【作者】 彭涛；

【导师】 滕军放； 贾延劼；

【作者基本信息】 郑州大学 ， 神经病学， 2011， 博士

【摘要】 研究背景骨髓间充质干细胞(mesenchymal stem cells, MSCs)是来源于中胚层的具有多向分化能力的干细胞,具有向软骨细胞、骨细胞、肌细胞、肌腱细胞、脂肪细胞、干细胞和造血细胞等多向分化及自我更新的功能,并且于一定条件下在体内及体外可横向分化为神经细胞和胶质细胞。近来,已经有很多动物实验研究报道,可以用移植骨髓间充质干细胞的方法来治疗各种神经系统的变性疾病、脑卒中及中枢损伤等,并取得了一定的效果。Rho/Rho激酶(ROCK)信号通路包括有三种成分：Rho蛋白、Rho激酶及Rho激酶的效应分子(ROCK),是体内一条重要的信号通路,主要参与了调控细胞骨架形成、细胞增殖、细胞迁移、基因转录和凋亡等生物行为及功能,主要通过小G蛋白GDP-GTP之间的转换,来调节细胞内肌动蛋白骨架的聚合状态,从而扮演着“分子开关”的角色,并参与调节细胞骨架蛋白的合成、降解、移动和收缩等,因此对细胞的分裂、黏附、收缩、迁移和分泌等活动具有非常重要的调节作用。我们观察了ROCK抑制剂法舒地尔在体外诱导大鼠MSCs向神经细胞分化中的作用。TRP (transient receptor potential)通道是一类六次跨膜的非选择性阳离子通道。它们在进化中高度保守,在哺乳动物体内广泛表达,参与了许多重要的生理学功能,如对温度、痛觉、听觉的感知。TRPM8通道是TRP的一个亚家族,在人的中枢神经系统中表达丰富,其主要生理功能是感知低温。令人惊奇的是,本研究在诱导MSCs分化为神经细胞时,首次发现TRPM8出现表达。microRNA-124a是脑组织中表达最为丰富的一类microRNA,约占脑组织microRNA总量的25%-48%。Lim等人的研究表明,将microRNA-124a感染到HeLa细胞中导致一系列非神经细胞转录物的表达受到抑制,HeLa细胞的基因组表达模式向神经方向转化。在小鼠脑组织,microRNA-124只限于在已经分化的和成熟的神经细胞中表达,而在神经前体细胞中表达很少。本研究通过构建携带含有绿色荧光蛋白(green fluorescence protein, GFP)报告基因的大鼠microRNA-124a慢病毒载体,将其感染至MSCs,并传代,观察其对骨髓间充质干细胞向神经细胞分化的影响。第一部分法舒地尔诱导大鼠骨髓间充质干细胞向神经细胞分化目的探讨Rho/Rho激酶(ROCK)抑制剂法舒地尔在体外诱导大鼠骨髓间充质干细胞(MSCs)向神经细胞分化中的可行性。方法全骨髓培养法分离培养大鼠MSCs；采用法舒地尔诱导MSCs;倒置显微镜观察诱导后各组的细胞形态学变化；AO-EB染色法鉴定诱导后各组细胞存活率；免疫荧光法鉴定诱导后NSE、NF200、GFAP的表达,判断其分化情况。结果AO-EB染色法鉴定诱导后细胞存活率诱导后存活细胞胞质呈绿色,核为亮绿色,核形态规则,为圆形或椭圆形,死亡细胞胞质呈红色,核呈亮红色,核皱缩或碎裂。随着诱导时间延长,死亡细胞数量增加。通过AO-EB染色法鉴定法舒地尔诱导30 min、90min、120min及180min,细胞的存活率分别为96.7±2.2%、95.3±1.9%、93.8±1.8%、92.5±2.1%及90.1±1.3%。诱导后免疫荧光鉴定法舒地尔诱导组,随着诱导时间延长, NSE、NF200表达显著增加,GFAP表达较少。诱导后60、90、120及180 min,通过免疫荧光鉴定,NSE阳性率(分别为66.5±1.9%、88.1±3.2%、93.6±1.9%、93.5±5.4%),NF200阳性率(分别为70.1±2.9%、89.5±1.3%、98.1±1.6%、98.3±1.9%)并呈逐渐增加的趋势,而各组GFAP表达率均小于5%。第二部分TRPM8在体外骨髓间充质干细胞分化为神经细胞中的表达变化瞬时受体电位(transient receptor potential, TRP)是一类广泛存在于细胞膜上的跨膜离子通道,可以辨别味觉、温度觉等特殊感觉。TRP通道亚型TRPM8主要存在于特定神经细胞的细胞膜上,当温度低于27℃或者薄荷醇存在的条件下,TRPM8通道开放,使Ca2+等带正电的粒子进入细胞,具有重要的生理意义。目的本研究探讨TRPM8在大鼠骨髓间充质干细胞(MSCs)分化为神经细胞中的表达变化和基本作用。方法在建立体外法舒地尔诱导大鼠MSCs分化为神经细胞的基础上,采用免疫细胞化学法、Western Blot法检测TRPM8的表达变化。结果：诱导前大鼠MSCs不表达TRPM8;诱导30min; MSCs开始表达TRPM8(45.3%±1.58%)；诱导60main,表达较前增加(57.50%±2.45%)；诱导后90min,TRPM8表达最高(89.56%±12.24%)；诱导120 min,TRPM8表达逐渐下降(59.25%±9.15%), Western Blot也有类似的趋势。第三部分microRNA-124a慢病毒载体的构建及感染大鼠骨髓间充质干细胞的研究目的探讨:microRNA-124a在法舒地尔诱导大鼠骨髓间充质干细胞(MSCs)向神经细胞分化中的作用。方法本研究构建携带含有绿色荧光蛋白(green fluorescence protein, GFP)报告基因的大鼠microRNA-124a慢病毒载体,将其感染至大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs),并传代,采用法舒地尔诱导大鼠MSCs分化为神经细胞。并于倒置荧光显微镜下观察MSCs感染后的荧光表达情况；采用免疫细胞化学染色和western印迹检测神经元烯醇化酶(NSE)、神经微丝蛋白(NF200)及胶质纤维酸性蛋白(GFAP)的表达变化,MTT方法检测细胞存活率。结果免疫细胞化学染色法感染组诱导1h后,NSE、NF200的表达率分别为83.2±2.0%,79.6±0.4%,显著高于其它两组(P<0.05)。各组GFAP的表达率都小于5%,无显著性差异。Western Blot结果三组分别诱导分化1h后均表达NSE、NF200,未感染组和阴性对照组无明显的统计学差异,而感染组表达的NSE、NF200较其它两组明显增高,有显著差异。结论1本研究发现Rho/Rho激酶(ROCK)抑制剂法舒地尔可以在体外快速,高效的诱导大鼠MSCs向神经细胞分化。2本研究首次报道TRPM8这种神经细胞特殊蛋白MSCs分化为神经细胞中出现表达,而且可能在其分化过程中起到一定的作用。3 microRNA-124a可促进大鼠骨髓间充质干细胞在向神经细胞分化更多还原

【Abstract】 BackgroundMesenchymal stem cells (mesenchymal stem cells, MSCs) are mesoderm derived multipotent stem cells with ability of differentiation to the bone cells, cartilage cells, tendon cells, muscle cells, stem cells, fat cells and hematopoietic cells and self-renewal capacity. In vivo and in vitro, MSCs can differentiate into neurons and glial cells under certain conditions. Recently, a number of animal studies reported therapy of a variety of neurodegenerative diseases, stroke and central nervous system injury by transplantation of bone marrow mesenchymal stem cells, and achieved certain results.Rho/Rho kinase (ROCK) signaling pathway is an important signaling pathway in vivo, including three components:Rho protein, Rho kinase, Rho effector kinase (ROCK), mainly involved in regulation of cytoskeleton formation, cell migration, gene transcription, cell proliferation, apoptosis and other biological behaviors and functions, which regulate intracellular actin cytoskeleton polymerization state through the small G protein GDP-GTP transition and plays a "molecular switch" role, involved in the regulation of cytoskeletal proteins synthesis, degradation, movement and shrinkage of cell division, contraction, adhesion, migration, secretion and other activities. We observed the role of the ROCK inhibitor fasudil in vitro induced MSCs differentiation into neuron-like cells. TRP (transient receptor potential) channels are a class of six transmembrane non-selective cation channels. They are highly conserved in evolution, widely expressed in mammals, involved in many important physiological functions, such as temperature, pain, auditory perception, and fertilization.TRPM8 is a TRP channel subfamily, express abundant in the human central nervous system, and its main function is to sense temperature. Surprisingly, this study found that when inducing MSCs to differentiate into neural cells, first detected the expression of TRPM8.microRNA-124a is most abundant expression in brain tissue of a class of microRNA, approximately 25%-48% of the total microRNA in brain tissue. Lim, whose studies have shown that the microRNA-124a infected HeLa cells and led to a series of expression of non-neuronal transcripts was inhibited, gene expression patterns of HeLa cells transform into the neuron. In the mouse brain, microRNA-124 is limited to expressed in already differentiated and mature neurons and very little in neural progenitor cells. This study was to construct the rat microRNA-124a lentiviral vector containing green fluorescent protein (green fluorescence protein, GFP) reporter gene, infection to the MSCs and passage, observed differentiation of bone marrow mesenchymal stem cells into nerve cells.Part 1 fasudil in vitro induce bone marrow mesenchymal stem cells into nerve cellsObjectiveResearch the feasiblity of Rho/Rho kinase (ROCK) inhibitor, fasudil in vitro induce rat bone marrow mesenchymal stem cells(MSCs) into neuron-like cell.MethodsRat MSCs were isolated from Wistar rats and cultured to 15-18 passages.We used fasudil (200μmol/L) as inducers to differentiate MSCs respectively, and then observed morphologic changes of differentiated cells,stained with AO/EB mixed fluorescent dye to estimate the apoptosis of MSCs, identified them by detecting NF200, NSE and GFAP by immunofluorescence.ResultsAO-EB staining identified cell survival after inductionCytoplasm of surviving cells after the induction of green, nuclear is bright green, nuclear shape rules for the round or oval-shaped, dead cells showed red cytoplasmic, nuclear was bright red, nuclear shrinkage or fragmentation. With the induction of prolonged increase in the number of dead cells. By AO-EB staining identified fasudil induced by 30 min,60min,90min,120min and 180min after the cell survival rates were 96.7±2.2%,95.3±1.9%,93.8±1.8%,92.5±2.1% and 90.1±1.3%.Immunofluorescence after inductionFasudil induced group, with the induction of prolonged, NSE, NF200 was significantly increased, GFAP expression less. After the induction of 60,90,120 and 180 min, identified by immunofluorescence, NSE-positive rates (respectively 66.5±1.9%,88.1±3.2%,93.6±1.9%,93.5±5.4%) and NF200-positive rates (70.1±2.9%, 89.5±1.3%,98.1±1.6%,98.3±1.9%) increased, while GFAP expression in each group were less than 5%.Part 2 in vitro Expression of TRPM8 of bone marrow mesenchymal stem cells differentiation into nerve cellsObjectivesThe transient receptor potential (TRP) channels are a large family of proteins have been involved in a wide range of processes ranging from sensing of thermal and chemical signals to reloading intracellular stores after responding to an extracellular stimulus. TRPM8 is conventionally reported as a cold- and menthol- sensing cation channel implicated in thermosensation. Here we surprisingly showed that expression of TRPM8 might incease greatly in neural cells derived from rat bone marrow mesenchymal stem cells (MSCs) in vitro.MethodsIn this study, we firstly characterized in vitro properties of neural cells derived from MSCs by immunocytochemistry, Western blot.ResultsBefore the induction of rat MSCs did not express TRPM8; induced 30min; MSCs began to express TRPM8 (45.3%±1.58%); induced 60min, possible increase in the expression (57.50%±2.45%); induced 90min, TRPM8 expression of the highest (89.56%±12.24%); induction of 120 min, TRPM8 expression decreased (59.25%±9.15%), Western Blot has a similar trend.Part 3 Construct the microRNA-124a lentiviral vectors and infection of rat bone marrow mesenchymal stem cells ObjectiveTo investigate the role of microRNA-124a in bone marrow mesenchymal stem cells differentiating into neurons.MethodsConstruction of preparations containing green fluorescent protein (green fluorescence protein, GFP) reporter gene in the rat lentiviral microRNA-124a, its infections and rat bone marrow mesenchymal stem cells (mesenchymal stem cells, MSCs), and passaged. Fasudil induced MSCs to differentiate into neuron. After infection fluorescence expression of MSCs was observed under fluorescence microscope; Expression of neuron-specific enolase (NSE), neurofilament protein (NF200) and glial fibrillary acidic protein (GFAP) was detected by western blot and immunocytochemistry. MTT was used to detect cell survival.ResultsImmunocytochemical staining1h after induction of infection, NSE, NF200 expression rates were 83.2±2.0%, 79.6±0.4%, significantly higher than the other two groups (P<0.05). GFAP expression in each group were lower than 5%, no significant difference.Western Blot ResultsThree groups were, respectively, after 1h inducing the expression of NSE, NF200, no infection and no positive control group statistically significant differences in the expression of the infected group NSE, NF200 was significantly higher than the other two groups, a significant difference. Conclusions1 Rho-kinase inhibitor fasudil can rapidly and efficiently induce rat bone marrow derived MSCs differentiating into neuron-like cells.2 These findings indicate that TRPM8 may play some important roles in MSCs differentiation into neural cells.3 microRNA-124a can promote bone marrow mesenchymal stem cells to differentiate into neural cells.更多还原