低氧对新生隐球菌生物膜的影响

Hypoxia promotes the formation of Cryptococcus neoformans biofilms

目的低氧对新生隐球菌毒力因子的影响尚不清楚。探讨低氧浓度对新生隐球菌生物膜形成的影响。方法采用DMEM培养基24孔板、96孔板培养的方法分别在正常氧浓度(21%)和1%氧浓度培养条件下建立生物膜体外模型;分别在2、4、8、12、24、48、72 h取新生隐球菌生物膜用光学显微镜、共聚焦激光扫描显微镜、MTT等方法观察2组氧浓度下新生隐球菌生物膜形成厚度、形态、生长动力学等指标。结果不同氧条件下新生隐球菌在体外模型中均能形成生物膜,且新生隐球菌生物膜的形成过程相似,均经历黏附、聚集、微菌落形成及生物膜成熟的过程,最终形成厚度约为16μm的成熟生物膜;生物膜的细胞密度及生长活性随培养时间延长而增加,达到一定成熟度后逐渐稳定;与正常氧浓度相比,1%氧浓度下各时间点生物膜细胞密度及生长活性相对较高。结论不同氧浓度下新生隐球菌形成生物膜的能力不同,低氧可能具有促进其生物膜形成的作用。

Objective There is a lack of information on the effect of hypoxia on the virulence of Cryptococcus neoformans . This study was to construct a Cryptococcus neoformans bilfilm model in vitro and observe the influence of hypoxia on the biofilm forma-tion. Methods We constructed a Cryptococcus neoformans biofilm model in vitro in 24-well and 96-well microculture plates using DMEM culture medium at the oxygen concentrations of 21%（ normoxia ） and 1% （ hypoxia ） .We collected the Cryptococcus neofor-mans biofilms at 2, 4, 8, 12, 24, 48, and 72 hours after culturing and observed their thickness , structure, and growth activity in the two different oxygen conditions by light microscopy , confocal laser scanning microscopy , and MTT assay . Results The Cryptococcus neoformans biofilm model was successfully constructed in the conditions of both hypoxia and normoxia .The processes of biofilm forma-tion in the two conditions were similar , involving adhesion , aggregation , micro-colony formation , and biofilm maturation , with the ulti-mate biofilm thickness of about 16 μm.The cell density and growth activity of the biofilms increased with the extension of incubation time, gradually stabilized with their maturity , and both were relatively higher at 1%than at 21%oxygen concentration . Conclusion The abilities of Cryptococcus neoformans biofilm formation vary with different oxygen concentrations , and hypoxia can promote the for-mation of Cryptococcus neoformans biofilms .