發(fā)布時(shí)間：2018-06-04 15:46

  本文選題：血管內(nèi)皮生長(zhǎng)因子 + 少突膠質(zhì)前體細(xì)胞��； 參考：《第三軍醫(yī)大學(xué)》2011年碩士論文

【摘要】：研究背景 近年來(lái)多項(xiàng)研究發(fā)現(xiàn)血管內(nèi)皮細(xì)胞生長(zhǎng)因子( vascular epthelial growth factor, VEGF)在神經(jīng)科學(xué)領(lǐng)域具有重要的作用。一方面,大量研究證據(jù)證明VEGF正常生長(zhǎng)發(fā)育過(guò)程中對(duì)神經(jīng)細(xì)胞、膠質(zhì)細(xì)胞具有神經(jīng)營(yíng)養(yǎng)和神經(jīng)保護(hù)作用,對(duì)神經(jīng)干細(xì)胞能夠起到促進(jìn)增殖和存活作用[1]。另一方面,VEGF在缺血性腦卒中等病理?xiàng)l件下能減輕腦損傷,促進(jìn)受損神經(jīng)細(xì)胞修復(fù)和神經(jīng)細(xì)胞再生[1]�？梢哉f(shuō)VEGF在中樞神經(jīng)系統(tǒng)中的復(fù)雜多樣作用使得人們對(duì)其研究的興趣日益濃厚。 自從1983年首次發(fā)現(xiàn)VEGF以來(lái),VEGF一直以來(lái)被認(rèn)為是脊椎動(dòng)物發(fā)育和一些常見(jiàn)慢性病中血管形成的重要調(diào)節(jié)因素[2]。最初發(fā)現(xiàn),VEGF在大鼠發(fā)育過(guò)程中以血管發(fā)生的方式促使血管內(nèi)皮細(xì)胞聚集形成血管網(wǎng)[3]。其生物學(xué)功能通過(guò)VEGFR2 (KDR/Flk-1)、VEGFR1 (Flt-1)兩種蛋白激酶受體[4]以及跨膜蛋白家族(NPs)[5]發(fā)揮作用。研究發(fā)現(xiàn)VEGF在大鼠胚胎發(fā)育第7天就可被檢測(cè)到,在隨后的中樞神經(jīng)系統(tǒng)發(fā)育過(guò)程中,VEGF主要表達(dá)于間充質(zhì)和頭部的神經(jīng)外胚層。隨著胚胎不斷發(fā)育,表達(dá)VEGF的細(xì)胞有神經(jīng)干細(xì)胞、神經(jīng)膠質(zhì)細(xì)胞和血管內(nèi)皮細(xì)胞,其中以分化中的神經(jīng)干細(xì)胞表達(dá)水平最高。成體階段中樞神經(jīng)系統(tǒng)發(fā)育成熟,VEGF表達(dá)逐漸減少,限制于脈絡(luò)叢、最后區(qū)、小腦顆粒細(xì)胞[6] [7-8]等區(qū)域。 盡管有研究認(rèn)為VEGF在胚胎發(fā)育期具有誘導(dǎo)新生血管形成和神經(jīng)發(fā)生(neurogenesis)的雙重作用,可能通過(guò)旁分泌和自分泌兩種方式作用共同影響胚胎神經(jīng)系統(tǒng)的生長(zhǎng)發(fā)育[9],但目前研究者對(duì)VEGF是否具有獨(dú)立的神經(jīng)營(yíng)養(yǎng)作用還存在爭(zhēng)議。而在成體的神經(jīng)發(fā)生中,一些研究證實(shí)內(nèi)皮細(xì)胞可以通過(guò)創(chuàng)造微環(huán)境或血管池[10]的方式來(lái)促進(jìn)特定腦區(qū)神經(jīng)干細(xì)胞的增殖。在體外培養(yǎng)環(huán)境下,VEGF則可以促進(jìn)星形膠質(zhì)細(xì)胞、雪旺細(xì)胞、小膠質(zhì)細(xì)胞以及皮質(zhì)神經(jīng)元的增殖;還可以保護(hù)海馬、皮層、周?chē)杏X(jué)神經(jīng)元和一些神經(jīng)細(xì)胞系免于缺血、缺氧、興奮性毒性導(dǎo)致的細(xì)胞凋亡。Jin等[11]通過(guò)在培養(yǎng)液中加入VEGF的方法使神經(jīng)前體細(xì)胞數(shù)量增加20%—30%,并運(yùn)用微管泵技術(shù)將VEGF持續(xù)注入大鼠側(cè)腦室內(nèi),發(fā)現(xiàn)4 d后海馬室下區(qū)Brdu標(biāo)記陽(yáng)性細(xì)胞明顯增加,提示VEGF可能具有獨(dú)立的促進(jìn)神經(jīng)干細(xì)胞增殖的作用。 在胚胎神經(jīng)元增殖中VEGF通過(guò)MEK/ERK, PLC-γ、PI3K、上調(diào)E2F轉(zhuǎn)錄因子[12]以及增加細(xì)胞周期蛋白A, D1和E的表達(dá)等方式來(lái)發(fā)揮促增殖作用。在多項(xiàng)成體研究中[13-14]發(fā)現(xiàn)VEGF對(duì)神經(jīng)前體細(xì)胞發(fā)揮遷移、存活和增殖作用主要是通過(guò)VEGFR2來(lái)調(diào)節(jié)的。而在另外一項(xiàng)研究中發(fā)現(xiàn)神經(jīng)干細(xì)胞的自我更新能力則依賴(lài)于FGF-2、內(nèi)皮細(xì)胞溶解成分以及Notch-1等信號(hào)物質(zhì)的共同作用。 1989年Martin等[15]首次發(fā)現(xiàn)少突膠質(zhì)前體細(xì)胞存在的證據(jù),他們認(rèn)為這種細(xì)胞具有表型可塑性,能夠?qū)Νh(huán)境作出反應(yīng),可以發(fā)展成少突膠質(zhì)細(xì)胞或膠質(zhì)纖維酸性蛋白GFAP陽(yáng)性的星形膠質(zhì)細(xì)胞。隨后的研究表明這種細(xì)胞表面存在一種硫酸軟骨素蛋白多糖(chondroitinsulp hate peoteoglycan ,CSPG)NG2特異抗原,同時(shí)在成體腦內(nèi)也發(fā)現(xiàn)了這種細(xì)胞,與胚胎期或新生組織一樣,這種細(xì)胞同樣具有表型可塑性,可以分化成少突膠質(zhì)細(xì)胞或星形膠質(zhì)細(xì)胞[16]。OPC是一種前體細(xì)胞,它能夠分化產(chǎn)生不同的細(xì)胞系。在不同的培養(yǎng)環(huán)境下OPC可分化為星形膠質(zhì)細(xì)胞、少突膠質(zhì)細(xì)胞甚至分化為神經(jīng)元[17]。OPC在生理發(fā)育過(guò)程中、疾病損傷中均有重要作用。在缺血性腦損傷研究[18]中就發(fā)現(xiàn)缺血損傷后大腦局部NG2陽(yáng)性細(xì)胞數(shù)增多,而吳波等[19]在脊髓機(jī)械損傷后移植OPC治療中發(fā)現(xiàn)OPC移植組大鼠的神經(jīng)功能恢復(fù)情況明顯優(yōu)于假手術(shù)對(duì)照組。這些研究均說(shuō)明了OPC在中樞神經(jīng)系統(tǒng)中的作用是獨(dú)特而又重要的。 Notch信號(hào)通路是廣泛存在于動(dòng)物體內(nèi)重要信號(hào)傳導(dǎo)通路,參與多種細(xì)胞的增殖和分化活動(dòng),尤其是在中樞神經(jīng)系統(tǒng)發(fā)生、發(fā)育和損傷后修復(fù)過(guò)程[20]。Notch基因在從無(wú)脊椎動(dòng)物到脊椎動(dòng)物的多個(gè)物種中表達(dá),其家族成員的結(jié)構(gòu)具有高度保守性,迄今研究人員認(rèn)為該通路實(shí)際上是十分復(fù)雜的調(diào)控網(wǎng)絡(luò)。具體到少突膠質(zhì)系發(fā)育過(guò)程,Notch信號(hào)可能起到抑制前體細(xì)胞向少突膠質(zhì)細(xì)胞方向發(fā)育,維持神經(jīng)前體細(xì)胞自我更新的作用。有研究稱(chēng)[9] VEGF促進(jìn)神經(jīng)干細(xì)胞增殖分化過(guò)程中有Notch蛋白變化。本研究的目的就是通過(guò)觀察VEGF對(duì)OPC增殖的影響,探索這種增殖變化與Notch信號(hào)通道上相關(guān)蛋白表達(dá)是否有關(guān)。 方法: 分離純化培養(yǎng)OPC,MTT法檢測(cè)不同濃度VEGF(50、100、200 ng/ml)促進(jìn)OPC增殖的作用以及加入Notch通路γ-分泌酶抑制劑DAPT后OPC增殖變化情況,RT-PCR、Western blot技術(shù)檢測(cè)VEGF處理OPC后Notch信號(hào)通道上Notch-1、Hes-1的基因表達(dá)和蛋白表達(dá)情況。 結(jié)果: 1、混合膠質(zhì)細(xì)胞培養(yǎng)10 d左右,經(jīng)兩次分離振蕩后去除小膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞,純化出OPCs。經(jīng)免疫熒光染色鑒定OPC特異性抗原NG2陽(yáng)性細(xì)胞占細(xì)胞總數(shù)90%。 2、50、100、200 ng/ml VEGF處理組的細(xì)胞增殖率分別為(107±2)%、(124±2)%、(142±7)%,與空白對(duì)照組相比差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。 3、加入γ-分泌酶抑制劑DAPT后100 ng/ml VEGF處理組的增殖率下降至(103±3)%(P0.05)。 4、RT-PCR及Western blot分析顯示VEGF處理OPC后, Notch-1、Hes-1蛋白在基因水平和蛋白水表達(dá)均增加(P0.05),加入γ-分泌酶抑制劑DAPT能抑制其表達(dá)(P0.05)。 結(jié)論 1、分離純化培養(yǎng)的細(xì)胞經(jīng)免疫熒光鑒定OPC特異性抗原NG2陽(yáng)性細(xì)胞占細(xì)胞總數(shù)≥90%; 2、VEGF對(duì)體外環(huán)境培養(yǎng)的OPC具有促進(jìn)增殖的作用,這種促增殖作用與Notch信號(hào)通路上相關(guān)蛋白的表達(dá)有關(guān)。
[Abstract]:Research background
In recent years, a number of studies have found that vascular epthelial growth factor (VEGF) plays an important role in the field of neuroscience. On the one hand, a lot of evidence has shown that the normal growth and development of VEGF have a neurotrophic and neuroprotective effect on neural cells and glial cells, and can be used for neural stem cells. On the other hand, [1]., on the other hand, can reduce brain damage in the middle pathological conditions of ischemic stroke, and promote the repair of damaged nerve cells and the regeneration of neural cells, [1]. can be said that the complex and diverse role of VEGF in the central nervous system makes people increasingly interested in the research.
Since the first discovery of VEGF in 1983, VEGF has been considered to be an important regulator of vascular formation in vertebrate development and some common chronic diseases. [2]. initially found that VEGF induced vascular endothelial cells to form blood pipe network [3]. in the development process of rat, and the biological function of [3]. was through VEGFR2 (KDR/Flk). -1), VEGFR1 (Flt-1) two protein kinase receptor [4] and the transmembrane protein family (NPs) [5] play a role. The study found that VEGF can be detected in the embryonic development of rats for seventh days. In the subsequent development of the central nervous system, VEGF is mainly expressed in the mesenchyme and the neuroectoderm of the head. With the development of the embryo, the VEGF cells are expressed. There are neural stem cells, glial cells and vascular endothelial cells, and the expression level of neural stem cells in differentiation is the highest. In adult stage, the central nervous system develops mature, the expression of VEGF decreases gradually, which is restricted to the choroid plexus, the final region, the cerebellar granular cell [6] [7-8] area and so on.
Although there is a study that VEGF has the dual role of inducing neovascularization and neurogenesis (neurogenesis) during embryonic development, it may affect the growth and development of the embryonic nervous system by two ways of paracrine and autocrine, but it is still controversial whether the researchers have independent neurotrophic effect on VEGF. In the adult neurogenesis, some studies have confirmed that endothelial cells can promote the proliferation of neural stem cells in specific brain regions by creating microenvironment or [10] in the vascular pool. In vitro culture, VEGF can promote the proliferation of astrocytes, Schwann cells, microglia and cortical neurons, and also protect the sea. The hippocampal, cortical, peripheral sensory neurons and some nerve cell lines are free from ischemia, hypoxia, and excitotoxicity caused by apoptosis.Jin and so on [11] increase the number of neural precursor cells by 20% to 30% by adding VEGF in the culture medium, and then use microtubule pump technique to inject VEGF into the lateral ventricle of the rat, and find the Brdu in the subventricular region after 4 d. Marked positive cells increased significantly, suggesting that VEGF may play an independent role in promoting the proliferation of neural stem cells.
In the proliferation of embryonic neurons, VEGF plays a role in promoting proliferation through MEK/ERK, PLC- gamma, PI3K, up regulation of E2F transcription factor [12], and increasing the expression of cell cycle protein A, D1 and E. In a number of adult studies, [13-14] shows that VEGF on neural precursor cells is migrated, and the survival and proliferation effects are mainly regulated by VEGFR2. In another study, the self-renewal capacity of neural stem cells was found to be dependent on the interaction of FGF-2, endothelial cells dissolving components and signal substances such as Notch-1.
In 1989, Martin and other [15] found evidence of the presence of oligodendrocyte precursor cells for the first time. They thought the cells were phenotypic plasticity and could react to the environment and could develop into oligodendrocytes or astrocytes positive for glial fibrillary acidic protein GFAP. The chondroitinsulp hate peoteoglycan (CSPG) NG2 specific antigen is also found in the adult brain. Like the embryonic or neonatal tissue, the cells also have phenotypic plasticity and can be differentiated into oligodendrocytes or astrocytes, [16].OPC, a precursor cell, which can differentiate and produce. OPC can differentiate into astrocytes in different culture environments. Oligodendrocytes and even neuron [17].OPC have an important role in the process of physiological development. In the study of ischemic brain damage [18], the number of NG2 positive cells in the brain is increased after ischemic injury, while Wu Bo, etc. The recovery of nerve function in the OPC transplantation group was obviously better than that of the sham control group in the treatment of the transplanted OPC after the spinal cord mechanical injury. These studies show that the role of OPC in the central nervous system is unique and important.
Notch signaling pathway, which is widely used in the important signaling pathways in animals, participates in the proliferation and differentiation of various cells, especially in the central nervous system, development and injury after repair, and the [20].Notch gene is expressed in a number of species from invertebrates to vertebrates, and the structure of family members is highly guaranteed. Conservatism, so far, researchers believe that the pathway is actually a very complex regulatory network. Specific to oligodendrocyte development, Notch signals may inhibit the development of precursor cells to oligodendrocytes and maintain self-renewal of neural precursor cells. There is a study called [9] VEGF to promote the proliferation and differentiation of neural stem cells. The aim of this study is to explore the effect of VEGF on the proliferation of OPC and to explore whether the proliferation changes are related to the expression of the associated protein on the Notch signaling pathway.
Method:
The effects of VEGF (50100200 ng/ml) on the proliferation of OPC and the proliferation of OPC with the Notch pathway gamma secretase inhibitor DAPT were detected by the isolation and purification of OPC. The expression of gene expression and protein expression were detected by RT-PCR and Western blot.
Result:
1, the mixed glial cells were cultured about 10 d, and after two isolation, the microglia and astrocytes were removed, and OPCs. was purified by immunofluorescence staining to identify the total number of OPC specific antigen NG2 positive cells (90%.).
The cell proliferation rates of the 2,50100200 ng/ml VEGF treatment group were (107 + 2)%, (124 + 2)%, (142 + 7)%, respectively, and the difference was statistically significant compared with the blank control group (P0.05).
3, the proliferation rate of the 100 ng/ml VEGF treatment group decreased to (103 + 3)% (P0.05) after the addition of the gamma secretase inhibitor DAPT.
4, RT-PCR and Western blot analysis showed that after VEGF treatment OPC, Notch-1, Hes-1 protein increased (P0.05) at gene level and protein water expression (P0.05), and DAPT could inhibit its expression (P0.05) with the addition of gamma secretase inhibitor DAPT.
conclusion
1, immunofluorescence assay showed that OPC specific antigen NG2 positive cells accounted for more than 90% of the total number of cells.
2, VEGF promotes proliferation of OPC cultured in vitro, which is related to the expression of related proteins in Notch signaling pathway.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R329

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 夏思文;神經(jīng)橋接導(dǎo)管內(nèi)GDNF緩釋微囊對(duì)大鼠面神經(jīng)的誘向再生作用[D];第二軍醫(yī)大學(xué);2012年

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