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Objective@#To preliminarily evaluate the effect of exosomes from human adipose-derived stem cells (hADSCs) on the migration of epidermal cells.@*Methods@#hADSCs were isolated from adipose tissues obtained from a healthy woman after liposuction, and subjected to culture. Exosomes were collected from the culture supernatant of hADSCs by using a modified method combining ultracentrifugation with ultrafiltration, and identified and isolated by transmission electron microscopy (TEM) , dynamic light scattering (DLS) and Western blot analysis. Some cultured HaCaT cells were divided into several groups to be treated with hydrogen peroxide (H2O2) at different concentrations of 0, 50, 100, 200, 250, 300, 400, 500, 600, 800 μmol/L for 1 hour, and cell counting kit-8 (CCK8) assay was performed to evaluate the effect of H2O2 on the survival rate of HaCaT cells. Some HaCaT cells were classified into 2 groups to be pretreated with phosphate-buffered saline (PBS) (normal group) or 100 μmol/L H2O2 (injury group) for 0.5 hour; then, HaCaT cells in the 2 groups were separately divided into treatment group and control group to be co-cultured with exosomes or not. Confocal fluorescence microscopy was conducted to confirm the uptake of PKH26-labelled exosomes by HaCaT cells, scratch assay to estimate the wound healing potential, and Transwell assay to evaluate the migratory activity of HaCaT cells. Statistical analysis was carried out by using one-way analysis of variance for comparison among groups, least significant difference (LSD) -t test for multiple comparisons, and Spearman correlation analysis for analyzing correlations.@*Results@#TEM showed that the exosomes isolated from hADSCs were saucer-like nanovesicles with diameters of 60-80 nm. DLS revealed that the purity of the isolated exosomes was 65.88%, and they were stained positively for CD63, Alix and TSG101, which coincided with the basic characteristics of exosomes. CCK8 assay showed that survival rates of HaCaT cells gradually decreased along with the increase of H2O2 concentrations after 1-hour treatment, and were negatively correlated with the concentration of H2O2 (r= -0.91, P < 0.01) . Confocal fluorescence microscopy showed that the isolated exosomes could be endocytosed into impaired HaCaT cells. Scratch assay showed that the gap-filling rates at 12 hours were 40.26% ± 0.64%, 69.57% ± 0.69%, 32.28% ± 0.31% and 69.62% ± 1.68% in the normal control group, normal treatment group, injury control group and injury treatment group respectively, and the injury treatment group showed a significantly increased gap-filling rate compared with the injury control group (t = 37.33, P < 0.01) . Transwell assay showed that the number of migratory cells per × 10 field was 20.85 ± 4.84, 44.8 ± 5.24, 14.95 ± 2.58 and 40.05 ± 7.66 in the normal control group, normal treatment group, injury control group and injury treatment group respectively, and was significantly larger in the injury treatment group than in the injury control group (t = 25.10, P < 0.01) .@*Conclusion@#Exosomes isolated from hADSCs can improve the migration of HaCaT cells after oxidative injury.
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Objective To preliminarily evaluate the effect of exosomes from human adipose-derived stem cells (hADSCs) on the migration of epidermal cells.Methods hADSCs were isolated from adipose tissues obtained from a healthy woman after liposuction,and subjected to culture.Exosomes were collected from the culture supernatant of hADSCs by using a modified method combining ultracentrifugatiou with ultrafiltration,and identified and isolated by transmission electron microscopy (TEM),dynamic light scattering (DLS) and Western blot analysis.Some cultured HaCaT cells were divided into several groups to be treated with hydrogen peroxide (H2O2) at different concentrations of 0,50,100,200,250,300,400,500,600,800 μmol/L for 1 hour,and cell counting kit-8 (CCK8) assay was performed to evaluate the effect of H2O2 on the survival rate of HaCaT cells.Some HaCaT cells were classified into 2 groups to be pretreated with phosphate-buffered saline (PBS) (normal group) or 100 μmol/L H2O2 (injury group) for 0.5 hour;then,HaCaT cells in the 2 groups were separately divided into treatment group and control group to be cocultured with exosomes or not.Confocal fluorescence microscopy was conducted to confirm the uptake of PKH26-labelled exosomes by HaCaT cells,scratch assay to estimate the wound healing potential,and Transwell assay to evaluate the migratory activity of HaCaT cells.Statistical analysis was carried out by using one-way analysis of variance for comparison among groups,least significant difference (LSD)-t test for multiple comparisons,and Spearman correlation analysis for analyzing correlations.Results TEM showed that the exosomes isolated from hADSCs were saucer-like nanovesicles with diameters of 60-80 nm.DLS revealed that the purity of the isolated exosomes was 65.88％,and they were stained positively for CD63,Alix and TSG101,which coincided with the basic characteristics of exosomes.CCK8 assay showed that survival rates of HaCaT cells gradually decreased along with the increase of H2O2 concentrations after 1-hour treatment,and were negatively correlated with the concentration of H2O2 (r =-0.91,P ＜ 0.01).Confocal fluorescence microscopy showed that the isolated exosomes could be endocytosed into impaired HaCaT cells.Scratch assay showed that the gap-filling rates at 12 hours were 40.26％ ± 0.64％,69.57％ ± 0.69％,32.28％ ± 0.31％ and 69.62％ ± 1.68％ in the normal control group,normal treatment group,injury control group and injury treatment group respectively,and the injury treatment group showed a significantly increased gap-filling rate compared with the injury control group (t =37.33,P ＜ 0.01).Transwell assay showed that the number of migratory cells per × 10 field was 20.85 ± 4.84,44.8 ± 5.24,14.95 ± 2.58 and 40.05 ± 7.66 in the normal control group,normal treatment group,injury control group and injury treatment group respectively,and was significantly larger in the injury treatment group than in the injury control group (t =25.10,P ＜ 0.01).Conclusion Exosomes isolated from hADSCs can improve the migration of HaCaT cells after oxidative injury.
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Objective To evaluate the immunogenicity of a recombinant adenovirus containing the porin IB (PIB) gene and Neisseria surface protein A (NspA) gene of N.gonorrhoeae in BALB/c mice.Methods A recombinant adenovirus containing the PIB gene and NspA gene of N.gonorrhoeae (pAdEasy-1-PN) was constructed in previous studies.Thirty BALB/c mice were randomly and equally divided into 5 groups:low-,medium-and high-dose experiment group intramuscularly immunized with 104,106,and 108 50％ tissue culture infective dose (TCID50) of the recombinant adenovirus pAdEasy-1-PN,respectively,pAdEasy-1 control group immunized with 106 TCID50 of pAdEasy-1,blank control group immunized with sodium chloride physiological solution.Immunization was carried out twice at a 4-week interval.Serum samples were collected at 0,3 and 5 weeks after the first immunization,and spleens were removed at 5 weeks followed by the isolation of spleen lymphocytes.Enzyme linked immunosorbent assay (ELISA) was performed to determine the serum levels of PIB-specific and NspA-specific antibodies,methyl thiazolyl tetrazolium (MTT) assay to evaluate the proliferaton activity of spleen lymphocytes after stimulation by the recombinant PIB and NspA antigens.A slide agglutination test was used to estimate the anti-bacterial activity of murine serum.The complement-dependent bacteriolytic activity of murine serum was also evaluated.Statistical analysis was carried out by t test.Results Both humoral and cellular immune response specific to PIB and NspA were elicited by the recombinant adenovirus in mice.At 3 and 5 weeks after the immunization,significant differences were observed in the serum levels of PIB-specific (F =285.72,564.83,respectively,both P ＜ 0.01) and NspA-specific (F =521.57,542.61,respectively,both P ＜ 0.01)antibodies.Also,the proliferation index was statistically different among these groups in spleen lymphocytes stimulated with PIB and NspA (F =171.61,233.96,respectively,both P ＜ 0.01).The vaccination efficiency was positively correlated with the inoculation dose of recombinant pAdEasy-1-PN,and 108 TCID50 per dose proved to be the optimal dose for immunization.The sera from mice immunized with pAdEasy-1-PN could agglutinate N.gonorrhoeae and kill it in the presence of complement.Conclusions The recombinant adenovirus pAdEasy-1-PN containing PIB and NspA genes could induce specific humoral and cellular immune response in mice,and may be a potential vaccine against N.gonorrhoeae.
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<p><b>OBJECTIVES</b>To investigate the DNA polymorphism of Sporothrix schenckii (S. schenckii) and to find the relationship between DNA patterns and geographic areas and clinical manifestations.</p><p><b>METHOD</b>The total DNA was extracted with hexadecyltrimethyl-ammonium bromide. Random Amplification of Polymorphic DNA (RAPD) assay was used to study DNA typing of 24 strains of S. schenckii collected from different areas and isolated from different clinical types.</p><p><b>RESULTS</b>Of seven random primers used, three primers (OPAA11, OPD18 and OPB07) gave good reactions, the sequences of which were 5'-ACCCGACCTG-3', 5'-GAGAGCCAAC-3', 5'-GGTGAC~GCAG-3' respectively. The RAPD patterns of the 24 isolates were not completely identical, showing certain degrees of hereditary variability. Different isolates showed a common conserved DNA band with the same primer. Different clinical types showed different genotypes.</p><p><b>CONCLUSION</b>RAPD analysis is useful in DNA typing of S. schenckii, the DNA band type of which is related to geographic origin and Clinical manifestation.</p>
Subject(s)
Humans , DNA, Fungal , Random Amplified Polymorphic DNA Technique , Sporothrix , GeneticsABSTRACT
Objective To investigate the differential expression of secretory aspartyl proteinase in patients with vaginal C.albicans infection and in asymptomatic candidal carriers.Methods Secretory aspartyl proteinase expression was determined with reverse transcription-PCR in vaginal specimens taken from 10 asymptomatic candidal carriers,14 patients with vulvovaginal candidiasis (VVC) and 10 patients with recurrent VVC (RVVC).Results SAP2 and SAP4 to SAP6 subfamily were detected in both candidal carriers and patients with vaginal candidiasis.SAP1 and SAP3 transcripts were not observed in 10 asymptomatic candidal carriers.All 9 SAP genes were differently expressed in VVC and RVVC patients.SAP1 and SAP3 transcripts were frequently amplified in some patients.Conclusion It is shown that C.albicans infection is associated with differential expression of individual SAP genes,which may be involved in the pathogenesis of vaginal candidiasis.
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Objective To investigate the clinical and laboratory profiles of a case of infantile chronic mucocutaneous candidiasis due to malnutrition.Methods Skin specimens were collected and examined by KOH preparation,fungus culture,biochemical tests and molecular biological study.The pathogenic strain and antimicrobial susceptibility were defined.Results A 5 month-old-girl was presented with big head,and erythema,erosion and crust on her head,auricles,nostrils,oral cavity,neck and buttocks,and itching,for 1 month.Dystrophic hair and nails were observed.Candida albicans was isolated from specimens of the buttocks and Candida glabrata from the scalp.The abnormalities,CD16+ (56 6.5%),serum IgG (6.74 g/L),IgA (0.321 g/L),and albumin (18 g/L) were found with flow cytometry and immunoassays.Conclusion The patient is diagnosed as infantile chronic mucocutaneous candidiasis,who is immunocompromised due to protein malnutrition.
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Objective To investigate the kinetic expression level of chemokines (MCP-1 and MIP-2) in vagina of a murine model of vulvovaginal candidiasis (VVC).Methods The estrogen-treated murine model of VVC was set up.Vaginal specimens were obtained in different duration after inoculation of C.albicans intravaginally.Semi-quantitative RT-PCR was applied to determine MCP-1 and MIP-2 mRNA levels in these tissues.Results Compared with the control mice treated with olive oil,persistent growth of C.albicans was found from the 2nd day to 21st day after inoculation in estrogen-treated mice.Significantly higher levels of MCP-1 mRNA were observed in vaginal tissues in infected estrogen-treated mice than those in other 2 groups,infected but non estrogen-treated mice and estrogen-treated but uninfected mice.The high level of MCP-1 mRNA maintained from the 4th day to 21st day in infected estrogen-treated mice.It was also found that levels of MIP-2 mRNA were significantly higher in the vagina in the 2nd day in 3 groups of mice than those in naive mice,however,no significant difference was shown among 3 groups throughout the study period.Conclusion High level of MCP-1,rather than MIP-2,may be associated with susceptibility to VVC.
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Objectives To analyse the antifungal susceptibility of Candida spp. isolated from the patients with recurrent vulvovaginal candidiasis (RVVC), vulvovaginal candidiasis (VVC) and asymptomatic carriers and to study the correlation between different Candida strains and antifungal susceptibility. Method According to the NCCLS-M27-A scheme, the antifungal susceptibility of Candida spp. isolated from the above different groups was measured. Results Almost all the MICs of C. glabrata and C. krusei to 8 antifungal agents were higher than those of C. albicans. The average MIC of C. albicans isolated from RVVC patients was higher than that from asymptomatic carriers. The resistant strains were mainly isolated from the RVVC group. No resistant strains against itraconazole, fluconazole, ketoconazole, econazole and nystatin was found in asymptomatic carriers. Conclusions These results indicate that more attention has to be paid to the low susceptibility of non-Candida albicans in the treatment of vulvovaginal candidiasis, and the resistant strains may result from long-term or irregular antifungal treatment.
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Objective To investigate the species and DNA polymorphism of Candida strains from different individuals and to find the relationship between the species or DNA patterns of strains and different patient groups. Methods The present study chose the isolates from 3 different sources groups(from the patients with vulvovaginal candidiasis, recurrent vulvovaginal candidiasis or asymptomatic carriers respectively). Germ tube test, chlamydospore test, CHROMagar Candida and API20 kit system were applied to separate non-Candida albicans strains from Candida albicans. Random amplification of polymorphic DNA (RAPD) analysis was used to study DNA typing of 97 strains of Candida collected from different patients and to determine whether isolates from different patients were genetically similar or dissimilar. The data were analyzed by SPSS10.0. Results The ninety-seven isolates consisted of 83 strains of Candida albicans and 14 of non-Candida albicans. Of 9 random primers used, two primers (primer 4 and primer 7) gave good reactions, the sequences of which were 5′-ACCCGACCTG-3′, 5′-GGTGACGCAG-3′ respectively. The 99 isolates could be classified into 19 and 21 genotypes by the two primers respectively. Different genotype was not shown in most isolates from different groups. A particular genotype associated with different conditions was seen in only a few isolates. Conclusion Candida albicans is the main pathogenic yeasts and most strains of non-Candida albicans are C.glabrata in the vulvovaginal candidiasis. Genotyping of most isolates didn′t show obvious correlation with different patient groups.