GAP-43在视网膜干细胞及神经干细胞定向分化过程中的作用机制研究及对比分析

发布时间：2018-07-23 20:36

【摘要】：在眼科领域内有许多难治性眼病如老年性黄斑变性、缺血性视神经病变、青光眼等因各种原因最终导致视网膜神经细胞损伤或丧失,从而造成不可逆的视力损伤甚至致盲。近年来视网膜干细胞(retinal stem cells, RSCs)的发现为治疗致盲性眼病开辟了广阔的应用前景。 RSCs具有多向分化潜能,目前研究认为通过体内外实验可将其诱导成为视网膜光感受器样细胞、视网膜神经节样细胞等多种视网膜细胞类型,而新近研究认为,RSCs经某些特定基因修饰后细胞将向特定类型的视网膜细胞分化,但目前其定向分化具体机制尚不明确。生长相关蛋白43(growth associated protein-43, GAP-43)是位于脊椎动物神经元细胞膜上的一种磷酸蛋白质,广泛分布于神经系统的神经元胞体及轴突内。现已证明GAP-43基因与神经系统及视网膜的发育、神经元轴突再生及神经突触建立均具有重要的作用。但对于其在神经干细胞(neural stem cell, NSCs)分化或其它种类的干细胞向神经细胞分化这一过程中的具体机制仍不为所知。因此本实验研究将GAP-43的研究与RSCs及NSCs的分化结合起来,希望探索出其在不同干细胞向神经细胞分化的可能机制并进行比较分析。首先,本实验就GAP-43在新生大鼠视网膜中的表达进行检测,应用免疫细胞化学染色法及RT-PCR法均检测到新生大乳鼠的视网膜细胞表达GAP-43,故而确认了GAP-43在视网膜细胞发育成熟过程中起一定作用。其次,通过无血清培养法于体外成功分离、培养并扩增大鼠RSCs及NSCs,两种干细胞均可在体外传代培养,并且第2代的RSCs及NSCs均表达神经干细胞表面标志物nestin及增殖标志物BrdU,并且用RT-PCR方法检测到RSCs表达Pax6。通过生长曲线可以看出,两种干细胞相比较,RSCs的体外培养、增殖等特点均与NSCs相似,但其自我更新的能力低于NSCs。在诱导分化方面,两种干细胞也表现出相似的特点,即均可在体外应用血清诱导分化为神经细胞,诱导分化后的细胞具有典型的神经细胞形态,并表达神经元标志物NSE、NF、βⅢ-tubulin及神经胶质细胞的标志物GFAP。此外,应用RT-PCR方法可检测到RSCs分化后的细胞表达视网膜神经节细胞标志物Thy1.1,提示RSCs分化后的细胞具有视网膜神经节细胞的特性。通过应用免疫细胞化学染色可以看出,两种干细胞在诱导分化后干细胞标志nestin表达阳性的细胞比例下降,提示其进入分化状态,比较两种干细胞诱导分化后表达NSE及GFAP阳性细胞比例可以看出RSCs组均明显低于NSCs组,结果提示RSCs相比NSCs其体外向神经元、神经胶质细胞分化的程度较低。在两种干细胞分化的这一过程中第3、7天应用免疫细胞化学染色及RT-PCR方法均检测到GAP-43持续表达,提示GAP-43基因在两种干细胞向神经细胞分化中发挥着重要作用。在接下来的实验中,首先通过测序确定GAP-43表达载体序列的正确性,并通过脂质体转染将GAP-43基因成功导入RSCs及NSCs中,通过G418的筛选富集了转染成功的干细胞,分别应用免疫细胞化学染色、RT-PCR及western blot方法证明了GAP-43基因转染到干细胞内并予表达。筛选一周后应用5%血清诱导其分化,表达外源性基因的干细胞在血清诱导的向神经细胞分化过程中表现出易于分化的趋势,具体表现为分化成神经细胞的突起数量及长度增加、突起更加舒展,经免疫细胞化学染色,两种基因修饰的干细胞分化后表达神经元标志物NSE的阳性细胞比例均明显提高(差异具有显著性,P0.05),表达神经胶质细胞标志物GFAP阳性细胞比例变化不明显(P0.05)。应用实时荧光定量PCR可检测到GAP-43基因修饰的细胞分化后表达标志物NSE、βⅢ-tubulin mRNA均有所增加(部分差异具有显著性,P0.05),GFAP mRNA水平增加不明显(P0.05)。实验证明GAP-43的表达促进了RSCs及NSCs向神经元细胞方向分化。从形态学分析,RSCs与NSCs相比后者对于GAP-43基因表达的影响更为明显。为了进一步研究GAP-43的作用,本研究通过基因转染将pGCsi-U6/Neo/GFP/ GAP-43shRNA成功导入RSCs及NSCs中,基因转染后应用G418筛选、纯化。并分别应用免疫细胞化学染色、RT-PCR及western blot方法证明了pGCsi-U6/Neo/GFP/GAP-43 shRN A转染到干细胞内并使细胞分化过程中的GAP-43基因沉默。研究发现,GAP-43表达抑制后干细胞在血清诱导的细胞分化过程中表现出形成神经细胞的突起明显受阻,同时分化后标志物NSE免疫细胞化学染色阳性细胞的比例及NSE、βⅢtubulin标志物mRNA相对含量均下降(部分差异具有显著性,P0.05),GFAP阳性细胞计数及mRNA水平变化无显著性(P0.05)。实验结果提示GAP-43沉默抑制了RSCs及NSCs向神经元细胞方向分化。从形态学分析,两种干细胞相比较NSCs所受GAP-43基因沉默的影响大于RSCs。综上所述,表达GAP-43将促进血清诱导的RSCs及NSCs向神经元细胞分化,相反,抑制该基因表达后RSCs及NSCs向神经元细胞分化明显受到抑制,因此,GAP-43基因在两种干细胞向神经元细胞分化过程中具有重要的正向导向性作用,此种调控作用在NSCs分化过程表现的更为明显。本研究为进一步探讨干细胞向神经细胞定向分化机理奠定了基础。
[Abstract]:In the field of Ophthalmology, there are many refractory eye diseases such as age-related macular degeneration, ischemic optic neuropathy, glaucoma, and so on, which eventually lead to retinal nerve cell damage or loss, resulting in irreversible visual impairment or even blindness. In recent years, the discovery of retinal stem cells (RSCs) for the treatment of blindness Eye disease has opened up a broad prospect of application.
RSCs has multiple differentiation potential. At present, it is believed that in vitro and in vivo experiments can be induced into retinal photoreceptor like cells, retinal ganglion like cells and many types of retinal cells. Recent studies suggest that RSCs can differentiate into a specific type of retinal cells after some specific gene modification, but it is currently determined. The specific mechanism for differentiation is not yet clear.
The growth related protein 43 (growth associated protein-43, GAP-43) is a phosphoric acid protein located on the membrane of the neuron cell of vertebrates. It is widely distributed in the neurons and axons of the nervous system. It has been proved that the GAP-43 gene is important to the development of the nervous system and the retina, the regeneration of the axons and the establishment of synapses in the neurons. The specific mechanism in the process of differentiation of neural stem cell (NSCs) or other kinds of stem cells to neural cells is still unknown. Therefore, this study combines the study of GAP-43 with the differentiation of RSCs and NSCs, and hopes to explore its differentiation into neural cells in different stem cells. The possible mechanism and comparative analysis.
First, we detected the expression of GAP-43 in the retina of newborn rats. The expression of GAP-43 in the retinal cells of newborn rats was detected by immunocytochemical staining and RT-PCR method. Therefore, it was confirmed that GAP-43 was used in the development of retinal cells. Secondly, it was successful in vitro by serum-free culture. Isolation, culture and amplification of RSCs and NSCs, two kinds of stem cells can be cultured in vitro, and the second generation of RSCs and NSCs all express the surface marker of neural stem cells nestin and the proliferation marker BrdU, and the RT-PCR method to detect RSCs expression Pax6. through the growth curve can be seen, two kinds of stem cells are compared, RSCs in vitro culture, The characteristics of proliferation are similar to that of NSCs, but the ability of self renewal is lower than that of NSCs. in inducing differentiation. The two kinds of stem cells also show similar characteristics, that is, they can be induced to differentiate into neural cells in vitro by using serum, and the differentiated cells have typical Neurocellular state and expression of neuron markers NSE, NF, and beta III -tubulin. And the marker of glial cells GFAP. in addition, RT-PCR method can be used to detect the expression of retinal ganglion cell marker Thy1.1 after RSCs differentiation, suggesting that the cells after RSCs differentiation have the characteristics of retinal ganglion cells. By using immunocytochemical staining, the two kinds of stem cells can be induced to be thin after differentiation. The proportion of cell marker nestin positive cells declined, suggesting that the cells entered the differentiation state. Comparing the expression of NSE and GFAP positive cells after differentiation of two kinds of stem cells, the proportion of NSE and GFAP positive cells showed that the RSCs group was significantly lower than that of the NSCs group. The results suggested that RSCs had a lower degree of differentiation of the neuroglial cells than the NSCs. In this process, the continuous expression of GAP-43 was detected by immunocytochemical staining and RT-PCR method on day 3,7, suggesting that the GAP-43 gene plays an important role in the differentiation of two kinds of stem cells to neural cells.
In the next experiment, the correctness of the GAP-43 expression vector sequence was determined by sequencing, and the GAP-43 gene was successfully transfected into RSCs and NSCs through liposome transfection. The successful transfected stem cells were enriched by G418 screening. The immunocytochemical staining, RT-PCR and Western blot methods were used to prove the GAP-43 gene transfection. After one week, 5% serum was used to induce differentiation. The stem cells expressing exogenous genes showed a tendency to differentiate easily during the differentiation of neural cells induced by serum. The specific expression was the increase in the number and length of the neurites that differentiated into nerve cells, the protruding and the immunocytochemical staining, two The proportion of the positive cells expressing the neuron marker NSE after the differentiation of the gene modified stem cells was significantly increased (the difference was significant, P0.05). The expression of GFAP positive cells in the expression of the neuroglial cell markers was not significant (P0.05). The application of real-time fluorescent quantitative PCR could detect the expression marker NS after the cell differentiation of the GAP-43 gene. E, beta III -tubulin mRNA increased (part difference was significant, P0.05), and GFAP mRNA level increased not significantly (P0.05). Experiments showed that the expression of GAP-43 promoted the differentiation of RSCs and NSCs into neuronal cell direction.
In order to further study the role of GAP-43, this study successfully transfected pGCsi-U6/Neo/GFP/ GAP-43shRNA into RSCs and NSCs by gene transfection. After gene transfection, the gene transfection was screened and purified by G418. Immunocytochemical staining was used, and RT-PCR and Western blot methods were used to prove that pGCsi-U6/ Neo/GFP/GAP-43 shRN was transfected into the stem cells and fined. The GAP-43 gene was silenced in the process of cell differentiation. It was found that the protuberances of the cells in the differentiation of serum induced by GAP-43 expression were obviously blocked, and the proportion of NSE immunocytochemical staining positive cells after differentiation and the relative content of NSE, and the relative content of the beta III tubulin marker mRNA were all decreased. Some of the differences were significant, P0.05), there was no significant change in GFAP positive cell count and mRNA level (P0.05). The experimental results suggest that GAP-43 silence inhibits the direction differentiation of RSCs and NSCs into neuron cells. From morphological analysis, the effect of GAP-43 gene silencing on NSCs is greater than that of NSCs, compared with the two types of stem cells.
To sum up, the expression of GAP-43 will promote the differentiation of serum induced RSCs and NSCs into neuron cells. On the contrary, the differentiation of RSCs and NSCs to neuronal cells is inhibited obviously after the inhibition of the gene expression. Therefore, the GAP-43 gene has an important positive direction in the process of differentiation of two kinds of stem cells to neuronal cells. This regulation is in N The differentiation process of SCs was more obvious. This study laid a foundation for further study on the mechanism of neural differentiation of stem cells.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.5

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