CN119193703A

CN119193703A - Central nervous system cell-specific miRNA expression vector, adeno-associated virus vector and use thereof

Info

Publication number: CN119193703A
Application number: CN202411199484.7A
Authority: CN
Inventors: 李天文; 朱剑虹; 王鹏
Original assignee: Huashan Hospital of Fudan University
Current assignee: Huashan Hospital of Fudan University
Priority date: 2024-08-29
Filing date: 2024-08-29
Publication date: 2024-12-27

Abstract

The invention relates to the technical field of genetic engineering, in particular to a central nervous system cell specific miRNA expression vector, an adeno-associated virus vector and application thereof, wherein the miRNA expression vector comprises an Ef1 alpha promoter sequence, an induction sequence, a Tre promoter, a fluorescent marker sequence, a tRNA sequence, a miRNA sequence and an SV40poly (A) termination signal sequence, and the specific miRNA over-expressed gene sequence is a combined sequence of tRNA-miRNA and tRNA incorporated at the downstream of the fluorescent marker sequence. Experiments show that the miRNA expression vector can be stably over-expressed in specific cells. The invention has the advantages of tracking the carrier and carrying out targeted treatment, and can be applied to the treatment of central nervous system nerve inflammatory diseases, such as neurodegenerative diseases, cerebral hemorrhage, apoplexy, cerebral trauma and the like.

Description

Central nervous system cell specific miRNA expression vector, adeno-associated virus vector and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a central nervous system cell specific miRNA expression vector, an adeno-associated virus vector and application thereof.

Background

The main constituent cells in the brain are neurons, astrocytes, oligodendrocytes and microglia. Neurons (neurons) are the basic functional units of the nervous system, responsible for the transfer and processing of information. They communicate with other neurons, muscle cells or gland cells by electrical and chemical signals. The normal function of neurons is critical to the health and functioning of the entire nervous system. In many neurodegenerative diseases (such as parkinson's disease and alzheimer's disease), neuronal damage and death are major pathological features. Astrocytes are the most abundant glial cells in the central nervous system, accounting for 20% -40% of the whole glial cells. They perform a wide range of homeostatic functions in the central nervous system, such as participation in synapse formation, maintenance of constant intracellular and extracellular ion concentrations, participation in neurotransmitter delivery, formation and maintenance of the blood brain barrier, support for other resident cells of the central nervous system, and the like. In pathological conditions, astrocytes can undergo morphological and molecular changes to adopt a so-called "reactive" state, which can alter their function and alter the balance between their neural support and neurotoxic properties. Oligodendrocytes (Oligodendrocytes) are an important type of glial cell in the Central Nervous System (CNS). Their main function is to form myelin sheath (MYELIN SHEATH), which is wrapped around the axons of neurons, and serves as an insulator, which can significantly increase the conduction velocity of nerve impulses. Microglia (Microglia) are resident immune cells in the Central Nervous System (CNS) that function similarly to macrophages in the peripheral immune system. They play a key role in maintaining the health of the nervous system, monitoring environmental changes, and responding to injury or infection.

Adeno-associated virus (AAV) is currently found to be the simplest, non-enveloped, single-stranded DNA virus consisting of a protein capsid and a single-stranded DNA genome of 4.7kb in length, the capsid length being 20-25 nm. The AAV genome has inverted terminal repeats at each end (INVERTED TERMINAL REPEAT, ITR) which play an important role in the viral life cycle, including key processes such as viral replication and packaging. The target gene sequence is inserted into the AAV genome coding region while retaining the ITR sequences at both ends. By such construction, the expression of the target gene will be finely regulated by AAV biological mechanisms, thereby achieving stable expression in the target cell.

Currently, gene delivery vectors derived from natural or genetically engineered adeno-associated viruses (AAV) have become the focus of human gene therapy, and have better safety due to their lower immunogenicity, stable gene expression levels and site-specific integration ability, so that the development of the field of gene therapy is increasingly vigorous, and more clinical trials are being applied to various therapies. In the central nervous system, AAV is currently the primary vector for in vivo delivery of transgenes to the central nervous system, with the ability to transduce transgenes efficiently, highly engineered cell targeting, low toxicity, and the potential to overcome physical barriers, including the blood brain barrier. In the current field of nervous system disease treatment, we often use AAV specific promoters to specifically infect certain specific cell types for the purpose of treating the disease. For example, an AAV using the GFAP promoter infects astrocytes, an AAV using the Synapsin promoter infects neurons, an AAV using the CD68 promoter infects microglia, and an AAV using the Olig2 promoter infects oligodendrocytes.

MicroRNA (miRNA) is an endogenous micronon-coding RNA, typically between 20-25 nucleotides in length, that regulates gene expression by base pairing with targeted messenger RNA in the 3' untranslated region, and that is widely involved in a variety of key biological and cellular processes such as apoptosis, differentiation, development, proliferation, metabolism, and signal transduction pathways. miRNA-based therapies are therefore likely promising potential tools for the treatment of various diseases.

Important physiological and pathological functions of micrornas are also gradually discovered in the central nervous system. 2017A Nature study confirmed that neural stem cells of the hypothalamus control the aging rhythm of the organism through microRNAs in exosomes. The microRNA with the most representative meaning is miR-124. The abundance of miR-124 accounts for 25-48% of total microRNAs in brain, and is the microRNA with the highest expression level in the central nervous system. miR-124 can promote nerve regeneration and nerve development, inhibit differentiation of neural stem cells, and provide an alternative strategy for nerve repair of stroke, brain trauma, neurodegenerative diseases and other diseases.

In the prior art, multiple nervous system cell subtype promoters are often used to drive expression of target gene mRNA with different cell specificities, however, no specific promoters are currently available to control expression of mirnas in specific cell types.

Disclosure of Invention

In view of the above technical problems, a first aspect of the present invention is to provide a central nervous system specific miRNA expression vector, which drives fluorescent marker gene expression under doxycycline (dox) induction by transfection into cells, thereby realizing specific expression of miRNA in transfected cells.

The second aspect of the invention provides an application of a central nervous system cell-specific miRNA expression vector in improving miRNA over-expression, wherein under the induction of doxycycline, a Tre promoter drives fluorescent marker gene expression so as to drive miRNA-specific expression.

The third aspect of the present invention provides an adeno-associated virus vector specific to cells of the central nervous system, wherein after the adeno-associated virus is injected, the cell-specific promoter is used to drive the expression of fluorescent genes, so that the specific expression of miRNA in specific cells is further realized, and the vector can be used for tracking the advantages of specific cells and targeted cell therapy.

In a fourth aspect, the invention provides the use of an adeno-associated viral vector specific for cells of the central nervous system for increasing the specific expression of a miRNA.

In a fifth aspect, the invention provides an application of an adeno-associated viral vector specific to cells of the central nervous system in preparing a medicament for treating diseases related to nervous inflammation of the central nervous system, such as nerve repair of cerebral hemorrhage, stroke, brain trauma, neurodegeneration and the like.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

The invention provides a central nervous system cell specific miRNA expression vector for constructing a specific over-expressed miRNA vector, which comprises an Ef1 alpha promoter sequence, an induction sequence, a Tre promoter, a fluorescent marker gene sequence, a tRNA sequence, a miRNA sequence, a tRNA sequence and an SV40 poly (A) termination signal sequence;

The Tre promoter sequence is shown as SEQ ID NO.1, the tRNA sequence is shown as SEQ ID NO.3, the SV40 poly (A) termination signal sequence is shown as SEQ ID NO.5, and the Ef1 alpha promoter sequence is shown as SEQ ID NO. 6;

The induction sequence is a doxycycline induction activation sequence, in particular The nucleotide sequence of the 3G transactivator protein factor is shown as SEQ ID NO. 7;

The specific miRNA over-expression gene sequence is formed by doping tRNA-miRNA-tRNA at the downstream of a doxycycline induction system, and the Tre promoter drives the fluorescent marker gene to express so as to realize miRNA expression.

In a preferred embodiment of the first aspect, the miRNA is miR-124-3P, and the sequence of the miRNA is shown in SEQ ID NO. 4.

In a preferred embodiment of the first aspect, the fluorescent marker gene sequence is an EGFP fluorescent marker gene sequence driven by a Tre promoter, and the sequence is shown in SEQ ID NO. 2.

In a second aspect, the invention provides the use of the central nervous system cell-specific miRNA expression vector of the first aspect for enhancing miRNA overexpression in specific cells (including but not limited to astrocytes, oligodendrocytes, neurons, microglia, etc.), and the cell-specific promoter drives expression of the fluorescent marker gene and thus drives miRNA-specific expression.

In a preferred embodiment of the second aspect, the miRNA-specific expression is specific expression in vivo or in vitro.

The third aspect of the invention provides a central nervous system cell-specific miRNA over-expression adeno-associated virus vector, which comprises an astrocyte-specific promoter, a fluorescent marker sequence and a tRNA-miRNA-tRNA sequence, wherein the tRNA-miRNA-tRNA sequence comprises a tRNA sequence and a miRNA sequence, the tRNA sequence is shown as SEQ ID NO.3, and the fluorescent marker sequence is an EGFP fluorescent marker gene sequence driven by the astrocyte-specific promoter GFAP, and the sequence is shown as SEQ ID NO. 2.

Further, the miRNA is miR-124-3P, and the sequence of the miRNA is shown as SEQ ID NO. 4.

In a fourth aspect, the invention provides an application of the central nervous system cell-specific miRNA over-expression adeno-associated virus vector in preparing a transgenic test animal model.

In a preferred embodiment of the fourth aspect, the cell-specific miRNA expression vector is miRNA expression in an astrocyte;

miRNA expression in astrocytes is used to prepare a therapeutic animal model for cerebral hemorrhage test.

In a fifth aspect, the invention provides an application of the miRNA over-expression adeno-associated virus vector specific to cells of the central nervous system in preparing a medicament for treating diseases related to central nervous system neuroinflammation, such as neurodegenerative diseases, cerebral hemorrhage, apoplexy, cerebral trauma and the like.

By adopting the technical scheme, the invention has the following advantages and positive effects compared with the prior art:

The cell-specific miRNA over-expression vector provided by the invention is characterized in that a fluorescent marker gene and tRNA-miRNA-tRNA are inserted at the downstream of a doxycycline induction system, and transcription is terminated by SV40 poly (A). The vector provided by the invention is transfected into cells, and under the induction of dox, the promoter drives the fluorescent marker gene to express, so that miRNA is specifically expressed in the cells.

Drawings

FIG. 1 is a simplified diagram of the overexpression of miRNA in the inducible miRNA expression vector of example 1 of the present invention;

FIG. 2 is a schematic diagram of a miRNA expression vector of the doxycycline induction system of example 1 of the present invention;

FIG. 3 is a diagram showing the identification of fluorescence of cells transfected with miRNA expression vectors of the doxycycline induction system of example 1 of the present invention;

FIG. 4 is a graph showing the expression level of miRNA after PCR amplification of transfected cells with miRNA expression vectors of the doxycycline induction system of example 1, wherein (a) is the expression level of miR-124-3p, (b) is the expression level of EGFP, and (c) is a graph showing the correlation between miR-124-3p and EGFP;

FIG. 5 is a graph showing the tracking of EGFP-positive cells in an animal model immunofluorescence test for cerebral hemorrhage test constructed in example 2 of the present invention, wherein (a) is a fluorescent photograph and (b) is a percentage count of EGFP-positive cell areas;

FIG. 6 shows the activation of neurotoxic astrocytes in animal models of experimental and control cerebral hemorrhage according to example 2 of the present invention, wherein (a) is an antibody fluorescence photograph, (b) is a comparison of C3 antibody coverage areas, and (C) is a comparison of C3 antibody expression;

FIG. 7 shows immunofluorescence of brain sections of animal models for experimental and control cerebral hemorrhage test in example 2 of the present invention, wherein (a) is a fluorescence chart and (b) is a dead cell count chart under a fluorescence microscope.

Detailed Description

The following describes in further detail a cell-specific miRNA expression vector, an adeno-associated viral vector, and uses thereof, according to the present invention, with reference to the accompanying drawings and specific examples. The advantages and features of the present invention will become more apparent from the following description.

Example 1

Overexpression verification of miRNA expression vector, taking miR-124-3p of doxycycline induction system as an example

1. A doxycycline-driven miRNA expression vector (target plasmid for short) comprises a Tre promoter, a fluorescent marker sequence, a tRNA sequence, a miRNA sequence, a tRNA sequence, an SV40 poly (A) termination signal sequence, an Ef1 alpha promoter sequence,A 3G sequence;

The Tre promoter sequence is shown as SEQ ID NO.1, the tRNA sequence is shown as SEQ ID NO.3, the SV40 poly (A) termination signal sequence is shown as SEQ ID NO.5, and the Ef1 alpha promoter sequence is shown as SEQ ID NO. 6; the 3G sequence is shown as SEQ ID NO. 7;

the fluorescent marker sequence is an EGFP fluorescent marker gene driven by a Tre promoter, the sequence is shown as SEQ ID NO.2, the miRNA sequence is a miR-124-3p sequence, and the specific sequence is shown as SEQ ID NO. 4;

as shown in fig. 1-2, the doxycycline-driven miRNA expression vector is a transcription termination signal that is incorporated into the tRNA-miRNA-tRNA downstream of the EGFP sequence. Doxycycline drives EGFP to express, and miR-124-3p expression is achieved.

2. The plasmid of interest described above was transfected into tool cells (293T)

The 293T cell line was placed in 24-well plates at a cell density of 1.5X10 ⁵ cells/well. 500ul of cell culture medium was added per well. 293T cells were identified under light to be grown to appropriate density and transfected with EZ trans cell transfection reagent, 50. Mu.g of plasmid of interest and 100. Mu.l of transfection reagent were added per 15cm dish of cells, as follows:

Two 50ml centrifuge tubes, tube A and tube B (blank), were prepared and each filled with Opti MEM transfection buffer with a volume of n (n=293T cell discs). Times.1 ml. Subsequently, a target plasmid amount of n×50 μg was added to the A-tube, and mixed by pipetting, wherein the Opti MEM transfection buffer was serum-free medium.

Add n×100. Mu.l of EZ trans cell transfection reagent to the B tube and blow mix. After the two pipes are kept stand for 3min to 5min, the liquid in the pipe B is added into the pipe A, and the two pipes are blown and evenly mixed. After further standing for 10-15 min, adding the mixed solution into the complete culture medium of 293T cells according to the amount of 2 ml/dish of cells for transfection, adding 300ul of fresh complete culture medium into each well after 48h of transfection, and continuously incubating the cells for 24-48h.

3. Evaluation of EGFP expression Using fluorescence identification of transfected cells

Cell supernatants were removed and washed 3 times with PBS. 4% paraformaldehyde (paraformaldehyde, PFA) was added and the mixture was allowed to stand for 10min. PFA was removed and washed three times with PBS. Soaking with 0.3% Triton X-100 (PBS solution) for 30min, and washing with PBS for 3 times each for 3min. EGFP antibodies were diluted with immunofluorescent primary anti-dilution and added to cell culture slides at 4℃overnight. The next day the primary antibody was removed and washed 3 times with PBS for 10min each. The donkey anti-mouse IgG (H+L) fluorescent secondary antibodies were labeled with diluted Alexa Fluor 488, added to the cell culture slide, and incubated on a shaker at room temperature for 2H. After that, the secondary antibody was removed, and washed 3 times with PBS for 10min each time. Anti-fluorescence quenching agent (containing DAPI) is dripped, cover glass and nail oil sealing sheet are covered. The results are shown in FIG. 3, where EGFP expression was substantially absent in the absence of doxycycline (dox), and addition of dox significantly upregulated EGFP expression, indicating that the Tre promoter driven EGFP expression under dox induction.

4. Real-time quantitative PCR detection of transfected cells

① RNA extraction using the selinum biological RNA extraction kit. The medium was removed, washed once with PBS, 500. Mu.l of lysate was added, the adherent 293T cells were blown down and transferred to a 1.5ml EP tube. Shake for 15s, stand at room temperature for 5min. 160 μl chloroform was added to the tube, shaken for 15s, and allowed to stand at room temperature for 3min. Centrifugal force at 13000rpm,4 ℃ for 10min. At this time, the liquid in the EP tube was found to be divided into 3 layers, and the upper layer was RNA. The upper liquid was transferred to a fresh EP tube, 1/2 volume of absolute ethanol was added, and the liquid was transferred to a centrifuge column in the kit by shaking for 15 s. Centrifuge at 13000rpm,4 ℃ for 45s, discard the liquid in the lower tube. 0.5ml of deproteinized solution RE was added, centrifuged at 13000rpm at 4℃for 45s, and the liquid in the lower tube was discarded. 0.5ml of rinse solution RW was added, centrifuged at 13000rpm at 4℃for 45s, the liquid in the lower tube was discarded, and the liquid in the lower tube was discarded by rinsing again with RW. Centrifugation was performed at 13000rpm for 3min at 4℃and the lower tube was discarded and a new EP tube was fitted to collect RNA. Adding 30 μl RNase-free water, standing for 2min, centrifuging at 13000rpm at 4deg.C for 3min to obtain RNA. RNA was quantified using DeNovix.

② RNA reverse transcription was performed using 1000ng of RNA. Reverse transcription was performed using the Norpran reverse transcription kit, briefly, using 4x DNA wiper to remove DNA followed by reverse transcription using 5x reverse transcriptase to give a final system of 20. Mu.l cDNA. qPCR was performed using kit HISCRIPT III RT SuperMix for qPCR (+ GDNA WIPER), R323-01, vazyme, and Norpraise.

③ QPCR the following qPCR primer sequences

EGFP-Q-F	GGACGACGGCAACTACAAGA	SEQ ID NO.8
			EGFP-Q-R	CTCGATGTTGTGGCGGATCT	SEQ ID NO.9
miR-124-3p-F	GCGTAAGGCACGCGGTG	SEQ ID NO.10
			miR-124-3p-R	AGTGCAGGGTCCGAGGTATT	SEQ ID NO.11
mGapdh-Q-F	CATGGCCTTCCGTGTTCCTA	SEQ ID NO.12
			mGapdh-Q-R	GCGGCACGTCAGATCCA	SEQ ID NO.13

The results are shown in FIG. 4, where miR-124-3p expression levels are significantly increased compared to the group without dox addition. Furthermore, correlation analysis results suggest that correlation analysis between miR-124-3p and EGFP mRNA levels reveals a surprisingly strong correlation between the two, with a p value of less than 0.0001 and an R2 value of 0.9427.

This example illustrates that inducible miRNA expression vectors can be used for the in vitro atopic expression of mirnas.

Example 2

Construction of central nervous system cell specific AVV-miR-124-3p and application thereof in cerebral hemorrhage test animal model

1. Construction of an adeno-associated viral vector for astrocyte miR-124-3 p-specific expression

EGFP expression was driven by the GFAP promoter (astrocyte-specific promoter), tRNA-miRNA-tRNA was incorporated downstream of EGFP, and the above-described double tRNA sandwich short sequence was used to release miR-124-3p expression, thus constructing an adeno-associated viral vector for astrocyte miR-124-3 p-specific expression. The vector structure was synthesized by the company Shanghai, inc., and was designated as AAV-PHP.eB-sGFAP-EGFP-tRNA-miR-124-3p-tRNA and AAV-PHP.eB-sGFAP-EGFP adeno-associated virus vector (control group)

2. Mouse model construction for cerebral hemorrhage test and AAV vector injection

The cerebral hemorrhage model adopts an IV type collagenase injection method, and the specific implementation process is as follows:

The IV type collagenase working solution is prepared, wherein the concentration of the IV type collagenase powder is more than or equal to 125CDU/mg. Deionized water was used to prepare a mother liquor having a final concentration of 3.75U/. Mu.l. The working solution used for mouse ICH modeling was 0.0375U/. Mu.l.

AAV vector injection for cerebral hemorrhage in mice 6-week-old male mice were selected for the experiments. The method comprises the steps of injecting sodium pentobarbital (45 mg/kg) into the abdominal cavity of a mouse for anesthesia, fixing the mouse by a brain stereotactic instrument, exposing the skull through a median incision, cleaning the tissue by using dry cotton and wiping the tissue dry, referencing the brain stereotactic map of the mouse, marking three AAV vector injection sites (x+2.0 mm, y+0.5 mm, z+2.9 mm) on the right brain of the mouse by taking a Bregma point as an origin, drilling the skull at the injection sites by using a cranium, injecting AAV vectors (titer is 6-9 multiplied by 10 ¹¹ vg/ml) into the brain parenchyma by using a microinjection pump at the injection speed of 0.1 mu L/min, injecting 1 mu L at each site, leaving the needle for 10min after the injection is finished, and removing the injection needle. ICH models were constructed 3 weeks after injection of mouse AAV vectors, which were divided into AAV-miR-124-3p positive virus groups and AAV-EGFP control virus groups.

The cerebral hemorrhage of mice is modeled by anesthetizing the mice with 0.3% pentobarbital, fixing the mice on a brain stereotactic apparatus, exposing the bregma and the postfontane of the mice. The glass electrode was sleeved into the programmable nanoliter microinjector tip. Mu.l (0.0375U) of type IV collagenase working fluid was aspirated using a microinjection pump. Leveling the brain plane of the mouse, namely aligning the tip of the glass electrode with the bregma point, and setting x, y and z as 0 point. The ICH molding site selects the position of striatum, and the coordinates are x: +2.0mm, y: +0.9mm and z: -2.9mm. After the glass electrode was entered to a prescribed depth, an injection procedure was started, and 1. Mu.l of type IV collagenase working solution was injected into the mouse striatum within 8 min. After the injection procedure was completed, the needle was left for 5min. Finally, slowly lifting the glass electrode to above the plane of the skull within 2 minutes. The boreholes were then sealed with bone wax and the skin of the mouse head was sutured. Mice were resuscitated in a resuscitation box at constant temperature (37 ℃) after the end of the surgery. After resuscitating the mice, the mice were transferred to clean-class mouse houses and observed daily.

3. Immunofluorescent staining of mouse brain sections:

After cerebral hemorrhage molding, the mice are anesthetized on the third day respectively, and perfusion materials are obtained, the specific method is that the mice are fixed on a perfusion table, the hearts of the mice are exposed by using scissors, a scalp needle connected with a perfusion pump is penetrated into the left ventricle of the mice, the right auricle of the hearts of the mice is sheared by using the scissors, physiological saline is used for perfusion firstly, then 4% PFA is used for perfusion, muscle spasm of the mice can be observed, and after 3min of perfusion, the perfusion is stopped. The mouse brain was removed. Placed in 4% pfa for 24h. The next day, 30% of sucrose was changed for sugar precipitation. After the brains of the mice were subjected to sugar precipitation, the surface sucrose was drained, transferred to an OCT embedding box for embedding, and put into a frozen microtome for slicing (30 μm/slice). The target area brain slice was transferred to a 24-well plate in a number of 8 slices per well. The brain slices were washed 3 times with 10min each using PBS. The corresponding antibodies were diluted with immunofluorescent primary antibody dilutions and added to 24-well plates at 4 ℃ overnight. The next day the primary antibody was removed and washed 3 times with PBS for 10min each. Diluted species corresponding to the fluorescent secondary antibodies are used, added into a 24-well plate and placed in a shaking table for reaction for 2 hours at room temperature. After that, the secondary antibody was removed, and washed 3 times with PBS for 10min each time. Brain pieces in 24 well plates were transferred to slides using toothpicks, DAPI solution was added dropwise, incubated for 20min at 37 ℃ in the dark, and washed 3 times with PBS for 10min each time.

The results are shown in FIG. 5, where EGFP-positive cells were tracked using various antibodies, including GFAP, neuN and Iba1, showing that over 90% of EGFP-positive cells were astrocytes. Furthermore, we compared the numbers of astrocytes, which were neurotoxic around hematoma in both groups of mice, and as shown in fig. 6, it was found that overexpression of miR-124-3p significantly inhibited astroneurotoxic activation.

4. Apoptosis staining of mouse brain sections

After cerebral hemorrhage molding, the materials are obtained in the third day, and the mice brain slices are washed and stained with primary antibody and secondary antibody, and the mice are subjected to immunofluorescence staining. After the secondary antibody was stained, TUNEL assay solutions were prepared, and 10. Mu.l of TdT enzyme, 90. Mu.l of fluorescent labeling solution, 100. Mu.l of TUNEL assay solution, and 100. Mu.l of TdT enzyme dilution were added to each well of the 24-well plate. Incubation was carried out at 37℃for 1h in the absence of light, after which washing was carried out twice with PBS for 10min each time. And finally sealing the sheet.

As shown in fig. 7, the results demonstrate a significant reduction in the number of hematoma marginal apoptotic cells in brain bleeding mice following AAV-miR-124-3p injection.

The AAV-miR-124-3p vector constructed in the embodiment can be used for preparing medicines for treating cerebral hemorrhage diseases.

The embodiments of the present invention have been described in detail with reference to the drawings, but the present invention is not limited to the above embodiments. Even if various changes are made to the present invention, including but not limited to various cell-specific promoters, various miRNA overexpression vectors, and the like, such changes are within the scope of the claims and the equivalents thereof, the changes are still within the scope of the present invention.

Claims

1. A central nervous system cell-specific miRNA expression vector for constructing a vector for overexpressing miRNA, characterized in that it comprises: an Ef1α promoter sequence, an inducible sequence, a Tre promoter, a fluorescent marker sequence, a tRNA sequence, a miRNA sequence, and an SV40 poly (A) termination signal sequence;

The Ef1α promoter sequence is shown in SEQ ID NO.6, and the inducible sequence is a doxycycline inducible activation sequence, specifically: 3G transactivator protein factor, the nucleotide sequence of which is shown in SEQ ID NO.7;

The Tre promoter sequence is shown in SEQ ID NO.1, the tRNA sequence is shown in SEQ ID NO.3, and the SV40 poly (A) termination signal sequence is shown in SEQ ID NO.5;

The gene sequence for overexpression of the specific miRNA is a tRNA-miRNA-tRNA combination sequence incorporated downstream of the fluorescent marker sequence.

2. The central nervous system cell-specific miRNA expression vector according to claim 1, characterized in that the miRNA is miR-124-3P, and the sequence is shown in SEQ ID NO.4.

3. The central nervous system cell-specific miRNA expression vector according to claim 1, characterized in that the fluorescent marker gene sequence is a doxycycline-driven EGFP fluorescent marker gene sequence, the sequence of which is shown in SEQ ID NO.2.

4. Use of the central nervous system cell-specific miRNA expression vector according to any one of claims 1 to 3 in improving miRNA overexpression, characterized in that under doxycycline induction, the Tre promoter drives the expression of the fluorescent marker gene, thereby driving the specific expression of the miRNA.

5. The use according to claim 4, characterized in that the miRNA specific expression is specific expression in vitro.

6. A central nervous system cell-specific miRNA overexpression adeno-associated virus vector, characterized in that it comprises: a cell-specific promoter, a fluorescent marker sequence, a tRNA-miRNA-tRNA sequence, wherein the fluorescent marker gene sequence is a doxycycline-driven EGFP fluorescent marker gene sequence, and the sequence is shown in SEQ ID NO.2;

The tRNA-miRNA-tRNA sequence includes a tRNA sequence and a miRNA sequence, and the tRNA sequence is shown as SEQ ID NO.3.

7. The central nervous system cell-specific miRNA overexpression adeno-associated virus vector according to claim 6, characterized in that the miRNA sequence is miR-124-3P, and the sequence is shown in SEQ ID NO.4.

8. Use of the central nervous system cell-specific miRNA overexpressing adeno-associated virus vector as claimed in claim 6 or 7 in preparing a transgenic experimental animal model.

9. The use according to claim 8, characterized in that the central nervous system cell-specific miRNA expression vector is miRNA expression in astrocytes;

The miRNA expression in astrocytes was used to prepare and validate the animal model for the treatment of cerebral hemorrhage.

10. Use of the central nervous system cell-specific miRNA overexpressing adeno-associated virus vector as claimed in claim 6 in the preparation of drugs for central nervous system neuroinflammation-related diseases.