Mash1基因過表達的骨髓間充質(zhì)干細胞治療癲癇的實驗研究
發(fā)布時間：2018-08-22 19:34
【摘要】：癲癇是發(fā)病率最高的中樞神經(jīng)系統(tǒng)功能障礙，表現(xiàn)為慢性、反復發(fā)作的大腦神經(jīng)元異常放電。世界衛(wèi)生組織（World Health Organization, WHO）的數(shù)據(jù)顯示，全球癲癇患者約為五千萬至一億，并且全球約5%的人群一生中具有罹患癲癇的風險。癲癇患者中有約30%行藥物治療效果欠佳，而藥物治療之外的手術(shù)治療以及神經(jīng)電刺激治療（迷走神經(jīng)電刺激術(shù)（vagus nerve stimulation, VNS）、腦深部電刺激治療（deepbrain stimulation, DBS））等并不適合于所有癲癇患者特別是無手術(shù)指征者，因此，有必要尋找更有效、安全、持久的新治療方法。 腦內(nèi)伽馬氨基丁酸（gamma-amino butyric acid, GABA）能神經(jīng)元減少會導致癲癇是目前了解的可能核心機制。研究顯示，源自不同類別干細胞（胚胎干細胞（embryonic stem cells, ESCs）、神經(jīng)干細胞（neural stem cells, NSCs）、間充質(zhì)干細胞（mesenchymal stem cells, MSCs）等）的GABA能神經(jīng)元可以抑制癇性發(fā)作、延長模型動物生存期，因此，大量學者把目光聚焦于基于干細胞的癲癇治療策略。但細胞來源無法滿足需求成為個中羈絆。 間充質(zhì)干細胞因具有自我更新和多向分化潛能的特性使其成為細胞治療策略的優(yōu)良候選細胞并逐漸成為熱點。作為間充質(zhì)干細胞中的一員，骨髓間充質(zhì)干細胞（bone morrow mesenchymal stem cells, BMSCs）來源于骨髓，它在人體內(nèi)分布廣泛，易于提取、分離，而且在應用于自體時不存在免疫排斥以及倫理學障礙，有研究顯示BMSCs移植治療可以抑制癲癇動物模型的自發(fā)性癇性發(fā)作（spontaneous recurrentseizure, SRS）的頻率。使用其進行移植治療并使之與宿主神經(jīng)元形成突觸聯(lián)系幫助其功能重建是有可能實現(xiàn)的。但針對在癲癇模型中起關(guān)鍵作用的GABA能神經(jīng)元的間充質(zhì)干細胞來源的特異性分化以及用其治療癲癇以促進療效的研究很少。盡管有研究顯示BMSCs可以經(jīng)條件誘導培養(yǎng)向GABA能神經(jīng)元樣細胞分化，但需使用化學成分（如氯化鉀（potassium chloride，Kcl），β-巰基乙醇（β-mercaptoethanol, BME），維甲酸（retinoic acid， RA）等）或細胞因子（如堿性成纖維細胞生長因子）等，上述方案如果應用于體內(nèi)則不僅難以實現(xiàn)而且可能產(chǎn)生無法預期的副作用。 堿性螺旋-環(huán)-螺旋（basic helix-loop-helix, bHLH）基因在干細胞的增殖與分化過程中起著重要的作用，其中，促分化轉(zhuǎn)錄因子哺乳動物無剛毛-鱗甲同源物（mammalian achaete-scute homologue，Mash1）基因作為bHLH家族中的一員，它不僅啟動神經(jīng)干細胞分化成神經(jīng)元的過程，而且同時決定著神經(jīng)元亞型的形成，與GABA能神經(jīng)元的分化形成有著密切的關(guān)系。本研究假設(shè)，過表達BMSCs中的Mash1基因?qū)⒖纱龠M其向GABA能神經(jīng)元樣細胞分化。本研究采用慢病毒轉(zhuǎn)染技術(shù)使BMSCs過表達Mash1基因，通過條件誘導培養(yǎng)技術(shù)誘導分化為GABA能神經(jīng)元樣細胞，對比BMSCs和Mash1過表達的BMSCs向GABA能神經(jīng)元樣細胞分化的比率以驗證假設(shè)，并且將Mash1過表達的BMSCs移植入癲癇大鼠模型體內(nèi)，觀察其是否具有更進一步抑制癲癇的治療效果。 第一部分大鼠來源的骨髓間充質(zhì)干細胞的提取、體外分離、培養(yǎng)和鑒定 目的：建立成熟高效的提取、分離與鑒定BMSCs的方法。 方法：無菌手術(shù)取材2~3周齡大鼠的雙側(cè)股骨與脛骨，提取骨髓，分別使用全骨髓貼壁培養(yǎng)法和應用Histopaque分離液的密度梯度離心分離法分離細胞并培養(yǎng)，采用流式細胞儀鑒定細胞表面分子標記并通過成脂肪、軟骨、骨誘導分化鑒定其多向分化潛能，使用噻唑藍（3-（4，5-dimethylthiazol-2-yl）-2，5-diphenyltetrazoliumbromide，MTT）檢測增殖曲線。 結(jié)果：與全骨髓貼壁培養(yǎng)法相比，密度梯度分離法獲得的BMSCs雜質(zhì)細胞少、細胞形態(tài)更標準并且均一，增殖曲線標準（接種第1~3天處于潛伏期，隨后進入對數(shù)生長期，，至第7~9天時進入平臺期），同時細胞表面分子標記表達（密度梯度分離法提取并傳代得到的用于本實驗研究的P3（passage3）代BMSCs低表達CD34抗原（（1.79±0.23）%，圖4-A）并且高表達CD105抗原（（98.6±0.68）%，圖4-B）、CD90抗原（（98.5±1.02）%，圖4-C）和CD73抗原（（98.8±0.98）%，圖4-D）及多向分化潛能均符合國際統(tǒng)一規(guī)范。 結(jié)論：密度梯度離心分離法可以高效、便捷地獲得純度高、擴增速度快、定向分化能力明確的符合國際統(tǒng)一規(guī)范的BMSCs，可以為下一步實驗研究提供優(yōu)良的細胞材料。 第二部分過表達Mash1基因促進骨髓間充質(zhì)干細胞成GABA能神經(jīng)元分化 目的：研究Mash1基因過表達對BMSCs成GABA能細胞分化的作用。 方法：提取2~3周Sprague-Dawley（SD）大鼠的BMSCs，傳至第3代；制備搭載Mash1基因的慢病毒載體，轉(zhuǎn)染BMSCs使其Mash1基因過表達，采用復合誘導培養(yǎng)基（1×B27+1mM N6, O2二丁酰腺苷3’,5’-環(huán)磷酸(N6, O2’-dibutyryladenosine3’,5’-cyclicmonophosphate, Bt2cAMP)+1μMATRA+Neurobasal）體外誘導Mash1+-BMSCs和BMSCs成GABA能神經(jīng)元分化并做免疫細胞化學染色、PCR檢測和Western blot檢測驗證它們的分化趨勢，同時行全細胞膜片鉗檢測了解分化細胞的電生理學特性。 結(jié)果：搭載Mash1基因的慢病毒轉(zhuǎn)染（感染指數(shù)MOI=100）BMSCs后Westernblot檢測結(jié)果顯示Mash1表達獲得顯著提高；成GABA能神經(jīng)元分化過程中Mash1+-BMSCs組（M-BMSCs）表現(xiàn)出更多的具有神經(jīng)元形態(tài)特征的細胞；誘導分化后Western blot和RT-PCR檢測顯示Mash1+-BMSCs組的細胞表達神經(jīng)元特異性核蛋白（neuron-specific nuclear protein，NeuN）和谷氨酸脫羧酶（glutamic aciddecarboxylase，GAD）67的水平顯著高于對照組細胞；分化后細胞的細胞免疫熒光檢測顯示M-BMSCs組（71.6±2.8）%中GAD67免疫熒光陽性比率明顯高于BMSCs組（61.4±5.3）%、Lv-con-BMSCs組（58.6±5.4）%和M-con-BMSCs組（0），并且，M-BMSCs組（72.0±2.0）%中GABA免疫熒光陽性比率明顯高于BMSCs組（61.4±2.9）%、Lv-con-BMSCs組（62.4±5.5）%和M-con-BMSCs組（0）；全細胞膜片鉗檢測到成神經(jīng)元分化的細胞具有動作電位和自發(fā)性抑制性突觸后電位提示其具有功能性。 結(jié)論：慢病毒介導的基因過表達具有良好的效果；Mash1基因過表達可以促進BMSCs向具有電生理活性的GABA能神經(jīng)元分化。 第三部分Mash1基因過表達的骨髓間充質(zhì)干細胞對大鼠癲癇模型的治療研究 目的：研究Mash1基因過表達的BMSCs（M-BMSCs）對癲癇模型大鼠的治療作用。 方法：60只SD大鼠行水迷宮訓練形成空間記憶后使用匹魯卡品誘導制作癲癇模型并入組達到RacineⅤ級發(fā)作的大鼠；使用BrdU體外標記M-BMSCs和BMSCs并通過立體定向聯(lián)合微量注射技術(shù)將M-BMSCs（M-BMSCs組）、BMSCs（BMSCs組）或大鼠成纖維細胞（陽性對照組）注射入癲癇大鼠右側(cè)腦室完成細胞移植（細胞數(shù)量5×105個/只）；其后選取不同時間點（移植后第7，14，21，28天）進行功能學（水迷宮檢測、自發(fā)性癲癇發(fā)作頻率監(jiān)測）、電生理（EEG）以及免疫組織化學（石蠟切片免疫熒光染色、尼氏染色、二氨基聯(lián)苯胺（diaminobenzidine，DAB）染色）檢測研究大鼠空間記憶能力、腦電圖變化和神經(jīng)元細胞層面在細胞移植之后產(chǎn)生的變化。 結(jié)果：細胞移植治療對大鼠因癲癇導致的空間記憶能力損害具有保護作用，在移植第7天細胞移植的保護作用開始出現(xiàn)，M-BMSCs組的保護作用的特點是出現(xiàn)早、效果明顯（逃避潛伏期時間恢復至接近正常大鼠水平），相比較而言，BMSCs組的效果與M-BMSCs組存在差異（P0.05）；細胞移植治療不僅可以降低大鼠造模后的死亡率（雖然各組數(shù)據(jù)間無統(tǒng)計學差異，但M-BMSCs組和BMSCs組的死亡數(shù)量仍是低于陽性對照組的），而且可以減低SRS的發(fā)生（雖然統(tǒng)計分析未見明顯差異，但是在M-BMSCs組和BMSCs組仍可見SRS頻率的減少），并且在EEG電生理水平也表現(xiàn)出促進恢復的作用：細胞移植治療后，與陽性對照組相比，M-BMSCs組和BMSCs組的棘波和棘慢波的數(shù)量顯著減少（P0.05），而且M-BMSCs組的效果優(yōu)于BMSCs組；免疫熒光雙標檢測發(fā)現(xiàn)M-BMSCs組的大鼠腦內(nèi)海馬旁皮層NeuN+/BrdU+共表達細胞和GAD67+/BrdU+共表達細胞比率高于BMSCs組大鼠；尼氏染色計數(shù)海馬旁皮層空泡核細胞（神經(jīng)元）密度結(jié)果顯示M-BMSCs組大鼠的神經(jīng)元密度顯著高于對照組（14D和28D，P0.05）；DAB染色定位移植細胞的遷移顯示，移植細胞在海馬和海馬旁皮層區(qū)域廣泛分布，提示了移植細胞促進癲癇大鼠功能恢復的結(jié)構(gòu)基礎(chǔ)。 結(jié)論：Mash1基因過表達的BMSCs移植治療能夠促進癲癇模型大鼠的功能恢復并且作用優(yōu)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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R742.1
本文編號：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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R742.1
【參考文獻】
相關(guān)期刊論文 前1條
1 陳紹良,方五旺,錢鈞,葉飛,劉煜昊,單守杰,張俊杰,林松,廖聯(lián)明,趙春華;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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