6．2)；CRIg在两组中均在24 h时表达上调且具有显著性差异（P<0．05)。结论：LPS/IL-4刺激3 h后，ANA-1细胞分别向M1、M2方向极化；不同补体组分mRNA的表达在两组中均上调但表达谱不同，其中C1q、C3和CRIg在M2极化组中上调更为显著，而CfB因子在M1极化组中显著上调；除CRIg外，C1q、C3及CfB因子均在刺激12 h时上调达高峰，上述结果提示补体组分的变化可能与极化的巨噬细胞功能有关。