本文關(guān)鍵詞： NF-κB 轉(zhuǎn)染 凋亡 存活 神經(jīng)前體細(xì)胞 永生化 基因 bcl-2 細(xì)胞培養(yǎng) 神經(jīng)前體細(xì)胞 細(xì)胞分化 時間特異性 空間特異性　出處：《華中科技大學(xué)》2007年博士論文　論文類型：學(xué)位論文

【摘要】： 研究背景和目的 缺血缺氧性腦損傷是嚴(yán)重威脅人類生命健康的常見疾病之一,其導(dǎo)致神經(jīng)組織缺失,傳統(tǒng)的藥物治療療效難以令人滿意。神經(jīng)干細(xì)胞或前體細(xì)胞的發(fā)現(xiàn)為組織再生修復(fù)帶來了希望。神經(jīng)干細(xì)胞或前體細(xì)胞是一類具有自我更新能力、增殖能力和多向分化潛能的未成熟細(xì)胞。它在神經(jīng)系統(tǒng)發(fā)育、正常腦功能的維護(hù)和腦損傷的修復(fù)中發(fā)揮了重要的作用。但是研究發(fā)現(xiàn),由于局部缺血缺氧性微環(huán)境的改變,內(nèi)源性遷移或外源性移植的神經(jīng)干細(xì)胞或前體細(xì)胞存活數(shù)量少、存活時間短,難以發(fā)揮有效的修復(fù)作用。核因子-κB (NF-κB)是一種核轉(zhuǎn)錄因子,能調(diào)控一系列基因的表達(dá)。它由5種亞基組成,p65和p50是其中最重要的兩個亞基。NF-κB在神經(jīng)系統(tǒng)中廣泛表達(dá),在缺血狀態(tài)下,缺血灶周圍組織中NF-κB被激活,但其發(fā)揮保護(hù)作用還是促凋亡作用,目前尚存爭議。以上這些現(xiàn)象使我們關(guān)注到這樣一個問題,缺血灶周圍NF-κB激活對神經(jīng)干細(xì)胞或前體細(xì)胞的存活有無影響?干預(yù)NF-κB能否成為改善神經(jīng)前體細(xì)胞腦損傷修復(fù)作用的新策略? 本課題擬以永生化神經(jīng)前體細(xì)胞株(INPC)為細(xì)胞模型,通過NF-κB亞基p50、p65基因轉(zhuǎn)染探討其對神經(jīng)前體細(xì)胞存活的影響及其機(jī)制,進(jìn)而明確NF-κB在神經(jīng)發(fā)生學(xué)和缺血缺氧性神經(jīng)前體細(xì)胞損傷中的作用,為進(jìn)一步探討神經(jīng)前體細(xì)胞修復(fù)治療缺血性腦損傷奠定理論基礎(chǔ)。 研究方法 1.介導(dǎo)外源基因?qū)胗郎窠?jīng)前體細(xì)胞株非病毒載體的選擇 用非病毒載體陽離子脂質(zhì)體Lipofectamine 2000,TRANSfection或陽離子聚合物Sofast分別負(fù)載質(zhì)粒EGFP-C1轉(zhuǎn)染INPC,轉(zhuǎn)染后24 h檢測轉(zhuǎn)染效率。對轉(zhuǎn)染效率最高的一種載體,觀察其轉(zhuǎn)染后12、24、48和72 h的轉(zhuǎn)染效率,確定EGFP表達(dá)最高峰。用臺盼藍(lán)排斥試驗檢測轉(zhuǎn)染和未轉(zhuǎn)染細(xì)胞的活力。質(zhì)粒EGFP-C1轉(zhuǎn)染INPC后,經(jīng)新霉素類似物G418篩選,挑選穩(wěn)定細(xì)胞克隆INPC/EGFP,并觀察其EGFP陽性率。應(yīng)用巢蛋白(Nestin)抗體鑒定INPC/EGFP。胎牛血清誘導(dǎo)INPC/EGFP分化,觀察分化后細(xì)胞的形態(tài)及EGFP表達(dá)。免疫細(xì)胞化學(xué)檢測脂質(zhì)體Lipofectamine 2000介導(dǎo)RcCMV-p65質(zhì)粒瞬時轉(zhuǎn)染INPC后p65的表達(dá)。 2.NF-κB亞基p50、p65基因修飾的永生化神經(jīng)前體細(xì)胞株的構(gòu)建 采用脂質(zhì)體Lipofectamine 2000將p50和p65編碼質(zhì)粒RcCMV-p50、RcCMV-p65和對照空質(zhì)粒Rc/CMV分別轉(zhuǎn)染永生化大鼠神經(jīng)前體細(xì)胞株(INPC)。經(jīng)G418篩選,挑選陽性克隆。采用有限稀釋法分離單細(xì)胞克隆并擴(kuò)大培養(yǎng)。采用免疫細(xì)胞化學(xué)和免疫印跡法挑選p50或p65表達(dá)量最高的單細(xì)胞克隆。瞬時轉(zhuǎn)染RcCMV-p50質(zhì)粒于已挑選出的p65表達(dá)量最高的單細(xì)胞克隆。RT-PCR檢測新霉素(Neo)基因、p50或p65基因轉(zhuǎn)錄水平的表達(dá)。免疫印跡法檢測p50或p65基因蛋白的表達(dá)。 3.轉(zhuǎn)染NF-κB亞基后不同二聚體的形成、分布及轉(zhuǎn)錄活性 將NF-κB亞基p50、p65基因修飾后的不同永生化神經(jīng)前體細(xì)胞株進(jìn)行凝膠電泳遷移實驗(EMSA)檢測胞核內(nèi)NF-κB的DNA結(jié)合活性。免疫印跡分析胞漿NF-κB抑制蛋白(IκBα)的表達(dá)。免疫細(xì)胞化學(xué)法檢測p50和p65分別在細(xì)胞中的含量及胞漿胞核的分布。運用熒光素酶報告系統(tǒng)檢測各細(xì)胞株NF-κB轉(zhuǎn)錄活性。 4.NF-κB對永生化神經(jīng)前體細(xì)胞株存活的影響及相關(guān)作用機(jī)理 在正常及缺氧缺糖1 h或3 h條件下,熒光素酶報告系統(tǒng)檢測各細(xì)胞株的NF-κB轉(zhuǎn)錄活性,Annexin V和碘化丙錠( PI)雙標(biāo)記法流式細(xì)胞儀檢測各細(xì)胞株凋亡率。缺氧缺糖6 h后,雙苯甲亞胺Hoechst 33342染細(xì)胞核,觀察細(xì)胞形態(tài)學(xué)改變,PI單染流式細(xì)胞術(shù)檢測細(xì)胞凋亡率。缺氧缺糖12h后,四甲基偶氮唑鹽比色試驗(MTT試驗)測定細(xì)胞存活率。在正常及缺氧缺糖6 h條件下,免疫印跡分析各細(xì)胞株NF-κB凋亡相關(guān)靶基因Bcl-2, Bax, Bcl-Xs/l, FAS-L,p53, XIAP和Actin蛋白的表達(dá),通過Amersham公司ImageQuant TL軟件分析條帶的光密度值,計算各細(xì)胞株Bax/Bcl-2比值。 5.統(tǒng)計學(xué)分析 采用SPSS11.5統(tǒng)計軟件分析數(shù)據(jù),計量資料以均數(shù)±標(biāo)準(zhǔn)差(±s)表示。組間多個樣本均數(shù)的比較采用重復(fù)測量的雙因素方差分析或單因素方差分析,P0.05為差異有統(tǒng)計學(xué)意義。 研究結(jié)果 1.Lipofectamine 2000,TRANSfection和Sofast轉(zhuǎn)染INPC后24 h,轉(zhuǎn)染效率分別為(25.5±2.9)%,(4.0±1.7)%,(7.9±1.4)%,Lipofectamine 2000的轉(zhuǎn)染效率最高。其轉(zhuǎn)染后12、24、48和72 h的轉(zhuǎn)染效率分別為(17.1±0.7)%,(25.5±2.9)%,(19.4±0.9)%,(15.6±1.4)%,EGFP在轉(zhuǎn)染后24 h表達(dá)最高。INPC/EGFP中,EGFP陽性率約為95%。INPC/EGFP巢蛋白表達(dá)陽性。其分化后呈神經(jīng)元或星形膠質(zhì)細(xì)胞樣,且胞體及突起中仍可見綠色熒光。Lipofectamine 2000介導(dǎo)RcCMV-p65質(zhì)粒的瞬時轉(zhuǎn)染后,部分細(xì)胞呈p65陽性染色,陽性率約為15%。 2.通過穩(wěn)定篩選和有限稀釋法分離得到穩(wěn)定轉(zhuǎn)染p65的單細(xì)胞克隆A12、A13、B11、C21、C22和E2。其p65免疫染色和免疫印跡分析均呈陽性,但C21的p65表達(dá)強(qiáng)于其它克隆。將C21克隆命名為INPC/p65細(xì)胞株,進(jìn)行后續(xù)實驗,并再次瞬時轉(zhuǎn)染RcCMV-p50質(zhì)粒,獲得INPC/p50p65細(xì)胞株。同樣得到穩(wěn)定轉(zhuǎn)染RcCMV-p50、RcCMV質(zhì)粒的單細(xì)胞克隆,分別命名為INPC/p50和INPC/CMV細(xì)胞株。Neo基因RT-PCR顯示,對照空質(zhì)粒已成功轉(zhuǎn)染至INPC/CMV。RT-PCR和免疫印跡分析顯示,p50和p65基因均能在穩(wěn)定轉(zhuǎn)染相應(yīng)質(zhì)粒的細(xì)胞株中高效表達(dá)。 3.EMSA表明,INPC/p50、INPC/p65和INPC/p50p65細(xì)胞胞核中分別形成p50同源二聚體、p65同源二聚體、p50p65異源二聚體和p50同源二聚體。INPC/p65和INPC/p50p65細(xì)胞株中,IκBα在胞漿中表達(dá)升高。免疫細(xì)胞化學(xué)顯示INPC/p50細(xì)胞株中主要為強(qiáng)的胞核p50陽性染色,然而INPC/p65細(xì)胞株中主要為胞漿p65陽性染色,但在一些細(xì)胞中也可見胞核p65陽性染色。NF-κB轉(zhuǎn)錄活性在INPC/p50細(xì)胞株中輕度增高,在INPC/p65、INPC/p50p65細(xì)胞株中明顯的升高,其中以INPC/p65細(xì)胞株最高(均P0.05)。 4.κB依賴性的熒光素酶活性在不同的NF-κB亞基轉(zhuǎn)染細(xì)胞株間及不同的缺氧缺糖處理組間(對照組,缺氧缺糖1 h組,缺氧缺糖3 h組)有顯著性差異。Annexin V-FITC凋亡檢測發(fā)現(xiàn),INPC/p65和INPC/p50p65細(xì)胞株發(fā)生了自發(fā)性的細(xì)胞凋亡,而且對缺氧缺糖刺激更為敏感。缺氧缺糖6 h后,各細(xì)胞株可觀察到凋亡形態(tài)學(xué)改變,流式細(xì)胞術(shù)檢測證實INPC/p65和INPC/p50p65細(xì)胞株的凋亡率升高(P0.05),并且缺氧缺糖12 h后,細(xì)胞存活率低于其它細(xì)胞株(P0.05)。在正常及缺氧缺糖處理6 h條件下, Bax和Bcl-2蛋白的表達(dá)及兩者的比值在INPC/p65和INPC/p50p65細(xì)胞株中升高(P0.05)。 研究結(jié)論 1.Lipofectamine 2000可高效簡便地轉(zhuǎn)染INPC。這為探討轉(zhuǎn)NF-κB基因?qū)τ郎窠?jīng)前體細(xì)胞的生物學(xué)特性影響奠定了實驗基礎(chǔ)。 2.成功構(gòu)建了NF-κB亞基p50、p65基因修飾的永生化大鼠神經(jīng)前體細(xì)胞株。 3.p50、p65的高表達(dá)可逃逸胞漿內(nèi)內(nèi)源性IκBα的阻滯作用,導(dǎo)致胞核內(nèi)形成不同的NF-κB二聚體,并可直接升高NF-κB轉(zhuǎn)錄活性。 4.轉(zhuǎn)染NF-κB不同亞基后升高的NF-κB轉(zhuǎn)錄活性導(dǎo)致神經(jīng)前體細(xì)胞發(fā)生自發(fā)性凋亡,并對凋亡性刺激更加敏感,而這一現(xiàn)象可能通過Bcl-2家族依賴性途徑介導(dǎo)。 研究總結(jié) 本課題通過脂質(zhì)體基因轉(zhuǎn)染技術(shù)構(gòu)建了NF-κB亞基p50、p65基因修飾的永生化神經(jīng)前體細(xì)胞株,并證實p50、p65的高表達(dá)可逃逸胞漿內(nèi)內(nèi)源性IκBα的阻滯作用,導(dǎo)致胞核內(nèi)形成不同的NF-κB二聚體,并直接升高NF-κB轉(zhuǎn)錄活性,從而觀察到升高的NF-κB轉(zhuǎn)錄活性導(dǎo)致神經(jīng)前體細(xì)胞自發(fā)凋亡,并對凋亡性刺激更加敏感,這一現(xiàn)象可能通過Bcl-2家族依賴性途徑介導(dǎo)。上述研究有助于我們明確NF-κB在神經(jīng)發(fā)生學(xué)和缺血缺氧性神經(jīng)干細(xì)胞或前體細(xì)胞損傷中的作用,抑制NF-κB活性可能成為改善神經(jīng)前體細(xì)胞腦損傷修復(fù)作用的新策略。 目的研究人胚不同胚齡或腦區(qū)神經(jīng)前體細(xì)胞(NPC)體外培養(yǎng)及增殖分化特性。方法取人胚腦組織原代細(xì)胞分為小胚齡全腦組、較大胚齡全腦組、較大胚齡新皮質(zhì)組、較大胚齡紋狀體組、較大胚齡間腦組、較大胚齡中腦組、較大胚齡后腦組和較大胚齡延髓組8組,懸浮培養(yǎng)。鑒定細(xì)胞球巢蛋白抗原的表達(dá),分化及自我更新能力。觀察各組培養(yǎng)細(xì)胞的生長、增殖狀況。運用免疫熒光細(xì)胞化學(xué)法比較小胚齡全腦組、較大胚齡全腦組、較大胚齡新皮質(zhì)組、較大胚齡紋狀體組及較大胚齡間腦組神經(jīng)球分化后,神經(jīng)元及星形膠質(zhì)細(xì)胞的比例。結(jié)果各組培養(yǎng)出的懸浮細(xì)胞球巢蛋白抗原陽性,可分化為微管相關(guān)蛋白2(MAP2)或膠質(zhì)纖維酸性蛋白(GFAP)陽性細(xì)胞,且5-溴-2-脫氧尿苷(BrdU)摻入實驗陽性。培養(yǎng)一周,較大胚齡紋狀體組的神經(jīng)球數(shù)目最多,形態(tài)最規(guī)則,其次分別為較大胚齡間腦組、小胚齡全腦組、較大胚齡全腦組、較大胚齡新皮質(zhì)組,其它組僅見個別神經(jīng)球。采用有限稀釋法可從較大胚齡紋狀體組挑選單細(xì)胞克隆球。小胚齡全腦組、較大胚齡全腦組、較大胚齡新皮質(zhì)組、較大胚齡紋狀體組及較大胚齡間腦組NPC誘導(dǎo)分化后,MAP2或GFAP陽性細(xì)胞率組間比較差異無顯著性。結(jié)論從不同胚齡和腦區(qū)的中樞神經(jīng)系統(tǒng)來源的人神經(jīng)前體細(xì)胞均能在體外擴(kuò)增,針對不同胚齡可采用不同的原代取材方法,小胚齡可取全腦組織培養(yǎng),而大胚齡則可取紋狀體等特定腦區(qū)進(jìn)行培養(yǎng)。不同胚齡或腦區(qū)來源的NPC星形膠質(zhì)細(xì)胞及神經(jīng)元分化比例一致。
[Abstract]:Background and purpose of research
Hypoxic ischemic brain damage is one of the common diseases of a serious threat to human life and health, which leads to the loss of neural tissue, the traditional drug treatment method is not satisfactory. The neural stem cells or progenitor cells for tissue regeneration and repair brings hope. Neural stem cells are self-renewal ability, immature the ability of cell proliferation and differentiation potential. It is in the development of nervous system, play an important role in the maintenance and repair of normal brain function and brain injury. But the study found that due to ischemia or hypoxia microenvironment change, migration of endogenous or exogenous transplanted neural stem cells or progenitor cell survival number the survival time is short, it is difficult to play an effective role. Repair of nuclear factor kappa B (NF- K B) is a nuclear transcription factor that can regulate the expression of a series of genes. It is composed of 5 subunits, p65 And p50 is one of the most important of the two subunits of.NF- kappa B is widely expressed in the nervous system, under ischemic condition, surrounding tissue ischemia in NF- kappa B was activated, but it still play a protective role in apoptosis, remains controversial. These phenomena make us pay attention to such a problem, ischemia around the focus of NF- kappa B activation on the survival of neural stem cells or progenitor cells have no effect? The intervention of NF- kappa B can become a new strategy to improve the neural precursor cells in brain injury?
This project is intended to immortalized neural progenitor cells (INPC) cells model by NF- kappa B subunit P50, p65 gene transfection to investigate its effects on neural precursor cell survival and its mechanism, and then clear NF- kappa B on neurogenesis and neural precursor cells in hypoxic ischemic injury. For the further study of neural precursor cells to repair in treatment of ischemic brain injury and laid a theoretical basis.
research method
Selection of 1. non viral vectors mediated by exogenous gene into immortalized neural precursor cell line
Using non viral vector cationic liposome Lipofectamine 2000, TRANSfection or Sofast were loaded cationic polymer transfection of plasmid EGFP-C1 INPC, 24 h after transfection. The transfection efficiency was detected on the transfection efficiency of a vector with the highest observed after transfection, the transfection efficiency of H and 12,24,48 72, to determine the expression peak of EGFP. Using the trypan blue exclusion test activity transfected and untransfected cells. After transfection of INPC plasmid EGFP-C1, the neomycin analogue G418 screening, selection of stable cell clones of INPC/EGFP, the positive rate of EGFP was observed. The application of nestin (Nestin) antibody INPC/EGFP INPC/EGFP. induced differentiation of fetal bovine serum, cell morphology and the expression of EGFP was observed after differentiation. The expression of immune cell chemistry detection of Lipofectamine 2000 liposome mediated RcCMV-p65 plasmid was transfected to INPC after p65.
Construction of an immortalized neural precursor cell line modified by 2.NF- kappa B subunit P50, p65 gene
Using liposome Lipofectamine 2000 P50 and p65 encoding plasmid RcCMV-p50, RcCMV-p65 and Rc/CMV respectively control empty plasmid transfected immortalized rat neural progenitor cells (INPC). After G418 screening, positive clones. With the method of limited dilution separation of single cell cloning and expand culture. The selection of P50 or p65 expression in single cell clone with the highest using immunocytochemistry and Western blotting. Transient transfection of RcCMV-p50 plasmid in selected p65 expression in single cell clone.RT-PCR detection with the highest amount of neomycin (Neo) gene, the expression of P50 or p65 gene transcription. To detect the expression of P50 or p65 protein by Western blotting.
3. the formation, distribution and transcriptional activity of different two polymer after transfection of NF- kappa B subunit
The NF- kappa B subunit P50, p65 gene modified different immortalized neural progenitor cells by electrophoretic mobility shift assays (EMSA) detected in the nucleus of NF- kappa B binding activity of DNA inhibitory protein. Analysis of cytoplasmic NF- kappa B immunoblotting (I kappa B alpha) expression of P50 and p65 detection. Immunocytochemical method respectively in the cell nucleus and cytoplasm content distribution. By using the luciferase reporter assay of each cell line NF- kappa B transcription activity.
The effect of 4.NF- kappa B on the survival of immortalized neural precursor cells and its related mechanism
Glucose deprivation for 1 h or 3 h in normal and hypoxic conditions, NF- kappa B transcription activity of luciferase reporter system to detect the cell lines Annexin, V and propidium iodide (PI) to detect the cell apoptosis rate of double labeling flow cytometry. OGD 6 h, Hoechst 33342 bisbenzimide staining the nucleus, cell morphological changes were observed by PI staining, cell apoptosis rate were detected by flow cytometry. 12h after OGD, four methyl thiazolyl tetrazolium colorimetric test (MTT test) to determine the cell survival rate. 6 h OGD in normal and hypoxia, B analysis of each cell line NF- kappa wither dead the related target gene Bcl-2, Bax, Bcl-Xs/l, FAS-L, p53 blotting, the expression of XIAP and Actin protein by ImageQuant TL analysis software, Amersham band densities, calculate the ratio of Bax/Bcl-2 cells.
5. statistical analysis
SPSS11.5 statistical software was used to analyze the data, and the measurement data were expressed by mean + standard deviation (+ s). Multiple sample mean comparisons between groups were repeated measure two factor ANOVA or one-way ANOVA, and P0.05 was statistically significant.
Research results
1.Lipofectamine 2000, TRANSfection 24 h and Sofast INPC after transfection, the transfection efficiency was (25.5 + 2.9)% and (4 + 1.7)% and (7.9 + 1.4)%, Lipofectamine 2000. The highest transfection efficiency after transfection, the transfection efficiency of H 12,24,48 and 72 respectively (17.1 + 0.7)%, (25.5 + 2.9)% and (19.4 + 0.9)% and (15.6 + 1.4)%, EGFP at 24 h after transfection was the highest in.INPC/EGFP, the positive rate of EGFP is about 95%.INPC/EGFP. The expression of nestin positive cells were neurons or astrocytes, transient transfection and cell bodies and processes are still visible green fluorescence 2000.Lipofectamine mediated RcCMV-p65 plasmid, some cells showed p65 positive staining, the positive rate is about 15%.
2. by stable screening and limited dilution method isolated single cell clones stably transfected with p65 A12, A13, B11, C21, C22 and E2. p65 staining and Western blot analysis were positive, but C21 p65 was stronger than other clones. The C21 clone named INPC/p65 cell lines, and for subsequent experiments. Again the transient transfection of RcCMV-p50 plasmid, INPC/p50p65 cell lines stably transfected with RcCMV-p50. Also, the single cell clone of RcCMV plasmid, named INPC/p50 and INPC/CMV cell strain.Neo gene RT-PCR showed that control empty plasmid was successfully transfected into INPC/ CMV.RT-PCR and Western blot analysis showed that P50 and p65 genes were highly expressed in transfected with the corresponding plasmid in cell lines.
3.EMSA INPC/p50, INPC/p65 and INPC/p50p65 showed that the cell nucleus were formed homodimer with two P50, two p65 homologous dimer, p50p65 heterologous dimer and two P50 homology two dimers.INPC/p65 and INPC/p50p65 cell lines, the expression of I kappa B alpha in the cytoplasm increased. Immunocytochemistry showed that as the main nuclear P50 strong positive staining of INPC/p50 cells, but INPC/p65 cells mainly in cytoplasm of p65 positive staining, but in some cells were also found in the nuclei of the positive staining of p65.NF- kappa B transcription activity in INPC/p50 cells increased slightly in INPC/p65, significantly increased INPC/p50p65 cells in the INPC/p65 cell line the highest (P0.05).
The 4. KB B dependent luciferase activity in NF- kappa B subunit in transfected cell lines between different and different hypoxia treatment group (control group, OGD 1 h group, OGD 3 h group) have detected significant differences.Annexin V-FITC apoptosis, INPC/p65 and INPC/p50p65 cell lines occurred spontaneous cell apoptosis, and to OGD stimulation is more sensitive to hypoxia and lack of glucose. After 6 h, the cells can be observed morphological changes of apoptosis, flow cytometry confirmed that the apoptosis of INPC/p65 and INPC/p50p65 cell lines was increased (P0.05), and 12 h after OGD, cell survival rate is lower than the other cell line (P0.05) in normal and hypoxia treatment under the condition of 6 h, the ratio of expression of Bax and Bcl-2 protein and both increased in INPC/p65 and INPC/p50p65 cell lines (P0.05).
research conclusion
1.Lipofectamine 2000 can efficiently and easily transfect INPC., which lays an experimental foundation for exploring the effect of NF- kappa B gene on the biological characteristics of immortalized neural progenitor cells.
2. the immortalized rat neural precursor cells modified by NF- kappa B subunit P50 and p65 gene were successfully constructed.
The high expression of 3.p50 and p65 can escape the blocking effect of endogenous I kappa B alpha in the cytoplasm, resulting in the formation of different NF- kappa B two dimers, and directly increase the transcriptional activity of NF- kappa B.
4., the activity of NF- kappa B transcriptional activity induced by different subunits of NF- kappa B can induce spontaneous apoptosis of neural precursor cells and is more sensitive to apoptotic stimuli, which may be mediated by Bcl-2 family dependent pathway.
Research Summary
This project constructed NF- kappa B subunit P50 by liposome transfection technique, p65 gene modified immortalized neural progenitor cells, and confirmed that P50, inhibition of the expression of p65 can run in the cytoplasm of endogenous I kappa B alpha in the nucleus, leads to the formation of different NF- kappa B two dimer and, the direct increase of NF- kappa B transcription activity, and observed elevated NF- kappa B transcription activity leads to spontaneous apoptosis of neural precursor cells, and more sensitive to apoptotic stimuli, this phenomenon may be the family of Bcl-2 dependent pathway. The study will help us to define NF- kappa B in neurogenesis and hypoxic ischemic neural stem cells or progenitor cells injury, inhibition of NF- K B activity may be a new strategy to improve the neural precursor cells in brain injury.
Objective to study the human embryo of different embryonic neural precursor cells or old brain regions (NPC) in vitro differentiation and proliferation characteristics. Methods the human fetal brain tissue cells into embryonic cerebral small groups, large embryo age whole brain group, large embryonic neocortex, larger embryonic striatum group, 14-17 the age group, large embryonic midbrain group, large embryo age group and cerebral medulla Group 8 large embryo age group, suspension culture. The expression of Nestin antigen identified cells, differentiation and self-renewal ability were observed. Cell growth, proliferation. Using immunofluorescence method to compare small embryo age of whole brain group the larger, embryonic whole brain group, large embryonic neocortex, larger embryo age group and larger embryonic striatum group diencephalic differentiation of neurospheres, neurons and astrocytes. The proportion of suspension cell culture results were nestin positive antigen, can differentiate into microtubule associated Protein 2 (MAP2) or glial fibrillary acidic protein (GFAP) positive cells, and 5- bromo -2- deoxyuridine (BrdU) incorporation test positive. One week culture, the number of neurospheres larger embryonic striatum group, the morphological rules, followed by the larger embryo age diencephalon group, small embryonic cerebral group the larger, embryonic whole brain group, large embryonic neocortex group, other groups were individual neurospheres. Using limited dilution method can choose single cell clone ball from the larger embryo age group. Small embryonic striatal brain group, large embryo age whole brain group, large embryonic neocortex, larger embryonic striatum the larger embryo age group and diencephalon group NPC after induction of differentiation, MAP2 or GFAP positive cell rate difference between groups was not significant. Conclusion from the human nerve central nervous system from different embryonic age and brain precursor cells were amplified in vitro and in different embryonic age can adopt different primary sampling method. Small embryo Age can be used for whole brain tissue culture, while large embryo age can be used to culture specific brain regions, such as striatum. NPC astrocytes and neurons differentiated from different embryonic ages or brain regions are in the same proportion.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R329

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