Background and Aims:Hepatocellular carcinoma(HCC)is a common primary liver neoplasm with high mortality.Dermcidin(DCD),an antimicrobial peptide,has been reported to participate in oncogenesis.This study assessed the e...Background and Aims:Hepatocellular carcinoma(HCC)is a common primary liver neoplasm with high mortality.Dermcidin(DCD),an antimicrobial peptide,has been reported to participate in oncogenesis.This study assessed the effects and underlying molecular events of DCD overexpression and knockdown on the regulation of HCC progression in vitro and in vivo.Methods:The serum DCD level was detected using enzyme-linked immunosorbent assay.DCD overexpression,knockdown,and Ras-related C3 botulinum toxin substrate 1(Rac1)rescue were performed in SK-HEP-1 cells using plasmids.Immunofluorescence staining,quantitative PCR,and Western blotting were used to detect the expression of different genes and proteins.Differences in HCC cell migration and invasion were detected by Transwell migration and invasion assays.A nude mouse HCC cell orthotopic model was employed to verify the in vitro data.Results:The level of serum DCD was higher in patients with HCC and in SK-HEP-1 cells.DCD overexpression caused upregulation of DCD,fibronectin,Rac1,and cell division control protein 42 homologue(Cdc42)mRNA and proteins as well as actin-related protein 2/3(Arp2/3)protein(but reduced Arp2/3 mRNA levels)and activated Rac1 and Cdc42.Phenotypically,DCD overexpression induced HCC cell migration and invasion in vitro,whereas knockout of DCD expression had the opposite effects.A Rac1 rescue experiment in DCD-knockdown HCC cells increased HCC cell migration and invasion and increased the levels of active Rac1/total Rac1,Wiskott-Aldrich syndrome family protein(WASP),Arp2/3,and fibronectin.DCD overexpression induced HCC cell metastasis to the abdomen and liver in vivo.Conclusions:DCD promotes HCC cell migration,invasion,and metastasis through upregulation of noncatalytic region of tyrosine kinase adaptor protein 1(Nck1),Rac1,Cdc42,WASP,and Arp2/3,which induce actin cytoskeletal remodeling and fibronectin-mediated cell adhesion in HCC cells.展开更多
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
基金supported in part by grants from Natural Science Foundation of Guangdong Province(#2021A1515010668)the National Natural Science Foundation of China(#81503424)+2 种基金the Guangzhou Science and Technology Innovation Commission(#201704020171)the Traditional Chinese Medicine Bureau of Guangdong Province(#20201263)the Guangzhou Municipal Health and Health Committee(#20191A011014,#20202A011006,#20192A010005).
文摘Background and Aims:Hepatocellular carcinoma(HCC)is a common primary liver neoplasm with high mortality.Dermcidin(DCD),an antimicrobial peptide,has been reported to participate in oncogenesis.This study assessed the effects and underlying molecular events of DCD overexpression and knockdown on the regulation of HCC progression in vitro and in vivo.Methods:The serum DCD level was detected using enzyme-linked immunosorbent assay.DCD overexpression,knockdown,and Ras-related C3 botulinum toxin substrate 1(Rac1)rescue were performed in SK-HEP-1 cells using plasmids.Immunofluorescence staining,quantitative PCR,and Western blotting were used to detect the expression of different genes and proteins.Differences in HCC cell migration and invasion were detected by Transwell migration and invasion assays.A nude mouse HCC cell orthotopic model was employed to verify the in vitro data.Results:The level of serum DCD was higher in patients with HCC and in SK-HEP-1 cells.DCD overexpression caused upregulation of DCD,fibronectin,Rac1,and cell division control protein 42 homologue(Cdc42)mRNA and proteins as well as actin-related protein 2/3(Arp2/3)protein(but reduced Arp2/3 mRNA levels)and activated Rac1 and Cdc42.Phenotypically,DCD overexpression induced HCC cell migration and invasion in vitro,whereas knockout of DCD expression had the opposite effects.A Rac1 rescue experiment in DCD-knockdown HCC cells increased HCC cell migration and invasion and increased the levels of active Rac1/total Rac1,Wiskott-Aldrich syndrome family protein(WASP),Arp2/3,and fibronectin.DCD overexpression induced HCC cell metastasis to the abdomen and liver in vivo.Conclusions:DCD promotes HCC cell migration,invasion,and metastasis through upregulation of noncatalytic region of tyrosine kinase adaptor protein 1(Nck1),Rac1,Cdc42,WASP,and Arp2/3,which induce actin cytoskeletal remodeling and fibronectin-mediated cell adhesion in HCC cells.
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.
