【作者】 刘洲；

【导师】 曾园山；

【作者基本信息】 中山大学 ， 人体解剖与组织胚胎学， 2013， 博士

【摘要】 本研究旨在研究督脉电针联合神经营养素-3(Neurotrophin3, NT-3)+维甲酸(Retinoid acid, RA)预诱导的MSCs(Bone marrow mesenchymal stem cells, MSCs)移植治疗溴化乙锭(Ethidium bromide, EB)诱导的大鼠脊髓脱髓鞘损伤，通过督脉电针提高病灶的NT-3浓度，促使经预诱导表达酪氨酸激酶受体C (tyrosine kinasereceptor C, TrkC)基因的MSCs移植后能更多地分化为少突胶质前体样细胞和少突胶质样细胞，参与髓鞘结构的重建，改善脊髓的神经传导功能。从而为MSCs移植治疗MS的神经替代机制提供实验依据，为临床应用提供治疗策略。为求证上述研究设想，现将实验内容分以下2个部分。1体外NT-3联合维甲酸预诱导MSCs表达TrkC的研究目的：用NT-3+RA预诱导MSCs，增加TrkC mRNA的转录，并翻译为TrkC蛋白。方法：贴壁法培养及纯化MSCs。分为对照组、NT-3组、RA组和NT-3+RA组对MSCs诱导分化。在预诱导1d、3d后提取RNA，反转录为cDNA后进荧光定量PCR反应检测TrkC mRNA的转录。用琼脂糖凝胶水平电泳检测MSCs TrkCmRNA Real-time PCR后的产物，原位杂交技术检测预诱导1d的TrkC mRNA在MSCs内的表达。免疫荧光细胞化学染色技术和Western blot检测预诱导1d、3d、7d TrkC蛋白在MSCs的表达。结果：NT-3+RA共同诱导后1d后就可以观察到TrkC mRNA相对表达量(3.54±0.87)明显升高，高于同一时间点内的对照组(1.20±0.39)，RA组(2.07±0.45)及NT-3组(1.24±0.13)，P<0.05；而且也高于3d后的RA组(2.15±0.81)、NT-3组(1.75±0.57)及NT-3+RA组(2.43±0.43)，P<0.05。琼脂糖凝胶水平电泳可见229bp的TrkC mRNA cDNAReal-time PCR扩增产物存在。原位杂交技术进行检测，发现对照组、RA组、NT-3组及NT-3+RA组MSCs均检测到TrkC mRNA转录。然而免疫荧光细胞化学染色技术和Western blot检测未能发现TrkC蛋白在MSCs表达。结论：NT-3+RA预诱导24h后可以明显增加MSCs TrkC mRNA转录，然而未能发现TrkC蛋白在MSCs表达。2`督脉电针与NT-3和RA预诱导的MSCs移植联合治疗EB诱导的大鼠脊髓脱髓鞘损伤模型目`的：利用预诱导1d的MSCs移植到EB诱导的大鼠脊髓脱髓鞘损伤模型，并联合督脉电针治疗，提高病灶处的NT-3水平，促使移植的MSCs更多地分化为少突胶质前体样细胞和少突胶质样细胞，参与髓鞘结构的重建，改善脊髓的神经传导功能。方`法：贴壁法培养及纯化带绿色荧光蛋白MSCs，NT-3+RA预诱导1d。应用SD成年雌鼠，注射EB制备脱髓鞘模型。分为7组：Sham组、1天EB组和EA组，4天EB组和EA组，14天EB组和EA组，用ELISA试剂盒检测脱髓鞘脊髓组织的内源性NT-3水平。另取动物造模后随机分为6组：EB组、EA组、MSCs组、NR-MSCs组、NR-MSCs+EA组和NR-MSCs+NP组。造模电针3d后，在脊髓脱髓鞘部位移植预诱导的MSCs。其中EA组、NR-MSCs+EA组和NR-MSCs+NP组在细胞移植后第2d开始进行电针治疗，隔天1次，持续29d。并且从移植后1d开始，每隔1d进行平衡木评分。29d后进行运动诱发电位的检查，然后分别灌注取出脊髓组织固定进行冰冻切片，进行免疫组化分析，观察TrkC、髓鞘碱性蛋白（BMP）的表达，计算MSCs存活数，硫酸软骨素蛋白多糖（NG2）和成熟少突胶质细胞标志物(APC)的阳性分化率。及固定后进行半薄切片髓鞘计数、透射电镜观察及超薄切片免疫电镜观察MSCs的分化。结果：督脉电针治疗14d后，EA组的NT-3浓度明显高于EB组。MSCs组、NR-MSCs组、NR-MSCs+EA组和NR-MSCs+NP组的MSCs细胞存活数差异无统计学意义。MSCs移植到体内后能表达TrkC蛋白。NR-MSCs+EA组的NG2阳性率和APC阳性率明显升高，差异有统计学意义。在NR-MSCs+EA组和NR-MSCs+NP组可见GFP阳性细胞与MBP阳性标记和Hoechest33342标记的细胞核重合，在前者，还可以观察到MBP+GFP+Hoechest33342阳性标记的细胞包绕宿主的NF神经纤维。GFP免疫电镜法观察可见NR-MSCs+EA组脊髓脱髓鞘区内MSCs分化为少突胶质样细胞并形成髓鞘包绕轴突。髓鞘计数情况，NR-MSCs+EA组溃变髓鞘明显减少，新生髓鞘和正常髓鞘明显增多，差异有统计学意义。皮质运动诱发电位，NR-MSCs+EA组潜伏服期明显缩短，峰峰值明显增加，差异有统计学意义。6组动物平衡木评分没有差异。结论：督脉电针治疗14d能明显提高EB脱髓鞘大鼠模型脊髓内的NT-3浓度。督脉电针与NT-3+RA预诱导的MSCs移植联合治疗EB脱髓鞘大鼠模型对MSCs存活没有明显影响，但能明显促进MSCs更多的向少突胶质前体样细胞及少突胶质样细胞分化，并形成髓鞘包绕轴突。联合治疗还能减少髓鞘溃变，促进更多的新生髓鞘形成，保留更多的正常髓鞘，改善脊髓神经传导功能，但对EB脱髓鞘大鼠的运动功能疗效不明显。更多还原

【Abstract】 The principal aim of this research is to explored whether Government vesselelectro-acupuncture(EA) combined with neurotrophin-3(NT-3)+retinoic acid(RA)pre-induced bone marrow mesenchymal stem cells (MSCs) transplantation treatmentcould promote differentiation of MSCs into oligodendrocyte precursor-like cells andoligodendrocyte-like cells in demyelinated spinal cord injury induced by ethidiumbromide(EB) through up-regulating the expression of TrkC in MSCs and increasingthe concentration of NT-3in injury area, and then to participate in the myelin structurereconstruction, improve the nerve conduction of spine cord. It will provide theexperimental evidence for the mechanism of neuronal cell replacement of MSCs andtherapeutic strategie for the clinical application.In order to validate the suppositions, the whole research was divided into two partsas follows: Part1: The study of TrkC expression in MSCs pre-induced withNT-3and RA in vitroObjective: To up-regulate the transcription of TrkC mRNA and translation of TrkCprotein in MSCs by pre-induced with NT-3+RA.Methods: MSCs were isolated, cultured and purified in vitro by adherent culture. Theexperiment groups are designed as control group, NT-3group, RA group andNT-3+RA group. Total mRNA in MSCs was extracted by Trizol, and reversetranscribed to cDNA at1day and3day after induced. The expression of TrkC mRNAwas detected by real-time PCR. The cDNA productions of the real-time PCR wereverified by agarose gel horizontal electrophoresis. The TrkC mRNA in MSCs after1dinduced was detected by in situ hybridization. The expression of TrkC protein inMSCs was validated by immunofluorescence staining and Western blot after1d,3dand7d induced.Results: The relative expression of TrkC mRNA in NT-3+RA group(3.54±0.87) washigher than control group(1.20±0.39)， RA group (2.07±0.45) and NT-3group(1.24±0.13) after1day induced(P<0.05). The cDNA productions of TrkC mRNAReal-time PCR were found by agarose gel horizontal electrophoresis. TrkC mRNAcould be found in the MSCs by in situ hybridization. The expression of TrkC proteinin MSCs was not detected by immunofluorescence staining and Western blot.Conclusions: The transcription of TrkC mRNA in MSCs was increased obviouslyafter1day pre-induced with NT-3+RA. However, the TrkC protein was not detected. Part2: Governor Vessel electro-acupuncture combined withneurotrophin-3and retinoic acid pre-induced MSCstransplantation treatment the rat demyelinated spinal cordinjury induced by EBObjective: To study whether MSCs transplantation, pre-induced with NT-3+RA1day, combined with Governor Vessel electro-acupuncture treatment, whichup-regulated the expression of NT-3in injury area of the rat demyelinated spinal cordinjury induced by EB, could promote the differentiation of MSCs intooligodendrocyte precursor-like cells and oligodendrocyte-like cells to participate inthe myelin structure reconstruction and improve the nerve conduction of spine cordand locomotion.Methods: GFP-MSCs were isolated, cultured and purified in vitro by adherent culture,and then pre-induced with NT-3+RA for1day. Demyelinated model of SD femaleadult rats were induced by EB injection and part of them were divided into7groups:sham group, one-day EB group and EA group, four-day EB group and EA group,14-day EB group and EA group. The concentration of NT-3was detected by ELISAmethod. The other model animals were divided into6groups at random: EB group,EA group, MSCs group, NR-MSCs group, NR-MSCs+EA group and NR-MSCs+NP group. The pre-induced MSCs were transplanted into the lesion site of the modelanimal3days after the modeling and Governor Vessel EA treatment. The animals inEA group, NR-MSCs+EA group and NR-MSCs+NP group were received GVelectro-acupuncture treatment once every other day after2day the transplantationperformed and last for29days. Behavior score evaluation (Balance Beam Score) wastested every other day. After the cortical motor evoked potential were detected, theanimals were perfuse and fix for frozen sections and immunofluorescence. Theexpression of TrkC and BMP in MSCs were evaluated. The survival rate of MSCs, thepositive rate of NG2and APC of MSCs were counted. Some animals were perfuseand fix for semi-thin sections to count the number of the myelin and differentiation ofMSCs by immune electron microscopy. Results: The concentration of NT-3was significantly higher in EA group after14-daytreatment. The survival rate of MSCs had no statistical significance betweenNR-MSCs group, NR-MSCs+EA group and NR-MSCs+NP group. The TrkC proteinin MSCs could be detected in vivo. The NR-MSCs+EA group exhibited higherNG2-positive rate and APC-positive rate and the difference was statisticallysignificant. In NR-MSCs+EA group and NR-MSCs+NP group, some cells weremarked by MBP, GFP and hocest33342simultaneously. Immune electron microscopyshowed that GFP-MSCs could differentiate into oligodendrocytes-like cells andformed myelin sheaths to wrap axon. Less degenerated myelin, more newborn myelinand normal myelin showed in NR-MSCs+EA group and the difference werestatistically significant. The NR-MSCs+EA group had shorter latency and higheramplitude in Cortical motor evoked potentials and the difference was statisticallysignificant. The Balance Beam Score had no statistical significant in all groups.Conclusion: Governor Vessel electro-acupuncture could increase the concentrationof NT-3in lesion site of the rat with spinal cord demyelination induced by EBinjection. Combined government vessel electro-acupuncture with MSCstransplantation pre-induced with NT-3+RA could promote differentiation of MSCsinto oligodendrocyte precursor-like cells and oligodendrocyte-like cells, and then toparticipate in the myelin structure reconstruction and improve the nerve conduction ofspine cord. But the combination therapy had no effect on the survival rate of MSCsand the recovery of locomotion.更多还原