本文選題：人腦脊液　切入點：人類脂肪間充質(zhì)干細胞　出處：《華中科技大學》2014年博士論文

【摘要】：對于中樞神經(jīng)系統(tǒng)多種難治性疾病而言,間充質(zhì)干細胞和神經(jīng)前體細胞被認為是理想的細胞資源,這兩類細胞具有促進組織修復、攜帶特殊靶向藥物、對損傷部位定向遷移的能力。然而在中樞神經(jīng)系統(tǒng)微環(huán)境的影響下,這些細胞資源原有的特殊性能是否會發(fā)生變化,目前尚不十分清楚。由于腦脊液是構(gòu)成人類中樞神經(jīng)系統(tǒng)微環(huán)境的重要組成部分,因此研究這兩類細胞在腦脊液的環(huán)境下,其特性的改變,將對于干細胞用于中樞系統(tǒng)的臨床試驗至關重要。本研究主要探索人腦脊液對人類脂肪間充質(zhì)干細胞及胎兒神經(jīng)前體細胞在增殖、凋亡、遷移等方面的影響,從而為優(yōu)化干細胞在中樞神經(jīng)系統(tǒng)的臨床應用提供參考。 人腦脊液對人類脂肪間充質(zhì)干細胞及胎兒神經(jīng)前體細胞增殖及凋亡的影響 目的：觀察人腦脊液(Human cerebrospinal fluid, H-CSF)對人脂肪間充質(zhì)干細胞(human adipose-derived mesenchymal stem cells, hAMSCs)及胎兒神經(jīng)前體細胞(human fetal-derived neural progenitor cells, hfNPCs)增殖及凋亡的影響。 方法：收集正常顱內(nèi)壓腦積水(normal pressure hydrocephalus)或先天性腦積水(congenital hydrocephalus)病人的腦脊液(H-CSF),同時配制人工腦脊液(Artificial cerebrospinal fluid, A-CSF)。用100%MSC完全培養(yǎng)基(對照組,CtrI)、25%A-CSF+75%MSC完全培養(yǎng)基(人工腦脊液組,A-CSF)以及25%H-CSF+75%MSC完全培養(yǎng)基(人腦脊液組,H-CSF)培養(yǎng)hAMSCs;用100%NPC完全培養(yǎng)基(對照組,Ctrl)、25%A-CSF+75%NPC完全培養(yǎng)基(人工腦脊液組,A-CSF)以及25%H-CSF+75%NPC完全培養(yǎng)基(人腦脊液組,H-CSF)培養(yǎng)hfNPCs。用MTT、免疫熒光染色(Ki67)測定hAMSCs和hfNPCs的增值能力；用流式細胞檢測Propidium iodide (PI)和Annexin V,并而分析兩類細胞的凋亡比例。 結(jié)果：與Ctrl和A-CSF相比,hAMSCs在H-CSF環(huán)境下,增殖力能明顯增加(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)。同時,相對于Ctrl和A-CSF組,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,用不同濃度的H-CSF (0%,25%,50%,75%,100%)培養(yǎng)hAMSCs和hfNPCs,其增殖能力有所不同。 結(jié)論：H-CSF可促進hAMSCs及hfNPCs的增殖,并抑制兩類細胞的凋亡。 人腦脊液對人類脂肪間充質(zhì)干細胞和胎兒神經(jīng)前體細胞運動及遷移的影響 目的：探討H-CSF對hAMSCs及hfNPCs運動速度、運動距離以及遷移能力的影響及其相關特點。 方法：將hAMSCs及hfNPCs在不同條件下(Ctrl, A-CSF及H-CSF)培養(yǎng),并種植于底面為納米材料的小憩室內(nèi),動態(tài)觀察兩類細胞的運動速度和運動距離。用MATLAB軟件分析不同培養(yǎng)條件對hAMSCs和hfNPCs運動能力的影響。同時用Boyden transwell chambers實驗測定兩類細胞在不同培養(yǎng)條件下,對膠質(zhì)母細胞瘤(Glioblastoma, GBM)條件培養(yǎng)基的遷移能力,并分析H-CSF對hAMSCs和hfNPCs遷移能力的影響。 結(jié)果：通過對兩類細胞在納米材料表面的運動狀態(tài)的觀察,并利用MATLAB軟件分析hAMSCs和hfNPCs的速度和距離。我們發(fā)現(xiàn),相對Ctrl和A-CSF組,H-CSF可增加hAMSCs的運動速度和運動距離(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)。同時H-CSF對hfNPCs的運動能力亦有促進作用(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)。通過Boyden transwell chambers實驗發(fā)現(xiàn),H-CSF可提高hAMSCs和hfNPCs的對GBM條件培養(yǎng)基的遷移能力(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,本研究發(fā)現(xiàn),在預處理時間一定的情況下(48hours),培養(yǎng)基中H-CSF的濃度越高,hAMSCs和hfNPCs對GBM條件培養(yǎng)基的遷移能力越強。 結(jié)論：H-CSF可提高hAMSCs及hfNPCs的運動速度和運動距離,并可促進hAMSCs和hfNPCs的遷移能力。人腦脊液中IGF-1對人類脂肪間充質(zhì)干細胞和胎兒神經(jīng)前體細胞特性的影響 目的：探討H-CSF中胰島素樣生長因子1(IGF-1)對hAMSCs及hfNPCs特性的影響及其機制。 方法：測定所收集H-CSF標本中IGF-1的含量,并用IGF-1拮抗劑(IGF-1inhibitor)拮抗H-CSF中的IGF-1。將hAMSCs及hfNPCs在不同條件下(Ctrl, Ctrl+IGF-1inhibitor, H-CSF及H-CSF+IGF-1inhibitor)培養(yǎng),觀察hAMSCs及hfNPCs的增殖、凋亡、運動及遷移能力,并探討其部分機制。 結(jié)果：H-CSF中的IGF-1可影響hAMSCs和hfNPCs的增殖能力及凋亡比例。MTT實驗證實,當IGF-1inhibiotr加入H-CSF時,hAMSCs及hfNPCs的增殖能力相對未加入IGF-1inhibitor組(H-CSF組)明顯下降(hAMSCs, H-CSF+IGF-1inhibitor vs.H-CSF:p0.05; hfNPCs, H-CSF+IGF-1inhibitor vs. H-CSF:p0.05)。流式細胞實驗證實,H-CSF中的IGF-1亦可影響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)。同時,本研究發(fā)現(xiàn),H-CSF中的IGF-1可調(diào)控hAMSCs及hfNPCs的遷移能力,H-CSF+IGF-1inhibitor組相對于H-CSF組而言,兩類細胞的遷移能力明顯降低(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相對于H-CSF組,其hAMSCs和hfNPCs細胞膜表面表達的基質(zhì)細胞衍生因子受體(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)。 結(jié)論：H-CSF中的IGF-1可影響hAMSCs及hfNPCs的增殖、凋亡及遷移能力,拮抗H-CSF中的IGF-1后,兩類細胞的增殖能力下降,凋亡比例上升,遷移能力減弱,細胞表面與遷移相關的CXCR4蛋白表達降低。
[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.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R741

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8 賈雪梅;逆轉(zhuǎn)錄病毒載體介導Fcy::Fur融合基因聯(lián)合5-FC對人卵巢癌細胞株HO-8910殺傷作用的初步研究[D];南京醫(yī)科大學;2004年

9 陳s，

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