角质形成细胞对红毛癣菌生长的体外抑制作用
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摘要
目的探讨角质形成细胞在体外对红毛癣菌的抑制作用。方法将红毛癣菌菌株分为实验组(红毛癣菌+HaCaT细胞共孵育)和对照组(单纯红毛癣菌),菌落计数法计算真菌生长抑制率、Keratin-azure法测量角蛋白酶活性、扫描电镜观察真菌形态。结果细胞和真菌共孵育比例1∶1、2∶1和4∶1时,菌落计数法测得菌生长抑制率分别为(19.6±7.2)%、(54.5±2.6)%和(66.6±1.5)%;细胞和真菌共孵育12 h、24 h,菌落计数法测得菌生长抑制率分别为(31.6±7.0)%和(70.9±5.2)%。角质形成细胞与真菌合适孵育浓度比例为角质形成细胞:红毛癣菌=4∶1,细胞与红毛癣菌合适共孵育时间为24 h,实验组真菌角蛋白酶活性明显下降,扫描电镜显示实验组菌丝出现损伤。结论角质形成细胞在体外能抑制红毛癣菌的生长并能引起真菌损伤。
Objective To investigate the inhibitory effect of keratinocyte on the growth of Trichophyton rubrum in vitro. Methods The fungus were divided into experimental group with HaCaT cells intervention and control group without HaCaT cells. The fungal viability was detected by enumeration of the colony-forming units and the growth inhibitory were calculated. In addition,fungal keratinase activity was detected by measuring dye release from keratin azure. Micromorphology was observed using scanning electron microscopy(SEM). Results Enumeration of the colony-forming units revealed that in the dose response assay,the inhibitory rates were(19.6±7.2)%,(54.5±2.6)% and(66.6±1.5)% at 1:1,2:1,4:1,respectively. And the inhibitory rates were(31.6±7.0)% and(70.9±5.2)% at 12,24 h,respectively. Therefore,a ratio of HaCaT cells to T. rubrum suspension was 4:1 and 24 h of co-cultured time was determined for the following experiment.The activity of fungal keratinase decreased significantly and the mycelia were damaged by SEM in the experimental group.Conclusion Keratinocyte can inhibit the growth of Trichophyton rubrum and lead to fungal injuries in vitro.
Objective To investigate the inhibitory effect of keratinocyte on the growth of Trichophyton rubrum in vitro. Methods The fungus were divided into experimental group with HaCaT cells intervention and control group without HaCaT cells. The fungal viability was detected by enumeration of the colony-forming units and the growth inhibitory were calculated. In addition,fungal keratinase activity was detected by measuring dye release from keratin azure. Micromorphology was observed using scanning electron microscopy(SEM). Results Enumeration of the colony-forming units revealed that in the dose response assay,the inhibitory rates were(19.6±7.2)%,(54.5±2.6)% and(66.6±1.5)% at 1:1,2:1,4:1,respectively. And the inhibitory rates were(31.6±7.0)% and(70.9±5.2)% at 12,24 h,respectively. Therefore,a ratio of HaCaT cells to T. rubrum suspension was 4:1 and 24 h of co-cultured time was determined for the following experiment.The activity of fungal keratinase decreased significantly and the mycelia were damaged by SEM in the experimental group.Conclusion Keratinocyte can inhibit the growth of Trichophyton rubrum and lead to fungal injuries in vitro.
引文
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[12]Arancibia SA,Beltrán CJ,Aguirre IM,et al. Tolllike receptors are key participants in innate immune responses[J]. Biol Res,2007,40(2):97-112.
[13]Harder J,Schr?der JM,Gl?ser R. The skin surface as antimicrobial barrier:present concepts and future outlooks[J]. Exp Dermatol,2013,22(1):1-5.
[14]Taylor PR,Tsoni SV,Willment JA,et al. Dectin-1is required for beta-glucan recognition and control of fungal infection[J]. Nat Immunol,2007,8(1):31-38.
[15]Brown GD,Gordon S. Immune recognition of fungal beta-glucans[J]. Cell Microbiol,2005,7(4):471-479.
[16]Da Silva CA,Chalouni C,Williams A,et al. Chitin is a size-dependent regulator of macrophage TNF and IL-10 production[J]. J Immunol,2009,182(6):3573-3582.
[17]Abdel-Rahman SM. Polymorphic protease expression inclinical isolates of Trichophyton tonsurans[J]. Mycopathologia,2000,150:117-120.
[18]Ouf SA,Moussa TA,Alshimaa M,et al. Anti-fungal potential of ozone against some dermatophytes[J].Brazilian Journal of Microbiology,2016,47(3):697-702
[19]Huang H, Lv W, Chen Y, et al. The role of NADPH oxidase in the inhibition of trichophyton rubrum by 420-nm intense pulsed light[J]. Front Microbiol,2017,8:2636.
[2] Akhtar N,Verma A,Pathak K. Topical delivery of drugs for the effective treatment of fungal infections of skin[J]. Curr Pharm Des,2015,21(20):2892-2913.
[3] de Koning HD,Simon A,Zeeuwen PL,et al. Pattern recognition receptors in infectious skin diseases[J]. Microbes Infect 2012,14(11):881-893.
[4] Brown GD. Dectin-1:a signalling non-TLR patternrecognition receptor[J]. Nat Rev Immunol,2006,6(1):33-43.
[5] Tani K,Adachi M,Nakamura Y,et al. The effect of dermatophytes on cytokine production by human keratinocytes[J]. Arch Dermatol Res,2007,299(8):381-387.
[6] Shiraki Y,Ishibashi Y,Hiruma M,et al. Cytokine secretion profiles of human keratinocytes during Trichophyton tonsurans and Arthroderma benhamiae infections[J]. J Med Microbiol,2006,55:1175-1185.
[7]陈辉,刘维达,吕桂霞,等. HaCaT细胞对红色毛癣菌生长的抑制作用[J].中华皮肤科杂志2007,40(2):95-97.
[8] Achterman RR,White TC. Dermatophytes[J]. Curr Biol,2013,3:551-552.
[9] Kantarcio_glu SA,Y€ucel A. Malassezia species:taxonomy, mycology, immunology, pathogenesis,distribution and related infections,laboratory diagnosis,antifungal susceptibility[J]. Cerrahpasa J Med,2005,36(20):134-154.
[10]Pacher P,Beckman JS,Liaudet L. Nitric oxide and peroxynitrite in health and disease[J]. Physiol Rev,2007,87(1):315-424.
[11]Mehrotra P,Mishra KP,Raman G,et al. Differential regulation of free radicals(reactive oxygenand nitrogen species)by contact allergens and irritants in human keratinocyte cell line[J]. Toxicology Mechanisms and Methods[J],2005,15(5):343-350.
[12]Arancibia SA,Beltrán CJ,Aguirre IM,et al. Tolllike receptors are key participants in innate immune responses[J]. Biol Res,2007,40(2):97-112.
[13]Harder J,Schr?der JM,Gl?ser R. The skin surface as antimicrobial barrier:present concepts and future outlooks[J]. Exp Dermatol,2013,22(1):1-5.
[14]Taylor PR,Tsoni SV,Willment JA,et al. Dectin-1is required for beta-glucan recognition and control of fungal infection[J]. Nat Immunol,2007,8(1):31-38.
[15]Brown GD,Gordon S. Immune recognition of fungal beta-glucans[J]. Cell Microbiol,2005,7(4):471-479.
[16]Da Silva CA,Chalouni C,Williams A,et al. Chitin is a size-dependent regulator of macrophage TNF and IL-10 production[J]. J Immunol,2009,182(6):3573-3582.
[17]Abdel-Rahman SM. Polymorphic protease expression inclinical isolates of Trichophyton tonsurans[J]. Mycopathologia,2000,150:117-120.
[18]Ouf SA,Moussa TA,Alshimaa M,et al. Anti-fungal potential of ozone against some dermatophytes[J].Brazilian Journal of Microbiology,2016,47(3):697-702
[19]Huang H, Lv W, Chen Y, et al. The role of NADPH oxidase in the inhibition of trichophyton rubrum by 420-nm intense pulsed light[J]. Front Microbiol,2017,8:2636.