【摘要】 目的探究巨噬细胞吞噬黑素化马尔尼菲青霉(PM)后的自噬水平变化,并探究雷帕霉素通过诱导巨噬细胞自噬杀灭该菌的可行性。方法采用含多巴培养基培养获得酵母相黑素化PM,蛋白质印记法检测黑素化及常规PM刺激后Ana-1细胞Ⅱ型微管相关蛋白1轻链3(LC3Ⅱ)的表达水平,并检测雷帕霉素预处理后Ana-1细胞LC3Ⅱ的表达水平,继而采用免疫荧光法显示LC3Ⅱ与黑素化PM在细胞内的定位;通过菌培养测算雷帕霉素的直接抗菌作用,并检测有无雷帕霉素预处理的Ana-1细胞对黑素化PM的杀菌效能。结果Ana-1细胞吞噬黑素化PM后自噬水平无明显变化(P>0.05),而雷帕霉素处理后染菌的Ana-1细胞LC3Ⅱ表达水平明显升高(P=0.009,P<0.05),并且菌体与LC3蛋白呈共定位关系。雷帕霉素处理后Ana-1细胞与黑素化PM共孵育3h(P=0.026)和6h(P=0.014)后菌存活率明显降低(P<0.05),而相同条件下单纯的Ana-1细胞或雷帕霉素无明显杀菌作用。结论黑素化PM抑制巨噬细胞的自噬活化,雷帕霉素可通过诱导自噬提高巨噬细胞对该菌的杀菌效能。更多还原

【Abstract】 Objective To investigate the autophagy level of Ana-1 cells after ingesting melanized Penicillium marneffei(PM),and to explore the feasibility of rapamycin in killing the bacteria by inducing macrophages autophagy.Methods Melanized PM was cultivated and isolated from the medium containing dopamine.The expression of microtubule-associated protein 1 light chain 3Ⅱ(LC3Ⅱ)in Ana-1 cells stimulated by conventional or melanized PM was detected by western blot.The expression levels of LC3Ⅱ protein in Ana-1 cells treated with rapamycin and incubated with melanized PM was detected.Then,the localization of LC3Ⅱin Ana-1 cells which contained melanized PM was observed by immunofluorescence staining.Finally,the direct sterilization effect of rapamycin on melanized PM were detected,and the sterilization effect of Ana-1 cells treated with or without rapamycin on melanized PM was measured.Results No significant change was found in the LC3Ⅱlevel of Ana-1 cells after ingesting melanized PM(P>0.05),while LC3Ⅱlevel in Ana-1 cells treated with rapamycin which contained melanized PM was significantly increased(P=0.009).The colocalization of LC3Ⅱ with melanized PM in cytoplasm of Ana-1 cells was observed.For Ana-1 cells treated with rapamycin,3 hand 6 hafter incubated with melanized PM,the survival rates of melanized PM both were significantly reduced(P=0.026,0.014).No significant sterilization effect of Ana-1 cells or rapamycin was observed under the same conditions.Conclusion Melanized PM can suppress the activation of macrophage autophagy,and rapamycin can improve sterilization effect of Ana-1 cells by inducing the activation of autophagy.更多还原