阅读说明：本技术 早期诊断心房颤动用的gan基因 (GAN gene for early diagnosis of atrial fibrillation ) 是由 曹月娟 崔丽 李阳春 张建艳 郭兆增 妥少勇 赵金艳 于 2019-11-05 设计创作，主要内容包括：本发明涉及早期诊断心房颤动用的GAN基因。本发明还涉及用于诊断心房颤动的试剂和方法,所述试剂包括测量GAN基因mRNA和蛋白水平的试剂,所述试剂可用于确定心房颤动的发病或发病的可能性。通过提供用于诊断心房颤动的标志物,可进行心房颤动发病的可能性的早期诊断。(The present invention relates to a GAN gene for early diagnosis of atrial fibrillation. The invention also relates to reagents and methods for diagnosing atrial fibrillation, including reagents that measure GAN gene mRNA and protein levels, which can be used to determine the onset or likelihood of onset of atrial fibrillation. By providing a marker for diagnosing atrial fibrillation, early diagnosis of the possibility of the onset of atrial fibrillation can be performed.)

The use of the GAN gene or its expression product in the preparation of products for the diagnosis of atrial fibrillation.

2. The use of claim 1, wherein the GAN gene or expression product thereof is down-regulated in patients with atrial fibrillation.

3. The use of claim 1 or 2, wherein the diagnostic product comprises a diagnostic agent at the gene level and a diagnostic agent at the protein level.

4. The use according to claim 3, wherein the gene level diagnostic reagent comprises a reagent for detecting the gene expression level by a real-time fluorescent quantitative PCR method or a gene chip method; diagnostic reagents at the protein level include reagents for detecting the expression level of the gene expression product by immunological methods.

5. A kit for detecting atrial fibrillation comprising primers that specifically amplify the GAN gene.

6. The kit of claim 5, wherein the primers comprise an upstream primer shown as SEQ ID No.1 and a downstream primer shown as SEQ ID No. 2.

7. The kit of claim 5 or 6, wherein the kit further comprises PCR buffer, Taq enzyme, and dNTPs.

8. A pharmaceutical composition for treating atrial fibrillation comprising an activator of a GAN gene or its expression product.

9. The pharmaceutical composition of claim 8, wherein the activator promotes GAN gene expression by constructing an overexpression vector.

Use of the GAN gene or its expression product in the preparation of a medicament for the treatment of atrial fibrillation.

Technical Field

The present invention relates to the field of biomedicine, and more particularly, to the early diagnosis of the GAN gene for atrial fibrillation.

Background

Atrial fibrillation, called atrial fibrillation for short, is the most common sustained arrhythmia in clinic. During atrial fibrillation, the atria lose normal and effective contraction function and are in a rapid and disordered fibrillation state, the frequency can be as fast as 300-. Atrial fibrillation is a common arrhythmia, and the incidence of atrial fibrillation increases with age. Approximately 1% of patients are <60 years of age, with an incidence rate of up to 12% in the 75-84 year age group. The actual incidence of atrial fibrillation may be higher because some patients do not have significant symptoms to treat. Atrial fibrillation can cause serious complications and place a heavy burden on families and society if not properly treated, and has become an increasingly serious public health problem.

Atrial fibrillation can be classified into the following according to its onset time:

paroxysmal atrial fibrillation, namely the attack of atrial fibrillation lasts for 7 days, mostly within 48 hours, and can be automatically converted into sinus rhythm.

Persistent atrial fibrillation, in which the attack lasts for more than 7 days, and a sinus rhythm can be recovered only by medicines or electric shocks.

Permanent atrial fibrillation, which cannot be stopped by self or recurred after termination.

Atrial fibrillation is not afraid, but stroke and cardiac function deterioration caused by atrial fibrillation are important causes of death and disability of patients. The atrial fibrillation causes palpitation, chest distress, dizziness, even faint and the like of a patient, and leads to obvious reduction of physical strength and life quality. Atrial fibrillation induces or aggravates comorbid heart disease, resulting in heart failure, pulmonary edema, angina pectoris, shock, sudden death, etc. The most serious problem with atrial fibrillation is that of causing thromboembolism. The incidence of stroke of patients with atrial fibrillation is 5 times 2 of that of healthy people. In atrial fibrillation, the atria lose their contractile function, and blood tends to stagnate in the atria to form thrombus. The thrombus can fall down at any time and circulate to all parts of the body along with the blood, once the thrombus is clamped at a certain position, the thrombus can cause the blockage of blood vessels, and the embolism of limb arteries, mesenteric arteries and even cerebral arteries can cause disability in mild cases and death in severe cases. Frequent medical visits are required and the expenditure is significantly increased.

In general, the simplest and most easily recognized sign of atrial fibrillation is the irregular pulse. The symptoms of the disease generally include palpitation, shortness of breath, dizziness, fainting, chest pain or discomfort, and many patients with atrial fibrillation have no symptoms or obvious and specific symptoms, and the atrial fibrillation is usually diagnosed after stroke. Thus, each year there are strokes due to potential atrial fibrillation, millions of people dying prematurely or being paralyzed for life. Which could otherwise be avoided by prophylaxis.

According to the application, biomarkers related to atrial fibrillation are screened on a gene level, the diagnosis purpose can be realized at the early stage of atrial fibrillation, and the probability of stroke of a patient is reduced.

Disclosure of Invention

One of the purposes of the present invention is to provide the application of GAN gene or its expression product in preparing products for diagnosing atrial fibrillation.

It is another object of the present invention to provide a kit for diagnosing atrial fibrillation.

The invention also aims to provide a medicament for treating atrial fibrillation.

It is a further object of the present invention to provide a method for diagnosing atrial fibrillation.

In order to achieve the above objects, the present invention provides, in a first aspect, a use of a GAN gene or an expression product thereof for the manufacture of a diagnostic product for atrial fibrillation.

Preferably, the GAN gene or expression product thereof is down-regulated in atrial fibrillation patients.

Further, the diagnostic product comprises a diagnostic agent at a gene level or a diagnostic agent at a protein level.

Further, the diagnostic reagent at the gene level includes a reagent for detecting the expression level of a gene by a real-time fluorescent quantitative PCR method or a gene chip method; diagnostic reagents at the protein level include reagents for detecting the expression level of the gene expression product by immunological methods.

Preferably, the real-time fluorescent quantitative PCR method includes a SYBRGreen method and a TaqMan probe method.

Preferably, the gene chip comprises a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, wherein the oligonucleotide probe comprises a part or all of a nucleotide sequence for specifically capturing the GAN gene.

Preferably, the immunological method is an ELISA detection method.

Preferably, the ELISA method is using an ELISA detection kit comprising: a solid phase carrier coated with the GAN monoclonal antibody, an enzyme labeled antibody, an enzyme substrate, a product standard substance, a negative control substance, a diluent, a washing liquid, an enzyme reaction stopping liquid and the like. The antibody may be a commercially available monoclonal antibody or may be obtained by a method known to those skilled in the art.

The invention also provides a kit for detecting atrial fibrillation, which comprises a primer for specifically amplifying the GAN gene.

Preferably, the primers comprise an upstream primer shown as SEQ ID NO.1 and a downstream primer shown as SEQ ID NO. 2.

Preferably, the kit further comprises PCR buffer, Taq enzyme, and dNTPs.

Preferably, the kit further comprises a negative control and a positive control.

The invention also provides a pharmaceutical composition for treating atrial fibrillation. The pharmaceutical composition comprises an activator of the GAN gene or its expression product.

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, such carriers including (but not limited to): diluents, buffers, suspensions, emulsions, granules, encapsulating agents, excipients, fillers, adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavors, or adsorptive carriers.

The pharmaceutical composition of the present invention can be prepared into various dosage forms as required, including, but not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories.

The route of administration of the pharmaceutical composition of the present invention is not limited as long as it can exert a desired therapeutic or prophylactic effect, and includes, but is not limited to, oral, intravenous, intramuscular, subcutaneous, sublingual, rectal infusion, nasal spray, oral spray, transdermal topical or systemic administration to the skin.

The pharmaceutical composition of the present invention can also be used in combination with other drugs for treating atrial fibrillation, and the combination of a plurality of drugs can greatly improve the success rate of treatment.

Preferably, the activator promotes the expression or enhances the activity of the GAN gene. Preferably, the activator promotes GAN gene expression by constructing an overexpression vector.

The activators of the present invention can be used, on the one hand, to supplement the absence or deficiency of the endogenous GAN gene product, and, by increasing the expression of the endogenous product, to treat atrial fibrillation resulting from the lack of the endogenous product. In another aspect, the compounds may be used to enhance the activity of endogenous products, thereby treating atrial fibrillation.

Preferably, the vector is any of a variety of vectors known in the art, such as commercially available vectors, including plasmids, cosmids, phages, viruses, and the like.

The present invention provides a method for diagnosing atrial fibrillation, which includes detecting the GAN gene expression level in a sample from a subject.

The invention also provides a method of treating atrial fibrillation comprising promoting GAN gene expression.

The invention also provides the application of the GAN gene or the expression product thereof in preparing the medicine for treating atrial fibrillation.

Definition of

The term "detection" is used herein in the broadest sense and includes both qualitative and quantitative measurements of a target molecule. Detection includes merely identifying the presence of the target molecule in the sample and determining whether the target molecule is present at a detectable level in the sample.

The term "biomarker" as used herein refers to an indicator that can be detected in a sample, e.g., predictive, diagnostic, and/or prognostic. Biomarkers can serve as indicators of particular subtypes of disease or disorder (e.g., cancer) characterized by certain molecular, pathological, histological, and/or clinical features. In some embodiments, the biomarker is a gene. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and/or RNA), polynucleotide copy number alterations (e.g., DNA copy number), polypeptides and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and/or glycolipid-based molecular markers.

The term "expression level" refers to the amount of a biomarker in a biological sample. "expression" generally refers to the process by which information (e.g., gene-encoded and/or epigenetic) is converted into structures present and operating in a cell. Thus, as used herein, "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., post-translational modifications of a polypeptide). Transcribed polynucleotides, translated polypeptides, or fragments of polynucleotide and/or polypeptide modifications (e.g., post-translational modifications of polypeptides) should also be considered expressed, whether they are derived from transcripts generated or degraded by alternative splicing, or from post-translational processing of polypeptides, e.g., by proteolysis.

"amplification" as used herein generally refers to the process of generating multiple copies of a desired sequence. "multiple copies" means at least two copies. "copy" does not necessarily imply complete sequence complementarity or identity to the template sequence. For example, the copies may include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced via primers comprising sequences that are hybridizable but not complementary to the template), and/or sequence errors that occur during amplification.

As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by synthetic reaction. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and their analogs. Modifications to the nucleotide structure, if present, may be made before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide building blocks. The polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, "caps", substitution of one or more naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with noCharge linkages (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.) and those with charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), those containing pendant moieties (pendantmoeity) such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radiometals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., α anomeric nucleic acids (anomeric nucleic acid), etc.), and unmodified forms of polynucleotides, moreover, any hydroxyl groups typically present in the sugar may be replaced with, for example, phosphonate groups, phosphate groups, protected with standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be attached to a solid or solid support or may be substituted with, for example, a phosphoribosyl phosphate group such as a phosphoribosyl-phosphate-O-ribosyl-phosphate group, or other sugar-substituted with a standard protecting group such as a phosphoribosyl-O-ribosyl sugar-phosphate group (e.g., as well as the art-O-phosphate-O-sugar-O ₂("amide ester"), P (O) R, P (O) OR', CO OR CH ₂(formacetal)) wherein each R or R' is independently H or a substituted or unsubstituted hydrocarbyl group (1-20C), optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aryl hydrocarbyl (araldyl). Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

As used herein, "oligonucleotide" generally refers to short, single-stranded polynucleotides that are less than about 250 nucleotides in length, although this is not required. The oligonucleotide may be synthetic. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description for polynucleotides is equally and fully applicable to oligonucleotides.

The term "primer" refers to a single-stranded polynucleotide capable of hybridizing to a nucleic acid and allowing polymerization of the complementary nucleic acid, typically by providing a free 3' -OH group.

The term "diagnosis" is used herein to refer to the identification or classification of a molecular or pathological state, disease or condition.

As used herein, the term "sample" refers to a composition obtained or derived from a subject and/or individual of interest that contains cells and/or other molecular entities to be characterized and/or identified, e.g., based on physical, biochemical, chemical and/or physiological characteristics. For example, the phrase "disease sample" or variants thereof refers to any sample obtained from a subject of interest that is expected or known to contain the cellular and/or molecular entities to be characterized. Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, and tissue culture fluids, tissue extracts such as homogenized tissue, cell extracts, and combinations thereof.

By "tissue sample" or "cell sample" is meant a collection of similar cells obtained from a tissue of a subject or individual. The source of the tissue or cell sample may be a solid tissue such as from a fresh, frozen and/or preserved organ, a tissue sample, a biopsy, and/or an aspirate; blood or any blood component such as plasma; bodily fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from a subject at any time during pregnancy or development. The tissue sample may also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a diseased tissue/organ. Tissue samples may contain compounds that are not naturally intermixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

As used herein, "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be performed during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating the disease state, and remission or improving prognosis.

Drawings

FIG. 1 shows the detection of GAN gene expression in patients with atrial fibrillation and normal persons by QPCR;

FIG. 2 shows a ROC curve for assessing the discriminatory power of the GAN gene.

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.