CN117244044A

CN117244044A - Application of DEK protein in preparation of drug for activating dormant neural stem cells

Info

Publication number: CN117244044A
Application number: CN202210650914.7A
Authority: CN
Inventors: 杨卫军; 蔡孙立
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-06-09
Filing date: 2022-06-09
Publication date: 2023-12-19

Abstract

The invention discloses an application of DEK protein in preparing a medicament for activating dormant neural stem cells, which utilizes the DEK protein to activate the dormant neural stem cells, promote nerve regeneration and generate new neurons, realize the effect of relieving the neurogenesis activity exhausted in the brain aging process, restart the neurogenesis to be equivalent to the neurogenesis level in the young brain, and in the cerebral apoplexy injury repair, the DEK protein can activate endogenous dormant neural stem cells, generate a large number of offspring neuron mother cells and astrocytes and accelerate the injury repair process. The application of relieving cerebral aging and repairing cerebral apoplexy injury is provided. Overexpression of DEK in adult resting neural stem cells does not disrupt the homeostasis of the neural stem cell pool, and does not trigger depletion of the neural stem cell pool for up to 8 months. The invention has great application value in clinic for treating various human neurodegenerative and injury diseases.

Description

Application of DEK protein in preparation of drug for activating dormant neural stem cells

Field of the art

The invention relates to an application of DEK protein in preparing a medicament for activating dormant neural stem cells, in particular to an application of the DEK protein in promoting neurogenesis to generate new neuron cells in neurodegenerative and injury diseases so as to realize the cure of cerebral aging and cerebral apoplexy and clinically cure various neurodegenerative and injury diseases.

(II) background art

Neurodegenerative (brain aging) and damaging diseases, such as Alzheimer's disease, parkinson's disease, cerebral apoplexy (apoplexy), craniocerebral and spinal cord injury, and the like, die in functional neurons in different regions of the nervous system and cause dysfunctions of movement, language, cognition and the like. After nerve injury lesions, apoptotic nerve cells cannot be supplemented, so that the nervous system diseases become one of the diseases with the highest disability rate. There is currently no effective way to treat the fully dead areas of cells, such as the dark areas of ischemic stroke. Whether or not there can be nerve regeneration by the newly generated neurons in the brain of an adult is controversial. Although neural stem cells exist in the brain of an adult, they are in a dormant state for a long period of time and their biological functions are not yet known. How to promote neurogenesis to generate new neurons, repair and supplement damaged or dead functional neurons by activating adult resting neural stem cells has been a major challenge for effective treatment of neurodegenerative and damaging diseases.

Mammalian neurogenesis originates mainly from the subventricular tubular region of the lateral ventricle of mammals and the neural stem cells underlying the granulosa cells of the dentate gyrus of the hippocampus. They can differentiate into different neuronal subtypes by proliferating and producing transitional-state neural precursor cells and neuroblast cells, migrating into the olfactory bulb by lateral migration flow and radially along the granulosa cell layer. However, adult neural stem cells are essentially in a dormant state, and their biological functions under various neurodegenerative and traumatic disease conditions and their regulatory mechanisms of dormancy and activation have not been clarified. Once activated, dormant neural stem cells can generate activated neural stem cells with proliferation and differentiation capabilities so as to maintain a stem cell bank, and functional neurons and glial cells are generated through cell proliferation, migration and differentiation to complete functional maintenance and reconstruction of a nervous system. Therefore, promotion of nerve regeneration by activating dormant neural stem cells is greatly expected in the treatment of nerve injury diseases. Recent studies have found that various molecules that regulate dormancy of neural stem cells are closely related to Notch signaling pathways. Wherein, the neural stem cells are regulated to enter into a dormant state by inhibiting Ascl1 gene expression and Ascl1 ubiquitination ligase HUWE1 degradation regulated by the Notch pathway effector HES family (abs, j.l., breunig, j.j., eisch, a.j., and Rakic, p. (2011). Not (ch) just development: notch signalling in the adult branch. Nature. Rev. Neurosci.12, 269-283.). However, sustained nerve regeneration cannot be achieved by Notch knockout which activates dormant neural stem cells but quickly leads to depletion of the stem cell bank.

In order to break through the research bottleneck of activation regulation of dormant nerves, we have studied using artemia (artemia) living in mountain salt lakes and capable of producing dormant embryos, and found that the DEK protein can promote the transition of heterochromatin to a euchromatin structure by binding to chromatin, thereby apparently regulating activation of dormant cells, resulting in the artemia breaking embryo dormancy (Wen-Huan Jia, an-Qi Li, jin-Yi Feng, yan-Fu din, sen Ye, jin-sho Yang and Wei-Jun Yang (2019), DEK terminates diapause by activation of quiescent cells in the crustacean artemia bio Journal 476 (12), 1753-1769).

According to the invention, by constructing a mouse research model for activating dormant neural stem cells by means of DEK conditional overexpression, the DEK conditional overexpression is found to activate the dormant neural stem cells and to show extremely obvious and durable nerve regeneration capability, and the nerve regeneration of the mice in the aged mice and cerebral apoplexy is found, so that the effects of relieving the brain aging of the aged mice and obviously damaging and repairing cerebral apoplexy are realized.

(III) summary of the invention

The invention aims to provide an application of DEK protein in preparing a medicament for activating dormant neural stem cells, in particular to an application of endogenous and exogenous DEK protein in activating adult dormant neural stem cells, promoting nerve regeneration of mice brains in adults, old people and cerebral apoplexy, and having remarkable effects in slowing down aging of the old people brains and repairing cerebral apoplexy injuries.

The technical scheme adopted by the invention is as follows:

in a first aspect, the invention provides an application of DEK protein in preparing a medicament for activating dormant neural stem cells.

Further, the DEK protein has a conserved nucleic acid sequence shown in SEQ ID NO. 1.

Further, the DEK protein has more than 95% of similarity with the amino acid sequence shown in SEQ ID NO. 2.

Furthermore, the DEK protein corresponds to SEQ ID NO.1, has a conserved amino acid sequence (380 amino acids) shown in SEQ ID NO.2, and has a coding gene sequence shown in SEQ ID NO.1 (the total length of DNA is 1143 bp).

The application of the DEK protein in preparing the medicine for activating the dormant neural stem cells is that the endogenous DEK protein is overexpressed in the dormant neural stem cells or the exogenous DEK protein is delivered into the dormant neural stem cells, so that the purpose of activating the dormant neural stem cells is achieved.

In a second aspect, the invention provides an application of the DEK protein in preparing a medicament for preventing or treating brain nerve injuries, wherein the brain nerve injuries comprise cerebral apoplexy, alzheimer's disease, parkinsonism and the like. The medicine is prepared by carrying DEK protein by exosomes which are purified by in vitro expression or carrying DEK by genetic engineering virus. The application is to use the exosome carried by the DEK protein purified by in vitro expression as a preparation or use the DEK carried by the genetically engineered virus as a means of gene therapy.

In a third aspect, the invention provides the use of a DEK protein in the preparation of an agent for activating dormant neural stem cells, said agent comprising endogenous and exogenous DEK proteins. The activation is to over-express endogenous DEK protein in the dormant neural stem cells or deliver exogenous DEK protein into the dormant neural stem cells, so as to achieve the purpose of activating the dormant neural stem cells. The reagent can be used for clinical treatment and basic scientific research of cerebral neurodegenerative diseases.

The mechanism of activating dormant neural stem cells by the DEK protein is as follows: overexpression of DEK protein in mouse neural stem cells can activate dormant neural stem cells and promote nerve regeneration, namely DEK protein activates dormant neural stem cells by downregulating Notch, FOXO3, P53 and P21 gene expression and upregulating MYC and Wnt3A gene expression, promotes proliferation of the dormant neural stem cells to generate a large number of neuroblast cells, and migrates to the olfactory bulb and radially along the granulosa cell layer through side migration flow, so that the dormant neural stem cells are differentiated into different neuron subtypes. These new neurons in turn improve aging of the elderly mouse brain and promote repair of stroke brain injury.

Compared with the existing clinical treatment method for cerebral aging and cerebral apoplexy, the invention has the following main beneficial effects: since the neural stem cells existing in the brain of an adult are in a dormant state for a long period of time, they cannot be activated and generate new neuron cells under the aging and injury conditions, which is a main cause of incomplete cure of neurodegenerative and injury diseases. At present, the treatment of neurodegenerative diseases in clinic still stays on the process of slowing down the injury and apoptosis of neuron cells, and the protection and recovery of cell injury by eliminating inflammation, rebuilding blood supply, repairing blood brain barrier, resisting oxidation and the like in nerve injury diseases are not available at present through the generation of a therapeutic effective way of new-born neurons. The invention utilizes DEK protein to activate dormant neural stem cells, promote nerve regeneration and generate new neurons, realize the effect of relieving the dead neurogenesis in the brain aging process, restart neurogenesis to ensure that the neurogenesis is equivalent to the neurogenesis level in young brain, and in the cerebral stroke injury repair, the DEK protein can activate endogenous dormant neural stem cells, generate a large number of offspring neuron mother cells and astrocytes and accelerate the application of the injury repair process. The application of relieving cerebral aging and repairing cerebral apoplexy injury is provided. Overexpression of DEK in adult resting neural stem cells does not disrupt the homeostasis of the neural stem cell pool, and does not trigger depletion of the neural stem cell pool for up to 8 months.

The invention can activate dormant neural stem cells through conditional overexpression of DEK protein in mice, promote nerve regeneration in the brain of aged and cerebral apoplexy mice, thereby realizing the alleviation of aged brain aging and the restoration of cerebral apoplexy nerve injury (figures 1-3). In this experiment, the significant generation of neogenesis neurons in the brain of aged mice and brain stroke mice was observed.

The invention relates to research and report of activating dormant neural stem cells by DEK protein and generating a large number of new neurons, and is also the first treatment of mice brain aging and cerebral apoplexy by using the DEK protein. The invention has great application value in clinic for treating various human neurodegenerative and injury diseases.

(IV) description of the drawings

FIG. 1 is a graph showing the distribution of DEK protein expression in neural stem progenitor cells in the subventricular tubular zone (SVZ zone) of the ventricles of the brain and immunofluorescence staining results. A represents a sagittal plane of the mouse brain and a coronal plane schematic of the SVZ zone; b represents the expression of DEK in proliferating neural stem cells (Sox 2+ Nestin + ki67+) with the right panel being a partially magnified image of the left dashed box; c represents the expression of DEK in transitional amplifying cells (ascl1+), where the right panel is a partially enlarged image of the left dashed box; d represents the expression of DEK in neuronal blast cells (dcx+) with the right panel being a partially enlarged image of the left dashed box; e represents DEK expression in BrdU-doped negative dormant neural stem cells (Sox2+GFAP+BrdU-), wherein the right panel is a partially enlarged image of the left dashed box; f represents the statistical graph of the expression distribution characteristics of DEK protein in the subventricular zone neural stem progenitor cells of the ventricles of the brain side. In the above-described partially enlarged image, the dotted circle indicates the nuclear position of a single cell.

FIG. 2 is a graph showing the distribution of DEK protein expression in neural stem progenitor cells in the granulosa cell lower layer region (SGZ region) of the dentate gyrus of the hippocampus of the brain and immunofluorescence staining results thereof. A represents a sagittal plane of the mouse brain and a coronal plane schematic of the SGZ region; b represents the expression of DEK in proliferating neural stem cells (gfap+nestin+ki67+), wherein the right panel is a partially enlarged image of the left dashed box; c represents the expression of DEK in activated neural stem cells (ascl1+), wherein the right panel is a partially enlarged image of the left dashed box; d represents the expression of DEK in neural progenitor cells (gfap+tbr2+) with the right panel being a partially enlarged image of the left dashed box; e represents DEK expression in neuronal blast cells (DCX+), where the right panel is a partially enlarged image of the left dashed box; f represents DEK expression in BrdU-doped negative dormant neural stem cells (Sox2+GFAP+BrdU-), wherein the right panel is a partially enlarged image of the left dashed box; g represents the statistical graph of the expression distribution of DEK protein in brain hippocampal dentate gyrus stem progenitor cells. In the above-described partially enlarged image, the dotted circle indicates the nuclear position of a single cell.

FIG. 3 is a graph showing the change in DEK protein in neural stem cell regions during brain aging; wherein SVZ represents the subventricular canal zone of the lateral ventricle, DG-SGZ represents the hippocampal dentate gyrus, LV represents the lateral ventricle, A1 represents the characteristic immunofluorescence map of expression of 2 month old mouse SVZ zone DEK in neural stem cells (gfap+sox2+), A2 represents the characteristic immunofluorescence map of expression of 6 month old mouse SVZ zone DEK in neural stem cells (gfap+sox2+), A3 represents the characteristic immunofluorescence map of expression of 12 month old mouse SVZ zone DEK in neural stem cells (gfap+sox2+), A4 represents the statistical map of the number of neural stem cells positive for different age mice SVZ zone DEK, A5 represents the characteristic immunofluorescence map of expression of 2 month old mouse DG-SGZ zone DEK in neural stem cells (gfap+sox2+), A6 month old mouse-SGZ zone DEK in neural stem cells (gfap+sox2+), A7 represents the characteristic immunofluorescence map of expression of 12 month old mouse DG-SGZ zone DEK in neural stem cells (gfap+sox2+), and A4 represents the statistical map of the number of neural stem cells positive for the different age mice DG-SGZ zone DEK; b1 is a schematic diagram of the brain of a mouse, SVZ represents a granulosa cell lower layer region of the dentate gyrus of the Hippocampus of the brain, LV represents a lateral ventricle, DG represents the dentate gyrus, hippocampus represents the Hippocampus, and protein extraction is derived from the SVZ region and the DG region; b2 represents a DEK expression amount Western blot image in total protein extracted from an SVZ region, B3 represents a DEK expression amount Western blot image in total protein extracted from a Hippocampus region, and B4 represents a relative protein expression level statistical image of the DEK protein in the SVZ region and the Hippocampus region.

FIG. 4 is a schematic diagram and verification of the construction principle of a Nestin-driven DEK conditional overexpression mouse; a represents a schematic diagram of the construction principle of a mouse with conditional overexpression of DEK. B represents a gel electrophoresis diagram for identifying the genotype of the double-positive offspring of the transgenic mice; lane 1 represents a DNA Standard molecular weight (100 to 2000 base pairs) marker, lane 2 represents a Luc-EGFP positive genotype, lane 3 represents a Luc-EGFP negative genotype, lane 4 represents a DNA Standard molecular weight (100 to 2000 base pairs) marker, lane 5 represents a DEK-EGFP positive genotype, lane 6 represents a DEK-EGFP negative genotype, lane 7 represents a DNA Standard molecular weight (100 to 2000 base pairs) marker, and Lane 8 represents Creer ^T2 Positive genotype, lane 9 represents Creer ^T2 Negative genotype. C represents the adult neural stem cell over-expression DEK strategy. D represents the expression of DEK protein in fluorescent control mice and conditional overexpressing DEK experimental mice brain SVZ zone neural stem cells (gfp+), panels are partial magnified images of the dashed boxes. E represents Western blot of DEK protein expression in total protein of brain SVZ region of fluorescent control mice and conditional over-expression DEK mice.

FIG. 5 shows DEK overexpression activates the hypothalamic area dormant neural stem cells of the ventricles of the brain side. A1 represents a characteristic immunofluorescence map of the expression of a cell proliferation marker Ki67 in the brain SVZ region of mice in a control group and a conditional overexpression DEK group after tamoxifen induction, and yellow arrow indicates Ki67 positive neural stem cells; a2 represents a statistical plot of the number of control and DEK overexpressing mouse SVZ region proliferating neural stem cells (gfp+ki67+); b1 represents a schematic diagram of a BrdU incorporation retention assay; b2 represents the characteristic immunofluorescence of BrdU still remained and marked after dilution in the brain SVZ region of the mice in the control group and the conditional overexpression DEK group, and the SVZ region is marked by a dotted line frame; b3 represents a statistical plot of the number of dormant neural stem cells (gfp+sox 2+brdu+) in the control group and the DEK overexpressing mouse SVZ region.

FIG. 6 shows the expression of DEK over-expressed and activated granular cell lower layer region dormant neural stem cells of the dentate gyrus of the brain hippocampus, A1 represents the characteristic immunofluorescence map of the cell proliferation marker Ki67 expressed in the SGZ region of the brain of the mice in the control group and the conditional over-expressed DEK group after the induction of tamoxifen, the yellow arrow indicates the Ki67 positive neural stem cells, and A2 represents the statistical map of the proliferation state neural stem cells (GFP+Ki67+) in the SGZ region of the mice in the control group and the DEK over-expressed group. B1 represents the characteristic immunofluorescence of BrdU in the brain SGZ region of the mice in the control group and the conditional overexpression DEK group, wherein the BrdU still stays in the mark after dilution in the retention experiment, and the SVZ region is marked by a dotted line frame; b2 represents a statistical plot of the number of dormant neural stem cells (gfp+sox 2+brdu+) in the SGZ zone of control and DEK overexpressing mice.

FIG. 7 shows AAV9-GFAP-DEK-GFP activated neural stem cells in the lower region of granulosa cells of the hippocampal dentate gyrus of aged mice, A1 represents a virus injection strategy, A2 represents the needle insertion position indicated by green arrows on the surface of the skull of the mice, A3 is a schematic view of coronal virus injection, A4 is an immunofluorescence image of virus injection on a coronal section of the brain, A5 represents an immunofluorescence result image of DEK protein expression in the SGZ neural stem cells (GFP+GFAP+) of the brain of a control virus injection group and an over-expressed DEK virus injection group after virus infection, and yellow arrows indicate single cells. B1 represents the characteristic immunofluorescence map expressed in the SGZ region of the brain of mice in the control virus-injected group and the over-expressed DEK virus-injected group after virus infection, and B2 represents the statistical map of the number of proliferating neural stem cells (gfp+ki67+) in the SGZ region of the mice in the control virus-injected group and the over-expressed DEK virus-injected group.

FIG. 8 is a graph showing that DEK overexpression promotes sustained neurogenesis in the subventricular tubular region of the ventricles of the brain and produces large numbers of neuroblast and neogenesis neurons. A represents an experimental strategy diagram. B1 represents the SVZ region expression profile immunofluorescence of DCX positive neuronal parent cells in brains of control and conditional over-expression DEK mice after 1 month induction with tamoxifen, B2 represents the SVZ region expression profile immunofluorescence of DCX positive neuronal parent cells in brains of control and conditional over-expression DEK mice after 4 months induction with tamoxifen, and B3 represents the SVZ region expression profile immunofluorescence of DCX positive neuronal parent cells in brains of control and conditional over-expression DEK mice after 8 months induction with tamoxifen; c1 represents the expression profile immunofluorescence of the mature neurons newly generated by gfp+neun+ in the olfactory bulb region in the brains of control and conditional overexpressed DEK mice after 1 month of tamoxifen induction, C2 represents the expression profile immunofluorescence of the mature neurons newly generated by gfp+neun+ in the SVZ region in the brains of control and conditional overexpressed DEK mice after 4 months of tamoxifen induction, C3 represents the expression profile immunofluorescence of the mature neurons newly generated by gfp+neun+ in the SVZ region in the brains of control and conditional overexpressed DEK mice after 8 months of tamoxifen induction; d1 represents a statistical plot of the number of SVZ regions in brains of control and conditional over-expression DEK mice following induction with tamoxifen for 1 month, 4 months, 8 months, and D2 represents a statistical plot of the number of olfactory bulb regions in brains of control and conditional over-expression DEK mice following induction with tamoxifen for 1 month, 4 months, 8 months, for gfp+neun+ newly produced mature neurons. D3 represents a statistical plot of the number of SVZ regions in brains of control and conditional over-expression DEK mice induced by tamoxifen for 1 month, 4 months, and 8 months after gfap+sox2+ total neural stem cells.

FIG. 9 is a graph showing that DEK overexpression promotes sustained neurogenesis in the granulosa cell lower layer region of the brain hippocampus dentate gyrus and produces a large number of neuroblast and neogenesis neurons. A1 represents an SGZ region expression distribution immunofluorescence map of DCX positive neuronal parent cells in brains of a control group and a conditional over-expression DEK mouse after being induced by tamoxifen for 1 month, A2 represents an SGZ region expression distribution immunofluorescence map of DCX positive neuronal parent cells in brains of the control group and the conditional over-expression DEK mouse after being induced by tamoxifen for 4 months, and A3 represents an SGZ region expression distribution immunofluorescence map of DCX positive neuronal parent cells in brains of the control group and the conditional over-expression DEK mouse after being induced by tamoxifen for 8 months; b1 represents the dentate gyrus region expression distribution immunofluorescence map of the mature neurons newly generated by BrdU+NeuN+ in the brain of a control group and a conditional over-expression DEK mouse after being induced by tamoxifen for 1 month, B2 represents the dentate gyrus region expression distribution immunofluorescence map of the mature neurons newly generated by BrdU+NeuN+ in the brain of the control group and the conditional over-expression DEK mouse after being induced by tamoxifen for 4 months, and B3 represents the dentate gyrus region expression distribution immunofluorescence map of the mature neurons newly generated by BrdU+NeuN+ in the brain of the control group and the conditional over-expression DEK mouse after being induced by tamoxifen for 8 months; c1 represents a statistical plot of the number of SGZ regions in brains of control and conditional overexpressed DEK mice after 1 month, 4 months, 8 months of tamoxifen induction, C2 represents a statistical plot of the number of dentate gyrus regions in brains of control and conditional overexpressed DEK mice after 1 month, 4 months, 8 months of tamoxifen induction, and C3 represents a statistical plot of the number of SGZ regions in brains of GFAP+Sox2+ total neural stem cells in control and conditional overexpressed DEK mice after 1 month, 4 months, 8 months of tamoxifen induction.

FIG. 10 shows the overexpression of DEK activating genes involved in the up-regulation and down-regulation of dormant neural stem cells. A represents a differential gene heat map of a neural stem cell transcriptome sequencing result analysis obtained by extracting a mouse stem cell region of a control group and a conditional over-expression DEK group, B represents a differential gene volcanic map of a neural stem cell transcriptome sequencing result analysis obtained by extracting a control group and a conditional over-expression DEK group, C represents an expression profile heat map of a gene related to cell dormancy activation regulation in the top 30 differential genes of a neural stem cell transcriptome sequencing result analysis obtained by extracting a control group and a conditional over-expression DEK group, D represents a signal path statistical map of up-regulation expression in an over-expression DEK group compared with the control group in the neural stem cell transcriptome sequencing result analysis obtained by extracting a control group and a conditional over-expression DEK group, and E represents an expression profile heat map of a near-upstream regulatory gene directly related to cell dormancy activation regulation in the differential genes of the neural stem cell transcriptome sequencing result analysis obtained by extracting a control group and a conditional over-expression DEK group.

FIG. 11 shows the activation of the signaling pathway such as Notch inhibition by dormant neural stem cells by overexpression of DEK. A1 represents a protein immunoblotting diagram representing the expression amount change of DEK protein, internal reference protein H3, beta-actin, signal pathway proteins Notch1, notch2, NICD, hes1, hes5, CSL, ASCL1, foxO3a, P-FoxO3a, P53, P21, MYC, wnt3a and the like in total protein of brain SVZ region of mice in a control group and a conditional over-expression DEK group after tamoxifen induction, A2 represents a protein immunoblotting result quantitative analysis statistical diagram; b1 Representing Notch1 expression of characteristic immunofluorescence patterns in the brain SVZ region of mice in the control group and the conditional over-expression DEK group after induction by tamoxifen, B2 representing expression of characteristic immunofluorescence patterns in the brain SVZ region of mice in the control group and the conditional over-expression DEK group after induction by tamoxifen, B3 representing expression of characteristic immunofluorescence patterns in the brain SGZ region of mice in the control group and the conditional over-expression DEK group after induction by tamoxifen, and B4 representing expression of characteristic immunofluorescence patterns in the brain SGZ region of mice in the control group and the conditional over-expression DEK group after induction by tamoxifen; c1 represents the characteristic immunofluorescence image expressed by ASCL1 in the brain SVZ region of mice in the control group and the conditional overexpressed DEK group after induction by tamoxifen, yellow arrow indicates ASCL1 positive cells, small image is a partially enlarged image of the dashed box in the large image, C2 represents the characteristic immunofluorescence image expressed by ASCL1 in the brain SGZ region of mice in the control group and the conditional overexpressed DEK group after induction by tamoxifen, yellow arrow indicates ASCL1 positive cells, small image is a partially enlarged image of the dashed box in the large image.

FIG. 12 is a schematic diagram of DEK overexpression activating the hypothalamic area dormant neural stem cells of the ventricles of the brain side of an aged mouse. A represents experimental strategy: aged 12 months old control and conditional over-expression DEK mice are induced by tamoxifen, brdU is doped with 7-day marks, and sampling analysis is carried out after continuous feeding for 1 month; b1 represents immunofluorescence of gfp+sox2+ki67+ proliferating active neural stem cells in the control group and the brain SVZ region of the conditional overexpressed DEK mice after 1 month of tamoxifen induction, and small image is a partially enlarged photograph of a dashed box in the large image, and B2 represents statistical image of the number of gfp+sox2+ki67+ proliferating active neural stem cells in the control group and the brain SVZ region of the conditional overexpressed DEK mice after 1 month of tamoxifen induction.

FIG. 13 shows DEK overexpression activation of resting neural stem cells in the granulosa cell lower layer region of the hippocampal dentate gyrus in aged mice. A represents immunofluorescence of gfp+sox2+ki67+ proliferating active neural stem cells in the control group and the brain SGZ region of the conditional over-expressed DEK mice after 1 month of tamoxifen induction, and B represents statistical figures of gfp+sox2+ki67+ proliferating active neural stem cells in the control group and the brain SGZ region of the conditional over-expressed DEK mice after 1 month of tamoxifen induction.

Fig. 14 is a graph showing that DEK overexpression promotes sustained neurogenesis in the subventricular tubular region of the ventricles of the brain side of aged mice and produces a large number of neuroblast and neonatal neurons. A1 represents immunofluorescence plots of gfp+dcx+ neuronal master cells in control and conditionally over-expressed DEK mice brain SVZ and kiss-side migration flow after 1 month induction with tamoxifen, RMS represents kiss-side migration flow, the right panel is a partially enlarged photograph of the dashed box in the large panel, A2 represents a statistical plot of the number of gfp+dcx+ neuronal master cells in control and conditionally over-expressed DEK mice brain SVZ and RMS after 1 month induction with tamoxifen. B1 represents immunofluorescence of gfp+neun+ newly generated mature neurons in the control group and the brain olfactory bulb region of the conditional over-expression DEK mice after 1 month of tamoxifen induction, and B2 represents statistical plot of gfp+neun+ newly generated mature neurons in the control group and the brain olfactory bulb region of the conditional over-expression DEK mice after 1 month of tamoxifen induction. C1 represents immunofluorescence of mature neurons newly generated in the brain olfactory bulb area BrdU+NeuN+ of a2 month old, 5 month old, 9 month old, 12 month old control group and 12 month old conditional overexpression DEK mouse after 1 month of tamoxifen induction, and C2 represents statistical figures of the number of mature neurons newly generated in the brain olfactory bulb area BrdU+NeuN+ of a2 month old, 5 month old, 9 month old, 12 month old control group and 12 month old conditional overexpression DEK mouse after 1 month of tamoxifen induction.

FIG. 15 is a graph showing that DEK overexpression promotes sustained neurogenesis in the granulosa cell lower layer region of the hippocampal dentate gyrus in aged mice and produces a large number of neuroblasts and neoneurons. A1 represents immunofluorescence of gfp+dcx+ neuronal blast cells in the control group and conditional overexpression DEK mice brain SGZ region after 1 month of tamoxifen induction, and A2 represents statistical figure of gfp+dcx+ neuronal blast cells in the control group and conditional overexpression DEK mice brain SGZ region after 1 month of tamoxifen induction; b1 Immunofluorescence of GFP+ novacells in the control and conditional overexpression of DEK mice brain hippocampal region after 1 month of tamoxifen induction, and B2 represents statistical plot of GFP+ novacells in the control and conditional overexpression of DEK mice brain hippocampal region after 1 month of tamoxifen induction; c1 represents immunofluorescence of gfp+neun+ newly generated mature neurons in control and conditional over-expressed DEK mice brain dentate gyrus region after 1 month of tamoxifen induction, and C2 represents statistics of gfp+neun+ newly generated mature neurons in control and conditional over-expressed DEK mice brain dentate gyrus region after 1 month of tamoxifen induction; d1 represents immunofluorescence of mature neurons newly generated in the dentate gyrus region brdu+neun+ of the brain of a 12 month old control group and a 12 month old conditional overexpression DEK mouse after 1 month induction by tamoxifen, and D2 represents statistical plot of the number of mature neurons newly generated in the dentate gyrus region brdu+neun+ of the brain of a 12 month old control group and a 12 month old conditional overexpression DEK mouse after 1 month induction by tamoxifen.

FIG. 16 is a diagram showing the construction of a mouse striatal hemorrhagic stroke disease model. A1 represents an experimental strategy, adult wild type C57BL/6 mice are injected with type IV collagenase through brain stereotactic, sampling analysis is carried out after 24 hours, and A2 represents a focus entity photograph of induction of intracranial hemorrhage by collagenase IV; b represents a Casp3 immunofluorescence staining chart of apoptosis markers of focal areas and undamaged areas; c represents the immunofluorescence staining pattern of mature neurons NeuN in the focal zone and the intact zone.

Fig. 17 is a graph showing that DEK overexpression promotes persistent neurogenesis in the subventricular tubular region of the brain side of stroke mice. A represents experimental strategy: adult control mice and conditional over-expressed DEK mice were subjected to brain stereotactic injection of collagenase type IV to induce hemorrhagic stroke, and after 7 days of continuous EdU drinking water and a total 30 days of injury repair period, sampled and analyzed; b represents immunofluorescence staining patterns of a control group and a conditional over-expression DEK mouse brain lesion area after the induction of tamoxifen and the occurrence of hemorrhagic cerebral apoplexy for 1 month, and a green dotted line frame marks the periphery of the lesion; c1 represents immunofluorescence staining patterns of gfp+dcx+neuronal parent cells in brain lesion areas of control and conditional overexpressed DEK mice after 1 month of hemorrhagic stroke induced by tamoxifen, and white dotted lines identify new-born neuronal parent cells, C2 represents statistical patterns of numbers of gfp+dcx+neuronal parent cells in brain lesion areas of control and conditional overexpressed DEK mice after 1 month of hemorrhagic stroke induced by tamoxifen; d1 represents immunofluorescent staining patterns of gfp+gfap+astrocytes from a control group and a conditional overexpressed DEK mouse brain focus area after 1 month of hemorrhagic stroke induced by tamoxifen, and right is a partially enlarged image of a dashed box area, D2 represents statistical patterns of numbers of gfp+gfap+astrocytes from a control group and a conditional overexpressed DEK mouse brain focus area after 1 month of hemorrhagic stroke induced by tamoxifen.

Fig. 18 is a graph showing that DEK overexpression promotes the production of large numbers of mature neurons in the subventricular tubular region and striatum of the brain side of stroke mice. A1 represents immunofluorescence staining patterns of GFP+EdU+neonatal cells in brain lesion areas of a control group and a conditional overexpressed DEK mouse after 1 month of hemorrhagic stroke induced by tamoxifen, and white dotted lines identify neonatal neuronal parent cells, and A2 represents statistical patterns of numbers of GFP+EdU+neonatal cells in brain lesion areas of a control group and a conditional overexpressed DEK mouse after 1 month of hemorrhagic stroke induced by tamoxifen; b1 represents immunofluorescence staining patterns of GFP+NeuN+ neonatal mature neurons in brain disease areas of a control group and a conditional overexpression DEK mouse after the induction of tamoxifen for 1 month after the occurrence of hemorrhagic cerebral apoplexy, and white dotted lines identify neonatal neuronal parent cells, and B2 represents statistical patterns of the GFP+NeuN+ neonatal mature neurons in brain disease areas of a control group and a conditional overexpression DEK mouse after the induction of tamoxifen for 1 month after the occurrence of hemorrhagic cerebral apoplexy; c1 represents immunofluorescence staining patterns of GFP+NeuN+ neonatal mature neurons in brain lesion areas of control and conditional overexpressed DEK mice after 1 month of hemorrhagic stroke induced by tamoxifen, and C2 represents statistics of numbers of GFP+NeuN+ neonatal mature neurons in brain lesion areas of control and conditional overexpressed DEK mice after 1 month of hemorrhagic stroke induced by tamoxifen.

FIG. 19 is a schematic diagram showing the progression, proliferation and differentiation of DEK-activated mouse dormant neural stem cells, contributing to neurogenesis, and involved in brain function maintenance and nerve injury repair.

(fifth) detailed description of the invention

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

the percentage concentrations in the examples of the present invention are all volume concentrations unless specifically indicated.

Phosphate Buffer (PBS) final concentration composition used in the present invention: 137mM sodium chloride, 2.7mM potassium chloride, 10mM disodium hydrogen phosphate dodecahydrate and 1.76mM potassium dihydrogen phosphate, the solvent was water, and the pH was adjusted to 7.4.

All mice of the invention are fed in a sterile environment, water and mouse grains are continuously supplied, and padding is replaced in time. All the experiments of mice are through ethical examination of animal experiments, and experimenters observe the welfare ethics principle of experimental animals when carrying out experiments.

Table 1, reagents and antibodies used in the examples of the invention:

example 1 expression profiling of DEK in adult neural Stem cell lineages

1. Mouse strain

Adult 8-week-old wild-type C57BL/6 mice (purchased from Shanghai Laike laboratory animal Co., ltd.) were fed with BrdU drinking water (1 mg/mL) for 14 consecutive days, and brain tissue samples were directly obtained, and the expression profile of DEK in the in vivo neural stem cell region was studied by detecting a series of neural stem/progenitor cell markers co-localization with DEK using immunofluorescence.

2. Immunofluorescence detection of DEK, sox2, nestin, GFAP, ki67, brdU, ASCL1, DCX, TBR2

a. Mouse heart perfusion and brain tissue fixation

(1) 4% paraformaldehyde fixing solution preparation: weighing a certain amount of paraformaldehyde, dissolving in PBS (phosphate buffer solution) in water bath at 60 ℃, occasionally mixing uniformly during the dissolving, preparing 4% paraformaldehyde solution, cooling to room temperature, standing at 4 ℃ for later use, and pre-cooling physiological saline.

(2) Isoflurane anesthesia was performed using the small animal anesthesia system until the experimental mice were touched with limbs without nerve reflex.

(3) After the mice were fully anesthetized, the mice were supinated, fixed with stainless steel syringe needles on foam plates with absorbent pads, the breasts were sterilized with 70% ethanol and the hair was wetted. The chest skin is gently clamped by using tissue forceps, the chest skin is gently lifted upwards by about 0.5cm, the chest skin and the left and right ribs are cut left and right by using anatomic scissors, and the heart and the liver are fully exposed.

(4) Using an injector with pre-cooled 0.9% normal saline, installing a venous indwelling needle, penetrating the needle tip into the left ventricle of the heart of the mouse, cutting a small opening by dissecting a right atrium valve, slowly pushing the injector after blood is found to flow out, and pouring the normal saline with a pouring amount of about 20mL, so that the liver is seen to be whitish and bloodless.

(5) After normal saline is filled, the injector filled with 4% paraformaldehyde solution is replaced for continuous filling, the filling amount is about 40mL, and after the mice are fixed by paraformaldehyde, the body is stiff, and the tail is stiff.

(6) The head skin is cut off by shearing from the shoulder and neck of the mouse by using a tissue scissors, the skull is peeled off by using a tissue forceps after repeatedly and lightly scratching the skull by using a blade, the brain is taken out, a brain tissue block with the thickness of 2-5 mm on the coronal plane or the sagittal plane is cut, and the brain tissue block is immersed into 4% paraformaldehyde solution and fixed in a refrigerator at the temperature of 4 ℃ for overnight.

b. Frozen section of brain tissue

(1) The fixed brain tissue was transferred to a PBS solution containing 30% sucrose for dehydration at 4 ℃. Brain tissue is dehydrated from floating in sucrose solution until it settles to the bottom. And (3) completely wrapping the treated brain tissue with an O.C.T. embedding agent in a mould box with proper size, balancing for 15min, transferring to dry ice to complete freezing embedding, and storing in an ultralow temperature refrigerator at-80 ℃.

(2) Pre-cooling the frozen microtome to-20 ℃ in advance, and placing the sample in the microtome to balance the temperature.

(3) Smearing O.C.T on a base matched with a microtome, adhering the embedded brain tissue sample on the base, placing the brain tissue sample into a quick freezing table of the frozen microtome to freeze the newly smeared O.C.T, and loading the sample on the base.

(4) The machine parameters were adjusted to 25 μm thickness patches and the slices of non-target areas were discarded until the target areas were found. Readjusting machine parameters to slice at a thickness of 10 μm, preparing slides of brain slices of subventricular zone (SVZ zone) of ventriculus of brain side ventricle and subgranulosa zone (SGZ zone) of dentate gyrus of hippocampus of brain respectively, slicing tissue of SVZ zone and SGZ zone with a thickness of 10 μm, adhering the obtained brain frozen slices on the slides, and storing the brain frozen slices in an ultralow temperature refrigerator for long term for later use.

c. Fluorescent staining of immune tissues

(1) Balance: and c, respectively airing the glass slides of the brain sections of the SVZ zone and the SGZ zone prepared in the step b for 30min at room temperature, and rinsing the glass slides with PBS for 5min to remove residual O.C.T.

(2) Secondary fixation: drawing a hydrophobic ring near the tissue by an immunohistochemical pen, adding a proper amount of 2% paraformaldehyde into the hydrophobic ring, treating for 5 minutes at room temperature, rinsing with PBS for 3 times, each time for 5 minutes, and preheating a water bath to 68 ℃.

(3) Penetration: a suitable amount of PBS containing 0.25% Triton X-100 was added to the hydrophobic ring using a pipette and the mixture was subjected to a permeation treatment, 15min incubation at room temperature, and PBS rinsing 3 times for 5min each.

(4) Antigen retrieval: the sections were immersed in a staining box containing a pH 9 antigen retrieval solution, incubated at 68 ℃ for 20min, transferred to cold water, cooled for 10min, and rinsed 3 times with PBS for 5min each.

(5) Closing: an appropriate amount of blocking solution (i.e., PBS containing 10% blocking donkey serum, 0.1% Triton X-100) was added to the hydrophobic ring using a pipette and incubated for 1h at room temperature.

(6) Incubation resistance: preparing an anti-dilution liquid (namely diluting an antibody stock solution into a sealing liquid according to a certain proportion), sucking and discarding the sealing liquid in the hydrophobic ring by using a liquid dispenser, adding the prepared anti-dilution liquid into the sealing liquid by using the liquid dispenser, and incubating overnight at 4 ℃. The volume ratio of antibody dilution is as follows: DEK antibody, 1:200; GFAP antibody, 1:1000; sox2 antibody, 1:500; nestin antibody, 1:500; ki67 antibody, 1:200; TBR2 antibody, 1:100; DCX antibody, 1:200; brdU antibody, 1:100; ASCL1 antibody, 1:100. After the primary antibody incubation was completed, PBS was rinsed 3 times for 5min each.

(7) Secondary antibody incubation: preparing a fluorescent secondary antibody diluent (namely diluting a fluorescein-coupled IgG/Y stock solution (Jackson, U.S.) in a sealing liquid according to the volume ratio of 1:200), and then adding the prepared secondary antibody diluent into the hydrophobic ring in the step (6) by using a pipette, and incubating for 2 hours at room temperature. After the secondary antibody incubation was completed, PBS was rinsed 3 times for 5min each.

(8) EdU staining (optional): the signal of the EdU marked tissue sample can be detected only by EdU staining treatment, and the specific method comprises the steps of preparing an EdU staining working solution according to the formula of an EdU Click kit, adding a hydrophobic ring after secondary incubation in the step (7) by a pipettor, fully covering the tissue section, and incubating for 45min at room temperature. The PBS was rinsed 3 times for 5min each.

(9) Nuclear dyeing: and (3) adding a proper amount of DAPI staining solution into the hydrophobic ring stained by the EdU in the step (7) or the step (8) by using a pipette, and incubating for 15min at room temperature to stain the cell nucleus. The PBS was rinsed 3 times for 5min each.

(10) Sealing piece: and adding a proper amount of anti-fluorescence quenching sealing liquid into the drain ring by using a liquid transfer device, and using tweezers to assist in clamping the glass slide, and slowly covering the glass cover along the edge of the liquid drop to avoid air bubble generation. And (5) preserving the sealed sample slice in a dark place for standby, and detecting fluorescent signals by a subsequent laser confocal scanning microscope.

We used adult wild-type mice and performed BrdU serial incorporation experiments to immunofluorescence detect a series of neural stem/progenitor markers co-localized with DEK. The result shows that: in the SVZ region (FIG. 1), DEK protein is abundantly expressed in proliferating neural stem cells (Sox2+nestin+Ki67+), transitional amplifying cells (ASCL1+) and neuronal blast cells (DCX+), but hardly expressed in dormant neural stem cells (Sox2+GFAP+BrdU-).

In the SGZ region (FIG. 2), DEK protein is also abundantly expressed in proliferating neural stem cells (GFAP+nestin+Ki67+), activated neural stem cells (ASCL1+), neural progenitor cells (GFAP+TBR2+), and neuronal blast cells (DCX+), while DEK is expressed in low amounts of 50% of cells in dormant neural stem cells (Sox2+GFAP+BrdU-).

Conclusion: these results indicate that the DEK protein is hardly expressed or low-expressed in dormant neural stem cells, but is abundantly expressed in proliferating neural stem cells, transitional amplifying cells and neuronal parent cells, and may be factors necessary for the activation of the neural stem cells, involved in the regulation of the dormancy and activation of the neural stem cells.

EXAMPLE 2 DEK Change in brain aging

1. Mouse strain

Adult 2 month old, 6 month old, 12 month old wild type C57BL/6 mice (purchased from Shanghai Laike laboratory animal Co., ltd.) were used to study the change in the expression profile of DEK during brain aging.

2. Immunofluorescence detection of DEK expression in neural stem cells of mice of different ages

DEK immunofluorescence assays (GFAP, DEK) were performed on the mice of different ages (step 2, example 1) and the results are shown in FIGS. 3, A1 to A8, in which the number of DEK-positive neural stem cells in the SVZ zone and the SGZ zone was continuously reduced by about 75% during aging from 2 months after birth to 12 months after birth.

3. Immunoblotting experiment to detect DEK protein level change

SVZ and hippocampal stem cell zone (hippocampal) tissue microdissection and protein extraction

The experimental mice were sacrificed by dislocation, fresh brain tissue was removed with dissecting scissors and forceps, vertically cut in PBS along the midbrain of the ventricles on the forebrain side with a scalpel, the section exposed the ventriculus bilateral cavity, and the tissues of the ventriculus lateral wall SVZ and hippocampus were cut using sharp-bent dissecting forceps tips (B1 in fig. 3). The dissected tissue is quickly filled into a centrifuge tube after absorbing excessive water, and is frozen in liquid nitrogen for temporary storage. The experimental study adopts TRIzol reagent extraction to obtain tissue total protein, and the specific method is as follows:

(1) Every 50-100mg of tissue is fully ground by a hand-held refiner with 1mL TRIzol reagent, and after being evenly mixed, 12000g is centrifugated for 10min at 4 ℃, the supernatant is transferred to a new tube, and the tube is placed for 5min at room temperature.

(2) 0.2mL of chloroform was added, shaken well for 15s, left at room temperature for 5min, and centrifuged at 12000g for 15min at 4℃with the upper clear aqueous phase available for total RNA extraction and the lower organic phase for the protein extraction step described below.

(3) Adding 0.3mL of absolute ethyl alcohol, uniformly mixing up and down until the solution is clear, standing at room temperature for 5min, centrifuging at 4 ℃ for 10min at 2000g, and discarding the supernatant.

(4) 1.5mL of isopropanol was added, and after 10min at room temperature, 12000g was centrifuged at 4℃for 10min, and the precipitate was collected.

(5) 2mL of 0.3M guanidine hydrochloride (dissolved in absolute ethanol) was added, the precipitate was triturated with a pipette nozzle, and after 20 min of shaking bed rinsing, 7500g was centrifuged at 4℃for 5min, and the precipitate was collected, and this step was repeated twice.

(6) The pellet was resuspended in 1mL absolute ethanol, allowed to stand at room temperature for 10min, centrifuged at 7500g for 5min at 4℃and the absolute ethanol removed, the pellet collected and the centrifuge tube lid opened until the pellet was completely dry.

(7) The dried precipitate is incubated with a proper amount (about 50-100 mu L) of 2% SDS aqueous solution in a water bath kettle at 55 ℃ for overnight according to the amount of the precipitate, 5X loading buffer (DTT) is added until the protein is completely dissolved, the mixture is fully mixed, the mixture is incubated in a boiling water bath for 10min to denature the protein, 12000g is centrifuged for 10min at room temperature, the supernatant is collected to obtain a protein extract, and the protein extract is preserved at minus 30 ℃ for standby after the protein concentration is measured by a Bradford method.

b. Detection of DEK by Western immunoblotting

(1) And (3) glue preparation: SDS-polyacrylamide gel is prepared according to the molecular weight of the target protein. The gel concentration used for detecting the target proteins with different molecular weights in the experiment is as follows: the molecular weight of the protein is less than or equal to 70kDa and is 12.5 percent; protein molecular weight less than or equal to 70 and less than or equal to 100kDa, 10%; the molecular weight of the protein is more than or equal to 100kDa and 8 percent.

(2) Electrophoresis: and (3) loading the prepared gel into an ATTO electrophoresis tank by using a 1X SDS electrophoresis buffer solution, lightly blowing the buffer solution in a gel hole by using a pipettor, ensuring that the gel hole is clean, adding a protein extract and a standard molecular weight protein sample into the gel hole, and carrying out electrophoresis separation in a mode of 15mA constant current per gel.

(3) Transferring: after electrophoresis, the upper concentrated gel is removed by using a gel plate, and the lower concentrated gel is temporarily stored in a prepared transfer buffer solution (0.01M glycine, 0.01M tris (hydroxymethyl) aminomethane, 10% methanol, and water as solvent). The membrane transfer filter paper and the PVDF membrane after methanol activation are soaked by using a membrane transfer buffer. Using a semi-dry film transfer instrument, placing the filter paper, PVDF film, gel and filter paper into film transfer 'sandwiches', wetting each component by a small amount of film transfer buffer solution, and using a push rod to enable the 'sandwiches' to be flat and bubble-free, wherein each film transfer 'sandwiches' is subjected to 7V constant pressure film transfer for 1h.

(4) Antibody incubation: rinsing PVDF membrane for 30s by using 1 XTBS after membrane transfer, and incubating for 1h at room temperature by using 1% blocking solution; incubating the PVDF membrane for 24 hours at the temperature of 4 ℃ by using the prepared primary anti-dilution solution after the sealing is finished; rinsing PVDF membrane 3 times with TBS containing 0.5% Tween 20 after the primary antibody incubation is finished, and incubating for 20min at room temperature with 0.5% blocking solution for 5min each time; after the sealing is finished, the prepared secondary antibody diluent corresponding to the primary antibody source is used for incubation for 40min at room temperature; after the secondary antibody incubation is finished, the PVDF membrane is rinsed 3 times for 5min each time by using TBS containing 0.5% Tween 20 for standby. Thanks to the characteristic of high analysis sensitivity of Western blotting, the dilution ratio of the primary antibody used in the experimental process is set to be 1:1000-1:5000 by referring to the antibody specification, and the dilution ratio of the secondary antibody coupled with HRP is 1:2500.

(5) Developing: the detection of the experimental result is based on horseradish peroxidase coupled to the secondary antibody molecule. The reaction solution was prepared using a chemiluminescent color development kit (ECL), and after the reaction solution was applied to a PVDF film, the reaction solution was placed in a chemiluminescent imager, and the experimental results were recorded, and as shown in fig. 3, B2 to B4, the DEK protein level was also decreased continuously with increasing age, and was decreased to about 40% in old mice (12 months) as young mice (2 months).

Conclusion: immunofluorescent staining and western blotting experiments revealed that DEK protein levels may be associated with brain aging, particularly neural stem cell aging.

EXAMPLE 3 DEK overexpression activates SVZ and SGZ regions dormant neural stem cells

1. Double positive offspring acquisition of transgenic mice

a. Construction of transgenic mice

R26-DEK-EGFP mice are entrusted with Hainan model organism limited company for construction, and the construction principle is that an ES cell (mouse embryonic stem cell) targeting method is adopted, and a CAG master-loxp-Neo-loxp-Dek-IRES-EGFP-polyA expression frame is inserted at a fixed point at a Rosa26 gene locus. The brief procedure is as follows: ES cell targeting vectors were constructed by seamless cloning, and contained a 1.1kb 5 'homology arm, CAG promoter, loxp-PGK-Neo-polyA-loxp, dek-IRES-EGFP coding region, a 4.3kb 3' homology arm, and MC1-DTA-polyA negative selection marker. After linearization of the vector, ES cells were transfected electrically. After screening with neomycin (G418) and propoxyguanosine (Ganc) drugs, 144 resistant ES cell clones were obtained in total; a total of 15 positive ES cell clones were obtained for the correct homologous recombination by long fragment PCR. Positive ES cell clones were amplified and injected into blastula of C57BL/6J mice to obtain chimeric mice. 8 positive F1 generation Neo-containing mice are obtained by mating the high-proportion chimeric mice with C57BL/6J mice, and the R26-DEK-EGFP mice are obtained. The development and reproduction of the R26-DEK-EGFP heterozygote mice are not obviously abnormal.

b. Double positive offspring of transgenic mice

Referring to FIG. 4, a schematic diagram of the construction principle of a conditional overexpressing DEK mouse, wherein the DEK-iresegFP sequence is inserted by transgenic technology into the rear of the Rosa26 promoter and is in an inactive state under the influence of the preceding termination sequence. In the double-positive offspring obtained by hybridizing the mice with a neural stem cell specific tool mouse (Nestin-Creet 2), after tamoxifen induction, the termination sequence in the genome of the neural stem cells is removed, and over-expression of DEK is started, and similarly, hybridization propagation is performed by using a Luc-EGFP fluorescent control mouse, so that a neural stem cell fluorescent report mouse is obtained as a control group for all experiments.

The presence of the loxp-PGK-Neo-polyA-loxp expression cassette prevents transcription of the downstream gene of interest Dek-IRES-EGFP. The expression site and efficiency of the target gene Dek-IRES-EGFP depend on the tissue type and efficiency of Cre expression. After the R26-DEK-EGFP mouse is mated with the Cre mouse, in the offspring double-positive mouse, an intracellular loxp-PGK-Neo-polyA-loxp expression frame expressing Cre recombinase tissue is knocked out, and the target gene is expressed in high degree under the drive of a CAG promoter.

The double positive laboratory mice used in this experiment were obtained by hybridization of 2 month old Nestin-CreERT2 (purchased from Jackson Laboratory, catalog number 016261) tool mice with 2 month old R26-DEK-EGFP mice (constructed in step a), and were designated as double positive laboratory mice Nestin-CreER ^T2 ::R26-DEK-EGFP。

The double positive fluorescence control mice were obtained by hybridization of 2 month old Nestin-CreERT2 tool mice with 2 month old R26-Luc-EGFP (available from Hainan model organism, catalog number NM-KI-00087) and were designated as double positive fluorescence control mice Nestin-CreER ^T2 ::R26-Luc-EGFP。

All experiments used genotyping double positive mice.

c. Genotyping

The genotype identification method of the neonatal mice comprises the following steps:

(1) And b, cutting the tail or toe of the double-positive experimental mice and the double-positive fluorescent control mice with proper lengths, placing the tail or toe into a microcentrifuge tube of which the pre-packaged size is 50 mu L DNA Extraction Buffer, and centrifuging the mixture by using a mini centrifuge to fully soak the tissues of the mice in the solution.

(2) Placing the centrifugal microcentrifuge tube obtained in the step (1) in a PCR instrument, and performing 15min at 55 ℃;95 ℃ for 5min; after digestion treatment at 16℃for 10min, 50. Mu.L Stop Solution was added to the centrifuge tube to terminate digestion without centrifugation, and a trace of supernatant was taken for subsequent detection, and the thus obtained genomic template was stored in a 4℃freezer for a short period of time.

(3) Genotyping

Performing genome identification PCR (polymerase chain reaction) on 1 mu L of supernatants of the double-positive experimental mice and the double-positive fluorescent control mice obtained in the step (2), wherein the supernatants of the double-positive experimental mice and the double-positive fluorescent control mice are respectively identified by PCR according to a reaction system and a program of a table 3 by using a primer Cre/Globin in the table 2 (lanes 2 and 3 corresponding to B in fig. 4); the supernatant from the double positive mice was subjected to PCR using the primers R26-DEK/P1, P2, and P3 in Table 2, and the genotype of R26-DEK-EGFP was identified by the reaction system and procedure in Table 4 (lanes 5 and 6 corresponding to B in FIG. 4); the supernatant from the fluorescent control mice was subjected to PCR using the primers R26-Luc/P1, P2, P3, and P4 in Table 2, and the genotype of R26-Luc-EGFP was identified according to the reaction system and the procedure of Table 5 (lanes 8 and 9 corresponding to B in FIG. 4).

The results are shown in FIG. 4B, and we successfully obtained the double positive experimental mice for subsequent experiments.

TABLE 2 genotyping primer sequences

TABLE 3 Cre genotype PCR reaction System and program

TABLE 4R 26-DEK-EGFP genotype PCR reaction system and program

TABLE 5R 26-Luc-EGFP genotype PCR reaction system and program

2. Acquisition of conditional over-expressed DEK mice and fluorescent control mice

And (3) weighing tamoxifen, taking corn oil and absolute ethyl alcohol in a volume ratio of 9:1 as solvents, firstly, re-suspending and dispersing tamoxifen powder by using the absolute ethyl alcohol, then adding the corn oil, heating and dissolving in a constant-temperature water bath at 37 ℃, occasionally uniformly mixing and accelerating dissolution during the period, and obtaining a clear and transparent 20 mg/mL tamoxifen solution. Freshly prepared tamoxifen solution was used for each injection.

Referring to the adult neural stem cell overexpression DEK strategy in FIG. 4C, the 2 month old experimental mice (double positive experimental mice and double positive fluorescent control mice) constructed by the method of step 1 were weighed, and tamoxifen solution was intraperitoneally injected into the mice at an injection dose of 10. Mu.L/g, once daily for 5 days, to obtain conditional overexpression DEK mice and fluorescent control mice. Three days after the last injection, samples (SVZ regions) were taken for immunofluorescence analysis (same as example 1, step 2) for the expression of DEK protein in neural stem cells (GFP+) in the brain SVZ regions of two groups of mice (D in FIG. 4), and the total protein expression in the brain SVZ regions of two groups of mice was analyzed by immunoblotting (E in FIG. 4).

3. Immunofluorescence assay DEK activation of dormant neural stem cells

The conditional overexpression DEK mice induced by tamoxifen in the step 2 and the fluorescence control mice were sampled three days after the end of the last injection, respectively in the subventricular canal area of the cerebral side and the subgranulosa cell area of the dentate gyrus of the hippocampus, and GFP/Ki67, GFP/Sox2/BrdU immunofluorescence analysis was performed (same as in example 1, step 2), wherein the antibody dilution ratio was as follows: GFP antibody, 1:1000; GFAP antibody, 1:1000; sox2 antibody, 1:500; ki67 antibody, 1:200; brdU antibody, 1:100.

We have found that over-expression of DEK directly activates dormant neural stem cells by immunofluorescence staining analysis of the cell proliferation marker Ki67, and that the number of proliferating neural stem cells (gfp+ki67+) in the SVZ region (A1 and A2 in fig. 5) and SGZ region (A1 and A2 in fig. 6) is significantly increased compared to the control group.

4. BrdU incorporation retention assay

Referring to FIG. 5, we performed a BrdU incorporation retention test simultaneously, and after feeding the double-positive experimental mice and the double-positive fluorescent control mice with BrdU drinking water for 14 consecutive days in step 1, tamoxifen was induced for 5 days in step 2, and after feeding with normal drinking water for 12 days, samples were taken in SVZ zone and SGZ zone, respectively, and fluorescent staining analysis was performed by the method of step 2 in example 1, and the results are shown in FIG. 5, B2, B3, and B1, B2 in FIG. 6.

As a result, it was found that after the overexpression of DEK, the number of BrdU-retention-labeled neural stem cells (GFP+Sox2+BrdU+) in the SVZ region and the SGZ region was significantly reduced, indicating that the overexpression of DEK activates dormant neural stem cells, promotes proliferation and division of neural stem cells, and dilutes BrdU labeling.

5. Overexpression of DEK Virus injection

The method for activating the neural stem cells by injecting the overexpression DEK virus and the fluorescence control virus through the brain stereotactic injection of the wild mice is as follows:

(1) Old mice C57BL/6J (12 months old) were anesthetized with 1.5% isoflurane gas using a small animal anesthesia machine.

(2) After the hair on the skin surface of the head of the mouse is removed by scissors, the head of the mouse is fixed on a brain stereotactic instrument, the mouth of the mouse is opened with the assistance of dissecting forceps, the teeth of the upper jaw of the mouse are mounted on the buckle of the brain stereotactic instrument, the fixing screw on the stereotactic instrument is rotated to enable the mouse to just fix the nose of the mouse, and finally, the two side fixing bars are embedded into the ears of the mouse to anchor the head of the mouse.

(3) The skin surface of the head is disinfected by wiping with iodine, a small wound is made by a surgical knife, and the wound is cut by a tissue shear.

(4) The sheared skin is pulled to two sides by using tissue forceps, physiological saline is smeared by using a cotton swab to remove the meninges, the front halogen position is found on the skull, and a marker pen is used as a coordinate origin.

(7) Fixed point coordinates: the former bittern is used as the origin (A2 and A3 in figure 7), the backward direction is 2.0mm, the right direction is 1.5mm, a marker pen is used for marking the coordinate position, a cranial drill is used for punching, the punching force is maintained in the handheld process, and the damage to brain tissues is avoided.

(8) 2. Mu.L of virus solution (AAV 9-GFAP-GFP or AAV9GFAP-DEK-GFP, purchased from Shandong View) was withdrawn, the microinjection pump was set at a flow rate of 1. Mu.L/10 min, the microinjection was fixed on a brain stereotactic apparatus, the needle was lowered to the coordinate hole on the skull, the syringe was lowered after the ordinate was reset to a depth of 2.3mm from the needle tip, and the microinjection pump procedure was started to start the injection.

(9) After the injection is finished, the injector is stopped for 10 minutes so as to be fully absorbed, the injector is slowly lifted, and the cotton swab dipped with physiological saline is used for cleaning up blood on the needle head, so that the needle head is prevented from bending.

(10) The skin on both sides of the wound is pulled together by tissue forceps, the wound is sewn by absorbable suture lines, the iodophor is sterilized, and the mouse is transferred to a heating pad at 37 ℃ so as to be convenient for awakening.

(11) All surgical instruments are cleaned by alcohol, and are placed in a high-temperature high-pressure sterilizing pot for sterilization, the operating table is sterilized, and ultraviolet irradiation is carried out for 30 minutes.

Referring to FIG. 7, after 7 days of injection of fluorescent control adeno-associated virus (AAV-Ctrl: AAV 9-GFAP-GFP) and over-expressed DEK adeno-associated virus (AAV-OE: AAV 9-GFAP-DEK-GFP) into striatum of aged wild-type C57BL/6 mice, immunofluorescence patterns of brain coronal sections were analyzed by sampling in SVZ region and SGZ region, respectively, and the expression pattern of DEK protein in control virus-injected group and over-expressed DEK virus-injected group in mouse brain SGZ region neural stem cells (GFP+GFAP+) (A4, A5 in FIG. 7), and statistics of the numbers of characteristic immunofluorescence patterns (B1 in FIG. 7) and proliferation-state neural stem cells (GFP+Ki67+) in the control virus-injected group and over-expressed DEK virus-injected group mouse brain SGZ region (B2 in FIG. 7) were examined, and whether hippocampal neural stem cells were activated was observed.

We also significantly increased the number of hippocampal proliferating neural stem cells using brain stereotactic over-expression of DEK virus injection (fig. 7). These results indicate that DEK is able to directly activate dormant neural stem cells of the SVZ and SGZ regions into the cell cycle.

Example 4 DEK activating dormant neural Stem cells to promote neurogenesis

Immunofluorescence assay of DEK promotes neural stem cell production of neuronal blast cells (DCX+) and mature neurons (NeuN)

To investigate whether or not neurogenesis can be promoted after activation of dormant neural stem cells by overexpression of DEK, we performed a series of lineage tracing experiments 1 month, 4 months and 8 months after tamoxifen induction using Nestin conditional overexpression mice and control groups, and detected and counted the changes in the number of various cells during neurogenesis, as follows:

referring to FIG. 8A, tamoxifen induction was performed on double-positive experimental mice and double-positive fluorescent control mice in the same manner as in example 3, step 1, for 5 days, three days after the end of the last injection, after feeding with BrdU drinking water (1 mg/mL) for 7 consecutive days, feeding with normal drinking water was continued for 1 month/4 month/8 months, and after sampling (SVZ zone and SGZ zone) was performed for brain tissue sections, and DCX positive neuronal blast cells, GFP were analyzed by immunofluorescence ⁺ NeuN ⁺ Or BrdU ⁺ NeuN ⁺ The newly generated mature neuron expression profile (as in example 1, step 2) is shown in FIG. 8 as B1, B2, B3, C1, C2, C3, D1, D2 and FIG. 9 as A1, A2, A3, B1, B2, B3, C1, C2, wherein the dilution ratio of the antibodies used is: GFP antibody, 1:1000; DCX antibody, 1:200; neuN antibody, 1:1000; brdU antibody, 1:100.

Immunofluorescence staining results showed that the numbers of neuronal blast cells (dcx+) in the SVZ and SGZ regions were increased after over-expression of DEK compared to the control group (fig. 8, fig. 9), as were the numbers of offspring neo-neurons (gfp+neun+ or brdu+neun+) detected in the olfactory bulb and dentate gyrus regions (fig. 8, fig. 9), indicating that over-expression of DEK promoted neurogenesis.

EXAMPLE 5 DEK does not disrupt the homeostasis of neural Stem cell banks

DEK is capable of promoting dormant neural stem cells into the cell cycle, producing a large number of proliferating neural stem cells, and promoting the neurogenesis process for a long period of time. In this process, it is of interest whether the homeostasis of the dormant neural stem cell pool is affected. Therefore, the immunofluorescence is adopted to detect the influence of DEK on the total quantity of the neural stem cells, and the specific steps are as follows:

the tamoxifen-induced conditional overexpressing mice and fluorescent control mice in step 2 of example 3 were subjected to brain tissue sections by sampling (SVZ region and SGZ region) after continuing normal drinking water feeding for 1 month/4 month/8 months three days after the end of the last injection, and the total number of neural stem cells (GFAP ⁺ Sox2 ⁺ ) Statistical analysis was performed (same asExample 1, step 2), results are shown in fig. 8, D3, and fig. 9, C3, wherein the antibody dilution ratio used is: GFAP antibody, 1:1000; sox2 antibody, 1:500.

The result shows that: overexpression of DEK in neural stem cells for a short period of time (1 month, 4 months) increased the number of neural stem cells (GFAP+Sox2+) compared to the control group, and fallen back to a level comparable to that of normal mice for a long period of time.

The above results indicate that overexpression of DEK in adult resting neural stem cells does not disrupt the homeostasis of the neural stem cell pool, and does not trigger depletion of the neural stem cell pool for up to 8 months.

Example 6 mechanism of DEK to regulate activation of dormant neural Stem cells

a. Overexpression of DEK protein causes changes in neural stem cell gene expression

To determine the molecular mechanism by which DEK regulates activation of dormant neural stem cells, we first performed a transcriptome sequencing analysis 24 hours after the overexpression of DEK on primary adult neural stem cells to obtain the change in gene expression at the transcriptional level (NCBI database GEO number: GSE 200000).

High throughput sequencing of transcriptomes (RNA-Seq): the SVZ zone tissue of example 2, step 3a was collected, primary neural stem cells were obtained using a neural tissue digestion kit (Meitian and Gentle, cat# 130-092-628), resuspended in 1 ml of Neuralbasal maintenance medium containing 2% B27 supplement, and 5uL 10 was added ¹³ The vg/mL AAV9-GFAP-GFP (control group) or AAV9-GFAP-DEK-GFP (experimental group) is incubated for 24 hours in a carbon dioxide incubator at 37 ℃, and then centrifuged, and the sediment is taken as a cell sample. By usingStranded RNA-Seq Kit (Clontech, cat# 634836), 1mL of TRIzol reagent (Thermo Fisher) was added to the above cell sample, total RNA was extracted, and mRNA with polyA tail was enriched by Oligo (dT) magnetic beads. Use of Illumina +.>UltraTM RNA Library Prep Kit construction of transcriptome library and sequencing on-machine. The sequence of the sequenced fragment was aligned to the mouse reference genome mm39 using HISAT2 software, and quantitative analysis of gene expression level was performed by counting the number of signals covered from the start to the end of each gene according to positional information of gene alignment on the reference genome. And further carrying out statistical analysis on the data of the gene expression level, and screening genes with obvious difference of the expression level between different samples.

Transcriptome outcome analysis found: overexpression of DEK in primary neural stem cells resulted in differential expression of 1730 genes, 636 genes of up-regulated genes and 1094 of down-regulated genes (B in FIG. 10); GO function query was performed on the top 30 of the differentially expressed genes to find that they all directly participated in cell cycle regulation (C in fig. 10); KEGG functional enrichment analysis found that the signal pathways (e.g., PI3K-Akt, MAPK, TGF- β, etc.) associated with the regulation of neural stem cell activation and cell cycle were significantly upregulated (D in fig. 10). Numerous studies have been reported that negative regulation of sleep maintenance factors is critical in the process of dormant neural stem cell activation. By differential analysis of expression of dormancy maintenance genes (e.g., notch1, notch2, hes5, foxo3, apoe, tp53, etc.) in transcriptome data, we found that overexpression of DEK significantly inhibited expression of dormancy maintenance genes, resulting in upregulation of expression of neural stem cell division-associated genes (e.g., mki67, pcna; E in fig. 10).

The results show that the neural stem cells inhibit the expression of dormancy maintenance genes after over-expressing DEK, so that cell division related regulatory molecules and signal channels are obviously up-regulated.

b. Immunofluorescence and western immunoblotting to verify DEK regulatory mechanism

To verify the findings, three days after the last injection, SVZ region tissues are obtained by microdissection from tamoxifen-induced conditional overexpression mice and fluorescence control mice in the step 2 of the example 3, total proteins are extracted, and Western blot is adopted to detect relevant signal channel molecular characteristics, and the method specifically comprises the following steps: (1) In situ brain tissue section immunofluorescence analysis (same as example 1 step 2), wherein the dilution ratio of the used antibody is GFP antibody, 1:1000; notch1 antibody, 1:400; notch2 antibody, 1:400; ASCL1 antibody, 1:100. (2) SVZ tissue analysis Western blotting (same as example 2, step 3) with a dilution ratio of DEK antibody of 1:1000; notch1 antibody, 1:1000; notch2 antibody, 1:1000; NICD antibody, 1:1000; hes1 antibody, 1:1000; hes5 antibody, 1:1000; CSL antibody, 1:1000; ASCL1 antibody, 1:1000; foxO3 antibody, 1:2000; phosphorylated FoxO3 antibody, 1:2000; p53 antibody, 1:500; p21 antibody, 1:500; MYC antibody, 1:500; wnt3a antibody, 1:2000; h3 antibody, 1:5000; beta-actin antibody, 1:5000.

The results showed that after over-expression of DEK, the protein expression level of key molecules (Notch 1, notch2, NICD, hes1, hes 5) in the sleep maintenance core signaling pathway-Notch signaling pathway was significantly reduced in the neural stem cells, whereas the ASCL1 protein expression downstream of the Notch signaling pathway was significantly increased. In addition, the expression level of the dormancy maintenance related factors FoxO3, P53 and P21 was also significantly reduced, while the expression of the activation related factors MYC and Wnt3a was significantly increased (A1 and A2 in fig. 11).

Based on this result, we found a consistent rule after in situ detection of overexpression of DEK in SVZ and SGZ regions by immunofluorescence staining technique at brain tissue slice level, the expression changes of Notch1, notch2 (B1, B2, B3, B4 in fig. 11) and ASCL1 (C1, C2 in fig. 11) in neural stem cells. It can be seen that DEK promotes dormant neural stem cell activation and neurogenesis by directly inhibiting Notch signaling pathways, epigenetic regulation of dormancy and activation-related factors.

EXAMPLE 7 DEK can activate dormant neural Stem cells in aged mice

1. Mouse strain

As shown in FIG. 12A, the present example uses the method of example 3, step 1, to construct a double positive experimental mouse Nestin-Creer for elderly (12 months old) ^T2 R26-DEK-EGFP and double positive fluorescence control mouse Nestin-Creer ^T2 R26-Luc-EGFP was tested as follows.

2. Immunofluorescence detection of DEK activated senile mouse dormant neural stem cells

To investigate whether DEK isCan directly activate dormant neural stem cells in the brains of aged mice, and the Nestin-Creet2 of a double-positive experimental mouse (12 months old) of the aged is R26-DEK-EGFP and the Nestin-CreetER of a double-positive fluorescent control mouse ^T2 R26-Luc-EGFP was induced with tamoxifen for 5 days by the method of step 2 of example 3, and after three days of the last injection, after feeding with BrdU drinking water (1 mg/mL) for 7 consecutive days, feeding with normal drinking water was continued for 1 month, and samples (SGZ region and SVZ region) were taken for brain tissue slice immunofluorescence analysis (same as step 2 of example 1) using the antibody dilution ratio: GFP antibody, 1:1000; sox2 antibody, 1:500; ki67 antibody, 1:200; DCX antibody, 1:200; neuN antibody, 1:1000; brdU antibody, 1:100.

Immunofluorescent staining by fluorescence lineage tracing found: over-expression of DEK in aged mouse neural stem cells compared to control group could activate dormant neural stem cells in SVZ and SGZ regions, resulting in a significant increase in the number of proliferating neural stem cells (gfp+sox 2+ki67+) (B1, B2 in fig. 12, A, B in fig. 13); after one month of over-expression of DEK by neural stem cells of aged mice, the number of neuronal blast cells (gfp+dcx+) in the SVZ and SGZ regions was also significantly increased (A1 and A2 in fig. 14, A1 and A2 in fig. 15). Wherein, under normal physiology: while only 1 or no detection of each SGZ region was possible in the tissue sections of aged mice, after the over-expression of DEK, we could detect about 40 proliferating neural stem cells (B1, B2 in fig. 12, A, B in fig. 13), and the same phenomenon was found in the distribution of neuronal blast cells (A1 and A2 in fig. 14, A1 and A2 in fig. 15), which was consistent with the rule of decreasing the neural stem cells in the SVZ region and SGZ region and their progeny in the brain aging process described in the previous study.

The above results indicate that DEK can activate dormant neural stem cells present in the brains of aged mice and significantly enhance neurogenesis.

EXAMPLE 8 DEK activating dormant neural Stem cells to "younger" the aged brain "

To further evaluate the effect of DEK activation on brain resting neural stem cells in aged mice, we compared and studied neurogenesis levels. Considering that the total number of neural stem cells in brains of mice of different ages was different, recombinant cells labeled with GFP were difficult to use for analytical comparison, we performed BrdU incorporation retention experiments in aged mice with tamoxifen induction to label newly generated mature neuronal cells for neurogenesis levels, detected the number of mature neurons generated after aged mice overexpressed DEK by immunofluorescent staining and compared to the nascent mature neuronal levels labeled with BrdU with control groups of different ages.

Double-positive experimental mice of different ages (2 months, 5 months, 9 months and 12 months) constructed by the method of the step 1 of the above example 3 and fluorescent control mice were continuously fed with BrdU drinking water (1 mg/mL) for 7 days, and then induced for 5 days by injecting tamoxifen by the method of the step 2 of the example 2, and after the last injection was completed, three days after feeding with normal drinking water for 1 month, were sampled for brain tissue sections, and were subjected to comparative analysis of brdu+neun+ immunofluorescent staining (same as in the step 2 of the example 1), wherein the antibody dilution ratio was used: neuN antibody, 1:1000; brdU antibody, 1:100.

The result shows that: overexpression of DEK in olfactory bulb and hippocampal regions activated neural stem cell-enhanced neurogenesis to produce more mature neuronal offspring (gfp+neun+) (B1, B2 in fig. 14, C1, C2 in fig. 15) than in the control group; with age aging, the number of newly generated mature neuronal cells marked by BrdU (brdu+neun+) in the olfactory bulb and the dentate gyrus of the hippocampus of the brain of the aged mice was continuously reduced compared with that of the young mice, and the number was remarkably increased after one month of over-expression of DEK, so that the neurogenesis level of the 5 month old mice was reached (C1, C2 in fig. 14, D1, D2 in fig. 15). The above results indicate that the restarting of neurogenesis by DEK "younger" the elderly brain.

Example 9 DEK activation of dormant neural Stem cells to promote repair of mouse nerve injury from cerebral apoplexy

a. Cerebral apoplexy mouse model construction

The method for inducing the cerebral arterial thrombosis by injecting collagenase into the brain stereotactic of a wild mouse comprises the following steps:

(1) Old mice (20 months old) were anesthetized with 1.5% isoflurane gas using a small animal anesthesia machine.

(7) Fixed point coordinates: the former bittern is used as an origin, a marker pen is used for marking the coordinate position of the original point by 0.5mm forwards and 1.5mm rightwards, a cranium drill is used for punching, and the punching force is maintained in the handheld process, so that the damage to brain tissues is avoided.

(8) 10 mu L of collagenase IV solution (150U/mL) is extracted, the flow rate of a microinjection pump is set to be 0.5 mu L/10min, a microinjection syringe is fixed on a brain stereotactic instrument, a needle head is lowered to a coordinate hole on a skull, the syringe is lowered after the ordinate is reset, the needle point enters the depth of 3.5mm, the microinjection pump program is started, and each mouse is injected with 0.5 mu L.

The striatum of an adult wild mouse is injected with 0.5 mu L collagenase IV solution (150U/mL) to induce hemorrhagic cerebral apoplexy, the focal region characterization is sampled and analyzed after 24 hours, whether the phenomenon of blood stasis after hemorrhage exists or not is observed, and the construction condition of the hemorrhagic cerebral apoplexy model is evaluated. This model was subsequently used to apply to the double positive offspring of the transgenic mice constructed in example 3.

b. Immunofluorescence analysis of the effect of overexpression of DEK on repair of mouse lesions in cerebral apoplexy

Cerebral apoplexy is a common sudden brain nerve injury disease, and has high mortality rate. In order to explore whether DEK can play a role in nerve injury repair as an activation regulating factor, a collagenase IV injection is used for initiating vascular rupture to construct a mouse hemorrhagic stroke injury model, and a transgenic mouse double-positive offspring constructed in the embodiment 3 is subjected to cerebral stroke injury repair pedigree tracing after tamoxifen induction.

Adult (2 months old) Nestin-CreER 3 days after injection of tamoxifen in example 3 step 2 using the procedure of step a ^T2 R26-Luc-EGFP and Nestin-Creer ^T2 R26-DEK-EGFP transgenic mice were injected with 0.5. Mu.L collagenase IV solution (150U/mL) to induce hemorrhagic stroke, after surgery, the mice were fed with EdU drinking water (0.25 mg/mL) for 7 consecutive days, and normal drinking water was used to feed the mice for 23 days, and after surgery, the mice were subjected to a one month period of injury repair, and samples were taken for brain tissue slice immunofluorescence analysis (same as in example 1, step 2), wherein the antibody dilution ratio used was: GFP antibody, 1:1000; GFAP antibody, 1:1000; DCX antibody, 1:200; neuN antibody, 1:1000.EdU signal detection was performed by using EDU-Click kit (Sigma, cat# BCK-EDU 594) to prepare staining solution, and incubating for 30 min at room temperature before immunofluorescence step DAPI staining.

By pathological observation, apoptosis detection and immunofluorescent staining, the results show that after collagenase IV is injected into the striatum region of the brain of the mouse for 24 hours, a large amount of blood stasis appears in the striatum region (A2 in fig. 16), and a large amount of cells in apoptosis (Casp3+) (B in fig. 16) exist in the region, and damaged striatum mature neurons (NeuN+) are remarkably absent (C in fig. 16), which indicates that the mouse hemorrhagic cerebral apoplexy model is successfully constructed.

Previous studies have shown that endogenous resting neural stem cells are only marginally activated in response to this injury stimulus during the repair of hemorrhagic stroke neural injury (fig. 19), whereas DEK overexpression can significantly enhance the endogenous neural stem cells in response to injury repair by activating endogenous resting neural stem cells, and the neuronal parent cells produced by the neural stem cells have the ability to migrate to the focal area, differentiate into mature neurons, and in addition, astrocytes also play an important role in the repair of injury, supporting and inhibiting inflammatory responses. After one month of injury repair phase (fig. 17 a), the over-expression of DEK activated endogenous neural stem cells significantly promoted injury repair, and the increased number of GFP-labeled neural stem cells and their progeny (gfp+) migrated to the injured area (fig. 17B), and the numbers of neuronal blast cells (gfp+dcx+), astrocytes (gfp+gfap+) and EdU-labeled neonatal total cells (gfp+edu+) generated by the neural stem cells were significantly increased, which indicates that DEK promoted activation of endogenous neural stem cells and promotion of nerve injury repair in mice in stroke (C1, C2, D1, D2 in fig. 17, A1, A2 in fig. 18) compared to the case of injury repair under normal physiology in the control group. In addition, analysis of the density of injured area mature neurons (neun+) found significant recovery of the density of mature neurons in the foci of the over-expressed DEK mice (B1, B2, C1, C2 in fig. 18), although only a small fraction of these neurons were lineage offspring generated after the presence of DEK-activated neural stem cells, we speculated that it might be the neuronal blast that had been present prior to tamoxifen induction that promoted the repair process of the mature neurons after the over-expression of DEK-activated neural stem cells to generate a large number of new-born neuronal blast.

Sequence listing

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Claims

1. An application of DEK protein in preparing medicament for activating dormant neural stem cells.

2. The use according to claim 1, wherein the DEK protein has the conserved nucleic acid sequence as shown in SEQ ID No. 1.

3. The use according to claim 1, wherein the DEK protein has more than 95% similarity to the amino acid sequence shown in SEQ ID No. 2.

4. The use according to claim 1, wherein the DEK protein has the amino acid sequence shown in SEQ ID No. 2.

5. The use of claim 1, wherein the use is for over-expression of endogenous DEK protein in dormant neural stem cells or for delivery of exogenous DEK protein into dormant neural stem cells for the purpose of activating dormant neural stem cells.

6. An application of DEK protein in preparing medicine for preventing or treating cerebral nerve injury is provided.

7. The use according to claim 6, wherein the brain nerve injury comprises stroke, alzheimer's disease, parkinson's disease.

8. The use according to claim 6, wherein the medicament is prepared by purifying the DEK protein by in vitro expression and by mounting exosomes or by mounting DEK with a genetically engineered virus.

9. An application of DEK protein in preparing a reagent for activating dormant neural stem cells.

10. The use of claim 9, wherein the agent comprises an endogenous or exogenous DEK protein.