CN116898971A - Application of SRPK3 gene in preparation of medicines for treating myocardial hypertrophy - Google Patents
Application of SRPK3 gene in preparation of medicines for treating myocardial hypertrophy Download PDFInfo
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- CN116898971A CN116898971A CN202310873792.2A CN202310873792A CN116898971A CN 116898971 A CN116898971 A CN 116898971A CN 202310873792 A CN202310873792 A CN 202310873792A CN 116898971 A CN116898971 A CN 116898971A
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- Prior art keywords
- srpk3
- myocardial hypertrophy
- gene
- hypertrophy
- gene expression
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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
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
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- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Public Health (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
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- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Cardiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Heart & Thoracic Surgery (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses application of an SRPK3 gene in preparing a medicament for treating myocardial hypertrophy, wherein the sequence of the SRPK3 gene is shown as SEQ ID NO.1, and an SRPK3 gene expression inhibitor is a substance which is prepared or screened by taking the SRPK3 gene as an action target and has an inhibiting effect on the SRPK3 gene. Compared with the normal group, the SRPK3 expression of the mice with the myocardial hypertrophy model is obviously up-regulated, and the heart weight, the lung weight, the myocardial cell cross-sectional area and the fibrosis of the mice with the SRPK3 gene knockout are obviously reduced, so that the heart contraction function is obviously improved. Based on the above findings, SRPK3 can be used as an action target of a drug for treating myocardial hypertrophy and heart failure, and its inhibitor can be used for preparing a drug for preventing, alleviating and/or treating pathological myocardial hypertrophy.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to application of SRPK3 genes in preparation of medicines for treating myocardial hypertrophy.
Background
Myocardial hypertrophy is a pathological change caused by long-term pressure or volume overload of the heart, is common in patients with cardiovascular diseases such as hypertension, heart valve lesions and the like, and is mainly characterized by heart volume increase and weight increase caused by myocardial cell hypertrophy and sarcomere increase. Early cardiac hypertrophy is characterized by hypertrophy of the heart muscle cells, an enhancement of the contractility of the heart muscle, as a compensatory mechanism to maintain cardiac output and peripheral circulatory perfusion, but long-term heart enlargement increases myocardial oxygen consumption and decreases ventricular wall compliance, resulting in decompensation of cardiac structure and function. Before early cardiac hypertrophy, the mitochondrial ultrastructural and metabolic levels of cardiomyocytes have been altered, while cardiomyocyte phenotypes undergo embryogenic transformation, accompanied by myocardial mesenchymal cell proliferation and deposition of cardiac extracellular matrix, leading to myocardial remodeling and cardiac insufficiency. Long-term cardiac hypertrophy is easy to induce cardiovascular diseases such as malignant arrhythmia, coronary heart disease, heart valve stenosis or insufficiency, and the like, and is closely related to other cardiovascular diseases such as cardiac metabolic diseases, sudden cardiac death, and the like.
Serine-arginine protein kinases (SRPKs) are a class of cell cycle dependent kinases that participate in the process of the shear regulation of precursor messenger RNAs (pre-mrnas) primarily by phosphorylation modification of proteins rich in serine-arginine sequences, while controlling subcellular localization of the shear regulatory factors; in addition, SRPKs are involved in regulating cell cycle and apoptosis, and as a result of these functions, SRPKs may play a role in tumorigenesis. In addition to splice-mediated factors, SRPKs are involved in the phosphorylation of various proteins, such as the phosphorylation of Tau protein by SRPK2 to cause the development and progression of Alzheimer's disease, and SRPKs phosphorylate viral proteins (e.g., HBV core proteins) and regulate their replication processes. SRPKs are a family of kinases that control alternative splicing, and are associated with many types of cancer. Targeting SRPKs presents a potential strategy to inhibit cancer cell proliferation and metastasis by modulating aberrant alternative splicing. Among them, SRPK3 is a kind of muscle-specific protein kinase, involved in the occurrence and development of diseases such as parkinson, but SRPK3 has not been studied and reported in heart diseases.
Disclosure of Invention
Applicants found Srpk fl/f After the SRPK3 gene is knocked out by the mice, srpk of the SRPK3 gene is compared with Srpk of the SRPK3 gene which is not knocked out fl/fl Compared with mice, heart weight/body weight, lung weight/body weight, myocardial cell cross-sectional area and fibrosis are all obviously reduced, and the heart contraction function is obviously improved, and the results indicate that SRPK3 gene knockout relieves the disease process of pathological myocardial hypertrophy. Based on the above findings, the SRPK3 gene can be used as a drug target for screening for the treatment of myocardial hypertrophy and heart failure, and inhibitors thereof can be used for preparing drugs for preventing, alleviating and/or treating pathological myocardial hypertrophy.
The technical scheme provided by the invention is as follows:
in a first aspect, the present invention provides an application of an SRPK3 gene expression inhibitor in preparing a medicament for preventing, alleviating and/or treating myocardial hypertrophy, wherein the sequence of the SRPK3 gene is shown as SEQ ID No.1, and the SRPK3 gene expression inhibitor is a substance having an inhibitory effect on the SRPK3 gene prepared or screened by using the SRPK3 gene as an action target.
Based on the technical scheme, the SRPK3 gene expression inhibitor is nucleic acid, a nucleic acid construct, adenovirus, protein or a small molecule compound.
Based on the technical scheme, the SRPK3 gene expression inhibitor comprises an interference plasmid, wherein the interference plasmid comprises a sequence shown as SEQ ID NO. 6.
Based on the above technical scheme, the interfering plasmid is packaged into adenovirus, and is transfected into cells through adenovirus.
On the basis of the technical scheme, the adenovirus is obtained by the following steps: the interference plasmid containing the sequence shown in SEQ ID NO.6 and an adenovirus skeleton vector pADEasy-1 are transfected into HEK293 cells to obtain recombinant adenovirus.
On the basis of the technical scheme, the myocardial hypertrophy is pathologic myocardial hypertrophy, and comprises myocardial hypertrophy or heart failure caused by one or more of hypertension, aortic stenosis and mitral insufficiency.
In a second aspect, the present invention provides a medicament for preventing, alleviating and/or treating myocardial hypertrophy comprising one or more of the above described inhibitors of SRPK3 gene expression.
In a third aspect, the present invention provides a combination therapeutic agent for myocardial hypertrophy comprising one or more inhibitors of SRPK3 gene expression as described above and at least one other agent for treating myocardial hypertrophy.
Based on the technical scheme, the myocardial hypertrophy treatment medicine combination is selected from any one of the following forms:
(i) Preparing SRPK3 gene expression inhibitor and other cardiac hypertrophy treating medicine into independent preparations;
(ii) The SRPK3 gene expression inhibitor and other medicines for treating cardiac hypertrophy are prepared into compound preparation.
In a fourth aspect, the present invention provides a myocardial hypertrophy detection system comprising:
a detection reagent or combination of detection reagents that specifically recognizes the SRPK3 protein; and
an apparatus for quantitatively detecting a detection reagent or a combination of detection reagents.
And when the myocardial hypertrophy detection system detects that the content of SRPK3 protein in myocardial cells is higher than a normal value, judging that the myocardial hypertrophy is caused.
Compared with the prior art, the invention has the following advantages and effects:
(1) The invention discovers that inhibiting the SRPK3 gene can improve myocardial hypertrophy and provides a new thought for preventing, relieving and/or treating myocardial hypertrophy by taking the SRPK3 gene as an inhibition target.
(2) The invention provides a plurality of SRPK3 gene inhibitors and application thereof in preparing medicaments for preventing, relieving and/or treating myocardial hypertrophy, and enriches a myocardial hypertrophy medicament library.
Drawings
FIG. 1 is a graph showing protein expression of BNP, beta-MHC, SRPK3 and statistical histogram of the protein in hearts of normal and clinical patients with myocardial hypertrophy (heart failure patients), beta-Tubulin is used as an internal reference, and the expression of SRPK3 in heart tissues of the patients with myocardial hypertrophy is up-regulated (p <0.05vs normal group).
FIG. 2 is a graph showing the expression of BNP, beta-MHC, SRPK3 and statistics of C57BL/6 mice in hearts 2 weeks, 4 weeks after sham surgery and aortic arch constriction surgery (TAC), with beta-Tubulin as an internal control, and with upregulation of the expression of cardiac SRPK3 in the event of myocardial hypertrophy (p <0.05vs sham surgery group).
FIG. 3 is a graph showing protein expression of SRPK3 in vitro in cultured primary cardiomyocytes of rats and after angiotensin II stimulation, with beta-Tubulin as an internal control, and with up-regulation of SRPK3 expression after angiotensin II stimulation (p <0.05vs control).
FIG. 4 is a structural map of the Ad-shSRPK3 plasmid, and a verification and statistical map of SRPK3 protein expression after SRPK3 gene knockdown, and the beta-Tubulin is taken as an internal reference, and the SRPK3 expression is found to be significantly reduced (p <0.05vsAd-shRNA group).
FIG. 5 is a graph showing immunofluorescence and cell surface area statistics of cultured primary cardiomyocytes in vitro infected with adenovirus Ad-shRNA and Ad-shSRPK3 stimulated with angiotensin II for 48 hours, interfering adenoviruses of SRPK3 inhibited cardiomyocyte hypertrophy (: p <0.05vsAd-shRNA control, #: p <0.05vsAd-shRNA angiotensin II group).
FIG. 6 is SRPK3 fl/fl Myh6-Cre and littermate wild-type SRPK3 fl/fl Statistical plots of heart mass, heart weight, lung weight, and heart weight/tibia length after 4 weeks of sham or TAC procedure modeling in mice (p <0.05 vssrpk3) fl/fl Sham group, #: p <0.05vs SRPK3 fl/fl Model group).
FIG. 7 is SRPK3 fl/fl Myh6-Cre mice and littermates wild-type SRPK3 fl/fl Cardiac tissue WGA staining and cardiomyocyte cross-sectional area statistics (p <0.05 vsSRPK3) after 4 weeks of sham or TAC surgery modeling in mice fl/fl Sham group, #: p <0.05vs SRPK3 fl/fl Model group).
FIG. 8 is SRPK3 fl/fl Myh6-Cre and littermate wild-type SRPK3 fl/fl Marsonian trichromatic (Masson) staining of heart tissue and left-hand collagen content statistical plots (p <0.05 vsSRPK3) after 4 weeks of sham or TAC operation modeling in mice fl/fl Sham group, #: p <0.05 vsSRPK3 fl/fl Model group).
FIG. 9 is a diagram ofSRPK3 fl/fl Myh6-Cre and littermate wild-type SRPK3 fl/fl Statistical graphs of M-type echocardiography and cardiac function measurements after 4 weeks of sham or TAC modeling including ejection fraction, left ventricular end systole inner diameter, left ventricular end diastole inner diameter and short axis shortening rate (p <0.05 vsSRPK3) fl/fl Sham group, #: p <0.05vs SRPK3 fl/fl Model group).
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
The invention uses myocardial cell specific SRPK3 gene knockout mice (SRPK 3) fl/fl Myh 6-Cre) and littermate wild-type SRPK3 fl/fl Mice are experimental subjects, and a myocardial hypertrophy animal model is induced by aortic arch constriction (TAC), so that the functions of SRPK3 genes are further researched, and the result shows that in the mouse myocardial hypertrophy model, the expression of the SRPK3 is obviously up-regulated compared with a normal group; with wild SRPK3 fl/fl SRPK3 compared to model group mice fl/fl The heart weight/body weight, lung weight/body weight, myocardial cell cross-sectional area and fibrosis of the mice in the Myh6-Cre model group are all obviously reduced, the heart contraction function is obviously improved, and the results indicate that the myocardial cell specific SRPK3 gene knockout can relieve the disease process of pathological myocardial hypertrophy. Therefore, the SRPK3 gene can be used as a drug target, and an in vitro cell model or an animal model for SRPK3 gene knockout is constructed, so that the aim of preventing, relieving and/or treating pathological myocardial hypertrophy is fulfilled by screening drugs and/or biological agents for preventing, relieving and/or treating myocardial hypertrophy through a genetic engineering technology. In addition, a small molecular compound inhibitor can be designed by taking SRPK3 as a target point, and a molecular capable of specifically inhibiting SRPK3 can be found by screening by utilizing an in vitro cell model or an animal model of SRPK3 gene overexpression, so that a novel therapeutic molecule is provided for the treatment of myocardial hypertrophy.
Experimental animals and feeding:
experimental animals: selecting 8-10 weeks old, weight of 25.5+ -2.0 g, SRPK3 fl/fl Myh6-Cre and littermate wildSRPK3 fl/fl Male mice were the subjects.
Feeding environment: all experimental mice were housed in a university of martial arts cardiovascular disease institute no specific pathogen (SpecificPathogenFree, SPF) class laboratory animal center.
Feeding conditions: the room temperature is between 22 and 24 ℃, the humidity is between 50 and 70 percent, the lighting time of light and dark alternation is 12 hours, and the food is ingested by free drinking water.
Example 1: cardiomyocyte-specific SRPK3 gene knockout (SRPK 3) fl/fl Construction of Myh 6-Cre) mice
SRPK3 at 8-10 weeks of age fl/fl Mice (purchased from Sai industry (Suzhou) biotechnology Co., ltd.) were mated with Myh6-Cre mice (purchased from Jackson laboratories, USA), and after mating, the mice were subjected to enzymatic genetic identification by shearing their tails, and screened to obtain SRPK3 fl /fl The primers used for the enzymolysis method of the Myh6-Cre mice are as follows:
F1(SEQ ID NO.2):5’-AAAGGCTACTTTTGAGGCCTACTT-3’；
R1(SEQ ID NO.3):5’-AGGAGCACTTAGACTCCAGAAATG-3’；
F2(SEQ ID NO.4):5’-AGCTGAGTACCTGAGAAGAGAGT-3’；
R2(SEQ ID NO.5):5’-CACTGTTCCTTGTCTACTCCCAGA-3’。
example 2
The expression of SRPK3 in hearts of normal people and cardiac hypertrophy patients is selected from normal hearts (individuals donated by non-cardiac death), hearts of cardiac hypertrophy patients (receptors replaced by heart transplant operation patients or heart biopsy tissues), SDS-PAGE-immunoblotting (Westernblot) is carried out on heart extract proteins, antibodies which specifically bind to SRPK3 proteins and myocardial cell hypertrophy markers BNP (Abclone, A2179) and beta-MHC (Abclone, A7564) are detected, and the expression of SRPK3 proteins (Invitrogen, PA 5-103255) and beta-Tubulin (Abmart, M2005L) are measured as internal references.
As shown in FIG. 1, the detection results are consistent with the up-regulation of the expression of BNP and beta-MHC markers of myocardial cell hypertrophy, and the expression of SRPK3 protein in hearts of patients suffering from myocardial hypertrophy is obviously up-regulated.
Example 3
In SRPK3 fl/fl Myh6-Cre and littermate wild SRPK3 fl/fl Male mice are experimental subjects, and a mouse myocardial hypertrophy model is constructed by adopting aortic arch constriction surgery (TAC), and the operation flow is as follows:
1. preoperative preparation
(1) Anesthesia: mice were first weighed, the amount of needed anesthetic (3% pentobarbital sodium) calculated as 90mg/kg body weight, injected intraperitoneally, and the injection time points recorded. The tail and toe clamping do not have obvious reaction, and the state of the mice is good as the anesthesia success standard (generally, about 10min after injection has no obvious reaction, about 50min after anesthesia has reaction, and about 30min after anesthesia is the optimal operation time).
(2) Preparing an operation area: the skin of the left chest, left side chest, left forelimb and underarm of the mice was dehaired. After shaving, the surgical field is wiped with wet gauze to remove the mouse hair, preferably without affecting the surgical field.
(3) Tracheal cannula: the upper incisors of the mice are fixed on the inclined planes of the V-shaped plates by rubber bands, the tracheal cannula is rapidly and accurately inserted into the trachea through the glottis, then the right lateral position is placed on a heating pad (the heating pad needs to be preheated in advance), and then the tracheal cannula is connected with a breathing machine to fix the mice. If the thoracic cavity fluctuation of the mice is consistent with the frequency of the breathing machine, the successful tracheal intubation is indicated.
Tac procedure
Model group: the right lateral position is taken, the left forelimb of the mouse is placed above the right forelimb, and the two forelimbs are fixed by using a medical adhesive tape. A cotton swab is padded under the right chest to raise the chest, and the skin of the operation area is disinfected by iodine and alcohol with the volume fraction of 75 percent. The left hand ophthalmic forceps hold the left chest skin up, the right hand ophthalmic scissors cut the skin about 1cm, the muscle and the soft tissue are separated in sequence, the chest is opened at the level of the 2 nd to 3 rd ribs, the left lung is slightly opened by a cotton swab, the aortic arch is free to descend, 7-0 surgical suture is passed through the blood vessel, a section of 26G (25.0-27.5G mice) or 27G (23.5-25.0G) syringe needle is placed above the blood vessel in parallel, the blood vessel and the needle are ligatured together, and the needle is withdrawn to achieve the corresponding degree of blood vessel constriction. After ligation, the chest was closed by suturing sequentially, inserting the chest through the incision with a syringe and withdrawing 1cc of gas to restore negative pressure in the chest, and rapidly suturing the skin incision after withdrawing the syringe.
Sham surgery group (Sham): after the descending branch of the aorta is released, only threading is performed without ligating, and the rest steps are the same as those of the myocardial hypertrophy model group.
3. Postoperative care
After TAC operation, when the mice breathe spontaneously and have strong reaction with toe clamping, the tracheal intubation is pulled out, and the mice are placed into a feeding cage filled with autoclave sterilized padding, feed and drinking water, and are continuously fed and observed in a feeding room. SRPK3 fl/fl Myh6-Cre mice and SRPK3 fl/fl The mice were examined for each index at 4 weeks post-surgery.
Example 4: myocardial hypertrophy pathology detection
1. Drawing materials
(1) Working in the early stage: urine cups with 20mL of 10% formaldehyde by volume were prepared in advance and labeled (mouse number, group, type of surgery and date of draw). Placing a culture dish filled with 10% KCl solution at a material sampling position. Opening the analytical balance and zeroing for standby. Mice were sacrificed by re-weighing.
(2) Drawing materials: the ophthalmic curved forceps clamp the vascular pedicle below the auricle, shear the heart and rapidly place the heart in 10% KCl solution by mass fraction. After the heart is stopped in diastole, the heart is placed on sterilized gauze, liquid in a heart cavity is lightly squeezed, surface liquid is dipped, the heart is weighed and recorded, and the heart is placed in a corresponding urine cup and fixed for 48 hours for pathological detection.
(3) Correlation measurement and calculation: the heart and lungs of the mice were removed, trimmed, and the filters blotted dry, weighed and recorded. Skin at the hind limb tibia of the mice was cut, and tibia length was measured and recorded. The ratio of heart weight to body weight (HW/BW), lung weight to body weight (LW/BW) and heart weight to tibia length (HW/TL) were calculated.
(4) SRPK3 expression in sham-operated and model group hearts: hearts of 2 weeks and 4 weeks after TAC surgery of a wild type C57BL/6 mouse sham surgery group and a model group are respectively selected, SDS-PAGE-immunoblotting (Westernblot) is carried out on heart extracted proteins, antibodies which specifically recognize SRPK3 proteins and myocardial cell hypertrophy markers BNP and beta-MHC are combined for detection, the expression of the SRPK3 proteins is determined, and beta-Tubulin is used as an internal reference. The results are shown in fig. 2, in which the expression of BNP, beta-MHC was significantly upregulated 4 weeks after TAC surgery, while the expression of SRPK3 protein was significantly upregulated after TAC surgery.
2. Pathology detection
2.1 preparation of Paraffin sample sections
The main operation procedure comprises: trimming heart, embedding frame treatment, running water flushing, dehydration, transparency, wax dipping, embedding, slicing, spreading, airing or baking for later use.
2.2 Wheat Germ Agglutinin (WGA) staining
The method mainly comprises the following steps: placing paraffin specimen slices baked at 60 ℃ for 30min in xylene for 5min x 3 times, 100% ethanol for 5min x 2 times, 95% ethanol for 5min, 70% ethanol for 5min, distilled water for 5min x 2 times, PBS for 5min, PBS for 10min, discarding PBS, dripping pancreatin working solution (DIG-3008, micnew, fuzhou) for 20min in dark place at 37 ℃, PBS for 5min x 3 times, taking out slices, wiping the liquid around the tissues with filter paper (the tissues are not dried), placing the slices in a wet box by using a combined pen loop, dripping WGA-AlexaFlour488 working solution (10 mug/mL) for 2h at 37 ℃, discarding dye liquor, PBS for 5min x 3 times, dripping the contrast agent with DAPI for dark place under a fluorescent microscope, observing, and taking a microscopic photograph.
2.3 Masson trichromatic (Masson) staining
The method mainly comprises the following steps: baking at 55deg.C for 30min, xylene for 2min,3 times, 100% alcohol for 1min, 95% alcohol for 1min, 70% alcohol for 1min, running water washing for 10min, double distilled water for 1min, weigert's iron hematoxylin dyeing for 5min, tap water washing for 5min, residual liquid removal, 1% hydrochloric acid alcohol differentiation for 4s, tap water washing for 5min returning blue, ponceau acid fuchsin liquid dyeing for 10min, distilled water washing for 5min, phosphomolybdic acid water solution treatment for about 5min, aniline blue liquid counterstain for 5min, 1% ice CH 3 COOH treatment for 1min, 70% alcohol for 1 time, 90% alcohol for 1 time, 100% alcohol for 30s,3 times, xylene for 2min,3 times, cover glass sealing immediately before xylene is dried, and microscopic photographing. SRPK3 fl/fl Myh6-Cre and littermates SRPK3 fl/fl After TAC operation molding of micePhenotypic results are shown in FIGS. 6-8. The heart is general in phenotype, the heart of the sham operation group has no obvious difference, the heart of the model group is increased compared with that of the sham operation group, and SRPK3 fl/fl The heart of the/Myh 6-Cre mice was significantly smaller than SRPK3 fl/fl A mouse; in addition, SRPK3 in sham surgery group fl/fl Mice and SRPK3 fl/fl The differences between HW/BW, LW/BW and HW/TL of the Myh6-Cre mice are not statistically significant; SRPK3 fl/fl HW/BW, LW/BW, HW/TL were higher than in sham surgery group 4 weeks after TAC surgery in mice; 4 weeks after TAC surgery, SRPK3 fl/fl HW/BW, LW/BW and HW/TL of Myh6-Cre mice are all compared with SRPK3 fl/fl Mice were lowered (fig. 6). WGA stained sections were observed: SRPK3 fl/fl the/Myh 6-Cre group is compared with SRPK3 fl/fl Group cell hypertrophy was significantly reduced, and the differences were statistically significant (fig. 7). After Masson staining, the increase of the ventricular myocardial interstitial collagen content of the model group is found to be more obvious than that of the sham operation group, the increase of the collagen around arterial blood vessels is more obvious, the collagen is thickened, and the arrangement is disordered into a network shape; SRPK3 fl/fl Myocardial interstitial collagen content and perivascular collagen content of the Myh6-Cre mice after TAC surgery are compared with SRPK3 fl/fl Mice were post-operative with TAC reduced (fig. 8). The above results demonstrate that after TAC operation molding, mice develop significant myocardial hypertrophy, SRPK3 fl/fl Myocardial hypertrophy and fibrosis levels in the/Myh 6-Cre mice were significantly less than those of SRPK3 fl/fl And (3) a mouse.
Example 5: ultrasonic cardiography for detecting heart function of mice
1. Early preparation
(1) Preparing an anesthesia machine: firstly connecting an oxygen bottle with an air inlet port on an anesthesia machine, unscrewing a sealing cover of a medicine adding port on the anesthesia machine, rapidly adding isoflurane to a safety scale, and then screwing the sealing cover. The total valve on the oxygen bottle is unscrewed, the knob of the flow control valve is adjusted, and the air outlet pressure is maintained to be 0.2-0.3mPa.
(2) Preparing a mouse to be tested: after the mice to be detected are rapidly anesthetized by isoflurane, the left chest area is shaved, the head of the mice after treatment is stretched into the anesthetic catheter sleeve head, and the stable anesthetic state of the mice is maintained by 1.5-2.0% isoflurane.
2. Cardiac function detection
The mouse takes the left lateral positionOr lying on the back, and uniformly applying ultrasonic coupling agent on the shaved area. And (3) using a high-frequency ultrasonic diagnostic apparatus with the frequency of 15MHz, selecting a standard left ventricular papillary muscle short axis section, and measuring the left ventricular end diastole inner diameter, the left ventricular end systole inner diameter, the left ventricular ejection fraction and the short axis shortening rate. FIG. 9 is SRPK3 fl/fl Myh6-Cre and SRPK3 fl/fl Results of heart function detection after TAC surgery in mice. SRPK3 of sham surgery group fl/fl SRPK3 compared to mice fl/fl The mice showed reduced cardiac function and cardiac hypertrophy at the 4-peripheral surface after TAC surgery, and were mainly characterized by an increase in left ventricular end diastole and left ventricular end systole, which are indicators of cardiac hypertrophy, while the index ejection fraction and short axis shortening rate, which reflect cardiac function, were decreased. 4 weeks after TAC surgery, SRPK3 fl/fl Cardiac dysfunction in Myh6-Cre mice compared to SRPK3 fl/fl Mice were relieved.
Example 6: isolation of SRPK3 expression in cardiomyocytes after control (PBS) or angiotensin II (AngII) stimulation
The method comprises culturing newborn Sprague-Dawley (SD) milk mice (1-3 days) cardiomyocytes, culturing primary cardiomyocytes for 48 hours, changing the liquid (the specific process of culturing primary newborn SD rat cardiomyocytes is described in example 7 below), starving the cardiomyocytes with serum-free DMEM/F12 for 12 hours to synchronize the cells, then respectively stimulating the cells with PBS and angiotensin II (AngII, 1 μM) for 48 hours, performing SDS-PAGE-immunoblotting (Westernblot) on the extracted proteins of the cardiomyocytes, detecting the extracted proteins in combination with antibodies specifically recognizing SRPK3 proteins, and measuring the expression of SRPK3 proteins of the detected protein. As shown in FIG. 3, the expression of SRPK3 in cardiomyocytes stimulated with angiotensin II (AngII) was significantly up-regulated.
Example 7: effect of SRPK3 interference (Ad-shSRPK 3) on AngII stimulated primary cardiomyocyte hypertrophy
1. Primary neonatal SD rat cardiomyocyte culture
(1) 10 Sprague-Dawley rats, which were newly born for 1-3 days, were sterilized with 75% alcohol below the neck, hearts were removed with an ophthalmic scissors and micro forceps, and placed in a glass plate containing 10ml of MEM/F12 solution. And taking the other one, and repeating the process.
(2) The heart was washed with DMEM/F12 medium and cut into 1-2mm pieces 3 Is a fragment of (c). Transfer to serum bottles with rotors, aspirate DMEM/F12, add pancreatin digest. The rotation speed is 120r/min, digestion is carried out for 15min, the reaction is stopped for a few seconds, and the supernatant is discarded.
(3) Adding pancreatin digestive juice, and digesting for 15min at a rotating speed of 120 r/min. After standing for several seconds, the supernatant was aspirated, and digestion was stopped with 20% calf serum in DMEM/F12 medium and stored in a refrigerator at 4 ℃. This step is repeated, and cycled several times. The supernatant should be removed as much as possible, and digestion is terminated when the tissue mass becomes white and significantly smaller.
(4) The collected cardiomyocyte suspension was centrifuged at 1500rpm for 8min and the supernatant was discarded. An appropriate amount of culture medium was added to the centrifuge tube, the resuspended cells were gently blown, concentrated into 150 mL centrifuge tube, and the cell suspension was filtered through a 40 μm filter screen.
(5) Cells were seeded in 100mm dishes and adherent for 90min at time difference, and the unadhered cell suspension was aspirated and filtered. Brdu (final concentration 0.1 mM) was added according to the total amount of cell suspension, and after mixing, added to a vessel coated with 0.1% gelatin.
(6) The cells were gently dispersed and not vortexed. 37 ℃ and 5% CO 2 Incubate 48 hours, wash 1 with PBS, change media.
SRPK3 expression interference (Ad-shSRPK 3)
The plasmid pAdeno-CMV-SRPK3-shRNA-EGFP-Puro (see figure 4) containing the target gene and an adenovirus skeleton vector pADEasy-1 are co-transfected into HEK293 cells to be recombined to obtain adenovirus Adeno-SRPK3-shRNA-EGFP-Puro, namely Ad-shSRPK3, and the adenovirus Adeno-SRPK3-shRNA-EGFP-Puro is subjected to mass amplification and purification, and the titer and the expression level of the target gene SRPK3 are detected (see figure 4) for later use.
The plasmid pAdeno-CMV-shRNA-EGFP-Puro without target genes and an adenovirus skeleton vector pADEasy-1 are co-transfected into HEK293 cells to be recombined to obtain adenovirus Adeno-shRNA-EGFP-Puro, ad-shRNA for short, and the adenovirus Adeno-shRNA is purified after a large amount of amplification and is used for later use after detecting titer.
The gene of interest: 5'-GAAAACUGCCUGUUUGUUU-3' (SEQ ID NO. 6);
shRNA：5’-UUCUCCGAACGUGUCACGUTT-3’(SEQ ID NO.7)。
primary cardiomyocytes cultured for 3 days were infected with virus solutions of Ad-shRNA and Ad-shrnpk 3 (10 moss) respectively for the AngII-induced cardiomyocyte hypertrophy model, stimulated with 1 μm angiotensin II (AngII) (purchased from ENZO corporation, ALX-151-039-M025) or PBS for 48 hours, followed by immunofluorescence assay. The results show that the surface area of the cardiomyocytes after Ad-shSRPK3 adenovirus infection is obviously reduced compared with that of an Ad-shRNA control group, namely, the interfering adenovirus of SRPK3 inhibits the cardiomyocyte hypertrophy (figure 5).
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
- Use of an inhibitor of srpk3 gene expression in the manufacture of a medicament for the prevention, alleviation and/or treatment of myocardial hypertrophy, characterized by: the sequence of the SRPK3 gene is shown as SEQ ID NO.1, and the SRPK3 gene expression inhibitor is a substance which is prepared or screened by taking the SRPK3 gene as an action target and has an inhibiting effect on the SRPK3 gene.
- 2. Use according to claim 1, characterized in that: the SRPK3 gene expression inhibitor is a nucleic acid, a nucleic acid construct, an adenovirus, a protein or a small molecule compound.
- 3. Use according to claim 2, characterized in that: the SRPK3 gene expression inhibitor comprises an interference plasmid, wherein the interference plasmid comprises a sequence shown as SEQ ID NO. 6.
- 4. Use according to claim 3, characterized in that: the interfering plasmid is packaged as an adenovirus, which is transfected into the cell.
- 5. Use according to claim 4, characterized in that: the adenovirus is obtained by the following method: the interference plasmid containing the sequence shown in SEQ ID NO.6 and an adenovirus skeleton vector pADEasy-1 are transfected into HEK293 cells to obtain recombinant adenovirus.
- 6. Use according to claim 1, characterized in that: myocardial hypertrophy is pathologic myocardial hypertrophy including myocardial hypertrophy or heart failure caused by one or more of hypertension, aortic stenosis, and mitral insufficiency.
- 7. A medicament for preventing, alleviating and/or treating myocardial hypertrophy, characterized by: comprising one or more of the SRPK3 gene expression inhibitors of claim 1.
- 8. A combination therapeutic agent for myocardial hypertrophy comprising: comprising one or more inhibitors of SRPK3 gene expression according to claim 1 and at least one other medicament for the treatment of myocardial hypertrophy.
- 9. The combination myocardial hypertrophy combination therapy as set forth in claim 8 wherein: the myocardial hypertrophy treatment drug combination is selected from any one of the following forms:(i) Preparing SRPK3 gene expression inhibitor and other cardiac hypertrophy treating medicine into independent preparations;(ii) The SRPK3 gene expression inhibitor and other medicines for treating cardiac hypertrophy are prepared into compound preparation.
- 10. A myocardial hypertrophy detection system characterized by: comprising the following steps:a detection reagent or combination of detection reagents that specifically recognizes the SRPK3 protein; andan apparatus capable of quantitatively detecting said detection reagent or said combination of detection reagents.
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