Function and application of adipocyte-enhanced binding protein 1 in treating tissue fibrosis

Abstract

The application provides functions and applications of adipocyte enhancer binding protein 1 (AEBP1) in treating tissue fibrosis, and specifically provides a use of a preparation in preparing a medicine, wherein the medicine is used for treating or preventing a fibrosis-related disease, and the preparation is used for improving the activity of AEBP1. In the application, the inventors find that low expression of AEBP1 promotes activation of fibroblasts, reduces the proliferation ability of the fibroblasts, reduces the function of tissues, and easily causes fibrosis-related diseases. After overexpression treatment of AEBP1, the fibroblasts maintain a resting state and the proliferation ability is improved. Therefore, the medicine prepared from the preparation for improving the activity of AEBP1 can effectively prevent or treat the fibrosis-related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular, the present application relates to the function and application of adipocyte enhancer binding protein 1 in treating tissue fibrosis. BACKGROUND

[0002] Tissue fibrosis is one of the main reasons for the occurrence and development of diseases, and the deterioration of diseases. For example, in heart diseases, myocardial fibrosis is a characteristic pathological change independent of myocardial hypertrophy in the development of hypertrophic cardiomyopathy (HCM), and is the pathological basis of ventricular arrhythmia and sudden death, which is closely related to the prognosis of patients. 60% of HCM patients have various forms of myocardial fibrosis, of which the most common subtypes are replacement fibrosis and interstitial fibrosis. So far, cardiac fibrosis is still an important target and difficulty in the clinical treatment of heart diseases. The manifestation of cardiac fibrosis is the excessive deposition of extracellular matrix in cardiac tissue, which leads to the destruction of physiological cardiac tissue structure, causes cardiac dysfunction, and eventually develops into heart failure, which seriously threatens human health and life.

[0003] Changes in gene expression in fibroblasts are the starting point and maintenance center of the cardiac fibrosis response. Existing studies have found that the transcription factor adipocyte enhancer binding protein 1 (AEBP1, also known as Aortic Carboxypeptidase-Like Protei, ACLP, Aortic Carboxypeptidase-Like Protein) is significantly up-regulated in activated fibroblasts and activated myofibroblasts, and both of these two cell subgroups highly express the characteristic genes COL1A2 and POSTN of tissue fibrosis. Conventional Bulk RNA-seq data also supports this finding, and spatial transcriptome data also confirms that the AEBP1 high expression area is severely fibrotic. Through GO function enrichment and transcription factor target gene prediction analysis, it is found that AEBP1 is a new regulator of cardiac fibrosis in HCM patients. However, so far it is not clear what role AEBP1 plays in cell life activities and diseases.

[0004] Myofibroblasts are mainly differentiated from fibroblasts. Myofibroblasts have important contraction and secretion functions, and are characterized by high expression of α-smooth actin (αSMA), fibronectin, and collagen I (Col I). TGFβ is a recognized agent in current research that can induce the activation of fibroblasts, and can be used to treat mice or fibroblasts, for example, to simulate the state of cardiac fibrosis.

[0005] Since treatment of fibrosis-related diseases can delay the occurrence and development of organ failure, finding targets and drugs for treating fibrosis-related diseases is an important problem to be solved. SUMMARY

[0006] The present application aims to at least partially solve one of the problems in the above-mentioned related art. The present inventors have found that a decrease in AEBP1 expression level promotes activation of fibroblasts, reduces their proliferation ability, leads to reduced tissue function, and causes or aggravates diseases such as tissue fibrosis, for example, cardiac fibrosis, myocardial hypertrophy, and the like, thereby providing a new idea for treatment and prevention of fibrosis-related diseases.

[0007] Therefore, in a first aspect of the present application, the present application provides a use of a preparation in the manufacture of a medicament for treating or preventing a fibrosis-related disease, wherein the preparation is used to increase the activity of AEBP1. According to some embodiments of the present application, a decrease in AEBP1 expression level promotes activation of fibroblasts, reduces their proliferation ability, leads to reduced tissue function, and causes or aggravates fibrosis-related diseases, such as hypertrophic cardiomyopathy and cardiac fibrosis accompanying the development of hypertrophic cardiomyopathy. Therefore, a preparation capable of increasing the activity of AEBP1 can effectively prevent or treat fibrosis-related diseases.

[0008] According to embodiments of the present application, the above-mentioned pharmaceutical use can further include at least one of the following additional technical features:

[0009] According to embodiments of the present application, the fibrosis-related disease includes at least one of myocardial hypertrophy, heart failure, liver cirrhosis, and tissue fibrosis.

[0010] According to some embodiments of the present application, the preparation capable of increasing the activity of AEBP1 can effectively maintain fibroblasts in a resting state and promote proliferation of fibroblasts, and can effectively treat or prevent fibrosis-related diseases, such as cardiac fibrosis and myocardial hypertrophy.

[0011] According to embodiments of the present application, the preparation includes at least one of the following: an AEBP1 small molecule agonist, a nucleic acid molecule that upregulates AEBP1 gene expression, and an antibody that specifically recognizes an AEBP1 antagonist. Those skilled in the art can understand that the preparation is not particularly limited, and substances capable of effectively increasing the activity of AEBP1 are within the scope of protection of the present application, and the substances can be at least one of a nucleic acid, a protein, and a compound.

[0012] According to an embodiment of the present application, the nucleic acid molecule for up-regulating the expression of the AEBP1 gene is carried on a viral vector, a liposome, or a self-replicating RNA vector. The nucleic acid molecule for up-regulating the expression of the AEBP1 gene can be carried on a vector that can deliver the nucleic acid molecule, and the type of the vector is not particularly limited.

[0013] According to an embodiment of the present application, the viral vector is an adeno-associated viral vector.

[0014] In a second aspect of the present application, the present application provides a use of a reagent in the preparation of a kit or device. According to an embodiment of the present application, the kit or device is used for diagnosing a fibrosis-related disease, and the reagent is used for detecting the activity of AEBP1. As described above, a decrease in the expression level of AEBP1 promotes the activation of fibroblasts, reduces the proliferation ability of the fibroblasts, and reduces the function of each tissue derived from the fibroblasts, such as a decrease in the function of the heart, which can easily cause a heart disease. After the overexpression of AEBP1, the fibroblasts maintain a resting state, the proliferation ability of the fibroblasts is increased, and the degree of fibrosis is reduced. Thus, AEBP1 can be used as a target for the diagnosis of a fibrosis-related disease, and the level of AEBP1 can be detected to determine whether an individual has or is susceptible to a fibrosis-related disease. Further, the individual who has a heart disease can be staged according to the quantitatively detected level of AEBP1, or the prognosis of the individual who has a fibrosis-related disease can be evaluated by monitoring the level of AEBP1 in the individual after treatment.

[0015] According to an embodiment of the present application, the use of the kit or device described above can further include at least one of the following additional technical features:

[0016] According to an embodiment of the present application, the fibrosis-related disease includes at least one of cardiac hypertrophy, heart failure, liver cirrhosis, and tissue fibrosis.

[0017] According to an embodiment of the present application, the tissue fibrosis includes at least one of cardiac fibrosis, pulmonary fibrosis, renal fibrosis, liver fibrosis, and tumor fibrosis.

[0018] According to an embodiment of the present application, the reagent includes a primer for amplifying the AEBP1 gene or a fragment thereof or an antibody that binds to AEBP1.

[0019] In a third aspect of the present application, a medicament for treating or preventing a fibrosis-related disease is provided. According to embodiments of the present application, the medicament comprises an agent that increases AEBP1 activity. According to some embodiments of the present application, the agent that increases AEBP1 activity is effective in maintaining fibroblasts in a quiescent state and promoting proliferation of fibroblasts, and is effective in treating or preventing a fibrosis-related disease, such as cardiac fibrosis and cardiac hypertrophy. Further, the medicament comprising the agent that increases AEBP1 activity is also effective in treating or preventing a fibrosis-related disease.

[0020] According to embodiments of the present application, the medicament can further comprise at least one of the following additional technical features:

[0021] According to embodiments of the present application, the agent comprises at least one of the following: an AEBP1 small molecule agonist, a nucleic acid molecule that upregulates AEBP1 gene expression, and an antibody that specifically recognizes an AEBP1 antagonist.

[0022] According to embodiments of the present application, the fibrosis-related disease comprises at least one of the following: cardiac hypertrophy, heart failure, liver cirrhosis, and tissue fibrosis.

[0023] According to embodiments of the present application, the tissue fibrosis comprises at least one of the following: cardiac fibrosis, lung fibrosis, kidney fibrosis, liver fibrosis, and tumor fibrosis.

[0024] According to embodiments of the present application, the nucleic acid molecule that upregulates AEBP1 gene expression is carried on a viral vector, a liposome, or a self-replicating RNA vector.

[0025] According to embodiments of the present application, the viral vector is an adeno-associated viral vector.

[0026] According to embodiments of the present application, the medicament further comprises a pharmaceutically acceptable carrier, except to the extent that any conventional adjuvant would not be compatible with the compound of the present application, for example, to the extent that use of any such adjuvant results in the production of an adverse biological effect or any other interaction which is detrimental to the efficacy of the composition of the present application.

[0027] For example, the agent that increases AEBP1 activity of the present application can be incorporated into a medicament suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These medicaments can be prepared in various forms. For example, liquid, semisolid, and solid dosage forms, etc., including but not limited to liquid solutions (e.g., injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. A typical medicament is in the form of an injection solution or an infusion solution. The agent that increases AEBP1 activity can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

[0028] The effective amount of the preparation for increasing the activity of AEBP1 according to the present application can vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art according to various factors (e.g. through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredients such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune condition of the patient, the route of administration, etc. For example, several separate doses can be administered daily, or the dose can be proportionally reduced, according to the exigencies of the therapeutic situation.

[0029] It should be noted that the medicament includes a combination of components separated in time and / or space, as long as it can act together to achieve the purpose of the present application. For example, the components contained in the medicament can be administered to the subject as a whole, or administered to the subject separately. When the components contained in the medicament are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0030] In a fourth aspect of the present application, a method for screening drugs is provided. According to an embodiment of the present application, the method comprises: contacting a candidate drug with a fibroblast; detecting the expression amount of AEBP1 in the fibroblast before and after the contacting, wherein the increase of the expression amount of AEBP1 in the fibroblast after the contacting relative to the contacting is an indication that the candidate drug is a target drug. As described above, the decrease of AEBP1 expression level promotes the activation of fibroblasts, reduces the proliferation ability of fibroblasts, and makes the function of fibroblast-derived tissues decrease, which is easy to cause diseases. After the overexpression treatment, the fibroblasts maintain the resting state, the proliferation ability is improved, and the degree of fibrosis is reduced. Therefore, AEBP1 can be used as a target for screening target drugs. Specifically, the target drug is determined according to the change of the expression amount of AEBP1 in the fibroblast before and after the contacting of the candidate drug with the fibroblast, for example, the target drug is determined according to the change of the expression amount of AEBP1 in the cardiac fibroblast before and after the contacting of the candidate drug with the cardiac fibroblast, and the target drug is not treated or prevented.

[0031] According to an embodiment of the present application, the method for screening drugs can further comprise at least one of the following additional technical features:

[0032] According to an embodiment of the present application, the fibrosis-related disease includes at least one of: cardiac hypertrophy, heart failure, liver cirrhosis, and tissue fibrosis.

[0033] According to an embodiment of the present application, the tissue fibrosis comprises at least one of cardiac fibrosis, lung fibrosis, kidney fibrosis, liver fibrosis and tumor fibrosis.

[0034] According to an embodiment of the present application, the fibroblast comprises at least one of cardiac fibroblast, lung fibroblast, kidney fibroblast, liver fibroblast and tumor-associated fibroblast.

[0035] According to an embodiment of the present application, the fibroblast carries a reporter gene in a fusion protein state with the AEBP1. Those skilled in the art can understand that the reporter gene is used to indicate the level of the AEBP1, and the reporter gene is not particularly limited, and conventional reporter genes in the art can be used.

[0036] In a fifth aspect of the present application, a method for constructing a cell model is provided. According to an embodiment of the present application, the cell model is used for screening drugs for treating or preventing fibrosis-related diseases, and the method comprises: down-regulating or knocking out AEBP1 gene in fibroblasts to obtain the cell model. As described above, AEBP1 can be used as a target to determine whether a sample source individual is susceptible to or has suffered from a disease, and further, according to some specific embodiments of the present application, low expression of AEBP1 promotes activation of fibroblasts, reduces proliferation ability of fibroblasts, reduces function of fibroblast-derived tissues, and causes diseases such as myocardial hypertrophy and myocardial fibrosis. Therefore, fibroblasts with down-regulated or knocked out AEBP1 gene can be used as a cell model for screening drugs that up-regulate AEBP1 gene, and the drugs can effectively treat or prevent fibrosis-related diseases.

[0037] According to an embodiment of the present application, the method for constructing a cell model can further comprise at least one of the following additional technical features:

[0038] According to an embodiment of the present application, the fibrosis-related diseases comprise at least one of myocardial hypertrophy, heart failure, liver cirrhosis and tissue fibrosis.

[0039] According to an embodiment of the present application, the tissue fibrosis comprises at least one of cardiac fibrosis, lung fibrosis, kidney fibrosis, liver fibrosis and tumor fibrosis.

[0040] According to an embodiment of the present application, the fibroblast comprises at least one of cardiac fibroblast, lung fibroblast, kidney fibroblast, liver fibroblast and tumor-associated fibroblast.

[0041] In a sixth aspect, the present application provides a cell model. According to embodiments of the present application, the cell model is constructed using the method of the fifth aspect. The cell model can be used to effectively screen drugs that up-regulate the AEBP1 gene, which can be used to treat or prevent fibrosis-related diseases.

[0042] In a seventh aspect, the present application provides a method for treating or preventing fibrosis-related diseases. According to embodiments of the present application, the method comprises administering a preparation that increases the activity of AEBP1 to a subject. According to some embodiments of the present application, the preparation that increases the activity of AEBP1 can effectively maintain fibroblasts in a quiescent state and promote the proliferation of fibroblasts, which can be used to treat or prevent fibrosis-related diseases, such as cardiac fibrosis and cardiac hypertrophy. Therefore, the method can be used to treat or prevent fibrosis-related diseases.

[0043] According to embodiments of the present application, the method for treating or preventing fibrosis-related diseases can further comprise at least one of the following additional technical features:

[0044] According to embodiments of the present application, the preparation that increases the activity of AEBP1 comprises at least one of the following: an AEBP1 small molecule agonist, a nucleic acid molecule that up-regulates the expression of the AEBP1 gene, and an antibody that specifically recognizes an AEBP1 antagonist.

[0045] According to embodiments of the present application, the fibrosis-related diseases comprise at least one of the following: cardiac hypertrophy, heart failure, liver cirrhosis, and tissue fibrosis.

[0046] According to embodiments of the present application, the tissue fibrosis comprises at least one of the following: cardiac fibrosis, pulmonary fibrosis, renal fibrosis, liver fibrosis, and tumor fibrosis.

[0047] According to embodiments of the present application, the nucleic acid molecule that up-regulates the expression of the AEBP1 gene is loaded on a viral vector, a liposome, or a self-replicating RNA vector.

[0048] According to embodiments of the present application, the viral vector is an adeno-associated viral vector.

[0049] In an eighth aspect, the present application provides a use of a preparation that increases the activity of AEBP1 for treating or preventing fibrosis-related diseases. According to some embodiments of the present application, the preparation that increases the activity of AEBP1 can effectively maintain fibroblasts in a quiescent state and promote the proliferation of fibroblasts, which can be used to treat or prevent fibrosis-related diseases, such as cardiac fibrosis and cardiac hypertrophy. Therefore, the method can be used to treat or prevent fibrosis-related diseases.

[0050] According to the embodiment of the present application, the use for treating or preventing the fibrosis-related disease can further include at least one of the following additional technical features:

[0051] According to the embodiment of the present application, the preparation for increasing the activity of AEBP1 includes at least one of the following: a small molecule agonist of AEBP1, a nucleic acid molecule for up-regulating the expression of AEBP1 gene, and an antibody for specifically recognizing an antagonist of AEBP1.

[0052] According to the embodiment of the present application, the nucleic acid molecule for up-regulating the expression of AEBP1 gene is carried on a viral vector, a liposome, or a self-replicating RNA vector.

[0053] According to the embodiment of the present application, the viral vector is an adeno-associated viral vector.

[0054] According to the embodiment of the present application, the fibrosis-related disease includes at least one of the following: cardiac hypertrophy, heart failure, liver cirrhosis, and tissue fibrosis.

[0055] According to the embodiment of the present application, the tissue fibrosis includes at least one of the following: cardiac fibrosis, pulmonary fibrosis, renal fibrosis, hepatic fibrosis, and tumor fibrosis. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 is a sequencing result diagram of m-AEBP1 shRNA1 after sequencing of a cloned sample according to the embodiment 1 of the present application;

[0057] Figure 2 is a sequencing result diagram of m-AEBP1 shRNA2 after sequencing of a cloned sample according to the embodiment 1 of the present application;

[0058] Figure 3 is a sequencing result diagram of m-AEBP1 shRNA3 after sequencing of a cloned sample according to the embodiment 1 of the present application;

[0059] Figure 4 is a sequencing result diagram of m-AEBP1 shRNA4 after sequencing of a cloned sample according to the embodiment 1 of the present application;

[0060] Figure 5 is an operation flow diagram for packaging and quality detection of an adeno-associated virus according to the embodiment 1 of the present application;

[0061] Figure 6 is a microscope observation diagram of the P1 generation of adeno-associated virus at 10*10 magnification according to the embodiment 2 of the present application;

[0062] Figure 7is a microscope observation image of P2 passage adenovirus obtained according to Embodiment 2 of the present application at 10*10 magnification;

[0063] Figure 8 is a microscope observation image of P3 passage adenovirus obtained according to Embodiment 2 of the present application at 10*10 magnification;

[0064] Figure 9 is a result image of PCR mycoplasma detection of purified P3 passage adenovirus according to Embodiment 2 of the present application, wherein 1, 2, 3, 6 represent mycoplasma contamination, and 4, 5 represent no mycoplasma contamination;

[0065] Figure 10 is a detection result image of the influence of knocking down the expression level of AEBP1 on fibroblast activation according to Embodiment 3 of the present application, wherein Relative protein expression represents the relative expression amount of protein;

[0066] Figure 11 is another detection result image of the influence of knocking down the expression level of AEBP1 on fibroblast activation according to Embodiment 3 of the present application, wherein Collagen-I represents collagen staining, and DAPI represents cell nucleus staining, NC in the left image is a negative control, and AEBP1-siRNA in the right image is an AEBP1 knockdown experimental group;

[0067] Figure 12 is still another detection result image of the influence of knocking down the expression level of AEBP1 on fibroblast activation and morphological change according to Embodiment 3 of the present application, wherein Phalloidin is a phalloidin, which mainly stains cytoskeleton, and DAPI represents cell nucleus staining;

[0068] Figure 13 is a detection result image of the influence of knocking down the expression level of AEBP1 on fibroblast proliferation ability according to Embodiment 3 of the present application, wherein Ki67 represents cells in a proliferation state, DAPI represents cell nucleus staining, and Number of Ki67-positive cells represents the number of Ki67 positive cells;

[0069] Figure 14 is a detection result image of the influence of overexpressing AEBP1 on fibroblast activation according to Embodiment 4 of the present application, wherein Phalloidin is a phalloidin, which mainly stains cytoskeleton, and DAPI represents cell nucleus staining;

[0070] Figure 15is a graph of detection results of the influence of overexpression of AEBP1 on the proliferation ability of fibroblasts according to Embodiment 4 of the present application, wherein Ki67 represents cells in a proliferative state, and DAPI represents cell nucleus staining;

[0071] Figure 16 is another graph of detection results of the influence of overexpression of AEBP1 on the activation of fibroblasts according to Embodiment 4 of the present application, wherein Relative protein expression represents the relative expression amount of proteins;

[0072] Figure 17 is a graph of detection results of the influence of knockdown of the expression level of AEBP1 on the fibrosis of a mouse heart according to Embodiment 5 of the present application, wherein the portion marked using a box-shaped figure is a portion in which there is a difference in fibrosis;

[0073] Figure 18 is a graph of detection results of the influence of knockdown of the expression level of AEBP1 on the size of a mouse heart according to Embodiment 5 of the present application; and

[0074] Figure 19 is a graph of detection results of the influence of knockdown of the expression level of AEBP1 on the ratio of heart-weight / body-weight (HW / BW) and the ejection fraction of a mouse heart according to Embodiment 5 of the present application, wherein Ratio of heart-weight / body-weight (HW / BW) represents the ratio of heart weight to body weight. DETAILED DESCRIPTION

[0075] Embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings.

[0076] It should be noted that the terms "first", "second", etc. are used only for the purpose of description, and should not be understood as indicating or implying relative importance or implying that the indicated technical features are limited to one or more. Further, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0077] In this document, the term "comprising" or "including" is an open expression, i.e., including the indicated content of the present application, but not excluding other aspects.

[0078] In this document, the term "optionally", "optional" or "optional" generally means that the event or condition described thereafter can but does not necessarily occur, and the description includes both cases where the event or condition occurs and cases where the event or condition does not occur.

[0079] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as the exact dimensions are not critical to the invention. Any numerical values are approximations only. Numerical ranges include all values from and including the lower and the upper values, in increments of one unit used in the respective scaling context. For ranges given herein, any lower limit can be combined with any upper limit to create a new range.

[0080] For the purposes of the present invention, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used in the present document have the meanings that are commonly understood by one of ordinary skill in the art in the field of the present invention. Abbreviations for amino acid residues are the standard three letter and / or one letter codes used in the art to designate one of the 20 common L-amino acids.

[0081] Herein, the "self-replicating RNA", "self-replicating RNA (SaRNA)", or "self-amplifying RNA" all refer to an RNA that is capable of replicating itself with its own RNA sequence as a template. For example, a sequence encoding an RNA polymerase and a sequence expressing a target protein are packed in the same linear mRNA; or a sequence encoding an RNA polymerase and a sequence encoding a target protein mRNA are divided into two parts of RNA, which are introduced into cells to perform trans-amplification. That is, more copies of the RNA are produced with its own RNA as a template. The sequence expressing a target protein can be the above-mentioned nucleic acid molecule that up-regulates the expression of the AEBP1 gene.

[0082] Herein, the "viral vector" refers to a virus that is modified by genetic engineering technology to become a carrier for transporting an exogenous gene, which brings the exogenous gene into a cell by infecting the cell and performs long-term gene expression. The viral vector carries the above-mentioned nucleic acid molecule that up-regulates the expression of the AEBP1 gene. When the above-mentioned nucleic acid molecule that up-regulates the expression of the AEBP1 gene is linked to the vector, the nucleic acid molecule that up-regulates the expression of the AEBP1 gene can be directly or indirectly linked to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule that up-regulates the expression of the AEBP1 gene, i.e., the nucleic acid molecule that up-regulates the expression of the AEBP1 gene is operably linked to the control elements. Of course, these control elements can be directly from the vector itself, or can be exogenous, i.e., not from the vector itself. The viral vector is not particularly limited, and conventional viral vectors in the art are all within the scope of the present application, such as: adeno-associated viral vectors, lentiviruses, adeno-associated viruses, etc.

[0083] "Operably linked" herein refers to the linkage of an exogenous gene to a vector, so that the control elements within the vector, such as transcription control sequences and translation control sequences, etc., can exert their expected functions of regulating the transcription and translation of the exogenous gene. Those skilled in the art can understand that, in addition to viral vectors, plasmids, bacteriophages, etc. can also be used.

[0084] The vector according to some embodiments of the present application, such as a viral vector, a liposome, a self-replicating RNA, etc., after being introduced into a suitable recipient cell, can effectively realize the expression of the aforementioned nucleic acid molecule for up-regulating the expression of AEBP1 gene under the mediation of a regulatory system, and further realize the in vitro mass acquisition of the protein expressed by the nucleic acid molecule for up-regulating the expression of AEBP1 gene.

[0085] Herein, the human AEBP1 gene (NM_001129.3) has the nucleotide sequence shown in SEQ ID NO: 1, and the human AEBP1 protein (NP_001120.3) expressed thereby has the amino acid sequence shown in SEQ ID NO: 2.

[0086]

[0087]

[0088] The mouse AEBP1 gene (NM_001291857.2) has a nucleotide sequence shown as SEQ ID NO: 22, and the mouse AEBP1 protein (NP_001278786) expressed thereby has an amino acid sequence shown as SEQ ID NO: 23.

[0089]

[0090]

[0091] Advantages of the present application:

[0092] The inventors of the present application experiment on a plurality of genes, expecting to obtain target genes of fibrosis-related diseases, and find the new function of AEBP1 gene after screening, which can be used as a target gene of fibrosis-related diseases. Specifically, the inventors take the research on cardiac fibrosis as an example, and find that the decrease of AEBP1 expression level can promote the activation of cardiac fibroblasts, reduce the proliferation ability, reduce the cardiac function, and easily cause cardiac diseases such as myocardial fibrosis and myocardial hypertrophy. After the AEBP1 overexpression treatment of cardiac fibroblasts, the cardiac fibroblasts maintain the resting state, the proliferation ability is improved, and the degree of cardiac fibrosis is reduced, that is, the high expression of AEBP1 gene has the effect of resisting fibrosis-related cardiac diseases. In addition, the fibrosis diseases in various tissues or organs also have fibrosis problems, therefore, the drugs prepared by AEBP1 small molecule agonists, nucleic acid molecules for up-regulating the expression of AEBP1 gene and antibodies specifically recognizing AEBP1 antagonists can effectively treat or prevent the fibrosis-related diseases of the above-mentioned tissues or organs. In addition, AEBP1 can be used as a target gene for preparing a cell model and screening drugs for treating or preventing fibrosis-related diseases of various tissues or organs, such as drugs for treating or preventing cardiac fibrosis and myocardial hypertrophy, thereby providing an effective new way for the treatment of fibrosis-related diseases.

[0093] The embodiments of the present application will be described in more detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below by referring to the accompanying drawings are exemplary, and are intended to explain the present application, and cannot be understood as a limitation of the present application. The specific techniques or conditions not mentioned in the embodiments are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be obtained by purchase.

[0094] It should be noted that the "plasmid" and "vector" described herein have the same meaning and can be used interchangeably.

[0095] Example 1 Construction of m-AEBP1 adeno-associated virus knockdown vector and virus packaging

[0096] This example is used to study the effect of AEBP1 gene knockdown on the cardiac function of mice. The specific experimental content is as follows:

[0097] 1.1 Construction of mouse AEBP1 (m-AEBP1) shRNA adeno-associated virus vector

[0098] In the present application, pHBAAV-U6-MCS-CMV-EGFP is selected as the vector, and 4 interference target points are designed, wherein:

[0099] (1) The siRNA sequence and shRNA sequence of the control virus vector (NC) are shown in Table 1.

[0100] Table 1

[0101]

[0102] (2) The siRNA sequence and shRNA sequence of the target gene are shown in Table 2.

[0103] Table 2

[0104]

[0105]

[0106] Suitable restriction enzymes are selected to cut the vector, and the purified linearized vector is recovered by agarose gel. The target fragment is obtained by PCR using the designed primers, and the correct size of the target fragment is recovered by agarose gel. The linearized vector and the target fragment are connected by homologous recombination or T4 ligation to obtain the connected plasmid. The above experiments are performed using conventional methods in the art, and will not be repeated here.

[0107] The prepared plasmid is transformed into competent cells DH5a or stbl3, and the bacterial solution is plated and cultured for 12-16 h. Then, single colonies are selected for colony verification, and the correct positive clones are selected for sequencing. Among them, Figure 1 The sequencing result of m-AEBP1 shRNA1 is shown below, Figure 2 The sequencing result of m-AEBP1 shRNA2 is shown below, Figure 3 The sequencing result of m-AEBP1 shRNA3 is shown below, Figure 4 The sequencing result of m-AEBP1 shRNA4 is shown below, Figures 1-4 The results show that the sequencing results are consistent with the target sequences, and the target plasmid is successfully constructed. Then, the correct sequencing clone sample is extracted.

[0108] 1.2 Adeno-associated virus packaging and quality detection

[0109] The packaging of adeno-associated virus is carried out by three plasmids, as shown in the flow chart of Figure 5 After high-purity endotoxin-free extraction of the vector plasmid carrying the target gene or shRNA, the pAAV-RC vector plasmid and the pHelper vector plasmid, co-transfection of 293T cells is carried out using LipofiterTM transfection reagent with a mass ratio of 4 μg plasmid: 15 μL transfection reagent. The cell precipitate is collected 72 h after transfection, and the target adeno-associated virus vector is obtained after virus purification and quality detection. The concentrations of the adeno-associated virus vectors are shown in Table 3.

[0110] Table 3

[0111] Viral vector Concentration AAV2 / 9-EGFP NC control 1.2*10^12vg / mL AAV2 / 9-m-AEBP1 shRNA-EGFP 1.4*10^12vg / mL

[0112] Construction of h-AEBP1 adenovirus overexpression vector and virus packaging

[0113] 2.1 Construction of h-AEBP1 adenovirus overexpression vector

[0114] (1) Select pAdEasy-EF1-M CS-3flag-CMV-EGFP as the vector, according to the sequence of the target gene AEBP1 and the restriction endonuclease KpnI and XhoI selected for the construction of the vector, and design the PCR primer of the target gene AEBP1 (NM_001129.3), and the primer information is shown in Table 4.

[0115] Table 4

[0116]

[0117] The operation steps of this experiment include:

[0118] The restriction endonuclease KpnI and XhoI were used to cut the vector, and the purified linearized vector was recovered by agarose gel; the target fragment PCR was performed according to the designed primer, and the correct size of the target fragment was recovered by agarose gel; the linearized vector and the target fragment were connected according to the method of homologous recombination or T4 connection; the competent DH5a was transformed, the bacterial liquid was plated, and cultured for 12-16h; the single colony was selected and the bacterial liquid was plated, and cultured for 12-16h; the correct positive clone was selected for sequencing; the correct clone sample was subjected to plasmid extraction and quality verification. Finally, the adenovirus vector was recombined: a. The shuttle plasmid was linearized and recovered by agarose gel; b. The prepared E. coli BJ5183 competent cells containing adenovirus backbone plasmid pAdeasy-1 were used to transform the recovered linearized shuttle plasmid, and intracellular recombination was performed; c. After transformation, plate, 37°C, culture for 12-16h; d. Select single colony for culture, extract plasmid for PacI enzyme digestion verification; e. Transform the correct recombinant plasmid into E. coli stbl3 competent cells to obtain high-purity recombinant plasmid; f. Linearize the recombinant plasmid with PacI for cell transfection.

[0119] 2.2 Adenovirus packaging and purification

[0120] The experimental operation steps of adenovirus packaging and purification are as follows:

[0121] (1) Cell seeding: One day before transfection, 293T cells were seeded in 60mm culture dishes with DMEM + 10% Hyclon fetal bovine serum as the culture medium and cultured overnight in an incubator containing 5% CO2 at 37℃.

[0122] (2) Cell transfection: When the cells have grown to 70%–80% confluency of the cell base area, take the linearized plasmid of the successfully recombined adeno-associated virus vector and transfect it using Lipofiter. TM Transfection was performed using transfection reagents. The specific steps are as follows:

[0123] a. Two hours before transfection, replace the complete culture medium, take 4 μg of the successfully recombined adeno-associated virus vector linearized plasmid, dilute it with 300 μL of DMEM culture medium, and incubate at room temperature for 5 min.

[0124] b. Take 15 μL of Lipofiter TM Dilute with 300 μL of DMEM culture medium and incubate at room temperature for 5 min.

[0125] c. Mix the two ingredients and let stand at room temperature in the dark for 20 minutes. Then add the mixture to a 60 mm petri dish, shake in a figure-eight motion, and incubate at 37°C in an incubator containing 5% CO2.

[0126] Note: Lipofiter TM The transfection reagent is a product of Hanheng Biotechnology. Please refer to Lipofiter for instructions on use. TM Instruction manual (Appendix 1).

[0127] (3) Change medium: Replace with fresh cell culture medium 6 hours after transfection.

[0128] (4) Collecting poison (P1):

[0129] Observe for signs of cellular toxicity daily. For example... Figure 6 As shown in the image, at a magnification of 10*10, the signs of toxicity include cell enlargement and rounding, resembling grapes, and the appearance of obvious plaques. Cells were collected after most of the cells had become diseased and detached from the bottom. All cells and culture medium in a 60mm culture dish were transferred to a 15mL centrifuge tube.

[0130] (5) Freeze-thaw:

[0131] Set the constant temperature water bath to 37°C, and repeatedly freeze and thaw 15mL centrifuge tubes in liquid nitrogen and a 37°C water bath three times. Centrifuge at 3000rpm for 5 minutes, collect the supernatant containing the virus, and discard the precipitate. This supernatant is the first-generation control virus (P1), which will be used as the virus for subsequent large-scale viral amplification.

[0132] (6) Amplification:

[0133] Take 2 mL of the P1 generation virus supernatant to infect cells in a 10 cm cell culture dish (ensuring a cell density of over 90%). Place the remaining virus supernatant in an externally screwed cryovial and store at -80°C as a seed culture.

[0134] (7) Collecting poison (P2):

[0135] like Figure 7 As shown, at a magnification of 10*10, after two days of virus amplification and once all cells have detached from the bottom, the virus can be harvested. The cells, along with the culture medium, are collected into a 15mL centrifuge tube. Following the freeze-thaw method described earlier, the tube is frozen and thawed three times. The supernatant is then used for the next generation of amplification or stored at -80℃. Subsequent generations of virus amplification and harvesting are repeated in this manner.

[0136] (8) P3 generation virus amplification and virus collection:

[0137] Based on 75cm each 2 Inoculate 4×10 in a square bottle 6 293 T cells were seeded into 6 75cm cells. 2 Culture flasks overnight. When cells reach 90% confluence, inoculate all of the P2 generation virus (except for a small amount of retained virus seed) into the culture flasks. Figure 8 As shown, 60% of the cells showed cytopathic effects after 24 hours of culture, and complete cytopathic effects were observed after 46 hours. After harvesting the cytopathic cell suspension, the cells were centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and 6 mL of ST buffer (culture medium + 10% serum + 2.5% glycerol) was added. The mixture was then votexed and thawed three times between liquid nitrogen and 37°C. The supernatant was collected after centrifugation at 3000 rpm for 5 min and used as the third-generation virus (P3).

[0138] (9) Adenovirus purification:

[0139] a. First ultracentrifugation

[0140] Pre-cool the centrifuge to 4°C. Slowly add 8 mL of CsCl 1.4 to the centrifuge tube, followed by 10 mL of CsCl 1.2. Then, slowly add approximately 20 mL of third-generation virus solution on top, for a total volume of approximately 37 mL. Centrifuge at 100,000 × g (24,000 rpm in SW31) for 90 min at 4°C with a deceleration of 0. After centrifugation, discard the waste liquid at the top of the centrifuge tube and wipe the tube wall with 75% alcohol. Cover the puncture site with adhesive tape. Using a 5 mL syringe fitted with a 1.22 (18G) needle, puncture below the blue-white band to aspirate the virus solution. Dilute the collected virus solution with at least one volume of TE buffer.

[0141] b. Second ultracentrifugation

[0142] Slowly add 12 mL of CsCl 1.4 to the centrifuge tube, then slowly add 14 mL of CsCl 1.2. Very slowly add 8–10 mL of the diluted virus solution after the first ultracentrifugation to the top, balance the volume, and centrifuge at 100,000 × g (24,000 rpm in SW31) for 16–20 hours at 4°C with a deceleration of 0. After centrifugation, discard the waste liquid at the top of the centrifuge tube and wipe the tube wall with 75% alcohol. Cover the puncture site with adhesive tape, and use a 5 mL syringe with a 0.8 (20G) needle to aspirate the blue-white band (method as above) or make a hole at the bottom of the centrifuge tube to allow the liquid to flow down naturally and collect the blue-white band (this can reduce the contamination of CsCl).

[0143] c. Dialysis

[0144] Use 200× volume of dialysis buffer each time, perform dialysis three times, and change the buffer every hour.

[0145] (10) The titer of the product after dialysis was tested and the virus was aliquoted and stored at -80℃.

[0146] 2.3 Adenovirus Quality Testing

[0147] Key aspects of adenovirus quality control include sterility testing, mycoplasma testing, and viral titer testing.

[0148] (1) Sterility test

[0149] Detection method: 10 μL of virus obtained from section 2.2 was mixed with HeLa cells in a 96-well plate and cultured for 24 h before microscopic examination.

[0150] QC Standard: The culture medium must be clear and transparent, with no obvious particles in the intercellular spaces and no bacterial or fungal contamination.

[0151] (2) Mycoplasma detection

[0152] Detection method: Take 10 μL of virus obtained from section 2.2, incubate at 96℃ for 15 min, and then prepare a PCR reaction system in a clean bench. Electrophoresis is performed after the PCR reaction to determine whether mycoplasma contamination is present.

[0153] QC standard: No obvious bands on PCR gel images.

[0154] like Figure 9 As shown, if there is a band at around 500bp, such as 1, 2, 3, or 6, it indicates that the sample is contaminated with mycoplasma. No bands at positions 4 and 5 indicate that there is no mycoplasma contamination.

[0155] 2.4 Titer Detection

[0156] In this embodiment, the adenovirus titer test uses TCID50 titer test, the specific process is as follows:

[0157] Culture 293T cells, when the cell growth density is about 80% to 90%, digest the cells and count the number of cells; prepare a cell suspension with DMEM culture solution containing 5% FBS; add 2 96-well plates at 100 μL (i.e. 1 x 10 4 cells) per well; then, prepare high concentration infection samples (10 6 Start 8 consecutive dilution gradients) and low concentration samples (10 4 Start 8 consecutive dilution times), among which the sample dilution gradient can be adjusted according to the specific situation, and in this experiment, the gradient dilution is 10 times.

[0158] Add 100 μL of 5% FBS DMEM to columns 11 and 12 in the 96-well plate to serve as negative controls. Add 100 μL of each of the 8 serially diluted sample solutions labeled in the 96-well plate to rows A-H, respectively. Cover the first plate and incubate it in a 37°C, CO2 incubator. Perform the same steps on the second plate. Cover the second plate and incubate it in a 37°C, CO2 incubator. The structure of the incubation plates in the experiment is shown in Table 5.

[0159] Table 5

[0160]

[0161] Then, place the 96-well plate in a 37°C CO2 incubator for 10 days. Observe the cell condition from the 3rd day to the 10th day.

[0162] Cytopathic effect (CPE) should appear within 10 days. On the 10th day, observe the CPE of each well under a microscope and compare it with the negative control row, and record the number of positive wells for each sample row. On the 10th day, (a) at least one sample dilution has obvious CPE in 12 wells. (b) at least one sample dilution has obvious CPE in at least 3 but not more than 9 wells. (c) at least one sample dilution has no CPE in 12 wells.

[0163] The formula for calculating virus activity is as follows:

[0164] For a 100 μL sample, the titer T = 10 1+d(s-0 . 5)

[0165] d = log 10 dilution = 1 (for a dilution of 10 times)

[0166] s = sum of positive rates (from the first 10-fold dilution)

[0167] Convert TCID50 / mL to PFU / mL:

[0168] T = a × 10 b TCID50 / mL=a×10 b-0.7 PFU / mL

[0169] The results of this experiment are shown in Table 6.

[0170] Table 6

[0171] AD-EGFP 3.16*10^10PFU / mL AD-Adeasy-h-AEBP1-3xflag-EGFP 1.26*10^10PFU / mL

[0172] Note: AD-EGFP represents the overexpression control virus, and AD-Adeasy-h-AEBP1-3xflag-EGFP represents the overexpression of AEBP1 virus.

[0173] Example 3: Effects of AEBP1 knockdown on human primary fibroblasts

[0174] Human primary fibroblasts were knocked down using AEBP1 siRNA (sequence information: GGCTCAGAGTTTGGGAACT, SEQ ID NO:21). 8 × 10⁸ cells were used. 4 Personal cardiac fibroblasts (cell passages 5-7) were seeded into 6-well plates. On the day of transfection, according to the transfection reagent instructions (stB0002601A, Ribobio), the human cardiac fibroblasts were treated with either 50 nM target human AEBP1 siRNA or 50 nM control scramble siRNA. The specific experimental steps were as follows: each well was transfected with 6 μL of Dharmafect 1 transfection reagent (T-2001-01, GE Healthcare Dharmacon) for 6 h, and then treated with TGFβ (6 ng / L, 100-21C-50UG, Peprotech). After that, the medium was replaced with maintenance medium containing 2% fetal bovine serum and transfection was continued for 48 hours. Cells were then collected, and mRNA was extracted for Bulk RNA-seq analysis. The mRNA extraction experiment was performed using standard methods in the art. Cells were also collected 72 h after transfection, and total protein was extracted for Western blot analysis.

[0175] Experimental results are as follows Figures 10-13 As shown, knocking down the expression level of the AEBP1 gene can promote fibroblast activation and reduce cell proliferation.

[0176] Example 4: Effects of AEBP1 overexpression on human primary fibroblasts

[0177] This example is used to study the effect of AEBP1 gene overexpression on human primary fibroblasts. The specific experimental content is as follows:

[0178] The overexpression vector of AEBP1 is constructed by using the adenovirus of Example 2, and fibroblasts are induced to overexpress AEBP1. 8x10 4 The individual cardiac fibroblasts (cell number 5-6) are implanted into a 6-well plate; on the transfection day, after the confluence rate reaches about 50%, the control virus vector and the target gene vector are respectively used for the experiment with a multiplicity of infection (MOI) of 400 or 300. In this experiment, the semi-volume infection method (i.e., 1 ml of culture medium and virus mixture is added first) is used to treat the cells, and 1 ml of fresh complete culture medium (i.e., culture medium containing 10% serum) is added after 4 hours of action; after 8 hours of culture, the cells are starved in 0.5% fetal bovine serum for 12 hours; TGFβ (6 ng / L, 100-21C-50UG, Peprotech) is used to treat the cells; after 48 hours of treatment, the total protein is extracted for western blot detection.

[0179] The experimental results are shown in Figures 14-16 The results show that overexpression of AEBP1 gene significantly inhibits the activation of fibroblasts and improves the proliferation ability of cells.

[0180] Example 5: Effect of AEBP1 gene knockdown on myocardial fibrosis and heart function of mice

[0181] In this example, wild-type mice and AEBP1 gene knockdown mice, each with 5 mice, are used for the experiment to study the effect of AEBP1 gene knockdown on myocardial fibrosis and heart function of mice. The specific experimental content is as follows:

[0182] 8-week-old male C57BL / 6 wild-type mice (WT) and heart-specific AEBP1 gene knockdown mice (AAV9-AEBP1-KD) are selected as experimental objects. 5 mice are injected with the adeno-associated virus vector obtained in Example 1 through the tail vein, 100 μL per mouse, AAV2 / 9-EGFPNC control and AAV2 / 9-m-AEBP1 shRNA-EGFP; after 4 weeks of injection, cardiac ultrasound detection is performed to observe the changes in heart function and fibrosis degree of the mice. Then, the heart specimens are taken, and the heart is divided into two parts from the front and back of the heart, one of which is used for pathological staining of the mouse heart, including HE staining and Masson staining, and the staining steps are conventional technical operations in the art. The experimental results are shown in Figures 17-19 The results show that the AEBP1 gene knockdown mice have problems such as myocardial hypertrophy and cardiac fibrosis, which lead to reduced heart function.

[0183] In addition, the terms "first", "second", etc. are used only for the purpose of description, and should not be understood as indicating or implying relative importance or a specific number of the technical features indicated. Therefore, the features defined as "first", "second" can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "a plurality of" is at least two, for example, two, three, etc., unless otherwise explicitly and specifically limited.

[0184] In the description of the present application, the description referring to the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples, without contradiction.

[0185] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. Use of a preparation for the manufacture of a medicament for the treatment or prevention of a fibrosis-related disease, the preparation being for increasing the activity of AEBP1, wherein, The nucleotide sequence of the AEBP1 is shown as SEQ ID NO: 1, the fibrosis-related disease is myocardial hypertrophy, the preparation is a nucleic acid molecule for up-regulating AEBP1 gene expression, and the nucleic acid molecule for up-regulating AEBP1 gene expression is loaded on a viral vector or a self-replicating RNA vector.

2. Use according to claim 1, characterized in that, The viral vector is an adeno-associated viral vector.

3. A method for screening drugs, characterized by, The method comprises: contacting a candidate drug with fibroblasts; detecting the expression amount of AEBP1 in the fibroblasts before and after the contacting, wherein the nucleotide sequence of the AEBP1 is shown as SEQ ID NO: 1; wherein the increase of the expression amount of the AEBP1 in the fibroblasts after the contacting relative to before the contacting is an indication that the candidate drug is a target drug; the drug is used for treating or preventing a fibrosis-related disease, and the fibrosis-related disease is myocardial hypertrophy.

4. The method of claim 3, wherein, The fibroblasts are cardiac fibroblasts.

5. The method of claim 3, wherein, The fibroblasts carry a reporter gene in a fusion protein state with the AEBP1.

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