CN115721735A - Application of CBS gene in preparation of medicine for treating diabetic retinopathy - Google Patents
Application of CBS gene in preparation of medicine for treating diabetic retinopathy Download PDFInfo
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- CN115721735A CN115721735A CN202211253920.5A CN202211253920A CN115721735A CN 115721735 A CN115721735 A CN 115721735A CN 202211253920 A CN202211253920 A CN 202211253920A CN 115721735 A CN115721735 A CN 115721735A
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Abstract
The invention discloses application of a CBS gene in preparation of a medicine for treating diabetic retinopathy, and animal experiments prove that the over-expression of the CBS gene can obviously delay cell aging, maintain the young state of cells, relieve vessel glycosylation, inhibit apoptosis of vascular endothelial cells and pericytes, has a good treatment effect on the diabetic retinopathy, provides a new idea, a new means and technical support for the research on the treatment of the diabetic retinopathy in the field, and has a good clinical application prospect.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an application of a CBS gene in preparation of a medicine for treating diabetic retinopathy.
Background
Diabetic Retinopathy (DR) is one of the most serious complications in Diabetic microangiopathy, and has become a globally important blinding eye disease at present. The onset of DR is primarily microvascular pathology, manifested by the formation of retinal microaneurysms, retinal hemorrhages, exudation, retinal neovascularization, vitreous blood and vitreoretinal proliferation, which ultimately can lead to irreversible loss of visual function in the patient. The generation of new blood vessels is a sign of Proliferative Diabetic Retinopathy (PDR) and is also an important reason for the visual prognosis. Clinically, the drug for interfering angiogenesis related molecules (targets) mainly through the whole retinal photocoagulation (PRP) and ocular injection achieves the effect of relieving the disease course of DR, but a treatment method for effectively controlling the occurrence and development of DR is still lacking. Previous studies have shown that the occurrence and development of DR are associated with high glucose environment, hypoxia, oxidative stress injury and related signaling pathways induced by chronic inflammatory processes, related gene expression and regulatory dysfunction, and although related researchers have conducted extensive studies in this field, the pathogenesis of DR is not completely understood.
Cystathionine- β -synthase (CBS) is one of the key enzymes in Homocysteine (Hcy) metabolism, and CBS is a Pyridoxal phosphate (PLP) dependent enzyme, which is a homotetramer composed of four identical subunits. Under the catalysis of vitamin B6, CBS can catalyze serine to react with Hcy to synthesize Cystathionine (Cystathionine, D- (P)). CBS plays an important role in the sulfation pathway of Hcy to cystathionine as a key enzyme affecting the level of Hcy, which is commonly used for the detection of homocysteine in blood in enzyme cycling methodologies. The reduced activity of CBS leads to Cystathionine- β -synthase deficiency (CBSD), which may cause Hyperhomocysteinemia (HHcy), and thus, damage to various organs and tissues, including eyes, bones, blood vessels, central nervous system, and the like. So far, no report related to the application of the CBS gene in the preparation of the medicine for treating diabetic retinopathy is available.
Disclosure of Invention
In order to make up for the technical blank existing in the field, the invention aims to provide the application of the CBS gene in preparing a medicament for treating diabetic retinopathy.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first aspect of the present invention provides the use of the CBS gene in the manufacture of a medicament for the treatment and/or prevention of angiogenic eye diseases.
Further, the amino acid sequence coded by the CBS gene is shown as SEQ ID NO. 1;
preferably, the nucleotide sequence of the CBS gene is shown as SEQ ID NO. 2;
preferably, the angiogenic eye disease comprises diabetic retinopathy, diabetic macular edema, age-related macular degeneration, macular edema following retinal vein occlusion, vascular retinopathy, choroidal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, corneal neovascularization;
more preferably, the angiogenic eye disease is diabetic retinopathy.
As used herein, "treating and/or preventing" refers to preventing, reversing, alleviating, inhibiting the progression of the disorder or condition to which the term applies, or one or more symptoms of such disorder or condition, treating a disease or condition including ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected, e.g., as used herein "treating and/or preventing an angiogenic eye disease" includes one or more of: (1) prevention of the development of angiogenic eye diseases; (2) inhibiting the development of angiogenic eye diseases; (3) curing angiogenic eye diseases; (4) alleviating symptoms associated with a patient with angiogenic eye disease; (5) reducing the severity of angiogenic eye disease; (6) prevention of recurrence of angiogenic eye disease.
In a second aspect of the invention, a recombinant viral vector is provided.
Further, the recombinant viral vector comprises a CBS gene;
preferably, the amino acid sequence coded by the CBS gene is shown as SEQ ID NO. 1;
preferably, the nucleotide sequence of the CBS gene is shown as SEQ ID NO. 2;
preferably, the recombinant viral vector includes adeno-associated virus (AAV) vector, adenoviral vector, helper-dependent adenoviral vector, retroviral vector, herpes simplex viral vector, lentiviral vector, poxvirus vector, japanese hemagglutinating virus-liposome (HVJ) complex vector, moloney murine leukemia viral vector, HIV-based viral vector;
more preferably, the recombinant viral vector is an adeno-associated viral vector;
most preferably, the adeno-associated viral vector comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and/or hybrids thereof;
most preferably, the recombinant viral vector is a recombinant AAV1 vector;
most preferably, the recombinant virus vector is an infectious virus particle AAV1-SFFV-CBS-T2A-mCherry obtained by co-transfecting a plasmid vector expressing a CBS gene and AAV1 into a cell and then harvesting;
most preferably, the cell is a human embryonic kidney cell line;
most preferably, the human embryonic kidney cell lines include 293FT, 293T, 293A;
most preferably, the human embryonic kidney cell line is 293FT;
most preferably, the plasmid vector for expressing the CBS gene is a pSFFV-CBS-T2A-mCherry plasmid vector;
most preferably, the pSFFV-CBS-T2A-mCherry plasmid vector is obtained by inserting a CBS gene sequence between pSFFV and T2A of pSFFV-T2A-mCherry plasmid.
In a third aspect, the present invention provides a method for producing the recombinant viral vector of the second aspect of the present invention.
Further, the method comprises the steps of:
(1) Constructing a pSFFV-CBS-T2A-mCherry plasmid vector;
(2) Co-transfecting the pSFFV-CBS-T2A-mCherry plasmid vector constructed in the step (1) and AAV1 into a cell, and then culturing the cell;
(3) Collecting cell supernatant to obtain infectious virus particles, namely recombinant virus vector AAV1-SFFV-CBS-T2A-mCherry;
preferably, the pSFFV-CBS-T2A-mCherry plasmid vector in the step (1) is constructed by inserting the CBS gene sequence between pSFFV and T2A of the pSFFV-T2A-mCherry plasmid;
preferably, the cells described in step (2) are human embryonic kidney cell lines;
more preferably, the human embryonic kidney cell lines comprise 293FT, 293T, 293A;
most preferably, the human embryonic kidney cell line is 293FT;
preferably, the culture conditions in step (2) are 5% CO 2 、37℃；
Preferably, the collection of cell supernatant in step (3) is started 48h after transfection.
In a fourth aspect, the present invention provides a gene therapy drug for the treatment and/or prevention of angiogenic eye diseases.
Further, the gene therapy drug comprises the recombinant viral vector according to the second aspect of the present invention;
preferably, the administration mode of the gene therapy medicine comprises subretinal injection of the recombinant virus vector, intravitreal injection of the recombinant virus vector;
more preferably, the gene therapy drug is administered by intravitreal injection of the recombinant viral vector;
preferably, the angiogenic eye disease comprises diabetic retinopathy, diabetic macular edema, age-related macular degeneration, macular edema following retinal vein occlusion, vascular retinopathy, choroidal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, corneal neovascularization;
more preferably, the angiogenic eye disease is diabetic retinopathy.
In a fifth aspect, the present invention provides a pharmaceutical composition for the treatment and/or prevention of angiogenic eye diseases.
Further, the pharmaceutical composition comprises a gene therapy agent according to the fourth aspect of the present invention;
preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant;
preferably, the pharmaceutical composition further comprises an additional therapeutic agent;
more preferably, the other therapeutic agent comprises a hypoglycemic agent, an anticoagulant agent, a vasoprotective agent;
most preferably, the hypoglycemic drugs comprise sulfonylureas drugs, meglitinides drugs, DPP-4 inhibitors, biguanides drugs, thiazolidinediones drugs, alpha-glycosidase inhibitors;
most preferably, the anticoagulant drug comprises aspirin, boli vitamin, heparin, warfarin;
most preferably, the vasoprotective drugs include lipid lowering drugs, antiplatelet drugs;
preferably, the angiogenic eye disease comprises diabetic retinopathy, diabetic macular edema, age-related macular degeneration, macular edema following retinal vein occlusion, vascular retinopathy, choroidal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, corneal neovascularization;
more preferably, the angiogenic eye disease is diabetic retinopathy;
in particular embodiments of the invention, the additional therapeutic agents include, but are not limited to: any presently disclosed agent for treating and/or preventing angiogenic eye diseases, and/or any presently disclosed agent for aiding in the treatment and/or prevention of angiogenic eye diseases, wherein the pharmaceutical composition comprising the combination of the above agent and the gene therapy agent of the fourth aspect of the present invention is within the scope of the present invention, and the combination of the agent and the gene therapy agent of the fourth aspect of the present invention can be used simultaneously, or sequentially (the combination of the agent and the gene therapy agent of the fourth aspect of the present invention, or the combination of the agent and the gene therapy agent of the fourth aspect of the present invention, and the administration mode can be the same or different, and the administration modes include, but are not limited to: intravitreal, subconjunctival, subretinal, intraocular, oral, rectal, vaginal, parenteral, pulmonary, intranasal, buccal, suppository, aerosol, or any combination thereof.
The "pharmaceutically acceptable carriers and/or adjuvants" mentioned herein are described in detail in Remington's pharmaceutical Sciences (19 th ed., 1995) and are used as needed to aid in the stability of the formulation or to enhance the activity or its bioavailability or to produce an acceptable taste or odor upon oral administration, and the formulations which may be used in such pharmaceutical compositions may be in the form of their original compounds themselves, or optionally in the form of their pharmaceutically acceptable salts. Preferably, the pharmaceutically acceptable carrier and/or adjuvant includes pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the mode of administration and the designed dosage form. Preferably, the pharmaceutical composition is in any pharmaceutically acceptable dosage form, including but not limited to: at least one of tablet, capsule, injection, granule, suspension and solution. Preferably, the appropriate dose of the pharmaceutical composition can be prescribed in various ways depending on factors such as formulation method, administration mode, age, body weight, sex, morbid state, diet, administration time, administration route, excretion rate and response sensitivity of the patient, and the skilled physician can easily determine the prescription and the dose prescribed to be effective for the desired treatment in general.
The sixth aspect of the present invention provides a use of the gene therapy drug according to the fourth aspect of the present invention for the production of a therapeutic and/or prophylactic agent for angiogenic eye diseases.
Further, the angiogenic eye disease includes diabetic retinopathy, diabetic macular edema, age-related macular degeneration, macular edema following retinal vein occlusion, vascular retinopathy, choroidal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, corneal neovascularization; in a specific embodiment of the present invention, the angiogenic eye disease refers to an ocular disease associated with angiogenesis, including but not limited to the angiogenic eye disease described above.
Preferably, the angiogenic eye disease is diabetic retinopathy.
The seventh aspect of the present invention provides the use of the pharmaceutical composition according to the fifth aspect of the present invention in the manufacture of a medicament for the treatment and/or prevention of angiogenic eye disorders.
Further, the angiogenic eye disease includes diabetic retinopathy, diabetic macular edema, age-related macular degeneration, macular edema following retinal vein occlusion, vascular retinopathy, choroidal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, corneal neovascularization;
preferably, the angiogenic eye disease is diabetic retinopathy.
The eighth aspect of the present invention provides the use of the CBS gene in the preparation of a cell senescence inhibitor, a vascular glycosylation inhibitor, a vascular endothelial apoptosis inhibitor and/or a pericyte apoptosis inhibitor.
Further, the amino acid sequence coded by the CBS gene is shown as SEQ ID NO. 1;
preferably, the nucleotide sequence of the CBS gene is shown in SEQ ID NO. 2.
The ninth aspect of the present invention provides the use of the gene therapy medicament according to the fourth aspect of the present invention in the preparation of a cell senescence inhibitor, a vascular glycosylation inhibitor, a vascular endothelial apoptosis inhibitor and/or a pericyte apoptosis inhibitor.
The tenth aspect of the present invention provides the use of the pharmaceutical composition according to the fifth aspect of the present invention in the preparation of a cell senescence inhibitor, a vessel glycosylation inhibitor, a vessel endothelial apoptosis inhibitor and/or a pericyte apoptosis inhibitor.
In the specific embodiment of the invention, experiments prove that the over-expression of the CBS gene can obviously delay cell aging and maintain the young state of the cell; the over-expression of the CBS gene can obviously relieve the blood vessel glycosylation; can obviously inhibit the apoptosis of vascular endothelial cells and pericytes; the verification results show that the over-expression CBS gene has a good treatment effect on diabetic retinopathy.
In addition, the invention also provides a method for treating and/or preventing diabetic retinopathy.
Further, the method comprises administering an effective amount of the recombinant viral vector of the second aspect of the present invention, the gene therapy drug of the fourth aspect of the present invention, and/or the pharmaceutical composition of the fifth aspect of the present invention to a subject in need thereof (e.g., diabetic retinopathy patient).
An "effective amount," as used herein, refers to an amount that has a therapeutic effect or is required to produce a therapeutic effect in a subject being treated. For example, a therapeutically or pharmaceutically effective amount of a drug refers to the amount of drug required to produce the desired therapeutic effect, which can be reflected by the results of clinical trials, model animal studies, and/or in vitro studies. The pharmaceutically effective amount will depend on several factors including, but not limited to, the characteristics of the subject (e.g., height, weight, sex, age and history of administration), and the severity of the disease.
In a specific embodiment of the present invention, the recombinant viral vector according to the second aspect of the present invention, the gene therapy medicament according to the fourth aspect of the present invention and/or the pharmaceutical composition according to the fifth aspect of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, and the appropriate dosage will be determined by clinical trials. The precise amount of the effective amount of the recombinant viral vector according to the second aspect of the present invention, the gene therapy drug according to the fourth aspect of the present invention and/or the pharmaceutical composition according to the fifth aspect of the present invention to be administered can be determined by a clinician, who can take into account the age, weight, severity of diabetes, severity of retinopathy and individual differences in the condition of the patient (subject), and can be administered using administration means well known to those skilled in the art. Optimal dosages and treatment regimens for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.
In particular embodiments of the invention, administration may be carried out in any convenient manner, including, for example, by intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via the carotid artery), intramuscular, intranasal, intraocular, topical or intradermal administration or spinal cord or brain delivery to effect administration of the recombinant viral vectors, gene therapy agents or pharmaceutical compositions of the invention. Aerosol formulations such as nasal spray formulations comprise a purified aqueous or other solution of the active agent together with a preservative and an isotonicity agent. Such formulations need to be adjusted to pH and isotonic conditions compatible with the conjunctiva of the eye for intraocular administration. Among the preparations for parenteral administration are sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, or fixed oils. Intravenous vehicles include liquid and nutritional supplements, electrolyte supplements, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention provides the application of the CBS gene in preparing the medicine for treating diabetic retinopathy for the first time, and provides a recombinant virus vector, a gene therapy medicine and a pharmaceutical composition for treating and/or preventing the diabetic retinopathy.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a diagram of the structure of pSFFV-CBS-T2A-mCherry plasmid.
FIG. 2 is a graph showing the results of the flow analysis of HUVEC cell senescence in the experimental group and the control group, wherein the control group: HUVEC cells not transformed with CBS gene, CBS group: HUVEC cells transformed with CBS gene;
fig. 3 is a graph of results of fluorescein imaging of rat eyes in a model group and an experimental group before administration, wherein the model control group: non-dosed diabetic retinal vasculopathy rats, experimental group: diabetic retinal vasculopathy rats injected intravitreally with AAV 1-CBS;
fig. 4 is a graph showing staining results of model control group and experimental group ZDF rat eyeball PAS at 3 weeks after administration, wherein the model control group: non-dosed diabetic retinal vasculopathy rats, experimental group: diabetic retinopathy rats injected intravitreally with AAV 1-CBS.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods in which specific conditions are not specified, and the detection is usually carried out according to conventional conditions or according to conditions recommended by the manufacturers. Reagents, materials and the like used in the following examples are commercially available unless otherwise specified.
Example 1 vector construction
1. Experimental materials and experimental methods
(1) pSFFV-OSK-T2A-mCherry plasmid (purchased from general organisms) is taken as a skeleton vector for gene expression;
(2) Construction of pSFFV-CBS-T2A-mCherry vector: synthesizing a CBS coding cDNA sequence, inserting the CBS coding cDNA sequence into a pAAV-SFFV-OSK-T2A-mCherry vector (namely the pSFFV-OSK-T2A-mCherry plasmid) through homologous recombination, replacing an OSK gene, transforming the OSK gene into stbl3 competent cells, picking out a single clone, and sequencing to ensure that the T2A-mCherry sequence has no mutation after insertion.
(3) The amino acid sequence of CBS is shown in SEQ ID NO. 1, and the nucleotide sequence of CBS is shown in SEQ ID NO. 2.
Amino acid sequence of CBS: <xnotran> MPSETPQAEVGPTGCPHRSGPHSAKGSLEKGSPEDKEAKEPLWIRPDAPSRCTWQLGRPASESPHHHTAPAKSPKILPDILKKIGDTPMVRINKIGKKFGLKCELLAKCEFFNAGGSVKDRISLRMIEDAERDGTLKPGDTIIEPTSGNTGIGLALAAAVRGYRCIIVMPEKMSSEKVDVLRALGAEIVRTPTNARFDSPESHVGVAWRLKNEIPNSHILDQYRNASNPLAHYDTTADEILQQCDGKLDMLVASVGTGGTITGIARKLKEKCPGCRIIGVDPEGSILAEPEELNQTEQTTYEVEGIGYDFIPTVLDRTVVDKWFKSNDEEAFTFARMLIAQEGLLCGGSAGSTVAVAVKAAQELQEGQRCVVILPDSVRNYMTKFLSDRWMLQKGFLKEEDLTEKKPWWWHLRVQELGLSAPLTVLPTITCGHTIEILREKGFDQAPVVDEAGVILGMVTLGNMLSSLLAGKVQPSDQVGKVIYKQFKQIRLTDTLGRLSHILEMDHFALVVHEQIQYHSTGKSSQRQMVFGVVTAIDLLNFVAAQERDQK (SEQ ID NO: 1); </xnotran>
Nucleotide sequence of CBS: <xnotran> ATGCCTTCTGAGACCCCCCAGGCAGAAGTGGGGCCCACAGGCTGCCCCCACCGCTCAGGGCCACACTCGGCGAAGGGGAGCCTGGAGAAGGGGTCCCCAGAGGATAAGGAAGCCAAGGAGCCCCTGTGGATCCGGCCCGATGCTCCGAGCAGGTGCACCTGGCAGCTGGGCCGGCCTGCCTCCGAGTCCCCACATCACCACACTGCCCCGGCAAAATCTCCAAAAATCTTGCCAGATATTCTGAAGAAAATCGGGGACACCCCTATGGTCAGAATCAACAAGATTGGGAAGAAGTTCGGCCTGAAGTGTGAGCTCTTGGCCAAGTGTGAGTTCTTCAACGCGGGCGGGAGCGTGAAGGACCGCATCAGCCTGCGGATGATTGAGGATGCTGAGCGCGACGGGACGCTGAAGCCCGGGGACACGATTATCGAGCCGACATCCGGGAACACCGGGATCGGGCTGGCCCTGGCTGCGGCAGTGAGGGGCTATCGCTGCATCATCGTGATGCCAGAGAAGATGAGCTCCGAGAAGGTGGACGTGCTGCGGGCACTGGGGGCTGAGATTGTGAGGACGCCCACCAATGCCAGGTTCGACTCCCCGGAGTCACACGTGGGGGTGGCCTGGCGGCTGAAGAACGAAATCCCCAATTCTCACATCCTAGACCAGTACCGCAACGCCAGCAACCCCCTGGCTCACTACGACACCACCGCTGATGAGATCCTGCAGCAGTGTGATGGGAAGCTGGACATGCTGGTGGCTTCAGTGGGCACGGGCGGCACCATCACGGGCATTGCCAGGAAGCTGAAGGAGAAGTGTCCTGGATGCAGGATCATTGGGGTGGATCCCGAAGGGTCCATCCTCGCAGAGCCGGAGGAGCTGAACCAGACGGAGCAGACAACCTACGAGGTGGAAGGGATCGGCTACGACTTCATCCCCACGGTGCTGGACAGGACGGTGGTGGACAAGTGGTTCAAGAGCAACGATGAGGAGGCGTTCACCTTTGCCCGCATGCTGATCGCGCAAGAGGGGCTGCTGTGCGGTGGCAGTGCTGGCAGCACGGTGGCGGTGGCCGTGAAGGCCGCGCAGGAGCTGCAGGAGGGCCAGCGCTGCGTGGTCATTCTGCCCGACTCAGTGCGGAACTACATGACCAAGTTCCTGAGCGACAGGTGGATGCTGCAGAAGGGCTTTCTGAAGGAGGAGGACCTCACGGAGAAGAAGCCCTGGTGGTGGCACCTCCGTGTTCAGGAGCTGGGCCTGTCAGCCCCGCTGACCGTGCTCCCGACCATCACCTGTGGGCACACCATCGAGATCCTCCGGGAGAAGGGCTTCGACCAGGCGCCCGTGGTGGATGAGGCGGGGGTAATCCTGGGAATGGTGACGCTTGGGAACATGCTCTCGTCCCTGCTTGCCGGGAAGGTGCAGCCGTCAGACCAAGTTGGCAAAGTCATCTACAAGCAGTTCAAACAGATCCGCCTCACGGACACGCTGGGCAGGCTCTCGCACATCCTGGAGATGGACCACTTCGCCCTGGTGGTGCACGAGCAGATCCAGTACCACAGCACCGGGAAGTCCAGTCAGCGGCAGATGGTGTTCGGGGTGGTCACCGCCATTGACTTGCTGAACTTCGTGGCCGCCCAGGAGCGGGACCAGAAG (SEQ ID NO: 2). </xnotran>
2. Results of the experiment
The pSFFV-CBS-T2A-mCherry vector is successfully constructed by adopting the method, and the structural schematic diagram of the pSFFV-CBS-T2A-mCherry vector is shown in figure 1.
Example 2 adeno-associated virus packaging
1. Adeno-associated virus packaging process
(1) Cell inoculation: inoculating 2X 10T 175 adherent cell culture bottle 7 293FT cells were cultured in 30mL of DMEM medium containing 10% FBS at 37 ℃ with 5% CO 2 Culturing overnight in an incubator for 16-24h, and then performing transfection.
(2) Cell transfection: when the cell growth confluency reaches 80% -90%, preparing to convertAnd (6) dyeing. The transfection system is shown in Table 1 below. Mixing solution A and B, and standing at room temperature for 5min. And dropwise adding the solution B into the solution A while shaking, and standing at room temperature of 22-26 deg.C for 20min. Dropwise adding into a culture dish, shaking gently, and adding 5% CO 2 And cultured overnight in an incubator at 37 ℃.
TABLE 1 transfection System
(3) And (3) transfection and liquid change: after 16-18h, the medium containing the transfection reagent was removed and 30mL of DMEM medium containing 10% FBS, 5% CO 2 And then the culture was continued at 37 ℃.
(4) Harvesting the virus for the first time: 48 hours after the start of transfection, the cell supernatant was harvested, transferred to a 50mL centrifuge tube, filtered through a 0.45 μm filter and stored at 4 ℃. Cells were added to 30mL DMEM medium containing 10% FBS, 5% CO 2 The culture was continued at 37 ℃.
(5) And (3) harvesting the virus for the second time: cell supernatants were harvested, transferred to 50mL centrifuge tubes, filtered through 0.45 μm filter and stored at 4 ℃. The cells were treated with 10% disinfectant (84 disinfectant) and discarded.
(6) And (3) concentrating the virus: the collected adeno-associated virus component was filtered by a 0.45 μm filter to remove bacterial contamination, and the filtered component was mixed with PEG8000 at a volume ratio of 3.
(7) Incubate at 4 ℃ for 30min or overnight.
(8) Centrifugation was carried out at 1500g for 45min at 4 ℃ and a white precipitate was observed at the bottom of the tube.
(9) The supernatant was carefully aspirated without destroying the white precipitate.
(10) Resuspending the pellet with an appropriate volume of adeno-associated virus preservative solution, performing qPCR assay on the virus to determine viral titer and separately packaging and preserving the harvested adeno-associated virus at-80 ℃.
2. Viral titer determination
qPCR detection is carried out on the adeno-associated virus to determine the virus titer, and the specific determination method comprises the following steps:
(1) Diluting the standard plasmid of virus to be detected by 10-fold gradient, and selecting 10 11 -10 7 copies/. Mu.L was used as a standard for the experimental standard curve.
(2) According to
Top Green qPCR Supermix instructions and fluorescent quantitative PCR instrument reaction systems require the preparation of qPCR reaction reagents, and the reaction systems are shown in Table 2 below.
TABLE 2 reaction System
| Components | Volume of |
| Forward Primer(10μM) | 1μL |
| Reverse Primer(10μM) | 1μL |
| 2xTransStar Top/Tip Green qPCR SuperMix | 10μL |
| Nuclease-free Water | 7μL |
| DNA | 1μL |
| Total volume | 20μL |
The sequence of the forward primer is as follows: 5 'GGAGTTGTGGCCCGTTGTT-3' (SEQ ID NO: 3);
the reverse primer sequence is as follows: 5 'GAGCCCCTGTCCAGCAGCAGC-3' (SEQ ID NO: 4).
(3) And (3) subpackaging the reaction mixed solution into 8-connecting tubes by taking 19 mu L/hole (20 mu L system), and sequentially adding 1 mu L/hole reaction standard substance and a sample to be detected into the holes.
(4) Instantly separating the 8-connection tube added with the reaction solution, slightly shaking and uniformly mixing, and placing the mixture after the instantaneous separation
96 In the System instrument, a reaction program was set to run, which is shown in table 3 below.
TABLE 3 reaction procedure
(5) After the reaction program is finished, the detection 8 connecting pipe is taken out and discarded, and data is copied out for storage and analysis.
3. Results of the experiment
The results showed that AAV1-SFFV-CBS-T2A-mCherry virus titer was 1.195X 10 after transferring CBS into adeno-associated virus 12 pfu/mL。
Example 3 HUVEC cell culture, viral infection, living marker of senescent cells and flow analysis
1. Experimental Material
The experimental materials referred to in this example are shown in table 4 below.
TABLE 4 materials of the experiments
| Name(s) | Source company | Goods number |
| Endothelial cell culture medium | ScienCell | BNCC342473 |
| Human umbilical vein endothelial cell HUVEC | ScienCell | #8000 |
| Neon TM Initial set of transfection system | Thermo Fisher | MPK5000S |
| DPBS (calcium and magnesium free) | Gibco | 2380005 |
| 0.25% pancreatin | Cytiva | J210027 |
| C12FDG reactive dyes | Abcam | 138777-25-0 |
| DMSO | Sigma | D2650 |
| Bafilomycin A1 | Abcam | 88899-55-2 |
2. Experimental methods
(1) HUVEC cell resuscitation
(1) The 1 vial was removed from the liquid nitrogen tank and immediately shaken continuously in water at 37 ℃ to completely thaw the frozen cell suspension.
(2) Once the cells in the cryopreservation tube are completely thawed (liquid), the cells are immediately taken out of the hot water bucket, the surface of the cryopreservation tube is completely disinfected by 75% of alcohol, and the frozen cryopreservation tube is placed in an ultra-clean workbench. When the frozen tube is about to melt, the centrifuge tube containing the endothelial culture medium preheated to 37 ℃ is sterilized by alcohol and then is placed in an ultra-clean workbench.
(3) Taking out the cell suspension in the freezing tube under strict aseptic operation conditions, adding the cell suspension into a 15mL centrifugal tube of a preheated E8 culture medium, gently blowing and beating for 2-3 times, centrifuging at 1300rpm for 5min, and discarding the supernatant after the centrifugation is finished.
(4) Adding endothelial culture medium, gently blowing and beating for 2-3 times, transferring cells to cell culture flask, and adding culture solution. Placing the culture flask in CO 2 Culturing in an incubator.
(2) HUVEC digestion passage
(1) The well plate/flask to be passaged was taken out of the incubator, the supernatant was aspirated and washed once with DPBS.
(2) Adding trypsin, spreading to the bottom of the bottle, removing trypsin, incubating in an incubator for 4-5min, and observing under a mirror to make the cells contract and become round and disperse.
(3) Gently tapping the culture bottle/plate to remove the cell wall, gently blowing and beating for several times with a gun head, adding endothelial culture medium to stop digestion, transferring the cell to a new culture bottle after blowing and beating, adding endothelial culture medium, standing at 37 deg.C and 5% CO 2 The incubator of (2) for cultivation.
(4) And (4) performing liquid changing operation every three days, and performing passage when the cell confluence is about 70-80%.
(3) HUVEC cell electrotransformation
HUVEC cells of generation 6, 20 ten thousand cells 1. Mu.g plasmid;
and (3) electrotransfer conditions: the voltage is 1350v, the pulse time is 30ms, and the pulse number is 1;
after 3 days of electrotransfer, the liquid was changed, and after 9 days, the aged cells were counted.
(4) In vivo labelling and flow analysis of senescent cells
Treating the cells with 100 mu M Bafilomycin for 1h, inhibiting the activity of beta-galactosidase in lysosomes, adding an active fluorescent substrate dye of the beta-galactosidase into an SH-SY5Y culture medium, culturing for 2h at 37 ℃, washing twice with DPBS, digesting with pancreatin, collecting the cells, and performing flow cytometry analysis. Comparison of the effects of over-expressed genes on reversal of senescence was made from mean fluorescence intensity and the proportion of cells stained less.
3. Results of the experiment
The results showed that the non-senescent cell fraction after CBS was transferred into HUVEC was 33.59% (1-66.41% = 33.59%), which is significantly higher than 5.99% (1-94.01% = 5.99%) of the control group (HUVEC cells not transferred with CBS gene) (see FIG. 2), and the results showed that over-expression of CBS gene could delay senescence of HUVEC cells and maintain the young state of cells.
Example 4 treatment of diabetic retinopathy with AAV1-SFFV-CBS rat model
1. Experimental materials
The experimental materials referred to in this example are shown in table 5 below.
TABLE 5 materials of the experiments
2. Experimental methods
(1) Model for diabetic retinopathy
Feeding ZDF rats with high fat for 2 months, measuring blood sugar once per month, selecting the rats with blood sugar values above 17mg/L for fluorescein radiography observation, and observing whether vascular leakage occurs. Rats with leakage were selected for treatment by intravitreal injection of AAV1-CBS (AAV 1-SFFV-CBS-T2A-mChery virus).
(2) Intravitreal injection for treating diabetic retinopathy
After confirming that the molding was successful, the high fat feeding was continued for two weeks, and then 5. Mu.L of the titer was 10 9 pfu/. Mu.L AAV1-CBS (AAV 1-SFFV-CBS-T2A-mCherry virus) was injected into the vitreous cavity, taking care not to touch the lens during the procedure. After injection, the animals were kept on high lipid, and 3 weeks later, the retinas were stained with PAS.
Experimental grouping: the model control group (5) and the experimental group (5) are respectively a diabetic retinopathy rat which is not administrated, and the experimental group is a diabetic retinopathy rat which is injected in a vitreous cavity with AAV1-CBS (AAV 1-SFFV-CBS-T2A-mCherry virus).
3. Results of the experiment
The results show that after 2 months of modeling, the ZDF rat model with the retina blood vessels before administration shows obvious leakage after the right eye of the control group and the left eye of the experimental group are subjected to fluorescein mapping; after PAS staining is carried out on the right eyes of rats in the model control group, the fact that the blood vessel glycosylation is serious and the blood vessel endothelial cells and the peripheral cell nuclei are lost is found; after the left eye of the rat in the experimental group is treated by AAV1-CBS (AAV 1-SFFV-CBS-T2A-mChery virus) for 3 weeks, the angioglycosylation is obviously relieved, and vascular endothelial cells and peripheral cell nuclei are not lost (see the figure 3 and the figure 4), and the result shows that the over-expressed CBS gene has a better treatment effect on diabetic retinopathy.
Example 5 Effect of AAV1-SFFV-CBS on high sugar-induced vascular aging
1. Experimental methods
The model of high sugar-induced vascular proliferation described in this example includes: human retinal microvascular endothelial cells hRMEC and human umbilical vein endothelial cells HUVEC, the phenotypes examined included: angiogenesis experiments, cell proliferation and colony formation experiments.
After culturing HUVEC of the 4 th generation under the condition of high sugar (ECM culture medium, 5.5mmol +22mmol glucose) for 3 days, AAV1-SFFV-CBS-T2A-mCherry virus constructed in the invention example 2 is added and cultured for 3 days, and the proportion of senescent cells is analyzed by living beta-galactosidase staining method and flow cytometry. The detailed experimental method is as follows:
(1) HUVEC cell resuscitation
(1) The 1 vial was removed from the liquid nitrogen tank and immediately shaken continuously in water at 37 ℃ to completely thaw the frozen cell suspension.
(2) Once the cells in the cryopreservation tube are completely thawed (liquid), the cells are immediately taken out of the hot water bucket, 75% of alcohol is used for completely disinfecting the surface of the cryopreservation tube, and the cryopreservation tube is placed in an ultra-clean workbench. When the frozen tube is about to melt, the centrifuge tube containing the endothelial culture medium preheated to 37 ℃ is sterilized by alcohol and then is placed in an ultra-clean workbench.
(3) Taking out the cell suspension in the freezing tube under strict aseptic operation conditions, adding the cell suspension into a centrifugal tube containing 15mL of preheated E8 culture medium, gently blowing and beating for 2-3 times, centrifuging at 1300rpm for 5min, and removing the supernatant after the centrifugation is finished.
(4) Adding endothelial culture medium, gently blowing and beating for 2-3 times, transferring cells to cell culture flask, and adding culture solution. Placing the culture flask in CO 2 Culturing in an incubator.
(2) HUVEC digestion passage
(1) The well plate/flask to be passaged was removed from the incubator, the supernatant was aspirated and washed once with DPBS.
(2) Adding trypsin, spreading the solution at the bottom of the bottle, removing trypsin, incubating in an incubator for 4-5min, and observing under a microscope to make the cells contract and become round and disperse.
(3) Gently tapping the culture flask/plate to remove the cell wall, gently blowing with a gun head for several times, adding endothelial medium to stop digestion, transferring the cell after blowing to a new culture flask, adding endothelial medium, standing at 37 deg.C and 5% CO 2 Culturing in an incubator.
(4) And performing liquid change operation every three days, and performing passage when the cell confluence is about 70-80%.
(3) High sugar induced endothelial cell senescence
The specific experimental groups and culture conditions for each group are shown in table 6 below.
TABLE 6 Experimental groups and culture conditions
Wherein the control group and the high sugar treatment group are at 37 deg.C and 5% CO 2 Culturing in incubator for 3 days, changing culture medium, culturing for 3 days, treating with high sugar and CBS virus at 37 deg.C and 5% CO 2 After 3 days of culture in the incubator, CBS virus (AAV 1-SFFV-CBS-T2A-mChery virus constructed in example 2) was added and the culture was continued for 3 days.
(4) In vivo labelling and flow analysis of senescent cells
Treating the cells with 100 mu M Bafilomycin for 1h, inhibiting the activity of beta-galactosidase in lysosomes, adding an active fluorescent substrate dye of the beta-galactosidase into an SH-SY5Y culture medium, culturing for 2h at 37 ℃, washing twice with DPBS, digesting with pancreatin, collecting the cells, and performing flow cytometry analysis. The effect of overexpressing genes on reversal of senescence was compared from mean fluorescence intensity and the proportion of cells stained for reduction.
2. Results of the experiment
The experimental result further shows the therapeutic effect of the over-expression CBS gene on diabetic retinopathy.
Example 6 Effect of AAV1-SFFV-CBS on high sugar-induced pericytes
1. Experimental methods
This example investigated the effect of AAV1-SFFV-CBS on the expression change and apoptosis of high-sugar-induced pericyte angiogenin Ang-2/Tie-2 system.
Culturing mouse retinal microvascular pericytes for 3 days in a normal culture medium and a high-sugar culture (17.5 mmol +10mmol glucose in a pericyte complete culture medium), culturing the mouse retinal microvascular pericytes for 3 days after infecting the mouse retinal microvascular pericytes by using the high-sugar culture plus AAV1-SFFV-CBS-T2A-mCherry virus (constructed in the example 2), carrying out flow detection on the apoptosis ratio of the cells in a mode of combining an apoptosis molecular marker phosphatidylserine and Annexin, and observing the change of the expression amount of Ang-2/Tie-2 by immunofluorescence.
(1) Pericyte culture
The specific experimental groups and culture conditions for each group are shown in table 7 below.
TABLE 7 Experimental groups and culture conditions
The control group, the high-sugar treated group, and the high-sugar treated + CBS virus group were treated at 37 ℃ with 5% CO 2 Culturing in an incubator for 3 days.
(2) Staining of samples
(1) Apoptosis is induced according to experimental requirements. The test sample should contain an untreated cell sample as a negative control.
In addition, during the flow detection, experimental groups are required to be respectively singly dyed with Annexin V-FITC and PI for adjustment and compensation. Collecting cells: collecting 1-5X 10 5 And (4) cells.
(2) The cells were digested with trypsin without EDTA, centrifuged at 1800rpm (300 Xg) at 4 ℃ for 5min, and the supernatant was discarded.
(3) Washing the cells: cells were washed twice with pre-chilled PBS and centrifuged at 1800rpm (300 Xg) for 5min at 4 ℃ each time.
(4) Cell resuspension: add 100. Mu.L of 1 XBinding Buffer and gently blow to make a single cell suspension.
(5) Cell staining: adding 5 mu L Annexin V-FITC and 5 mu L PI stabilizing Solution, and gently blowing uniformly; incubating for 10min at room temperature (20-25 deg.C) in the dark; add 400. Mu.L of 1 × Binding Buffer and mix gently. After staining the samples were detected by flow cytometry within 1 h.
(3) Flow analysis
The excitation wavelength of the flow cytometer is 488nm; the green fluorescence of FITC was detected on the FL1 channel; red fluorescence of PI was detected on FL2 or FL3 channel, suggesting that FL3 was used and 10000events were collected for each sample. And (3) performing data analysis by using software such as FlowJo and the like, wherein FL1 is an abscissa, FL3 is an ordinate, and determining the yin-yang boundary of two fluorescence parameters according to FITC and PI fluorescence values to define a cross gate. Typical experiments can be divided into three subgroups of cells: the living cells are double negative (Annexin V-FITC-/PI-); the early apoptosis cell is Annexin V-FITC single positive (Annexin V-FITC +/PI-); the late apoptotic cells were Annexin V-FITC and PI double positive (Annexin V-FITC +/PI +).
2. Results of the experiment
The experimental result further shows the therapeutic effect of the over-expression CBS gene on diabetic retinopathy.
Example 7 Effect of AAV1-SFFV-CBS on microglial angiogenesis promotion under hypoxic conditions 1, experimental method
The amount of VEGF secreted by microglia under hypoxic conditions was measured by ELISA. The blood vessel endothelium is cultured by the microglia cell culture fluid cultured under the anoxic condition, and whether the blood vessel is formed or not is observed.
(1) Cell culture
Specific cell culture conditions are shown in table 8 below.
TABLE 8 cell culture conditions
After the HUVEC cells were passed to the fourth passage, 3-5 million cells were seeded into a Matrigel-coated 24-well plate with a DPBS wash and treated with virus/DPBS.
37℃、5％ CO 2 Culturing microglial cells in a 6-pore plate 10% FBS DMEM culture medium in an incubator for 2 days, changing the culture medium to a 3 rd day, placing the culture medium in an anoxic/normal-oxygen incubator for culturing for 24h, taking the culture medium, and centrifuging at 13000rpm for 5min. Taking supernatant culture medium to determine VEGF content and replacing HUVEC culture medium, 37 deg.C, 5% CO 2 The vessel formation condition is recorded by observing and photographing under a microscope after 4, 6, 8 and 20 hours of the incubator.
The amount of VEGF secreted by endothelial cells and microglia was determined by ELISA.
(2) Preparation of VEGF assay
(1) The culture solution should be centrifuged at high speed (13000rpm, 5 min) and the supernatant taken for detection.
(2) Preparing a washing solution: diluted with distilled water 1.
(3) Preparing a standard substance: taking 8 1.5mL centrifuge tubes, respectively marking as S1, S2, S3, S4, S5, S6, S7 and blank, adding 900 μ L of standard/sample diluent into the first tube S1, respectively adding 200 μ L of standard/sample diluent into the second tube to the eighth tube, adding 100 μ L (20 ng/mL) of standard solution into the first tube, placing the first tube on a vortex mixer, uniformly mixing, sucking 200 μ L by using a sample injector, transferring to the second tube, repeatedly performing double dilution, sucking 200 μ L from the seventh tube, and discarding, wherein the eighth tube is a blank control. The standard curve concentrations are: 2000. 1000, 500, 250, 125, 62.5, 31.25 and 0pg/mL (the dosage of the standard substance and the range of the standard curve can be configured according to actual needs).
(4) Preparing a biotinylated antibody working solution: 20 minutes before use, 100x biotinylated antibody was diluted to 1x working solution with biotinylated antibody diluent, prepared according to the required dose, used the day, and discarded.
(5) Preparation of TMB color development liquid: for 10 minutes before use, the TMB developing solution A and B1:1, mixing and placing in the dark for standby.
(6) If the concentration of the target protein in the detected sample is higher than the highest value of the standard substance, the re-detection is recommended, and proper fold dilution is carried out according to the actual experimental condition.
(3) Detection of VEGF
(1) Sample adding: adding 50 μ L of the standard substance/sample diluent into the blank hole, adding 50 μ L of the standard substance or the sample to be detected into the other holes, uniformly mixing the reaction plates, and placing the reaction plates at 37 ℃ for 40min.
(2) Washing the plate: the reaction plate was washed thoroughly 4-6 times with 1 Xwashing solution, 350. Mu.L of 1 Xwashing solution was added to each well, shaking/soaking was performed for 1-2min each time, and the filter paper was printed dry.
(3) mu.L of biotinylated antibody diluent is added into blank wells, 100 mu.L of 1 Xbiotinylated antibody working solution is added into the rest wells respectively, and the mixture is placed for 30min at 37 ℃ after being mixed uniformly.
(4) Washing the plate: as above.
(5) Adding 100 μ L of SABC compound working solution into each well, mixing well, and standing at 37 deg.C for 20min.
(6) Washing the plate: as above.
(7) Adding 100 μ L of TMB mixed solution prepared in advance into each hole, mixing, and reacting at 37 deg.C in dark for 10-20min (the specific color development time is determined according to color development result).
(8) Adding 100 μ L stop solution into each well, mixing, and measuring absorbance value at 450nm with enzyme labeling instrument within 30min.
2. Results of the experiment
The experimental result further shows the therapeutic effect of the over-expressed CBS gene on diabetic retinopathy.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.
Claims (10)
- The application of CBS gene in preparing medicine for treating and/or preventing angiogenic eye disease is characterized in that the amino acid sequence coded by the CBS gene is shown in SEQ ID NO. 1;preferably, the nucleotide sequence of the CBS gene is shown as SEQ ID NO. 2;preferably, the angiogenic eye disease comprises diabetic retinopathy, diabetic macular edema, age-related macular degeneration, macular edema following retinal vein occlusion, vascular retinopathy, choroidal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, corneal neovascularization;more preferably, the angiogenic eye disease is diabetic retinopathy.
- 2. A recombinant viral vector, wherein the recombinant viral vector comprises a CBS gene;preferably, the amino acid sequence coded by the CBS gene is shown as SEQ ID NO. 1;preferably, the nucleotide sequence of the CBS gene is shown as SEQ ID NO. 2;preferably, the recombinant viral vector includes adeno-associated virus (AAV) vector, adenoviral vector, helper-dependent adenoviral vector, retroviral vector, herpes simplex viral vector, lentiviral vector, poxvirus vector, japanese hemagglutinating virus-liposome (HVJ) complex vector, moloney murine leukemia viral vector, HIV-based viral vector;more preferably, the recombinant viral vector is an adeno-associated viral vector;most preferably, the adeno-associated viral vector comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and/or hybrids thereof;most preferably, the recombinant viral vector is a recombinant AAV1 vector;most preferably, the recombinant virus vector is an infectious virus particle AAV1-SFFV-CBS-T2A-mCherry obtained by harvesting after a plasmid vector for expressing the CBS gene and AAV1 are co-transfected into cells;most preferably, the cell is a human embryonic kidney cell line;most preferably, the human embryonic kidney cell lines comprise 293FT, 293T, 293A;most preferably, the human embryonic kidney cell line is 293FT;most preferably, the plasmid vector for expressing the CBS gene is a pSFFV-CBS-T2A-mCherry plasmid vector;most preferably, the pSFFV-CBS-T2A-mCherry plasmid vector is obtained by inserting a CBS gene sequence between pSFFV and T2A of pSFFV-T2A-mCherry plasmid.
- 3. A method for producing the recombinant viral vector according to claim 2, comprising the steps of:(1) Constructing a pSFFV-CBS-T2A-mCherry plasmid vector;(2) Co-transfecting the pSFFV-CBS-T2A-mCherry plasmid vector constructed in the step (1) and AAV1 into a cell, and then culturing the cell;(3) Collecting cell supernatant to obtain infectious virus particles, namely recombinant virus vector AAV1-SFFV-CBS-T2A-mCherry;preferably, the pSFFV-CBS-T2A-mCherry plasmid vector in the step (1) is constructed by inserting the CBS gene sequence between pSFFV and T2A of the pSFFV-T2A-mCherry plasmid;preferably, the cells in step (2) are human embryonic kidney cell lines;more preferably, the human embryonic kidney cell lines comprise 293FT, 293T, 293A;most preferably, the human embryonic kidney cell line is 293FT;preferably, the conditions of the culturing in step (2) are 5% 2 、37℃；Preferably, the collection of cell supernatant in step (3) is started 48h after transfection.
- 4. A gene therapeutic agent for treating and/or preventing angiogenic eye disease, characterized in that it comprises the recombinant viral vector according to claim 2;preferably, the administration mode of the gene therapy medicament comprises subretinal injection of the recombinant virus vector, intravitreal injection of the recombinant virus vector;more preferably, the gene therapy drug is administered by intravitreal injection of the recombinant viral vector;preferably, the angiogenic eye disease comprises diabetic retinopathy, diabetic macular edema, age-related macular degeneration, macular edema following retinal vein occlusion, vascular retinopathy, choroidal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, corneal neovascularization;more preferably, the angiogenic eye disease is diabetic retinopathy.
- 5. A pharmaceutical composition for treating and/or preventing angiogenic eye diseases, which comprises the gene therapy drug of claim 4;preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant;preferably, the pharmaceutical composition further comprises an additional therapeutic agent;more preferably, the other therapeutic agent comprises a hypoglycemic agent, an anticoagulant agent, a vasoprotective agent;most preferably, the hypoglycemic agents include sulfonylurea agents, meglitinide agents, DPP-4 inhibitors, biguanide agents, thiazolidinedione agents, alpha-glucosidase inhibitors;most preferably, the anticoagulant drug comprises aspirin, borrelidin, heparin anticoagulant drug, warfarin;most preferably, the vasoprotective drugs include lipid lowering drugs, antiplatelet drugs;preferably, the angiogenic eye disease comprises diabetic retinopathy, diabetic macular edema, age-related macular degeneration, macular edema following retinal vein occlusion, vascular retinopathy, choroidal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, corneal neovascularization;more preferably, the angiogenic eye disease is diabetic retinopathy.
- 6. Use of the gene therapeutic drug of claim 4 for the preparation of a medicament for the treatment and/or prevention of angiogenic eye diseases, wherein the angiogenic eye diseases include diabetic retinopathy, diabetic macular edema, age-related macular degeneration, macular edema after retinal vein occlusion, vascular retinopathy, choroidal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, corneal neovascularization;preferably, the angiogenic eye disease is diabetic retinopathy.
- 7. Use of a pharmaceutical composition according to claim 5 for the preparation of a medicament for the treatment and/or prevention of angiogenic eye diseases, wherein said angiogenic eye diseases include diabetic retinopathy, diabetic macular edema, age-related macular degeneration, macular edema following retinal vein occlusion, vascular retinopathy, choroidal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, corneal neovascularization;preferably, the angiogenic eye disease is diabetic retinopathy.
- Application of CBS gene in preparing cell aging inhibitor, blood vessel glycosylation inhibitor, vascular endothelial cell apoptosis inhibitor and/or pericyte apoptosis inhibitor, wherein the amino acid sequence coded by CBS gene is shown in SEQ ID NO 1;preferably, the nucleotide sequence of the CBS gene is shown as SEQ ID NO. 2.
- 9. Use of the gene therapy agent of claim 4 for the preparation of a cell aging inhibitor, a blood vessel glycosylation inhibitor, a vascular endothelial apoptosis inhibitor and/or a pericyte apoptosis inhibitor.
- 10. Use of the pharmaceutical composition of claim 5 for the preparation of a cell senescence inhibitor, a vessel glycosylation inhibitor, a vessel endothelial apoptosis inhibitor and/or a pericyte apoptosis inhibitor.
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