| Objective:To isolate and obtain human adipose derived mesenchymal stem cells(hADSCs)from human fat suction suspension by collagenase digestion,analyze the expression of CD29,CD31,CD34 and CD45 on the cell surface,induce osteogenic and adipogenic differentiation,and test whether the obtained cells meet the immunophenotype of mesenchymal stem cells,Whether it has the ability of multi-directional differentiation;Different concentrations(0,0.01,0.1,1,10,50,100μg/mL)of lipopolysaccharide(LPS)were used to pretreat hADSCs,to explore the effects of different concentrations of LPS on the proliferation and apoptosis of hADSCs,and to screen the optimal pretreatment concentration;After pretreatment of hADSCs with the optimal concentration of LPS,the cell culture supernatant,conditioned medium(CM),was collected and compared with 3×103 CFU Escherichia coli/Staphylococcus aureus coculture,observe the bacteriostatic effect,and explore its potential mechanism,provide a theoretical basis for inducing or enhancing the antibacterial activity of hADSCs,and provide a new way of thinking for the treatment of infectious wounds and infectious diseases.Methods:(1)hADSCs in adipose tissue were isolated by collagenase digestion.The morphological characteristics of different generations of cells were observed by inverted microscope,photographed and archived.The cells were passaged and frozen by conventional methods.(2)The expression of CD29,CD31,CD34 and CD45 on the cell surface was identified by flow cytometry,and osteogenic and adipogenic differentiation was induced.The cell proliferation curve was drawn by CCK-8 colorimetry.(3)Adopt different concentrations of LPS(0,0.01,0.1,1.0,10.0,50.0,100 μg/mL)pretreatment of hADSCs for 24 hours.CCK-8 colorimetry was used to detect the cell proliferation activity after pretreatment with different concentrations of LPS;AO/EB double fluorescence staining and flow cytometry(FCM)were used to detect apoptosis,and the optimal pretreatment concentration of LPS with the most obvious proliferation activity and no effect on apoptosis was selected;(4)Method for obtaining cell culture supernatant:add the cells to the medium containing 10%fetal bovine serum for culture for a period of time.When the cells are in good growth condition,replace them with serum-free basic medium.After continuous culture for a period of time,the cell culture supernatant can be collected;(4)The bacteriostatic effect was determined by plate colony counting method:3×103 CFU Escherichia coli/Staphylococcus aureus isolated from chronic refractory wounds were mixed with the cell culture supernatant of untreated hADSCs,pretreatment LPS hADSCs and control normal human fibroblast(NHF)respectively,and incubated in 37℃incubator for 2h.Then,the co culture solution of each group was diluted in multiple ratio,and the diluent was coated on agar plate,After overnight culture at 37℃,the plate colony count was carried out to compare the antibacterial effect;(6)Antimicrobial peptides LL-37 andβ-defensin-2concentrations in the cell culture supernatants of each group were determined using the Elisa method.(7)RT-PCR was used to detect the expression of CAMP gene(LL-37)and HBD2(β-defensin-2)genes in each group.result:(1)The cells isolated from human fat suction suspension by collagenase digestion showed positive expression of surface marker CD29 and negative expression of CD31,CD34 and CD45,and could successfully induce osteogenic and adipogenic differentiation.Combined with the cell proliferation curve,it was suggested that the obtained cells were in line with the biological characteristics of mesenchymal stem cells.(2)CCK-8 colorimetric results showed that LPS at low concentration levels(0.01-10μg/mL)promoted the proliferation of hADSCs compared with the control group(0 μg/mL LPS),of which,at 1.0 μg/mL LPS level treatment,the proliferation of hADSCs cells peaked,and the difference was statistically significant(P<0.05);as the LPS concentration continued to increase(50.0-100 μg/mL),the proliferative activity of hADSCs cells showed an arrest or even a decreasing trend.(3)AO-EB fluorescence double staining revealed that the cells in the 0,0.01,0.1,1.0,and 10.0μg/mL LPS treatment groups were mainly stained with AO,showing green fluorescence,normal cell morphology and structure,and no obvious apoptotic cells were observed;while with the increase of LPS concentration,the cells were mainly stained with EB,showing orange fluorescence,apoptotic cells or dead cells,and gradually increased;(4)Flow cytometry showed that:The apoptosis rates of hADSCs treated with 0,1.0,10,50 and 100.0 μg/mL LPS for 24 hours were(4.15±0.14)%,(5.10±0.39)%,(6.31±0.2)%,(11.12±1.75)%,(14.29±0.27)%.respectively,Compared with the control group(0μg/mL LPS),There was no significant difference in the apoptosis rate of 1.0 μg/mL and 10 μg/mL LPS intervention group(P>0.05).But as the concentration continues to rise,The apoptosis rate of50 and 100 μg/mL LPS intervention group was significantly higher than that of the control group(P<0.05);(5)In the bacteriostatic test of Staphylococcus aureus,the bacteriostatic rates of hADSCs-CM in untreated group and LPS-hADSCs-CM in pretreatment group were(8.48±1.90)and(61.64±0.77),respectively.Compared with NHF-CM in control group(7.75±0.42),the difference was statistically significant(P<0.05);In the bacteriostatic test of Escherichia coli,the bacteriostatic rates of untreated group and pretreatment group were(23.23±3.9)and(55.12±1.75),respectively.Compared with the control group(9.22±1.65),the difference was statistically significant(P<0.05);(6)Elisa was used to detect the concentrations of antimicrobial peptides LL-37 and β-defensin-2 in the cell culture supernatants of each group,and the results suggested that the expressions of the two antimicrobial peptides in hADSCs-CM were higher than those in the control group(NHF-CM),and the differences were statistically significant(P<0.01);moreover,the expressions of the two antimicrobial peptides in LPS-hADSCs-CM pretreated with 1μg/mL LPS were further enhanced compared with hADSCs-CM,and the differences were statistically significant(P<0.01),and the pairwise comparisons of each group were also statistically significant(P<0.01);(7)RT-PCR results showed thatThe CAMP gene and HBD2 gene expression of hADSCs were higher than those of the control group(human skin fibroblasts),and the difference was statistically significant(P<0.05);and,compared with hADSCs without LPS pretreatment,the CAMP gene and HBD2 gene expression of LPS-hADSCs pretreated with LPS was further enhanced,and the difference was statistically significant(P<0.05).Conclusion:(1)The collagenase digestion method used in this experiment can successfully separate and obtain hADSCs from human fat suction suspension;(2)low concentrations of LPS can promote hADSCs proliferation and help to improve cell viability;high concentrations of LPS can inhibit hADSCs proliferation and negatively affect cell viability.Treatment with low concentrations of LPS had no significant effect on apoptosis of hADSCs,and high concentrations of LPS could cause apoptosis of hADSCs,which may be one of the factors affecting cell survival.(3)hADSCs can release antimicrobial peptides LL-37 and β-defensin-2 into their culture supernatant in vitro and inhibit the growth of Staphylococcus aureus and Escherichia coli;(4)1μg/mL LPS pretreatment can effectively enhance the expression levels of CAMP and HBD2 genes in hADSCs and improve the expression of LL-37 andβ-defensin-2 in cell culturesupernatant,further strengthened the antibacterial ability of cells in vitro. |
