| Objective:Mouse bone marrow mesenchymal stem cells(BMSCs)are non-hematopoietic tissue mesenchymal stem cells obtained from the isolation of mouse bone marrow stroma.These cells have the ability to secrete multiple cytokines.As a cell model with great potential,it is widely used to study and identify in vitro immune responses.Lipopolysaccharide(LPS),as a component of the cell wall of Gram-negative bacteria,is widely used in the induction of inflammation models.The purpose of this experiment was to assess the secretion state of inflammatory cytokines stimulated by LPS in vitro of mouse BMSCs and to discuss the possible element of affecting the secretion of inflammatory cytokines.Methods:BMSCs were isolated and cultured by whole bone marrow adherent method.The expression of CD34,CD29 and CD44 on the cell surface was identified by flow cytometry.The growth curve was measured by CCK-8 method and the proliferation-toxicity of BMSCs with different concentrations was observed.Using RT-q PCR method to analyze the expression of TNF-?,IL-1?,IL-6,IL-10 and ARG-1m RNA in BMSCs at different time points(1h,4h,8h,24 h,3d,7d)under the inflammatory environment induced by LPS.The Western-Blot assay was used to assess the expression of TNF-?,IL-1? and IL-10 protein in cells at different time points(1h,8h,24h)and the ELISA method was uesd to detect the secretion of TNF-?,IL-1? and IL-10 protein in the culture supernatant of BMSCs at different time points(1h,4h,8h,24 h,3d,7d)in the inflammatory environment induced by LPS,so as to explore the effect of LPS-induced in vitro inflammatory environment on the immune status of BMSCs and its possible reasons.Results:The primary cultured BMSCs were fusiform and polygonal,and their growth curves were obviously "S" shaped.The results of flow cytometry showed as follow: CD34(-),CD29(+),CD44(+).CCK-8 results showed that chemical stimulation with different concentrations of LPS has no obvious toxic effect on BMSCs,and there may be a dose-dependent relationship between the concentration of LPS and the proliferation of BMSCs.LPS of 0.1,1,10ug/m L concentration can promote the proliferation of BMSCs.Among them,1ug/m L LPS stimulation has the most obvious effect on promoting the proliferation of BMSCs.As the concentration gradually increased,the proliferation activity of BMSCs showed a downward trend.RT-q PCR results showed that the expression of TNF-?,IL-1?,IL-6,IL-10,ARG-1 m RNA at different time points all changed significantly(P<0.05).As time went by,the trend of up-regulation of gene expression gradually declined.The expression of IL-10 and IL-1? reached the maximum at 8h.TNF-?,IL-6,and ARG-1 reached the maximum at1,4,and 24 h,respectively.Among them,the expression of ARG-1 was down-regulated at 4h.The results of ELISA showed that the expression of TNF-?,IL-1 ? and IL-10 showed a tendency to rise first and then decreased over time(P<0.05).Among them,the expression of TNF-? protein was in the peak reached at8 h,and the expression of IL-1? and IL-10 peaked at 3d.Western-Blot results showed that within 1-24 hours,the expression of IL-1 ? and TNF-? protein in the cells increased first and then decreased,and the expression of IL-10 protein continued to increase(P<0.05).Among them,the expression of IL-1? and TNF-? protein reached the maximum at 8h,and the expression of IL-10 protein reached the maximum at 24 h.Conclusions:Mouse BMSCs cultured by whole bone marrow adherence method have good proliferation ability and high expression of bone marrow stem cell surface markers.Proper concentration of LPS stimulation has no toxic effect on BMSCs,and can appropriately promote the proliferation of mouse BMSCs.LPS stimulation at a concentration of 1ug/m L not only enhances the expression of BMSCs' pro-inflammatory factors IL-1 ?,TNF-?,and IL-6,but also the expression of anti-inflammatory factor IL-10.The expression of different factors has a sequential order in time,which may be related to the regulation of inflammation by BMSCs secreting cytokines through autoimmune regulation. |
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