Inducing Condition And Mechanism Of Differentiation From Mouse Mdscs To Schwann Cells-like In Vitro

【Background】peripheral nerve injury is a very frequent diseases in clinicalsurgery. Clinicians and scientist devoted to improve their surgical technique and method torepair injured peripheral nerves. But the result is still disaffect with regard to those severenerve stem injury and the functional rehabilitation after treatment; autoallergic nervetransplantation usually caused sensory disorders and motor dysfunction; And it is very toobtain enough donor tissue and allotransplantation faced with immunologic rejection.Therefore, Tissue Engineering become to be the hotspot. Schwann cell is a kind of TissueEngineering cell which is effective and well studied. Schwann cells can survive,proliferate, differentiate and secrete kinds of neurotrophic factors,cellular adhesionmolecules and extracellular matrix to promote the regeneration of axon and themedullation after transportation, thereby promote the restoration of peripheral nerves.However as seed cells, Schwann cells have a very limited differentiation potency,theyare terminal-stage cells and hard to obtain. Therefore we need another kind of cell which isyounger and could differentiate to schwann cells. According to the papers, many kinds ofstem cells could differentiate to schwann cells include bone marrow mesenchyma stemcells(BMSCs),bone marrow stroma stem cells(MSCs),adipose-derived stem cells(ADSCs), nerve stem cells(NSCs)and etc.Muscle drived stem cells(MDSCs)reside in skeletal muscles,and could differentiateto muscle cells, hematopoietic cells, osteoblasts, chondrocytes,endotheliocytes,endotheliocytes, hepatocytes; nerve cells and ect. But we had hardly any heard about thenews that MDSCs induced to schwann cells. If we could successfully induce MDSCs toschwann cells-like, it will be a new thread and provide a new choice for peripheral nerveinjury treatment of peripheral nerve injury.【Objective】(1)To master the method and technique of digestion, isolation,cultivation and identification of mouse muscle drived stem cells;(2)prepare Schwann cell conditioned medium, make sure that it could induce mouse MDSCs to schwann cells-like;(3) to detect the expression of several kinds of NTFs in Schwann cell conditioned mediumaccording to our available condition and relevant papers；choose the higher-express onesto induce MDSCs;(4)mouse MDSCs were induced by NTFs,then morphocytology,immunohistochemistry and flow cytometry was detected to make sure that the cells afterinduction are schwann cells-like; to provide a new alternative choise for laboratory studiedand clinical treatment.【Methods】(1) obtain the pure mouse skeletal muscle tissue under microscope,usethe enzyme digestion and improved pre-plating technique to digest, isolate and purify thetissue,when we got the cells(tSCs), use trypan blue staining to evaluate the activity ofthem;observe the appearance and growth of tSCs；then immunohistochemistry and flowcytometry was detected to make sure that the cells after induction are MDSCs use theMarker(sSca-1,Desmin)of mouse MDSCs;(2)Schwann cells were isolated from mousesciatic nerve and Dorsal Root Ganglia(DRG),and were identified byimmunohistochemistry and flow cytometry method use the commonly used makers(S100,GFAP,p75) of Schwann cell. Schwann cell conditioned medium was obtainedby half-harvest method;(3) Detect the expression of NTFs in Schwann cell conditionedmedium culture with ELISA method and choose the higher express one induce mouseMDSCs;(4) Induce mouse MDSCs with NTFs by add it in stem cell growth medium; thenobserve the appearance and growth of tSCs； immunohistochemistry and flow cytometrywas detected to evaluate the cells after induction use the commonly used makers(S100,GFAP,p75) of Schwann cell.【Results】(1) trypan blue staining showed that the activity of MDSCs was95%；mouse MDSCs under inverted microscope were little round-shaped or shotspindle-shaped; differentiation didn't happen and differentiation is fast; thenimmunohistochemistry showed that Sca-1and Desmin were both positive expressed；theflow cytometry showed that the positive rate of Sca-1and Desmin expression was93.23±0.93%and94.18±0.38%,Double positive rate was90.1±1.28%；(2)mouseMDSCs was successfully induced to Schwann cells-like by Schwann cell conditionedmedium,after72h induction,the cells presented spindle or trabs shape and appeared synapses in both side；immunohistochemistry showed that S100was positive expressed；the flow cytometry showed that S100,GFAP and P75was positive expressed,thepositive rate was65.48±6.20%,39.84±1.66%and41.08±0.78%respectively,the triplepositive rate was about25.86±5.37%；(3) the ELISA detection showed that among theNTFs,besides FGF and GDNF were undetected, NGF, BDNF, NT-3,PDGF and IGF-2were expressed in different level,so we decided to induce MDSCs with these five NTFs;(4)After72h induction by NT-3(500pg/ml), PDGF(1000pg/ml) and IGF-2(200pg/ml), thecells presented spindle or trabs shape and appeared synapses in both side；immunohistochemistry showed that S100was positive expressed；the flow cytometryshowed that S100,GFAP and P75was positive expressed,the positive rate was58.64±4.38%,47.38±0.84%and44.33±2.39%respectively,the triple positive rate wasabout27.89±5.98%.【Conclusions】(1)we obtained high-activity and high-purity MDSCs from mouseskeletal muscle tissue through improved pre-plating technique and the use of microsurgicaltechnique；successfully induced mouse MDSCs to Schwann cells-like both with Schwanncell conditioned medium and NTFs(NT-3,PDGF and IGF-2),therefore provide a newalternative choise and for laboratory studied and clinical treatment of peripheral nerveinjury.