摘要
目的建立稳定发光的类中性粒细胞-大肠杆菌-金属片双荧光三相共培养动态模型,并研究其可行性和应用价值。方法抽提pUC57-mCherry质粒,电转入大肠杆菌临床株ST131,构建稳定发光的ST131-mCherry大肠杆菌。通过活体成像(IVIS)系统对构建的ST131-mCherry大肠杆菌进行筛选。通过稀释涂板法比较ST131-mCherry与ST131的生长曲线,通过结晶紫染色比较ST131-mCherry与ST131的生物膜形成能力。将ST131-mCherry与钛片共培养,荧光显像观察不同时间点成膜情况,并通过超声震荡稀释涂板法对钛片表面生物膜计量。采用全反式维甲酸(ARTA)诱导HL60-eGFP细胞分化为类中性粒细胞。使用诱导分化的HL60-eGFP细胞、ST131-mCherry与金属片共培养,构建双荧光三相共培养模型,通过荧光显微镜观察以及稀释涂板计数评价不同金属表面的类中性白细胞抗菌能力。使用独立样本t检验对OD值和活菌数等定量数据进行统计。结果经临床株筛选,质粒电转获得红色荧光菌株"ST131-mCherry",在体外连续培养48 h,ST131-mCherry可稳定发光。ST131-mCherry与野生株ST131相比,两者的生长曲线没有统计学差异。在胰蛋白胨大豆肉汤培养基(TSB)中,ST131与ST131-mCherry生物膜结晶紫染色490nm的OD值分别为(1. 04±0. 06)和(1. 04±0. 05)(t=0. 890,P> 0. 05);在0. 5%葡萄糖胰蛋白胨大豆肉汤培养基(TSBG)中,分别为(1. 55±0. 06)和(1. 49±0. 17)(t=0. 822,P> 0. 05);在10%滑液(SF)中,分别为(2. 13±0. 16)和(2. 124±0. 13)(t=0. 833,P>0. 05)。ST131-mCherry可以在钛片表面成膜,其荧光强度随着成膜量的增加而变强。诱导分化的HL60-eGFP细胞、ST131-mCherry与钛片或钽片共培养结果显示,经过60 min的共培养,钽金属体系内的HL60-eGFP细胞能够吞噬更多的ST131-mCherry大肠杆菌,钛片体系和钽片体系内细菌存活率分别为(33±2. 5)%和(15. 7±1. 2)%(t=10. 47,P <0. 01)。结论本研究成功构建了大肠杆菌-类中性粒细胞-金属片双荧光三相动态共培养模型,该模型是1种实用的PJI相关抗菌材料体外研究方法。
Objective To establish a stable fluorescent neutrophil-Escherichia coli( E. coli)-metals binary fluorescence dynamic model,and to evaluate the practicability and application value of this model.Methods p UC57-mCherry plasmid was isolated and transferred to clinical E. coli isolates( ST131) through electroporation,ST131-mCherry was then constructed. ST131-mCherry was screened using in-vivo imaging( IVIS) system. Dilution plate method was utilized to compare the growth curve of ST131-mCherry and ST131,crystal violet staining was used to compare the biofilms in 96-well plates. After the co-culture of ST131-mCherry with titanium,the biofilms at different time points were visualized by fluorescence microscope and quantified by ultrasonic concussion and dilution plate method. All-trans-retinoid acid( ARTA) was used to induce HL60-enhanced green fhcorescent protein( e GFP) to differentiate into neutrophil like cells.Induced differentiated HL60-eGFP,ST131-mCherry and metals were co-cultured to establish the binary fluorescence triple components dynamic model. The antibacterial efficiencies of neutrophil like cells on different metal-surfaces were compared through fluorescent observation and bacterial count. Independent t test was used to evaluate the statistical significance. Results Red fluorescence clinical E. coli( ST131-mCherry)was constructed through clinical strains screening and plasmid electroporation. The fluorescence of ST131-m Cherry was proved to be stable in the 48 h consecutive culture. There was no significant difference of growth curve between ST131-mCherry and ST131 wild strain. OD value of biofilms stained with crystal violet in tryptic soy broth( TSB) at 490 nm was( 1. 04 ± 0. 06) and( 1. 04 ± 0. 05)( t = 0. 890,P > 0. 05) for ST131 and ST131-mCherry respectively,it was( 1. 55 ± 0. 06) and( 1. 49 ± 0. 17)( t = 0. 822,P > 0. 05) in 0. 5%glucose tryptic soy broth( TSBG),( 2. 13 ± 0. 16) and( 2. 124 ± 0. 13)( t = 0. 833,P > 0. 05) in 10%synovial fluid( SF). ST131-mCherry can form biofilms on the surface of titanium,the fluorescent intensity of bacteria was enhanced with the growth of biofilm. The co-culture of differentiated HL60-eGFP,ST131-m Cherry and titanium/tantalum revealed that neutrophils could devour more E. coli in the tantalum system after60 min,bacterial survival rate was( 33 ± 2. 5) % and( 15. 7 ± 1. 2) % for Ti and Ta system respectively( t =10. 47,P < 0. 01). Conclusion E. coli-fluorescent neutrophil-metals binary fluorescence dynamic model is constructed in current study,which is a practical tool to investigate antimicrobial biomaterials in vitro.
引文
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