非黏附骨髓间充质干细胞分化为神经细胞的实验研究
发布时间：2019-07-01 13:48
【摘要】： 干细胞移植是近年来医学领域乃至整个生命科学领域的研究热点和前沿,其具有高度的自我更新能力和多向分化潜能,并能在体内外表达多种外源目的基因,因而作为种子细胞被广泛用于细胞疗法和基因疗法的研究中。随着功能基因组学和蛋白质组学的进一步发展,干细胞的基础以及临床应用研究也取得了突破性进展。 骨髓来源的间充质干细胞是起步较早研究较深入的成体干细胞,长期以来人们一致认为它是骨髓中一类高度黏附的成纤维细胞样细胞,然而Miao等最近的实验结果显示:大鼠骨髓中存在一种非黏附状态的间充质干细胞,体外培养条件下能够贴壁生长形成克隆,给予以往用于骨髓间充质干细胞的诱导条件也可以分化为三种间质终末期细胞,注射到亚致死剂量射线照射的小鼠体内可以参与其造血重建,表现出一定的多分化潜能。 本文将在此理论基础上通过反复转移的非黏附细胞培养法分离小鼠骨髓中的非黏附间充质干细胞;观察表皮生长因子对非黏附间充质干细胞克隆形成效率的调节作用;并深入探讨非黏附骨髓间充质干细胞在体外、体内尤其是缺血损伤的微环境中分化为神经细胞的潜能,为这些细胞进一步用于细胞移植治疗相关疾病提供技术参数。 第一部分小鼠非黏附骨髓间充质干细胞的分离培养、鉴定以及表皮生长因子对其克隆形成效率的影响 目的分离培养并鉴定小鼠骨髓中的非黏附间充质干细胞(Non-adherent Bone-Marrow-derived Mesenchymal Stem Cells,NA-BM-MSC);观察表皮生长因子(Epidermal Growth Factor,EGF)对NA-BM-MSC产生成纤维细胞集落形成单位(colony-formingunits-fibroblast,CFU-F)效率的影响。方法分离小鼠总骨髓细胞,分两组体外培养,一组在总骨髓细胞中加入10ng/mL的EGF,另一组为对照组,每天转移未贴壁的非黏附骨髓细胞(Non-adherent Bone MarrowCells,NA-BMC)到新的培养皿中继续培养,每组中各皿细胞长满12天后,亚甲基蓝染色显示形成的克隆,计数克隆个数;在总骨髓细胞培养到第四天时收集未贴壁的NA-BMC到新的培养皿中,待细胞贴壁生长达融合后传代纯化,取第四代细胞接种于向脂肪细胞、软骨细胞和成骨细胞诱导分化的培养液中,2周后分别进行油红染色、抗Ⅱ型胶原的免疫组织化学染色以及茜素红染色检测脂肪滴、Ⅱ型胶原和钙化结节的形成和表达情况;流式细胞检测表面标记物的表达。结果反复转移未贴壁NA-BMC到新培养皿继续培养的过程中,不仅总骨髓细胞可以贴壁形成CFU-F,而且反复转移的未贴壁NA-BMC也能不断贴壁并形成CFU-F;总骨髓细胞培养到第四天时收集的NA-BMC经脂肪细胞诱导液诱导后,油红染色可见细胞内产生大量油滴;经软骨细胞诱导液诱导后有软骨细胞特异性Ⅱ型胶原表达;经成骨细胞诱导液诱导后能够形成钙化结节;这些细胞高表达CD105、CD13,而不表达CD34、CD45等造血干细胞的特异性标记。加EGF刺激组每次转移的非黏附细胞贴壁生长后得到的细胞数和克隆数均比对照组多(P＜0.05),是对照组的3倍。结论小鼠骨髓细胞培养过程中,24小时内未贴壁的NA-BMC转移到新培养皿中继续培养能够重新贴壁并形成CFU-F,并且NA-BMC所形成的克隆具有向脂肪细胞、软骨细胞和成骨细胞分化的能力,具备了干细胞自我复制更新和多分化潜能的两大特性,并表达间充质细胞的特异性标记物,因此称其为非黏附骨髓间充质干细胞(NA-BM-MSC)。EGF能有效促进NA-BM-MSC的增殖,进一步提高了该细胞的富集效率。多分化潜能;表皮生长因子;增殖 第二部分非黏附骨髓间充质干细胞体外向神经细胞分化的实验研究 目的探讨骨髓中存在的NA-BM-MSC在体外向神经细胞分化的能力。方法分离小鼠骨髓总细胞进行培养,于第四天收集未贴壁的NA-BMC到新的培养环境中继续培养,待细胞贴壁融合后传代纯化,取第四代细胞用于诱导实验;细胞按照1X10~3/cm~2的密度接种于预置盖玻片的六孔板中,细胞贴壁后,更换培养液为含20ng/ml的EGF和20ng/ml的碱性成纤维细胞生长因子-2(b-FGF)的诱导液进行诱导,2周后,甲苯胺蓝染色和抗神经细胞特异性核蛋白、抗神经中间丝蛋白的免疫细胞化学染色检测神经细胞特异性蛋白的表达。结果总骨髓细胞和NA-BM-MSC经过EGF和b-FGF的诱导后都能表达神经特异性蛋白:NeuN/NF-200/尼氏体。结论NA-BM-MSC在适当的条件下能够被诱导分化成神经细胞,为下一步用于体内实验的研究提供了保证。 第三部分非黏附骨髓间充质干细胞在缺血损伤脑内向神经细胞分化的实验研究 目的探讨骨髓非黏附间充质干细胞(non-adherent bonemorrow mesenchymal stem cells,NA-BM-MSC)在缺血损伤的脑内存活以及向神经细胞分化的能力。方法从β-Gal转基因小鼠分离总骨髓细胞,培养到第四天时,收集未贴壁的NA-BMC到新的培养环境中继续培养,贴壁增殖,待生长到融合时传代纯化,取第4代NA-BM-MSC定向移植到小鼠大脑中动脉阻塞脑缺血模型脑内,8周后LacZ组织化学染色和免疫组织化学染色法观察供体细胞在缺血脑内微环境中的存活情况以及神经元特异性核蛋白(NeuN)、神经胶质细胞特异性酸性蛋白(GFAP)的表达。结果LacZ组织化学染色发现在缺血侧供体细胞能够存活,免疫组织化学单染和双染后发现在缺血模型的坏死区及坏死边缘区都检测到β-Gal阳性的供体细胞,部分细胞还同时表达神经元和神经胶质细胞特异性蛋白NeuN和GFAP。结论小鼠骨髓内的NA-BM-MSC在缺血损伤的脑内能够存活、迁移,部分细胞还能分化为成熟的神经元或神经胶质细胞参与脑损伤的修复,为下一步作为种子细胞用于移植修复实验提供了保证。
[Abstract]:Stem cell transplantation is a hot spot and a leading edge in the field of medical science and the whole life science in recent years. It has a high self-renewal ability and a multi-directional differentiation potential, and can express multiple foreign object genes in vitro. Thus, as a seed cell, is widely used in the study of cell therapy and gene therapy. With the further development of functional genomics and proteomics, the foundation of stem cells and the research of clinical application have made breakthrough progress. The bone marrow-derived mesenchymal stem cells are the more in-depth adult stem cells, which have long been thought to be a class of highly adherent fibroblast-like cells in the bone marrow, but Miao et al. It is shown that there is a non-adherent mesenchymal stem cell in the bone marrow of the rat, and under the condition of in vitro culture, the bone marrow mesenchymal stem cells can be grown to form a clone, and the induction conditions for the bone marrow mesenchymal stem cells can also be differentiated into three kinds of mesenchymal stem cells. The cells, injected into the sublethal dose-ray irradiated mice, can participate in the hemopoietic reconstruction of the mouse and show a certain multi-differentiation. Potential. The non-adherent mesenchymal stem cells in the bone marrow of mice were isolated by the non-adherent cell culture method of repeated transfer on the basis of this theory, and the formation efficiency of the non-adherent mesenchymal stem cells was observed by observing the growth factor of the epidermal growth factor. The role of non-adherent bone marrow mesenchymal stem cells in the microenvironment of the in vitro and in vivo, especially in the ischemic injury, is discussed, and the cells are further used for cell transplantation for the treatment of related diseases. Technical parameters: isolation, culture, identification, and epidermal growth factor for non-adherent bone marrow mesenchymal stem cells of the first part of the mouse The effect of cloning and forming efficiency is to separate and culture and identify the non-adherent mesenchymal stem cells (Non-adhert Bone-Marrow-derivable Mesenchymal Stem Cells) in the bone marrow of the mouse. (NA-BM-MSC); epidermal growth factor (EGF) was observed to form a colony-forming unit-fibreblast for NA-BM-MSC. The effect of the efficiency of the CFU-F was studied. The total bone marrow cells of the mice were isolated and cultured in vitro. One group was added with 10 ng/ mL of EGF in the total bone marrow cells, and the other group was the control group. The non-adherent bone marrow cells (NA-BMC) were transferred to the new culture dish every day. and collecting the unadhered NA-BMC into a new culture dish when the total bone marrow cells are cultured to a fourth day, The generation and expression of fat, type II collagen and calcified nodules were detected by the immunohistochemical staining of the anti-II collagen and the red staining of the collagen in the medium of the differentiation of the adipocytes, the chondrocyte and the osteoblast, and the flow was fine. In the process of repeated transfer of unadherent NA-BMC to a new culture dish, not only the total bone marrow cells could be attached to the CFU-F, but the non-adherent NA-BMC which was repeatedly transferred could be adhered to and form the CFU-F; the total bone marrow cells were cultured to the NA-BMC collected at the fourth day After induction of the fat cell-induced liquid, a large amount of oil droplets are generated in the oil red-stained visible cells, and the chondrocyte-specific type II collagen expression is produced after the induction of the chondrocyte-induced liquid, and the calcified nodules can be formed after the induction of the osteoblast-inducing liquid; and the cells express the CD105, the CD, 13, and not CD34, CD45, etc. The number of cells and the number of clones obtained after the adherent growth of non-adherent cells transferred by the EGF-stimulated group were more than that in the control group (P <0. Conclusion In the process of bone marrow cell culture, the non-adherent NA-BMC was transferred to the new culture dish for 24 hours to continue to culture and form the CFU-F, and the clone formed by the NA-BMC had the following advantages: The ability of the differentiation of the cell and the osteoblast has two characteristics of self-replication and multi-differentiation potential of the stem cells, and the specific marker of the mesenchymal cells is expressed, so that the non-adherent bone marrow mesenchymal stem cell (NA-BM-MSC) is called a non-adherent bone marrow mesenchymal stem cell (NA-BM-MSC). The EGF can effectively promote the proliferation of the NA-BM-MSC, further and the enrichment efficiency of the cell is improved, and the multi-differentiation potential; epidermal growth factor; proliferation of second part of non-adherent bone marrow mesenchymal stem An experimental study of the differentiation of the cells in vitro to the nerve cells in order to explore the presence of NA-BM in the bone marrow -the ability of the MSC to differentiate into the nerve cells in vitro. The method separates the mouse bone marrow total cells for culture, and collects the unadhered NA-BMC in the fourth day to continue to culture in a new culture environment, the cells are passaged and purified after the cell is adhered and fused, and the fourth-generation cells are used for inducing the experiment; and the cells are cultured according to 1X10-3/ cm- 2, inoculating in a six-well plate of a preset cover glass, after the cells are adhered, the culture solution is changed into an induction liquid containing 20 ng/ ml of EGF and 20 ng/ ml of basic fibroblast growth factor-2 (b-FGF), and after 2 weeks, the cells immunocytochemical of aniline blue staining and anti-nerve cell-specific nucleoprotein and anti-neurointermediate silk protein Results Total bone marrow cells and NA-BM-MSCs can express nerve-specific egg after the induction of EGF and b-FGF. Conclusion NA-BM-MSC can be induced to differentiate into nerve cells under appropriate conditions. The next step is to provide assurance for the study of in vivo experiments. The third part of the non-adherent bone marrow mesenchymal stem The purpose of this study is to study the non-adhermal bone marrow mesenchymal stem cells (NA-BM-MSC) of bone marrow in the brain of ischemic injury. ) Survival in the brain of the ischemic injury and the ability to differentiate into the nerve cells. The method is to separate the total bone marrow cells from the bone marrow-Gal transgenic mice and to collect the unadhered NA-BMC to the new culture ring at the fourth day The culture, the proliferation of the adherent cells and the passage and purification of the cells to be grown to the fusion were continued. The 4-generation NA-BM-MSC was transplanted to the brain of the cerebral artery of the mice. After 8 weeks, the microenvironment of the donor cells in the ischemic brain was observed by the immunohistochemical staining and the immunohistochemical staining. Survival and Neuron-specific Nuclear Protein (NeuN) in Neuron The expression of glial cell-specific acidic protein (GFAP) was found in the expression of glial cell-specific acidic protein (GFAP). In the area of the area of necrosis and the area of necrosis, the donor cells of the antigen-Gal positive are detected, and some of the cells also express the gods at the same time. Conclusion The NA-BM-MSC in the bone marrow of the mouse can survive and migrate in the brain of the ischemic injury, and the partial cells can also be differentiated into mature neurons or glial cells to participate in the repair of the brain injury.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329
本文编号：2508532
[Abstract]:Stem cell transplantation is a hot spot and a leading edge in the field of medical science and the whole life science in recent years. It has a high self-renewal ability and a multi-directional differentiation potential, and can express multiple foreign object genes in vitro. Thus, as a seed cell, is widely used in the study of cell therapy and gene therapy. With the further development of functional genomics and proteomics, the foundation of stem cells and the research of clinical application have made breakthrough progress. The bone marrow-derived mesenchymal stem cells are the more in-depth adult stem cells, which have long been thought to be a class of highly adherent fibroblast-like cells in the bone marrow, but Miao et al. It is shown that there is a non-adherent mesenchymal stem cell in the bone marrow of the rat, and under the condition of in vitro culture, the bone marrow mesenchymal stem cells can be grown to form a clone, and the induction conditions for the bone marrow mesenchymal stem cells can also be differentiated into three kinds of mesenchymal stem cells. The cells, injected into the sublethal dose-ray irradiated mice, can participate in the hemopoietic reconstruction of the mouse and show a certain multi-differentiation. Potential. The non-adherent mesenchymal stem cells in the bone marrow of mice were isolated by the non-adherent cell culture method of repeated transfer on the basis of this theory, and the formation efficiency of the non-adherent mesenchymal stem cells was observed by observing the growth factor of the epidermal growth factor. The role of non-adherent bone marrow mesenchymal stem cells in the microenvironment of the in vitro and in vivo, especially in the ischemic injury, is discussed, and the cells are further used for cell transplantation for the treatment of related diseases. Technical parameters: isolation, culture, identification, and epidermal growth factor for non-adherent bone marrow mesenchymal stem cells of the first part of the mouse The effect of cloning and forming efficiency is to separate and culture and identify the non-adherent mesenchymal stem cells (Non-adhert Bone-Marrow-derivable Mesenchymal Stem Cells) in the bone marrow of the mouse. (NA-BM-MSC); epidermal growth factor (EGF) was observed to form a colony-forming unit-fibreblast for NA-BM-MSC. The effect of the efficiency of the CFU-F was studied. The total bone marrow cells of the mice were isolated and cultured in vitro. One group was added with 10 ng/ mL of EGF in the total bone marrow cells, and the other group was the control group. The non-adherent bone marrow cells (NA-BMC) were transferred to the new culture dish every day. and collecting the unadhered NA-BMC into a new culture dish when the total bone marrow cells are cultured to a fourth day, The generation and expression of fat, type II collagen and calcified nodules were detected by the immunohistochemical staining of the anti-II collagen and the red staining of the collagen in the medium of the differentiation of the adipocytes, the chondrocyte and the osteoblast, and the flow was fine. In the process of repeated transfer of unadherent NA-BMC to a new culture dish, not only the total bone marrow cells could be attached to the CFU-F, but the non-adherent NA-BMC which was repeatedly transferred could be adhered to and form the CFU-F; the total bone marrow cells were cultured to the NA-BMC collected at the fourth day After induction of the fat cell-induced liquid, a large amount of oil droplets are generated in the oil red-stained visible cells, and the chondrocyte-specific type II collagen expression is produced after the induction of the chondrocyte-induced liquid, and the calcified nodules can be formed after the induction of the osteoblast-inducing liquid; and the cells express the CD105, the CD, 13, and not CD34, CD45, etc. The number of cells and the number of clones obtained after the adherent growth of non-adherent cells transferred by the EGF-stimulated group were more than that in the control group (P <0. Conclusion In the process of bone marrow cell culture, the non-adherent NA-BMC was transferred to the new culture dish for 24 hours to continue to culture and form the CFU-F, and the clone formed by the NA-BMC had the following advantages: The ability of the differentiation of the cell and the osteoblast has two characteristics of self-replication and multi-differentiation potential of the stem cells, and the specific marker of the mesenchymal cells is expressed, so that the non-adherent bone marrow mesenchymal stem cell (NA-BM-MSC) is called a non-adherent bone marrow mesenchymal stem cell (NA-BM-MSC). The EGF can effectively promote the proliferation of the NA-BM-MSC, further and the enrichment efficiency of the cell is improved, and the multi-differentiation potential; epidermal growth factor; proliferation of second part of non-adherent bone marrow mesenchymal stem An experimental study of the differentiation of the cells in vitro to the nerve cells in order to explore the presence of NA-BM in the bone marrow -the ability of the MSC to differentiate into the nerve cells in vitro. The method separates the mouse bone marrow total cells for culture, and collects the unadhered NA-BMC in the fourth day to continue to culture in a new culture environment, the cells are passaged and purified after the cell is adhered and fused, and the fourth-generation cells are used for inducing the experiment; and the cells are cultured according to 1X10-3/ cm- 2, inoculating in a six-well plate of a preset cover glass, after the cells are adhered, the culture solution is changed into an induction liquid containing 20 ng/ ml of EGF and 20 ng/ ml of basic fibroblast growth factor-2 (b-FGF), and after 2 weeks, the cells immunocytochemical of aniline blue staining and anti-nerve cell-specific nucleoprotein and anti-neurointermediate silk protein Results Total bone marrow cells and NA-BM-MSCs can express nerve-specific egg after the induction of EGF and b-FGF. Conclusion NA-BM-MSC can be induced to differentiate into nerve cells under appropriate conditions. The next step is to provide assurance for the study of in vivo experiments. The third part of the non-adherent bone marrow mesenchymal stem The purpose of this study is to study the non-adhermal bone marrow mesenchymal stem cells (NA-BM-MSC) of bone marrow in the brain of ischemic injury. ) Survival in the brain of the ischemic injury and the ability to differentiate into the nerve cells. The method is to separate the total bone marrow cells from the bone marrow-Gal transgenic mice and to collect the unadhered NA-BMC to the new culture ring at the fourth day The culture, the proliferation of the adherent cells and the passage and purification of the cells to be grown to the fusion were continued. The 4-generation NA-BM-MSC was transplanted to the brain of the cerebral artery of the mice. After 8 weeks, the microenvironment of the donor cells in the ischemic brain was observed by the immunohistochemical staining and the immunohistochemical staining. Survival and Neuron-specific Nuclear Protein (NeuN) in Neuron The expression of glial cell-specific acidic protein (GFAP) was found in the expression of glial cell-specific acidic protein (GFAP). In the area of the area of necrosis and the area of necrosis, the donor cells of the antigen-Gal positive are detected, and some of the cells also express the gods at the same time. Conclusion The NA-BM-MSC in the bone marrow of the mouse can survive and migrate in the brain of the ischemic injury, and the partial cells can also be differentiated into mature neurons or glial cells to participate in the repair of the brain injury.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329
【引证文献】
相关期刊论文 前1条
1 肖龙艳;傅晋翔;张学光;陈秋;张宏;王盼君;孙谕;王明元;古彦铮;;胎源性非黏附骨髓基质细胞的特性[J];中国组织工程研究;2012年01期
，本文编号：2508532
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