阻断间充质干细胞向神经细胞分化与肿瘤发生相关性研究
发布时间：2019-04-01 13:25
【摘要】：骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)是一类存在于骨髓中的非造血干细胞,具有自我更新、增殖及多向分化的潜能,在体外可诱导分化为骨、软骨、肌肉、脂肪、神经细胞和心肌细胞等。肿瘤干细胞(cancer stem cell, CSC)是指存在于肿瘤组织中的一小部分具有干细胞性质的细胞群体,具有无限增殖和自我更新的能力,是肿瘤发生的根源。目前已有研究者在白血病、乳腺癌及脑瘤中发现CSC,但它的起源仍存在诸多争议没有定论。随着对CSC研究的深入,BMSCs与CSC起源的关系成为了研究的关键点。 本文旨在从细胞增殖分化的角度研究CSC与干细胞的相关性。实验选用大鼠BMSCs为研究对象,建立大鼠BMSCs的神经细胞分化模型,以此为基础观察不同时间点阻断正常分化过程后细胞发生的变化,探讨干细胞与肿瘤发生的相关性。具体方法如下： 1、采用全骨髓培养法分离大鼠BMSCs,实现体外培养和扩增； 2、建立诱导分化模型：用β-巯基乙醇诱导BMSCs向神经细胞分化,通过免疫组化检测神经元特异性烯醇化酶(NSE),神经丝蛋白(NF),胶质纤维酸性蛋白(GFAP)的表达,鉴定诱导后的细胞； 3、建立分化阻断模型,探讨干细胞与肿瘤发生的相关性：BMSCs分为分化组和阻断组,分化组细胞正常诱导与此同时阻断组选择不同的时间点阻断正常的诱导过程,利用形态学观察,免疫组化,核型分析等手段检测阻断诱导后的细胞。通过对正常BMSCs、正常分化的神经细胞及阻断组中各时间点细胞的比对研究,探讨干细胞分化阻断与肿瘤发生的相关性。 研究结果如下： 1、成功分离了大鼠BMSCs,并实现体外扩增培养。传代培养的BMSCs生长状态良好,克隆形成能力强。 2、成功诱导大鼠BMSCs分化为神经细胞。诱导后的细胞显示典型的神经细胞形态特征,经免疫组化检测后NSE、NF阳性表达,GFAP阴性表达。 3、进行了大鼠BMSCs分化阻断与肿瘤相关性的探索研究。分化受阻的BMSCs发生了恶性转变,阻断分化的时间点不同,细胞的神经细胞特定蛋白表达量及恶性程度均不同。受阻的干细胞分化程度越低,相应产生的细胞分化程度越低、增殖速度越快,恶性程度越高,反之亦然。 综上所述,干细胞分化过程受到阻断,细胞停止在分化的中间阶段,大量增殖可能发生了干细胞向CSC的转化。通过对正常干细胞分化阻断的研究,本实验为CSC起源于正常干细胞提供了新的参考实验数据。
[Abstract]:Bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) are non-hematopoietic stem cells that exist in bone marrow. They have the potential of self-renewal, proliferation and multidirectional differentiation. They can be induced to differentiate into bone, cartilage, muscle and fat in vitro. Nerve cells and cardiomyocytes, etc. Tumor stem cell (cancer stem cell, CSC) is a small group of stem cells that exist in tumor tissue. It has the ability of unlimited proliferation and self-renewal. It is the origin of tumorigenesis. At present, researchers have found CSC, in leukemia, breast cancer and brain tumors, but there are still many controversies about its origin. With the deepening of the study of CSC, the relationship between BMSCs and the origin of CSC has become the key point of the study. The aim of this study is to study the relationship between CSC and stem cells from the perspective of cell proliferation and differentiation. The neural cell differentiation model of rat BMSCs was established by using rat BMSCs as the object of study. Based on this model, the changes of cell formation after blocking the normal differentiation process at different time points were observed, and the correlation between stem cells and tumorigenesis was discussed. The specific methods were as follows: (1) isolation of rat BMSCs, by whole bone marrow culture to achieve in vitro culture and amplification; 2. To establish differentiation model: BMSCs was induced to differentiate into nerve cells by 尾-mercaptoethanol. The expression of neuron-specific enolase (NSE), neurofilament protein (NF), glial fibrillary acidic protein (GFAP) was detected by immunohistochemistry. The induced cells were identified. (3) to establish a differentiation-blocking model to explore the correlation between stem cells and tumorigenesis: BMSCs was divided into two groups: differentiation group and blocking group. At the same time, the differentiation group selected different time points to block the normal induction process, and the differentiation group was divided into differentiation group and blocking group. Morphological observation, immunohistochemistry and karyotype analysis were used to detect the cells induced by blocking. The relationship between stem cell differentiation blocking and tumorigenesis was studied by comparing the normal BMSCs, differentiated nerve cells and the cells in the blocking group at different time points in order to explore the relationship between stem cell differentiation blocking and tumorigenesis. The results were as follows: 1. Rat BMSCs, was successfully isolated and cultured in vitro. The BMSCs cultured in subculture had good growth condition and strong clone forming ability. 2. The rat BMSCs was successfully induced to differentiate into nerve cells. The induced cells showed typical morphological features of nerve cells, and the expression of NSE,NF was positive and GFAP negative was detected by immunohistochemistry. 3. The correlation between BMSCs differentiation blocking and tumor in rats was studied. The differentiation-blocked BMSCs had a malignant transformation. The expression of neuron-specific protein and the degree of malignancy of the cells were different at different time points of blocking differentiation. The lower the differentiation degree of the blocked stem cells, the lower the differentiation degree of the corresponding cells, the faster the proliferation rate, the higher the malignant degree, and vice versa. In conclusion, stem cell differentiation process is blocked, cells stop in the intermediate stage of differentiation, a large number of proliferation may occur stem cells to CSC transformation. Through the study of normal stem cell differentiation blocking, this experiment provides new experimental data for the origin of CSC from normal stem cells.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
本文编号：2451589
[Abstract]:Bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) are non-hematopoietic stem cells that exist in bone marrow. They have the potential of self-renewal, proliferation and multidirectional differentiation. They can be induced to differentiate into bone, cartilage, muscle and fat in vitro. Nerve cells and cardiomyocytes, etc. Tumor stem cell (cancer stem cell, CSC) is a small group of stem cells that exist in tumor tissue. It has the ability of unlimited proliferation and self-renewal. It is the origin of tumorigenesis. At present, researchers have found CSC, in leukemia, breast cancer and brain tumors, but there are still many controversies about its origin. With the deepening of the study of CSC, the relationship between BMSCs and the origin of CSC has become the key point of the study. The aim of this study is to study the relationship between CSC and stem cells from the perspective of cell proliferation and differentiation. The neural cell differentiation model of rat BMSCs was established by using rat BMSCs as the object of study. Based on this model, the changes of cell formation after blocking the normal differentiation process at different time points were observed, and the correlation between stem cells and tumorigenesis was discussed. The specific methods were as follows: (1) isolation of rat BMSCs, by whole bone marrow culture to achieve in vitro culture and amplification; 2. To establish differentiation model: BMSCs was induced to differentiate into nerve cells by 尾-mercaptoethanol. The expression of neuron-specific enolase (NSE), neurofilament protein (NF), glial fibrillary acidic protein (GFAP) was detected by immunohistochemistry. The induced cells were identified. (3) to establish a differentiation-blocking model to explore the correlation between stem cells and tumorigenesis: BMSCs was divided into two groups: differentiation group and blocking group. At the same time, the differentiation group selected different time points to block the normal induction process, and the differentiation group was divided into differentiation group and blocking group. Morphological observation, immunohistochemistry and karyotype analysis were used to detect the cells induced by blocking. The relationship between stem cell differentiation blocking and tumorigenesis was studied by comparing the normal BMSCs, differentiated nerve cells and the cells in the blocking group at different time points in order to explore the relationship between stem cell differentiation blocking and tumorigenesis. The results were as follows: 1. Rat BMSCs, was successfully isolated and cultured in vitro. The BMSCs cultured in subculture had good growth condition and strong clone forming ability. 2. The rat BMSCs was successfully induced to differentiate into nerve cells. The induced cells showed typical morphological features of nerve cells, and the expression of NSE,NF was positive and GFAP negative was detected by immunohistochemistry. 3. The correlation between BMSCs differentiation blocking and tumor in rats was studied. The differentiation-blocked BMSCs had a malignant transformation. The expression of neuron-specific protein and the degree of malignancy of the cells were different at different time points of blocking differentiation. The lower the differentiation degree of the blocked stem cells, the lower the differentiation degree of the corresponding cells, the faster the proliferation rate, the higher the malignant degree, and vice versa. In conclusion, stem cell differentiation process is blocked, cells stop in the intermediate stage of differentiation, a large number of proliferation may occur stem cells to CSC transformation. Through the study of normal stem cell differentiation blocking, this experiment provides new experimental data for the origin of CSC from normal stem cells.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
【参考文献】
相关期刊论文 前2条
1 李晓峰;赵劲民;苏伟;崔向荣;罗世兴;马爱国;;大鼠骨髓间充质干细胞的培养与鉴定[J];中国组织工程研究与临床康复;2011年10期
2 李慕,贺祥瑞;Wistar系大鼠染色体核型及分带研究[J];兽医大学学报;1993年02期
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