体外呼吸道上皮细胞细菌黏附模型的建立
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摘要
目的基于流动小室的构架,建立一种用以研究细菌在呼吸道上皮细胞黏附的体外模型。方法以2 mg/mL牛胶原蛋白预包被流动小室后,接种1×10~5个HBE细胞,含20%血清的RPMI1640培养基,37℃,5%CO_2孵箱培养24 h铺满底部后用于实验;以流动小室中细胞数量为评价指标,正交设计考察流速、流动时长和流动相成分对细胞模型的影响,筛选最优实验条件;以正交实验结果为条件,细菌黏附量为指标,与常规用于细菌细胞黏附的培养板方法相比较,分别于2×10~8、10~8、5×10~7、2.5×10~7CFU/mL接种浓度下与细胞进行黏附实验,判定流动小室是否为细菌细胞黏附研究的可靠模型;以SYTO9荧光标记细菌的方法表征黏附于细胞的细菌量。结果优化得到最佳实验条件,影响因素主次为流速>流动时长>流动相组成,方差分析结果显示,流速和流动时长影响因素有显著差异(P<0.05),流动相组成无显著差异(P>0.05)。荧光染色后,该模型可实现细菌在呼吸道上皮细胞上黏附的荧光实时观察;细菌黏附的定量结果显示,随着感染复数增大,流动小室模型与常规培养板方法均检测出黏附细菌的增多,且呈线性关系;但同一感染复数下常规培养板方法所检测出的黏附菌量与流动小室模型相比显著增多(P<0.05)。结论在适宜的流速、流动时长下,该模型可用于研究细菌在呼吸道上皮细胞上黏附,并且较常规培养板方法具有更贴近体内环境、准确度高、可实时观察等优点。
Objective To establish an in vitro model to study the adhesion of bacteria to respiratory epithelial cells based on the construction of flow-chamber. Methods After 1 × 10~5 HBE cells were seeded in the flow chamber pre-coated with 2 mg/mL bovine collagen,the chamber was cultured in RPMI 1640 medium containing 20% serum and incubated at 37 ℃ in a 5% CO_2 incubator for 24 h. Once the bottom was covered with HBE cells,the chamber could be used for further experiments. When the number of cells in the flow chamber was taken as the evaluation index,the effects of the flow rate,the flow duration and the mobile phase composition on the cellular model were investigated by orthogonal design,and the optimal experimental conditions were screened. Then under the obtain optimal conditions,after the bacteria were adhered to the cells at 2 × 10~8,10~8,5 × 10~7,and 2. 5 × 10~7 CFU/mL inoculated concentrations respectively,the amount of bacterial adhesion was taken as an index,and the results were compared with the conventional plate culture for bacterial adhering to cells,so as to determine whether the flow chamber is a viable model of bacteria adhesion to cells. The amount of bacteria adhering to cells was characterized by SYTO9 fluorescently labeled bacteria.Results The influencing factors in optimal experimental conditions in order of their impacts were as follows:flow rate > flow duration > mobile phase composition. The ANOVA study showed that the impacts of flow rate and flow duration were significant( P < 0. 05),and while those of mobile phase composition were not. After fluorescent staining,the model realized the real-time fluorescence observation of the bacteria adhesion to the respiratory epithelial cells. According to the quantitative results of bacterial adhesion,with the increase of multiplicity of infection,both the flow chamber and the conventional culture plate detected the elevated amount of adhering bacteria,in a linear manner. However,with the same multiplicity of infection,the conventional culture plate model detected significantly larger amount of adhering bacteria when compared to the flow chamber( P < 0. 05). Conclusion Under appropriate flow rate and flow duration,our model is an alternative to the conventional plate for studying the adhesion of bacteria to the airway epithelial cells,with the advantages of being more close to the in vivo environment,high accuracy and real-time observation.
Objective To establish an in vitro model to study the adhesion of bacteria to respiratory epithelial cells based on the construction of flow-chamber. Methods After 1 × 10~5 HBE cells were seeded in the flow chamber pre-coated with 2 mg/mL bovine collagen,the chamber was cultured in RPMI 1640 medium containing 20% serum and incubated at 37 ℃ in a 5% CO_2 incubator for 24 h. Once the bottom was covered with HBE cells,the chamber could be used for further experiments. When the number of cells in the flow chamber was taken as the evaluation index,the effects of the flow rate,the flow duration and the mobile phase composition on the cellular model were investigated by orthogonal design,and the optimal experimental conditions were screened. Then under the obtain optimal conditions,after the bacteria were adhered to the cells at 2 × 10~8,10~8,5 × 10~7,and 2. 5 × 10~7 CFU/mL inoculated concentrations respectively,the amount of bacterial adhesion was taken as an index,and the results were compared with the conventional plate culture for bacterial adhering to cells,so as to determine whether the flow chamber is a viable model of bacteria adhesion to cells. The amount of bacteria adhering to cells was characterized by SYTO9 fluorescently labeled bacteria.Results The influencing factors in optimal experimental conditions in order of their impacts were as follows:flow rate > flow duration > mobile phase composition. The ANOVA study showed that the impacts of flow rate and flow duration were significant( P < 0. 05),and while those of mobile phase composition were not. After fluorescent staining,the model realized the real-time fluorescence observation of the bacteria adhesion to the respiratory epithelial cells. According to the quantitative results of bacterial adhesion,with the increase of multiplicity of infection,both the flow chamber and the conventional culture plate detected the elevated amount of adhering bacteria,in a linear manner. However,with the same multiplicity of infection,the conventional culture plate model detected significantly larger amount of adhering bacteria when compared to the flow chamber( P < 0. 05). Conclusion Under appropriate flow rate and flow duration,our model is an alternative to the conventional plate for studying the adhesion of bacteria to the airway epithelial cells,with the advantages of being more close to the in vivo environment,high accuracy and real-time observation.
引文
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[13]MCCONNELL M J,ACTIS L,PACHON J.Acinetobacter baumannii:human infections,factors contributing to pathogenesis and animal models[J].FEMS Microbiol Rev,2013,37(2):130-155.DOI:10.1111/j.1574-6976.2012.00344.x.
[15]BAKKER D P,VAN DER PLAATS A,VERKERKE G J,et al.Comparison of velocity profiles for different flow chamber designs used in studies of microbial adhesion to surfaces[J].Appl Environ Microbiol,2003,69(10):6280-6287.DOI:10.1128/aem.69.10.6280-6287.2003.
[16]KHAN O F,CHAMBERLAIN M D,SEFTON M V.Toward an in vitro vasculature:differentiation of mesenchymal stromal cells within an endothelial cell-seeded modular construct in a microfluidic flow chamber[J].Tissue Eng Part A,2012,18(7/8):744-756.DOI:10.1089/ten.TEA.2011.0058.
[2]SMANI Y,MCCONNELL M J,PACHON J.Role of fibronectin in the adhesion of Acinetobacter baumannii to host cells[J].PLo S ONE,2012,7(4):e33073.DOI:10.1371/journal.pone.0033073.
[3]PEREZ A,MERINO M,RUMBO-FEAL S,et al.The Fha B/Fha C two-partner secretion system is involved in adhesion of Acinetobacter baumannii Ab H12O-A2 strain[J].Virulence,2017,8(6):959-974.DOI:10.1080/21505594.2016.1262313.
[4]VAN DER FLIER M,CHHUN N,WIZEMANN T M,et al.Adherence of Streptococcus pneumoniae to immobilized fibronectin[J].Infect Immun,1995,63(11):4317-4322.
[5]ANDERSEN T E,KINGSHOTT P,PALARASAH Y,et al.A flow chamber assay for quantitative evaluation of bacterial surface colonization used to investigate the influence of temperature and surface hydrophilicity on the biofilm forming capacity of uropathogenic Escherichia coli[J].J Microbiol Methods,2010,81(2):135-140.DOI:10.1016/j.mimet.2010.02.009.
[6]ARAI T,OCHIAI K,SENPUKU H.Actinomyces naeslundii Gro EL-dependent initial attachment and biofilm formation in a flow cell system[J].J Microbiol Methods,2015,109:160-166.DOI:10.1016/j.mimet.2014.12.021.
[7]YU C,LI X,ZHANG N,et al.Inhibition of biofilm formation by D-tyrosine:effect of bacterial type and D-tyrosine concentration[J].Water Res,2016,92:173-179.DOI:10.1016/j.watres.2016.01.037.
[8]QIN L,KIDA Y,ISHIWADA N,et al.The relationship between biofilm formations and capsule in Haemophilus influenzae[J].J Infect Chemother,2014,20(3):151-156.DOI:10.1016/j.jiac.2013.06.001.
[9]NEIL R B,SHAO J Q,APICELLA M A.Biofilm formation on human airway epithelia by encapsulated Neisseria meningitidis serogroup B[J].Microbes Infect,2009,11(2):281-287.DOI:10.1016/j.micinf.2008.12.001.
[10]黎皓思,潘频华.鲍曼不动杆菌黏附相关机制研究进展[J].中国感染与化疗杂志,2015,15(5):496-500.LI H S,PAN P H.Research advances on adherence mechanism of Acinetobacter baumannii.[J].Chin J Infect Chemother,2015,15(5):496-500.
[11]GUZEK A,RYBICKI Z,KORZENIEWSKI K,et al.Etiological factors causing lower respiratory tract infections isolated from hospitalized patients[J].Adv Expl Med Biol,2015,835:37-44.DOI:10.1007/5584_2014_23.
[12]NAMENDYS-SILVA S A,CORREA-GARCIA P,GARCIAGUILLIN F J,et al.Outcomes of critically ill cancer patients with Acinetobacter baumannii infection[J].World J Crit Care Med,2015,4(3):258-264.DOI:10.5492/wjccm.v4.i3.258.
[13]MCCONNELL M J,ACTIS L,PACHON J.Acinetobacter baumannii:human infections,factors contributing to pathogenesis and animal models[J].FEMS Microbiol Rev,2013,37(2):130-155.DOI:10.1111/j.1574-6976.2012.00344.x.
[15]BAKKER D P,VAN DER PLAATS A,VERKERKE G J,et al.Comparison of velocity profiles for different flow chamber designs used in studies of microbial adhesion to surfaces[J].Appl Environ Microbiol,2003,69(10):6280-6287.DOI:10.1128/aem.69.10.6280-6287.2003.
[16]KHAN O F,CHAMBERLAIN M D,SEFTON M V.Toward an in vitro vasculature:differentiation of mesenchymal stromal cells within an endothelial cell-seeded modular construct in a microfluidic flow chamber[J].Tissue Eng Part A,2012,18(7/8):744-756.DOI:10.1089/ten.TEA.2011.0058.