人脑脊液对人类脂肪间充质干细胞及胎儿神经前体细胞增殖、凋亡及迁移特性的影响

发布时间：2018-03-26 03:01

  本文选题：人脑脊液　切入点：人类脂肪间充质干细胞　出处：《华中科技大学》2014年博士论文

【摘要】：对于中枢神经系统多种难治性疾病而言,间充质干细胞和神经前体细胞被认为是理想的细胞资源,这两类细胞具有促进组织修复、携带特殊靶向药物、对损伤部位定向迁移的能力。然而在中枢神经系统微环境的影响下,这些细胞资源原有的特殊性能是否会发生变化,目前尚不十分清楚。由于脑脊液是构成人类中枢神经系统微环境的重要组成部分,因此研究这两类细胞在脑脊液的环境下,其特性的改变,将对于干细胞用于中枢系统的临床试验至关重要。本研究主要探索人脑脊液对人类脂肪间充质干细胞及胎儿神经前体细胞在增殖、凋亡、迁移等方面的影响,从而为优化干细胞在中枢神经系统的临床应用提供参考。 人脑脊液对人类脂肪间充质干细胞及胎儿神经前体细胞增殖及凋亡的影响 目的：观察人脑脊液(Human cerebrospinal fluid, H-CSF)对人脂肪间充质干细胞(human adipose-derived mesenchymal stem cells, hAMSCs)及胎儿神经前体细胞(human fetal-derived neural progenitor cells, hfNPCs)增殖及凋亡的影响。 方法：收集正常颅内压脑积水(normal pressure hydrocephalus)或先天性脑积水(congenital hydrocephalus)病人的脑脊液(H-CSF),同时配制人工脑脊液(Artificial cerebrospinal fluid, A-CSF)。用100%MSC完全培养基(对照组,CtrI)、25%A-CSF+75%MSC完全培养基(人工脑脊液组,A-CSF)以及25%H-CSF+75%MSC完全培养基(人脑脊液组,H-CSF)培养hAMSCs;用100%NPC完全培养基(对照组,Ctrl)、25%A-CSF+75%NPC完全培养基(人工脑脊液组,A-CSF)以及25%H-CSF+75%NPC完全培养基(人脑脊液组,H-CSF)培养hfNPCs。用MTT、免疫荧光染色(Ki67)测定hAMSCs和hfNPCs的增值能力；用流式细胞检测Propidium iodide (PI)和Annexin V,并而分析两类细胞的凋亡比例。 结果：与Ctrl和A-CSF相比,hAMSCs在H-CSF环境下,增殖力能明显增加(MTT assay, H-CSF vs.Ctrl:p0.05; H-CSF vs. A-CSF:p0.05; Ki67assay, H-CSF vs. Ctrl:p0.05; H-CSF vs. A-CSF:p0.05),凋亡比例下降(H-CSF vs. Ctrl:p0.05; H-CSF vs. A-CSF:p0.05)。同时,相对于Ctrl和A-CSF组,H-CSF可增加hfNPCs的增殖能力(MTT assay, H-CSF vs. Ctrl: p0.05; H-CSF vs. A-CSF:p0.05; Ki67assay, H-CSF vs. Ctrl:p0.05; H-CSF vs. A-CSF:p0.05),降低其凋亡比例(H-CSF vs. Ctrl:p0.05; H-CSF vs. A-CSF:p0.05)。此外,用不同浓度的H-CSF (0%,25%,50%,75%,100%)培养hAMSCs和hfNPCs,其增殖能力有所不同。 结论：H-CSF可促进hAMSCs及hfNPCs的增殖,并抑制两类细胞的凋亡。 人脑脊液对人类脂肪间充质干细胞和胎儿神经前体细胞运动及迁移的影响 目的：探讨H-CSF对hAMSCs及hfNPCs运动速度、运动距离以及迁移能力的影响及其相关特点。 方法：将hAMSCs及hfNPCs在不同条件下(Ctrl, A-CSF及H-CSF)培养,并种植于底面为纳米材料的小憩室内,动态观察两类细胞的运动速度和运动距离。用MATLAB软件分析不同培养条件对hAMSCs和hfNPCs运动能力的影响。同时用Boyden transwell chambers实验测定两类细胞在不同培养条件下,对胶质母细胞瘤(Glioblastoma, GBM)条件培养基的迁移能力,并分析H-CSF对hAMSCs和hfNPCs迁移能力的影响。 结果：通过对两类细胞在纳米材料表面的运动状态的观察,并利用MATLAB软件分析hAMSCs和hfNPCs的速度和距离。我们发现,相对Ctrl和A-CSF组,H-CSF可增加hAMSCs的运动速度和运动距离(speed, H-CSF vs. Ctrl: p0.05; H-CSF vs. A-CSF:p0.05; distance, H-CSF vs. Ctrl:p0.05; H-CSF vs. A-CSF:p0.05)。同时H-CSF对hfNPCs的运动能力亦有促进作用(speed, H-CSF vs. Ctrl:p0.05; H-CSF vs. A-CSF:p0.05; distance, H-CSF vs. Ctrl: p0.05; H-CSF vs. A-CSF:p0.05)。通过Boyden transwell chambers实验发现,H-CSF可提高hAMSCs和hfNPCs的对GBM条件培养基的迁移能力(hAMSCs, H-CSF vs. Ctrl:p0.05; H-CSF vs. A-CSF:p0.05; hfNPCs, H-CSF vs. Ctrl:p0.05; H-CSF vs. A-CSF:p0.05)。此外,本研究发现,在预处理时间一定的情况下(48hours),培养基中H-CSF的浓度越高,hAMSCs和hfNPCs对GBM条件培养基的迁移能力越强。 结论：H-CSF可提高hAMSCs及hfNPCs的运动速度和运动距离,并可促进hAMSCs和hfNPCs的迁移能力。人脑脊液中IGF-1对人类脂肪间充质干细胞和胎儿神经前体细胞特性的影响 目的：探讨H-CSF中胰岛素样生长因子1(IGF-1)对hAMSCs及hfNPCs特性的影响及其机制。 方法：测定所收集H-CSF标本中IGF-1的含量,并用IGF-1拮抗剂(IGF-1inhibitor)拮抗H-CSF中的IGF-1。将hAMSCs及hfNPCs在不同条件下(Ctrl, Ctrl+IGF-1inhibitor, H-CSF及H-CSF+IGF-1inhibitor)培养,观察hAMSCs及hfNPCs的增殖、凋亡、运动及迁移能力,并探讨其部分机制。 结果：H-CSF中的IGF-1可影响hAMSCs和hfNPCs的增殖能力及凋亡比例。MTT实验证实,当IGF-1inhibiotr加入H-CSF时,hAMSCs及hfNPCs的增殖能力相对未加入IGF-1inhibitor组(H-CSF组)明显下降(hAMSCs, H-CSF+IGF-1inhibitor vs.H-CSF:p0.05; hfNPCs, H-CSF+IGF-1inhibitor vs. H-CSF:p0.05)。流式细胞实验证实,H-CSF中的IGF-1亦可影响hAMSCs和hfNPCs的凋亡比例(hAMCs, H-CSF+IGF-1inhibitor vs.H-CSF:p0.05; hfNPCs, H-CSF+IGF-1inhibitor vs. H-CSF:p0.05)。同时,本研究发现,H-CSF中的IGF-1可调控hAMSCs及hfNPCs的迁移能力,H-CSF+IGF-1inhibitor组相对于H-CSF组而言,两类细胞的迁移能力明显降低(hAMCSs,H-CSF+IGF-1inhibitor vs. H-CSF:p0.05; hfNPCs, H-CSF+IGF-1inhibitor vs. H-CSF: p0.05)。此外,H-CSF+IGF-1inhibitor相对于H-CSF组,其hAMSCs和hfNPCs细胞膜表面表达的基质细胞衍生因子受体(Cxc Chemokin Receptor4, CXCR4)亦有下降(hAMSCs, H-CSF+IGF-1inhibitor vs. H-CSF:p0.05; hfNPCs, H-CSF+IGF-1inhibitorvs. H-CSF:p0.05)。 结论：H-CSF中的IGF-1可影响hAMSCs及hfNPCs的增殖、凋亡及迁移能力,拮抗H-CSF中的IGF-1后,两类细胞的增殖能力下降,凋亡比例上升,迁移能力减弱,细胞表面与迁移相关的CXCR4蛋白表达降低。
[Abstract]:For a variety of central nervous system refractory disease, mesenchymal stem cells and neural precursor cells is considered to be an ideal cell resource, these two kinds of cells can promote tissue repair, carrying special drug targeting ability on the site of injury. However, in the directional migration of the central nervous system effects of micro environment. Whether these cells are the original special properties will change, it is not very clear. The cerebrospinal fluid is an important part of the human central nervous system micro environment, so the research on the two kinds of cells in the cerebrospinal fluid environment, the characteristics of the change, for stem cells in clinical trials is the central nervous system. This study the main exploration of human cerebrospinal fluid on human adipose derived mesenchymal stem cells and fetal neural precursor cells in proliferation, apoptosis, migration and other aspects of the impact, so that the stem cells in the central God for optimization It provides reference for the systematic clinical application.
The effect of human cerebrospinal fluid on the proliferation and apoptosis of human adipose mesenchymal stem cells and fetal neural precursor cells
Objective: To observe the effects of cerebrospinal fluid (Human cerebrospinal, fluid, H-CSF) on human adipose derived mesenchymal stem cells (human adipose-derived mesenchymal stem cells, hAMSCs) and fetal neural precursor cells (human fetal-derived neural progenitor cells, hfNPCs) on proliferation and apoptosis.
Methods: normal intracranial pressure hydrocephalus (normal pressure hydrocephalus) or congenital hydrocephalus (congenital hydrocephalus) the patient's cerebrospinal fluid (H-CSF), at the same time, the artificial cerebrospinal fluid (Artificial cerebrospinal, fluid, A-CSF). Using 100%MSC complete medium (control group, CtrI), 25%A-CSF+75%MSC complete medium (ACSF group, A-CSF) and 25%H-CSF+75%MSC complete medium (CSF group, H-CSF) cultured hAMSCs; with 100%NPC complete culture medium (control group, Ctrl), 25%A-CSF+75%NPC complete medium (artificial cerebrospinal fluid group, A-CSF) and 25%H-CSF+75%NPC complete medium (CSF group, H-CSF hfNPCs.) cultured by MTT, immunofluorescence staining (Ki67) assay of hAMSCs hfNPCs and Propidium iodide capability; flow cytometry (PI) and Annexin V, and analysis the apoptosis rate of two cell.
Results: compared with Ctrl and A-CSF, hAMSCs in the H-CSF environment, the proliferation significantly increased (MTT assay, H-CSF vs.Ctrl:p0.05; H-CSF vs. A-CSF:p0.05; Ki67assay H-CSF, vs. Ctrl:p0.05; H-CSF vs. A-CSF:p0.05 (H-CSF), vs. Ctrl:p0.05 decreased the apoptosis rate of H-CSF; vs. A-CSF:p0.05). At the same time, compared with Ctrl and A-CSF group, H-CSF increase the proliferation of hfNPCs (MTT assay, H-CSF vs. Ctrl: P0.05; H-CSF vs. A-CSF:p0.05; H-CSF vs. Ctrl:p0.05; H-CSF Ki67assay, vs. A-CSF:p0.05), decreased the apoptosis ratio (H-CSF vs. Ctrl:p0.05 H-CSF; vs. A-CSF:p0.05). In addition, with different concentrations of H-CSF (0%, 25%, 50%, 75%, 100%) and hAMSCs training hfNPCs, the proliferation ability is different.
Conclusion: H-CSF can promote the proliferation of hAMSCs and hfNPCs and inhibit the apoptosis of two types of cells.
The effect of human cerebrospinal fluid on the movement and migration of human adipose mesenchymal stem cells and fetal neural precursor cells
Objective: To investigate the effects of H-CSF on the speed of movement, distance and mobility of hAMSCs and hfNPCs and their related characteristics.
Methods: hAMSCs and hfNPCs in different conditions (Ctrl, A-CSF and H-CSF) culture, and planted in the bottom to the nano nap room, movement speed and distance dynamic observation of two kinds of cells. Analysis of the influence of different culture conditions on hAMSCs and hfNPCs can use MATLAB software. At the same time, two kinds of cells under different culture conditions was determined by Boyden Transwell chambers experiment of glioblastoma (Glioblastoma, GBM) migration of conditioned medium, and analyze the effect of H-CSF on hAMSCs and hfNPCs migration.
Results: the two kinds of cells in the state of motion of nano materials surface observation and analysis of the hAMSCs and hfNPCs velocity and distance by using the MATLAB software. We found that, compared with Ctrl and A-CSF group, H-CSF hAMSCs can increase the movement speed and distance (speed, H-CSF vs. Ctrl: P0.05; H-CSF vs. A-CSF:p0.05; distance, H-CSF vs. Ctrl:p0.05; H-CSF vs. A-CSF:p0.05). At the same time exercise the ability of H-CSF to hfNPCs also had a role in promoting (speed, H-CSF vs. Ctrl:p0.05; H-CSF vs. A-CSF:p0.05; distance H-CSF, vs. Ctrl: P0.05; H-CSF vs. A-CSF:p0.05 Boyden Transwell chambers). The experiment shows that H-CSF can improve hAMSCs and hfNPCs migration of GBM medium conditions (hAMSCs, H-CSF vs. Ctrl:p0.05; H-CSF vs. A-CSF:p0.05; H-CSF vs. Ctrl:p0.05; H-CSF hfNPCs, vs. A-CSF:p0.05). In addition, the study found that in pretreatment In a certain period of time (48hours), the higher the concentration of H-CSF in the medium, the stronger the migration ability of hAMSCs and hfNPCs to the GBM conditioned medium.
Conclusion: H-CSF can increase the movement speed and distance of hAMSCs and hfNPCs, and promote the migration ability of hAMSCs and hfNPCs. The effect of IGF-1 in human cerebrospinal fluid on the characteristics of human adipose derived mesenchymal stem cells and fetal neural progenitor cells.
Objective: To investigate the effect of insulin like growth factor 1 (IGF-1) on the characteristics of hAMSCs and hfNPCs in H-CSF and its mechanism.
Methods: to determine the content of IGF-1 H-CSF were collected, and IGF-1 antagonist (IGF-1inhibitor) antagonist H-CSF in IGF-1. hAMSCs and hfNPCs in different conditions (Ctrl, Ctrl+IGF-1inhibitor, H-CSF and H-CSF+IGF-1inhibitor) culture, proliferation, observation of hAMSCs and the apoptosis of hfNPCs, motility and migration ability, and to explore its mechanism.
Results: H-CSF IGF-1 can affect the ratio of proliferation and apoptosis in hAMSCs and hfNPCs.MTT experiments confirmed that when IGF-1inhibiotr joined H-CSF, hAMSCs and hfNPCs of the relative proliferation without IGF-1inhibitor group (group H-CSF) were significantly decreased (hAMSCs H-CSF+IGF-1inhibitor, vs.H-CSF:p0.05 hfNPCs, H-CSF+IGF-1inhibitor vs.; H-CSF:p0.05). Flow cytometry experiments confirmed that H-CSF the IGF-1 can affect hAMSCs and hfNPCs (hAMCs, H-CSF+IGF-1inhibitor, apoptosis rate of vs.H-CSF:p0.05; hfNPCs, H-CSF+IGF-1inhibitor vs. H-CSF:p0.05). At the same time, the study found that H-CSF in IGF-1 migration regulation of hAMSCs and hfNPCs, H-CSF+IGF-1inhibitor group compared with H-CSF group, the migration ability of two kinds of cells was significantly decreased (hAMCSs, H-CSF+IGF-1inhibitor vs. H-CSF:p0.05 hfNPCs; H-CSF+IGF-1inhibitor vs. H-CSF:, P0.05). In addition, H-CSF+ IGF-1inhibitor relative to H-CSF group, the expression of Cxc Chemokin Receptor4 (CXCR4) on the surface of hAMSCs and hfNPCs cell membrane also decreased (hAMSCs, H-CSF+IGF-1inhibitor vs., hAMSCs).
Conclusion: IGF-1 in H-CSF can affect the proliferation, apoptosis and migration ability of hAMSCs and hfNPCs, and antagonize IGF-1 in H-CSF. After that, the proliferation ability of two kinds of cells decreases, the proportion of apoptosis increases, the migration ability decreases, and the expression of CXCR4 related to migration is reduced.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R741

【共引文献】

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9 陈s，

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