CNTF Combined With Small Molecule Compounds(Fsk-IBMX)induced MDSCs To Differentiate Into SCs Phenotype Through CAMP Signaling Pathway

ObjectiveThrough the adoption of ciliary neurotrophic factor(CNTF)combined with small molecular compounds(forskolin and 3-isobutyl-1-methylxanthine,Fsk-IBMX)to induce muscle-derived stem cells(MDSCs)step by step in vitro,the changes in cell phenotypes at different stages of differentiation were detected,and to explore the role of c AMP signaling pathways in the differentiation process and significance,in order to supplement the theory and research basis for the differentiation of MDSCs into Schwann cells(SCs)phenotype,and to improve the mechanism of action.MethodsMDSCs were extracted from hind limbs skeletal muscle of one-week-old SD rats with mixed digestive enzymes,and purified by differential adherent method.The MDSCs were identified by cellular immunofluorescence,and the changes in proliferative activity of MDSCs of each generation were detected by CCK-8 method at different culture time points.The MDSCs were induced in vitro by CNTF combined with FSK-IBMX step by step.The experimental grouping and treatment methods of the dedifferentiation process are the control group(dedifferentiation medium treatment for 72h),the dedifferentiation group(dedifferentiation medium + CNTF treatment for 72h),the inhibition group(dedifferentiation medium + CNTF + SQ22536 treatment for 72h),the medium should be replaced every 24 h.The experimental grouping and treatment methods of the SCs-like differentiation process are the control group(nerve growth medium treatment for 120h),the induction group(nerve growth medium+ Fsk + IBMX treatment for 120h),the inhibition group(nerve growth medium +Fsk + IBMX + SQ22536 treatment for 120h),the medium should be replaced every 60 h.The dedifferentiated cells and SCS-like cells were identified and analyzed by cell morphology,CCK-8,Western Blot,RT-q PCR and other techniques,and the expression of c AMP signaling pathway-related proteins in the process of dedifferentiation and SCS-like differentiation were detected.ResultsCell immunofluorescence staining showed that primary cells taken from the hindlimb skeletal muscle of one-week-old SD rats could be labeled with Desmin,one of the specific markers of MDSCs.CCK-8 showed that the proliferation activity of P1～P3 MDSCs gradually increased at the same culture time point(P<0.0 001),and the proliferation activity of P3～P8 MDSCs tended to stabilize.Western Blot showed that the expression of myogenic differentiation-related proteins p21 and Myo G decreased after CNTF induction(P<0.05;P<0.01),and the expression of c AMP-related protein p-CREB increased(P<0.001).After Fsk-IBMX induction,the expressions of SCs specific markers S100β,GFAP,p75 and p-CREB increased(P<0.05;P<0.05;P<0.05;P<0.01),while the c AMP inhibitor SQ22536 can inhibit the effects of CNTF and Fsk-IBMX,and the expression level of Desmin was significantly decreased after induction(P<0.01).The results of RT-q PCR results are consistent with the Western Blot results,that is,the expression levels of S100β,GFAP and p75 m RNA increase after Fsk-IBMX induction(P<0.001;P<0.001;P<0.0 001),and SQ22536 can inhibit this effect(P<0.01;P<0.001;P<0.0 001).The results of CCK-8 showed that the cell activity was reduced after Fsk-IBMX induction(P<0.0 001).ConclusionsCNTF combined with Fsk-IBMX can induce MDSCs to differentiate into SCs phenotype,and both the CNTF-induced dedifferentiation process and the Fsk-IBMX-induced SCs-like differentiation process activate the c AMP signaling pathway.