VEGF促进少突胶质前体细胞增殖及其机制的初步研究

发布时间：2018-06-04 15:46

本文选题：血管内皮生长因子 + 少突胶质前体细胞　；参考：《第三军医大学》2011年硕士论文

【摘要】：研究背景近年来多项研究发现血管内皮细胞生长因子( vascular epthelial growth factor, VEGF)在神经科学领域具有重要的作用。一方面,大量研究证据证明VEGF正常生长发育过程中对神经细胞、胶质细胞具有神经营养和神经保护作用,对神经干细胞能够起到促进增殖和存活作用[1]。另一方面,VEGF在缺血性脑卒中等病理条件下能减轻脑损伤,促进受损神经细胞修复和神经细胞再生[1]。可以说VEGF在中枢神经系统中的复杂多样作用使得人们对其研究的兴趣日益浓厚。自从1983年首次发现VEGF以来,VEGF一直以来被认为是脊椎动物发育和一些常见慢性病中血管形成的重要调节因素[2]。最初发现,VEGF在大鼠发育过程中以血管发生的方式促使血管内皮细胞聚集形成血管网[3]。其生物学功能通过VEGFR2 (KDR/Flk-1)、VEGFR1 (Flt-1)两种蛋白激酶受体[4]以及跨膜蛋白家族(NPs)[5]发挥作用。研究发现VEGF在大鼠胚胎发育第7天就可被检测到,在随后的中枢神经系统发育过程中,VEGF主要表达于间充质和头部的神经外胚层。随着胚胎不断发育,表达VEGF的细胞有神经干细胞、神经胶质细胞和血管内皮细胞,其中以分化中的神经干细胞表达水平最高。成体阶段中枢神经系统发育成熟,VEGF表达逐渐减少,限制于脉络丛、最后区、小脑颗粒细胞[6] [7-8]等区域。尽管有研究认为VEGF在胚胎发育期具有诱导新生血管形成和神经发生(neurogenesis)的双重作用,可能通过旁分泌和自分泌两种方式作用共同影响胚胎神经系统的生长发育[9],但目前研究者对VEGF是否具有独立的神经营养作用还存在争议。而在成体的神经发生中,一些研究证实内皮细胞可以通过创造微环境或血管池[10]的方式来促进特定脑区神经干细胞的增殖。在体外培养环境下,VEGF则可以促进星形胶质细胞、雪旺细胞、小胶质细胞以及皮质神经元的增殖;还可以保护海马、皮层、周围感觉神经元和一些神经细胞系免于缺血、缺氧、兴奋性毒性导致的细胞凋亡。Jin等[11]通过在培养液中加入VEGF的方法使神经前体细胞数量增加20%—30%,并运用微管泵技术将VEGF持续注入大鼠侧脑室内,发现4 d后海马室下区Brdu标记阳性细胞明显增加,提示VEGF可能具有独立的促进神经干细胞增殖的作用。在胚胎神经元增殖中VEGF通过MEK/ERK, PLC-γ、PI3K、上调E2F转录因子[12]以及增加细胞周期蛋白A, D1和E的表达等方式来发挥促增殖作用。在多项成体研究中[13-14]发现VEGF对神经前体细胞发挥迁移、存活和增殖作用主要是通过VEGFR2来调节的。而在另外一项研究中发现神经干细胞的自我更新能力则依赖于FGF-2、内皮细胞溶解成分以及Notch-1等信号物质的共同作用。 1989年Martin等[15]首次发现少突胶质前体细胞存在的证据,他们认为这种细胞具有表型可塑性,能够对环境作出反应,可以发展成少突胶质细胞或胶质纤维酸性蛋白GFAP阳性的星形胶质细胞。随后的研究表明这种细胞表面存在一种硫酸软骨素蛋白多糖(chondroitinsulp hate peoteoglycan ,CSPG)NG2特异抗原,同时在成体脑内也发现了这种细胞,与胚胎期或新生组织一样,这种细胞同样具有表型可塑性,可以分化成少突胶质细胞或星形胶质细胞[16]。OPC是一种前体细胞,它能够分化产生不同的细胞系。在不同的培养环境下OPC可分化为星形胶质细胞、少突胶质细胞甚至分化为神经元[17]。OPC在生理发育过程中、疾病损伤中均有重要作用。在缺血性脑损伤研究[18]中就发现缺血损伤后大脑局部NG2阳性细胞数增多,而吴波等[19]在脊髓机械损伤后移植OPC治疗中发现OPC移植组大鼠的神经功能恢复情况明显优于假手术对照组。这些研究均说明了OPC在中枢神经系统中的作用是独特而又重要的。 Notch信号通路是广泛存在于动物体内重要信号传导通路,参与多种细胞的增殖和分化活动,尤其是在中枢神经系统发生、发育和损伤后修复过程[20]。Notch基因在从无脊椎动物到脊椎动物的多个物种中表达,其家族成员的结构具有高度保守性,迄今研究人员认为该通路实际上是十分复杂的调控网络。具体到少突胶质系发育过程,Notch信号可能起到抑制前体细胞向少突胶质细胞方向发育,维持神经前体细胞自我更新的作用。有研究称[9] VEGF促进神经干细胞增殖分化过程中有Notch蛋白变化。本研究的目的就是通过观察VEGF对OPC增殖的影响,探索这种增殖变化与Notch信号通道上相关蛋白表达是否有关。方法: 分离纯化培养OPC,MTT法检测不同浓度VEGF(50、100、200 ng/ml)促进OPC增殖的作用以及加入Notch通路γ-分泌酶抑制剂DAPT后OPC增殖变化情况,RT-PCR、Western blot技术检测VEGF处理OPC后Notch信号通道上Notch-1、Hes-1的基因表达和蛋白表达情况。结果: 1、混合胶质细胞培养10 d左右,经两次分离振荡后去除小胶质细胞和星形胶质细胞,纯化出OPCs。经免疫荧光染色鉴定OPC特异性抗原NG2阳性细胞占细胞总数90%。 2、50、100、200 ng/ml VEGF处理组的细胞增殖率分别为(107±2)%、(124±2)%、(142±7)%,与空白对照组相比差异具有统计学意义(P0.05)。 3、加入γ-分泌酶抑制剂DAPT后100 ng/ml VEGF处理组的增殖率下降至(103±3)%(P0.05)。 4、RT-PCR及Western blot分析显示VEGF处理OPC后, Notch-1、Hes-1蛋白在基因水平和蛋白水表达均增加(P0.05),加入γ-分泌酶抑制剂DAPT能抑制其表达(P0.05)。结论 1、分离纯化培养的细胞经免疫荧光鉴定OPC特异性抗原NG2阳性细胞占细胞总数≥90%; 2、VEGF对体外环境培养的OPC具有促进增殖的作用,这种促增殖作用与Notch信号通路上相关蛋白的表达有关。
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

【引证文献】