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...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 tissues of full-term healthy fetuses after cesarean section and then cultured. Cells in the third passage were chosen for the use of experiment after identification. SA strains isolated from wounds of burn patients in our burn wards were used in the following experiment. Cells were divided into 0, 10, 100 and 1,000 ng/ml lipopolysaccharide (LPS) groups by use of the random number table (similarly hereinafter). Cells were cultured with culture medium containing mesenchymal stem cells (MSCs) after being treated with medium containing corresponding mass concen-trations 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 (ELISA). Ninety blood agar culture plates were divided into ciprofloxacin control group (CC), ciprofloxacin + supernatant group (CS), and ciprofloxacin + supernatant+ LL-37 antibody group (CSL), with 30 blood agar culture plates in each group. Blood agar culture plates in group CC were coated with 1.5 × 108 colony forming unit (CFU)/ml bacteria solution prepared with normal saline. Blood agar culture plates in group CS were coated with 1.5 × 108 CFU/ml bacteria solution prepared with normal saline and hUCMSC culture supernatant (cultured by MSC culture medium, the same below) in double volume of normal saline. Blood agar culture plates in group CSL were coated with 1.5 × 108 CFU/ml bacteria solution prepared with normal saline, hUCMSC culture supernatant in double volume of normal saline, and 2.6 μL of LL-37 antibodies at the concentration of 2 μg/ml. At PCH 12, 24 and 48, 10 blood agar culture plates were taken out from each group to observe the distribution of SA colonies on blood agar culture plates and to measure diameters of zones of inhibition of ciprofloxacin. The minimum inhibitory concentration (MIC) of ciprofloxacin against SA in each group was recorded. Fractional inhibitory concentration (FIC) indexes of ciprofloxacin in group CS and group CSL at PCH 12, 24 and 48 were calculated, with the synergistic effect evaluated. Data were processed with factorial design ANOVA, one way ANOVA, LSD-t test, Kruskal-Wallis test and Mann-Whitney U test. Results: (1) At each PCH, the content of LL-37 in cell culture supernatant in 10 ng/ml LPS group, 100 ng/ml LPS group or 1,000 ng/ml LPS group was higher than that in 0 ng/ml LPS group (with t values ranging from 11.22 to 33.36, p values all below .01);the content of LL-37 in cell culture supernatant in either 100 ng/ml LPS group or 1,000 ng/ml LPS group was higher than that in 10 ng/ml LPS group (with t values ranging from 2.24 to 18.73, p < .05 or p < .01);the content of LL-37 in cell culture supernatant in 1,000 ng/ml LPS group was higher than that in 100 ng/ml LPS group (with t values ranging from 12.46 to 14.70, p values all below .01). (2) At PCH 12, 24 and 48, the bacterial colonies in groups CC, CS and CSL became integrated over time. In CC group, diameters of zones of inhibition of ciprofloxacin at PCH 12, 24 and 48 were 26 mm, 24 mm and 23 mm respectively, with no obvious changes. At PCH 12, 24 and 48, diameters of zones of inhibition of ciprofloxacin in groups CS and CSL were 82 mm, 71 mm, 68 mm and 74 mm, 59 mm, 56 mm respectively, which were significantly larger than those in group CC. (3) At each PCH, MIC of ciprofloxacin against SA in group CC was significantly higher than that in groups CS and CSL respectively (with Z values ranging from 6.22 to 6.71, p values all below .01);MIC of ciprofloxacin against SA in group CSL was significantly higher than that in group CS (with Z values all equal to 6.72, p values all below .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 hUCMSC culture supernatant had a synergistically antibacterial effect when combined with ciprofloxacin. Conclusions: hUCMSCs can secrete LL-37, and the secretion level is improved with increase of LPS concentration. The com-bination of hUCMSC culture supernatant with ciprofloxacin can decrease the dosage of ciprofloxacin in resisting SA effectively. Once LL-37 is neutralized, the synergistically antibacterial effect of hUCMSC culture supernatant is decreased thereby.展开更多
文摘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 tissues of full-term healthy fetuses after cesarean section and then cultured. Cells in the third passage were chosen for the use of experiment after identification. SA strains isolated from wounds of burn patients in our burn wards were used in the following experiment. Cells were divided into 0, 10, 100 and 1,000 ng/ml lipopolysaccharide (LPS) groups by use of the random number table (similarly hereinafter). Cells were cultured with culture medium containing mesenchymal stem cells (MSCs) after being treated with medium containing corresponding mass concen-trations 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 (ELISA). Ninety blood agar culture plates were divided into ciprofloxacin control group (CC), ciprofloxacin + supernatant group (CS), and ciprofloxacin + supernatant+ LL-37 antibody group (CSL), with 30 blood agar culture plates in each group. Blood agar culture plates in group CC were coated with 1.5 × 108 colony forming unit (CFU)/ml bacteria solution prepared with normal saline. Blood agar culture plates in group CS were coated with 1.5 × 108 CFU/ml bacteria solution prepared with normal saline and hUCMSC culture supernatant (cultured by MSC culture medium, the same below) in double volume of normal saline. Blood agar culture plates in group CSL were coated with 1.5 × 108 CFU/ml bacteria solution prepared with normal saline, hUCMSC culture supernatant in double volume of normal saline, and 2.6 μL of LL-37 antibodies at the concentration of 2 μg/ml. At PCH 12, 24 and 48, 10 blood agar culture plates were taken out from each group to observe the distribution of SA colonies on blood agar culture plates and to measure diameters of zones of inhibition of ciprofloxacin. The minimum inhibitory concentration (MIC) of ciprofloxacin against SA in each group was recorded. Fractional inhibitory concentration (FIC) indexes of ciprofloxacin in group CS and group CSL at PCH 12, 24 and 48 were calculated, with the synergistic effect evaluated. Data were processed with factorial design ANOVA, one way ANOVA, LSD-t test, Kruskal-Wallis test and Mann-Whitney U test. Results: (1) At each PCH, the content of LL-37 in cell culture supernatant in 10 ng/ml LPS group, 100 ng/ml LPS group or 1,000 ng/ml LPS group was higher than that in 0 ng/ml LPS group (with t values ranging from 11.22 to 33.36, p values all below .01);the content of LL-37 in cell culture supernatant in either 100 ng/ml LPS group or 1,000 ng/ml LPS group was higher than that in 10 ng/ml LPS group (with t values ranging from 2.24 to 18.73, p < .05 or p < .01);the content of LL-37 in cell culture supernatant in 1,000 ng/ml LPS group was higher than that in 100 ng/ml LPS group (with t values ranging from 12.46 to 14.70, p values all below .01). (2) At PCH 12, 24 and 48, the bacterial colonies in groups CC, CS and CSL became integrated over time. In CC group, diameters of zones of inhibition of ciprofloxacin at PCH 12, 24 and 48 were 26 mm, 24 mm and 23 mm respectively, with no obvious changes. At PCH 12, 24 and 48, diameters of zones of inhibition of ciprofloxacin in groups CS and CSL were 82 mm, 71 mm, 68 mm and 74 mm, 59 mm, 56 mm respectively, which were significantly larger than those in group CC. (3) At each PCH, MIC of ciprofloxacin against SA in group CC was significantly higher than that in groups CS and CSL respectively (with Z values ranging from 6.22 to 6.71, p values all below .01);MIC of ciprofloxacin against SA in group CSL was significantly higher than that in group CS (with Z values all equal to 6.72, p values all below .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 hUCMSC culture supernatant had a synergistically antibacterial effect when combined with ciprofloxacin. Conclusions: hUCMSCs can secrete LL-37, and the secretion level is improved with increase of LPS concentration. The com-bination of hUCMSC culture supernatant with ciprofloxacin can decrease the dosage of ciprofloxacin in resisting SA effectively. Once LL-37 is neutralized, the synergistically antibacterial effect of hUCMSC culture supernatant is decreased thereby.
