CN107022635B - ACARDL gene and application of expression product thereof in preparation of abdominal aortic aneurysm diagnosis and treatment product - Google Patents
ACARDL gene and application of expression product thereof in preparation of abdominal aortic aneurysm diagnosis and treatment product Download PDFInfo
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Abstract
The invention discloses an ACARDL gene and application of an expression product thereof in preparing a product for diagnosing and treating abdominal aortic aneurysm. The gene chip and QPCR experiments prove that compared with normal tissues, ACARDL gene expression in abdominal aortic aneurysm tissues is different. According to the correlation between the two, the invention provides a product for diagnosing abdominal aortic aneurysm, which can assist in diagnosing abdominal aortic aneurysm by detecting the level of ACADL gene or its expression product in tissue. In addition, the invention also provides an ACARDL gene and an expression product thereof, which can be used as a target for developing a medicament for treating abdominal aortic aneurysm and assist clinicians in formulating personalized treatment schemes.
Description
Technical Field
The invention relates to the field of tumor diagnosis and treatment, in particular to a tumor diagnosis method by taking ACARDL abnormality detection as a means; and a tumor therapeutic agent which activates the ACADL gene or protein.
Background
Abdominal Aortic Aneurysm (AAA) refers to a disease in which localized expansion and expansion of the abdominal aorta, mainly accompanied by pulsatile mass, are caused by lesions or loss of the abdominal aorta wall. The continuous expansion of the three-layer structure of the artery wall of the abdominal aorta section to more than 1.5 times the diameter of the abdominal aorta at the renal arteries is usually defined as AAA, and is a common arterial degenerative disease with high fatality rate. At present, the specific pathogenesis of the abdominal aortic aneurysm is not completely clear, and the reported reports show that the pathogenesis of the abdominal aortic aneurysm is closely related to genetic factors, inflammation, protease degradation, smooth muscle cell apoptosis and other factors, and the pathogenesis of the abdominal aortic aneurysm is different from that of other tumors due to chemical radiation, gene mutation and the like. Human abdominal aortic smooth muscle cells (HVSMCs) serve as the major constituent of the mesentery of the abdominal aorta and play an important role in maintaining the structural and functional integrity of the arterial wall. In recent years, several reports have shown that a reduction in the number of VSMCs in abdominal aortic aneurysm tissue is a key factor in its development. How to effectively inhibit the apoptosis of VSMC is expected to provide a new treatment idea for delaying the formation and development of AAA.
Disclosure of Invention
An object of the present invention is to provide a method for diagnosing abdominal aortic aneurysm by detecting ACADL gene or protein expression difference.
It is another object of the present invention to provide a method for treating abdominal aortic aneurysm by activating ACADL gene or ACADL protein.
The invention also aims to provide a method for screening a medicament for treating the abdominal aortic aneurysm.
The fourth purpose of the invention is to provide a medicine for treating abdominal aortic aneurysm.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an application of a product for detecting ACARDL in preparing an abdominal aortic aneurysm diagnosis tool.
Further, the products for detecting ACARDL comprise products for detecting the expression level of ACARDL genes.
Still further, the product for detecting ACARDL includes a product capable of quantifying mRNA of the ACARDL gene, and/or a product capable of quantifying ACARDL protein.
The product for quantifying the mRNA of the ACARDL gene of the present invention can exert its function based on a known method using a nucleic acid molecule: such as PCR, e.g., Southern hybridization, Northern hybridization, dot hybridization, Fluorescence In Situ Hybridization (FISH), DNA microarray, ASO methods, high throughput sequencing platforms, etc. The product can be used to conduct the assay qualitatively, quantitatively, or semi-quantitatively.
The nucleic acid contained in the above-mentioned products can be obtained by chemical synthesis, or by preparing a gene containing a desired nucleic acid from a biological material and then amplifying it using a primer designed to amplify the desired nucleic acid.
Further, the PCR method is a known method, for example, ARMS (Amplification Mutation System) method, RT-PCR (reverse transcriptase-PCR) method, nested PCR method, or the like. The amplified nucleic acid can be detected by using a dot blot hybridization method, a surface plasmon resonance method (SPR method), a PCR-RFLP method, an in situ RT-PCR method, a PCR-SSO (sequence specific oligonucleotide) method, a PCR-SSP method, an AMPFLP (amplifiable fragment length polymorphism) method, an MVR-PCR method, and a PCR-SSCP (single strand conformation polymorphism) method.
The product capable of quantifying the mRNA of the ACARDL gene comprises a primer used in real-time quantitative PCR for specifically amplifying the ACARDL gene, and the sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO. 2.
The primers included in the product can be prepared by chemical synthesis, appropriately designed by referring to known information using a method known to those skilled in the art, and prepared by chemical synthesis.
The above-mentioned nucleic acids may further include a probe which can be prepared by chemical synthesis, appropriately designed by referring to known information using a method known to those skilled in the art, and prepared by chemical synthesis, or can be prepared by preparing a gene containing a desired nucleic acid sequence from a biological material and amplifying it using a primer designed for amplifying the desired nucleic acid sequence.
The product of the present invention for quantifying ACADL protein can exert its function based on a known method using an antibody: for example, ELISA, radioimmunoassay, immunohistochemistry, Western blotting, etc. may be included.
The product for quantifying the ACADL protein of the present invention comprises an antibody or a fragment thereof that specifically binds to the ACADL protein. An antibody or fragment thereof of any structure, size, immunoglobulin class, origin, etc., may be used so long as it binds to the target protein. The antibodies or fragments thereof included in the assay products of the invention may be monoclonal or polyclonal. An antibody fragment refers to a portion of an antibody (partial fragment) or a peptide containing a portion of an antibody that retains the binding activity of the antibody to an antigen. Antibody fragments may include F (ab')2Fab', Fab, single chain fv (scfv), disulfide-bonded fv (dsfv) or polymers thereof, dimerized V regions (diabodies), or CDR-containing peptides. The product for quantifying ACADL protein of the present invention may include an isolated nucleic acid encoding an amino acid sequence of an antibody or encoding an antibody fragment, a vector comprising the nucleic acid, and a cell carrying the vector.
Antibodies can be obtained by methods well known to those skilled in the art. For example, mammalian cell expression vectors that retain all or part of the target protein or incorporate polynucleotides encoding them are prepared as antigens. After immunizing an animal with an antigen, immune cells are obtained from the immunized animal and myeloma cells are fused to obtain hybridomas. The antibody is then collected from the hybridoma culture. Finally, a monoclonal antibody against the ACADL protein can be obtained by subjecting the obtained antibody to antigen-specific purification using the ACADL protein or a portion thereof used as an antigen. Polyclonal antibodies can be prepared as follows: an animal is immunized with the same antigen as above, a blood sample is collected from the immunized animal, serum is separated from the blood, and then antigen-specific purification is performed on the serum using the above antigen. The antibody fragment can be obtained by treating the obtained antibody with an enzyme or by using sequence information of the obtained antibody.
Binding of the label to the antibody or fragment thereof can be carried out by methods generally known in the art. For example, proteins or peptides may be fluorescently labeled as follows: the protein or peptide is washed with phosphate buffer, a dye prepared with DMSO, a buffer, or the like is added, and the solution is mixed and left at room temperature for 10 minutes. In addition, labeling may be carried out using commercially available labeling kits, such as biotin labeling kit, e.g., biotin labeling kit-NH 2, biotin labeling kit-SH (Dojindo laboratories); alkaline phosphatase labeling kits such as alkaline phosphatase labeling kit-NH 2, alkaline phosphatase labeling kit-sh (dojindo laboratories); peroxidase labeling kits such as peroxidase labeling kit-NH 2, peroxidase labeling kit-NH 2(Dojindo Laboratories); phycobiliprotein labeling kits such as phycobiliprotein labeling kit-NH 2, phycobiliprotein labeling kit-SH, B-phycoerythrin labeling kit-NH 2, B-phycoerythrin labeling kit-SH, R-phycoerythrin labeling kit-NH 2, R-phycoerythrin labeling kit SH (dojindo laboratories); fluorescent labeling kits such as fluorescein labeling kit-NH 2, HiLyte Fluor (TM)555 labeling kit-NH 2, HiLyte Fluor (TM)647 labeling kit-NH 2(Dojindo Laboratories); and DyLight 547 and DyLight647(Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody labeling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation), and EZ-marker protein labeling kit (Funakoshi Corporation). For proper labeling, a suitable instrument can be used to detect the labeled antibody or fragment thereof.
As a sample of the detection product according to the present invention, a tissue sample or fluid obtained from a biopsy subject, for example, can be used. The sample is not particularly limited as long as it is suitable for the assay of the present invention; for example, it may comprise tissue, blood, plasma, serum, lymph, urine, serosal cavity fluid, spinal fluid, synovial fluid, aqueous humor, tears, saliva, or fractions or treated materials thereof.
In a specific embodiment of the invention, the sample is from a tissue of a subject.
Further, the product of the quantitative ACADL gene or ACADL protein may be a reagent for detecting ACADL gene or ACADL protein, or a kit, a chip, a test paper, etc. containing the reagent, or a high-throughput sequencing platform using the reagent.
The present invention also provides a tool for diagnosing abdominal aortic aneurysm, which can detect ACADL gene expression level.
Further, the means comprise agents capable of quantifying the mRNA of the ACARDL gene, and/or agents capable of quantifying the ACARDL protein.
Furthermore, the reagent capable of quantifying the mRNA of the ACARDL gene is a primer for specifically amplifying the ACARDL gene used in real-time quantitative PCR, and the sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO. 2.
Further, the means for diagnosing abdominal aortic aneurysm includes, but is not limited to, a chip, a kit, a strip, or a high throughput sequencing platform; the high-throughput sequencing platform is a special tool for diagnosing the abdominal aortic aneurysm, and with the development of a high-throughput sequencing technology, the construction of a gene expression profile of a person becomes very convenient work. By comparing the gene expression profiles of patients with diseases and normal people, the abnormality of which gene is related to the disease can be easily analyzed. Therefore, the finding that the abnormality of ACARDL gene is related to abdominal aortic aneurysm in high throughput sequencing is also included in the application of ACARDL and is also within the scope of the present invention.
The number of amino acids recognized by the anti-ACADL antibody or a fragment thereof used in the detection product, the diagnostic tool of the present invention is not particularly limited as long as the antibody can bind to ACADL.
The present invention also provides a method of diagnosing an abdominal aortic aneurysm, the method comprising the steps of:
(1) obtaining a sample from a subject;
(2) detecting the expression level of an ACADL gene or protein in a sample from the subject;
(3) correlating the measured expression level of the ACARDL gene or protein with the presence or absence of disease in the subject.
(4) If the ACADL gene or protein expression level is decreased compared to the control, the subject is judged to have or at risk of having abdominal aortic aneurysm, or a patient with abdominal aortic aneurysm is judged to have recurrence, or a patient with abdominal aortic aneurysm is judged to have poor prognosis.
The present invention also provides a method for treating abdominal aortic aneurysm, which comprises activating ACADL gene or ACADL protein.
Further, the method comprises promoting the expression of an ACADL gene, or promoting the expression of an ACADL protein or enhancing the activity of an ACADL protein.
The present invention also provides a screening method of tumor drug, which can determine the effect of tumor drug in improving tumor prognosis by measuring the expression level of ACARDL gene or ACARDL protein after adding test drug to cancer cells or at a certain period after administering test drug to tumor model animals. More specifically, when the expression level of ACADL gene or ACADL protein is elevated or restored to a normal level after addition or administration of a test drug, the drug can be selected as a therapeutic drug for improving the prognosis of tumor.
The invention also provides a medicament for treating abdominal aortic aneurysm, which comprises an activator of ACADL.
The activator of ACADL of the invention is not limited as long as the activator can promote or enhance the expression or activity of ACADL or a substance involved in the upstream or downstream pathway of ACADL, and is a drug effective for treating tumors.
The invention also provides application of the activator in preparing a medicament for treating abdominal aortic aneurysm.
Further, the activator comprises an ACADL gene, an ACADL protein, a promoting miRNA, a promoting transcription regulatory factor, or a promoting targeting small molecule compound.
The activator can be used for supplementing the deletion or deficiency of endogenous ACADL protein and treating abdominal aortic aneurysm caused by ACADL protein deficiency by improving the expression of the ACADL protein. On the other hand, the protein can be used for enhancing the activity of the ACARDL protein, thereby treating abdominal aortic aneurysm.
The agonist drugs of the present invention may be used by formulating pharmaceutical compositions in any manner known in the art. Such compositions comprise the active ingredient in admixture with one or more pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the mode of administration and the dosage form envisaged. Therapeutically inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols such as polyethylene glycol, water, sucrose, ethanol, glycerol and the like, various preservatives, lubricants, dispersants, flavoring agents. Moisturizers, antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like may also be added as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration, formulations which may be used in such compositions may be in the form of their original compounds as such, or optionally in the form of their pharmaceutically acceptable salts, and the agonist drugs of the present invention may be administered alone, or in various combinations, as well as in combination with other therapeutic agents. The compositions so formulated may be administered in any suitable manner known to those skilled in the art for administration of agonist drugs as desired. In using the pharmaceutical compositions, a safe and effective amount of an inhibitor of the present invention is administered to a human, wherein the safe and effective amount is typically at least about 100 micrograms per kilogram of body weight for oral administration. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The medicine of the present invention may be prepared into various preparation forms. Including, but not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories for transdermal, mucosal, nasal, buccal, sublingual or oral use.
The route of administration of the drug of the present invention is not limited as long as it exerts the desired therapeutic or prophylactic effect, and includes, but is not limited to, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intratracheal, subcutaneous, transdermal, transpleural, topical, inhalation, transmucosal, dermal, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, intrahepatic, intratumoral. In some cases, the administration may be systemic. In some cases topical administration.
The dose of the drug of the present invention is not limited as long as the desired therapeutic effect or prophylactic effect is obtained, and can be appropriately determined depending on the symptoms, sex, age, and the like. The dose of the therapeutic agent or prophylactic agent of the present invention can be determined using, for example, the therapeutic effect or prophylactic effect on a disease as an index.
In the context of the present invention, "diagnosing an abdominal aortic aneurysm" includes determining whether a subject has had an abdominal aortic aneurysm, determining whether a subject is at risk of having an abdominal aortic aneurysm, determining whether a patient with an abdominal aortic aneurysm has relapsed and metastasized, determining responsiveness of a patient with an abdominal aortic aneurysm to treatment with a drug, or determining a prognosis of a patient with an abdominal aortic aneurysm.
As used herein, "treatment" encompasses treatment-related diseases or disease states in a mammal, such as a human, having the associated disease or disorder, and includes:
(1) preventing the occurrence of a disease or condition in a mammal, particularly when the mammal is susceptible to said disease condition but has not been diagnosed as having such a disease condition;
(2) inhibiting a disease or disease state, i.e., preventing its occurrence; or
(3) Alleviating the disease or condition, i.e., causing regression of the disease or condition.
The term "treatment" generally refers to the treatment of a human or animal (e.g., as applied by a veterinarian) wherein some desired therapeutic effect is achieved, e.g., inhibiting the progression of a condition (including slowing the progression, stopping the progression), ameliorating the condition, and curing the condition. Treatment as a prophylactic measure (e.g., prophylaxis) is also included. The use of a patient who has not yet developed a condition but who is at risk of developing the condition is also encompassed by the term "treatment".
The invention has the advantages and beneficial effects that:
the invention discovers the molecular marker for diagnosing the abdominal aortic aneurysm, can be used for judging the early occurrence of the abdominal aortic aneurysm by using the molecular marker, and provides the survival rate of patients.
The therapeutic agent of the present invention comprising an activator of ACADL gene or protein can be used as a novel therapeutic agent for abdominal aortic aneurysm.
Drawings
FIG. 1 shows the detection of differential expression of ACARDL gene in abdominal aortic aneurysm tissue and normal control tissue using QPCR;
FIG. 2 shows the differential expression of ACARDL protein in abdominal aortic aneurysm tissue and normal control tissue detected by Western blot experiment;
FIG. 3 shows the detection of the inhibition rate of ACARDL gene expression by Western blot experiment.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.
Example 1 Gene chip screening for differentially expressed genes
1. Tissue acquisition and processing: collecting 2 parts of AAA artery wall middle layer living body specimen and 2 parts of infrarenal normal aorta control tissue, cutting the aorta wall at a position about 4cm below the flat renal artery under the conditions of sterility and no RNase, washing with sterilized physiological saline to remove blood stain, removing the artery adventitia from the taken tissue, and rapidly (for <5min) freezing in liquid nitrogen for standby.
2. RNA extraction, cDNA Synthesis
Respectively placing aneurysm tissues preserved by liquid nitrogen in a ceramic mortar, grinding the aneurysm tissues into powder in a liquid nitrogen low-temperature environment, adding Trizol reagent for homogenizing, centrifuging, taking supernate, extracting twice by using 1: 1 acidic phenol-chloroform, extracting by using sodium acetate and 5: 1 acidic phenol-chloroform, precipitating by using isopropanol with the same volume, dissolving the precipitate by using Milli-Q water after centrifuging, measuring an OD value (A260/A280) by using an ultraviolet spectrophotometer, and detecting 28S and 18S rRNA bands by using formaldehyde denaturing gel electrophoresis.
3. Chip processing
The HGEC40s type expression profiling chip was manufactured by Shanghai Boxing Gene chip Co. 4096 full-length genes are subjected to Polymerase Chain Reaction (PCR) amplification by using universal primers at two ends of a carrier, the length of a product is 1000-3000 bp, agarose electrophoresis is used for detecting the PCR product, the concentrated amplification product is used as a target gene to be dissolved in 3 XSSC (x sequence number) sample application liquid, and a Cartesian Pixsys 7500 sample application instrument is used for carrying out sample application on a 18mm x 18mm glass slide according to the distance of 0.28 mm. After spotting, the slide glass is hydrated, dried at room temperature, UV-crosslinked, treated with 0.2% SDS, water and 0.2% sodium borohydride solution for 10min, and dried for later use.
4. Probe preparation
Oligolex mRNA Midi Kits (Qiagen, USA) purified mRNA, and appropriate amount of total RNA was collected according to the concentration of RNA solution, and purified by Oligold T affinity column to obtain pure mRNA. cDNA probes were synthesized and purified by reverse transcription using the method described in reference [ SchenaM, Shalon D, Davis RW, et al.quantitative monitoring of gene expression patterns with a complementary DNA micro array, science,1995,270: 467-. After ethanol precipitation, Cy3-dUTP and Cy5-dUTP labeled probes were mixed and dissolved in 20. mu.l of 5 XSSC + 0.2% SDS hybridization solution.
5. Hybridization and washing: the HGEC40s expression spectrum chip and the hybridization probe are respectively denatured at 95 ℃ and then placed in a hybridization chamber, the mixed probe is added, and the hybridization chamber is sealed without air bubbles. The hybridization was carried out in a constant humidity hybridization chamber at 60 ℃ for 16 h.
6. Detection and molecular biological information analysis of differential genes: after scanning the chip with a GenePix 4000B fluorescence scanner, the GenePix Pro3.0 software analyzed the intensity and ratio of 2 fluorescent signals. The equalization of Cy3 and Cy5 was performed with 40 housekeeping genes. Subtracting the foreground value and the background value of all data to obtain intensity values marked by Cy3 and Cy5, and calculating a natural logarithmic average value, namely a homogenization coefficient, of the genes Cy5/Cy3 according to the natural logarithmic value lnRi of Cy5/Cy3 of the effective genes with the total number of n, i.e. lnCy5/Cy3, and taking the effective genes with the Ri value of 0.1-10.0 as a homogenization basis. The Cy3 labeled intensities for all data items were multiplied by a normalization factor to give adjusted Cy3 values. All intensity values with Cy3 values less than 200 were substituted for bioinformatics analysis results by 200: the intensity and ratio of 2 fluorescent signals were analyzed using GenePix Pro3.0 software.
7. Bioinformatics analysis results
The intensity and ratio of 2 fluorescent signals were analyzed using GenePix Pro3.0 software. The results showed 432 differentially expressed genes, 278 of which was up-regulated and 154 of which was down-regulated.
Example 2 validation of differentially expressed genes in Large samples
The ACARDL which expresses differentially and is suggested by the expression selection gene chip in the abdominal aortic aneurysm tissue is selected as a research target for reverse verification.
1. Tissue acquisition and processing: 30 AAA artery wall media biopsy specimens and 40 infrarenal normal aortic control tissues were collected as described in example 1.
2. RNA extraction
RNA extraction was performed according to the method of example 1.
3. Reverse transcription
Mu.g of total RNA was reverse transcribed with reverse transcription buffer to synthesize cDNA. A25-mu-l reaction system is adopted, 1 mu g of total RNA is taken from each sample as template RNA, and the following components are respectively added into a PCR tube: DEPC water, 5 Xreverse transcription buffer, 10mmol/L dNTP, 0.1mmol/L DTT, 30. mu. mmol/L Oligo dT, 200U/. mu. L M-MLV, template RNA. Incubate at 42 ℃ for 1h, 72 ℃ for 10min, and centrifuge briefly.
4、QPCR
A25. mu.l reaction was used, with 3 parallel channels per sample. The following reaction system was prepared: 12.5 ul of SYBR Green polymerase chain reaction system, 1 ul of forward primer (5 uM), 1 ul of reverse primer (5 uM), 2.0 ul of template cDNA2, and 8.5 ul of enzyme-free water; the forward primer sequence for amplifying the ACARDL gene is 5'-TAGTATTCATTCAGGTATTGTC-3' (SEQ ID NO.1), and the reverse primer sequence is 5'-GCTCTGTCATTGCTATTG-3' (SEQ ID NO. 2); the forward primer sequence for amplification of the GAPDH gene was 5'-AAAGGGTCATCATCTCTG-3' (SEQ ID NO.3), and the reverse primer sequence was 5'-GCTGTTGTCATACTTCTC-3' (SEQ ID NO.4), all on ice. The amplification procedure was: 95 ℃ for 5min, (95 ℃ 10s, 60 ℃ 55s) 45 cycles. SYBR Green is used as a fluorescent marker, PCR reaction is carried out on a Light Cycler fluorescent real-time quantitative PCR instrument, a target band is determined through melting curve analysis and electrophoresis, and relative quantification is carried out through a delta CT method.
5. Western blot detection
Shearing the tissue, mechanically homogenizing, and centrifuging at 4 deg.C for 10min at 12000 r min. Total protein concentrations were determined by coomassie brilliant blue R250 staining, and each histone concentration was adjusted to the same level. A10% SDS-polyacrylamide gel was prepared, and 100. mu.g of the protein sample was added to each well, and after electrophoresis, the protein was transferred to a nitrocellulose filter by a semi-dry type electrotransfer (Bio-Rad, USA). Ponceau S staining determines the transmembrane status and marks the protein Marker position. 5% skimmed milk powder TBS buffer solution is sealed in a refrigerator at 4 ℃ for overnight; diluting the primary antibody at a ratio of 1: 1000, shaking for 2h at room temperature, and washing the membrane for 3 times with TBS; adding peroxidase-labeled goat anti-rabbit IgG-HRP at a ratio of 1: 1000 for 60min, washing with TBS for 3 times, adding ECL for 2-3min, developing in dark room for 2min, and washing the film. And (5) performing camera shooting analysis by using a gel imaging analysis system.
And analyzing the gray value of the protein band by using Image J software, and normalizing the gray value of the ACARDL protein band by using beta-actin as an internal reference. The results were expressed as mean ± sd, statistically analyzed using SPSS13.0 statistical software, and the difference between the two was considered statistically significant when P <0.05 using the t-test.
6. Results
(1) QPCR results
As shown in fig. 1, ACADL gene mRNA levels were significantly reduced in abdominal aortic aneurysm tissues compared to normal control tissues, with the difference statistically significant (P < 0.05).
(2) Western blot results
As shown in fig. 2, ACADL protein levels were significantly reduced in abdominal aortic aneurysm tissue compared to normal control tissue, with statistical significance for the difference (P < 0.05).
Example 3 overexpression of ACARDL Gene expression
1. ACARDL gene recombinant plasmid construction
(1) Amplifying the coding sequence of the ACARDL gene;
(2) designing an amplification primer;
(3) the amplified ACADL gene is connected to expression vector pcDNA3.0 to construct pcDNA3.0-ACADL recombinant expression vector.
2. Culture of human aortic smooth muscle cells
T/G HA-VSMC (HVSMC) cells were cultured in DMEM high-glucose medium containing 10% Fetal Bovine Serum (FBS) with penicillin 100units/ml and streptomycin 100. mu.g/ml at 37 ℃ in 5% CO2The culture medium is changed for 1 time every 24 hours, and the culture medium is subcultured for 1 time in 48 hours. Cells in logarithmic growth phase were taken for subsequent experiments.
3. Cell transfection
Liposome Lipofectamine2000 was used as the transfection reagent. Experiment was divided into 2 groups: negative control group (pcDNA3.0 transfected); experimental group (pcDNA3.0-ACARDL transfection). HVSMC cells were harvested in logarithmic growth phase and plated in 6-well cell culture plates. After 24h, the coverage rate of the cell culture plate is about 70-80%. The transfection protocol was performed according to Lipofectamine2000 instructions.
4. Western blot experiment for detecting overexpression condition of pcDNA3.0-ACADL
The procedure is as in example 2.
5. Results
As shown in FIG. 3, compared with the negative control group (pcDNA3.0 transfected), the ACADAL protein expression level in the cells of the experimental group (pcDNA3.0-ACADAL transfected) was significantly increased, and the difference was statistically significant (P < 0.05).
Example 4 Effect of ACARDL Gene expression on apoptosis of human aortic smooth muscle cells
The influence of the ACARDL gene expression on the smooth muscle cell apoptosis is detected by utilizing an Annexin V-PE/7-AAD apoptosis detection kit (purchased from KTK102-020, Beijing Keyue Biotech Co., Ltd.).
1. Cell culture and transfection
The procedure is as in example 3.
2. Apoptosis induction and detection
After 24 hours of cell transfection, each group of cells was cultured in serum-free DMEM medium to induce apoptosis. After 48 hours, the cells were collected by trypsinization, washed 2 times with PBS, analyzed according to the instructions, added with binding buffer (200. mu.l), Annexin V-PE (10. mu.l) and 7-AAD (5. mu.l), incubated for 15min in the dark, finally added with 300. mu.l of ending buffer, and the machine was operated to detect the change in apoptosis.
3. Results
The results show that after each group of cells are starved and stimulated for 48 hours in serum-free culture medium, the apoptosis rate detected by a flow cytometer shows that the apoptosis rate of pcDNA3.0-ACADL transfected group cells is (13.428 +/-1.196)%; the apoptosis rate of the negative control group is (31.627 +/-1.513)%; the apoptosis rate of pcDNA3.0-ACADL transfection group is obviously reduced compared with that of negative control group, the difference has statistical significance (P <0.05), and the result shows that the promotion of ACADL gene expression can inhibit the apoptosis of human aortic smooth muscle cells. Since the occurrence of abdominal aortic aneurysm is caused by the increase of apoptosis of human aortic smooth muscle cells, the promotion of ACADL gene expression can achieve the purpose of treating abdominal aortic aneurysm.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
SEQUENCE LISTING
<110> Beijing, the deep biometric information technology GmbH
<120> ACARDL gene and application of expression product thereof in preparation of abdominal aortic aneurysm diagnosis and treatment product
<160> 4
<170> PatentIn version 3.5
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<213> Artificial sequence
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Claims (2)
1. Application of ACARDL expression promoting agent in preparing medicine for treating abdominal aortic aneurysm is disclosed.
2. Use according to claim 1, wherein said agent is an overexpression vector of ACADL.
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