Zhou B, Tu H L, Ba T, Wang L F, Wang S J, Nie S Y
Department of Burns, the Third Affiliated Hospital of Inner Mongolia Medical University, Burn Institute of Inner Mongolia, Baotou 014010, China.
Zhonghua Shao Shang Za Zhi. 2017 Jun 20;33(6):355-360. doi: 10.3760/cma.j.issn.1009-2587.2017.06.014.
To explore the effects of combined application of culture supernatant of human umbilical cord mesenchymal stem cells (hUCMSCs) and ciprofloxacin on (SA) in vitro. 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- test, Kruskal-Wallis test, and Mann-Whitney test. (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 values from 11.22 to 33.36, 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 values from 2.24 to 18.73, <0.05 or <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 values from 12.46 to 14.70, 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 values from 6.22 to 6.71, values below 0.01); the MIC of ciprofloxacin against SA was significantly higher in group CSL than in group CS (with values all equal to 6.72, 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. 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.
探讨人脐带间充质干细胞（hUCMSCs）培养上清液与环丙沙星联合应用对体外金黄色葡萄球菌（SA）的影响。剖宫产术后从足月健康胎儿脐带组织中分离hUCMSCs并进行培养。经鉴定后，选用第3代细胞进行实验。实验采用从我院烧伤病房烧伤患者创面分离的SA菌株。根据随机数字表将细胞分为0、10、100和1000 ng/mL脂多糖（LPS）组（以下分组方法相同）。细胞用含相应质量浓度LPS的培养基处理12 h后，再用间充质干细胞（MSCs）培养基培养。在培养后6、12和24小时（PCH），每组取6孔细胞培养上清液，采用酶联免疫吸附测定法检测LL-37含量。将90个血琼脂平板分为环丙沙星对照组（CC）、环丙沙星+上清液组（CS）和环丙沙星+上清液+LL-37抗体组（CSL），每组30个血琼脂平板。CC组血琼脂平板接种用生理盐水配制的1.5×10⁸菌落形成单位（CFU）/mL菌液。CS组血琼脂平板接种用生理盐水和双倍体积生理盐水配制的hUCMSCs培养上清液（用MSCs培养基培养，下同）配制的1.5×10⁸CFU/mL菌液。CSL组血琼脂平板接种用生理盐水、双倍体积生理盐水配制的hUCMSCs培养上清液和浓度为2 μg/mL的2.6 μL LL-37抗体配制的1.5×10⁸CFU/mL菌液。在PCH 12、24和48小时，每组取10个血琼脂平板，观察SA菌落在血琼脂平板上的分布情况，并测量环丙沙星的抑菌圈直径。记录环丙沙星对每组SA的最低抑菌浓度（MIC）。计算PCH 12、24和48小时CS组和CSL组中环丙沙星的部分抑菌浓度（FIC）指数，并评估协同作用效果。数据采用析因设计方差分析、单因素方差分析、LSD检验、Kruskal-Wallis检验和Mann-Whitney检验进行处理。（1）在各PCH，10、100和1000 ng/mL LPS组细胞培养上清液中LL-37含量高于0 ng/mL LPS组（P值为11.22至33.36，P值均<0.01）；100和1000 ng/mL LPS组细胞培养上清液中LL-37含量高于10 ng/mL LPS组（P值为2.24至18.73，P<0.05或P<0.01）；1000 ng/mL LPS组细胞培养上清液中LL-37含量高于100 ng/mL LPS组（P值为12.46至14.70，P值均<0.01）。（2）在PCH 12、24和48小时，CC、CS和CSL组细菌菌落随时间开始融合。在PCH 12、24和48小时，CC组环丙沙星的抑菌圈直径分别为26、24和23 mm，无明显变化。在PCH 12、24和48小时，CS组和CSL组环丙沙星的抑菌圈直径分别为82、71、68 mm和74、59、56 mm，明显长于CC组。（3）在各PCH，CC组中环丙沙星对SA的MIC显著高于CS组和CSL组（P值为6.22至6.71，P值均<0.01）；CSL组中环丙沙星对SA的MIC显著高于CS组（P值均等于6.72，P值均<0.01）。（4）PCH 12、24和48小时CS组和CSL组中环丙沙星的FIC指数分别为0.011、0.032、0.032和0.122、0.350、0.350。结果表明，hUCMSCs培养上清液对环丙沙星有协同抗菌作用。hUCMSCs可分泌LL-37，且分泌水平随LPS浓度升高而增加。hUCMSCs培养上清液与环丙沙星联合应用可降低环丙沙星抗SA的用药剂量。一旦LL-37被中和，hUCMSCs培养上清液的协同抗菌作用减弱。
