【作者】 任志午；

【导师】 卢世璧；

【作者基本信息】 南开大学 ， 外科学， 2012， 博士

【摘要】 目的在建立大鼠周围神经损伤模型的基础上，研究Wallerian变性与干细胞归巢之间的关系，周围神经损伤后干细胞向神经断端归巢的现象及潜在机制，以及干细胞归巢后的转归，为周围神经损伤的修复探索新的思路。利用蛋白组学技术建立感觉神经和运动神经的双向电泳（2-DE）图，比较周围感觉神经和运动神经内蛋白质的表达差异，为周围神经损伤后趋化性再生的机制研究提供依据。方法1.使用PCR，Western blot，免疫荧光等方法鉴定趋化因子SDF-1在Wallerian变性的损伤神经远端及其特异性受体CXCR4在BMSCs中的表达情况。2.用Transwell细胞迁移模型，检验离断后发生Wallerian变性的远端神经组织匀浆对BMSCs迁移的趋化作用，使用SDF-1/CXCR4轴特异性阻断剂AMD3100检验SDF-1在其中扮演的角色。3.通过向大鼠坐骨神经切断/吻合模型的尾静脉注射移植表达RFP的BMSCs，模拟BMSCs向神经组织归巢的过程，使用荧光成像和病理学方法观察BMSCs是否通过机体血液循环归巢至损伤的神经组织局部，并进一步使用特异性阻断剂AMD3100验证SDF-1/CXCR4轴对BMSCs归巢的动员机制。4.建立大鼠坐骨神经节段性冰冻损伤和CEANA修复节段性缺损两种模型，将表达RFP的BMSCs添加到损伤的神经组织周围，观察BMSCs在神经损伤局部的迁移对神经修复的促进效果并探索其作用机制，重点观察干细胞在神经微环境中的分化和转归。5.正常雌性Wistar大鼠，深度麻醉后取双侧隐神经及股神经肌支，打开椎管及硬脊膜，显微镜下分离取出背侧神经根及腹侧神经根，分别提取蛋白质。在蛋白质充分匀浆、超声裂解并定量后，在相同条件下，对每组蛋白质样品进行3次双向电泳，对重复3次实验的隐神经与股神经肌支，背侧神经根与腹侧神经根两两进行分析，比较两对感觉和运动神经电泳图中的蛋白质表达的变化。将差异表达的蛋白质点从凝胶上切下来并溶解为多肽，使用纳升超高效液相色谱－电喷雾串联质谱（nanoUPLC-nano-ESI-MS/MS）进行分析，根据所得的质谱数据通过Mascot搜索引擎进行蛋白质查询鉴定。结果1.切断后1d、3d和7d组发生Wallerian变性的远端组织SDF-1在mRNA的表达均明显高于正常对照组。CXCR4在分离培养扩增后的BMSCs和表达RFP的BMSCs中均稳定表达。2. Wallerian变性的神经远端组织匀浆可以在体外Transwell模型中吸引BMSCs的迁移，该趋化作用可以被SDF-1/CXCR4轴AMD3100阻断。3.尾静脉注射RFP-BMSCs向切断并吻合后发生Wallerian变性的神经组织局部迁移。Wallerian变性的神经对BMSCs的趋化作用，可以被SDF-1/CXCR4轴阻断剂AMD3100阻断。4.在节段性冰冻损伤神经或移植的CEANA内部或周围移植RFP-BMSCs后12周，再生神经纤维间均有RFP-BMSCs分布，并显示Nestin、S100、P0阳性。在节段性冰冻损伤的神经、桥接节段性神经缺损的CEANA内注射或周围局部添加BMSCs，可以提高小腿三头肌收缩力和湿重的恢复率，缩短CMAPs潜伏期，增高CMAPs波幅，增加神经有髓纤维密度以及髓鞘的厚度，促进周围神经损伤修复。5.应用双向电泳体系，成功建立周围感觉神经和运动神经蛋白质组双向电泳图谱，隐神经与股神经肌支，背侧神经根与腹侧神经根等四种神经双向电泳图蛋白匹配率均>80%。通过比较双向电泳图，共计有13个蛋白质斑点在隐神经与股神经肌支的蛋白质组中差异表达，其中隐神经表达水平较股神经肌支高的有8个点，股神经肌支较隐神经表达水平高有5个点；共计有16个蛋白质斑点在腹侧神经根组与背侧神经根组表达差异，其中背侧神经根较腹侧神经根表达水平高的有6个点，表达水平低的有10个点。将选取的29个蛋白差异点进行nanoUPLC-nano-ESI-MS/MS分析得到质谱数据并在Mascot搜索引擎中查询并排除角蛋白污染后，共成功鉴定隐神经与股神经肌支的11个蛋白差异点（涉及7种蛋白），以及腹侧神经根与背侧神经根的9个蛋白差异点（涉及7种蛋白）。结论Wallerian变性的周围神经组织在体内和体外，对BMSCs均有趋化动员作用。SDF-1/CXCR4轴在Wallerian变性神经组织对BMSCs的趋化动员过程中发挥重要作用。在节段性损伤的神经或桥接节段性神经缺损的CEANA周围局部添加BMSCs，BMSCs可以迁移到再生神经内，并向神经干细胞及雪旺细胞方向分化，通过促进髓鞘再生和引导轴索生长，达到与直接向神经内注射BMSCs相当的修复效果。成功构建大鼠周围感觉神经与运动神经双向电泳图，并联合使用质谱技术鉴定了其中的大部分蛋白质。这些蛋白质有可能成为区分感觉和运动神经的特异性标记，同时可能在周围神经损伤后的趋化性再生过程中发挥重要作用。更多还原

【Abstract】 OBJECTIVE: To observe the homing and migration of bone marrow derivedstem cell to Wallerian degenerated peripheral nerve；To investigate the characteristicsand mechanism of the phenomenon，especially the fate of migrated stem cell. Todetect differential protein expression in rat between saphenous nerve and motorbranch of femoral nerve, as dorsal root and ventral root, using two-dimensional gelelectrophoresis and mass spectrum and to determine specific biological markers ofsensory and motor nerve.METHODS:1. We measured the expression of CXCR4in BMSCs and theexpression of SDF-1in degenerated rat sciatic nerve with RT-PCR, Western blot,immunofluorescence methods.2. By using Transwell in vitro migration assay system, the effects of SDF-1andtissular extract from normal and degenerated rat sciatic nerve on BMSCs’ migrationwere observed, the influence of AMD3100treatment on BMSCs’ migration inducedby nerve tissular extract were also evaluated.3. The RFP transfected BMSCs were injected through tail vein into the SD ratswith sciatic nerve cut and anastomosised, and the migration of BMSCs to the injurednerve3days later. The influence of AMD3100treatment on BMSCs’ migrationinduced by degenerated nerve, were also evaluated.4. The RFP transfected BMSCs were injected in or applied around the segmentalfreeze-treated nerve or CEANA bridged to the segmental defect of rat sciatic nerve.12weeks later, the fate of transplanted RFP-BMSCs was observed and its effect ofpromoting nerve regeneration and repair was evaluated through functional assessmentand histological observation.5. A total of9Wistar rat were sacrificed. The segments of bilateral motorbranches to the quadriceps muscles and the saphenous nerves, ventral roots and dorsal roots of spinal at the level of L1~5were collected, separately. Total protein wasextracted from four kinds of nerve tissue, followed by two-dimensional gelelectrophoresis and silver staining, and the differential protein expression wasanalyzed using imagemaster2D platinum software. Protein peptide massspectrometry data of differential protein spots was obtained by nano ultra highperformance liquid chromatography electrospray ionization mass spectrometrytandem mass spectrometry. The National Center for Biotechnology Information(NCBI) protein database was retrieved by Mascot to identify protein type.RESULT:1. Western blot and Immunofluorescence analysis revealed thatBMSCs were CXCR4positive. The mRNA of CXCR4in BMSCs and SDF-1inWallerian degenerated nerve were successfully reversely transcribed by RT-PCR.2. Wallerian degenerated nerve tissular extract can induce the migration ofBMSCs, and this effect can be suppressed by AMD3100which is a blocker ofCXCR4.3. Injured and Wallerian degenerated nerve can induce the migration oftransplanted BMSCs, and this effect can be suppressed by AMD3100which is ablocker of CXCR4.4. The RFP-BMSCs transplanted around the freezed nerve or CEANA hadmigrated into regenerated nerve, with Nestin, S100, P0positive, as the cells directlyinjected into the freezed nerve or CEANA. Transplanting BMSCs in or around thefreezed nerve or CEANA, had equally increased contraction of triceps muscle,recovery rate of muscle, the number of sciatic nerve myelinated fibers and myelinthickness.5. Compared with the motor branches of femoral nerve,13protein spotsexhibited obviously differential expression in saphenous nerves (P<0.05). Comparedwith in ventral roots,16protein spots exhibited obviously differential expression indorsal roots (P<0.05). A total of26out of29protein spots with obvious differentialexpression were identified successfully, with peptide of matching score>34(P <0.05). Protein types were identified and included transgelin, Ig kappa chain precursor,plasma glutathione peroxidase precursor, aldose reductase, a glyceraldehyde- 3-phosphate dehydrogenase-like protein, lactoylglutathione lyase, adenylate kinaseisozyme1, Pol(yrC)-binding protein1, rCG31027and two unnamed protein product(gi|55628and gi|1334163).CONCLUSION: Wallerian degenerated nerve can induce the migration ofBMSCs, SDF-1/CXCR4axis play an important role in this process.Compared to directly injected BMSCs into acellular nerve, BMSCs suppliedaround injured nerve have the similar effect, by differentiated into Schwann cell andpromoting myelin formation.Through the proteomics comparison with2DGE andnanoUPLC-nano-ESI-MS/MS analysis between motor branch of femoral nerve andsaphenous nerve, as ventral roots and dorsal roots, a few proteins were found for thefirst time to be differentially expressed in sensory and motor nerve. These proteinsmay play roles in specific nerve regeneration.更多还原