| [Background] Muscle-derived stem cells (MDSCs) are a class of newly discovered adultstem cells with self-renewal and differentiation potential, which can be isolated from theskeletal muscle of animals. MDSCs can differentiate into muscle cells in vivo/vitro withoutspecific stimulation, but may require specific growth factors or suitable environment todifferentiate into other lineage cells. A growing number of studies have shown the greatpotential of MDSCs in tissue development, regeneration and repair, and gene therapy.Evidence indicate that MDSCs can promote the regeneration of muscle fibers, enhance thefunction of regenerative muscle tissue, and have the potential to differentiate intoepineurium tissue cells and Schwann cells (SCs) like cells, which would bring hope toserve as an alternative candidate for commonly studied Schwann cells in tissue engineeringnerve graft. Neuregulin-1(NRG-1) is a peptide carried or secreted by membrane, whichbelongs to the EGF family. NRG-1is mainly produced by neurons, and can combine withErbB receptor to cause a wide range of biological effects. According to the differentN-terminal domain, NRG-1can be divided intoⅠ, II, and III subtypes. Recent studiesindicate that NRG1type III (sensory and motor neuron-derived factor, SMDF) presentedon the axonal surface not only promotes the proliferation and differentiation of Schwanncells, but also determines the myelination fate of axions and controls the thickness of themyelin sheath. The secretion of SMDF peaks during the embryonic and neonatal period,but significantly decreased in adulthood. After peripheral nerve injury, SMDF can be foundin Schwann cells and innervated organ–muscle tissues. However the expression of SMDFis not sufficient to complete the remyelination of adult axons, resulting in the formation ofthinner myelin sheathes and shorter internodes compared with normal nerves.Studies havealso shown that Schwann cells themselves can produce NRG1(III α2homolog) in the formof autocrine or paracrine to promote Schwann cells proliferation, and participate in theremoval of damaged axons and myelin. Differentiation and proliferation of Schwann cellsis a prerequisite for axonal regeneration after peripheral nerve injury, and SMDF is a keyregulator of the myelin sheath thickness.Therefore, to improve the expression of SMDF atthe site of peripheral nerve injury provides a potential therapeutic strategy for improvingmyelination by Schwann cells.[Objective](1) Collect the conditioned of mature Schwann cells to induce MDSCsdifferentiate into Schwann cells, to bring hope to find new substitutes for commonlystudied Schwann cells in tissue engineering nerve graft.(2) Clone SMDF gene and construct of eukaryotic expression vector of pEGFP-N2-SMDF to provide a fundamentaltool to further study.(3) The pEGFP-N2-SMDF was transfected into MDSCs, to observeand study the SMDF expression at the mRNA and protein levels in order to confirme thatthe recombinant plasmid vector can effectively transduction of target gene.(4) Confirmedthe SMDF gene can promote Schwann cells-like cells proliferation、differentiation, andfurther verify the function of SMDF protein, in order to provide a new basis for furtherstudy of Schwann cells remylination after peripheral nerve injury.[Methods](1) To isolate MDSCs from newborn mice skeletal muscle by a modifiedpreplate technique, and identify these cells by immunocytochemistry with antibodiesDesmin and Sca-1.(2) Separate and purify Schwann cells from newborn mice sciatic nerve,identify these cells by immunocytochemistry with antibodies P75NTR、S-100βand collectthe conditioned medium(CM) of mature Schwann cells to induce MDSCs differentiate intoSchwann cells.(3) Western blot analysis of SMDF protein expression in different tissuesand immunohistochemistry detection of SMDF expression in DRG.(4) Fresh newbornmouse DRG total RNA were extracted with Trizol and the SMDF gene was amplied byRT-PCR and construct the eukaryotic expression vector of pEGFP-N2-SMDF, identified ofthe recombinant plasmid pEGFP-N2-SMDF by PCR, restriction enzyme analysis andsequencing;(5) Two different plasmids including a recombination pEGFP-N2-SMDF andan empty pEGFP-N2were respectively transferred into MDSCs cultured in vitro byliposome-mediated gene transferred method. Real time PCR and Western blot methodswere used to analyze the expression of SMDF and protein in MDSCs.(6) Induced thetransfected MDSCs differentiate into Schwann cells with CM, and identify these cells byimmunocytochemistry.[Results](1) MDSCs were obtained from mouse skeletal muscle by using a modifiedpreplate technique and was verified by the positive results of Desmin and Sca-1.(2)Schwann cells were purified from newborn mouse sciatic nerve and MDSCs weresuccessfully induced to express the Schwann cell-specific marker P75NTR, S-100β in theCM.(3) Restriction enzyme analysis and sequencing result showed that the sequence ofthis gene fragment from the mouse DRG was identical with SMDF gene sequence reportedin GenBank.(4) The pEGFP-N2-SMDF eukaryotic expression vector was transfected intoMDSCs, and green fluorescent protein was seen under fluorescence microscopy. Theresults of PCR and Western blot showed that the level of SMDF both on mRNA andprotein were higher in cells transfected with pEGFP-N2-SMDF than in those un-transfected or empty vector-transfected cells and there in no significant differencebetween the latter two groups. The transfected MDSCs induce by CM can be divided intoSchwann cells-like cells, and the positive rate of S-100β was significantly higher than thedirectly induced.[Conclusions](1) MDSCs were able differentiate into Schwann cells-like cells under theinduction of CM, which can be served as optimal autogenous cell source for nerve tissueengineering.(2) The recombinant eukaryotic vector was transfected successfully intoMDSCs with liposome, the expression of SMDF mRNA and protein are significantlyincreased in the transfected cells. And also confirmed the SMDF gene can promoteSchwann cells-like cells proliferation、differentiation. All of these results provided a newbasis for further study of Schwann cells remyelination after peripheral nerve injury. |
PDF Full Text Request