巨噬细胞来源外泌体对白念珠菌形态转换的影响

doi:10.35541/cjd.20230696

中华皮肤科杂志 ›› 2024, Vol. 57 ›› Issue (6): 539-546.doi: 10.35541/cjd.20230696

巨噬细胞来源外泌体对白念珠菌形态转换的影响

李硕¹ 孙袁媛² 郝瑞英¹ 徐艳艳³ 刘钊³ 景婷婷³ 李小静³ 张秀娟⁴

¹河北工程大学临床医学院，邯郸 056000；²河北医科大学，石家庄 050000；³河北工程大学附属医院皮肤科，邯郸 056000；⁴河北工程大学附属医院检验科，邯郸 056000
李小静和张秀娟对本文有同等贡献

收稿日期:2023-11-29 修回日期:2024-03-15 发布日期:2024-06-03
通讯作者: 李小静；张秀娟 E-mail:zlmdsh@126.com; 12357414@qq.com
基金资助:
河北省政府资助临床医学优秀人才培养项目（ZF2023222、ZF2023222）；河北省自然科学基金（H2021402009）

Effect of macrophage-derived exosomes on the morphological transformation of Candida albicans

Li Shuo¹, Sun Yuanyuan², Hao Ruiying¹, Xu Yanyan³, Liu Zhao³, Jing Tingting³, Li Xiaojing³, Zhang Xiujuan⁴

¹Clinical Medical College of Hebei University of Engineering, Handan 056000, Hebei, China; ²Hebei Medical University, Shijiazhuang 050000, China; ³Department of Dermatology, Affiliated Hospital of Hebei University of Engineering, Handan 056000, Hebei, China; ⁴Department of Laboratory, Affiliated Hospital of Hebei University of Engineering, Handan 056000, Hebei, China
Li Xiaojing and Zhang Xiujuan contributed equally to the article

Received:2023-11-29 Revised:2024-03-15 Published:2024-06-03
Contact: Li Xiaojing; Zhang Xiujuan E-mail:zlmdsh@126.com; 12357414@qq.com
Supported by:
Hebei Government-funded Training Program for Excellent Talents in Clinical Medicine （ZF2024205, ZF2023222）; Natural Science Foundation of Hebei Province （H2021402009）

摘要/Abstract

摘要： 【摘要】目的探讨巨噬细胞来源外泌体对白念珠菌（CA）形态转换的影响及其作用机制。方法体外培养人急性单核细胞白血病细胞系THP-1，采用佛波酯诱导其分化成M0巨噬细胞。活化CA并配置其菌悬液，感染M0巨噬细胞，并于高内涵成像分析系统下观察细胞吞噬现象。采用差速离心法分别提取M0巨噬细胞来源的外泌体（外泌体组）及CA感染M0巨噬细胞后分泌的外泌体（CA外泌体组），并通过透射电镜观察、纳米颗粒跟踪分析、Western印迹法检测外泌体表面标志蛋白来鉴定并比较两组外泌体。将这两组外泌体分别与CA共培养（外泌体处理组、CA外泌体处理组），CA独立培养作为空白对照组，倒置显微镜下观察上述3组CA的形态变化，酶联免疫吸附试验检测胞内环磷酸腺苷（cAMP）含量，实时荧光定量PCR检测cAMP相关基因RAS1及CDC35（也称Cyr1）的表达水平。结果 Western印迹检测显示，外泌体组和CA外泌体组外泌体均表达外泌体标志物肿瘤易感基因101蛋白（TSG101），均不表达阴性标志蛋白钙联蛋白（calnexin）；透射电镜观察及纳米颗粒跟踪分析显示，两组外泌体形态、大小无明显差异。倒置显微镜观察显示，与空白对照组相比，外泌体处理组及CA外泌体处理组CA由酵母相向菌丝相的转换明显受到抑制，其菌丝长度也明显缩短。酶联免疫吸附试验结果显示，外泌体处理组及CA外泌体处理组CA细胞内cAMP含量［分别为（16.70 ± 0.84）和（16.82 ± 0.87） pmol/ml］均显著低于空白对照组［（21.82 ± 1.08） pmol/ml；t = 6.45、6.23，均P = 0.003］。实时荧光定量PCR结果显示，外泌体处理组及CA外泌体处理组CA cAMP相关基因RAS1及CDC35表达均较空白对照组下调（均P < 0.01），且CA外泌体处理组RAS1 mRNA表达量低于外泌体处理组（t = 7.43，P = 0.002）。结论 M0巨噬细胞外泌体以及M0巨噬细胞感染CA后外泌体均能有效抑制CA菌丝的生长，且后者抑制作用更强，其作用机制可能是通过下调cAMP/蛋白激酶A通路中的cAMP实现。

关键词: 白色念珠菌, 巨噬细胞, 外泌体, 形态转换, 环磷酸腺苷, 菌丝相

Abstract: 【Abstract】 Objective To investigate the effect of macrophage-derived exosomes on the morphological transformation of Candida albicans （CA）, and to explore the underlying mechanisms. Methods In vitro cultured human acute monocytic leukemia cell line THP-1 was induced and differentiated into M0 macrophages using the phorbol ester PMA. CA was activated and prepared as the fungal suspension. M0 macrophages were infected with the CA suspension, and the process of cell phagocytosis was observed under a high-content imaging analysis system. M0 macrophage-derived exosomes （exosome group） and CA-infected M0 macrophage-derived exosomes （CA exosome group） were extracted by differential centrifugation; transmission electron microscopy, nanoparticle tracking analysis, and Western blot analysis were performed to identify and compare exosomes in the two groups. The exosomes from the two groups were separately co-cultured with CA （exosome-treated group and CA exosome-treated group）, and independently cultured CA served as the blank control group; the morphological changes of CA were observed under an inverted microscope, the intracellular cyclic adenosine monophosphate （cAMP） contents were detected by the enzyme-linked immunosorbent assay （ELISA）, and the expression levels of cAMP-related genes, RAS1 and CDC35 （also known as Cyr1）, were detected by real-time quantitative PCR （RT-qPCR）. Results Western blot analysis showed that exosomes from the exosome group and CA exosome group both expressed the tumor susceptibility gene 101 protein （TSG101, an exosome marker）, and did not express calnexin （a negative marker）; transmission electron microscopy and nanoparticle tracking analysis showed no significant differences in the morphology or size of the exosomes between the two groups. Compared with the blank control group, the exosome-treated group and CA exosome-treated group both showed obvious inhibition of the yeast-to-mycelial phase transition of CA, with a noticeable reduction in the length of the hyphae under the inverted microscope. ELISA revealed that the intracellular cAMP content in CA significantly decreased in the exosome-treated group and CA exosome-treated group （16.70 ± 0.84 pmol/ml, 16.82 ± 0.87 pmol/ml, respectively） compared with the blank control group （21.82 ± 1.08 pmol/ml; t = 6.45, 6.23, respectively, both P = 0.003）. RT-qPCR revealed that the expression of the cAMP-related genes, RAS1 and CDC35, was down-regulated in the exosome-treated group and CA exosome-treated group compared with the blank control group （all P < 0.01）, and the RAS1 mRNA expression was significantly lower in the CA exosome-treated group than in the exosome-treated group （t = 7.43, P = 0.002）. Conclusion Both M0 macrophage-derived exosomes and CA-infected M0 macrophage-derived exosomes could effectively inhibit the mycelial growth of CA, and the latter one exhibited a stronger inhibitory effect, possibly by down-regulating cAMP in the cAMP/protein kinase A pathway.

Key words: Candida albicans, Macrophages, Exosomes, Morphological transformation, cAMP, Mycelial phase

李硕孙袁媛郝瑞英徐艳艳刘钊景婷婷李小静张秀娟. 巨噬细胞来源外泌体对白念珠菌形态转换的影响[J]. 中华皮肤科杂志, 2024,57(6):539-546. doi:10.35541/cjd.20230696

Li Shuo, Sun Yuanyuan, Hao Ruiying, Xu Yanyan, Liu Zhao, Jing Tingting, Li Xiaojing, Zhang Xiujuan. Effect of macrophage-derived exosomes on the morphological transformation of Candida albicans[J]. Chinese Journal of Dermatology, 2024, 57(6): 539-546.doi:10.35541/cjd.20230696

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巨噬细胞来源外泌体对白念珠菌形态转换的影响

Effect of macrophage-derived exosomes on the morphological transformation of Candida albicans

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