人诱导多能干细胞来源的神经干细胞对缺血性脑损伤的修复作用

发布时间：2018-07-11 14:08

  本文选题：诱导性多能干细胞 + 神经干细胞　； 参考：《南昌大学》2012年硕士论文

【摘要】：研究背景与目的： 诱导多能干细胞(induced pluripotent stem cells, iPSCs)是一类与胚胎干细胞（embryonic stem cells, ESC）具有相似全向分化潜能的干细胞，体外可分化为神经干细胞（Neural Stem Cells，NSC）、神经元等用于中枢神经系统疑难疾病的细胞替代治疗，临床应用前景广阔，有望成为干细胞移植治疗的新的种子细胞来源。然而，目前人们对iPSCs向NSC分化的调控机制知之甚少，要获得数量丰富且来源稳定的iPSCs源性的NSC用于临床治疗还存在相当的技术困难。因此，进一步了解iPSCs向NSC定向分化的具体调控机制，对于iPSCs将来的临床推广应用意义重大。 由于iPSCs与ESC生物学特性极其相似,而ESC的自我更新和定向分化机制又一直是生命科学领域的研究热点和重点,这就为我们深入探讨诱导iPSCs向NSC分化的调控机制提供了必要的前提和基础。研究表明ESC的自我更新和定向分化受Notch、Wnt等外部信号通路、转录因子、和表观遗传修饰等多种因素的调控。Notch通路是生物体极其重要的信号传导通路之一，其在干细胞的增殖和分化等方面均起着重要的作用。Notch受体与配体结合激活时，干细胞就表现为增殖；当Notch信号通路被抑制时，干细胞就分化为功能细胞。微小RNA（microRNA，miRNA）在生物体发生发育、干细胞增殖分化和肿瘤发生发展中同样发挥着极其重要的调控作用，在人类，microRNA能够调控至少30%的基因，一个microRNA可负调控数百个靶基因的蛋白表达，几乎可参与调控所有的生物学过程。诸多文献研究提示在ESC的NSC定向分化过程中，microRNA能够调控可下调干细胞内维持其未分化状态的基因表达水平，同时激活干细胞谱系特异性基因，从而促进ESC定向分化。 然而目前尚未见iPSCs神经定向分化相关调控机制的研究报道,为探讨iPSCs向NSC定向分化的机制以及iPSCs来源的NSC对缺血性脑损伤疾病的修复效果，本课题拟分为体外实验及体内实验两部分：体外实验首先将iPSCs定向分化为NSC，利用实时定量PCR、Western blot等方法检测分化过程中各时间点Notch信号分子、miRNAs的表达变化，初步探讨Notch信号分子、miRNAs与iPSCs向NSC定向分化过程的关系；体内实验拟通过将iPSCs来源的NSC立体定向移植至大鼠中动脉栓塞模型（middle cerebral arterial occlusion，MCAO）模型中，观察其对大鼠缺血性脑损伤所致神经功能缺陷的修复作用，为利用干细胞和再生医学技术治疗中枢神经系统疑难疾病提供新的理论依据和技术准备。 研究内容和方法： 1体外实验 1.1人iPSCs的培养及向NSC的诱导分化：采用本课题组已建立的培养及分化体系培养人iPSC细胞并诱导其向NSC分化，应用免疫荧光染色对分化后的细胞进行NSC标志物Nestin、Sox2表达的鉴定。 1.2采用实时荧光定量PCR检测分化过程中mir-9，mir-34a、mir-200b的表达，与iPS组比较，观察mir-9，mir-34a、mir-200b的动态表达变化。 1.3采用实时荧光定量PCR检测分化过程中Notch1、Hes1的基因表达水平，与iPS组比较，观察Notch1、Hes1基因的动态表达变化。 1.4采用免疫荧光染色和Western blot法检测诱导过程中Notch1、Hes1的蛋白表达水平，与iPS组比较，观察诱导过程中Notch1、Hes1蛋白的表达变化。 1.5利用Notch信号阻断剂DAPT进一步验证Notch信号通路在iPSCs向NSC定向分化过程中的作用。实验分为RA对照组，DAPT诱导组，，RA+DAPT诱导组，RA+DMSO对照组；倒置显微镜下观察各组细胞的形态变化，实时荧光定量PCR诱导第7天的各组细胞Nestin、β-tubulinШ、GFAP、Notch1和Hes1基因的表达情况，免疫荧光染色同时检测Nestin、β-tubulinШ、和GFAP蛋白的表达。 2体内实验 2.1参考Longa法制作大脑中动脉栓塞（MCAO）模型，CM-DiI标记iPS来源的NSC进行大鼠纹状体立体定向移植，观察移植的NSC在大鼠脑内的存活、迁移状况，并以免疫荧光染色法检测神经细胞相关标志物Nestin，β-tublinIII，GFAP的变化，以观察其分化情况。同时利用平衡木行走实验、抓握实验及水迷宫实验对大鼠进行神经功能测评。 结果： 1体外实验 1.1成功将iPSCs诱导分化为NSC。 iPSCs经RA结合无血清培养基诱导3天后，可观察到细胞球形成神经rosette结构，免疫荧光染色显示细胞球呈Nestin及Sox2阳性表达，贴壁培养1个月后，细胞形成神经网络状结构，免疫荧光鉴定提示细胞高表达NSC标志物Nestin及Sox2。 1.2实时荧光定量PCR结果显示，与iPSCs组相比较，mir-9、-34a、-200b的表达水平在iPSCs的NSC定向分化过程中显著上调。 1.3实时荧光定量PCR结果显示，在iPSCs形成EB的过程中，与iPSCs组相比较， Notch1和Hes1mRNA的表达明显上调，随着RA及无血清培养基诱导iPSCs分化的开始，Notch1和Hes1mRNA的表达下调。 1.4免疫荧光染色结果显示，与自然分化组比较，诱导28d后RA无血清培养基诱导组的Notch1以及Hes1蛋白表达的阳性细胞率显著下降，Western blot结果也显示，在iPSCs向NSC的分化过程中, Notch1、Hes1蛋白的表达随着RA结合无血清培养基诱导分化，Notch1、Hes1蛋白的表达逐渐降低，诱导分化后贴壁培养14d时最低，而后表达水平逐渐增高。 1.5DAPT加入后，大部分细胞球容易贴壁，细胞球周围可见爬出细长的神经丝样触角；第3d左右，贴壁的细胞球内可见大量神经rosette结构，培养2W后，细胞球分化形成神经网络状结构。结合实时荧光定量PCR和免疫荧光鉴定各诱导组神经标志物Nestin、β-tubullinIII及GFAP的表达结果表明， DAPT能够促进iPSC向NSC的定向分化；与RA对照组及RA+DMSO对照组比较，加入DAPT后，iPSC向NSC的分化速度加快，同时神经元细胞及少突胶质细胞所占分化后细胞的比例也增加，说明加入DAPT后，Notch信号被抑制，使得iPSC能够快速的向NSC定向分化，而由于DAPT对Notch信号的抑制作用是持续的，使得部分NSC继续分化为神经元及少突胶质细胞。 2体内实验 2.1移植后iPSCs来源的NSC在大鼠脑组织内能够长期存活，移植1周和2周后免疫荧光染色提示移植区和脑缺血区分别可见移植细胞，且细胞分别表达神经细胞标志物β-tubulinШ、GFAP，表明移植后的细胞在脑组织内能够向缺血区迁移并进一步分化为神经细胞；分别利用抓握实验、平衡木行走实验及水迷宫实验在模型制备后0，1，2，3W对各组SD大鼠进行评分，与正常组比较，大鼠脑缺血损伤后各项神经功能检测指标均明显降低；与对照组比较，NSC细胞移植组在移植2周后大鼠的抓握力、平衡行走能力和记忆功能均有一定程度的恢复。 结论： 1. RA结合无血清培养基诱导法能够有效将人iPSCs定向分化为NSC； 2. Notch信号通路参与了人iPSCs向NSC定向分化过程的调控； 3. mir-9、-34a及mir-200b有可能通过调控Notch信号参与了人iPSCs向NSC定向分化的调控； 4. iPSCs来源的NSC定向移植至MCAO大鼠脑组织后能够长期存活，定向迁移至脑缺血区并分化为神经细胞，一定程度促进大鼠脑缺血损伤后的神经功能恢复。
[Abstract]:Research background and purpose:
Induced pluripotent stem cells (iPSCs) is a kind of stem cells with similar omnidirectional differentiation potential with embryonic stem cells (embryonic stem cells, ESC). In vitro it can be differentiated into neural stem cells (Neural Stem Cells), neurons and other cell replacement therapy for the central nervous system, before clinical application. It is promising to be a new source of seed cells for stem cell transplantation. However, little is known about the regulatory mechanism of iPSCs to NSC differentiation. There are considerable technical difficulties in obtaining a rich and stable source of iPSCs derived NSC for clinical treatment. Therefore, further understanding of the specific differentiation of iPSCs to NSC is specific. The regulation mechanism is of great significance for the clinical popularization and application of iPSCs in the future.
As the biological characteristics of iPSCs and ESC are very similar, the mechanism of self renewal and orientation differentiation of ESC has been the focus and focus in the field of life science. This provides the necessary premise and basis for us to explore the regulation mechanism of inducing iPSCs to NSC differentiation. The self-renewal and directional differentiation of ESC is determined by Notch, Wnt. The regulatory.Notch pathway, such as external signaling pathways, transcription factors, and epigenetic modification, is one of the most important signaling pathways in organisms. It plays an important role in the proliferation and differentiation of stem cells. When the.Notch receptor is activated by the ligand binding to the ligand, the stem cells are proliferated, and when the Notch signaling pathway is used. When suppressed, stem cells differentiate into functional cells. Small RNA (microRNA, miRNA) also plays an extremely important regulatory role in the development of organisms, proliferation and differentiation of stem cells and the development of tumor. In humans, microRNA can regulate at least 30% of the gene, and a microRNA can negatively regulate the expression of hundreds of target genes. It is possible to participate in the regulation of all biological processes. Many literature studies suggest that microRNA can regulate the level of gene expression that can maintain its undifferentiated state in stem cells during the NSC directional differentiation of ESC, and activates the specific genes of stem cell lineage, thus promoting the ESC differentiation.
However, there is no research report on the regulation mechanism of iPSCs neurodirectional differentiation. In order to explore the mechanism of iPSCs directed differentiation to NSC and the effect of iPSCs source NSC on the repair of ischemic brain damage, this subject is divided into two parts: in vitro experiment and in vivo experiment: in vitro, iPSCs is first differentiated into NSC, and the real time is used in real time. Quantitative PCR, Western blot and other methods were used to detect the expression of Notch signal molecules and miRNAs in the process of differentiation. The relationship between Notch signal molecules, miRNAs and iPSCs to NSC directional differentiation was preliminarily investigated. In vivo experiments were designed to transplant NSC stereotaxis of iPSCs sources to the rat middle artery embolism model. In the occlusion, MCAO) model, the repair of neural functional defects caused by ischemic brain injury in rats was observed and a new theoretical basis and technical preparation were provided for the use of stem cells and regenerative medicine to treat the difficult diseases of the central nervous system.
Research contents and methods:
1 in vitro experiment
The culture of 1.1 iPSCs and induction of differentiation into NSC: the culture and differentiation system established by this group were used to cultivate human iPSC cells and induce it to differentiate into NSC. Immunofluorescence staining was used to identify the NSC marker Nestin and Sox2 expression of the differentiated cells.
1.2 real-time fluorescence quantitative PCR was used to detect the expression of miR-9, miR-34a and mir-200b in the differentiation process. Compared with iPS group, the dynamic expression of miR-9, miR-34a and mir-200b was observed.
1.3 real-time fluorescence quantitative PCR was used to detect the expression level of Notch1 and Hes1 in differentiation. Compared with group iPS, the dynamic expression of Notch1 and Hes1 genes was observed.
1.4 the protein expression level of Notch1 and Hes1 during induction was detected by immunofluorescence staining and Western blot, and the expression of Notch1 and Hes1 protein in the induction process was observed.
1.5 Notch signal blocking agent DAPT was used to further verify the role of Notch signaling pathway in the directional differentiation of iPSCs to NSC. The experiments were divided into RA control group, DAPT induction group, RA+DAPT induction group and RA+DMSO control group, and the morphological changes of each cell were observed under the inverted microscope, and the real-time fluorescence quantitative PCR induced seventh days of Nestin, beta -tubu. The expressions of Lin, GFAP, Notch1 and Hes1 genes were detected, and the expression of Nestin, beta -tubulin and GFAP proteins were detected by immunofluorescence staining.
2 in vivo experiment
2.1 Longa method was used to make the middle cerebral artery embolism (MCAO) model, and the CM-DiI labeled iPS derived NSC for the stereotactic transplantation of the rat striatum. The survival and migration of the transplanted NSC in the rat brain were observed and the changes of the neurons related markers Nestin, beta -tublinIII, and GFAP were detected by immunofluorescence staining, in order to observe the differentiation. At the same time, we used the balance beam walking test, grasping experiment and water maze test to evaluate the neurological function of rats.
Result:
1 in vitro experiment
1.1 the iPSCs induced differentiation into NSC. iPSCs was induced by RA and serum-free medium for 3 days. The rosette structure of the cell ball was observed. The immunofluorescence staining showed that the cells showed positive expression of Nestin and Sox2. After 1 months of adherent culture, the cells formed a neural network structure, and the immunofluorescence identification suggested that the cell expressed the NSC marker. Nestin and Sox2.
1.2 the results of real-time quantitative PCR showed that the expression levels of miR-9, -34a and -200b in iPSCs were significantly higher than those in iPSCs group.
1.3 real time fluorescence quantitative PCR results showed that in the process of iPSCs formation, the expression of Notch1 and Hes1mRNA was obviously up-regulated compared with the iPSCs group. The expression of Notch1 and Hes1mRNA decreased with the initiation of iPSCs differentiation induced by RA and serum-free medium.
1.4 the results of immunofluorescence staining showed that, compared with the natural differentiation group, the Notch1 and the positive cell rate of Hes1 protein expression in the RA serum-free medium induction group decreased significantly after the induction of 28d, and the Western blot results also showed that the expression of Notch1, Hes1 protein was induced by RA combined with serum-free medium during the differentiation of iPSCs to NSC. The expression of H1 and Hes1 protein decreased gradually, and the lowest expression level was observed after adherent culture, and the expression level of 14d increased gradually.
After the addition of 1.5DAPT, most of the cell spheres were easily adhered to the wall, and the long nerve filament like tentacles were found around the cell spheres. A large number of nerve rosette structures were visible in the cell spheres on the wall of the cell. After the culture of 2W, the cell spheres were differentiated into neural network structure. The neural markers of the induced groups were identified by real-time fluorescent quantitative PCR and immunofluorescence. The expression of Nestin, beta -tubullinIII and GFAP showed that DAPT could promote the directional differentiation of iPSC to NSC. Compared with the RA control group and RA+DMSO control group, the differentiation rate of iPSC to NSC increased after the addition of DAPT, while the proportion of neuron cells and oligodendrocytes also increased. Inhibited, iPSC can rapidly differentiate into NSC, and the inhibition of DAPT to Notch signals is continuous, making some NSC continue to differentiate into neurons and oligodendrocytes.
2 in vivo experiment
After 2.1 transplantation, the NSC derived from iPSCs could survive in the rat brain for a long time. After 1 and 2 weeks, immunofluorescence staining showed the transplanted cells in the transplanted area and the cerebral ischemia area, respectively, and the cells expressed the nerve cell marker, beta -tubulin, respectively, GFAP, indicating that the transplanted cells could migrate into the ischemic region in the brain tissue and further further migrate to the ischemic region. The rats were divided into nerve cells, and the SD rats were scored by 0,1,2,3W after the model was prepared by grasping the grip experiment, the balance Wood Walking experiment and the water maze test. Compared with the normal group, the nerve function indexes of the rats were obviously decreased after the cerebral ischemia injury. Compared with the control group, the NSC cell transplantation group was transplanted 2 weeks after the transplantation. Grip strength, balance walking ability and memory function were restored to some extent.
Conclusion:
1. RA combined with serum-free medium induction method can effectively differentiate human iPSCs into NSC.
2. the Notch signaling pathway is involved in the regulation of the directional differentiation of human iPSCs into NSC.
3., miR-9, -34a and mir-200b may participate in the regulation of NSC induced differentiation by regulating Notch signaling.
4. iPSCs derived NSC can be transplanted to the brain tissue of MCAO rats for a long time and migrate to the cerebral ischemia area and differentiate into nerve cells, to a certain extent, to promote the recovery of nerve function after cerebral ischemia injury in rats.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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相关期刊论文 前1条

1 冯年花;谢安;娄远蕾;阮琼芳;郭菲;杨阳;潘长福;邓志锋;汪泱;;人诱导性多能干细胞向神经干细胞分化的方法探讨[J];中国病理生理杂志;2010年08期

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