非黏附骨髓間充質(zhì)干細胞分化為神經(jīng)細胞的實驗研究
發(fā)布時間：2019-07-01 13:48
【摘要】： 干細胞移植是近年來醫(yī)學領(lǐng)域乃至整個生命科學領(lǐng)域的研究熱點和前沿,其具有高度的自我更新能力和多向分化潛能,并能在體內(nèi)外表達多種外源目的基因,因而作為種子細胞被廣泛用于細胞療法和基因療法的研究中。隨著功能基因組學和蛋白質(zhì)組學的進一步發(fā)展,干細胞的基礎(chǔ)以及臨床應用研究也取得了突破性進展。 骨髓來源的間充質(zhì)干細胞是起步較早研究較深入的成體干細胞,長期以來人們一致認為它是骨髓中一類高度黏附的成纖維細胞樣細胞,然而Miao等最近的實驗結(jié)果顯示:大鼠骨髓中存在一種非黏附狀態(tài)的間充質(zhì)干細胞,體外培養(yǎng)條件下能夠貼壁生長形成克隆,給予以往用于骨髓間充質(zhì)干細胞的誘導條件也可以分化為三種間質(zhì)終末期細胞,注射到亞致死劑量射線照射的小鼠體內(nèi)可以參與其造血重建,表現(xiàn)出一定的多分化潛能。 本文將在此理論基礎(chǔ)上通過反復轉(zhuǎn)移的非黏附細胞培養(yǎng)法分離小鼠骨髓中的非黏附間充質(zhì)干細胞;觀察表皮生長因子對非黏附間充質(zhì)干細胞克隆形成效率的調(diào)節(jié)作用;并深入探討非黏附骨髓間充質(zhì)干細胞在體外、體內(nèi)尤其是缺血損傷的微環(huán)境中分化為神經(jīng)細胞的潛能,為這些細胞進一步用于細胞移植治療相關(guān)疾病提供技術(shù)參數(shù)。 第一部分小鼠非黏附骨髓間充質(zhì)干細胞的分離培養(yǎng)、鑒定以及表皮生長因子對其克隆形成效率的影響 目的分離培養(yǎng)并鑒定小鼠骨髓中的非黏附間充質(zhì)干細胞(Non-adherent Bone-Marrow-derived Mesenchymal Stem Cells,NA-BM-MSC);觀察表皮生長因子(Epidermal Growth Factor,EGF)對NA-BM-MSC產(chǎn)生成纖維細胞集落形成單位(colony-formingunits-fibroblast,CFU-F)效率的影響。方法分離小鼠總骨髓細胞,分兩組體外培養(yǎng),一組在總骨髓細胞中加入10ng/mL的EGF,另一組為對照組,每天轉(zhuǎn)移未貼壁的非黏附骨髓細胞(Non-adherent Bone MarrowCells,NA-BMC)到新的培養(yǎng)皿中繼續(xù)培養(yǎng),每組中各皿細胞長滿12天后,亞甲基藍染色顯示形成的克隆,計數(shù)克隆個數(shù);在總骨髓細胞培養(yǎng)到第四天時收集未貼壁的NA-BMC到新的培養(yǎng)皿中,待細胞貼壁生長達融合后傳代純化,取第四代細胞接種于向脂肪細胞、軟骨細胞和成骨細胞誘導分化的培養(yǎng)液中,2周后分別進行油紅染色、抗Ⅱ型膠原的免疫組織化學染色以及茜素紅染色檢測脂肪滴、Ⅱ型膠原和鈣化結(jié)節(jié)的形成和表達情況;流式細胞檢測表面標記物的表達。結(jié)果反復轉(zhuǎn)移未貼壁NA-BMC到新培養(yǎng)皿繼續(xù)培養(yǎng)的過程中,不僅總骨髓細胞可以貼壁形成CFU-F,而且反復轉(zhuǎn)移的未貼壁NA-BMC也能不斷貼壁并形成CFU-F;總骨髓細胞培養(yǎng)到第四天時收集的NA-BMC經(jīng)脂肪細胞誘導液誘導后,油紅染色可見細胞內(nèi)產(chǎn)生大量油滴;經(jīng)軟骨細胞誘導液誘導后有軟骨細胞特異性Ⅱ型膠原表達;經(jīng)成骨細胞誘導液誘導后能夠形成鈣化結(jié)節(jié);這些細胞高表達CD105、CD13,而不表達CD34、CD45等造血干細胞的特異性標記。加EGF刺激組每次轉(zhuǎn)移的非黏附細胞貼壁生長后得到的細胞數(shù)和克隆數(shù)均比對照組多(P＜0.05),是對照組的3倍。結(jié)論小鼠骨髓細胞培養(yǎng)過程中,24小時內(nèi)未貼壁的NA-BMC轉(zhuǎn)移到新培養(yǎng)皿中繼續(xù)培養(yǎng)能夠重新貼壁并形成CFU-F,并且NA-BMC所形成的克隆具有向脂肪細胞、軟骨細胞和成骨細胞分化的能力,具備了干細胞自我復制更新和多分化潛能的兩大特性,并表達間充質(zhì)細胞的特異性標記物,因此稱其為非黏附骨髓間充質(zhì)干細胞(NA-BM-MSC)。EGF能有效促進NA-BM-MSC的增殖,進一步提高了該細胞的富集效率。多分化潛能;表皮生長因子;增殖 第二部分非黏附骨髓間充質(zhì)干細胞體外向神經(jīng)細胞分化的實驗研究 目的探討骨髓中存在的NA-BM-MSC在體外向神經(jīng)細胞分化的能力。方法分離小鼠骨髓總細胞進行培養(yǎng),于第四天收集未貼壁的NA-BMC到新的培養(yǎng)環(huán)境中繼續(xù)培養(yǎng),待細胞貼壁融合后傳代純化,取第四代細胞用于誘導實驗;細胞按照1X10~3/cm~2的密度接種于預置蓋玻片的六孔板中,細胞貼壁后,更換培養(yǎng)液為含20ng/ml的EGF和20ng/ml的堿性成纖維細胞生長因子-2(b-FGF)的誘導液進行誘導,2周后,甲苯胺藍染色和抗神經(jīng)細胞特異性核蛋白、抗神經(jīng)中間絲蛋白的免疫細胞化學染色檢測神經(jīng)細胞特異性蛋白的表達。結(jié)果總骨髓細胞和NA-BM-MSC經(jīng)過EGF和b-FGF的誘導后都能表達神經(jīng)特異性蛋白:NeuN/NF-200/尼氏體。結(jié)論NA-BM-MSC在適當?shù)臈l件下能夠被誘導分化成神經(jīng)細胞,為下一步用于體內(nèi)實驗的研究提供了保證。 第三部分非黏附骨髓間充質(zhì)干細胞在缺血損傷腦內(nèi)向神經(jīng)細胞分化的實驗研究 目的探討骨髓非黏附間充質(zhì)干細胞(non-adherent bonemorrow mesenchymal stem cells,NA-BM-MSC)在缺血損傷的腦內(nèi)存活以及向神經(jīng)細胞分化的能力。方法從β-Gal轉(zhuǎn)基因小鼠分離總骨髓細胞,培養(yǎng)到第四天時,收集未貼壁的NA-BMC到新的培養(yǎng)環(huán)境中繼續(xù)培養(yǎng),貼壁增殖,待生長到融合時傳代純化,取第4代NA-BM-MSC定向移植到小鼠大腦中動脈阻塞腦缺血模型腦內(nèi),8周后LacZ組織化學染色和免疫組織化學染色法觀察供體細胞在缺血腦內(nèi)微環(huán)境中的存活情況以及神經(jīng)元特異性核蛋白(NeuN)、神經(jīng)膠質(zhì)細胞特異性酸性蛋白(GFAP)的表達。結(jié)果LacZ組織化學染色發(fā)現(xiàn)在缺血側(cè)供體細胞能夠存活,免疫組織化學單染和雙染后發(fā)現(xiàn)在缺血模型的壞死區(qū)及壞死邊緣區(qū)都檢測到β-Gal陽性的供體細胞,部分細胞還同時表達神經(jīng)元和神經(jīng)膠質(zhì)細胞特異性蛋白NeuN和GFAP。結(jié)論小鼠骨髓內(nèi)的NA-BM-MSC在缺血損傷的腦內(nèi)能夠存活、遷移,部分細胞還能分化為成熟的神經(jīng)元或神經(jīng)膠質(zhì)細胞參與腦損傷的修復,為下一步作為種子細胞用于移植修復實驗提供了保證。
[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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R329
本文編號：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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R329
【引證文獻】
相關(guān)期刊論文 前1條
1 肖龍艷;傅晉翔;張學光;陳秋;張宏;王盼君;孫諭;王明元;古彥錚;;胎源性非黏附骨髓基質(zhì)細胞的特性[J];中國組織工程研究;2012年01期
，本文編號：2508532
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