内皮祖细胞对神经干细胞增殖分化及Notch信号分子表达的影响

发布时间：2018-05-16 03:35

  本文选题：内皮祖细胞 + 神经干细胞　； 参考：《南昌大学》2008年硕士论文

【摘要】： 目的和意义: 缺血性脑血管疾病已成为人类致死致残的最主要的原因之一,它主要包括两大病理损害,即局部血循环结构的缺失和神经元的损失。故而血供的再恢复及神经修复是缺血性脑损伤临床治疗的两个关键环节。 传统观念认为,成年哺乳动物中枢神经系统不可能再生,如果出现神经元丢失或缺损则难以修复,神经功能的重建被视为几乎不可能。但上世纪90年代,神经新生与神经干细胞(neural stem cells,NSCs)的发现改变了脑损害后神经不可修复的传统观念。近年亦发现,各种脑损害如脑缺血、癫痫和脑创伤等能产生各种可溶性细胞损伤信号激活NSCs,通过神经新生,选择性补充脑皮层神经元与神经胶质的丢失。有关NSCs的增殖及分化的调控机制随之成为神经科学的研究热点。现已知NSCs的增殖与分化的作用机制复杂而多样,其中Notch信号通路是调节细胞增殖分化的一条古老的途径,在神经发生过程中,它决定着NSCs是选择继续增殖还是向神经元分化,并与NSCs分化为神经元与胶质细胞的比例密切相关。 随着血管内皮祖细胞(endothelial progenitor cells, EPCs)参与体内血管新生理论的建立,利用内源性EPCs或移植EPCs促进脑缺血区域血管重建也开始受到关注。研究发现缺血性脑损伤发生时, EPCs可从骨髓动员释放到外周血循环或经人为移植到体内,这些细胞迁移到缺血部位参与缺血组织的血管重建和损伤血管的再内皮化过程,使缺血区域中毛细血管密度上调,脑梗死面积缩小,神经功能改善。值得重视的是,神经发生与脑血管发生具有密切关系,血供的恢复是神经修复的基础,EPCs不仅参与血供的恢复,而且还可促进内源性NSCs迁入缺血脑组织参与修复,提示EPCs移植或NSCs与EPCs共移植治疗脑缺血性中风具有良好前景。但EPCs对NSCs的具体作用及影响还不尽了解。因此我们应用体外的共培养模型,研究在不同的共培养条件下,例如NSCs分别在SU5416及DAPT作用后与EPCs共培养等,观察骨髓来源的EPCs对NSCs增殖分化的影响及对其Notch信号通路的影响,探讨EPCs调控NSCs增殖分化的分子机制,为EPCs、NSCs移植或两者联合移植治疗缺血性脑损伤等中枢神经系统疾病奠定理论基础。 研究内容和方法: 1.贴壁筛选法分离培养大鼠EPCs,形态学、免疫细胞染色法检测VIII因子、FLK-1、CD133、CD34的表达及RT-PCR检测eNOS、VIII因子、FLK-1、CD133、Tie-2、CD34的基因表达鉴定EPCs。 2.神经球悬浮法培养小鼠NSCs,免疫细胞染色法检测nestin的表达以鉴定NSCs。 3.建立transwell共培养体系,实验共分为5组:①EPCs与NSCs共培养组(EPCs+NSCs):实验所用EPCs为原代,NSCs为第3代,共培养时,transwell上层皿为EPCs,下层皿为NSCs;②EPCs与经SU5416处理后的NSCs共培养组[EPCs+(SU5416+NSCs)]:transwell下层皿的NSCs经SU5416(10μM)作用6h后再与上层皿的EPCs共培养;③EPCs与经DAPT处理后的NSCs共培养组[EPCs+(DAPT+NSCs)]:transwell上层皿为EPCs,下层皿为已经DAPT(10μM)作用6h后的NSCs;④经DAPT处理后的NSCs对照组(DAPT+NSCs):transwell上层皿为混合培养培养基,下层皿为已经DAPT(10μM)作用6h后的NSCs;⑤单纯NSCs对照组(NSCs):transwell上层皿为混合培养培养基,下层皿为NSCs。 4.采用RT-PCR方法观察各培养组共培养2h、6h、12h后notch信号通路相关基因notch1、Hes1、mash1以及NSCs增殖分化相关基因ki67、神经巢蛋白(nestin)、β-微管蛋白(β-Tubulin)、神经胶质酸性蛋白(GFAP)的基因表达的变化。 结果: 1.EPCs呈典型的“鹅卵石”排列生长,并经细胞化学染色结果显示VIII因子、CD34、FLK-1和CD133阳性,RT-PCR检测有eNOS、VIII因子、CD34、FLK-1、CD133和Tie-2的基因表达,符合EPCs特征。 2.成球悬浮法培养的小鼠NSCs增殖旺盛,nestin呈阳性表达。 3.各共培养组NSCs增殖分化相关基因及notch信号通路相关基因的变化: (1)EPCs可促进NSCs的ki67及nestin基因表达。(EPCs+NSCs)组与[EPCs+(SU5416+NSCs)]组比较,ki67及nestin基因表达明显上调,说明EPCs可以促进NSCs的ki67、nestin基因表达且与EPCs分泌的内皮生长因子有关。 (2)EPCs使NSCs的β-Tubulin基因表达上调,同时GFAP基因表达下降。(EPCs+NSCs)组、[EPCs+(SU5416+NSCs)]组及[EPCs+(DAPT+NSCs)]组的NSCs的向神经元分化相关基因β-Tubulin表达均不同程度上调,同时向胶质细胞分化相关基因GFAP基因下调。其中尤以(EPCs+NSCs)明显,说明EPCs可以促进NSCs向神经元方向转化,并与Notch信号通路的调节及内皮生长因子有关。 (3)(EPCs+NSCs)组及[EPCs+(SU5416+NSCs)]组NSCs的hes1基因较其他组表达下调,而mash1基因表达上调。 结论: EPCs可以促进NSCs的增殖及其向神经元方向的分化,这种作用与EPCs对NSCs的Notch信号通路相关基因的调节以及EPCs的内皮生长因子密切相关。
[Abstract]:Purpose and significance:
Ischemic cerebrovascular disease has become one of the most important causes of death and disability of human being. It mainly includes two major pathological damage, that is, the loss of local blood circulation structure and the loss of neurons. Therefore, the re recovery of blood supply and nerve repair are the two key links in the clinical treatment of ischemic brain injury.
Traditionally, the central nervous system of adult mammals is not possible to regenerate, and it is difficult to repair if neuron loss or defect occurs, and the reconstruction of neural function is considered almost impossible. In 90s last century, the discovery of neurogenesis and neural stem cells (neural stem cells, NSCs) changed the non repairable transmission of nerve after brain damage. In recent years, it is also found that all kinds of brain damage, such as cerebral ischemia, epilepsy and brain trauma, can produce a variety of soluble cell damage signals to activate NSCs, through neurogenesis, and selectively supplement the loss of cerebral cortex neurons and neuroglia. The regulatory mechanism related to the proliferation and differentiation of NSCs has become a hot topic in neuroscience. Now NS is known. The mechanism of proliferation and differentiation of Cs is complex and diverse, in which the Notch signaling pathway is an ancient way to regulate cell proliferation and differentiation. In the process of neurogenesis, it determines whether NSCs is selected to continue to proliferate or differentiate into neurons, and it is closely related to the proportion of NSCs into neurons and the proportion of gelatin cells.
As vascular endothelial progenitor cells (endothelial progenitor cells, EPCs) are involved in the establishment of angiogenesis theory in vivo, the use of endogenous EPCs or transplantation of EPCs to promote vascular reconstruction in ischemic regions is also concerned. The study found that when ischemic brain injury occurred, EPCs could be released from bone marrow mobilization to peripheral blood circulation or human transplantation. In vivo, these cells migrate to the ischemic site to participate in the vascular reconstruction of ischemic tissue and to damage the endothelialization of blood vessels. The capillary density is up-regulated in the ischemic region, the area of cerebral infarction is reduced, and the nerve function is improved. It is worth paying attention to that the neurogenesis is closely related to the occurrence of cerebral vasculogenesis, and the recovery of blood supply is the basis of nerve repair. EPCs not only participates in the recovery of blood supply, but also promotes the migration of endogenous NSCs into the ischemic brain tissue to participate in the repair. It is suggested that EPCs transplantation or NSCs and EPCs have good prospects for the treatment of cerebral ischemic stroke. But the specific effects and effects of EPCs on NSCs are not well understood. Therefore, we apply a co culture model in vitro to study in different ways. Under co culture conditions, such as NSCs and EPCs co culture after SU5416 and DAPT respectively, the effects of EPCs on the proliferation and differentiation of NSCs and its influence on the Notch signaling pathway were observed, and the molecular mechanism of EPCs regulating NSCs proliferation and differentiation was discussed, and EPCs, NSCs transplantation or combined transplantation for the treatment of ischemic brain injury and other central nervous systems The theoretical foundation of the disease is established.
Research contents and methods:
1. adherent wall screening method was used to isolate and culture EPCs, morphology, immune cell staining method was used to detect VIII factor, FLK-1, CD133, CD34 expression and RT-PCR detection eNOS, VIII factor, FLK-1, CD133, Tie-2, CD34 gene expression identification
2. mouse NSCs was cultured by neurosphere suspension. The expression of nestin was detected by immuno cell staining to identify NSCs..
3. the Transwell co culture system was established. The experiment was divided into 5 groups: (1) EPCs and NSCs co culture group (EPCs+NSCs): the experimental EPCs was the original generation and the NSCs was the third generation. When co culture, the upper Transwell Petri dish was EPCs, the lower layer was NSCs; (2) EPCs and SU5416 treated NSCs co culture group] EPCs co culture after the action of 6h; (3) EPCs and DAPT treated NSCs co culture group [EPCs+ (DAPT+NSCs)]: the upper layer of Transwell is EPCs, the lower layer is after DAPT (10 mu M) action 6h. 6h after NSCs; NSCs simple control group (NSCs): Transwell upper layer was mixed culture medium, and the lower layer was NSCs..
4. RT-PCR method was used to observe the gene expression of Notch1, Hes1, Mash1 and NSCs proliferation and differentiation related genes Notch1, Hes1, Mash1, and NSCs proliferation and differentiation genes, Notch1, Hes1, Mash1, and NSCs proliferation and differentiation related genes after 2h, 6h, 12h, and the gene expression of glial acidic protein (beta -Tubulin) and glial acidic protein.
Result:
1.EPCs showed a typical "cobblestone" arrangement, and the results of cytochemical staining showed that VIII, CD34, FLK-1 and CD133 were positive. RT-PCR detected the gene expression of eNOS, VIII factor, CD34, FLK-1, CD133 and Tie-2.
2. the proliferation of mouse NSCs cultured in suspension culture was strong and nestin was positive.
3. changes in NSCs proliferation and differentiation related genes and Notch signaling pathway related genes in co culture group:
(1) EPCs could promote the expression of Ki67 and nestin gene in NSCs. (EPCs+NSCs) the expression of Ki67 and nestin genes was obviously up-regulated compared with [EPCs+ (SU5416+NSCs) group, indicating that EPCs can promote NSCs Ki67, and it is related to endothelial growth factor secreted.
(2) EPCs increased the expression of NSCs's beta -Tubulin gene and decreased the expression of GFAP gene. (EPCs+NSCs), the beta -Tubulin expression of the neuron differentiation related genes in the [EPCs+ (SU5416+NSCs)] group and [EPCs+ (DAPT+NSCs)] group were up regulated in varying degrees, and the GFAP genes of the glial cell differentiation related genes were downregulated. It shows that EPCs can promote the transformation of NSCs into neurons, and is related to the regulation of Notch signaling pathway and endothelial growth factor.
(3) (EPCs+NSCs) and [EPCs+ (SU5416+NSCs)] group NSCs Hes1 gene expression was down regulated compared with other groups, while Mash1 gene expression was upregulated.
Conclusion:
EPCs can promote the proliferation of NSCs and its differentiation into the direction of neuron, which is closely related to the regulation of EPCs related genes related to the Notch signaling pathway of NSCs and the endothelial growth factor of EPCs.

【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

【引证文献】

中国期刊全文数据库 前1条

1 龚婷;贾素洁;张毕奎;;内皮祖细胞生物学特性及其进展[J];现代生物医学进展;2012年21期

中国硕士学位论文全文数据库 前1条

1 龚婷;冠心病患者DDAH2启动子甲基化对内皮祖细胞功能的调节及机制[D];中南大学;2012年

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