Research Of H₂S Synthetase And H₂S Content Changes In The Process Of ICS? Induction HAMSCs Differentiation

Objective: Explore epimedium glucoside ?(Icariside ?,ICS ?)ectomesenchymal Stem Cells in the induction of Human amniotic membrane(Human Amnion Mesenchymal Stem Cells,h AMSCs)into neural differentiation in the process of hydrogen sulfide(hydrogen sulfide,H2S)synthase and the influence of H2 S content,explore the ICS ?h AMSCs induction differentiation survive for a long time after the possible mechanism.Methods:(1)The P3 h AMSCs provided by the Key Laboratory of Cell Engineering in Guizhou Province,our hospital were selected to meet the research requirements,and the phenotypes were identified by flow cytometry and seeded into the culture plate as the subject cells.(2)according to the preliminary experimental results of the same team,a cells were divided into:(1)ICS ? 0 group,the ICS ? 3 groups and ICS ? 0 solvent control group,respectively,to contain the ICS ? concentration of 0 mu mol/L,3 mu mol/L and DMSO volume fraction of 3 ‰ and DMEM medium for culture.(2)all trans retinoic acid(all-trans retinoic acid,ATRA)groups: replace ICS with ATRA ? 3 mu mol/L as positive control.(3)H2S synthetase inhibitors groups: ICS ? 3 + AOOA(H2S synthetase inhibitors-Carboxymethyl hydroxylamine half hydrochloric acid(O-(Carboxymethyl)hydroxylamine hemihydrochloride,AOOA)group and ATRA + AOOA group,in ICS ? 3group and to join AOOAATRA group of medium.Change liquid at 48 h interval.(3)H2S content in the superfluid of the medium was induced by methylene blue at 6h,12 h,24h,36 h and 48 h,and morphological changes of each group were observed by inverted phase contrast microscope.Induced 72 h(4)when using Western Blot method to detect each group of hydrogen sulphide synthetase(thioether-beta synthase(cystathionine-beta-synthase,CBS)and 3-mercapto pyruvate sulfonium transferase(3-Mercaptopyruvatesulfurtransferase 3 MST)expression.(5)Immunofluorescence assay was used to detect map-2 expression in each group at 12 days of induction.(6)using molecular docking virtual,observe the ICS ? and positive drug ATRA and 3 MST and the affinity of CBS.Results:(1)In DMEM/F12 medium containing 10% serum,h AMSCs of P3 generation proliferated rapidly and arranged in nests or whirlpool.Flow cytometry was used to detect high expression of CD90,CD44,CD105 and CD73,but no expression of CD45,CD34,CD19,CD11 b or HLA-DR.(2)In 0 h to 48 h,groups of H2 S concentration increasing over time,ICS ? 3 groups appear obvious peak in 24 h,significantly higher than the rest of the group(P < 0.05),but no statistically significant difference between 48 h group.With ICS ? 0 group and ICS ?0 solvent control group,6 h ICS ? 3 + AOOA and ATRA + AOOA group decrease(P <0.05),12 h ICS ? 3 group and ATRA group increased,24 h when ICS ? 3 groups and ICS ? 3 + AOOA group increased,36 h were increased,no difference between groups of48 h.Compared with ATRA group,ICS ? three groups increased 24 h,36 h is reduced,the rest of the time difference.Compared with ICS ? 3 + AOOA group,ICS ? 3 groups,6 h,12 h,24 h were increased,compared with ATRA + AOOA group,ATRA group increased at6 h and 12 h.(3)Western Blot results showed that with the ICS ? 0,ICS ? 0 solvent control group,in addition to the ICS ? CBS increased expression,group three groups had no difference,With ICS ? 3 + AOOA and ATRA + AOOA group comparison,ICS ? 3groups and ATRA CBS show no statistical significance.With ICS ? 0 group and ICS ? 0solvent control group comparison,ICS ? 3 groups there was no statistically significant difference 3 MST expression,groups increased group with statistical significance(P <0.05),ICS ? 3 + AOOA group is higher than the ICS ? three groups with statistical significance(P < 0.05).(4)The induction training 12 d,were observed under inverted phase contrast microscope showed that ICS ? 0,ICS ? 0 solvent control group cells form a spindle,cell density increases,the rise in the Numbers;At high magnification,no nuclear morphology,no bipolar neuron-like cells.ICS ? 3,ICS ? 3 + AOOA,ATRA,ATRA + AOOA group are bipolar cells called neurons.Immunofluorescence results showed that the ICS ? 3,ICS ?3 + AOOA,ATRA,ATRA+ AOOA group were higher expression with MAP-2,ICS ? 0,ICS ? 0 solvent control group almost no expression.(5)Microscopic observation showed that after induction to 14 days,cells in ATRA and ATRA+AOOA groups showed nucleus migration,cell body enlargement,and interrupted envelop continuity,etc.,and cell morphology could not be maintained at 16 days.The rest of the cells were basically normal in shape.Induced to 25 days,ICS ? 0 group,the ICS ?0 cell cell index decline solvent control group,the cell body width,cell fusion,round or oval nuclei at high magnification is visible,ICS ? 3,ICS ? 3 + AOOA group of cells of refraction sex decline,a bipolar neurons,no cell fusion phenomenon,seen here at high magnification is round or oval nuclei,ATRA,ATRA + AOOA group cell free existence.(6)Molecular docking technology evaluation suggested that ICS II could directly bind3 MST and CBS proteins,and its binding force with 3MST and CBS proteins was stronger than that of ATRA.Conclusion: ICS? can promote the expression of CBS in h AMSCs and increase the secretion of H2 S,and ATRA can promote the expression of 3mst in h AMSCs and the secretion of H2 S,which may be the reason for the difference of h AMSCs and cell survival time after induction.