Experimental Study On The Prevention Of Cataplasia Of End Plate And Myatrophy With Muscle Satellite Cells Transfected With Acid Fibroblast Growth Factor Gene

[Background]Many researches show that the normal architecture of muscle and the maintenance of its function depend on the contributions of some neurotrophic factors. When motor nerve is damaged, the greatest change of denervated skeletal muscle is the loss of such kind of factors. Meanwhile, skeletal muscle is disused, which finally results in the change of muscles in morphosis and physiological biochemistry. What displays in histomorphology is the cataplasia of end plate, the loss of the muscle cytoplasm and the diminution of anteroposterior diameter. What’s more, the symptom is gradually getting more and more with the time elapsed. Presently, the re-establishment of motor nerve function after its injury focused on the recovery of the succession of nerve while ignores the protection of its target organs. Such factors as the lengthy reactivation process of nerve with re-cataplasia of nerve during this process, the baffling effect of injury region, the immobilization of limbs after its reparation, even if the nerve is promptly repaired, the process that muscles with denervation lasts for a long time, and the trophic action of nerve lost and gradual cataplasia of end plate bring great difficulty to the re-establishment of myoneural junction. The features that muscle presents beads shaped degeneration, muscle horizontal grain abolishment, myatrophy, the death and disappearance of muscle are found.These changes also affects the shape of motor end plate. Then, the apraxia and atrophy of muscle have influence on the morphology of end plate, which will lead to an vicious circle between them. This is one of the most important factors nowadays which hinder the recovery of function after nerve repair. As a result, how to prevent the cataplasia of endplate and myatrophy becomes an urgent and tough problem nowadays which awaits to be solved urgently in clinic once motor nerve is damaged.In recent years, many domestic or foreign scholar, have brought up a series of methods to prevent or delay motor endplate degeneration and myatrophy from multiple perspectives, in order to improve the curative effect of peripheral nerve injury. The methods included:①passive exercise;②The nerve fibers or neurons implanted in muscle;③pulsed magnetic therapy,electrical stimulation therapy;④drugs:Various kinds of nerve growth factor,hormones and so on. Passive exercise is the most commonly used method by clinician.Clinical studies have confirmed that a passive activity can make paralyze muscles shorter and stretch exercise. This may on the one hand promoted the muscle blood circulation, reduce tissue edema, shortened oxygen and nutrients and muscle cells in the blood the dispersion distance between, on the other hand through mechanical muscle fibers spun and shorten, keep muscle certain elasticity, and prevent joint stiffness, and disuse osteoporosis and muscle atrophy, in preparation for the regeneration of nerve fibers reaches the target organ.However clinically often meet some difficulties:fracture repair often need to early fixed body, and patients patients do not follow the doctor’s advice after training for a long time.All these caused degeneration is inevitable. Use pulsed magnetic therapy and electrical stimulation therapy to prevent degenerative ideas had been accepted by more and more clinical doctor, and they had make electrical therapy stimulation and magnetic therapy gradually apply in clinical. Shimada, Boonyarom et al.found that magnetic therapy, electrical stimulation therapy can obviously relieve muscle cell apoptosis, increased after nerve injury Ⅰ fiber and Ⅱ fiber diameter, slow muscle weight reduction, these may delay degeneration and atrophy. However the equipment is difficult to popularize, clinical curative effect is slow and not accurate, the clinical promotion was not well accepted. In recent years, the drug research has achieved some significant results:studies which from domestic or foreign have shown that nerve growth factor (NGF) or hepatocyte growth factor (HGF) was intermittently used can reduce motor endplate degeneration, and its mechanism may be through axoplasmic transport and corresponding receptor target organs to implement.However, these factors were only suitable for Seddon category Ⅲ degree of nerve injury, non-surgical therapy or Seddon classification Ⅰ, Ⅱ degree were not suitable.What’s more intermittently use slow-release system is difficult to implement and there is no guarantee that drugs in the long-term effect, so the research prospect is poor. There have the effect of the protective film structure, the authors reported that injecting dexamethasone to rat abdominal cavity can reduce mouses which were non-severed nerve damage motor endplate degeneration.However,dexamethasone was belong to hormone drugs, it had problem of high side-effect after used for a long time. So dexamethasone is confined to the experimental study, should not be appropriate for patients with nerve injury for a long time. So the clinical application prospect is also poor.Gene therapy is an emerging subject in recent years,it is defined as inserting foreign genes into cells and making it expression effectively, then transplant cells in body to achieve the purpose of treating diseases. Gene therapy usually carries exogenous gene with the help of stem cells. MSCs were one kind of stem cells which are usually used. It was found in the frog skeletal muscle for the first time in1961by Mauro et al.They were precursor muscle cells or muscle stem cells which had proliferation and self-renewal ability,plays an important role in repair and maintenance skeletal muscle injury after postnatal days. Studies have reported:the number of skeletal muscle satellite cells (muscle satellite cells, MSCs) were increased in the early time after nerve injury, while their number and volume were significantly decreased in the later time. The proliferation potential of MSCs were decreased significantly and their number was gradually decline after denervation a long time. Some studies suggest this state of progressive decrease is due to the loss of innervation, cell apoptosis had happened.The reason was that the nutrition of nerve function is very important to maintain MSCs content and function. Viguie speculate that the reason for this decline such as:in the long-term loss of innervation, MSCs have no renewable alternative after death. The speed of MSCs merged to atrophic muscle fibers or new muscle fibers faster than the speed of proliferation. According to this change, many scholars believe that the number of MSCs was gradually reduced affect long-term loss of skeletal muscle after denervation was an important reason for nerve injury functional recovery. Through research on skeletal muscle regeneration process, scholars found that growth factors acted on different cell cycle of MSCs. By cell culture technology, researchers had detected a variety of growth factors influenced the growth of MSCs, they acted on separate or combinate. there are several growth factors:many research found that IGF (IGF-I) and IGF-II can increase the proliferation and differentiation of MSCs in vitro.Injecting cells with IGF-1, can promoted the number and the ability of proliferation of MSCs; Hepatocyte growth factor (HGF) on the regulation of MSCs in multiple aspects, including as a potential chemokines, the activation of MSCs and the inhibition of fibroblast cells, FGF can activate and selectively proliferate of MSCs; Yablonka-Reuveni et al. confirmed FGF can promote young and adult mouse MSCs proliferation; McFarland et al. found FGF family detailed function of MSC proliferation in research of culter MSCs. In nine subtypes of FGF family, they found that FGF-1(named aFGF), FGF-2, FGF-4and FGF-9can stimulate the proliferation of MSCs, HGF and FGF-2or FGF4or FGF-6or FGF-9can improve the proliferation of MSCs. Kastner, Dong respectively reported aFGF can promote proliferation and division of MSCs in different periods. We also observed that aFGF can maintain the number of MSCs in the research of aFGF slow-release system protect motor endplate and prevent muscle atrophy in the early time.Based on the above theoretical and experimental basis, we believe that MSCs and aFGF have a close relation.We prepare MSCs transfected with aFGF gene for transplanting to prevent motor endplate degeneration and myatrophy,with the purpose of persistently maintaining the number of MSCs during lacking of innervation. This will be try to prevent motor endplate degeneration and myatrophy fundamentally, which will improve the effect of motor nerve repair.[Objective]1. To explore the approach to isolate and culture muscle satellite cells(MSCs) in vitro and identify it;2. To observe the expression of aFGF gene transfected MSCs;3. To transplant MSCs into mouses which had nerve injury and and detect the effect of preventing motor endplate degeneration and myatrophy.[Methods]1. MSCs solation, culture and identification:MSCs were extracted from adult Wister rat and purified by difference-speed adherence method and identified by immunohistochemical assay;2. To construct the eukaryotic expression vector:the aFGF gene was cloned from human total RNA which was obtained from human skeletal muscle tissue by RT-PCR method. Human interleukin2(IL-2) signal peptide sequence (SPS) was obtained by direct chemosynthesis method. Then aFGF and SPS were fused to obtain SPS-aFGF. Finally, directional cloning SPS-aFGF into pEGFP-N1, the recombinant (pEGFP-N1-SPS-aFGF) was obtained; 3. Cells groups, gene transfection and detect expression in vitro:the recombinant was confirmed by endonuclease digestion and DNA sequencing. MSCs were purified by difference-speed adherence method and were ideontified by immunofluorescence assay. The correct cells were divided into3groups: Experimental group (aFGF+N1), Control group(N1), Blank group(Blank). All the groups were transfected by Lipofectamine2000TM Reagent, and pEGFP-N1-SPS-aFGF, pEGFP-N1were respectively added in Experimental group and Control groups while Blank group was added none plasmid.The transfected cells were selected by G418for stable growth. Fluorescence microscope was employed to detect transfection efficiency tendency along with time changes. The expression of target gene was detected by fluorescent quantitation PCR and Western blot;4.To build animal neurological damage model and test the transplanted cells curative effect:40adult inbred line Wister rats were selected and abscised their right tibial muscle branch of the common peroneal nerves(the distance between abscising point and the point where nerve entering into muscle is1.5cm) and performed epineurial repair with complete anesthesia which was through10%chloral hydrate through intraperitoneal injection. The models were randomly divided into four groups:group A:the rats were transplanted experimental group(aFGF+N1) into tibial muscle; Group B:the rats were transplanted control group(N1) into tibial muscle; Group C:the rats were transplanted blank group(Blank) into tibial muscle; Group D:The rats were transplanted same volume of saline into tibial muscle; Group. A was experimental group, B, C and and D were control groups. All rats were observed for4weeks postoperation. Observe the appearance of the muscle through morphological method with naked eyes and measure humid weight of tibial muscle. Staining with auric chloride was deployed to display end plate, Stimulate it repetitively for9times with the frequency from3to5Hz pulsating current and observe whether there is decrement or not. Masculine was based on the fact that if the fifth myoelectric wave amplitude is lower10%than that of the first one. Statistical analysis on the average decrement rate of the fifth myoelectric wave amplitude was conducted.[Results]l.The results of identification by immunohistochemistry showed that cultivated cells were MSCs;2.Thesequencing of pEGFP-Nl-SPS-aFGF was completely consistent with published GenBank sequences and the outcome of endonuclease was equal to actual ban size. The expression of GFP was appeared as early as6h after transfection, and the amount and intensity peaked at72h. GFP was still seen in the subculture cells. The optimal concentration of G418was300μg/ml and the positive clones still existed after28days selected. Real-time fluorescent quantitation PCR proved strong aFGF mRNA expression in transfected cells(the average relative expression of Experimental group was1464.95) with aFGF gene, while it was detected a little in the other groups(the average relative expression of Control group was1.016and Blank group was1.000)(P<0.05). Western blot also proved strong expression in Experimental group then the other two groups.3.Morphological observation with naked eyes:group A:compared wit self-control, it could be observed that there was symmetry between bilateral tibial muscles and normal muscular tension while no manifest muscular atrophy in this group.There were similar performances in group B and group C:there was p muscular atrophy in the right tibial muscular and descending muscular tensions,but not so manifest as in group D.Group D:There was manifest muscular atrophy in the right tibial muscle and no muscular tension in this group. RNS detecting results:There was no obvious decrement of myoelectricity wave in group A when there was low frequency RNS. The decrement rate was lower than10%and no positive sample was observed while there was obvious decrement of myoeleetricity wave in group B, group Cand group D. The positive rate was90%in these three groups and the group D was more obvious than any goups. Chromoscopy of end plate:majority end plates in group A were with integrated structure and normal morphology. There were similar performances in B and group C:the morphology of end plates in denervated muscle fiber was anomalistic. The branched ending of axon was decreasing and tangling as well. End plate was in the form of striga or punctiform. Group D:no axon was observed, the morphology of majority end plates was basically normal, only the end plates region reduced. Group D:axon was observed, the quantity of end plate decreased manifestly and the morphology of end plate was anomalistic[Conclusion]1.The method of differential sticking was feasible to extract the rat muscle satellite cell walls;2. The construct of eukaryotic expression vector was correct, gene expression is normally.3. The method of transplanting MSCs which were transfected with aFGF gene to prevent motor endplate degeneration and myatrophy method is feasible.