Objective: To explore the effects of combined application of culture supernatant of human umbilical cord mesenchymal stem cells (hUCMSCs) and ciprofloxacin on Staphylococcus aureus (SA) in vitro. Methods: hUCMSCs were isolated from umbilical cord tissue of full-term healthy fetus after cesarean section and cultured. Cells in the third passage were used in the experiments after identification. SA strains isolated from wounds of burn patients in our burn wards were used in the experiments. Cells were divided into 0, 10, 100, and 1 000 ng/mL lipopolysaccharide (LPS) groups according to the random number table (the same dividing method below). Cells were cultured with culture medium of mesenchymal stem cells (MSCs) after being treated with medium containing the corresponding mass concentrations of LPS for 12 h. At post culture hour (PCH) 6, 12, and 24, 6 wells of culture supernatant of cells in each group were obtained to measure the content of LL-37 with enzyme-linked immunosorbent assay. Ninety blood agar plates were divided into ciprofloxacin control group (CC), ciprofloxacin+ supernatant group (CS), and ciprofloxacin+ supernatant+ LL-37 antibody group (CSL), with 30 blood agar plates in each group. Blood agar plates in group CC were coated with 1.5×10(8) colony forming unit (CFU)/mL bacteria solution prepared with normal saline. Blood agar plates in group CS were coated with 1.5×10(8) CFU/mL bacteria solution prepared with normal saline and culture supernatant of hUCMSCs (cultured by culture medium of MSCs, the same below) in double volume of normal saline. Blood agar plates in group CSL were coated with 1.5×10(8) CFU/mL bacteria solution prepared with normal saline, culture supernatant of hUCMSCs in double volume of normal saline, and 2.6 μL LL-37 antibody in the concentration of 2 μg/mL. At PCH 12, 24, and 48, 10 blood agar plates of each group were harvested to observe the distribution of SA colony on blood agar plate and to measure the diameter of bacterial inhibition ring of ciprofloxacin. The minimum inhibitory concentration (MIC) of ciprofloxacin against SA of each group was recorded. Fractional inhibitory concentration (FIC) indexes of ciprofloxacin in groups CS and CSL at PCH 12, 24, and 48 were calculated, and the effect of synergy was evaluated. Data were processed with analysis of variance of factorial design, one-way analysis of variance, LSD-t test, Kruskal-Wallis test, and Mann-Whitney U test. Results: (1) At each PCH, the content of LL-37 in culture supernatant of cells in 10, 100, and 1 000 ng/mL LPS groups was higher than that in 0 ng/mL LPS group (with t values from 11.22 to 33.36, P values below 0.01); the content of LL-37 in culture supernatant of cells in 100 and 1 000 ng/mL LPS groups was higher than that in 10 ng/mL LPS group (with t values from 2.24 to 18.73, P<0.05 or P<0.01); the content of LL-37 in culture supernatant of cells in 1 000 ng/mL LPS group was higher than that in 100 ng/mL LPS group (with t values from 12.46 to 14.70, P values below 0.01). (2) At PCH 12, 24, and 48, the bacterial colonies in groups CC, CS, and CSL began to integrate over time. At PCH 12, 24, and 48, the diameters of bacterial inhibition ring of ciprofloxacin in group CC were 26, 24, and 23 mm, respectively, with no obvious change. At PCH 12, 24, and 48, the diameters of bacterial inhibition ring of ciprofloxacin in groups CS and CSL were 82, 71, 68 mm, and 74, 59, 56 mm, respectively, significantly longer than those of group CC. (3) At each PCH, the MIC of ciprofloxacin against SA was significantly higher in group CC than in groups CS and CSL (with Z values from 6.22 to 6.71, P values below 0.01); the MIC of ciprofloxacin against SA was significantly higher in group CSL than in group CS (with Z values all equal to 6.72, P values below 0.01). (4) FIC indexes of ciprofloxacin in groups CS and CSL at PCH 12, 24, and 48 were 0.011, 0.032, 0.032, and 0.122, 0.350, 0.350, respectively. The results indicated that culture supernatant of hUCMSCs had synergistically antibacterial effect on ciprofloxacin. Conclusions: hUCMSCs can secrete LL-37, and the secretion level is increased with increase of LPS concentration. Combination of culture supernatant of hUCMSCs and ciprofloxacin can decrease the dosage of ciprofloxacin in resisting SA. Once LL-37 is neutralized, the synergistically antibacterial effect of culture supernatant of hUCMSCs is decreased.
目的： 探讨联合应用人脐带间充质干细胞(hUCMSC)培养上清液与环丙沙星对金黄色葡萄球菌的作用。 方法： 取足月剖宫产健康胎儿的脐带组织，分离、培养、鉴定hUCMSC后选取第3代细胞进行实验；取笔者单位烧伤患者创面分离培养的金黄色葡萄球菌菌株用于以下实验。按随机数字表法(下同)将细胞分为0、10、100、1 000 ng/mL LPS组，添加相应质量浓度LPS处理12 h后，换为间充质干细胞(MSC)培养液培养，培养6、12、24 h，每组收集6孔细胞培养上清液，ELISA法测定LL－37含量。将90个血琼脂平板培养基分为环丙沙星对照组、环丙沙星＋上清液组、环丙沙星＋上清液＋LL－37抗体组，每组30个。环丙沙星对照组培养基涂布1.5×10(8) CFU/mL生理盐水配制的菌液；环丙沙星＋上清液组培养基涂布1.5×10(8) CFU/mL由生理盐水和2倍生理盐水体积的hUCMSC培养上清液(用MSC培养液培养，下同)配制的菌液；环丙沙星＋上清液＋LL－37抗体组培养基涂布1.5×10(8) CFU/mL由生理盐水和2倍生理盐水体积的hUCMSC培养上清液配制的菌液，菌液中加入2 μg/mL LL－37抗体2.6 μL。培养12、24、48 h，每组取10个血琼脂平板培养基，观察血琼脂平板培养基上金黄色葡萄球菌菌落分布情况，测量环丙沙星抑菌环直径，读取环丙沙星对金黄色葡萄球菌的MIC。计算环丙沙星＋上清液组和环丙沙星＋上清液＋LL－37抗体组培养12、24、48 h时环丙沙星的部分抑菌浓度(FIC)指数并评定协同效果。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 U检验。 结果： (1)培养各时相点，10、100、1 000 ng/mL LPS组细胞培养上清液中LL－37含量均较0 ng/mL LPS组高(t值为11.22～33.36，P值均小于0.01)，100、1 000 ng/mL LPS组细胞培养上清液中LL－37含量均较10 ng/mL LPS组高(t值为2.24～18.73，P<0.05或P<0.01)，1 000 ng/mL LPS组细胞培养上清液中LL－37含量均较100 ng/mL LPS组高(t值为12.46～14.70，P值均小于0.01)。(2)培养12、24、48 h，环丙沙星对照组、环丙沙星＋上清液组、环丙沙星＋上清液＋LL－37抗体组血琼脂平板培养基中细菌菌落随时间延长逐渐融合。环丙沙星对照组培养12、24、48 h环丙沙星抑菌环直径变化不大，分别为26、24、23 mm。环丙沙星＋上清液组、环丙沙星＋上清液＋LL－37抗体组培养12、24、48 h环丙沙星抑菌环直径分别为82、71、68 mm和74、59、56 mm，明显大于环丙沙星对照组。(3)培养各时相点，环丙沙星对照组环丙沙星对金黄色葡萄球菌的MIC明显大于环丙沙星＋上清液组和环丙沙星＋上清液＋LL－37抗体组(Z值为6.22～6.71，P值均小于0.01)；环丙沙星＋上清液＋LL－37抗体组环丙沙星对金黄色葡萄球菌的MIC均明显大于环丙沙星＋上清液组(Z值均为6.72，P值均小于0.01)。(4)环丙沙星＋上清液组、环丙沙星＋上清液＋LL－37抗体组培养12、24、48 h时环丙沙星的FIC指数分别为0.011、0.032、0.032，0.122、0.350、0.350，即hUCMSC培养上清液对环丙沙星产生了协同抗菌作用。 结论： hUCMSC能够分泌LL－37且分泌水平随着LPS浓度的增加而提高。hUCMSC培养上清液联合环丙沙星抗金黄色葡萄球菌，可以有效降低环丙沙星用量，中和LL－37后，hUCMSC培养上清液的协同抗菌作用降低。.
Keywords: Ciprofloxacin; Dermcidins; Mesenchymal stem cells; Staphylococcus aureus.