Sertoli细胞促进神经前体细胞分化信号转导相关分子作用机理研究
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摘要
很多研究将Sertoli细胞在神经前体细胞分化中的作用归结为Sertoli细胞分泌的细胞因子和营养因子等分泌物上,而从Notch信号转导系统角度,通过Notch受体与配体的相互作用,来探讨细胞与细胞间的相互接触在神经前体细胞分化中的意义,国内外尚未见报道。因而我们从Notch信号转导相关分子方面,对Sertoli细胞在神经前体细胞分化作用机理进行了深入研究。现将四部分内容分述如下:
第一部分:大鼠大脑皮质神经前体细胞的分离培养及其增殖分化特性鉴定
目的:神经前体细胞是一类具有多向分化潜能和自我复制能力,在特定条件下,能够向特定类型神经元或神经胶质细胞分化的细胞。本实验通过体外分离和培养大鼠大脑皮质神经前体细胞,对神经前体细胞增殖和分化特性进行鉴定。
方法:分离2周龄大鼠大脑皮质神经前体细胞,在含EGF、bFGF和GDNF的NSC培养基中进行体外培养,使用免疫细胞荧光染色技术对细胞的分化特性进行鉴定。
结果:诱导神经前体细胞增殖的有丝裂原包括碱性成纤维细胞生长因子和表皮生长因子等。我们的实验结果表明EGF、bFGF和GDNF可促进神经前体细胞的增殖及神经球的克隆形成,并获得了Nestin阳性的神经前体细胞,其可分化为分别表达β-III tubulin、GFAP和GalC的阳性细胞。
结论:神经前体细胞在体外大量扩增后,植入脑组织内或通过基因重组后再植入脑内,可用来治疗神经系统疾病,如帕金森病或阿尔茨海默病。中枢神经系统中神经前体细胞的增殖与分化是一个复杂的进程,存在细胞自身基因调控和外来信号调控两种机制。因而,目前需要积极寻找可靠的方法来有效地分离和扩增神经前体细胞,并精确地了解其增殖与分化特性。本研究第一部分利用大鼠的大脑皮质分离培养神经前体细胞,观察了bFGF、EGF和GDNF三种生长因子对神经前体细胞在体外培养增殖和分化的影响,体外分离和培养的大脑皮质神经前体细胞,在含EGF、bFGF和GDNF的NSC培养基中培养,具有不断增殖和多向分化的能力,符合神经前体细胞的特点,为神经前体细胞的定向分化研究奠定了基础。
第二部分:Sertoli细胞对大鼠大脑皮质神经前体细胞的诱导分化作用研究
目的:同一来源的神经前体细胞移植到不同部位后,其分化结果也不相同,但往往与接受移植部位的细胞相似,提示外来信号调控作用的重要性。而且,在不同因素影响下,神经干细胞还具有横向分化潜能。本部分主要研究Sertoli细胞对体外培养大鼠大脑皮质神经前体细胞生长分化的作用特点,及向多巴胺能神经元分化的诱导作用。
方法:将大鼠Sertoli细胞与14d大鼠皮质神经前体细胞体外共培养,免疫组化检测神经干细胞标记物Nestin ,神经元、星形胶质细胞和小胶质细胞标记物β-Ⅲtubulin、GFAP和GalC,及多巴胺能神经元标记物酪氨酸羟化酶(TH)的表达情况。
结果:随着共培养时间的延长,神经干细胞中GFAP阳性细胞数量逐渐减少,而β-Ⅲtubulin阳性细胞数增多,并出现TH阳性的神经元。
结论:把胎鼠或成年鼠前脑神经干细胞移植给经亚致死量照射破坏了造血系统的同种宿主的骨髓,发现后者出现了供者源性的骨髓样细胞、淋巴细胞和早期的造血干细胞;把从人胚胎和成年小鼠快速分离的神经干细胞与成肌细胞共培养,结果神经前体细胞分化为骨骼肌细胞。证实了神经干细胞有着更大的分化潜能。Clarke等发现,成年鼠脑的干细胞能在鸡胚环境中被诱导形成所有的胚层。这些研究结果显示,环境的指导性信号对神经干细胞分化的重要作用。我们的实验结果表明:Sertoli细胞抑制体外培养的大鼠皮质神经前体细胞的生长,但具有显著的促神经元分化的作用,并能够诱导与其共培养的大鼠大脑皮质神经前体细胞分化为多巴胺能神经元。
第三部分:Sertoli细胞共培养大鼠大脑皮质神经前体细胞分化中Notch信号相关基因表达分析
目的:对那些在发育期调控细胞决定和细胞分化的基因进行了深入研究,发现Notch信号转导通路是具备这些功能的重要家族之一。它在大量的组织和器官的发育中发挥重要作用,调控着组织类型及形态的发生。本部分研究主要检测与Srtoli细胞共培养条件下大鼠大脑皮质神经前体细胞分化过程中Notch信号相关分子的表达变化,分析Notch信号途径在神经前体细胞分化过程中的可能作用。
方法:本实验通过神经前体细胞与Srtoli细胞共培养,用Real-time PCR相对定量法,以β-actin为内对照,分别检测Notch受体Notch1、Notch2、Notch3、Notch4,及其配体Delta1、Delta3、Jagged1、Jagged2,以及Hes1、Ngn1基因的表达变化。
结果:养细胞汇片后第5d, sertoli细胞Notch1～4基本表达维持不变;其配体Jagged2表达较高,但不表达Delta1;同时也不表达Hes1、Ngn1。神经前体细胞Notch表达水平较高,尤以Notch1为高;而其配体以Jagged1、Jagged2表达较高,前神经因子Hes1相对有较高的表达,而Ngn1表达较低。细胞共培养条件下,Notch受体、配体以及Hes1与神经前体细胞组相比都有明显的下调,而Ngn1却相对有所提高。
结论:在干细胞发育过程中,细胞膜表面分子Notch通过结合其配体Delta或Jagged而在干细胞和周围细胞间传递信号,这种细胞间的相互作用可以调控干细胞的分化。在哺乳动物,Notch信号通过CBF1激活E(spl)的同源物Hes[Hairy and E(spl)]1/5,并影响Ngn(Neurogenins)等前神经因子的表达,从而调节神经干细胞的分化。Notch在细胞间传导相互作用的信号,并通过这种邻近细胞间的信号传递来精确调控各谱系细胞分化。Notch信号通路在哺乳动物CNS发育中参与神经发生过程,决定着CNS祖细胞是选择继续增殖还是向神经元分化。一般而言,Notch信号转导通路的激活维持神经干细胞的稳定状态,抑制定向分化;但另一方面,也有证据表明激活Notch信号转导通路又可以加速并不可逆地促进神经干细胞向成胶质细胞分化。本研究第三部分应用实时荧光定量PCR法,检测了与sertoli细胞共培养神经前体细胞分化过程中Notch相关基因Notch1～4、Delta1、Delta3、Jagged1、Jagged2、hes1和ngn1的表达变化,实验表明在与sertoli细胞共培养条件下,神经前体细胞的分化特点与Notch信号相关分子的表达变化相关。Sertoli细胞可通过Notch配体的作用,调节神经前体细胞Notch信号分子的表达。在共培养条件下,Sertoli细胞对神经前体细胞Hes1表达有抑制作用,但对Ngn1有正向调节作用,从而影响神经前体细胞的分化。
第四部分:Hes1基因真核表达载体的构建及瞬时表达在大脑皮质神经前体细胞分化中的作用
目的:随着神经前体细胞分离、提纯及体外培养等研究的深入,对内在和外在因子控制神经前体细胞增殖和分化的研究也取得了显著进步。目前研究证明,发生在不同组织内的同一机制调控着该关键步骤,而其中涉及最多者为bHLH基因家族。bHLH是一组转录因子,通过一个碱性区域和一个HLH域的异二聚化与DNA形成特定的接触。Notch信号的下游基因E(spl)/Hes就是其中之一。本实验通过克隆大鼠Hes1基因并构建真核表达载体pCDNA3.1-Hes1,将重组质粒转染大鼠大脑皮质神经前体细胞,获得高表达Hes1基因的神经前体细胞克隆。以探讨Hes1基因高表达对神经前体细胞分化的影响。
方法:从大鼠脑组织中提取总RNA,用RT-PCR方法获得Hes1基因的全长cDNA,插入pGEM-T-Easy克隆载体中进行序列测定,测序正确后将其亚克隆至表达载体pCDNA3.1,利用脂质体将重组质粒转染原代培养的神经前体细胞,经G418筛选获得抗性细胞克隆,采用RT-PCR方法鉴定Hes1基因在神经前体细胞基因组中的存在,实时荧光定量PCR检测Hes1基因的过表达情况。
结果:经限制性内切酶酶切图谱分析和DNA序列测定证实目的基因已插入重组质粒,RT-PCR证明经G418筛选得到的转基因神经前体细胞克隆的基因组DNA中存在Hes1基因,实时荧光定量PCR进一步证明转基因神经前体细胞Hes1基因的mRNA呈高表达。
结论:不同的bHLH因子具有不同的决定和分化的功能,经常作为一个相关蛋白质家族而存在,按时空顺序短暂表达于细胞系分化的不同阶段。早期表达因子参与祖细胞的决定,而晚期表达因子则参与分裂细胞的终期分化,早期表达蛋白质激活晚期蛋白质表达。研究显示,Notch和它的下游效应器Hes基因,以及同源基因Emx2具有维持脑内多潜能干细胞状态的功能,而Ngn1/2和Mash1和Pax6是神经前体细胞向神经元分化的必要条件。Kabos等发现阻断Hes1表达促进中枢神经干细胞分化为γ-氨基丁酸神经元。神经元形成后,Hes因子的激活促进星形胶质细胞的分化,而前神经bHLH因子Ngn抑制星形胶质细胞的分化。本实验通过克隆大鼠Hes1基因,构建真核表达载体pCDNA3.1-Hes1,并将重组质粒转染大鼠大脑皮质神经前体细胞,研究观察了Hes1基因过表达对神经前体细胞Ngn1表达的影响,以及这些变化在神经前体细胞分化的作用。实验成功构建了大鼠Hes1基因的真核表达载体,获得了稳定表达Hes1基因的神经前体细胞克隆,细胞培养实验表明:Hes1基因高表达可抑制神经前提细胞Ngn1的表达,并促进神经前提细胞向神经胶质细胞的分化。
Many studies on the functions of Sertoli cells in the differentiation of precursor cells have credit it to the cytokines and nutrient factors that Sertoli cells secreted. But there are no reports about the studies on its functions from the respects of Notch-related genes and Notch receptors and ligands through which the cells contact one another. So we studied the expression of Notch-related genes in the differentiation of precursor cells co-cultured with Sertoli cells, and we hope furthermore to understand the mechanism of neural precursor cells that Sertoli cells exerted. Now the four parts are summarized as follows now:
PartⅠ:Isolation and differentiation of neural precursor cells from cerebral cortex of Wistar rats
Objective: Neural precursor cell is a type of cells that has a capability of multiple differentiation potential and self-renewal, it can differentiate to a particular type of neurons or glial cells under a particular condition. Our research is to isolate and culture neural precursor cells(NPCs) from brain cortex of rats in vitro and to identify the differentiations of the cells.
Methods: NPCs in cortex of twe weeks born rats were isolated and cultured with the NSC medium which contains EGF、bFGF and GDNF in vitro. Immuno-fluorescence staining was used to study the differentiation characters of the cells.
Results: The EGF、bFGF and GDNF could enhance the proliferations of the NPCs and cloning formations of neural spheres,The Nestin positive NPCs were obtained at the same time and it could becomeβ- III tubulin, GFAP and GalC positive cells (neurons, astrocytes and oligodendrocytes).
Conclusion: The mitogens can induce proliferation of neural precursors, which include basic fibroblast growth factor(bFGF) and epidermal growth factor(EGF) etc. The neural precursor cells propagated in great quantities in vitro or through gene transformation, then transplanted into brains. It can be used to cure the nervous system disease, such as Parkinson’s diseases or Alzheimer’s diseases. But the proliferation and differentiation of the neural precursor cells in central nervous system is a complicated progress. And there are two kinds of mechanisms to adjust and control the proliferation and differentiation of the neural precursor cells, the one is signals outside the cells, the other is genes inside the cells. Therefore, we need to look for the dependable methods to separate and propagate the neural precursor cells availably and find out by the square the characteristics of its proliferation and differentiation. NPCs isolated from brain cortex and cultured in the NSC medium contained EGF、bFGF and GDNF in vitro have the ability of proliferation and differentiation. So they may be used as graft for the cell transplantation therapy in neuronal handicap and injury. In the light of the purpose above mentioned, The research of the part one made use of the cerebral cortex of rat to separate and develop neural precursor cells, and observed the influences of three kinds of growth factors bFGF, EGF and glial fibrillary acid protein (GDNF) on the proliferation and differentiation of the neural precursors cells in vitro. The research was laying a foundation on the studies for oriented differentiation of neural precursor cells.
PartⅡ:The study on Sertoli cells inducing the differentiation of neural progenitor cells from cerebral cortex of rat
Objective:Once neural precursor cells com from one part transplanted to different part, the result of its differentiation is also different. It is usually similar to the cells of the part accepted transplantation. It hints that the signals outside cells have an important functions in regulating cell differentiation. Moreover, with the influence of different factors, the neural stem cells have potential ability of horizontal differentiation. In this part we want to investigate the effect of Sertoli cell on proliferation and differentiation of cultured neural prigenitor cells(NPCs) from cerebral cortex of the rat.
Methods:Sertoli cells from the testis and the neural precursors cells(NPCs) from the cerebral cortex of Wistar rat were co-cultured. The detections ofβ- III tubulin, GFAP, GalC and tyrosine hydroxylase (TH) were made by immunocytochemistry in vitro.
Results:The numbers of NPs with nestin-positive was decreasing along the time of the co-culture, so were the GFAP and GalC. While theβ- III tubulin-positive cells increased, and TH-positive cells appeared in the co-culture.
Conclusion:if the neural stem cell from forebrain of adulthood or embryonic rat transplanted to the marrow of the same race host its blood system had broken down through second death-dosage of irradiation, we will find there were marrow-like cells, lymphoid cells and earlier periods blood stem cells that derived from the cells of provider source in the host marrow. One experiment testified that if the neural stem cells separated quickly from person's embryo and adulthood mice co-cultured with myoblast cells, as a result, the neural stem cells will differentiated into the skeleton muscle cell. Clarke and his colleague detected that adult stem cell from rat brain can be induced to become all layers of embryonic strata in the chicken embryonic environment. These researches show that the leading signals of the environment have an important effect on the differentiation of neural precursor cells, meanwhile it can confirm that neural stem cells have more potential differentiation. Fanigaki discovered that Notch play an important part in the neural precursor cells differentiating into glial cells. Through up-regulation of the bHLH protein Hes1 and Hes5, the signal of Notch can adjust and control proliferation and differentiation of neural precursor cells. Notch signaling regulates the differentiation of different cells through a kind of by-pass repression. When a group of same cells is stimulated by a certain and special signal at the same time, only a small parts of cells is induced by the signal and appear the differentiation toward particular direction. But the Adjacent cells that express more proteins of Notches can't differentiate toward particular direction, and behave a repression of differentiation. The function of by-pass repression depended on dosages of Notch proteins have already got the confirmation in the Drosophila and mice. Through the direct contact between cells, the expression of different Notch ligands together with the limited activation of proteins of Notch can induce cells differentiating toward dissimilarity directions. According to the research above, this experiment of the part two using the precursors cells from cerebral cortex of rat co-cultured with Sertoli cells, investigate the functions of interactions of two kinds of cells as well, and explore the characteristics of Sertoli cells inducing the neural precursors cells proliferation and differentiation to definite direction. Sertoli cells exert the negative effect on proliferation of the NPCs,but have distinctly the positive effect on the NPCs for its differentiation into neurons in vitro, The neural precursors from the cortex of rat can be directly induced to differentiation into the dopaminergic progenitors by co-cultured with the Sertoli cells.
PartⅢ:Expression of Notch-related Genes in the Differentiation of the neural precursor cells(NPCs) from brain cortex of rats co-cultured with sertoli cells
Objective: If a stable precursor cells pool needs to be maintained then the balance between asymmetrical and symmetrical division must be tightly regulated. Carried on the thorough research for those genes that determine the cell decision and differentiation, it has been discovered that the signal of Notch turns to be one of the important families that has the functions. Notch may be important molecules in specifying cell fate, and play an important role in the growth of much tissues and organs, and regulate the occurrence and morphogenetic processes of the special tissue types. This research of part three applies the fluorescence quantitative real time RT-PCR method examining the expressing variety of the Notch related gene Notch1～4, Delta1, Delta3, Jagged1, Jagged2, Hes1 and Ngn1,and investigates the expression of Notch-related genes in the neural precursor cells(NPCs) from brain cortex of rats in vitro and analyze the possible function of Notch pathway in the differentiation of neural precursor cells co-cultured with sertoli cells.
Methods: NPCs in cortex of twe weeks born rats were isolated and cultured with the NPC medium contained EGF、bFGF and GDNF in vitro. At the same time to isolate its sertoli cells, and co-culture the neural precursor cells with sertoli cells. The expressions of notch-related genes were identified in cells of different groups. Total RNA was isolated at 5 day after confluence of cells. Fluorescent quantitative real-time RT-PCR(FQ-PCR) was used to detect the expression of both Notch(Notch1,Notch2,Notch3 and Notch4) and its ligand (Delta1,Delta3,Jagged1 and Jagged2); expression of ngn1and hes1 genes was also examined at the control ofβ-actin.
Results: Under the NPC medium contained EGF、bFGF and GDNF culture condition, the expression of the four notches in the sertoli cells group were no different, and the level of Jagged2 showed slightly high, but not express Delta1. In the neural precursor cells group the four notch genes especially the Notch1 had high expressions, So Its Jagged1 and Jagged2. Its Hes1 gene was high level, but the expression of its Ngn1 gene was lower respectively. Interestingly, the expression of four notch genes and its four ligands plus Hes1 exhibited down-regulation in the co-cultured cells group than the neural precursor cells group, but its Ngn1 gene showed up-regulation.
Conclusion: The Notch conducts the signals of cell-to-cell interaction between the cells, and precisely regulates the differentiation of each group of cells through adjusting the signals between adjacent cells. The Notch signaling participate in the neural occurrence process in the CNS development, and give the progenitor cell of CNS a choice to continue to propagation or to differentiate into neurons. Generally speaking, the activation of Notch signaling maintain the stable state of neural stem cell, and repress the definite oriented differentiation; But on the other hand, there are evidences prove that activation of the signal of Notch can accelerate and against the ground promote neural precursors cells become glial cells. Some studies manifest that in the process of neural precursors cells development, The Notch on the surface of cell membrane can combine its ligand Delta or Jaggeds and deliver the signals between stem cells and surroundings cells. The cell-to-cell interaction of this kind can adjust and control differentiation of stem cells. For mammals, the signal of Notch activates Hes1/5 homologic to the E(spl) through a CBF1 together, and affect the expression of the pre-neural factor Ngn( Neurogenins), and regulate differentiation of the neural stem cell. The experiment indicate that the specific properties of neural precursors cells have relationship with the varieties of Notch signaling related genes expression under the condition of co-cultured with Sertoli cells.Notch pathway is involved in the differentiation of the neural precursor cells, Sertoli cells can suppress the expression of the Hes1 gene, meanwhile enhance the expression of the Ngn1 gene in the neural precursor cells co-cultured with it. The Sertoli cells may have a positive effect on neural precursor cells differentiation through affecting Notch signaling.
PartⅣ:Construction of eukaryotic expression vector containing rat Hes1 genes and expression in neural precursor cells for the study of its differentiation functions
Objective: Along with the development of Separation, purification and culture for the neural precursor cells in vitro, the study of research for the neural precursor cells controlling its proliferation and differentiation under the influence of cell intrinsic mechanisms or extrinsic signals also obtained great progress. The current studies demonstrate the same mechanism occurred in different tissues which control the key step, and among them involved the most is genes of bHLH families of proteins. The experiment of part four is to clone hes1 gene, and set up eukaryotic expressing victor pCDNA3.1- Hes1, then transfected it to the neural precursors cells from cerebral cortex of rat , we construct its eukaryotic expression vector and obtain positive neural precursor cell clones expressing Hes1 genes stably through transfectation,then to study their effects to the differentiation of neural precursor cells.
Methods: The total RNA was extracted from 14 day Wistar rat brain. The full-length cDNA encoding Hes1 genes were obtained using RT-PCR method and inserted into pGEM T Easy cloning vector. After the sequencing was confirmed, the gene was subcloned to pCDNA3.1 to construct recombinant eukaryotic expression vectors of pCDNA3.1-Hes1. The recombinant plasmid was transfected into neural precursor cells from cerebral cortex of 14 day Wistar rat by lipofectamine method and positive cell clones were screened with G418. The existence of Hes1 genes in the transfected cells genomic DNA ,and the over-expression of their mRNA in the transfected cells were confirmed with Fluorescent quantitative real-time RT-PCR(FQ-PCR).
Results: Enzyme digestion analysis and sequencing showed that the target genes were cloned into recombinant vector. The Existence and over-expression of Hes1 genes in the transfected neural precursor cells was identified with FQ-PCR and it display that Hes1 mRNA had high expressed in the transfected neural precursor cells.
Conclusion: bHLH is the exact subset of transcription factors, containing an alkaline district and a HLH domain. The homologues of many of these transcription factors often act in cascades to control the successive steps in neuronal determination and differentiation as well as neuronal-subtype identity. These transcription factors often construct heterodimers each other, which can band to particular sequences of DNA, and act at particular stage of stem cell development. The downstream gene E(spl)/ Hes of the Notch signaling pathways is one of them, the different factors of bHLH have different function that decide cell fate decision or differentiation. As a related family of proteins, they usually transiently express in a proper time and space order and at the different stage of cell development. The factors that early expressed participated in the cell decision, but the later expressed participated in the cell differentiation eventually, the proteins that expressed in early stages activated the expression of proteins in later stages. Many study manifest that Notch and its downstream target Hes gene have important functions on the potentials maintenance of stem cell in brain. Ngn1/2 and Mash1 are essential for the neural precursor cells differentaition into neurons. Kabos discovered that the repression of Hes1 promote the neural stem cells in central nervous system differentiation intoγ-GABA neurons. Moreover, after the formation of neurons, the activity of the Hes factor often promotes the glial differentiation, the bHLH factors Ngn inhibits a glial fate. The eukaryotic expression plasmid containing Hes1 genes were successfully constructed. The positive neural precursor cell clones expressing Hes1 genes stably were obtained, which may be a promising cell model for studying the biological function of Hes1 genes and the role of Hes1 genes in the differentiation of neural precursor cells. The experiment manifested that the high expression of Hes1 can restrain the expression of Ngn1 gene,and enhance the neural precursor cells differentiation to glial cells. From the research we observed how the high expression of Hes1 gene influence on the differentiation of neural precursors cells.
第一部分:大鼠大脑皮质神经前体细胞的分离培养及其增殖分化特性鉴定
目的:神经前体细胞是一类具有多向分化潜能和自我复制能力,在特定条件下,能够向特定类型神经元或神经胶质细胞分化的细胞。本实验通过体外分离和培养大鼠大脑皮质神经前体细胞,对神经前体细胞增殖和分化特性进行鉴定。
方法:分离2周龄大鼠大脑皮质神经前体细胞,在含EGF、bFGF和GDNF的NSC培养基中进行体外培养,使用免疫细胞荧光染色技术对细胞的分化特性进行鉴定。
结果:诱导神经前体细胞增殖的有丝裂原包括碱性成纤维细胞生长因子和表皮生长因子等。我们的实验结果表明EGF、bFGF和GDNF可促进神经前体细胞的增殖及神经球的克隆形成,并获得了Nestin阳性的神经前体细胞,其可分化为分别表达β-III tubulin、GFAP和GalC的阳性细胞。
结论:神经前体细胞在体外大量扩增后,植入脑组织内或通过基因重组后再植入脑内,可用来治疗神经系统疾病,如帕金森病或阿尔茨海默病。中枢神经系统中神经前体细胞的增殖与分化是一个复杂的进程,存在细胞自身基因调控和外来信号调控两种机制。因而,目前需要积极寻找可靠的方法来有效地分离和扩增神经前体细胞,并精确地了解其增殖与分化特性。本研究第一部分利用大鼠的大脑皮质分离培养神经前体细胞,观察了bFGF、EGF和GDNF三种生长因子对神经前体细胞在体外培养增殖和分化的影响,体外分离和培养的大脑皮质神经前体细胞,在含EGF、bFGF和GDNF的NSC培养基中培养,具有不断增殖和多向分化的能力,符合神经前体细胞的特点,为神经前体细胞的定向分化研究奠定了基础。
第二部分:Sertoli细胞对大鼠大脑皮质神经前体细胞的诱导分化作用研究
目的:同一来源的神经前体细胞移植到不同部位后,其分化结果也不相同,但往往与接受移植部位的细胞相似,提示外来信号调控作用的重要性。而且,在不同因素影响下,神经干细胞还具有横向分化潜能。本部分主要研究Sertoli细胞对体外培养大鼠大脑皮质神经前体细胞生长分化的作用特点,及向多巴胺能神经元分化的诱导作用。
方法:将大鼠Sertoli细胞与14d大鼠皮质神经前体细胞体外共培养,免疫组化检测神经干细胞标记物Nestin ,神经元、星形胶质细胞和小胶质细胞标记物β-Ⅲtubulin、GFAP和GalC,及多巴胺能神经元标记物酪氨酸羟化酶(TH)的表达情况。
结果:随着共培养时间的延长,神经干细胞中GFAP阳性细胞数量逐渐减少,而β-Ⅲtubulin阳性细胞数增多,并出现TH阳性的神经元。
结论:把胎鼠或成年鼠前脑神经干细胞移植给经亚致死量照射破坏了造血系统的同种宿主的骨髓,发现后者出现了供者源性的骨髓样细胞、淋巴细胞和早期的造血干细胞;把从人胚胎和成年小鼠快速分离的神经干细胞与成肌细胞共培养,结果神经前体细胞分化为骨骼肌细胞。证实了神经干细胞有着更大的分化潜能。Clarke等发现,成年鼠脑的干细胞能在鸡胚环境中被诱导形成所有的胚层。这些研究结果显示,环境的指导性信号对神经干细胞分化的重要作用。我们的实验结果表明:Sertoli细胞抑制体外培养的大鼠皮质神经前体细胞的生长,但具有显著的促神经元分化的作用,并能够诱导与其共培养的大鼠大脑皮质神经前体细胞分化为多巴胺能神经元。
第三部分:Sertoli细胞共培养大鼠大脑皮质神经前体细胞分化中Notch信号相关基因表达分析
目的:对那些在发育期调控细胞决定和细胞分化的基因进行了深入研究,发现Notch信号转导通路是具备这些功能的重要家族之一。它在大量的组织和器官的发育中发挥重要作用,调控着组织类型及形态的发生。本部分研究主要检测与Srtoli细胞共培养条件下大鼠大脑皮质神经前体细胞分化过程中Notch信号相关分子的表达变化,分析Notch信号途径在神经前体细胞分化过程中的可能作用。
方法:本实验通过神经前体细胞与Srtoli细胞共培养,用Real-time PCR相对定量法,以β-actin为内对照,分别检测Notch受体Notch1、Notch2、Notch3、Notch4,及其配体Delta1、Delta3、Jagged1、Jagged2,以及Hes1、Ngn1基因的表达变化。
结果:养细胞汇片后第5d, sertoli细胞Notch1～4基本表达维持不变;其配体Jagged2表达较高,但不表达Delta1;同时也不表达Hes1、Ngn1。神经前体细胞Notch表达水平较高,尤以Notch1为高;而其配体以Jagged1、Jagged2表达较高,前神经因子Hes1相对有较高的表达,而Ngn1表达较低。细胞共培养条件下,Notch受体、配体以及Hes1与神经前体细胞组相比都有明显的下调,而Ngn1却相对有所提高。
结论:在干细胞发育过程中,细胞膜表面分子Notch通过结合其配体Delta或Jagged而在干细胞和周围细胞间传递信号,这种细胞间的相互作用可以调控干细胞的分化。在哺乳动物,Notch信号通过CBF1激活E(spl)的同源物Hes[Hairy and E(spl)]1/5,并影响Ngn(Neurogenins)等前神经因子的表达,从而调节神经干细胞的分化。Notch在细胞间传导相互作用的信号,并通过这种邻近细胞间的信号传递来精确调控各谱系细胞分化。Notch信号通路在哺乳动物CNS发育中参与神经发生过程,决定着CNS祖细胞是选择继续增殖还是向神经元分化。一般而言,Notch信号转导通路的激活维持神经干细胞的稳定状态,抑制定向分化;但另一方面,也有证据表明激活Notch信号转导通路又可以加速并不可逆地促进神经干细胞向成胶质细胞分化。本研究第三部分应用实时荧光定量PCR法,检测了与sertoli细胞共培养神经前体细胞分化过程中Notch相关基因Notch1～4、Delta1、Delta3、Jagged1、Jagged2、hes1和ngn1的表达变化,实验表明在与sertoli细胞共培养条件下,神经前体细胞的分化特点与Notch信号相关分子的表达变化相关。Sertoli细胞可通过Notch配体的作用,调节神经前体细胞Notch信号分子的表达。在共培养条件下,Sertoli细胞对神经前体细胞Hes1表达有抑制作用,但对Ngn1有正向调节作用,从而影响神经前体细胞的分化。
第四部分:Hes1基因真核表达载体的构建及瞬时表达在大脑皮质神经前体细胞分化中的作用
目的:随着神经前体细胞分离、提纯及体外培养等研究的深入,对内在和外在因子控制神经前体细胞增殖和分化的研究也取得了显著进步。目前研究证明,发生在不同组织内的同一机制调控着该关键步骤,而其中涉及最多者为bHLH基因家族。bHLH是一组转录因子,通过一个碱性区域和一个HLH域的异二聚化与DNA形成特定的接触。Notch信号的下游基因E(spl)/Hes就是其中之一。本实验通过克隆大鼠Hes1基因并构建真核表达载体pCDNA3.1-Hes1,将重组质粒转染大鼠大脑皮质神经前体细胞,获得高表达Hes1基因的神经前体细胞克隆。以探讨Hes1基因高表达对神经前体细胞分化的影响。
方法:从大鼠脑组织中提取总RNA,用RT-PCR方法获得Hes1基因的全长cDNA,插入pGEM-T-Easy克隆载体中进行序列测定,测序正确后将其亚克隆至表达载体pCDNA3.1,利用脂质体将重组质粒转染原代培养的神经前体细胞,经G418筛选获得抗性细胞克隆,采用RT-PCR方法鉴定Hes1基因在神经前体细胞基因组中的存在,实时荧光定量PCR检测Hes1基因的过表达情况。
结果:经限制性内切酶酶切图谱分析和DNA序列测定证实目的基因已插入重组质粒,RT-PCR证明经G418筛选得到的转基因神经前体细胞克隆的基因组DNA中存在Hes1基因,实时荧光定量PCR进一步证明转基因神经前体细胞Hes1基因的mRNA呈高表达。
结论:不同的bHLH因子具有不同的决定和分化的功能,经常作为一个相关蛋白质家族而存在,按时空顺序短暂表达于细胞系分化的不同阶段。早期表达因子参与祖细胞的决定,而晚期表达因子则参与分裂细胞的终期分化,早期表达蛋白质激活晚期蛋白质表达。研究显示,Notch和它的下游效应器Hes基因,以及同源基因Emx2具有维持脑内多潜能干细胞状态的功能,而Ngn1/2和Mash1和Pax6是神经前体细胞向神经元分化的必要条件。Kabos等发现阻断Hes1表达促进中枢神经干细胞分化为γ-氨基丁酸神经元。神经元形成后,Hes因子的激活促进星形胶质细胞的分化,而前神经bHLH因子Ngn抑制星形胶质细胞的分化。本实验通过克隆大鼠Hes1基因,构建真核表达载体pCDNA3.1-Hes1,并将重组质粒转染大鼠大脑皮质神经前体细胞,研究观察了Hes1基因过表达对神经前体细胞Ngn1表达的影响,以及这些变化在神经前体细胞分化的作用。实验成功构建了大鼠Hes1基因的真核表达载体,获得了稳定表达Hes1基因的神经前体细胞克隆,细胞培养实验表明:Hes1基因高表达可抑制神经前提细胞Ngn1的表达,并促进神经前提细胞向神经胶质细胞的分化。
Many studies on the functions of Sertoli cells in the differentiation of precursor cells have credit it to the cytokines and nutrient factors that Sertoli cells secreted. But there are no reports about the studies on its functions from the respects of Notch-related genes and Notch receptors and ligands through which the cells contact one another. So we studied the expression of Notch-related genes in the differentiation of precursor cells co-cultured with Sertoli cells, and we hope furthermore to understand the mechanism of neural precursor cells that Sertoli cells exerted. Now the four parts are summarized as follows now:
PartⅠ:Isolation and differentiation of neural precursor cells from cerebral cortex of Wistar rats
Objective: Neural precursor cell is a type of cells that has a capability of multiple differentiation potential and self-renewal, it can differentiate to a particular type of neurons or glial cells under a particular condition. Our research is to isolate and culture neural precursor cells(NPCs) from brain cortex of rats in vitro and to identify the differentiations of the cells.
Methods: NPCs in cortex of twe weeks born rats were isolated and cultured with the NSC medium which contains EGF、bFGF and GDNF in vitro. Immuno-fluorescence staining was used to study the differentiation characters of the cells.
Results: The EGF、bFGF and GDNF could enhance the proliferations of the NPCs and cloning formations of neural spheres,The Nestin positive NPCs were obtained at the same time and it could becomeβ- III tubulin, GFAP and GalC positive cells (neurons, astrocytes and oligodendrocytes).
Conclusion: The mitogens can induce proliferation of neural precursors, which include basic fibroblast growth factor(bFGF) and epidermal growth factor(EGF) etc. The neural precursor cells propagated in great quantities in vitro or through gene transformation, then transplanted into brains. It can be used to cure the nervous system disease, such as Parkinson’s diseases or Alzheimer’s diseases. But the proliferation and differentiation of the neural precursor cells in central nervous system is a complicated progress. And there are two kinds of mechanisms to adjust and control the proliferation and differentiation of the neural precursor cells, the one is signals outside the cells, the other is genes inside the cells. Therefore, we need to look for the dependable methods to separate and propagate the neural precursor cells availably and find out by the square the characteristics of its proliferation and differentiation. NPCs isolated from brain cortex and cultured in the NSC medium contained EGF、bFGF and GDNF in vitro have the ability of proliferation and differentiation. So they may be used as graft for the cell transplantation therapy in neuronal handicap and injury. In the light of the purpose above mentioned, The research of the part one made use of the cerebral cortex of rat to separate and develop neural precursor cells, and observed the influences of three kinds of growth factors bFGF, EGF and glial fibrillary acid protein (GDNF) on the proliferation and differentiation of the neural precursors cells in vitro. The research was laying a foundation on the studies for oriented differentiation of neural precursor cells.
PartⅡ:The study on Sertoli cells inducing the differentiation of neural progenitor cells from cerebral cortex of rat
Objective:Once neural precursor cells com from one part transplanted to different part, the result of its differentiation is also different. It is usually similar to the cells of the part accepted transplantation. It hints that the signals outside cells have an important functions in regulating cell differentiation. Moreover, with the influence of different factors, the neural stem cells have potential ability of horizontal differentiation. In this part we want to investigate the effect of Sertoli cell on proliferation and differentiation of cultured neural prigenitor cells(NPCs) from cerebral cortex of the rat.
Methods:Sertoli cells from the testis and the neural precursors cells(NPCs) from the cerebral cortex of Wistar rat were co-cultured. The detections ofβ- III tubulin, GFAP, GalC and tyrosine hydroxylase (TH) were made by immunocytochemistry in vitro.
Results:The numbers of NPs with nestin-positive was decreasing along the time of the co-culture, so were the GFAP and GalC. While theβ- III tubulin-positive cells increased, and TH-positive cells appeared in the co-culture.
Conclusion:if the neural stem cell from forebrain of adulthood or embryonic rat transplanted to the marrow of the same race host its blood system had broken down through second death-dosage of irradiation, we will find there were marrow-like cells, lymphoid cells and earlier periods blood stem cells that derived from the cells of provider source in the host marrow. One experiment testified that if the neural stem cells separated quickly from person's embryo and adulthood mice co-cultured with myoblast cells, as a result, the neural stem cells will differentiated into the skeleton muscle cell. Clarke and his colleague detected that adult stem cell from rat brain can be induced to become all layers of embryonic strata in the chicken embryonic environment. These researches show that the leading signals of the environment have an important effect on the differentiation of neural precursor cells, meanwhile it can confirm that neural stem cells have more potential differentiation. Fanigaki discovered that Notch play an important part in the neural precursor cells differentiating into glial cells. Through up-regulation of the bHLH protein Hes1 and Hes5, the signal of Notch can adjust and control proliferation and differentiation of neural precursor cells. Notch signaling regulates the differentiation of different cells through a kind of by-pass repression. When a group of same cells is stimulated by a certain and special signal at the same time, only a small parts of cells is induced by the signal and appear the differentiation toward particular direction. But the Adjacent cells that express more proteins of Notches can't differentiate toward particular direction, and behave a repression of differentiation. The function of by-pass repression depended on dosages of Notch proteins have already got the confirmation in the Drosophila and mice. Through the direct contact between cells, the expression of different Notch ligands together with the limited activation of proteins of Notch can induce cells differentiating toward dissimilarity directions. According to the research above, this experiment of the part two using the precursors cells from cerebral cortex of rat co-cultured with Sertoli cells, investigate the functions of interactions of two kinds of cells as well, and explore the characteristics of Sertoli cells inducing the neural precursors cells proliferation and differentiation to definite direction. Sertoli cells exert the negative effect on proliferation of the NPCs,but have distinctly the positive effect on the NPCs for its differentiation into neurons in vitro, The neural precursors from the cortex of rat can be directly induced to differentiation into the dopaminergic progenitors by co-cultured with the Sertoli cells.
PartⅢ:Expression of Notch-related Genes in the Differentiation of the neural precursor cells(NPCs) from brain cortex of rats co-cultured with sertoli cells
Objective: If a stable precursor cells pool needs to be maintained then the balance between asymmetrical and symmetrical division must be tightly regulated. Carried on the thorough research for those genes that determine the cell decision and differentiation, it has been discovered that the signal of Notch turns to be one of the important families that has the functions. Notch may be important molecules in specifying cell fate, and play an important role in the growth of much tissues and organs, and regulate the occurrence and morphogenetic processes of the special tissue types. This research of part three applies the fluorescence quantitative real time RT-PCR method examining the expressing variety of the Notch related gene Notch1～4, Delta1, Delta3, Jagged1, Jagged2, Hes1 and Ngn1,and investigates the expression of Notch-related genes in the neural precursor cells(NPCs) from brain cortex of rats in vitro and analyze the possible function of Notch pathway in the differentiation of neural precursor cells co-cultured with sertoli cells.
Methods: NPCs in cortex of twe weeks born rats were isolated and cultured with the NPC medium contained EGF、bFGF and GDNF in vitro. At the same time to isolate its sertoli cells, and co-culture the neural precursor cells with sertoli cells. The expressions of notch-related genes were identified in cells of different groups. Total RNA was isolated at 5 day after confluence of cells. Fluorescent quantitative real-time RT-PCR(FQ-PCR) was used to detect the expression of both Notch(Notch1,Notch2,Notch3 and Notch4) and its ligand (Delta1,Delta3,Jagged1 and Jagged2); expression of ngn1and hes1 genes was also examined at the control ofβ-actin.
Results: Under the NPC medium contained EGF、bFGF and GDNF culture condition, the expression of the four notches in the sertoli cells group were no different, and the level of Jagged2 showed slightly high, but not express Delta1. In the neural precursor cells group the four notch genes especially the Notch1 had high expressions, So Its Jagged1 and Jagged2. Its Hes1 gene was high level, but the expression of its Ngn1 gene was lower respectively. Interestingly, the expression of four notch genes and its four ligands plus Hes1 exhibited down-regulation in the co-cultured cells group than the neural precursor cells group, but its Ngn1 gene showed up-regulation.
Conclusion: The Notch conducts the signals of cell-to-cell interaction between the cells, and precisely regulates the differentiation of each group of cells through adjusting the signals between adjacent cells. The Notch signaling participate in the neural occurrence process in the CNS development, and give the progenitor cell of CNS a choice to continue to propagation or to differentiate into neurons. Generally speaking, the activation of Notch signaling maintain the stable state of neural stem cell, and repress the definite oriented differentiation; But on the other hand, there are evidences prove that activation of the signal of Notch can accelerate and against the ground promote neural precursors cells become glial cells. Some studies manifest that in the process of neural precursors cells development, The Notch on the surface of cell membrane can combine its ligand Delta or Jaggeds and deliver the signals between stem cells and surroundings cells. The cell-to-cell interaction of this kind can adjust and control differentiation of stem cells. For mammals, the signal of Notch activates Hes1/5 homologic to the E(spl) through a CBF1 together, and affect the expression of the pre-neural factor Ngn( Neurogenins), and regulate differentiation of the neural stem cell. The experiment indicate that the specific properties of neural precursors cells have relationship with the varieties of Notch signaling related genes expression under the condition of co-cultured with Sertoli cells.Notch pathway is involved in the differentiation of the neural precursor cells, Sertoli cells can suppress the expression of the Hes1 gene, meanwhile enhance the expression of the Ngn1 gene in the neural precursor cells co-cultured with it. The Sertoli cells may have a positive effect on neural precursor cells differentiation through affecting Notch signaling.
PartⅣ:Construction of eukaryotic expression vector containing rat Hes1 genes and expression in neural precursor cells for the study of its differentiation functions
Objective: Along with the development of Separation, purification and culture for the neural precursor cells in vitro, the study of research for the neural precursor cells controlling its proliferation and differentiation under the influence of cell intrinsic mechanisms or extrinsic signals also obtained great progress. The current studies demonstrate the same mechanism occurred in different tissues which control the key step, and among them involved the most is genes of bHLH families of proteins. The experiment of part four is to clone hes1 gene, and set up eukaryotic expressing victor pCDNA3.1- Hes1, then transfected it to the neural precursors cells from cerebral cortex of rat , we construct its eukaryotic expression vector and obtain positive neural precursor cell clones expressing Hes1 genes stably through transfectation,then to study their effects to the differentiation of neural precursor cells.
Methods: The total RNA was extracted from 14 day Wistar rat brain. The full-length cDNA encoding Hes1 genes were obtained using RT-PCR method and inserted into pGEM T Easy cloning vector. After the sequencing was confirmed, the gene was subcloned to pCDNA3.1 to construct recombinant eukaryotic expression vectors of pCDNA3.1-Hes1. The recombinant plasmid was transfected into neural precursor cells from cerebral cortex of 14 day Wistar rat by lipofectamine method and positive cell clones were screened with G418. The existence of Hes1 genes in the transfected cells genomic DNA ,and the over-expression of their mRNA in the transfected cells were confirmed with Fluorescent quantitative real-time RT-PCR(FQ-PCR).
Results: Enzyme digestion analysis and sequencing showed that the target genes were cloned into recombinant vector. The Existence and over-expression of Hes1 genes in the transfected neural precursor cells was identified with FQ-PCR and it display that Hes1 mRNA had high expressed in the transfected neural precursor cells.
Conclusion: bHLH is the exact subset of transcription factors, containing an alkaline district and a HLH domain. The homologues of many of these transcription factors often act in cascades to control the successive steps in neuronal determination and differentiation as well as neuronal-subtype identity. These transcription factors often construct heterodimers each other, which can band to particular sequences of DNA, and act at particular stage of stem cell development. The downstream gene E(spl)/ Hes of the Notch signaling pathways is one of them, the different factors of bHLH have different function that decide cell fate decision or differentiation. As a related family of proteins, they usually transiently express in a proper time and space order and at the different stage of cell development. The factors that early expressed participated in the cell decision, but the later expressed participated in the cell differentiation eventually, the proteins that expressed in early stages activated the expression of proteins in later stages. Many study manifest that Notch and its downstream target Hes gene have important functions on the potentials maintenance of stem cell in brain. Ngn1/2 and Mash1 are essential for the neural precursor cells differentaition into neurons. Kabos discovered that the repression of Hes1 promote the neural stem cells in central nervous system differentiation intoγ-GABA neurons. Moreover, after the formation of neurons, the activity of the Hes factor often promotes the glial differentiation, the bHLH factors Ngn inhibits a glial fate. The eukaryotic expression plasmid containing Hes1 genes were successfully constructed. The positive neural precursor cell clones expressing Hes1 genes stably were obtained, which may be a promising cell model for studying the biological function of Hes1 genes and the role of Hes1 genes in the differentiation of neural precursor cells. The experiment manifested that the high expression of Hes1 can restrain the expression of Ngn1 gene,and enhance the neural precursor cells differentiation to glial cells. From the research we observed how the high expression of Hes1 gene influence on the differentiation of neural precursors cells.
引文
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