The Study On The Mechanism Of The Related Genes In The Differentiation Of The Neural Precursor Cells(NPCs) Co-cultured With Sertoli Cells

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 ratsObjective: 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 ratObjective: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 cellsObjective: 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 functionsObjective: 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.