【作者】 赵文勇；

【导师】 万立华；

【作者基本信息】 重庆医科大学 ， 组织工程与细胞工程， 2013， 博士

【摘要】 外周神经损伤后骨骼肌发生严重萎缩抑或损伤神经修复后肌肉功能难以恢复的患者在临床上较为常见,并且治疗相当困难。深入阐明失神经性骨骼肌萎缩的机理，有效地延缓或防治其萎缩，最大程度地维持骨骼肌原有功能，是近年来研究的重点和亟待解决的难题。显微外科技术的广泛应用尽管能提高外周神经损伤的修复水平，但术后骨骼肌功能恢复并不十分理想，其根源在于周围神经损伤后，运动神经再生非常缓慢，当受损神经通过再植或重新长入所支配的靶肌肉时，长时间失神经支配所致的肌肉不可逆变化（如肌肉萎缩、纤维化等）使骨骼肌无法恢复正常功能。为防治失神经性骨骼肌萎缩，国内外学者采取了许多方法，包括功能性电刺激、被动运动、药物疗法、神经元植入、生长因子治疗、基因治疗等，尽管实验研究均取得了一定成功，但临床应用的疗效却有限。干细胞(stem cell，SC)是一类具有自我增殖能力与多向分化潜能的细胞,既能产生基因型与自己完全相同的子代细胞,也能分化为祖细胞，具有再生为各种组织、器官的潜能,故医学界称其为“万用细胞”。研究表明，成体干细胞（adult stem cells，ASCs）在自然条件下通常倾向于分化为所属组织的各种细胞类型，主要用于替换衰老死亡的细胞和维持机体新陈代谢的相对稳定，但在特定的外界诱导条件下，一种组织的成体干细胞可以“横向分化”为其他组织的功能细胞，参与组织的损伤修复。此外，成体干细胞还可取自于患者的自身组织,通过定向诱导分化后重新植入患者体内避免了免疫排斥的问题。正是由于成体干细胞具有可塑性、旺盛的自我增殖能力及多向分化潜能，且疾病或损伤均能刺激成体干细胞的增殖和分化，因此一些学者认为成体干细胞对于各种神经系统损伤将是一种重要的治疗措施，并具有广阔的应用前景。近年来，一些学者将神经干细胞（neural stem cells，NSCs）及间充质干细胞（Mesenchymal stem cells,MSCs）移植于离断的外周神经或直接注入靶肌肉内并有效延缓了失神经性骨骼肌萎缩，动物实验确实获得了成功，但由于足量的神经干细胞获得途径相对有限且扩增困难，体外培养的小鼠间充质干细胞又极易遭受到造血干细胞的污染而使其增殖效果不理想，寻找新的移植细胞尤为迫切。新近研究发现，间充质祖细胞(mesenchymal progenitor cell,MPCs)可体外定向诱导分化为神经元样细胞，同时间充质祖细胞具有易获取、易生长、增殖能力强等特点，且来源于密质骨，体外培养时可避免造血干细胞的污染，故有望成为神经干细胞及间充质干细胞的替代种子细胞。因此，本课题拟培养GFP转基因C57小鼠密质骨碎片来源MPCS，体外定向诱导分化为神经元样细胞后进行神经断离处移植或直接注入靶肌肉内，初步探讨移植细胞体内原位定植存活情况以及延缓骨骼肌萎缩的作用与机制，为进一步探明其分子机制、后期临床应用提供理论指导和实验依据，同时也为法医物证学的个体识别方法、神经损伤后鉴定时限及其损伤程度的把握提供了新的思路。本研究内容主要包括两部分：第一部分：间充质祖细胞源性神经元样细胞肌内移植后自身特性的维持。目的：体外培养绿色荧光蛋白转基因C57小鼠密质骨碎片来源的间充质祖细胞，通过体外定向诱导分化为神经元及神经胶质样细胞后移植于神经断离处及直接注入靶肌肉中，探讨肌内移植后能否定植存活并维持其移植前的特性。方法：取3周龄健康GFP转基因C57小鼠后肢长骨进行MPCs培养，利用流式细胞术及成骨、成脂定向诱导分化检测其纯度及多向分化潜能。选取生长良好的第三代MPCs，神经元原代培养上清液进行神经元及神经胶质样细胞诱导分化后，采用免疫荧光染色检测神经元特异性标志物NSE、NeuN及神经胶质特异性标志物GFAP的表达情况并收集细胞，用生理盐水调整细胞浓度至5×105/μl细胞悬液备用；选取12周龄健康C57小鼠24只，按随机数字表法分为神经断离处+MPC移植组、神经断离处+生理盐水组、肌肉+MPC移植组、肌肉+生理盐水组，其中上述各组小鼠右后肢作为实验侧，左后肢为假手术侧。参照文献操作方法，4组小鼠右后肢均切断坐骨神经建立小鼠失神经腓肠肌萎缩模型，左后肢仅分离坐骨神经但不做切断处理。神经断离处+MPC移植组和肌肉+MPC移植组：右侧坐骨神经切断处、右侧腓肠肌内及左侧对应部位分别注入5μl MPCs悬液；神经断离处+生理盐水组和肌肉+生理盐水组：右侧坐骨神经切断处、右侧腓肠肌内及左侧对应部位均注入5μl生理盐水。术后4周，荧光显微镜下观察肌内移植细胞存活情况，并采用免疫荧光染色检测移植细胞神经元特异性标志物NSE、NeuN及神经胶质特异性标志物GFAP的表达情况。结果：1、通过小鼠密质骨培养获得的MPCs主要表达间质细胞免疫表型CD29、CD44、CD90、CD106，不表达造血干细胞免疫表型CD31和CD45，且在体外能够诱导分化为骨细胞及脂肪细胞，提示通过小鼠密质骨培养获得的MPCs是细胞同源性好、纯度高、排除了造血干细胞干扰且具有多向分化潜能的成体干细胞。2、MPCs经神经元原代培养上清液诱导24h后，大部分细胞阳性表达NSE、NeuN，少数细胞阳性表达GFAP，而对照组未见确切阳性表达细胞，提示MPCs经体外定向诱导分化后具备神经元或神经胶质的某些特性。3、术后4周，荧光显微镜下观察发现移植细胞均匀分布于肌细胞间隙；免疫荧光检测发现，神经断离处+MPC移植组和肌肉+MPC移植组右后肢注射部位周围肌肉组织中大部分移植细胞阳性表达NSE、NeuN，少量细胞阳性表达GFAP，而左侧对应部位肌肉组织以及另外两组均未见明显阳性细胞表达，提示MPCs源性神经元样细胞移植于神经断离处及失神经支配靶肌中能够定植存活并很好地维持其移植前特性。结论：1、成功培养出生长良好、纯度高且具有多向分化潜能的MPCs细胞。2、MPCs体外可定向诱导分化为神经元及神经胶质样细胞。3、MPCs源性神经元样细胞肌内移植后能够在原位定植存活并很好地维持其移植前特性。第二部分：间充质祖细胞源性神经元样细胞体内移植延缓骨骼肌萎缩的研究目的：初步探讨间充质祖细胞源性神经元样细胞移植于神经断离处和靶肌肉中延缓失神经性骨骼肌萎缩作用及其机制。方法：取GFP转基因C57小鼠后肢长骨进行MPCs培养及鉴定，选取生长良好的第三代MPCs，采用神经元原代培养上清液进行神经元及神经胶质样细胞诱导分化后收集细胞，生理盐水调整细胞浓度至5×105/μl细胞悬液备用。选取C57小鼠48只，随机分为对照组、神经断离组、神经断离处移植组、肌肉移植组，每组12只。参照文献操作方法，建立小鼠失神经腓肠肌萎缩模型，其中对照组不做任何处理。神经断离处移植组和肌肉移植组分别于坐骨神经断离处、腓肠肌内注入5μl MPCs悬液，神经断离组于腓肠肌内注入等量生理盐水，对照组不作任何处理。观察小鼠后肢活动能力，术后2和4周测量腓肠肌湿重、肌纤维横截面积维持率及观察超微结构，用Western blot检测α-actin、MHC及RT-PCR检测Myogenin、MyoD的表达情况。结果：1、术后2和4周，神经断离处移植组和肌肉移植组腓肠肌湿重及肌纤维横截面积维持率显著高于神经断离组，提示两种治疗方法均可有效延缓失神经性骨骼肌萎缩，其中神经断离处移植组治疗效果更显著。2、术后4周，神经断离处移植组和肌肉移植组肌细胞核、线粒体、内质网的退变及肌肉纤维化程度明显低于神经断离组，且部分细胞表现出旺盛的增殖活性的特点（细胞核内移，核增大，核仁明显，异染色质发达、线粒体数量增多等），表明两种治疗方法均可有效抑制失神经性骨骼肌纤维化；3、术后4周，Western blot检测发现，神经断离处移植组和肌肉移植组腓肠肌内α-actin及MHC的表达程度均显著高于神经断离组和对照组，表明两种治疗方法既可有效抑制失神经性骨骼肌蛋白质的降解，又能够促进蛋白质的加快合成。4、术后4周，RT-PCR检测发现，神经断离处移植组和肌肉移植组腓肠肌内Myogenin及MyoD的基因表达丰度显著高于神经断离组和对照组，其中以MyoD的高表达更为显著。结论：1、MPCs源性神经元样细胞移植于神经断离处及靶肌肉内均可有效延缓失神经性骨骼肌萎缩及肌肉纤维化的发生，其中以神经断离处细胞移植的疗效更为显著；2、MPCs源性神经元样细胞移植于神经断离处及靶肌肉内均可有效抑制失神经支配靶肌肉蛋白质的降解；3、初步得出MPCs源性神经元样细胞移植于神经断离处及靶肌肉内延缓失神经性骨骼肌萎缩的机制。更多还原

【Abstract】 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.更多还原