Experimental Study On Delay Of Skeletal Muscle Atrophy After Transplantation Of Neuron-like Cells Derived From Mesenchymal Progenitor Cells Invivo

Patients whose skeletal muscle appears severe atrophy or injury torecovery of muscle function after nerve repair are very common inclinical， and the treatment is very difficult. Further elaboration ofmechanisms of denervated skeletal muscle atrophy, effectively delaying orpreventing skeletal muscle atrophy，to the greatest extent to maintain theoriginal function of skeletal muscle,is the focus of research in recent yearsand the problems to be solved urgently. Although microsurgical techniqueof widely application can improve the repair level of peripheral nerve injury,recovery of skeletal muscle function after the operation is not very ideal.when the damaged nerve was replanted or grown back into the targetmuscle,long time inreversible changes (such as muscle atrophy,myofibrosis) after denervation makes it unable to recover the normalfunction of skeletal muscle as a result of slow regeneration of motor nerve.,For the prevention and treatment of denervated skeletal muscle atrophy,scholars at home and abroad have adopted many methods, including functional electrical stimulation, passive motion, drug therapy, neuralimplantation, growth factor therapy, gene therapy, ect, and achieved somesuccess in experimental studies, but the clinical efficacy was limited.Stem cell (SC) is a type of self-proliferation ability and multipotencyof cells, can both produce the descendant cells with their genotype identicaland differentiate into progenitor cells,and have be potential to regenerateinto various tissue and organ,then are called “universal cells” in themedical profession. Studys have shown that adult stem cells (ASCs)usually prefer to differentiate into all kinds of cells of autochthonous tissuesnuder natural conditions to mainly used to replace to aging death cells andmaintain relative stability of the metabolism of organism,but underspecified induction conditions, ASCs of an organization can be "transversal differentiation " as functional cells of other organizations toparticipate in injury and recovery of tissues. In addition, ASCs taken fromthe patient ’s own tissues through directional differentiation in vivo andthen implant patients to avoid problems of immune rejection. Due toplasticity, strong self proliferation and multilineage differentiation potentialof ASCs, and the injury or disease be able to induce the cell proliferationand differentiation of ASCs, some scholars argued that ASCs for treatmentof various neurological damage will be an important measure, and hasbroad application prospects.In recent years, some scholars study found that neural stem cells (NSCs) and mesenchymal stem cells (MSCs) transplanted into thetransected peripheral nerve or injected directly into the target muscleeffectively prevented denervated skeletal muscle atrophy. Although theyhave obtained success in animal experiment, the way to obtain plenty ofNSCs was relatively limited and amplification of them was difficult, andthe proliferation effect of MSCs was not ideal due to be vulnerable to bepolluted by hematopoietic stem cells in vitro culture, so the finding of newcells for transplantation is particularly urgent. Recent research found thatmesenchymal progenitor cells (MPCs) coming from the compact bone ofmice could be induced to differentiate into neuron-like cells, and was thecharacteristics of easy access, easy to grow, powerful reproductive activity,and could avoid the contamination of hematopoietic stem cells during becultured in vitro, therefore，therefore,it is expected to become the seed cellreplacing the NSCs and MSCs.Therefore,this topics proposed to cultivate MPCs from compactbone fragments of GFP transgenic C57mice,and then be transplanted intothe transected position or directly injected into the target muscle topreliminary study the survival situation of transplanted cells in situ and theeffect and mechanisms delaying skeletal muscle atrophy after be induceddifferentiation into neuron-like cells in vitro.This will provide thetheoretical guidance and experimental basis for further proving itsmolecular mechanism and clinical application of later, at the same time, and also provide a new way of thinking for the individual identificationmethod for forensic science and the grasp of the identification time andextent of the damage after nerve injury.Our study mainly consisted of three parts:The first part: Study on their own characteristic maintenance ofneuron-like cells from mesenchymal progenitor cells after beingtransplanted into muscle.Objective:MPCs from compact bone fragments of green fluorescent proteintransgenic C57mice was cultivated and induced differentiation intoneuron-like cells and neuroglia-like cells in vitro,and then weretransplanted into the transected position and directly injected into the targetmuscle to study the survival situation of transplanted cells in situ and themaintenance of pretransplant characteristics.Methods:MPCS were isolated from Bones of hind limbs of3week old healthyGFP transgenic C57mice mechanically and cultivated, and its purity andmulti-directional differentiation potency were detected by flow cytometryand induced differentiation of osteogenic and adipogenic cellsrespectively. After MPCs of the third generation(P3) in good growth wereinduced directionally into neuron-like cells and neuroglia-like cells by the supernatant cultured with primary neuron in vitro, detected its expressionof neuronal specific markers(NSE, NeuN) and glial specific marker(GFAP) by immunofluorescence staining,and collected it to prepared intocell suspension (5×105/μl) with physiological saline.24healthy C57miceaged12week old were divided into4groups evenly in random, thetransected position+MPC transplantation group, the transected position+physiological saline group, the muscle+MPC transplantation group and themuscle+physiological saline group, right leg as experimental side and leftleg for sham operation side among mice. According to the literature method,the sciatic nerves of right hind limbs of4groups of mice were transected toestablish the denervation atrophy model of gastrocnemius muscle while thesciatic nerves of the left hind were only isolated but not transected.5μl ofMPCs suspension were injected into the right sciatic nerve transectedposition，the right gastrocnemius muscle and the corresponding parts ofleft legs in the transected position+MPC transplantation group and themuscle+MPC transplantation group respectively while5μ L physiologicalsaline were injected into the right sciatic nerve transected position，theright gastrocnemius muscle and the corresponding parts of left legs in thetransected position+physiological saline group and the muscle+physiological saline group. At4weeks postoperatively, the survivalsituation of transplanted cells in situ were observed under fluorescentmicroscope, and the expression of neuronal specific markers(NSE, NeuN) and glial specific marker (GFAP) of transplanted cells were detected byimmunofluorescence staining.Results:1，Studies have shown that MPCs from the compact bone of mice didnot express immune phenotype of hematopoietic stem cell CD31andCD45but expressed immune phenotype of interstitial cells CD29, CD44,CD90and CD106Detection of flow cytometry,and could be induceddifferentiation into osteocyte and adipocyte. Therefore, it suggested thatMPCs from the compact bone of mice were a type of adult stem cells withgood homology, high purity, exclusion of hematopoietic stem cells andmulti-directional potency.2，Studies have shown that the majority of MPCs after being inducedby the supernatant cultured with primary neuron for24h expressedpositively NSE, NeuN and a few cells expressed positively GFAP byimmune fluorescence detection while positive expression cells were notfound in the control group. It suggested that MPCs after being directionlyinduced in vitro are some characteristics of neuronal or glial.3，At4weeks postoperatively,studies have shown that transplantedcells distributed evenly in the muscle fiber gap and the majority oftransplanted cells in right hindlimb muscles around the injection siteexpressed positively NSE, NeuN and a few cells expressed positivelyGFAP in the transected position+MPC transplantation group and the muscle+MPC transplantation group while no obvious positive cells werefound in the corresponding site of muscle tissue of left hindlimb in theother two groups.This indicates that neuron-like cells derived from MPCscould survive in situ and maintain the pretransplant characteristics afterbeing transplanted into the transected position and the denervated targetmuscle.Conclusion:1， MPCs with good growth, high purity and multilineagedifferentiation potentiality were successful cultivanted.2， MPCs could directiongly induced and differentiated intoneuron-like cells and neuroglia-cells in vitro.3，The fact was revealed that neuron-like cells derived from MPCscould survive in situ and well maintain the pretransplant characteristicsafter transplantation.The second part: Study on Delay of denervated Skeletal MuscleAtrophy After Transplantation of Neuron-like Cells derived fromMesenchymal Progenitor Cells in vivoObjective:To study the effect and mechanism delaying denervated skeletalmuscle atrophy after neuron-like cells derived from MPCs weretransplanted into the transected position and the target muscle. Methods:MPCS were isolated from bones of hind limbs of GFP transgenic C57mice for cultivation and identification. After being directionally inducedinto neuron-like cells and neuroglia-like cells by the supernatant culturedwith primary neuron in vitro，P3MPCs with good growth were collectedand prepared into cell suspension (5×105/μl) with physiological saline.48C57mice were divided into4groups evenly in random, the control group,the transected group, the transected transplantation group and the muscletransplantation group. According to the literature method, the sciaticnerves of mice were transected to establish the denervation atrophy modelof gastrocnemius muscle while nothing was treated in the control group.5μl of MPCs suspension was injected into the sciatic nerve transectedposition and the gastrocnemius muscle in the transected transplantationgroup and the muscle transplantation group respectivelyand5μl ofphysiological saline was injected into the gastrocnemius muscle in thetransected group while nothing was injected in the control group. Theactivity ability of hind limbs of mice were observed. At2and4weekspostoperatively, the retain ratio of wet weight of gastrocnemius muscleand cross sectional area of muscle fiber was measured and theultrastructural organization was observed. The expression of α-actin,myoglobulin (MHC) were detected by western blot and the expression ofMyogenin and MyoD were detected by RT-PCR. Results：1，At2and4weeks postoperatively, the retain ratio of the wet weightof gastrocnemius muscle and the cross sectional area of muscle fiber ofmice of the transected transplantation group and the muscletransplantation group were higher than that of the transected groupsignificantly (P <0.01), which suggested that two kinds of treatmentmethods could effectively delay denervated skeletal muscle atrophy and thecurative effect of the transected transplantation group was moresignificant.2，At4weeks postoperatively, compared with the degeneration ofmyocyte, mitochondria and sarcoplasmic reticulum and the extent ofmusculus fibrosis of the transected group, that of the transectedtransplantation group and the muscle transplantation group were lowersignificantly and some cells showed the characteristics of vigorousproliferation activity (nuclear ingression, nuclear enlargement, nucleolusmanifestation, developed heterochromatin and the number of mitochondriaincreased,ect), which suggested that two kinds of treatment methodscould effectively could inhibit the fibrosis of denervated skeletal muscle.3，At4weeks postoperatively, compared with the the expression ofα-actin, MHC of the transected group, that of the transected transplantationgroup and the muscle transplantation group were higher significantly byWestern blot,which suggested that two kinds of treatment methods not only could effectively inhibit the degradation of skeletal muscle proteinafter denervation,but also accelerate the synthesis of protein.4，At4weeks postoperatively, compared with the the expression ofMyogenin and MyoD of the transected group, that of the transectedtransplantation group and the muscle transplantation group were highersignificantly by RT-PCR,and the expression of MyoD were the mostsignificant.Conclusion:1,That neuron-like cells derived from MPCs were transplanted intothe transected position and the target muscle could effectively delaydenervated skeletal muscle atrophy and inhibit muscle fibrosis, and theeffect of cell transplantation into the transected position was moresignificant.2,That neuron-like cells which derived from MPCs were transplantedinto the transected position and the target muscle could effectively inhibitthe degradation of skeletal muscle protein after denervation.3, It was drawn the preliminary mechanism delaying skeletal muscleatrophy that neuron-like cells derived from MPCs were transplanted intothe transected position and the target muscle.