An Experimental Study Adult Rat Mesenchymal Stem Cells Express A Variety Of Neuro-regulatory Molecules And Delay Denervated Muscular Atrophy And Mechanism

Objective:Bone marrow stromal stem cells (MScs), which normally differentiate into mesenchymal derivatives, have recently reported to transdifferentiate into neurons. However, the findings from different groups and interpretations have been challenged.The purpose of this paper is to re-evaluate the phenomenon of neuronal trans-differentiation of MSCs and compare the expression levels of neurotrophins in rMSCs and neuronal-like phenotypes derived from rMSCs.Method:rMSCs were obtained from both bilateral femurs and tibias. rMSCs were differentiated in pre-induction media and then transferred to neuronal induction media for 7 days and 14days. RT-PCR and ELISA were employed to determine the expression level of Brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT3), ciliary neurotrophic factor (CNTF) and glial derived neurotrophic factor (GDNF) in rMSCs and neuronal-like phenotype derived from rMSCs.Results:The adherent rMSCs exhibited spindle-shaped or large flattened morphology, and expressed CD90 and CD44 with a positive ratio of 95.37%±2.38% and 94.35%±1.73% respectively. After neuronal induction,, neuronal-like phenotypes accounted for about 80% of the total population. Immunological labelling showed that neuronal-like phenotypes cells expressed mature neuronal markers (GFAP 40.17%±2.95%, Tubulin-Ⅲ19.00%±1.58%, CHAT 19.30%±1.38%), NF 16%±1.23%, MAP-2 36.30%±1.78%) RT-PCR and ELISA analysis confirmed expression of neurotrophins in rMSCs and neuronal-like phenotypes. Moreover, the level of BDNF,NGF,NT3,CNTF and GDNF of rMSCs is remarkably higher in rMSCs than the neuronal-like phenotypes, especially CNTF. The expression level of these neurotrophins did not change significantly after enduring induction.Conclusion:Under certain conditions, rMSCs can transdifferentiate into neurons. The non-induced rMSCs has a dynamic expression profile of neurotrophins and may serves as a better tool than the trans-differentiated rMSCs for transplant therapy.Objective:Although there are many therapies, it is still difficulty to reverse post-denervation muscle atrophy. Recently, stem cells transplantation seems to be as a method of delaying muscle atrophy. The findings from different groups and interpretations have shown that MSCs from adult bone marrow have the potential to differentiate into a variety of cell types and express many cytokines and cellular factors. The purpose of this paper is to evaluate whether delivery of neurotrophins to denervated gastronemius muscles improved the function of these muscles when adult rat mesenchymal stem cells (rMSCs) were transplanted into the distal tibial nerve. Further, we observed whether rMSCs by silencing CNTF, the maxlmum in neurotrophins, with RNA interference changed its role in delaying muscular atrophy.Method:After anesthesia, the right leg of each rat (Wistar, about 120g) was denervated by sectioning the tibial never in 1cm above gastrocnemius muscle. The tibial nerve was tightly ligated with surgical silk in two places, cut between the sutures, and a 0.4cm section of the nerve was removed. Both proximal and distal nerve stumps were implanted in the surrouding tissue as far away from each other as possible. After 1 week of muscle denervation, animals were divided into the following four experimental groups:Ⅰ, control group (n=24);Ⅱ,CNTF injection group(n=24);Ⅲ,rMSCs by silencing CNTF with RNA interference transplantation group (n=24);Ⅳ, rMSCs transplantation group (n=24). They were respectively injected with 5ul of culture media,5ul of CNTF,2.5×105 rMSCs by silencing CNTF with RNA interference and 2.5×105 rMSCs at the site of tibial nerve just proximal to the nerve branch of the medial gastrocnemius. The animals were euthanized again at 4,8and 12 weeks after denervation. Tissue samples of tibial nerve and gastrocnemius muscle were taken. Muscle strength assay,gastrocnemius muscle wet,weight,richrome staining and muscle fiber areas from media gastrocnemius muscle were performed. Meanwhile, Electron microscope examination was done to demonstrate the NMJ. All the data was analyzed by Stata.Results:Fluorescence immunocytochemistry confirmed that rMSCs expressed the neurotrophins, and scarcely differentiate into neuronal-like phenotypes. Further, tissue examination found that mean±SE wet weight ratio was significantly different in all groups. Mean ofⅢandⅣgroup was about 70% higher than that ofⅠgroup, and 30% higher than mean ofⅡgroup. However, mean betweenⅢandⅣgroup was nearly approximate. Moreover, wet weight ratio in all group declined gradually with lasting denervation, especiallyⅠgroup. The variation of Muscle strength, muscle fiber areas and neuromuscular junctions was nearly consistent with wet weight ratio.Conclusion:rMSCs could improve the function of denervated gastronemius muscles by delivery of neurotrophins when rMSCs were transplanted into the distal tibial nerve. Furthermore, rMSCs lacking CNTF, the maxlmum in neurotrophins, could not reduce remarkably its role in delaying muscular atrophy.Objective:Bone marrow mesenchymal stem cells(MSCs) contributes to gene therapy and muscle repair. Neuregulins(NRGs) thought to regulate acetylcholine receptor(AChR) expression, which is a hallmark of the inductive events of synapse formation. This present study investigated effect of rMSCs on Sol 8 cells and which signal transduction pathway involve in it.Method:Lentivirus (LVTHM) was performed according to instruction manual. Sol 8 cells were transfectioned by AChR and ERBB3 gene through LVTHM. RT-PCR was performed to show the mRNA of AChR and ERBB3. Then, we co-culture rMSCs and Sol 8 cells for 48 hours. Compared with the normal group, Western blot showed protein of AChR raised and protein of AChR is up-regulated. The phosphorylation of Raf,Ras and MAPK were indicated also.Results:The result was consistent with the precious. After 48 hours, protein of AChR and ERBB3 was up-regulated in experimental group. Meanwhile, the protein of Raf,Ras and MAPK were increased.Conclusion:rMSCs is able to up-regulate the protein of AChR. We infer that Ras→Raf→MAPK pathway may participate in this process.