人脐带间充质干细胞培养上清液与阿莫西林体外联合应用对金黄色葡萄球菌的结构影响

Bacteriostatic effect of human umbilical cord mesenchymal stem cell secretion combined with amoxicillin on staphylococcus aureus

目的探讨联合应用人脐带间充质干细胞(h UCMSC)上清液与阿莫西林对烧伤创面分离的金黄色葡萄球菌的抗菌活性研究。方法 (1)取足月剖宫产健康胎儿的脐带组织,分离、培养、鉴定h UCMSC后选取第3代细胞接种于4个6孔板,对应培养后分别提取其上清液用于以下实验,其中1组为100 ng/m L脂多糖预处理h UCMSC培养上清液。选取内蒙古烧伤研究所患者创面分离培养的金黄色葡萄球菌菌株进行体外增殖培养,按随机数字表法分为阿莫西林对照组(简称对照组)、阿莫西林+上清液组(简称上清液组)、阿莫西林+上清液+LL-37抗体组(简称抗体组)、阿莫西林+100 ng/m L脂多糖预处理上清液组(简称预处理组)。于7、12、24、48 h读取各组阿莫西林E-Test药敏试条的最低抑菌浓度(MIC)值,计算上清液组、抗体组、预处理组阿莫西林的部分抑菌浓度(FIC)指数。(2)细胞、细菌分组和培养同实验(1),选取金黄色葡萄球菌菌株进行体外增殖培养24 h,每组在抑菌环附近取菌,涂片固定,扫描电镜检测。(3)对数据行析因设计方差分析、单因素方差分析、LSD-t检验、Kruskal-Wallis检验、Mann-Whitney检验。结果 (1)阿莫西林E-Test药敏试条结果:在不同时相点(7、12、24、48 h),对照组阿莫西林对金黄色葡萄球菌的MIC大于上清液组、抗体组和预处理组(Z值为6.609~6.655,P值均小于0.05),抗体组阿莫西林对金黄色葡萄球菌的MIC大于上清液组和预处理组(Z值为5.797~6.656,P值均小于0.05),上清液组阿莫西林对金黄色葡萄球菌的MIC大于预处理组(Z值为5.923~6.656,P值均小于0.05)。在不同时相点(7、12、24、48 h),上清液组、预处理组阿莫西林的FIC指数均小于0.5,抗体组小于或接近0.5且小于1.0。(2)扫描电镜结果:对照组金黄色葡萄球菌的排列变得松散,无规律,不能聚集成葡萄状,细胞间的界限略变模糊,部分胞体间界限明显。上清液组细菌形状无圆形结构,部分胞体壁、细胞膜已经被破坏,可见少量穿膜孔道,细胞壁的降解物多糖类物质在菌体细胞附近聚集,从而使细胞黏结成团块状。抗体组细菌形状变形,细胞壁和细胞膜界限敏明显,细胞膜凹陷。预处理组细胞膜已经被完全破坏,可见大量穿膜孔道,可见部分细胞崩解。结论 h UCMSC上清液联合阿莫西林协同抗菌,可以有效增强抗菌作用,中和敲除LL-37后,h UCMSC上清液的协同抗菌作用明显降低。适宜浓度脂多糖预处理后h UCMSC上清液的协同抗菌作用增强。

Objective To study the antimicrobial activity of human umbilical cord mesenchymal stem cell( h UCMSC) supernatant and amoxicillin in staphylococcus aureus which isolated from burn wounds.Methods( 1) h UCMSC were isolated from umbilical cord tissue of full-term healthy fetus after cesarean section and cultured. The third generation h UCMSC was inoculated into four 6-well plates,lipopolysaccharide was 100 ng/m L in one 6-well plate medium while no lipopolysaccharide in the other 6-well plates. The supernatant was extracted after corresponding culture for the following experiments.Staphylococcus aureus strains that come from Inner Mongolia Burn Medical Research Institute were selected for in vitro proliferation culture. As the random number table,divided into four groups: amoxicillin + control( referred to as control group),amoxicillin + h UCMSC supernatant( referred to as supernatant group),amoxicillin + h UCMSC supernatant + LL-37 antibody group( referred to as antibody group),amoxicillin+ 100 ng/m L lipopolysaccharide pretreatment h UCMSC supernatant group( referred to as pretreatment group). At post culture hour 7,12,24 and 48,ten blood agar plates in each group. The minimum inhibitory concentration of amoxicillin against staphylococcus aureus of each group was recored. The fractional inhibitory concentration index of amoxicillin in this four group was calculated at post culture hour,and the effect of synergy was evaluated.( 2) The experiment was the same as( 1),ten blood agar plates in each group. When staphylococcus aureus strains were inoculated in vitro for 24 h, and each group staphylococcus aureus was stained near the antibacterial ring,than fixed,scanned by electron microscopy.( 3) Data were processed with analysis of variance of factorial design,one-way analysis of variance,LSD-t test,Kruskal-Wallis test,Mann-Whitney test. Results( 1) At each post culture hour,the minimum inhibitory concentration of amoxicillin against staphylococcus aureus was higer in control group than those of supernatant group,antibody group and pretreatment group( with Z values from 6. 609 to 6. 655,P values below 0. 05); The minimum inhibitory concentration of amoxicillin against staphylococcus aureus was higer in antibody group than those of supernatant group and pretreatment group( with Z values from 5. 797 to6. 656,P values below 0. 015); The minimum inhibitory concentration of amoxicillin against staphylococcus aureus was higer in supernatant group than those of pretreatment group( with Z values from 5. 923 to 6. 656,P values below 0. 05).( 2) Scanning electron microscopy results: the control group of staphylococcus aureus arranged to become loose,irregular,can not be gathered into grapes,the boundaries between cells slightly blurred,some of the boundaries between the cell body is obvious. Supernatant group of bacteria shape no circular structure,part of the cell wall,the membrane has been destroyed,showing a small amount of perforation membrane,cell wall degradation of polysaccharides in the vicinity of bacteria cells gathered,so that cells adhere to the lumps. The Antibody group of bacterial deformation,cell wall and cell membrane limit sensitive, cell membrane depression. Pretreatment of the cell membrane has been completely destroyed,showing a large number of perforation through the membrane, showing some of the cells disintegrated. Conclusion h UCMSC supernatant combined with amoxicillin antibacterial,can effectively reduce the amount of amoxicillin,h UCMSC supernatant synergistic antibacterial effect was significantly reduced after knockout LL-37. While,synergistic antibacterial action of lipopolysaccharide pretreatment h UCMSC supernatant is surpass h UCMSC supernatant.