法舒地尔、TRPM8及microRNA-124a在骨髓间充质干细胞分化为神经细胞中的作用
发布时间：2018-08-23 08:00
【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(P0.05)。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
[Abstract]:Research background
Bone marrow mesenchymal stem cells (MSCs) are mesenchymal stem cells derived from the mesoderm and have the ability to differentiate into chondrocytes, osteocytes, muscle cells, tendon cells, adipocytes, stem cells and hematopoietic cells, and self-renewal. Under certain conditions, MSCs can be transverse in vivo and in vitro. Recently, many animal experiments have reported that bone marrow mesenchymal stem cells can be transplanted to treat various degenerative diseases of the nervous system, stroke and central injury, and some results have been achieved.
Rho/Rho kinase (ROCK) signaling pathway consists of three components: Rho protein, Rho kinase and Rho kinase effector molecule (ROCK), which is an important signaling pathway in vivo, mainly involved in the regulation of cytoskeleton formation, cell proliferation, cell migration, gene transcription and apoptosis, and other biological behaviors and functions, mainly through the transduction of small G protein GDP-GTP. In other words, it regulates the aggregation of actin cytoskeleton, thus acting as a "molecular switch" and regulating the synthesis, degradation, movement and contraction of cytoskeleton proteins. Therefore, it plays an important role in regulating cell division, adhesion, contraction, migration and secretion. In vitro, the effects of MSCs on the differentiation of rat neurons into neurons were observed.
TRP (transient receptor potential) channels are a class of six-time transmembrane nonselective cationic channels. They are highly conserved in evolution, widely expressed in mammals, and participate in many important physiological functions, such as temperature, pain, and auditory perception. TRPM8 channels are a subfamily of TRP, which are found in the human central nervous system. Surprisingly, TRPM8 was found for the first time in this study when MSCs were induced to differentiate into neural cells.
MicroRNA-124a is one of the most abundant microRNAs in brain tissues, accounting for about 25-48% of the total microRNA in brain tissues. Lim et al. showed that infection of microRNA-124a into HeLa cells resulted in a series of non-neuronal transcripts being inhibited, and the genomic expression pattern of HeLa cells was transformed to neural direction. Tissue, microRNA-124 is only expressed in differentiated and mature neural cells, but rarely in neural precursor cells. In this study, rat microRNA-124a lentiviral vector containing green fluorescence protein (GFP) reporter gene was constructed to infect MSCs and pass it on to observe its expression in bone marrow. The effect of mesenchymal stem cells on differentiation of neurons.
The first part is fasudil induced differentiation of rat bone marrow mesenchymal stem cells into neurons.
objective
Objective To investigate the feasibility of fasudil, a Rho/Rho kinase inhibitor, in inducing rat bone marrow mesenchymal stem cells (MSCs) to differentiate into neural cells in vitro.
Method
Rat MSCs were isolated and cultured by whole bone marrow culture, induced by fasudil, observed by inverted microscope, identified by AO-EB staining, and identified by immunofluorescence staining the expression of NSE, NF200 and GFAP after induction.
Result
AO-EB staining was used to identify cell viability after induction.
After induction, the cytoplasm of the surviving cells was green, the nucleus was bright green, the nucleus was round or oval, the dead cells were red, the nucleus was bright red, and the number of the dead cells increased with the induction time. It is 96.7 + 2.2%, 95.3 + 1.9%, 93.8 + 1.8%, 92.5 + 2.1% and 90.1 + 1.3%. respectively.
Immunofluorescence identification after induction
The expression of NSE, NF200 and GFAP increased significantly and GFAP decreased with the prolongation of induction time in the fasudil-induced group. After induction, the positive rates of NSE (66.5 -1.9%, 88.1 -3.2%, 93.6 -1.9%, 93.5 -5.4%) and NF200 (89.5 -1.3%, 98.1 -1.6%, 98.3 -1.9% and 98.3 -1.9%) were gradually increased by immunofluorescence assay. The expression rate of GFAP in each group was less than 5%..
The second part is the expression change of TRPM8 in the differentiation of bone marrow mesenchymal stem cells into nerve cells in vitro.
Transient receptor potential (TRP) is a kind of transmembrane ion channel which widely exists on the cell membrane. It can distinguish taste, temperature and other special sensations. TRP channel subtype TRPM8 mainly exists on the cell membrane of specific nerve cells. When the temperature is lower than 27 C or the presence of menthol, TRPM8 channel opens, so that Ca2+ and other positively charged particles enter cells and have important physiological significance.
objective
This study was designed to investigate the expression and role of TRPM8 in the differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neural cells.
Method
The expression of TRPM8 was detected by immunocytochemistry and Western Blot assay on the basis of inducing rat MSCs to differentiate into neural cells by stereotactic external fasudil.
Result:
Before induction, MSCs did not express TRPM8; 30 minutes after induction; MSCs began to express TRPM8 (45.3% + 1.58%); 60 main cells were induced and the expression increased (57.50% + 2.45%); 90 minutes after induction, the expression of TRPM8 was the highest (89.56% + 12.24%); 120 minutes after induction, the expression of TRPM8 gradually decreased (59.25% + 9.15%) and Western Blot had a similar trend.
The third part is the construction of microRNA-124a lentiviral vector and the infection of rat bone marrow mesenchymal stem cells.
objective
Objective: To investigate the role of microRNA-124a in the differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neural cells induced by fasudil.
Method
In this study, rat microRNA-124a lentiviral vector containing GFP reporter gene was constructed and infected into rat bone marrow mesenchymal stem cells (MSCs) for passage. Rat MSCs were induced to differentiate into nerve cells by Fasudil and then subjected to an inverted fluorescence microscope. The expression of neuron enolase (NSE), neurofilament protein (NF200) and glial fibrillary acidic protein (GFAP) were detected by immunocytochemical staining and Western blot, and the cell viability was detected by MTT assay.
Result
Immunocytochemical staining
One hour after induction, the expression rates of NSE and NF200 were 83.2 (+ 2.0%) and 79.6 (+ 0.4%) respectively, which were significantly higher than those of the other two groups (P 0.05). The expression rates of GFAP in each group were less than 5%, and there was no significant difference.
Western Blot results
The expression of NSE and NF200 was not significantly different between the non-infected group and the negative control group, but the expression of NSE and NF200 was significantly higher in the infected group than in the other two groups.
conclusion
In this study, we found that fasudil, an inhibitor of Rho/Rho kinase (ROCK), could induce rat MSCs to differentiate into neural cells quickly and efficiently in vitro.
This is the first report that TRPM8, a special neuronal protein MSCs, is expressed in differentiated neurons and may play a role in the process of differentiation.
3 microRNA-124a can promote the differentiation of rat bone marrow mesenchymal stem cells into neurons.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R329
本文编号：2198403
[Abstract]:Research background
Bone marrow mesenchymal stem cells (MSCs) are mesenchymal stem cells derived from the mesoderm and have the ability to differentiate into chondrocytes, osteocytes, muscle cells, tendon cells, adipocytes, stem cells and hematopoietic cells, and self-renewal. Under certain conditions, MSCs can be transverse in vivo and in vitro. Recently, many animal experiments have reported that bone marrow mesenchymal stem cells can be transplanted to treat various degenerative diseases of the nervous system, stroke and central injury, and some results have been achieved.
Rho/Rho kinase (ROCK) signaling pathway consists of three components: Rho protein, Rho kinase and Rho kinase effector molecule (ROCK), which is an important signaling pathway in vivo, mainly involved in the regulation of cytoskeleton formation, cell proliferation, cell migration, gene transcription and apoptosis, and other biological behaviors and functions, mainly through the transduction of small G protein GDP-GTP. In other words, it regulates the aggregation of actin cytoskeleton, thus acting as a "molecular switch" and regulating the synthesis, degradation, movement and contraction of cytoskeleton proteins. Therefore, it plays an important role in regulating cell division, adhesion, contraction, migration and secretion. In vitro, the effects of MSCs on the differentiation of rat neurons into neurons were observed.
TRP (transient receptor potential) channels are a class of six-time transmembrane nonselective cationic channels. They are highly conserved in evolution, widely expressed in mammals, and participate in many important physiological functions, such as temperature, pain, and auditory perception. TRPM8 channels are a subfamily of TRP, which are found in the human central nervous system. Surprisingly, TRPM8 was found for the first time in this study when MSCs were induced to differentiate into neural cells.
MicroRNA-124a is one of the most abundant microRNAs in brain tissues, accounting for about 25-48% of the total microRNA in brain tissues. Lim et al. showed that infection of microRNA-124a into HeLa cells resulted in a series of non-neuronal transcripts being inhibited, and the genomic expression pattern of HeLa cells was transformed to neural direction. Tissue, microRNA-124 is only expressed in differentiated and mature neural cells, but rarely in neural precursor cells. In this study, rat microRNA-124a lentiviral vector containing green fluorescence protein (GFP) reporter gene was constructed to infect MSCs and pass it on to observe its expression in bone marrow. The effect of mesenchymal stem cells on differentiation of neurons.
The first part is fasudil induced differentiation of rat bone marrow mesenchymal stem cells into neurons.
objective
Objective To investigate the feasibility of fasudil, a Rho/Rho kinase inhibitor, in inducing rat bone marrow mesenchymal stem cells (MSCs) to differentiate into neural cells in vitro.
Method
Rat MSCs were isolated and cultured by whole bone marrow culture, induced by fasudil, observed by inverted microscope, identified by AO-EB staining, and identified by immunofluorescence staining the expression of NSE, NF200 and GFAP after induction.
Result
AO-EB staining was used to identify cell viability after induction.
After induction, the cytoplasm of the surviving cells was green, the nucleus was bright green, the nucleus was round or oval, the dead cells were red, the nucleus was bright red, and the number of the dead cells increased with the induction time. It is 96.7 + 2.2%, 95.3 + 1.9%, 93.8 + 1.8%, 92.5 + 2.1% and 90.1 + 1.3%. respectively.
Immunofluorescence identification after induction
The expression of NSE, NF200 and GFAP increased significantly and GFAP decreased with the prolongation of induction time in the fasudil-induced group. After induction, the positive rates of NSE (66.5 -1.9%, 88.1 -3.2%, 93.6 -1.9%, 93.5 -5.4%) and NF200 (89.5 -1.3%, 98.1 -1.6%, 98.3 -1.9% and 98.3 -1.9%) were gradually increased by immunofluorescence assay. The expression rate of GFAP in each group was less than 5%..
The second part is the expression change of TRPM8 in the differentiation of bone marrow mesenchymal stem cells into nerve cells in vitro.
Transient receptor potential (TRP) is a kind of transmembrane ion channel which widely exists on the cell membrane. It can distinguish taste, temperature and other special sensations. TRP channel subtype TRPM8 mainly exists on the cell membrane of specific nerve cells. When the temperature is lower than 27 C or the presence of menthol, TRPM8 channel opens, so that Ca2+ and other positively charged particles enter cells and have important physiological significance.
objective
This study was designed to investigate the expression and role of TRPM8 in the differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neural cells.
Method
The expression of TRPM8 was detected by immunocytochemistry and Western Blot assay on the basis of inducing rat MSCs to differentiate into neural cells by stereotactic external fasudil.
Result:
Before induction, MSCs did not express TRPM8; 30 minutes after induction; MSCs began to express TRPM8 (45.3% + 1.58%); 60 main cells were induced and the expression increased (57.50% + 2.45%); 90 minutes after induction, the expression of TRPM8 was the highest (89.56% + 12.24%); 120 minutes after induction, the expression of TRPM8 gradually decreased (59.25% + 9.15%) and Western Blot had a similar trend.
The third part is the construction of microRNA-124a lentiviral vector and the infection of rat bone marrow mesenchymal stem cells.
objective
Objective: To investigate the role of microRNA-124a in the differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neural cells induced by fasudil.
Method
In this study, rat microRNA-124a lentiviral vector containing GFP reporter gene was constructed and infected into rat bone marrow mesenchymal stem cells (MSCs) for passage. Rat MSCs were induced to differentiate into nerve cells by Fasudil and then subjected to an inverted fluorescence microscope. The expression of neuron enolase (NSE), neurofilament protein (NF200) and glial fibrillary acidic protein (GFAP) were detected by immunocytochemical staining and Western blot, and the cell viability was detected by MTT assay.
Result
Immunocytochemical staining
One hour after induction, the expression rates of NSE and NF200 were 83.2 (+ 2.0%) and 79.6 (+ 0.4%) respectively, which were significantly higher than those of the other two groups (P 0.05). The expression rates of GFAP in each group were less than 5%, and there was no significant difference.
Western Blot results
The expression of NSE and NF200 was not significantly different between the non-infected group and the negative control group, but the expression of NSE and NF200 was significantly higher in the infected group than in the other two groups.
conclusion
In this study, we found that fasudil, an inhibitor of Rho/Rho kinase (ROCK), could induce rat MSCs to differentiate into neural cells quickly and efficiently in vitro.
This is the first report that TRPM8, a special neuronal protein MSCs, is expressed in differentiated neurons and may play a role in the process of differentiation.
3 microRNA-124a can promote the differentiation of rat bone marrow mesenchymal stem cells into neurons.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R329
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