【作者】 唐乙；

【导师】 江华； 何成；

【作者基本信息】 第二军医大学 ， 外科学， 2012， 博士

【摘要】 【研究背景】周围神经损伤是常见的外科疾病，临床医生和科学工作者长期致力于提高外科技术和手术方法以促进周围神经损伤的修复，取得了一定效果，但其术后功能恢复仍不满意。自体神经移植会造成供区感觉、运动功能障碍，且可供移植的神经极其有限；供体神经多细小，与缺损的神经常难以匹配；异体神经移植又面临免疫排斥问题。组织工程学的发展为此提供了一种新的解决途径，其中雪旺细胞是周围神经组织工程中研究较多并且有效的种子细胞，在神经再生中发挥重要的作用。移植的雪旺细胞可在周围神经内存活、增殖、迁移和分化，并分泌多种神经营养因子、细胞黏附分子和细胞外基质，促进轴突的再生和髓鞘形成，从而促进周围神经的修复。但是作为种子细胞，雪旺细胞的分化能力较低，是一种终末期细胞，来源有限，并且存在移植排斥问题，所以需要一种更为年轻化的细胞，通过其诱导分化成为雪旺细胞。已报道成功诱导分化为雪旺细胞的干细胞包括：骨髓间充质千细胞、脂肪源性干细胞、神经干细胞、皮肤源性祖细胞等。但关于肌源干细胞诱导分化为雪旺细胞的研究却鲜有报道。肌源干细胞是新近发现的干细胞，具有较好的自我更新和多分化潜能，可以分化成肌肉细胞、造血细胞、成骨细胞、软骨细胞、内皮细胞、脂肪细胞、肝细胞和神经细胞等。如果能成功诱导肌源干细胞分化为雪旺细胞，将具有组织来源多，获取较容易，对机体损伤相对较小，具有免疫优越性，较容易获取和扩增等优点，能为临床治疗周围神经损伤提供一种新的思路和选择。本课题在应用雪旺细胞条件培养液诱导小鼠肌源干细胞分化为类雪旺细胞的基础上，进一步研究其诱导机制。雪旺细胞能分泌多种神经营养因子，并且与其有密切的相互作用，在周围神经再生中发挥重要作用。因此我们确定以神经营养因子作为研究因素，在成功分离、培养小鼠肌源干细胞，以雪旺细胞条件培养液诱导其分化为类雪旺细胞的基础上，进一步以神经营养因子作为诱导因素，用其体外诱导小鼠肌源干细胞，观察诱导效果，从而进一步明确神经营养因子在小鼠肌源干细胞的诱导和分化中的作用。【目的】（1）掌握小鼠肌源干细胞的分离、纯化、培养及鉴定技术。尤其是利用显微镜和显微手术器械分离获取较纯净的肌肉组织，提高分离纯度，减少组织损伤；摸索并掌握差速贴壁法的贴壁时间，从而提高培养效率；（2）获取小鼠雪旺细胞条件培养液并以其诱导小鼠肌源干细胞，确定其诱导效果；（3）对小鼠雪旺细胞条件培养液中的神经营养因子成分有选择的进行检测，挑选表达较高的神经营养因子作为诱导条件进行研究；（4）神经营养因子诱导小鼠肌源干细胞，观察细胞形态学变化并检测其特异性标记物的表达以确定诱导效果，从而进一步明确小鼠肌源干细胞诱导分化为类雪旺细胞的机制，为肌源干细胞诱导为类雪旺细胞并治疗周围神经损伤提供实验室基础。【方法】（1）显微镜下获取较纯净的新生小鼠骨骼肌肌组织，应用酶消化法和改良的pre-plating差速贴壁法，消化、分离、纯化出小鼠原代肌源干细胞；用台盼蓝染色法鉴定所获小鼠肌源干细胞的活性；倒置相差显微镜下观察历次贴壁细胞和肌源干细胞的细胞形态改变和生长状态；以Sca-1（干细胞表面标记物）和Desmin（肌源性标记物）进行western、免疫组织化学荧光染色及流式细胞检测进行鉴定；（2）显微镜下取小鼠坐骨神经和背根神经节经消化、培养获得小鼠雪旺细胞，并获得小鼠雪旺细胞条件培养液；以小鼠雪旺细胞条件培养液诱导小鼠肌源干细胞，倒置显微镜下观察细胞形态改变，并以S100，GFAP和p75等雪旺细胞表面标记物进行western bolt、免疫组织化学荧光染色及流式细胞学检测进行鉴定；（3）用ELISA方法检测雪旺细胞条件培养液中相关神经营养因子的表达情况，选取表达量相对较高的神经营养因子作为进一步诱导MDSCs向雪旺细胞方向分化的的诱导因子加以研究；(4)将选择的神经营养因子加入干细胞培养液对小鼠肌源干细胞进行体外诱导，倒置显微镜下观察细胞的形态学改变，并以S100，GFAP和p75等雪旺细胞表面标记物进行western blot、免疫组织化学荧光染色及流式细胞学检测进行鉴定。【结果】（1）台盼蓝染色法显示分离、纯化所得小鼠肌源干细胞的活性为95%；倒置显微镜下观察历次贴壁细胞和肌源干细胞的细胞形态和生长状态，发现细胞呈小圆形或短梭形的形态特征，未发生分化，增殖速度快， Sca-1和Desmin的细胞免疫组织化学荧光染色均呈现阳性；流式细胞检测结果亦显示Sca-1和Desmin阳性表达，阳性率分别为93.23±0.93%和94.18±0.38%，双阳性率为90.1±1.28%；（2）小鼠雪旺细胞条件培养液成功诱导小鼠肌源干细胞向类雪旺细胞分化，发现小鼠肌源干细胞诱导72h后细胞形态发生改变，成梭形或条索形，两端有突起形成，成片聚集，诱导后细胞免疫组织化学荧光检测特异性标记物S100的表达，结果显示为阳性；流式细胞检测结果亦显示雪旺细胞特异性标记物S100，GFAP和p75阳性表达，阳性率分别为65.48±6.20%、39.84±1.66%和41.08±0.78%，三阳性率约为25.86±5.37%；（3）用ELISA方法检测小鼠雪旺细胞条件培养液中神经营养因子的表达情况，发现除FGF、GDNF基本检测不到外，神经生长因子(NGF)、脑源性神经生长因子(BDNF)、神经营养素-3(NT-3)、血小板源性生长因(PDGF)、胰岛素样生长因子-2(IGF-2)均有不同量的表达，因此确定以上述有表达的神经营养因子为研究对象；（4）5种NTFs单独诱导小鼠肌源干细胞，结果无作用；尝试5种NTFs两两组合，共10组进行诱导，结果也未发现明显效果；尝试5种NTFs三三组合，共9组进行诱导小鼠肌源干细胞，结果神经营养素-3(NT-3)（500pg/ml）、血小板源性生长因(PDGF)（1000pg/ml）、胰岛素样生长因子-2(IGF-2)（200pg/ml）组成功诱导其分化为类雪旺细胞，其余组无效。诱导72h后小鼠肌源性干细胞细胞形态发生改变，成梭形或条索形，两端有突触形成，成片聚集，分布较散乱；诱导后细胞免疫荧光检查雪旺细胞特异性标记物S100的表达为阳性；流式细胞检测结果亦显示雪旺细胞特异性标记物S100，GFAP和p75阳性表达，阳性率分别为58.64±4.38%、47.38±0.84%和44.33±2.39%；三阳性百分比约为27.89±5.98%。【结论】对传统的pre-plating差速贴壁法加以改良，应用显微外科技术，从小鼠骨骼肌组织中分离、纯化获得活性良好、纯度较高的肌源干细胞；在明确小鼠雪旺细胞条件培养液能成功诱导小鼠肌源干细胞分化为类雪旺细胞的前提下，进一步明确小鼠肌源干细胞分化为类雪旺细胞的分子机制，从而更准确的探讨肌源干细胞的分化条件；为雪旺细胞作为种子细胞提供更广阔的选择空间；为周围神经损伤修复的临床治疗和实验室研究提供更多的参考。更多还原

【Abstract】 【Background】peripheral nerve injury is a very frequent diseases in clinicalsurgery. Clinicians and scientist devoted to improve their surgical technique and method torepair injured peripheral nerves. But the result is still disaffect with regard to those severenerve stem injury and the functional rehabilitation after treatment; autoallergic nervetransplantation usually caused sensory disorders and motor dysfunction; And it is very toobtain enough donor tissue and allotransplantation faced with immunologic rejection.Therefore, Tissue Engineering become to be the hotspot. Schwann cell is a kind of TissueEngineering cell which is effective and well studied. Schwann cells can survive,proliferate, differentiate and secrete kinds of neurotrophic factors,cellular adhesionmolecules and extracellular matrix to promote the regeneration of axon and themedullation after transportation, thereby promote the restoration of peripheral nerves.However as seed cells, Schwann cells have a very limited differentiation potency，theyare terminal-stage cells and hard to obtain. Therefore we need another kind of cell which isyounger and could differentiate to schwann cells. According to the papers, many kinds ofstem cells could differentiate to schwann cells include bone marrow mesenchyma stemcells（BMSCs），bone marrow stroma stem cells(MSCs)，adipose-derived stem cells(ADSCs)， nerve stem cells（NSCs）and etc.Muscle drived stem cells（MDSCs）reside in skeletal muscles，and could differentiateto muscle cells, hematopoietic cells, osteoblasts, chondrocytes,endotheliocytes,endotheliocytes, hepatocytes; nerve cells and ect. But we had hardly any heard about thenews that MDSCs induced to schwann cells. If we could successfully induce MDSCs toschwann cells-like, it will be a new thread and provide a new choice for peripheral nerveinjury treatment of peripheral nerve injury.【Objective】(1)To master the method and technique of digestion, isolation,cultivation and identification of mouse muscle drived stem cells;(2)prepare Schwann cell conditioned medium, make sure that it could induce mouse MDSCs to schwann cells-like;(3) to detect the expression of several kinds of NTFs in Schwann cell conditioned mediumaccording to our available condition and relevant papers；choose the higher-express onesto induce MDSCs;(4)mouse MDSCs were induced by NTFs,then morphocytology,immunohistochemistry and flow cytometry was detected to make sure that the cells afterinduction are schwann cells-like; to provide a new alternative choise for laboratory studiedand clinical treatment.【Methods】(1) obtain the pure mouse skeletal muscle tissue under microscope，usethe enzyme digestion and improved pre-plating technique to digest, isolate and purify thetissue,when we got the cells（tSCs）, use trypan blue staining to evaluate the activity ofthem;observe the appearance and growth of tSCs；then immunohistochemistry and flowcytometry was detected to make sure that the cells after induction are MDSCs use theMarker（sSca-1，Desmin）of mouse MDSCs;（2）Schwann cells were isolated from mousesciatic nerve and Dorsal Root Ganglia（DRG），and were identified byimmunohistochemistry and flow cytometry method use the commonly used makers（S100,GFAP,p75） of Schwann cell. Schwann cell conditioned medium was obtainedby half-harvest method;(3) Detect the expression of NTFs in Schwann cell conditionedmedium culture with ELISA method and choose the higher express one induce mouseMDSCs;(4) Induce mouse MDSCs with NTFs by add it in stem cell growth medium; thenobserve the appearance and growth of tSCs； immunohistochemistry and flow cytometrywas detected to evaluate the cells after induction use the commonly used makers（S100,GFAP,p75） of Schwann cell.【Results】(1) trypan blue staining showed that the activity of MDSCs was95%；mouse MDSCs under inverted microscope were little round-shaped or shotspindle-shaped; differentiation didn’t happen and differentiation is fast; thenimmunohistochemistry showed that Sca-1and Desmin were both positive expressed；theflow cytometry showed that the positive rate of Sca-1and Desmin expression was93.23±0.93%and94.18±0.38%，Double positive rate was90.1±1.28%；（2）mouseMDSCs was successfully induced to Schwann cells-like by Schwann cell conditionedmedium，after72h induction，the cells presented spindle or trabs shape and appeared synapses in both side；immunohistochemistry showed that S100was positive expressed；the flow cytometry showed that S100，GFAP and P75was positive expressed，thepositive rate was65.48±6.20%、39.84±1.66%and41.08±0.78%respectively，the triplepositive rate was about25.86±5.37%；(3) the ELISA detection showed that among theNTFs，besides FGF and GDNF were undetected, NGF, BDNF, NT-3,PDGF and IGF-2were expressed in different level，so we decided to induce MDSCs with these five NTFs;(4)After72h induction by NT-3(500pg/ml), PDGF(1000pg/ml) and IGF-2(200pg/ml), thecells presented spindle or trabs shape and appeared synapses in both side；immunohistochemistry showed that S100was positive expressed；the flow cytometryshowed that S100，GFAP and P75was positive expressed，the positive rate was58.64±4.38%、47.38±0.84%and44.33±2.39%respectively，the triple positive rate wasabout27.89±5.98%.【Conclusions】(1)we obtained high-activity and high-purity MDSCs from mouseskeletal muscle tissue through improved pre-plating technique and the use of microsurgicaltechnique；successfully induced mouse MDSCs to Schwann cells-like both with Schwanncell conditioned medium and NTFs(NT-3,PDGF and IGF-2),therefore provide a newalternative choise and for laboratory studied and clinical treatment of peripheral nerveinjury.更多还原