LU505371B1 - Oxygen-regulated cell growth factor and application thereof - Google Patents
Oxygen-regulated cell growth factor and application thereof Download PDFInfo
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- LU505371B1 LU505371B1 LU505371A LU505371A LU505371B1 LU 505371 B1 LU505371 B1 LU 505371B1 LU 505371 A LU505371 A LU 505371A LU 505371 A LU505371 A LU 505371A LU 505371 B1 LU505371 B1 LU 505371B1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/50—Fibroblast growth factor [FGF]
- C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
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- Molecular Biology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Biomedical Technology (AREA)
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- General Engineering & Computer Science (AREA)
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Abstract
The invention provides an oxygen-regulated cell growth factor and application thereof. The gene sequence of the oxygen-regulated basic fibroblast growth factor is shown in SEQ ID No.1. The gene sequence of the oxygen-regulated cell growth factor (bFGF) is shown in SEQ ID No.1, and the method for modifying cells by using the gene sequence comprises the following steps: S1, providing the gene sequence SEQ ID No.1; S2, constructing the gene sequence SEQ ID No.1 as a recombinant adeno-associated virus vector; S3, fusing the constructed recombinant adeno-associated virus vector into stem cells to obtain an oxygen-regulated basic fibroblast growth factor modified and controlled bFGF gene expression vector, so as to realize controllable expression under specific conditions, and different expressions in normoxic and hypoxic environments can avoid many side effects such as tissue hyperproliferation and tumour risk caused by long-term over-expression of bFGF by traditional gene therapy vectors.
Description
OXYGEN-REGULATED CELL GROWTH FACTOR AND HUS0557
APPLICATION THEREOF
The invention belongs to the medical field, and relates to a method for modifying cells by growth factors, in particular to an oxygen-regulated cell growth factor and application thereof.
Spinal cord injury (SCI) is a serious trauma to the central nervous system, which has the characteristics of high incidence, high disability rate and high cost. It will form necrotic cysts, spinal cord tissue softening or glial scars, resulting in the loss of permanent sexual function of the spinal cord, seriously affecting people's quality of life and bringing heavy burdens to society and families.
In recent years, stem cell transplantation for the treatment of spinal cord injury has become a hot spot in the research of nerve trauma rehabilitation. Stem cells can be induced to differentiate into nerve cells and blend into the injured tissues of patients, and form synapse-like structures between neurons with normal nerve cells, which improves the function of the central nervous system to some extent and makes it a new means to treat spinal cord injury. At present, there are embryonic stem cells, neural stem cells, umbilical cord blood stem cells, bone marrow mesenchymal stem cells and so on, among which Shenjing stem cells are considered to be the most ideal transplant cells for the treatment of spinal cord injury.
Neural stem cells can be used for gene therapy of nervous system diseases through gene modification, expressing exogenous neurotransmitters, neurotrophic factors and metabolic enzymes. Transplanting this pluripotent stem cell into the injured part of the central nervous system can not only survive and integrate into the host tissue, differentiate into neurons, astrocytes and oligodendrocytes, and fill the cavity created by the injury, but also form a synapse-like structure with the host cell, so that the tissue structure of the injured area of the nerve tissue can be repaired and the function of the central nervous system can be restored. However, at present, the 17005871 research on the effect of differentiation and proliferation of neural stem cells on autophagy level in spinal cord microenvironment is still very scarce. The expression of bFGF is low in adult mammalian spinal cord tissue, but it is significantly up-regulated after spinal cord injury. Numerous studies have shown that bFGF can promote angiogenesis, axonal regeneration, nerve protection, tissue repair and endogenous neural stem cells, which plays an important role in the development and injury repair of the central nervous system. In the rat model of acute spinal cord injury, bFGF can inhibit the apoptosis of nerve cells, increase the blood supply in the ischemic area of spinal cord, significantly repair the injured area of spinal cord and improve the neurological dysfunction.
However, as a key nutritional factor in stem cell differentiation, bFGF has a short half-life in vivo and degrades rapidly after intravenous administration, so it is difficult to maintain its biological activity. In the application of spinal cord injury, it is also a biological molecule that is difficult to penetrate the blood-spinal barrier; At the same time, FGF receptors are widely distributed in various tissues and organs of the whole body, and the side effects of intravenous systemic administration also limit its application.
Aiming at the defects in the prior art, the invention provides a gene sequence of oxygen-regulated bFGF expression and a method for modifying cells by using the gene sequence.
The first aspect of the present invention is to provide an application of an oxygen-regulated basic fibroblast growth factor in preparing a product for treating spinal cord injury, wherein the gene sequence of the oxygen-regulated basic fibroblast growth factor (SHRE-CMVmp- bFGF) is SEQ ID No.1.
The second aspect of the present invention is to provide a gene sequence
SHRE-CMVmp (SEQ ID No.2).
The third aspect of the present invention is to provide a plasmid vector 17005871 constructed by using the gene sequence of SEQ ID No.1, which is any one or a combination of recombinant adeno-associated virus (AAV), recombinant lentivirus and other available recombinant plasmid vectors, preferably recombinant adeno-associated virus.
Optionally, the preparation method of the recombinant adeno-associated virus includes: transfection of the adeno-associated virus vector of SEQ ID No.1 with the pHelper plasmid in a culture medium containing cells, collection of transfected cells, and extraction to obtain the recombinant adeno-associated virus.
Optionally, the preparation method of the recombinant adeno-associated virus plasmid comprises the following steps:
S1, using plasmid pCMV-bFGF as a template, and performing PCR amplification to obtain gene sequence bFGF-poly À;
S2, designing primers, using plasmid pShuttle-SHRE-CMVmp-Luc as a template, and performing PCR amplification to obtain the gene sequence SHRE-CMVMP;
S3, pairing the gene sequence bFGF-polyA obtained in S2 with the gene sequence SHRE-CMVmp obtained in S3, and amplifying the gene sequence
SHRE-CMVmp-bfgf-polya by overlapping PCR;
S4, using the upstream primer SEQ ID No.4 and the downstream primer SEQ ID
No.5 as primers, and using the plasmid pShuttle-IRES-hrGFP-1 as a template for PCR amplification to obtain the IRES-hrGFP-polyA gene sequence; connecting the
IRES-hrGFP-polyA gene sequence with pENTR S'TOPO vector by topo recombination, transforming the connected product into TOP10 competent bacteria, and digesting the plasmid by Sall, and recovering the DNA fragment
IRES-HRGFP-Polya by gel;
SS, preparing the recombinant shuttle plasmid PShuttle/SHRE-CMVmp-bFGF of
SHRE-CMVMP-bFGF, performing Sall single enzyme digestion, performing CIAP dephosphorylation on pShuttle/SHRE-CMVmp-bFGF after Sall single enzyme digestion, ligating pshuttle/SHRE-CMVmp-bFGF after dephosphorylation by CIAP with rubber-recovered DNA fragment IRES-hrGFP- polyA at 4°C for 16 h,
transforming the ligated product into TOP10 competent bacteria, and extracting and 17005871 identifying the plasmid by multiple enzyme digestion to screen the correct recombinant plasmid PShuttle-SHRE-bFGF-IRES-HRGFP-Polya;
S6, using the upstream primer 5’-CGCGTAATACTGGTACCGAG-3’ (SEQ ID
No.8) and the downstream primer 5’- TCACTATTACACCCACTCGTGCAG-3’ (SEQ
ID No.7) as primers and the plasmid
PShuttle/SHRE-CMVMP-bFGF-IRES-HRGFP-Polya as a template, and performing
PCR amplification on the SHRE-bFGF-IRES-hrGFP-polyA sequence; connecting the PCR product SHRE-bFGF-IRES-hrGFP-polyA seamlessly with
AAV-CAG-MCS-3FLAG vector, transforming the obtained connection product into
TOP10 competent bacteria, extracting and identifying the plasmid by multiple enzyme digestion to screen the correct recombinant adeno-associated virus plasmid
AAV-5HRE-bFGF-IRES-hrGFP-polyA.
The fourth aspect of the present invention is to provide a method for modifying cells with bFGF, which comprises the following steps:
S1, providing a gene sequence of SEQ ID No.1;
S2, constructing the gene sequence SEQ ID No.l obtained in step 1 into a recombinant adeno-associated virus vector;
S3, the constructed recombinant adeno-associated virus vector is fused into stem cells to obtain stem cells modified by oxygen-regulated basic fibroblast growth factor.
As a preferred embodiment of the present invention, the stem cells are any one or a combination of totipotent stem cells, pluripotent stem cells and pluripotent stem cells, preferably one or more of totipotent stem cells and pluripotent stem cells, such as any one or a combination of bone marrow mesenchymal stem cells, neural stem cells, umbilical cord stem cells, olfactory ensheathing cells and adipose stem cells, and more preferably neural stem cells.
As a preferred embodiment of the present invention, S2 further includes packaging the constructed virus particles.
As a preferred embodiment of the present invention, 293TN cells are co-transfected with recombinant adeno-associated virus plasmid mixed with auxiliary plasmid by liposome transfection technology, the culture medium is changed for 12 h, 17005871 the fluorescence intensity is observed to be greater than 90% at 36 h, the culture medium is collected after 72 h, the supernatant is discarded after centrifugation at 5%104 rpm, and 50 ul PBS is added to resuspend the recombinant adeno-associated 5 virus.
As a preferred embodiment of the present invention, the method of fusing the constructed recombinant adeno-associated virus vector into stem cells in S3 is as follows: adding the packaged plasmid vector into the culture solution containing stem cells, assisting infection with 6 ug/mL polybrene (poly (polyamine)), and changing the culture medium after 24 h to obtain stem cells modified by bFGF.
As a preferred embodiment of the present invention, the modified stem cells are induced to express bFGF under hypoxia for 36 h, and the screening results are determined by fluorescence and WB detection.
As a preferred embodiment of the present invention, the culture solution of stem cells in S3 includes DMEM/F12 basal medium, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and B27.
As a preferred embodiment of the present invention, the hypoxic environment in
S3 is that the Oz concentration is less than 1%.
As a preferred embodiment of the present invention, the time of the hypoxia treatment in S3 is 6-72 h, preferably 12- 60 h, more preferably 24-48 h, such as 36 h.
As a preferred embodiment of the present invention, the plasmid vector is any one or a combination of recombinant adeno-associated virus, recombinant lentivirus and other available recombinant plasmid vectors, preferably recombinant adeno-associated virus.
As a preferred embodiment of the present invention, the recombinant adeno-associated virus is AAV-5he-bFGF, and the packaging method of the recombinant adeno-associated virus AAV-She-bFGF is as follows: the recombinant virus AAV-5he-bFGF and the recombinant adeno-associated virus packaging plasmid pHelper are co-transfected into 293 TN cells.
As a preferred embodiment of the present invention, the recombinant 17005870 adeno-associated virus is AAV-SHRE-bFGF.
The invention creatively proposes a method for treating spinal cord tissue injury by modifying neural stem cells with oxygen-regulated bFGF, which not only retains the advantages that bFGF inhibits nerve cell apoptosis, increases blood supply in ischemic areas of spinal cord, significantly repairs the injured area of spinal cord and improves neurological dysfunction, but also avoids the defects that bFGF is rapidly decomposed due to short half-life in vivo and intravenous injection in the prior art.
In the invention, the expression vector of oxygen-regulated bFGF gene is constructed for the first time, and the controllable expression of therapeutic genes under specific pathological conditions is realized. The vector can be effectively induced to start expressing bFGF in ischemia and hypoxia environment, and play a role in promoting vascular proliferation, nerve protection and tissue repair locally.
When new blood vessels are formed, collateral circulation is established, and local blood supply is gradually restored, the expression of bFGF is gradually decreased accordingly. In normal oxygen environment, only a very low level of bFGF expression is maintained, which can avoid many side effects such as excessive tissue proliferation and the risk of tumour caused by long-term over-expression of bFGF by traditional gene therapy vectors.
Hypoxia-regulated bFGF gene therapy combined with neural stem cell transplantation can complement each other and provide a new idea for the treatment of acute spinal cord injury. Promote the proliferation, migration and differentiation of neural stem cells into neurons through gene level regulation, W\while improving the survival rate after transplantation, it can also affect inflammatory reaction, oxidative stress, angiogenesis and other factors, improve the microenvironment of spinal cord injury area and promote repair.
Fig. 1 shows the effect of oxygen-regulated recombinant adeno-associated virus on the expression of hrGFP gene;
Fig. 2-1 shows the gene expression of recombinant adeno-associated virus in 17005870 hypoxic environment;
Fig. 2-2 shows the gene expression of recombinant adeno-associated virus
AAV-SHRE-bFGF at different times in hypoxic environment;
Fig. 2-3 shows the expression of hrGFP gene in PC12 cells infected by
AAV-SHRE-hrGFP;
Fig. 2-4 shows the expression of bFGF gene in PC12 cells infected by
AAV-SHRE-hrGFP;
Fig. 3-1 is the PI staining image of recombinant adeno-associated virus;
Fig. 3-2 shows the cell content of PI staining of recombinant adeno-associated virus;
Fig. 3-3 is Hoechst staining image of recombinant adeno-associated virus;
Fig. 3-4 shows the results of complete nuclear counting;
Fig. 4 shows the effect of recombinant adeno-associated virus on the differentiation of primary neural stem cells;
Fig. 5 is a fluorescence microscope photograph of the prepared neural stem cells;
Fig. 6 shows the protein expression level of bFGF under different conditions;
Fig. 7 shows the concentration of bFGF in cell fluid under hypoxia;
Fig. 8 is a fluorescence microscope photograph of the cell culture solution;
Fig. 9 shows the effect of bFGF gene protein on the treatment of spinal cord injury;
Fig. 10 1s a light microscope photograph of spinal cord injury tissue.
1 recombinant adeno-associated virus 1.1 preparation of recombinant adeno-associated virus plasmid
S1, using the recombinant plasmid pCMV-bFGF as a template, and performing
PCR amplification to obtain the gene sequence bFGF-poly À;
S2, using the upstream primer CGCGTAATACTGGTACCGAG (SEQ ID No.8) and the downstream primer GATCTGACGGTTACTAAAC (SEQ ID No.9) as primers,
and using the plasmid pShuttle-SHRE-CMVmp-Luc as a template to perform PCR 17005871 amplification to obtain the gene sequence SHRE-CMV MP;
S3, pairing the gene sequence bFGF-polyA obtained in S1 with the gene sequence SHRE-CMVmp obtained in S2, and amplifying by overlapping PCR to obtain the gene sequence SHRE-CMVMP-intron-bFGF-Polya;
S4, after the gene sequence SHRE-CMVmp-Intron-bFGF-polyA obtained in S3 is digested, it is inoculated with cytoplasmic AAV-MCS to obtain recombinant adeno-associated virus AAV-SHRE-bFGF.
The recombinant adeno-associated virus AAV-SHRE-hrGFP is prepared by similar preparation method. 1.2 Packaging recombinant adeno-associated virus 1.2.1 Preparation of plasmid and clone the foreign gene into a suitable vector.
The constructed virus vector, pHelper (carrying genes from adeno-associated virus) and pAAV-RC (carrying AAV replication and capsid genes) are extracted in large quantities. 1.2.2 AAV-293 cell culture 1.2.2.1 AAV-293 cell resuscitation 1) Preheating the culture medium; 2) Taking out the cell cryopreservation tube from the liquid nitrogen tank, quickly putting into a water bath containing 37°C water, shaking from time to time, thawing as soon as possible, wiping with 70% alcohol and disinfecting, and then moving to clean bench; 3) Sucking out the cell suspension and adding to 3 ml of culture medium, then adding 5 ml of culture medium for dilution and gently blowing away; 4) Collecting cells by centrifugation at 1000 rpm for 3 min; 5) Inoculating suspension cells with proper amount of culture. 1.2.2.2 Passage of AAV-293 cells (taking a 10 cm cell dish as an example) 1) Discarding the old culture medium, adding 10 ml of sterilized PBS, gently shaking, washing the cell growth surface, and then discarding the PBS;
2) Adding 1 ml of trypsin digestive juice and digesting for 1-2 min until the cells 17005870 become round and start to fall off;
3) Adding 2 ml of complete culture medium to stop digestion, and blowing the cells at the bottom of the cell dish into single cells;
4) Inoculating by centrifugation and resuspension counting as required, or directly dividing the mixed cells into new culture bottles and continuing the culture. 1.2.2.3 Cryopreservation of AAV-293 cells
1) Taking cells that have been cultured for 2-3 days and grow vigorously, digesting them and blowing them into single cells according to the method described inS1.222.
2) Centrifuging at 1000 rpm for 3 min to collect cells.
3) Adding a proper amount of PBS to resuspend the cells and counting them.
4) Collecting cells by centrifugation at1000 rpm for 3 min.
5) Resuspending the cells to 2x10°-5x10° cells /ml with cryopreservation medium, and then subpackaging the cell suspension into 2 ml frozen tubes and 1 ml frozen tubes.
6) Placing the tubule in the programmed cooling box, then putting the programmed cooling box into -80°C for freezing overnight, and transferring the tubule of frozen cells to liquid nitrogen for long-term storage the next day.
1.2.3 Inoculation of AAV-293 Cells Transfected with Cells
1) Inoculating AAV-293 cells in a 10 cm cell dish for later use.
2) Taking out the cell culture plate 1 h before transfection, removing the original cell culture medium, and adding 10 ml Opti-MEM medium.
3) Preparing the compound of transfection reagent and plasmid.
a.
Dissolving 32 pg of recombinant adeno-associated virus vector plasmid to be transfected (volume ratio:PHelper:PAAV-RC:shuttle plasmid=1:1:1) in Opti-MEM medium, with a total volume of 500 pl, mixing gently and letting stand for 5 min.
b.
Dissolving the Obio transfection reagent in Opti-MEM medium, with a total volume of 500 ul, mixing gently and letting stand for 5 min.
c.
Dropwise adding the diluent of Obio transfection reagent into the plasmid 17005871 diluent, gently mixing while adding, and standing at room temperature for 20 min, so that DNA and Obio transfection reagent can be fully combined to form a stable DNA- transfection complex. 4) Taking out the cell dish, adding the prepared DNA- transfection reagent complex into the cell culture plate, and changing the solution after transfection. 5) After 6 h, sucking the medium off, washing once with PBS, and adding with ml of fresh complete medium for culture. 1.2.4 Virus harvesting and purification 10 1) After transfection for 60 h, scraping the cells off with a cell scraper and collecting in a centrifuge tube. 2) Centrifuging the collected cells at 4°C for 5 min, and resuspending the cell lysate precipitate in 7 ml of 10 mM Tris-HCI buffer. 3) Storing the cell lysate in a dry ice/ethanol bath, and freeze-thawing for 8 times ina water bath at 37°C. 4) Using 10 mM Tris-HCI to fix the cell lysate to 21 ml. 5) Ultrasonic instrument for 7 min. 6) Adding 2 ml DNase I (3 mg/ml) into the cell lysate after ultrasound, and incubating at 37°C for 30 min. 7) Ultrasounding again for 7 min. 8) Adding 2.5 ml of 10% sodium deoxycholate and 2.1 ml 0.25% trypsin -EDTA, mixing them evenly, incubating at 37°C for 30 min, and placing them on ice for 20 min. 9) Adding 16.9 g CsCl, mixing well, incubating at 37°C for 20 min, and shaking continuously during incubation at 37°C until CsCl is completely dissolved. 10) Centrifuging 3000 g of the treated cell lysate for 30 min at 4°C, and carefully transferring the cell lysate to an ultracentrifuge tube (about 5 ml/tube). 11) Centrifuging at a speed of 53,000 rpm at 4°C for 30 h. 12) Using a homemade needle to collect the virus layer.
13) Dialysis at 4°C, changing the dialysing for 12-16 h, and using 4 L buffer 17005870 every time. 1.2.5 Titre determination
The titre of AAV is determined by quantitative PCR to detect the genome copy number of AAV vector in the genome to determine the number of virus particles of
AAV. 1) Prepare samples and standards, and dilute the standard quality granules and the sample to be tested to the original concentrations of 10°, 10° 107 and 10% in gradient; 2) Calculate the volume of the reaction manifold according to the reaction number (x) (make two holes for each gradient, and prepare one more for every 10 reactions); 3) Add 15 ul of reaction solution into each reaction hole, and then add 5 pl of template; 4) On the computer, set the annealing temperature to 60°C, and obtain the Ct value according to the standard operation, and calculate the number of copies in AAV samples. 2. Extraction of neural stem cells and identification of infection
Materials and reagents
Embryo of Sprague-Dawley rats at 14 - 16 days' gestation, DMEM/F12 medium, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), blue chain antibody, neuron specific enolase (NSE) antibody, glial fibrillary acidic protein (GFAP) antibody, mouse anti-rat monoclonal nestin, Hochest, Merge, B27, foetal calf serum (FCS) and stem cell culture medium; among them, GFAP, Hochest, Merge and nestin are all purchased from Sigma
Company in the United States, and B27 and FCS are all purchased from Gibco
Company.
The preparation method of stem cell culture medium is to add 20 pl EGF, 10 g/l or 20 g/l of bFGF, 20 mg/l of B27 and 1% of blue chain antibody to the basic liquid medium.
2.1 Extraction of neural stem cells 17005870
Rat embryos with gestational age of 14 - 16 days were isolated under aseptic conditions, meninges and superficial blood vessels were stripped under microscope, mechanically digested, cut into chyme, added with DMEM/F12 medium, and passed through a 200-mesh stainless steel screen. After washing with basic culture medium and centrifugation, the centrifuged rat nerve cells were resuspended with the prepared stem cell culture solution, and the living cells were counted to a concentration of 5x10° cells /ml with the improved abalone counting disk, and then 2 mL of treated rat nerve cells were sucked, inoculated into a 50mL glass cell culture bottle, and continuously cultured in an incubator with 37°C, 5% CO; and saturated humidity for 2 - 3 days, and the solution was changed by half. After 7-10 days of culture, according to the growth status and density of cells, half of the culture medium was sucked off, the stem cell balls were blown away by mechanical method, the cells were suspended again with stem cell culture solution, and then transferred to a new culture bottle to continue culture. 2.2 Identification of neural stem cells
Stain the cultured neural stem cells with Nestin and Hochest immunofluorescence, suck an appropriate amount of NSC balls, centrifuge them at a speed of 800 r/Smin for 5 min, add a culture solution containing 5% FCS and NSC to them, and inoculate them into a Petri dish coated with poly-lysine cover glass. After 4 days of culture, it was washed with PBS for 3 times, each time for 5 minutes, then fixed with 4% formaldehyde for 15 min at room temperature, and washed with PBS for 3 times, each time for 5 min. 0.2% rat TritonX-100 is added into it for 15min, washed with PBS for 3 times, Smin/ time, and sealed with goat serum sealing solution in a wet box at 37°C for 30 min. Add primary antibody Nestin (1:50), stay overnight at 4°C, and wash with PBS twice, 5 min each time, add secondary antibody (sheep anti-mouse IgG labelled with TRITC) diluted by rats at 1:100, incubate for 4 h at room temperature, wash with PBS for 5 min each time, and seal the tablets after washing. Observe and photograph the results under a fluorescent microscope, and the results are shown in Fig. 5.
2.3 bFGF modified cells 17005870
The packaged recombinant adeno-associated viruses AAV-She-bFGF and
AAV-/She-hrgfp are taken, and the supernatant of the two recombinant adeno-associated viruses is added to the primary neural stem cells of rats cultured in 24-well plates with 25MOI. After 18 h of culture, the recombinant adeno-associated viruses AAV-She-bFGF and AAV-/She-hrgfp are equally divided into two groups, respectively. One group continued to be cultured in an incubator with normal oxygen (O2 concentration of 21%) for 24 h, and the other group continued to be cultured in an incubator with low oxygen (O» concentration below 1%) for 24 h. After the culture, they are observed under a fluorescence microscope, and the results were shown in Fig. 1.
As can be seen from Fig. 1, in the normal oxygen environment, only a few cells express the hrGFP gene in the cultured recombinant adeno-associated viruses
AAV-SHRE-bFGF and AAV-5HRE- hrGFP. However, the recombinant adeno-associated viruses AAV-SHRE-bFGF and AAV-SHRE-hrGFP are cultured in hypoxia with Oz concentration below 1%. hrGFP gene expression is observed in more than 95% of cells, and the concentration of hrGFP gene in primary neural stem cells infected with AAV-SHRE-hrGFP gradually increased with the extension of induction time, reaching the peak at 24 h, and hrGFP/GAPDH=1.1 With the prolongation of induction time, the concentration of bFGF gene in primary neural stem cells infected by AAV-SHRE-bFGF gradually increased, reaching the peak at 24 h, with bFGF/GAPDH = 1.5.
After primary neural stem cells were infected with recombinant adeno-associated viruses AAV-SHRE-bFGF and AAV-SHRE-hrGFP for 18 h, they were treated under hypoxia for 1-24 h respectively, and the normoxic control group was set up. The expression of bFGF gene and hrGFP gene was detected by western blot. The results are shown in the following Figures 2-1 - 2-4:
As can be seen from Figures 2-1 - 2-4, in hypoxic environment, the expression of bFGF gene and hrGFP gene can be detected after 3 hours of induction, and it reaches the peak at 24 hours. However, under normal oxygen conditions, the gene expressions of bFGF and hrGFP were not detected for 3 hours, and only a trace of bFGF and 17005871 hrGFP were detected for 24 hours after induction. 2.4 Effect of recombinant adeno-associated virus on inhibiting apoptosis of primary neural stem cells
PC12 cells were infected with recombinant adenovirus vectors
AAV-SHRE-bFGF and AAV-SHRE-hrGFP for 18 hours, respectively, and then treated for 6 hours in a hypoxic environment with oxygen gas volume concentration lower than 1% to induce transgenic expression. Then, the conventional culture medium containing 15% serum was replaced by serum-free culture medium, and the culture was continued for 48 hours. AAV was evaluated by propidium iodide (PI) staining,
Hoechst staining and complete cell nuclear counting. The results of PI staining are shown in Figure 3-1 and Figure 3-2. The cell survival rate of AAV- SHRE-bFGF group is 76.1%, while the cell survival rates of control group (adding PC buffer salt) and AAV-SHRE-hrGFP group are 24.8% and 25.3% respectively.
Hoechst staining results are shown in Figure 3-3. PC12 cells infected with
AAV-SHRE-hrGFP have apoptosis characteristics such as nuclear condensation and nuclear fragmentation after serum deprivation, while the cell nucleus morphology of cells infected with AAV-SHRE-bFGF remains normal after serum deprivation. As shown in Figures 3 and 4, the number of complete nuclei in AAV-SHRE-bFGF group increased significantly compared with the control group (PBS added) and
AAV-SHRE-hrGFP group, with significant difference (p<0.01). 2.5 Effect of recombinant adeno-associated virus on differentiation of primary neural stem cells
Recombinant adeno-associated viruses AAV-SHRE-bFGF and
AAV-S5HRE-hrGFP infected primary neural stem cells for 18 h, respectively, and then treated them in a hypoxic environment with oxygen content less than 1% for 6 h to induce the transgenic expression of bFGF and hrGFP. Then, the cell culture medium containing 15% serum was replaced with the cell culture medium containing 1% serum, and the cell morphology was observed after 6 days of continuous culture, and the neuron-specific antigen NeuN was detected by immunofluorescence staining. The results are shown in Figure 4. Among them, most cell bodies in the primary neural 17005871 stem cell group infected by AAV-SHRE- bFGF were spindle-shaped or triangular, with protrusions of different numbers and lengths, and NeuN protein was positive by immunofluorescence staining. However, the primary neural stem cells in the control group (added PBS) and AAV-SHRE-hrGFP infected primary nerve trunk group remained round or oval, with no process growing out and negative NeuN staining. 3. Treatment of spinal cord injury by recombinant cells of oxygen-regulated bFGF gene
This test was divided into control group, model group and experimental group, all of which were 10-week-old SD rats weighing 280-320 g, with 32 rats in each group randomly assigned. Rats in blank group were exposed to T9-11 spinal cord.
Rats in model group exposed T9-11 spinal cord, and used arterial clamp (30 g, 2 min) to compress the spinal cord to cause acute spinal cord injury in SD rats. In the experimental group, rats were exposed to T9-11 spinal cord, and acute spinal cord injury was induced in SD rats by compressing the spinal cord with arterial vascular clamp (30 g, 2 min), and oxygen-regulated bFGF gene recombinant cells were continuously injected subcutaneously.
Basso-Beattie-Bresnahan (BBB) scoring method was used to evaluate the hind limb walking and limb activity of rats in each group, and the spinal cord function was evaluated on the 1st, 3rd and 7th day after operation, respectively. The results are shown in Figure 9.
As can be seen from Figure 9, one day after operation, the hind limb movement of the control group was normal, and the hind limb movement of the model group and the experimental group had different degrees of obstacles. One day after operation, there was no significant difference in BBB score between model group and experimental group. Three days after operation, the BBB score of the experimental group was significantly higher than that of the model group (P<0.05). Seven days after operation, it was obvious that the experimental group rats could walk in harmony with their front and rear limbs, while the model group rats still could not walk with load (P<0.05).
Seven days after operation, HE staining method was used to stain the tissue 17005871 slices of spinal cord injury in each group, and the results were observed under the light microscope. As shown in Figure 10, the tissue slices of spinal cord in the control group were dense and clear, and no obvious apoptosis and necrosis were found. HE staining showed that 7 days after operation, compared with the model group, the spinal cord tissue structure of SD rats in the experimental group was denser and clearer, cell loss was reduced, and the number of vacuoles in white matter was significantly reduced. These results indicate that oxygen-regulated bFGF gene recombinant cells can promote the repair of spinal cord injury in rats.
The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. It will be obvious to those skilled in the art that any equivalent modifications and substitutions are also within the scope of the invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the invention should be included in the scope of the invention.
Claims (6)
1. An application of an oxygen-regulated basic fibroblast growth factor in preparing a product for treating spinal cord injury, wherein the gene sequence of the oxygen-regulated basic fibroblast growth factor is shown in SEQ ID No.1.
2. A gene sequence shown in SEQ ID No.2.
3. A recombinant plasmid vector constructed by the gene sequence of SEQ ID
No.1, wherein the recombinant plasmid vector is any one or a combination of recombinant adeno-associated virus, recombinant lentivirus and other available plasmid vectors.
4. A preparation method of a recombinant plasmid vector, wherein the recombinant plasmid vector is a recombinant adeno-associated virus, and the preparation method of the recombinant adeno-associated virus comprises: transfecting the pAAV vector of SEQ ID No.1 and pHelper plasmid in a culture medium containing cells, and collecting and purifying the transfected cells to obtain the recombinant adeno-associated virus.
5. The preparation method according to claim 4, wherein the Obio transfection reagent is slowly added into the virus vector plasmid to be transfected, and is left stand for 5-50 min, so that the virus vector plasmid to be transfected is combined with the Obio transfection reagent to form a stable transfection complex, the transfection complex is added into a cell culture dish containing a culture medium, the fresh culture medium is replaced 18 h, and after 72 h, the transfected cells are collected and the recombinant adeno-associated virus is extracted; wherein the virus vector plasmid to be transfected is a mixed solution with the volume ratio of pAAV vector: pHelper plasmid = 1: 1.
6. The preparation method according to claim 4, comprising: S1, using plasmid pCMV-bFGF as a template, and performing PCR amplification to obtain gene sequence bFGF-poly À; S2, designing primers, using plasmid pShuttle-SHRE-CMVmp-Luc as a template, and performing PCR amplification to obtain the gene sequence SHRE-CMVMP;
S3, pairing the gene sequence bFGF-polyA obtained in S2 with the gene 17005871 sequence SHRE-CMVmp obtained in S3, and amplifying the gene sequence SHRE-CMVmp-bFGF-poly A by overlapping PCR; S4, using the upstream primer SEQ ID No.4 and the downstream primer SEQ ID
No.5 as primers, and using the plasmid pShuttle-IRES-hrGFP-1 as a template for PCR amplification to obtain the IRES-hrGFP-polyA gene sequence; connecting the IRES-hrGFP-polyA gene sequence with pENTR S'TOPO vector by topo recombination, transforming the connected product into TOP10 competent bacteria, and digesting the plasmid by Sall, and recovering the DNA fragment IRES-HRGFP-polyA by gel; SS, preparing the recombinant shuttle plasmid PShuttle/SHRE-CMVmp-bFGF of SHRE-CMVMP-bFGF, performing Sall single enzyme digestion, performing CIAP dephosphorylation on pShuttle/SHRE-CMVmp-bFGF after Sall single enzyme digestion, ligating pShuttle/SHRE-CMVmp-bFGF after dephosphorylation by CIAP with rubber-recovered DNA fragment IRES-hrGFP-polyA at 4°C for 16 h, transforming the ligated product into TOP10 competent bacteria, and extracting and identifying the plasmid by multiple enzyme digestion to screen the correct recombinant plasmid PShuttle-SHRE-bFGF-IRES-hrGFP-polyA; S6, using the upstream primer SEQ ID No.8 and the downstream primer SEQ ID
No.7 as primers and the plasmid PShuttle/SHRE-CMVmp-bFGF-IRES-hrGFP-poly A as a template, and performing PCR amplification on the SHRE-bFGF-IRES-hrGFP-polyA sequence; connecting the PCR product SHRE-bFGF-IRES-hrGFP-polyA seamlessly with AAV-CAG-MCS-3FLAG vector, transforming the obtained connection product into TOPI10 competent bacteria, extracting and identifying the plasmid by multiple enzyme digestion to screen the correct recombinant adeno-associated virus plasmid AAV-5HRE-bFGF-IRES-hrGFP-polyA.
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