The Experimental Study Of Governor Vessel Electro-acupuncture Combined With Neurotrophin-3and Retinoic Acid Pre-induced Mscs Transplantation For The Treatment Of Rat Demyelinated Spinal Cord Injury Induced By EB

The principal aim of this research is to explored whether Government vesselelectro-acupuncture(EA) combined with neurotrophin-3(NT-3)+retinoic acid(RA)pre-induced bone marrow mesenchymal stem cells (MSCs) transplantation treatmentcould promote differentiation of MSCs into oligodendrocyte precursor-like cells andoligodendrocyte-like cells in demyelinated spinal cord injury induced by ethidiumbromide(EB) through up-regulating the expression of TrkC in MSCs and increasingthe concentration of NT-3in injury area, and then to participate in the myelin structurereconstruction, improve the nerve conduction of spine cord. It will provide theexperimental evidence for the mechanism of neuronal cell replacement of MSCs andtherapeutic strategie for the clinical application.In order to validate the suppositions, the whole research was divided into two partsas follows: Part1: The study of TrkC expression in MSCs pre-induced withNT-3and RA in vitroObjective: To up-regulate the transcription of TrkC mRNA and translation of TrkCprotein in MSCs by pre-induced with NT-3+RA.Methods: MSCs were isolated, cultured and purified in vitro by adherent culture. Theexperiment groups are designed as control group, NT-3group, RA group andNT-3+RA group. Total mRNA in MSCs was extracted by Trizol, and reversetranscribed to cDNA at1day and3day after induced. The expression of TrkC mRNAwas detected by real-time PCR. The cDNA productions of the real-time PCR wereverified by agarose gel horizontal electrophoresis. The TrkC mRNA in MSCs after1dinduced was detected by in situ hybridization. The expression of TrkC protein inMSCs was validated by immunofluorescence staining and Western blot after1d,3dand7d induced.Results: The relative expression of TrkC mRNA in NT-3+RA group(3.54±0.87) washigher than control group(1.20±0.39)， RA group (2.07±0.45) and NT-3group(1.24±0.13) after1day induced(P<0.05). The cDNA productions of TrkC mRNAReal-time PCR were found by agarose gel horizontal electrophoresis. TrkC mRNAcould be found in the MSCs by in situ hybridization. The expression of TrkC proteinin MSCs was not detected by immunofluorescence staining and Western blot.Conclusions: The transcription of TrkC mRNA in MSCs was increased obviouslyafter1day pre-induced with NT-3+RA. However, the TrkC protein was not detected. Part2: Governor Vessel electro-acupuncture combined withneurotrophin-3and retinoic acid pre-induced MSCstransplantation treatment the rat demyelinated spinal cordinjury induced by EBObjective: To study whether MSCs transplantation, pre-induced with NT-3+RA1day, combined with Governor Vessel electro-acupuncture treatment, whichup-regulated the expression of NT-3in injury area of the rat demyelinated spinal cordinjury induced by EB, could promote the differentiation of MSCs intooligodendrocyte precursor-like cells and oligodendrocyte-like cells to participate inthe myelin structure reconstruction and improve the nerve conduction of spine cordand locomotion.Methods: GFP-MSCs were isolated, cultured and purified in vitro by adherent culture,and then pre-induced with NT-3+RA for1day. Demyelinated model of SD femaleadult rats were induced by EB injection and part of them were divided into7groups:sham group, one-day EB group and EA group, four-day EB group and EA group,14-day EB group and EA group. The concentration of NT-3was detected by ELISAmethod. The other model animals were divided into6groups at random: EB group,EA group, MSCs group, NR-MSCs group, NR-MSCs+EA group and NR-MSCs+NP group. The pre-induced MSCs were transplanted into the lesion site of the modelanimal3days after the modeling and Governor Vessel EA treatment. The animals inEA group, NR-MSCs+EA group and NR-MSCs+NP group were received GVelectro-acupuncture treatment once every other day after2day the transplantationperformed and last for29days. Behavior score evaluation (Balance Beam Score) wastested every other day. After the cortical motor evoked potential were detected, theanimals were perfuse and fix for frozen sections and immunofluorescence. Theexpression of TrkC and BMP in MSCs were evaluated. The survival rate of MSCs, thepositive rate of NG2and APC of MSCs were counted. Some animals were perfuseand fix for semi-thin sections to count the number of the myelin and differentiation ofMSCs by immune electron microscopy. Results: The concentration of NT-3was significantly higher in EA group after14-daytreatment. The survival rate of MSCs had no statistical significance betweenNR-MSCs group, NR-MSCs+EA group and NR-MSCs+NP group. The TrkC proteinin MSCs could be detected in vivo. The NR-MSCs+EA group exhibited higherNG2-positive rate and APC-positive rate and the difference was statisticallysignificant. In NR-MSCs+EA group and NR-MSCs+NP group, some cells weremarked by MBP, GFP and hocest33342simultaneously. Immune electron microscopyshowed that GFP-MSCs could differentiate into oligodendrocytes-like cells andformed myelin sheaths to wrap axon. Less degenerated myelin, more newborn myelinand normal myelin showed in NR-MSCs+EA group and the difference werestatistically significant. The NR-MSCs+EA group had shorter latency and higheramplitude in Cortical motor evoked potentials and the difference was statisticallysignificant. The Balance Beam Score had no statistical significant in all groups.Conclusion: Governor Vessel electro-acupuncture could increase the concentrationof NT-3in lesion site of the rat with spinal cord demyelination induced by EBinjection. Combined government vessel electro-acupuncture with MSCstransplantation pre-induced with NT-3+RA could promote differentiation of MSCsinto oligodendrocyte precursor-like cells and oligodendrocyte-like cells, and then toparticipate in the myelin structure reconstruction and improve the nerve conduction ofspine cord. But the combination therapy had no effect on the survival rate of MSCsand the recovery of locomotion.