中国修复重建外科杂志

中国修复重建外科杂志

人脐带间充质干细胞上清液与阿莫西林联合应用对金黄色葡萄球菌的抗菌研究

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目的 探讨联合应用人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)培养上清液与阿莫西林,对烧伤创面分离的金黄色葡萄球菌的抗菌活性。 方法 取足月剖宫产健康胎儿的脐带组织分离培养 hUCMSCs,选取第 3 代 hUCMSCs 分为 4 组,分别添加 0、10、100、1 000 ng/mL 脂多糖(lipopolysaccharide,LPS),于培养 6、12、24、48 h 采用 ELISA 法测定抗菌肽 LL-37 含量。取 160 个血琼脂平板培养基分 4 组,每组 40 个。分别于血琼脂平板培养基上涂布金黄色葡萄球菌菌液(对照组、A 组)、hUCMSCs 培养上清液配制的金黄色葡萄球菌菌液(上清液组、B 组)、hUCMSCs 培养上清液配制的金黄色葡萄球菌菌液+200 ng/mL 鼠抗人 LL-37 抗体(抗体组、C 组)、100 ng/mL LPS 预处理 hUCMSCs 培养上清液配制的金黄色葡萄球菌菌液(预处理组、D 组);各组 15 min 后于培养基中间平贴阿莫西林 E-Test 药敏试条,培养箱培养 7、12、24、48 h 时每组分别取 10 个培养基,观察金黄色葡萄球菌的菌落分布情况及阿莫西林的抑菌环大小,读取阿莫西林对金黄色葡萄球菌的最小抑菌浓度(minimal inhibitory concentration,MIC),计算 B、C、D 组培养 7、12、24、48 h 时阿莫西林的部分抑菌浓度(fractional inhibitory concentration,FIC)指数并评定协同抗菌效果。 结果 培养各时间点,随 LPS 浓度升高,细胞培养上清液中 LL-37 含量也逐渐升高,各 LPS 浓度组间比较差异均有统计学意义(P<0.05)。培养各时间点,各组血琼脂平板培养基中均出现明显抑菌环,呈“彗星拖尾”状,且细菌菌落随时间延长逐渐融合,24 h 后抑菌环趋于稳定。各时间点 D 组抑菌环的短轴直径均显著大于 A、B、C 组,B 组大于 A、C 组,C 组大于 A 组,差异均有统计学意义(P<0.05);A 组阿莫西林对金黄色葡萄球菌的 MIC 显著大于 B、C、D 组,C 组大于 B、D 组,B 组大于 D 组,差异均有统计学意义(P<0.05)。培养各时间点阿莫西林的 FIC 指数 C 组大于 B 、D组,B 组大于 D 组。各时间点 B、D 组 FIC 指数均<0.5,产生了有效的协同作用;C 组在 24 h 和 48 h 时 FIC 指数>0.5,hUCMSCs 培养上清液中的 LL37 中和沉默后,其协同抗菌作用降低。 结论 hUCMSCs 上清液能够分泌 LL-37,且分泌水平随 LPS 浓度增大不断提高;hUCMSCs 上清液联合阿莫西林协同抗金黄色葡萄球菌,可有效降低阿莫西林用量,中和 LL-37 后,hUCMSCs 上清液的协同抗菌作用明显降低。

Objective To study the antibacterial activity of human umbilical cord mesenchymal stem cells (hUCMSCs) culture supernatant and amoxicillin on Staphylococcus aureus isolated from burn wounds. Methods The hUCMSCs were isolated from fetal umbilical cord tissue of full-term healthy fetus after cesarean section and cultured. The 3rd generation cells were divided into 4 groups which were added 0, 10, 100, and 1 000 ng/mL lipopolysaccharide (LPS), respectively; the contents of antibacterial peptide LL-37 in each group were detected by ELISA method after 6, 12, 24, and 48 hours of culture. One hundred and sixty blood agar culture mediums were divided into 4 groups (40 in each group). The Staphylococcus aureus bacteria liquid (control group, group A), Staphylococcus aureus bacteria liquid prepared by hUCMSCs culture supernatant (supernatant group, group B), Staphylococcus aureus bacteria liquid prepared by hUCMSCs culture supernatant + 200 ng/mL mouse anti human LL-37 antibody (antibody group, group C), and Staphylococcus aureus bacteria liquid prepared by hUCMSCs culture supernatant which were pretreated by 100 ng/mL LPS (preconditioning group, group D) were coated on the blood agar culture mediums respectively. The E-Test amoxicillin drug sensitivity test strip were stricked in the middle medium in each group after 15 minutes. And after 7, 12, 24, and 48 hours, 10 mediums in each group were taked out to observe the distribution of colony of Staphylococcus aureus and the size of the bacteriostatic ring of amoxicillin, and observe the minimum inhibitory concentration (MIC) of amoxicillin against Staphylococcus aureus; the fractional inhibitory concentration (FIC) index of amoxicillin of groups B, C, and D were calculated after 7, 12, 24, and 48 hours of culture and the synergistic antibacterial effects were evaluated. Results At various time points, with the increase of LPS concentration, LL-37 content in hUCMSC supernatant was gradually increased, all showing significant differences among groups (P<0.05). At various time points, blood agar plate culture medium had obvious bacteriostatic ring with a " tail”-shape in each group, and the bacterial colonies gradually fused and the bacterial inhibition ring tended to be stable after 24 hours. The short axis diameter of bacterial inhibition ring in group D was significant larger than groups A, B, and C, in group B than in groups A and C, and in group C than in group A, all showing significant differences (P<0.05); the MIC of amoxicillin againstStaphylococcus aureus in group A was significant larger than groups B, C, and D, in group C than in groups B and D, and in group B than in group D, all showing significant differences (P<0.05). At various time points, the FIC index in group C was larger than groups B and D, and in group B than in group D. The FIC indexes in groups B and D were less than 0.5, producing the effective synergy; the FIC indexes in group C at 24 and 48 hours were more than 0.5, showing after the silence of LL-37 in hUCMSCs supernatant, the synergistic antibacterial effect decreased. Conclusion hUCMSCs supernatant can secrete LL-37, and the secretion level increases with the increase of LPS concentration. hUCMSCs supernatant combined with amoxicillin against Staphylococcus aureus, can effectively reduce the amount of amoxicillin; but after knockout LL-37, hUCMSCs supernatant synergistic antibacterial effect was significantly reduced.

关键词: 人脐带间充质干细胞; 金黄色葡萄球菌; 抗菌肽类; 阿莫西林

Key words: Human umbilical cord mesenchymal stem cells; Staphylococcus aureus; antibacterial peptide; amoxicillin

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