酰化高丝氨酸内酯对巨噬细胞模型影响初探

Preliminary study on the effect of acyl-homoserine lactones on macrophage model

酰化高丝氨酸内酯类(Acyl-homoserine lactones,AHLs)化合物是革兰氏阴性菌群体感应系统中最重要的一类信号分子,本文模拟淡水鱼革兰氏阴性腐败菌产生的5种主要AHLs(C_4-HSL、C_6-HSL、C_8-HSL、C_10-HSL和3OC_12-HSL)。通过构建Ana-1和RAW 264.7两种巨噬细胞模型,筛选敏感识别特定AHLs的巨噬细胞株,为构建基于细胞传感的淡水鱼腐败菌的检测方法进行前期初步研究。结果表明,5种AHLs均能造成Ana-1和RAW 264.7细胞的活性降低;胞内Ca^(2+)荧光检测发现,在C_10-HSL的刺激作用下,Ana-1细胞的活性与胞内Ca^(2+)水平变化呈现一定的相关性,由此推测C_10-HSL具有诱导Ana-1细胞胞内Ca^(2+)上升,进而引发细胞内钙离子探针的荧光反应。同时采用流式细胞术和电镜分析对Ana-1细胞识别C_10-HSL的敏感性进行了验证,为构建基于Ana-1细胞的淡水鱼腐败菌信号分子C_10-HSL的荧光传感检测方法提供了前期研究基础。

Acyl-homoserine lactones（AHLs） compound is a class of the most important signaling molecule in quorum sensing system of gram- negative bacterium, this paper simulates 5 dominant AHLs （ C4 - HSL, C6 - HSL, C8- HSL, C10 - HSL, and 3OC12-HSL） generated from gram negative bacteria in freshwater fish, and screens the macrophage strain with sensitive identification of specific AHLs by constructing two macrophage models of Ana-1 and RAW 264.7.The results showed that each of the 5 AHLs could cause the decrease of Ana-land RAW 264.7 cell activeness.Through the intracellular Ca2＋ fluorescence detection, under C10 -HSL stimulation, the activity of Ana-1 cells showed a certain correlation with intracellular Ca2＋ , it was speculated that C10-HSL could induce the intracellular Ca2＋ increase in Ana-1 cells, thus trigger the fluorescence reaction of intracellular Ca2＋ probe.Meanwhile the sensitivity of Ana-1 cells to C10- HSL was verified by flow cytometry and electron microscopy, which provided the basis and guidance for the construction of ANA- 1 cell based fluorescent sensing method for detection of signaling molecule C10 -HSL in freshwater fish spoilage organisms.