| OBJECTIVE: To observe the homing and migration of bone marrow derivedstem cell to Wallerian degenerated peripheral nerve；To investigate the characteristicsand mechanism of the phenomenon，especially the fate of migrated stem cell. Todetect differential protein expression in rat between saphenous nerve and motorbranch of femoral nerve, as dorsal root and ventral root, using two-dimensional gelelectrophoresis and mass spectrum and to determine specific biological markers ofsensory and motor nerve.METHODS:1. We measured the expression of CXCR4in BMSCs and theexpression of SDF-1in degenerated rat sciatic nerve with RT-PCR, Western blot,immunofluorescence methods.2. By using Transwell in vitro migration assay system, the effects of SDF-1andtissular extract from normal and degenerated rat sciatic nerve on BMSCs’ migrationwere observed, the influence of AMD3100treatment on BMSCs’ migration inducedby nerve tissular extract were also evaluated.3. The RFP transfected BMSCs were injected through tail vein into the SD ratswith sciatic nerve cut and anastomosised, and the migration of BMSCs to the injurednerve3days later. The influence of AMD3100treatment on BMSCs’ migrationinduced by degenerated nerve, were also evaluated.4. The RFP transfected BMSCs were injected in or applied around the segmentalfreeze-treated nerve or CEANA bridged to the segmental defect of rat sciatic nerve.12weeks later, the fate of transplanted RFP-BMSCs was observed and its effect ofpromoting nerve regeneration and repair was evaluated through functional assessmentand histological observation.5. A total of9Wistar rat were sacrificed. The segments of bilateral motorbranches to the quadriceps muscles and the saphenous nerves, ventral roots and dorsal roots of spinal at the level of L1~5were collected, separately. Total protein wasextracted from four kinds of nerve tissue, followed by two-dimensional gelelectrophoresis and silver staining, and the differential protein expression wasanalyzed using imagemaster2D platinum software. Protein peptide massspectrometry data of differential protein spots was obtained by nano ultra highperformance liquid chromatography electrospray ionization mass spectrometrytandem mass spectrometry. The National Center for Biotechnology Information(NCBI) protein database was retrieved by Mascot to identify protein type.RESULT:1. Western blot and Immunofluorescence analysis revealed thatBMSCs were CXCR4positive. The mRNA of CXCR4in BMSCs and SDF-1inWallerian degenerated nerve were successfully reversely transcribed by RT-PCR.2. Wallerian degenerated nerve tissular extract can induce the migration ofBMSCs, and this effect can be suppressed by AMD3100which is a blocker ofCXCR4.3. Injured and Wallerian degenerated nerve can induce the migration oftransplanted BMSCs, and this effect can be suppressed by AMD3100which is ablocker of CXCR4.4. The RFP-BMSCs transplanted around the freezed nerve or CEANA hadmigrated into regenerated nerve, with Nestin, S100, P0positive, as the cells directlyinjected into the freezed nerve or CEANA. Transplanting BMSCs in or around thefreezed nerve or CEANA, had equally increased contraction of triceps muscle,recovery rate of muscle, the number of sciatic nerve myelinated fibers and myelinthickness.5. Compared with the motor branches of femoral nerve,13protein spotsexhibited obviously differential expression in saphenous nerves (P<0.05). Comparedwith in ventral roots,16protein spots exhibited obviously differential expression indorsal roots (P<0.05). A total of26out of29protein spots with obvious differentialexpression were identified successfully, with peptide of matching score>34(P <0.05). Protein types were identified and included transgelin, Ig kappa chain precursor,plasma glutathione peroxidase precursor, aldose reductase, a glyceraldehyde- 3-phosphate dehydrogenase-like protein, lactoylglutathione lyase, adenylate kinaseisozyme1, Pol(yrC)-binding protein1, rCG31027and two unnamed protein product(gi|55628and gi|1334163).CONCLUSION: Wallerian degenerated nerve can induce the migration ofBMSCs, SDF-1/CXCR4axis play an important role in this process.Compared to directly injected BMSCs into acellular nerve, BMSCs suppliedaround injured nerve have the similar effect, by differentiated into Schwann cell andpromoting myelin formation.Through the proteomics comparison with2DGE andnanoUPLC-nano-ESI-MS/MS analysis between motor branch of femoral nerve andsaphenous nerve, as ventral roots and dorsal roots, a few proteins were found for thefirst time to be differentially expressed in sensory and motor nerve. These proteinsmay play roles in specific nerve regeneration. |
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