Mash1基因过表达的骨髓间充质干细胞治疗癫痫的实验研究
发布时间：2018-08-22 19:34
【摘要】：癫痫是发病率最高的中枢神经系统功能障碍，表现为慢性、反复发作的大脑神经元异常放电。世界卫生组织（World Health Organization, WHO）的数据显示，全球癫痫患者约为五千万至一亿，并且全球约5%的人群一生中具有罹患癫痫的风险。癫痫患者中有约30%行药物治疗效果欠佳，而药物治疗之外的手术治疗以及神经电刺激治疗（迷走神经电刺激术（vagus nerve stimulation, VNS）、脑深部电刺激治疗（deepbrain stimulation, DBS））等并不适合于所有癫痫患者特别是无手术指征者，因此，有必要寻找更有效、安全、持久的新治疗方法。 脑内伽马氨基丁酸（gamma-amino butyric acid, GABA）能神经元减少会导致癫痫是目前了解的可能核心机制。研究显示，源自不同类别干细胞（胚胎干细胞（embryonic stem cells, ESCs）、神经干细胞（neural stem cells, NSCs）、间充质干细胞（mesenchymal stem cells, MSCs）等）的GABA能神经元可以抑制痫性发作、延长模型动物生存期，因此，大量学者把目光聚焦于基于干细胞的癫痫治疗策略。但细胞来源无法满足需求成为个中羁绊。 间充质干细胞因具有自我更新和多向分化潜能的特性使其成为细胞治疗策略的优良候选细胞并逐渐成为热点。作为间充质干细胞中的一员，骨髓间充质干细胞（bone morrow mesenchymal stem cells, BMSCs）来源于骨髓，它在人体内分布广泛，易于提取、分离，而且在应用于自体时不存在免疫排斥以及伦理学障碍，有研究显示BMSCs移植治疗可以抑制癫痫动物模型的自发性痫性发作（spontaneous recurrentseizure, SRS）的频率。使用其进行移植治疗并使之与宿主神经元形成突触联系帮助其功能重建是有可能实现的。但针对在癫痫模型中起关键作用的GABA能神经元的间充质干细胞来源的特异性分化以及用其治疗癫痫以促进疗效的研究很少。尽管有研究显示BMSCs可以经条件诱导培养向GABA能神经元样细胞分化，但需使用化学成分（如氯化钾（potassium chloride，Kcl），β-巯基乙醇（β-mercaptoethanol, BME），维甲酸（retinoic acid， RA）等）或细胞因子（如碱性成纤维细胞生长因子）等，上述方案如果应用于体内则不仅难以实现而且可能产生无法预期的副作用。 碱性螺旋-环-螺旋（basic helix-loop-helix, bHLH）基因在干细胞的增殖与分化过程中起着重要的作用，其中，促分化转录因子哺乳动物无刚毛-鳞甲同源物（mammalian achaete-scute homologue，Mash1）基因作为bHLH家族中的一员，它不仅启动神经干细胞分化成神经元的过程，而且同时决定着神经元亚型的形成，与GABA能神经元的分化形成有着密切的关系。本研究假设，过表达BMSCs中的Mash1基因将可促进其向GABA能神经元样细胞分化。本研究采用慢病毒转染技术使BMSCs过表达Mash1基因，通过条件诱导培养技术诱导分化为GABA能神经元样细胞，对比BMSCs和Mash1过表达的BMSCs向GABA能神经元样细胞分化的比率以验证假设，并且将Mash1过表达的BMSCs移植入癫痫大鼠模型体内，观察其是否具有更进一步抑制癫痫的治疗效果。 第一部分大鼠来源的骨髓间充质干细胞的提取、体外分离、培养和鉴定 目的：建立成熟高效的提取、分离与鉴定BMSCs的方法。 方法：无菌手术取材2~3周龄大鼠的双侧股骨与胫骨，提取骨髓，分别使用全骨髓贴壁培养法和应用Histopaque分离液的密度梯度离心分离法分离细胞并培养，采用流式细胞仪鉴定细胞表面分子标记并通过成脂肪、软骨、骨诱导分化鉴定其多向分化潜能，使用噻唑蓝（3-（4，5-dimethylthiazol-2-yl）-2，5-diphenyltetrazoliumbromide，MTT）检测增殖曲线。 结果：与全骨髓贴壁培养法相比，密度梯度分离法获得的BMSCs杂质细胞少、细胞形态更标准并且均一，增殖曲线标准（接种第1~3天处于潜伏期，随后进入对数生长期，，至第7~9天时进入平台期），同时细胞表面分子标记表达（密度梯度分离法提取并传代得到的用于本实验研究的P3（passage3）代BMSCs低表达CD34抗原（（1.79±0.23）%，图4-A）并且高表达CD105抗原（（98.6±0.68）%，图4-B）、CD90抗原（（98.5±1.02）%，图4-C）和CD73抗原（（98.8±0.98）%，图4-D）及多向分化潜能均符合国际统一规范。 结论：密度梯度离心分离法可以高效、便捷地获得纯度高、扩增速度快、定向分化能力明确的符合国际统一规范的BMSCs，可以为下一步实验研究提供优良的细胞材料。 第二部分过表达Mash1基因促进骨髓间充质干细胞成GABA能神经元分化 目的：研究Mash1基因过表达对BMSCs成GABA能细胞分化的作用。 方法：提取2~3周Sprague-Dawley（SD）大鼠的BMSCs，传至第3代；制备搭载Mash1基因的慢病毒载体，转染BMSCs使其Mash1基因过表达，采用复合诱导培养基（1×B27+1mM N6, O2二丁酰腺苷3’,5’-环磷酸(N6, O2’-dibutyryladenosine3’,5’-cyclicmonophosphate, Bt2cAMP)+1μMATRA+Neurobasal）体外诱导Mash1+-BMSCs和BMSCs成GABA能神经元分化并做免疫细胞化学染色、PCR检测和Western blot检测验证它们的分化趋势，同时行全细胞膜片钳检测了解分化细胞的电生理学特性。 结果：搭载Mash1基因的慢病毒转染（感染指数MOI=100）BMSCs后Westernblot检测结果显示Mash1表达获得显著提高；成GABA能神经元分化过程中Mash1+-BMSCs组（M-BMSCs）表现出更多的具有神经元形态特征的细胞；诱导分化后Western blot和RT-PCR检测显示Mash1+-BMSCs组的细胞表达神经元特异性核蛋白（neuron-specific nuclear protein，NeuN）和谷氨酸脱羧酶（glutamic aciddecarboxylase，GAD）67的水平显著高于对照组细胞；分化后细胞的细胞免疫荧光检测显示M-BMSCs组（71.6±2.8）%中GAD67免疫荧光阳性比率明显高于BMSCs组（61.4±5.3）%、Lv-con-BMSCs组（58.6±5.4）%和M-con-BMSCs组（0），并且，M-BMSCs组（72.0±2.0）%中GABA免疫荧光阳性比率明显高于BMSCs组（61.4±2.9）%、Lv-con-BMSCs组（62.4±5.5）%和M-con-BMSCs组（0）；全细胞膜片钳检测到成神经元分化的细胞具有动作电位和自发性抑制性突触后电位提示其具有功能性。 结论：慢病毒介导的基因过表达具有良好的效果；Mash1基因过表达可以促进BMSCs向具有电生理活性的GABA能神经元分化。 第三部分Mash1基因过表达的骨髓间充质干细胞对大鼠癫痫模型的治疗研究 目的：研究Mash1基因过表达的BMSCs（M-BMSCs）对癫痫模型大鼠的治疗作用。 方法：60只SD大鼠行水迷宫训练形成空间记忆后使用匹鲁卡品诱导制作癫痫模型并入组达到RacineⅤ级发作的大鼠；使用BrdU体外标记M-BMSCs和BMSCs并通过立体定向联合微量注射技术将M-BMSCs（M-BMSCs组）、BMSCs（BMSCs组）或大鼠成纤维细胞（阳性对照组）注射入癫痫大鼠右侧脑室完成细胞移植（细胞数量5×105个/只）；其后选取不同时间点（移植后第7，14，21，28天）进行功能学（水迷宫检测、自发性癫痫发作频率监测）、电生理（EEG）以及免疫组织化学（石蜡切片免疫荧光染色、尼氏染色、二氨基联苯胺（diaminobenzidine，DAB）染色）检测研究大鼠空间记忆能力、脑电图变化和神经元细胞层面在细胞移植之后产生的变化。 结果：细胞移植治疗对大鼠因癫痫导致的空间记忆能力损害具有保护作用，在移植第7天细胞移植的保护作用开始出现，M-BMSCs组的保护作用的特点是出现早、效果明显（逃避潜伏期时间恢复至接近正常大鼠水平），相比较而言，BMSCs组的效果与M-BMSCs组存在差异（P0.05）；细胞移植治疗不仅可以降低大鼠造模后的死亡率（虽然各组数据间无统计学差异，但M-BMSCs组和BMSCs组的死亡数量仍是低于阳性对照组的），而且可以减低SRS的发生（虽然统计分析未见明显差异，但是在M-BMSCs组和BMSCs组仍可见SRS频率的减少），并且在EEG电生理水平也表现出促进恢复的作用：细胞移植治疗后，与阳性对照组相比，M-BMSCs组和BMSCs组的棘波和棘慢波的数量显著减少（P0.05），而且M-BMSCs组的效果优于BMSCs组；免疫荧光双标检测发现M-BMSCs组的大鼠脑内海马旁皮层NeuN+/BrdU+共表达细胞和GAD67+/BrdU+共表达细胞比率高于BMSCs组大鼠；尼氏染色计数海马旁皮层空泡核细胞（神经元）密度结果显示M-BMSCs组大鼠的神经元密度显著高于对照组（14D和28D，P0.05）；DAB染色定位移植细胞的迁移显示，移植细胞在海马和海马旁皮层区域广泛分布，提示了移植细胞促进癫痫大鼠功能恢复的结构基础。 结论：Mash1基因过表达的BMSCs移植治疗能够促进癫痫模型大鼠的功能恢复并且作用优于BMSCs移植。
[Abstract]:Epilepsy is the highest incidence of central nervous system dysfunction, characterized by chronic, recurrent abnormal discharge of brain neurons. World Health Organization (WHO) data show that about 50 million to 100 million people worldwide with epilepsy, and about 5% of the world's population has a lifetime risk of epilepsy. About 30% of epilepsy patients were not treated well with drugs. Surgical treatment, vagus nerve stimulation (VNS) and deep brain stimulation (DBS) were not suitable for all epilepsy patients, especially those without surgical indications. Find a more effective, safe and lasting new treatment.
Reduced gamma-amino butyric acid (GABA) neurons in the brain can lead to epilepsy, which is a potential core mechanism. Studies have shown that it is derived from different types of stem cells (embryonic stem cells, ESCs), neural stem cells (NSCs), mesenchymal stem cells (MSCs). (3) GABAergic neurons can inhibit seizures and prolong the survival of model animals. Therefore, many researchers focus on stem cell-based epilepsy therapy strategies. However, the lack of cell sources to meet the needs becomes a constraint.
As a member of mesenchymal stem cells, bone marrow mesenchymal stem cells (BMSCs) are derived from bone marrow, which are widely distributed in human body and easy to be extracted. There is no immune rejection or ethical barrier in autologous application. Studies have shown that BMSCs transplantation can inhibit the frequency of spontaneous epileptic seizures (SRS) in animal models of epilepsy. Reconstitution is possible. However, few studies have focused on the specific differentiation of GABAergic neurons derived from mesenchymal stem cells, which play a key role in epilepsy models, and the use of BMSCs for treatment of epilepsy to promote efficacy. Potassium chloride (Kcl), beta-mercaptoethanol (BME), retinoic acid (RA) or cytokines (basic fibroblast growth factor) are the main components of this regimen. These regimens are not only difficult to achieve in vivo, but also may produce unexpected side effects.
The basic helix-loop-helix (bHLH) gene plays an important role in the proliferation and differentiation of stem cells. As a member of the bHLH family, the mammalian achaete-scute homologue (Mash1) gene not only initiates the differentiation of neural stem cells, but also promotes the differentiation of neural stem cells. This study hypothesized that overexpression of Mash1 gene in BMSCs would promote the differentiation of BMSCs into GABAergic neuron-like cells. Lentiviral transfection was used to overexpress Mash1 gene in BMSCs. Induction culture technique was used to induce differentiation into GABAergic neuron-like cells. The hypothesis was verified by comparing the ratio of BMSCs overexpressed by BMSCs and Mash1 to GABAergic neuron-like cells. BMSCs overexpressed by Mash1 were transplanted into epileptic rat models to observe whether they had better therapeutic effect on inhibiting epilepsy.
The first part is the extraction, isolation, culture and identification of rat bone marrow mesenchymal stem cells.
Objective: to establish a mature and efficient method for isolation, identification and identification of BMSCs.
METHODS: Bone marrow was extracted from bilateral femurs and tibia of 2-3 weeks old rats by aseptic operation. Cells were isolated and cultured by whole bone marrow adherent culture and density gradient centrifugation with Histopaque. Cell surface molecular markers were identified by flow cytometry and identified by adipogenesis, cartilage and bone induction. Multidimensional differentiation potential was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).
Results: Compared with the whole bone marrow adherent culture, BMSCs obtained by density gradient method had fewer impurity cells, more standard and uniform cell morphology, and standard proliferation curve (incubation 1-3 days, then into the logarithmic growth period, to 7-9 days into the plateau phase), and the expression of cell surface molecular markers (density gradient separation method extraction). CD34 antigen ((1.79 +0.23)%, Fig. 4-A) and CD105 antigen ((98.6 +0.68)%, Fig. 4-B, CD90 antigen ((98.5 +1.02)%, Fig. 4-C and C D73 antigen ((98.8 +0.98)%, Fig. 4-D) and multidirectional differentiation potential were all in accordance with the international standard.
CONCLUSION: Density gradient centrifugation is an efficient and convenient method to obtain BMSCs with high purity, rapid amplification and well-defined directional differentiation, which can provide excellent cell materials for the next experimental study.
Second, overexpression of Mash1 gene promotes the differentiation of bone marrow mesenchymal stem cells into GABA neurons.
Objective: To study the effect of Mash1 gene overexpression on the differentiation of BMSCs into GABA cells.
METHODS: BMSCs from Sprague-Dawley (SD) rats were extracted for 2-3 weeks and passed to the third generation. Lentiviral vector carrying Mash1 gene was prepared and transfected into BMSCs to overexpress Mash1 gene. The Mash1 gene was overexpressed in composite induction medium (1 *B27+1 mMN6, O2-dibutyryladenosine 3', 5'-cyclic monophosphate, Bt2cAMP) +1. Mash-1 +-BMSCs and BMSCs were induced to differentiate into GABA-ergic neurons by mu-MATRA+Neurobasal in vitro and immunocytochemical staining, PCR and Western blot were performed to verify their differentiation trend, and whole-cell patch-clamp assay was performed to investigate the electrophysiological characteristics of differentiated cells.
Results: Western blot and RT-PCR showed that the expression of Mash1 was significantly increased in BMSCs transfected with Mash1 gene (infection index MOI = 100), and more neuronal morphological cells were found in the Mash1 + - BMSCs group during the differentiation of GABAergic neurons. The expression levels of neuron-specific nuclear protein (NeuN) and glutamic acid decarboxylase (GAD) 67 were significantly higher in the sh1 + - BMSCs group than those in the control group, and the immunofluorescence assay of differentiated cells showed that the positive rate of GAD67 was significantly higher in the M-BMSCs group (71.6 [2.8]%). The positive rate of GABA immunofluorescence was significantly higher in BMSCs group (61.4+5.3)%, Lv-con-BMSCs group (58.6+5.4)% and M-con-BMSCs group (0), and in M-BMSCs group (72.0+2.0)% than that in BMSCs group (61.4+2.9)%, Lv-con-BMSCs group (62.4+5.5)% and M-con-BMSCs group (0). Inhibitory postsynaptic potentials suggest it is functional.
Conclusion: Lentivirus-mediated gene overexpression has a good effect, and Mash1 gene overexpression can promote BMSCs to differentiate into GABAergic neurons with electrophysiological activity.
The third part is the treatment of rat epilepsy model with Mash1 gene overexpressed bone marrow mesenchymal stem cells.
Objective: To study the therapeutic effect of Mash1 gene overexpression BMSCs (M-BMSCs) on epileptic rats.
METHODS: 60 SD rats were trained in water maze to form spatial memory, then induced by pilocarpine to form epileptic models and then incorporated into Racine V-grade seizure rats; M-BMSCs (M-BMSCs group), BMSCs (BMSCs group) or rat fibroblasts (positive) were labeled with BrdU in vitro and injected with BMSCs by stereotactic microinjection technique. The control group was injected into the right ventricle of epileptic rats to complete cell transplantation (5 *105 cells per rat), and then selected at different time points (7,14,21,28 days after transplantation) for functional (water maze test, spontaneous seizure frequency monitoring), electrophysiology (EEG) and immunohistochemistry (paraffin section immunofluorescence staining, Nissl staining, II). Diaminobenzidine (DAB) staining was used to study the spatial memory ability, electroencephalogram (EEG) changes and neuronal cell level changes after cell transplantation in rats.
Results: Cell transplantation had protective effect on spatial memory impairment induced by epilepsy in rats. The protective effect of cell transplantation began to appear on the 7th day after transplantation. The protective effect of M-BMSCs group was early and obvious (escape latency time was restored to near normal level). Compared with BMSCs group, the protective effect of BMSCs group was better. There was a significant difference between M-BMSCs group and M-BMSCs group (P 0.05). Cell transplantation therapy could not only reduce the mortality of rats after modeling (although there was no statistical difference among the groups, the mortality of M-BMSCs group and BMSCs group was still lower than that of the positive control group), but also reduce the incidence of SRS (although there was no statistical difference, but there was no significant difference in M-BMSCs). Compared with the positive control group, the number of spikes and slow waves in M-BMSCs group and BMSCs group was significantly decreased (P 0.05), and the effect of M-BMSCs group was better than that of BMSCs group. The ratio of NeuN+/BrdU+ co-expressing cells and GAD67+/BrdU+ co-expressing cells in the parahippocampal cortex of rats in MSCs group was higher than that in BMSCs group; the density of vacuolar nucleus cells (neurons) in the parahippocampal cortex of rats in M-BMSCs group was significantly higher than that in control group (14D and 28D, P 0.05). Cell migration showed that the transplanted cells were widely distributed in the hippocampus and paracortex, suggesting that the transplanted cells could promote the functional recovery of epileptic rats.
CONCLUSION: BMSCs transplantation with overexpression of Mash1 gene can promote the functional recovery of epileptic rats and is superior to BMSCs transplantation.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742.1
本文编号：2198100
[Abstract]:Epilepsy is the highest incidence of central nervous system dysfunction, characterized by chronic, recurrent abnormal discharge of brain neurons. World Health Organization (WHO) data show that about 50 million to 100 million people worldwide with epilepsy, and about 5% of the world's population has a lifetime risk of epilepsy. About 30% of epilepsy patients were not treated well with drugs. Surgical treatment, vagus nerve stimulation (VNS) and deep brain stimulation (DBS) were not suitable for all epilepsy patients, especially those without surgical indications. Find a more effective, safe and lasting new treatment.
Reduced gamma-amino butyric acid (GABA) neurons in the brain can lead to epilepsy, which is a potential core mechanism. Studies have shown that it is derived from different types of stem cells (embryonic stem cells, ESCs), neural stem cells (NSCs), mesenchymal stem cells (MSCs). (3) GABAergic neurons can inhibit seizures and prolong the survival of model animals. Therefore, many researchers focus on stem cell-based epilepsy therapy strategies. However, the lack of cell sources to meet the needs becomes a constraint.
As a member of mesenchymal stem cells, bone marrow mesenchymal stem cells (BMSCs) are derived from bone marrow, which are widely distributed in human body and easy to be extracted. There is no immune rejection or ethical barrier in autologous application. Studies have shown that BMSCs transplantation can inhibit the frequency of spontaneous epileptic seizures (SRS) in animal models of epilepsy. Reconstitution is possible. However, few studies have focused on the specific differentiation of GABAergic neurons derived from mesenchymal stem cells, which play a key role in epilepsy models, and the use of BMSCs for treatment of epilepsy to promote efficacy. Potassium chloride (Kcl), beta-mercaptoethanol (BME), retinoic acid (RA) or cytokines (basic fibroblast growth factor) are the main components of this regimen. These regimens are not only difficult to achieve in vivo, but also may produce unexpected side effects.
The basic helix-loop-helix (bHLH) gene plays an important role in the proliferation and differentiation of stem cells. As a member of the bHLH family, the mammalian achaete-scute homologue (Mash1) gene not only initiates the differentiation of neural stem cells, but also promotes the differentiation of neural stem cells. This study hypothesized that overexpression of Mash1 gene in BMSCs would promote the differentiation of BMSCs into GABAergic neuron-like cells. Lentiviral transfection was used to overexpress Mash1 gene in BMSCs. Induction culture technique was used to induce differentiation into GABAergic neuron-like cells. The hypothesis was verified by comparing the ratio of BMSCs overexpressed by BMSCs and Mash1 to GABAergic neuron-like cells. BMSCs overexpressed by Mash1 were transplanted into epileptic rat models to observe whether they had better therapeutic effect on inhibiting epilepsy.
The first part is the extraction, isolation, culture and identification of rat bone marrow mesenchymal stem cells.
Objective: to establish a mature and efficient method for isolation, identification and identification of BMSCs.
METHODS: Bone marrow was extracted from bilateral femurs and tibia of 2-3 weeks old rats by aseptic operation. Cells were isolated and cultured by whole bone marrow adherent culture and density gradient centrifugation with Histopaque. Cell surface molecular markers were identified by flow cytometry and identified by adipogenesis, cartilage and bone induction. Multidimensional differentiation potential was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).
Results: Compared with the whole bone marrow adherent culture, BMSCs obtained by density gradient method had fewer impurity cells, more standard and uniform cell morphology, and standard proliferation curve (incubation 1-3 days, then into the logarithmic growth period, to 7-9 days into the plateau phase), and the expression of cell surface molecular markers (density gradient separation method extraction). CD34 antigen ((1.79 +0.23)%, Fig. 4-A) and CD105 antigen ((98.6 +0.68)%, Fig. 4-B, CD90 antigen ((98.5 +1.02)%, Fig. 4-C and C D73 antigen ((98.8 +0.98)%, Fig. 4-D) and multidirectional differentiation potential were all in accordance with the international standard.
CONCLUSION: Density gradient centrifugation is an efficient and convenient method to obtain BMSCs with high purity, rapid amplification and well-defined directional differentiation, which can provide excellent cell materials for the next experimental study.
Second, overexpression of Mash1 gene promotes the differentiation of bone marrow mesenchymal stem cells into GABA neurons.
Objective: To study the effect of Mash1 gene overexpression on the differentiation of BMSCs into GABA cells.
METHODS: BMSCs from Sprague-Dawley (SD) rats were extracted for 2-3 weeks and passed to the third generation. Lentiviral vector carrying Mash1 gene was prepared and transfected into BMSCs to overexpress Mash1 gene. The Mash1 gene was overexpressed in composite induction medium (1 *B27+1 mMN6, O2-dibutyryladenosine 3', 5'-cyclic monophosphate, Bt2cAMP) +1. Mash-1 +-BMSCs and BMSCs were induced to differentiate into GABA-ergic neurons by mu-MATRA+Neurobasal in vitro and immunocytochemical staining, PCR and Western blot were performed to verify their differentiation trend, and whole-cell patch-clamp assay was performed to investigate the electrophysiological characteristics of differentiated cells.
Results: Western blot and RT-PCR showed that the expression of Mash1 was significantly increased in BMSCs transfected with Mash1 gene (infection index MOI = 100), and more neuronal morphological cells were found in the Mash1 + - BMSCs group during the differentiation of GABAergic neurons. The expression levels of neuron-specific nuclear protein (NeuN) and glutamic acid decarboxylase (GAD) 67 were significantly higher in the sh1 + - BMSCs group than those in the control group, and the immunofluorescence assay of differentiated cells showed that the positive rate of GAD67 was significantly higher in the M-BMSCs group (71.6 [2.8]%). The positive rate of GABA immunofluorescence was significantly higher in BMSCs group (61.4+5.3)%, Lv-con-BMSCs group (58.6+5.4)% and M-con-BMSCs group (0), and in M-BMSCs group (72.0+2.0)% than that in BMSCs group (61.4+2.9)%, Lv-con-BMSCs group (62.4+5.5)% and M-con-BMSCs group (0). Inhibitory postsynaptic potentials suggest it is functional.
Conclusion: Lentivirus-mediated gene overexpression has a good effect, and Mash1 gene overexpression can promote BMSCs to differentiate into GABAergic neurons with electrophysiological activity.
The third part is the treatment of rat epilepsy model with Mash1 gene overexpressed bone marrow mesenchymal stem cells.
Objective: To study the therapeutic effect of Mash1 gene overexpression BMSCs (M-BMSCs) on epileptic rats.
METHODS: 60 SD rats were trained in water maze to form spatial memory, then induced by pilocarpine to form epileptic models and then incorporated into Racine V-grade seizure rats; M-BMSCs (M-BMSCs group), BMSCs (BMSCs group) or rat fibroblasts (positive) were labeled with BrdU in vitro and injected with BMSCs by stereotactic microinjection technique. The control group was injected into the right ventricle of epileptic rats to complete cell transplantation (5 *105 cells per rat), and then selected at different time points (7,14,21,28 days after transplantation) for functional (water maze test, spontaneous seizure frequency monitoring), electrophysiology (EEG) and immunohistochemistry (paraffin section immunofluorescence staining, Nissl staining, II). Diaminobenzidine (DAB) staining was used to study the spatial memory ability, electroencephalogram (EEG) changes and neuronal cell level changes after cell transplantation in rats.
Results: Cell transplantation had protective effect on spatial memory impairment induced by epilepsy in rats. The protective effect of cell transplantation began to appear on the 7th day after transplantation. The protective effect of M-BMSCs group was early and obvious (escape latency time was restored to near normal level). Compared with BMSCs group, the protective effect of BMSCs group was better. There was a significant difference between M-BMSCs group and M-BMSCs group (P 0.05). Cell transplantation therapy could not only reduce the mortality of rats after modeling (although there was no statistical difference among the groups, the mortality of M-BMSCs group and BMSCs group was still lower than that of the positive control group), but also reduce the incidence of SRS (although there was no statistical difference, but there was no significant difference in M-BMSCs). Compared with the positive control group, the number of spikes and slow waves in M-BMSCs group and BMSCs group was significantly decreased (P 0.05), and the effect of M-BMSCs group was better than that of BMSCs group. The ratio of NeuN+/BrdU+ co-expressing cells and GAD67+/BrdU+ co-expressing cells in the parahippocampal cortex of rats in MSCs group was higher than that in BMSCs group; the density of vacuolar nucleus cells (neurons) in the parahippocampal cortex of rats in M-BMSCs group was significantly higher than that in control group (14D and 28D, P 0.05). Cell migration showed that the transplanted cells were widely distributed in the hippocampus and paracortex, suggesting that the transplanted cells could promote the functional recovery of epileptic rats.
CONCLUSION: BMSCs transplantation with overexpression of Mash1 gene can promote the functional recovery of epileptic rats and is superior to BMSCs transplantation.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742.1
【参考文献】
相关期刊论文 前1条
1 陈绍良,方五旺,钱钧,叶飞,刘煜昊,单守杰,张俊杰,林松,廖联明,赵春华;Improvement of cardiac function after transplantation of autologous bone marrow mesenchymal stem cells in patients with acute myocardial infarction[J];Chinese Medical Journal;2004年10期
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