CN115957301A - Cell secretion factor for promoting myocardial infarction repair and application - Google Patents

Abstract

The invention provides a cell secretion factor for promoting myocardial infarction repair and application thereof. The application of insulin-like growth factor binding protein 7 (IGFBP 7) in a composition or a medicament for promoting myocardial repair after myocardial infarction (myocardial infarction, MI), improving cardiac function after myocardial infarction and protecting myocardial ischemia injury is disclosed for the first time. Based on the above functions of IGFBP7, the substance can be inhibited as a drug screening target spot, and substances for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction and protecting myocardial ischemia injury are screened.

Description

Cell secretion factor for promoting myocardial infarction repair and application

Technical Field

The invention belongs to the field of biotechnology and pharmacology, and particularly relates to a cell secretion factor for promoting myocardial infarction repair and application thereof.

Background

Cardiovascular disease is one of the most serious diseases threatening human health today. Among cardiovascular diseases, ischemic Heart Disease (IHD) is undoubtedly the most serious problem. Ischemic heart disease is characterized by ischemic injury of the myocardium due to imbalanced demand of oxygen and energy substances from the myocardium and blood supply to the coronary arteries. Acute myocardial infarction (myocardial infarction) is usually due to the sudden occlusion of coronary arteries, resulting in the interruption of blood flow, resulting in myocardial ischemia. Continuous ischemia causes a great deal of myocardial cells to die, and the dead myocardial cells cannot be effectively supplemented due to the lack of regeneration capacity of adult myocardial cells, so that the proliferation of fibroblasts causes excessive fibrosis and scar formation, ventricular remodeling occurs, and the cardiac function is reduced.

How to rebuild blood supply for ischemic myocardium is a key problem for solving myocardial infarction, and the blood supply of ischemic part can be realized again through means of drug thrombolysis, bypass and the like clinically, so that the myocardial infarction area is effectively reduced, the long-term cardiac function is improved, and the death rate is reduced. However, studies have shown that in the process of restoring blood supply to an ischemic heart, new damage is caused to the heart, which together with the damage caused by Ischemia is collectively referred to as Ischemia/Reperfusion (I/R) damage.

Myocardial infarction is based on coronary artery pathological changes, coronary blood supply is sharply reduced or interrupted, acute and persistent myocardial ischemia occurs to myocardium at a coronary supply part, severe cell energy metabolic disorder is caused, and a large number of myocardial cells are damaged and killed. Since the cardiomyocytes are terminally differentiated cells and have very limited regeneration capacity, the lost cardiomyocytes are replaced by fibrous scars, resulting in irreversible myocardial injury, and in severe cases, arrhythmia, ventricular aneurysm formation, heart rupture and the like. Heart failure is a complex group of clinical symptoms of cardiac insufficiency caused by various heart diseases; in most cases, the myocardial contractility is reduced, so that the heart blood output cannot meet the metabolic needs of the organism, and the blood perfusion of organs and tissues is insufficient.

The death modes of myocardial cells after myocardial ischemia injury mainly comprise autophagy, apoptosis, scorching, iron death, necrosis and the like. Where necrosis is considered to be the major type of myocardial cell death caused by the myocardial infarction. Ischemic injury causes dead cardiomyocytes to die about 30% of the time from apoptosis and 70% of the time from necrosis. Further studies have shown that there is a different form of death in cells from apoptosis and traditionally necrotic cells, a process with typical necrosis-like morphological features such as loss of membrane integrity and initiation of inflammation, but which is tightly regulated by a series of molecules called programmed necrosis. The proportion of cells undergoing programmed necrosis after myocardial infarction is about 50%. Activation of death receptors is the major mechanism by which programmed necrosis occurs. The regulation of Receptor Interacting Protein kinase 1 (RIP 1) and RIP3 activation mediated by Tumor Necrosis Factor Receptor (TNFR) is a major feature of death Receptor-dependent apoptosis. TNFR activation promotes dimerization activation of RIP1, activated RIP1 is ubiquitinated by PELI1 and then forms a complex with RIP3, mixed lineage kinase domain-like protein (MLKL) is recruited, and a programmed necrosis process is performed.

In view of the wide and multiple occurrence of myocardial infarction, there is a need in the art to find more new approaches to relieving myocardial infarction and its symptoms after occurrence.

Disclosure of Invention

The invention aims to provide a cell secretion factor for promoting myocardial infarction repair and application thereof.

In a first aspect of the invention there is provided the use of insulin-like growth factor binding protein 7 (IGFBP 7) or a modulator thereof in the manufacture of a medicament or composition having the following functions: promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, and relieving myocardial ischemia injury.

In one or more embodiments, the promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, and protecting myocardial ischemic injury comprises: reducing the area of the myocardial infarction area after myocardial infarction; reducing the area of fibrous scars after myocardial infarction; increasing the number of blood vessels in the myocardial infarction area and the myocardial infarction edge area after myocardial infarction; promoting myocardial cells to resist damage; the method can inhibit programmed necrosis of myocardial infarction, preferably inhibit activation of RIP1/RIP3 signal pathway of programmed necrosis, and further inhibit programmed necrosis of injured myocardial cell.

In one or more embodiments, the up-regulator of insulin-like growth factor binding protein 7 comprises a peptide selected from the group consisting of: (a) A substance that enhances the activity of insulin-like growth factor binding protein 7; (b) A substance that enhances the expression, stability or effective duration of action of insulin-like growth factor binding protein 7.

In one or more embodiments, the up-regulator of insulin-like growth factor binding protein 7 comprises a peptide selected from the group consisting of: an expression construct (including an expression vector) for recombinantly expressing insulin-like growth factor binding Protein 7, a polypeptide or compound that enhances the inhibitory effect of insulin-like growth factor binding Protein 7 on the cellular programmed necrosis RIP1 (Receptor Interacting Protein Kinase 1)/RIP 3 signal pathway, a chemical up-regulator of insulin-like growth factor binding Protein 7, an up-regulator that promotes the driving ability of the insulin-like growth factor binding Protein 7 gene promoter, a down-regulator of insulin-like growth factor binding Protein 7 gene-specific microRNA, or a combination thereof.

In one or more embodiments, insulin-like growth factor binding protein 7 is selected from the group consisting of: (a) a polypeptide with an amino acid sequence shown as SEQ ID NO: 1; (b) An insulin-like growth factor binding protein 7 derivative having the polypeptide function of (a) or (b) formed by substituting, deleting or adding one or more (such as 1-20, 1-10, 1-5, 1-3 or 1-2) amino acid residues to the amino acid sequence shown in (a), or an active fragment thereof; (c) The sequence is more than or equal to 80 percent (such as homology is more than or equal to 85 percent, more than or equal to 90 percent, more than or equal to 95 percent, more than or equal to 98 percent or more than or equal to 99 percent) of the insulin-like growth factor binding protein 7 derivative or the active fragment thereof compared with the amino acid sequence shown in SEQ ID NO. 1.

In one or more embodiments, the expression construct (expression vector) comprises: viral vectors, non-viral vectors; preferably, the expression vector comprises: adeno-associated virus vectors, lentiviral vectors, and adenoviral vectors.

In one or more embodiments, the inhibition of apoptosis RIP1/RIP3 signaling pathway comprises inhibition of RIP3.

In one or more embodiments, inhibiting RIP3 comprises: reduce RIP3 mRNA stability in an IGF-independent manner, thereby inhibiting myocardial cell programmed necrosis after myocardial infarction.

In another aspect of the invention, there is provided the use of insulin-like growth factor binding protein 7 for screening (i.e. as a drug screening target) drugs or compounds having the following functions: promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, and protecting myocardial ischemia injury.

In another aspect of the present invention, there is provided a method for screening a drug or a compound (or a potential drug or a compound) for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, and protecting myocardial ischemic injury, comprising: (1) Treating an expression system expressing insulin-like growth factor binding protein 7 with a candidate substance; and, (2) detecting the expression or activity of insulin-like growth factor binding protein 7 in said system; if the candidate substance statistically increases the expression or activity of insulin-like growth factor binding protein 7, it is indicative that the candidate substance is the desired (interesting) drug or compound.

In one or more embodiments, step (1) comprises: in the test group, adding a candidate substance to the expression system; and/or, the step (2) comprises: detecting the expression or activity of insulin-like growth factor binding protein 7 in said system and comparing said expression or activity to a control, wherein said control is an expression system without said candidate substance; if the candidate substance statistically increases (e.g., by more than 20%, preferably by more than 50%, more preferably by more than 80%) the expression or activity of insulin-like growth factor binding protein 7, the candidate substance is indicative of the desired (interesting) drug or compound.

In one or more embodiments, the system in step (1) further comprises a cell programmed necrosis RIP1/RIP3 signal pathway or pathway protein RIP3; and, step (2) also includes: and detecting the interaction condition of the insulin-like growth factor binding protein 7 and the programmed necrosis RIP1/RIP3 signal pathway or pathway protein RIP3 in the system, wherein if the insulin-like growth factor binding protein 7 inhibits (such as inhibits more than 20%, preferably inhibits more than 50%, and more preferably inhibits more than 80%) the programmed necrosis RIP1/RIP3 (especially inhibits RIP 3) signal pathway or pathway protein RIP3, the candidate substance is the required (interested) drug or compound.

In one or more embodiments, in the system comprising a apoptotic RIP1/RIP3 signal pathway, IGFBP7 acts on RIP3.

In one or more embodiments, the system is selected from the group consisting of: a cell system (or cell culture system), a subcellular system (or subcellular culture system), a solution system, a tissue system, an organ system, or an animal system.

In one or more embodiments, the candidate substances include (but are not limited to): overexpression molecules such as constructs, activity promoting molecules, chemical small molecules, interacting molecules and the like designed aiming at insulin-like growth factor binding protein 7, fragments or variants thereof, encoding genes thereof or upstream and downstream molecules thereof or signal paths.

In one or more embodiments, the method further comprises: the obtained drug or compound (potential drug or compound) is subjected to further cell experiments and/or animal experiments to further select and identify a composition useful for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, and protecting (alleviating) myocardial ischemic injury from the candidate substance.

In another aspect of the invention there is provided the use of an agent that specifically recognizes or amplifies insulin-like growth factor binding protein 7 for the preparation of a reagent or kit for assessing or prognosing disease progression after myocardial infarction.

In one or more embodiments, if insulin-like growth factor binding protein 7 is under-expressed (i.e., expressed below the average or conventional value for the population of species), then the subject has a poor (relatively poor) prognosis following myocardial infarction.

In one or more embodiments, normal or high expression (i.e., expression equal to, equivalent to, or higher than the average or conventional value for the population of the species; wherein the higher is, e.g., greater than 5%, 10%, 15%, 20%, 30%, 50%, 60%, 80%, 90% or more), of insulin-like growth factor binding protein 7 is indicative of a normal or good (relatively better) prognosis after myocardial infarction in the subject.

In one or more embodiments, the agent includes (but is not limited to): a binding molecule that specifically binds to insulin-like growth factor binding protein 7; primers for specifically amplifying the insulin-like growth factor binding protein 7 gene; a probe that specifically recognizes insulin-like growth factor binding protein 7 gene; or a chip that specifically recognizes the insulin-like growth factor binding protein 7 gene.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, human IGFBP7 domain.

FIG. 2, distribution of IGFBP7 in human organs and expression and localization in mouse myocardium and fibroblasts.

(A) IGFBP7 expression in different tissues of the human body, where the heart is the only muscle tissue expressing IGFBP7 (data from the proteomics db database); (B) Immunofluorescence staining of adult mouse cardiomyocytes shows that IGFBP7 in the cardiomyocytes is widely distributed and has no obvious positioning preference; in fibroblasts, IGFBP7 is spotted, partially co-localized with the vimentin positive spots (white arrows); (C) WB results showed that the expression of IGFBP7 was significantly higher in adult mouse cardiomyocytes than in adult mouse fibroblasts. Scale bar =50 μm.

FIG. 3, expression of IGFBP7 in hCGPCs supernatant and myocardial infarction mouse heart tissue.

(A) The content of IGFBP7 in the CVPC-CdM of the antibody chip result is far higher than that of DMEM of a control; (B) detecting the content of IGFBP7 in hCGPC-CdM by an Elisa test; (C) WB results showed a gradual increase in IGFBP7 protein levels in heart tissue of infarct mice, n =3; (D) QPCR results showed a sustained increase in IGFBP7 mRNA levels in myocardial infarction mouse heart tissue, n =6; (E) Elisa measures IGFBP7 levels in serum from infarcted mice, which also increased after the infarct, with n =5.* P <0.001vs.

FIG. 4, IGFBP7 myocardial-specific knockout exacerbates post-myocardial infarction myocardial injury.

(A)IGFBP7 ^CM-/- Constructing a mouse; (B) IGFBP7 ^CM -/-does not affect the cardiac function and cardiomyocyte size of mice in physiological states, IGFBP7 after myocardial infarction ^CM -/-mice with increased myocardial damage, n =8.* p is a radical of formula<0.05，**p<0.01，***p<0.001。

FIG. 5 IGFBP7 injected into myocardium can significantly improve cardiac function of myocardial infarction mice and reduce fibrous scar area.

(A) Myocardial infarction surgery mouse survival curve; (B) The cardiac function index of the mouse, namely the IGFBP7 myocardial infarction can obviously improve the cardiac function and the Left Ventricular Ejection Fraction (LVEF) 1 week later; (C-D) Masson staining pattern and statistical plot of scar region, n =10; (E) The number of Tunel positive cells in the myocardial infarction area of the mouse is obviously reduced by injecting IGFBP7 myocardium, and n =5; (F) Myocardial injection of IGFBP7 significantly increased the number of myocardial infarct border zone vessels in the myocardial infarct zone of mice, n =5 × p was restricted to 0.05,. × p <0.01.

FIG. 6, myocardial IGFBP7 injection significantly improves cardiac function of myocardial infarction mice, reduces fibrous scar area.

(A) A pattern diagram; (B) The heart function index of the mouse, namely, the heart function improvement of IGFBP7 after myocardial infarction, namely 7, 14 and 28, can be detected, and the Left Ventricular Ejection Fraction (LVEF); (C) Masson staining and statistical plot of scar region, n =10.* p <0.05.

FIG. 7 IGFBP7 protects adult mouse cardiomyocytes against injury.

(A) Morphology before and after OGD injury of adult mouse cardiac myocytes, the rod cell proportion of the group treated by IGFBP7 is obviously higher than that of the OGD control group, and the group added with NBI31772 is also obviously higher than that of the OGD control group, but has no difference with the IGFBP7 group; (B) statistics of the ratio of the rod cells in each group, wherein n =6; (C) LDH release detection, n =5; (D) Morphology of wild type and IGFBP7 knockout mouse myocardial cells before and after OGD injury; (E) statistics of WT and KO rod cell ratios, n =5.* p <0.05. Scale bar: 200 μm.

Figure 8, IGFBP7 inhibits RIP3 protein levels in cardiac tissue.

RIP3 protein expression in heart tissue samples 3 days after myocardial infarction, n =3.* P <0.001.

Fig. 9, IGFBP7 significantly inhibited RIP3 mRNA levels.

RIP3 mRNA levels were measured in heart tissue samples 3 days after myocardial infarction, n =8-10.* P <0.01, p <0.001.

FIG. 10 IGFBP7 decreased RIP3 mRNA stability.

Adult mouse cardiomyocytes were treated with actinomycin D (10 ug/mL) and RIP3 mRNA levels were measured at 0h, 1h, 3h, 6h, 8h, n =5.* p <0.05.

Detailed Description

Based on intensive research, the inventor firstly discloses application of insulin-like growth factor binding protein 7 (IGFBP 7) in a composition or a medicament for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction and protecting myocardial ischemia injury. Based on the above functions of IGFBP7, the inhibitor can be used as a drug screening target spot to screen substances for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction and protecting myocardial ischemia injury.

IGFBP7

The IGFBPs family is a class of secreted proteins, which have conserved IGFBP motifs at their N-terminus, which play a critical role in the metabolism and bioactivity of IGFs by forming a complex with insulin-like growth factor I (IGF-I) and IGF-II. Currently, there are 7 members of the IGFBPs family, which are different in function and involved in biological processes, and most of the IGFBPs 1-3 and 6 are involved in the metabolism of obesity-related diseases and the regulation of tumors. IGFBP7 is a newly discovered member of the IGFBPs family, and has a specific Kazal-type serine protease inhibition domain and an immunoglobulin-like C2 domain besides a conserved N-terminal domain similar to IGFBPs 1-6, and the binding capacity of the specific Kazal-type serine protease inhibition domain and the immunoglobulin-like C2 domain with IGFs is far lower than that of other IGFBPs, which indicates that the IGFBP7 has different functions and modes of action from other IGFBPs. At present, researches show that IGFBP7 is involved in biological processes such as the regulation of proliferation, differentiation, cell aging, apoptosis and the like of tumor cells such as breast cancer, lung cancer and the like besides the regulation of bioavailability of IGFs and insulin, but the exact action of the IGFBP is controversial and different actions can be exerted in specific tissues, cell types and environments. As in the senescence regulation study, wajapeye et al found that activation of the Proto-Oncogene BRAF (B-Raf Proto-Oncogene) in human melanoma cells and fibroblasts induces cellular senescence and apoptosis by inhibiting the BRAF-MEK-ERK signaling pathway by up-regulating IGFBP7 synthesis and secretion. Scurr et al demonstrated that the BRAF signal could neither induce IGFBP7 expression nor affect BRAF target gene expression; in the aspect of angiogenesis regulation, IGFBP7 is highly expressed in endothelial cells of malignant glioma vessels, and promotes endothelial cell proliferation and angiogenesis. It has been found that IGFBP7 inhibits normal vascular endothelial cell proliferation and angiogenesis by inhibiting vascular endothelial growth factor expression. The role of IGFBP7 in myocardial infarction, myocardial ischemia/reperfusion, and on damaged cardiomyocytes has not been reported.

The IGFBP7 of the invention may be naturally occurring, e.g., it may be isolated or purified from a mammal. In addition, the IGFBP7 may also be artificially prepared, for example, recombinant IGFBP7 may be produced according to conventional genetic engineering recombination techniques for experimental or clinical applications. In use, recombinant IGFBP7 may be used. The IGFBP7 includes full-length IGFBP7 or a biologically active fragment thereof. Preferably, the amino acid sequence of IGFBP7 may be substantially identical to the sequence shown in SEQ ID NO. 1. The corresponding nucleotide coding sequence is conveniently derived from the amino acid sequence of IGFBP7, and may, for example, be substantially identical to the sequence shown in SEQ ID NO. 1. Proteins from other species having homology to the IGFBP7 may also be included in the invention.

The amino acid sequence of IGFBP7 formed by substitution, deletion or addition of one or more (e.g., 1-30) amino acid residues is also included in the present invention. IGFBP7 or a biologically active fragment thereof includes a partial substitution of conserved amino acids that does not affect its activity or retains some of its activity. Appropriate substitutions of amino acids are well known in the art and can be readily made and will ensure that the biological activity of the resulting molecule is not altered. These techniques allow one of skill in the art to recognize that, in general, altering a single amino acid in a non-essential region of a polypeptide does not substantially alter biological activity.

Biologically active fragments isolated from IGFBP7 may also be used in the present invention. As used herein, a biologically active fragment of IGFBP7 is meant to be a polypeptide that still retains all or part of the function of full-length IGFBP7. Typically, the biologically active fragment retains at least 50% of the activity of full-length IGFBP7. Under more preferred conditions, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99% or 100% of the activity of full-length IGFBP7.

The invention may also employ modified or improved IGFBP7, e.g., IGFBP7 modified or improved to promote its half-life, effectiveness, metabolism, and/or protein potency may be employed. The modified or improved IGFBP7 may be a conjugate of IGFBP7, or it may comprise substituted or artificial amino acids. The modified or modified IGFBP7 may have less commonality with naturally occurring IGFBP7, but may also promote myocardial repair following myocardial infarction, improve cardiac function following myocardial infarction, and more particularly include: reducing the area of fibrous scar after myocardial infarction, reducing the area of myocardial infarction area after myocardial infarction, increasing the number of blood vessels in the myocardial infarction marginal area after myocardial infarction, promoting myocardial cell to resist injury, and inhibiting programmed necrosis after myocardial infarction.

As used herein, the terms "myocardial infarction" or "myocardial infarction" are used interchangeably.

IGFBP7, signal pathway involved therein and use of modulators thereof

In the research work of the present inventors, it was found that cardiovascular precursor cells (hCVPCs) secrete high-abundance IGFB7 factors, and the effect of IGFBP7 in promoting myocardial repair after myocardial infarction was demonstrated. Specifically, IGFBP7 can protect cardiac function of myocardial infarction animals, reduce fibrous scar after myocardial infarction, and promote myocardial repair of ischemia injury. The mechanism research shows that the action mechanism of the compound at least partially reduces the stability of RIP3 mRNA in an IGF independent mode, and further inhibits programmed necrosis of myocardial cells after myocardial infarction.

Based on the new discovery of the inventor, the invention provides the application of IGFBP7 or the regulator thereof, which is used for preparing the composition or the medicine for promoting the myocardial repair after myocardial infarction, improving the cardiac function after myocardial infarction and protecting myocardial ischemia injury; the method specifically comprises the following steps: reducing the area of fibrous scar after myocardial infarction, reducing the area of myocardial infarction area after myocardial infarction, increasing the number of blood vessels in the myocardial infarction marginal area after myocardial infarction, promoting myocardial cell to resist injury, and inhibiting programmed necrosis after myocardial infarction. The invention also provides the use of IGFBP7 as a target for screening substances that inhibit the above-mentioned diseases or conditions.

As used herein, the up-regulation of IGFBP7 includes accelerants, agonists, and the like. Any substance that increases the activity of IGFBP7, maintains the stability of IGFBP7, promotes the expression of IGFBP7, promotes the secretion of IGFBP7, prolongs the effective duration of IGFBP7, or promotes the transcription and translation of IGFBP7 may be used in the present invention as an effective substance having an up-regulating function.

In a preferred embodiment of the present invention, the IGFBP7 up-regulator includes (but is not limited to): an expression vector or expression construct that expresses (preferably overexpresses) IGFBP7 following transfer into a cell. Typically, the expression vector comprises a gene cassette containing a gene encoding IGFBP7 and expression control sequences operably linked thereto. The term "operably linked" or "operably linked" refers to a condition in which certain portions of a linear DNA sequence are capable of modulating or controlling the activity of other portions of the same linear DNA sequence. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.

In the present invention, the IGFBP7 polynucleotide sequence may be inserted into a recombinant expression vector, so that it may be transferred into a cell and overexpressed to produce IGFBP7. Any plasmid and vector can be used in the present invention as long as it can replicate and is stable in the host. An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translation control elements. For example, the expression vector includes: viral vectors, non-viral vectors; preferably, the expression vector includes (but is not limited to): adeno-associated virus, lentiviral vectors, adenoviral vectors, and the like.

Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence of IGFBP7 and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.

The present invention also provides a composition comprising an effective amount (e.g., 0.000001-20wt%, preferably 0.00001-10 wt%) of said IGFBP7, or a upregulating agent thereof (e.g., an expression vector overexpressing said IGFBP 7), or an analog thereof, and a pharmaceutically acceptable carrier.

The composition can be directly used for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction and protecting myocardial ischemia injury. In addition, it may be used in combination with other therapeutic agents or adjuvants.

Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8.

As used herein, the term "comprising" means that the various ingredients can be applied together in a mixture or composition of the invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "comprising. As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity in, and is acceptable to, a human and/or an animal.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The compositions of the present invention comprise a safe and effective amount of IGFBP7, or a upregulating agent thereof (e.g., an expression vector that overexpresses IGFBP 7), or an analog thereof, and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical composition of the present invention may be prepared in the form of injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the invention can also be prepared into a sustained release preparation.

The effective amount of IGFBP7 or upregulated dosage of the invention may vary with the mode of administration and the severity of the condition being treated, among other things. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of said IGFBP7 or its up-regulator 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 status of the patient, the route of administration, and the like. In general, satisfactory results are obtained when IGFBP7 of the invention or a formulation thereof is administered at a daily dosage of about 0.00001mg to about 10mg per kg of animal body weight

The mode of administration of IGFBP7 of the invention or its up-regulator, or its analogs is not particularly limited and may be systemic or local. For example, IGFBP7 of the invention or a upregulation thereof may be administered by means of local tissue injection, preferably myocardial injection. In addition, other modes of injection are possible, such as, but not limited to, intraperitoneal injection, intravenous injection, oral administration, subcutaneous injection, intrathecal injection, intradermal injection, and the like.

Given the use of IGFBP7, various methods well known in the art can be used to administer IGFBP7 or its encoding gene, or its pharmaceutical composition to a mammal. Preferably, gene therapy is used, such as administering IGFBP7 directly to the subject by methods such as injection; alternatively, expression units (e.g., expression vectors or viruses) carrying the IGFBP7 gene may be delivered to the target in a route that allows expression of the active IGFBP7.

In one embodiment of the present invention, the IGFBP7 may be administered directly to a mammal (e.g., human), or a gene encoding IGFBP7 may be cloned into an appropriate vector (e.g., a conventional prokaryotic or eukaryotic expression vector, or a viral vector such as a herpesvirus vector or an adenovirus vector) by a conventional method, and the vector may be introduced into a cell expressing IGFBP7, so that the cell expresses IGFBP7. Expression of IGFBP7 may be achieved by introducing appropriate amounts of the cells into appropriate parts of the mammalian body.

The mode of administration of an up-regulator or analog of IGFBP7 will depend primarily on the type and nature of the up-regulator, as can be assessed by one skilled in the art.

IGFBP7 as drug screening target

After the function and mechanism of action of IGFBP7 are known, substances that promote the expression or activity of IGFBP7 can be screened based on this characteristic.

Accordingly, the present invention provides a method for screening a potential substance useful for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, and protecting myocardial ischemia damage, the method comprising: contacting the candidate substance with a system expressing IGFBP 7; and detecting the effect of the candidate substance on IGFBP 7; if the candidate substance can improve the expression or activity or secretion of IGFBP7, the candidate substance is indicated to be used for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction and protecting myocardial ischemia injury.

In the mechanism research of the invention, the expression of RIP3, which is a key protein of programmed necrosis after the treatment of IGFBP7, is obviously reduced, and the RIP3 inhibition by IGFBP7 comprises the regulation and control of the transcription level. Based on this new discovery, in a preferred embodiment of the present invention, the system further comprises: programmed necrosis RIP1/RIP3 signal pathway or pathway protein RIP3. And the method further comprises: and detecting the interaction condition of the insulin-like growth factor binding protein 7 and the programmed necrosis RIP1/RIP3 signal pathway or pathway protein RIP3 in the system, wherein if the insulin-like growth factor binding protein 7 inhibits the programmed necrosis RIP1/RIP3 (especially inhibits RIP 3) signal pathway or pathway protein RIP3, the candidate substance is the required (interested) drug or compound.

In a preferred embodiment of the present invention, in order to more easily observe the change in expression or activity of IGFBP7 and its involved signal pathway protein or its upstream and downstream proteins during screening, a control group may be provided, which may be a system expressing IGFBP7, RIP1/RIP3 signal pathway or pathway protein RIP3 without adding the candidate substance.

The expression system may be, for example, a cell (or cell culture) system, the cell may be a cell endogenously expressing IGFBP7, RIP1/RIP3 signaling pathway or pathway protein RIP3; or may be cells recombinantly expressing IGFBP7, RIP1/RIP3 signal pathway or RIP3 pathway protein. The system for expressing IGFBP7, RIP1/RIP3 signal pathway or RIP3 pathway protein can also be (but is not limited to) a subcellular system, a solution system, a tissue system, an organ system or an animal system (such as an animal model) and the like.

As a preferred mode of the present invention, the method further comprises: the obtained potential substance is subjected to further cell experiments and/or animal experiments to further select and identify a substance which is truly useful for performing the function or the like claimed by the present invention.

The method of detecting the expression, activity, amount of IGFBP7 or secretion of IGFBP7 is not particularly limited in the present invention. Conventional protein quantitative or semi-quantitative detection techniques may be employed, such as (but not limited to): SDS-PAGE, western-Blot, ELISA, etc.

In another aspect, the invention also provides compounds, compositions or medicaments, or potential substances, obtained by the screening method. Some preliminarily screened substances can form a screening library so that people can finally screen substances which are really useful for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, protecting myocardial ischemia injury and the like, thereby being used for clinic.

IGFBP7 as a diagnostic target

Based on the above-mentioned new findings of the present inventors, IGFBP7 can be used as a marker for evaluating/prognosing the disease progression after myocardial infarction: (i) Performing disease typing, differential diagnosis, and/or susceptibility analysis after myocardial infarction; (ii) Evaluating the treatment medicine, the medicine curative effect and the prognosis of the myocardial infarction of related people, and selecting a proper treatment method; (iii) Early evaluating the disease risk of related population, and early monitoring the early prevention and treatment. For example, a person at high risk for having an abnormal IGFBP7 gene expression can be isolated, and more targeted prevention or treatment can be performed.

Accordingly, the present invention provides the use of IGFBP7 for the preparation of a reagent or kit for the assessment or prognosis of disease progression after myocardial infarction.

The presence, expression level or activity of the IGFBP7 gene may be detected using a variety of techniques known in the art and are encompassed by the present invention. For example, the conventional techniques such as Southern blotting, western blotting, DNA sequencing, PCR and the like can be used, and these methods can be used in combination.

The invention also provides reagents for detecting the presence and expression of the IGFBP7 gene in an analyte. Preferably, when the detection at the gene level is performed, a primer that specifically amplifies IGFBP 7; or a probe that specifically recognizes IGFBP7 to determine the presence or absence of the IGFBP7 gene; when detecting protein levels, antibodies or ligands that specifically bind to proteins encoded by IGFBP7 may be used to determine the expression of IGFBP7 protein. In a preferred embodiment of the present invention, the reagent is a primer that specifically amplifies IGFBP7 gene or gene fragment. The design of a specific probe for IGFBP7 gene is well known to those skilled in the art, and for example, a probe is prepared which specifically binds to a specific site on IGFBP7 gene but not to genes other than IGFBP7 gene and which carries a detectable signal. In addition, methods for detecting the expression of IGFBP7 protein in an analyte using antibodies that specifically bind IGFBP7 protein are well known to those skilled in the art.

The present invention also provides a kit for detecting the presence or absence and expression of the IGFBP7 gene in an analyte, the kit comprising: primers for specifically amplifying IGFBP7 gene; a probe that specifically recognizes the IGFBP7 gene; or an antibody or ligand that specifically binds to an IGFBP7 protein.

The kit may further comprise various reagents required for DNA extraction, PCR, hybridization, color development, and the like, including but not limited to: an extraction solution, an amplification solution, a hybridization solution, an enzyme, a control solution, a color development solution, a washing solution, and the like. In addition, the kit may further comprise instructions for use and/or nucleic acid sequence analysis software, and the like.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Sequence information

Human IGFBP7 CDS sequence (SEQ ID NO: 2):

atggagcggccgtcgctgcgcgccctgctcctcggcgccgctgggctgctgctcctgctcctgcccctctcctcttcctcctcttcggacacctgcggcccctgcgagccggcctcctgcccgcccctgcccccgctgggctgcctgctgggcgagacccgcgacgcgtgcggctgctgccctatgtgcgcccgcggcgagggcgagccgtgcgggggtggcggcgccggcagggggtactgcgcgccgggcatggagtgcgtgaagagccgcaagaggcggaagggtaaagccggggcagcagccggcggtccgggtgtaagcggcgtgtgcgtgtgcaagagccgctacccggtgtgcggcagcgacggcaccacctacccgagcggctgccagctgcgcgccgccagccagagggccgagagccgcggggagaaggccatcacccaggtcagcaagggcacctgcgagcaaggtccttccatagtgacgccccccaaggacatctggaatgtcactggtgcccaggtgtacttgagctgtgaggtcatcggaatcccgacacctgtcctcatctggaacaaggtaaaaaggggtcactatggagttcaaaggacagaactcctgcctggtgaccgggacaacctggccattcagacccggggtggcccagaaaagcatgaagtaactggctgggtgctggtatctcctctaagtaaggaagatgctggagaatatgagtgccatgcatccaattcccaaggacaggcttcagcatcagcaaaaattacagtggttgatgccttacatgaaataccagtgaaaaaaggtacacaataa

human IGFBP7 amino acid sequence (SEQ ID NO: 1):

MERPSLRALLLGAAGLLLLLLPLSSSSSSDTCGPCEPASCPPLPPLGCLLGETRDACGCCPMCARGEGEPCGGGGAGRGYCAPGMECVKSRKRRKGKAGAAAGGPGVSGVCVCKSRYPVCGSDGTTYPSGCQLRAASQRAESRGEKAITQVSKGTCEQGPSIVTPPKDIWNVTGAQVYLSCEVIGIPTPVLIWNKVKRGHYGVQRTELLPGDRDNLAIQTRGGPEKHEVTGWVLVSPLSKEDAGEYECHASNSQGQASASAKITVVDALHEIPVKKGTQ

the human IGFBP7 domains are shown in FIG. 1.

Example 1 obtaining of IGFBP7

The inventor analyzes the secretion spectrum of cardiovascular precursor cells (hCGCs) by using a cytokine chip, and finds that the hCGCs secrete at least 500 factors including cell growth factors, chemokines, inflammatory response factors and the like.

After large-scale research and analysis, the inventor finds that the increase of the existence of Insulin-like Growth Factor Binding Protein 7 (IGFBP7) in the hCGCs conditioned medium is more than 500 times that of the hCGCs conditioned medium, and preliminary research indicates that the Insulin-like Growth Factor Binding Protein may be an important effect Factor for hCGCs to play a myocardial repair role.

Example 2 study of IGFBP7 inhibition of apoptosis necrosis of ischemia damaged cardiomyocytes

(I) animal model

IGFBP7 myocardial specific knockout (IGFBP 7) ^CM-/- ) Preparation of mice:

constructing a gRNA aiming at a mouse Igfbp7 gene by using a Cas9/RNA system gene targeting technology, and guiding a Cas9 protein to cut a DNA double chain at a specific site of a crRNA guide sequence target; meanwhile, a Donor vector carrying a target site homology arm and a conditional knockout element is prepared and injected together with the Cas9system, and after the Cas9system cuts a DNA chain, loxp elements are recombined to two sides of a target site No. 3 exon through homologous recombination to obtain the igfbp7 flox mouse. After mating with myh6-Cre mice, the mice induced by tamoxifen to induce igfbp7 knockout.

Mouse myocardial infarction model: male C57BL/6 mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution (45 mg/kg), and after fixation, the mice were depilated from the left anterior chest and were intubated with a ventilator. Opening chest at the left side of chest 1cm above xiphoid process, opening 2-3 intercostals, transversely placing chest expander, and fully expanding intercostals to expose heart. The left anterior descending branch of the coronary artery is ligated by using a slipknot, and the part of the left ventricle of the heart becomes white and the ST segment of the electrocardiogram is lifted after the blood vessel is successfully ligated.

Oxygen deprivation (OGD) injury model: an oxygen deprivation model (OGD) injury model was established on neonatal rat cardiomyocytes using a hypoxia/anaerobic workstation. Setting the parameters of the workstation: the oxygen concentration in the low-oxygen mode is less than 0.1 percent, and the carbon dioxide concentration is 5 percent. In the hypoxia treatment process, the cell culture solution is an ischemia solution and normal oxygen control group, and is always in a normal culture solution and cultured in an incubator. For cell damage assays, cells were treated anaerobically for 1 hour.

(II) design of cell experiments

Cell preparation

Isolation of adult wild-type, IGFBP7 myocardial-specific knockout (IGFBP 7) ^CM-/- ) Mouse myocardial cell, utilize oxygen sugar deprivation (OGD) damage model simulation myocardial ischemia damage, hypoxia chamber parameter sets up: the concentration of oxygen in the low-oxygen mode is less than 0.1 percent, and the concentration of carbon dioxide is 5 percent. The cell culture solution in the hypoxia treatment process is ischemia solution (125mM NaCl,8mM KCl,1.2mM KH) ₂ PO ₄ ，1.25mM MgSO ₄ ，6.25mM NaHCO ₃ ，20mM HEPES-Na Salt，5mM lactate-Na，1.2mM CaCl ₂ And pH 6.6), the normoxic control group was always in normal culture broth (M199 1% penillilin-streptomycin, 10mM 2, 3-robust monooxime, 1% insulin-transferrin-selenium,1% chemicall defined lipid concentration and 5% bovine serum albumin) and cultured in a normal incubator.

Preparation of IGFBP 7-overexpressing adenovirus (Ad-IGFBP 7): the pAd-igfbp7 plasmid and the adenovirus backbone plasmid were co-expressed in 293A cells using Lipofectamine 2000 as transfection reagent. After 5-7 days of culture, the cells were observed to confirm the formation of virus clones, and after about 12 days, the cells in the centers of the clones were lysed to form lesions, and the virus solution in the lesions was aspirated and added to 500. Mu.L DMEM, which was the primary virus. 293A cells were plated on 100mm dishes and 100. Mu.L of primary virus-infected cells were added. Significant cell lysis was usually seen 2-3 days after infection. The virus is collected when one third to half of the cells float (usually 3-5 days after infection). The cells were gently scraped, transferred to a 15mL centrifuge tube, frozen in liquid nitrogen, thawed in a 37 ℃ water bath, and shaken vigorously. This step was repeated 3 times. Centrifuging at 6000 deg.C and 4 deg.C for 5 min, collecting supernatant, and storing. The product is not repeatedly frozen and thawed during storage, and can be stored at-20 deg.C; for further amplification of adenovirus, the viral supernatant obtained in the previous step was again infected into cells of a 100mm dish, and the virus was collected in the same manner, and 293A cells in a 150mm cell dish were further infected, whereby a sufficient amount of virus was obtained.

The recombinant adenovirus is used for infecting the myocardial cells to obtain the recombinant myocardial cells of over-expression IGFBP7.

Detecting the index

For cell damage assay, cells were treated with hypoxia for 1 hour, and cell morphology, lactate Dehydrogenase (LDH) release, ATP synthesis, mitochondrial membrane potential were measured to assess cell damage.

(II) animal experiment design

1. Laboratory animal

8-12 weeks of C57BL6 WT mice;

IGFBP7 for 8-12 weeks ^CM-/- A mouse.

Experimental grouping:

a: sham surgery + vehicle group;

b: surgical group + solvent group;

c: surgical group + IGFBP7 (1 ng/kg);

d: surgical group + IGFBP7 (30 ng/kg).

2. Detection indexes are as follows:

(1) Survival rate: detecting the survival rates of a, b, c and d groups of mice;

(2) Ultrasonic cardiac function: cardiac functional ultrasound examination of Left Ventricular Ejection Fraction (LVEF) was performed at 2, 7, 14 and 28 days post-surgery, respectively;

(3) Scar area: the area of a fibrous scar of the myocardial tissue is evaluated by adopting Masson dyeing on a section after OCT embedding;

(4) Apoptosis of myocardial cells: the section of the myocardial tissue is subjected to OCT embedding and then TUNEL staining is used for evaluating the apoptosis condition of the damaged myocardial cells;

(5) And (3) forming blood vessels: the myocardial tissue is cut into slices after being embedded by OCT and the blood vessel forming conditions of the myocardial infarction area and the marginal area are evaluated by a-SMA staining;

(6) Collecting myocardial tissue samples to extract RNA, detecting protein and gene expression conditions and carrying out subsequent experiments.

Example 3 expression of IGFBP7 in cardiomyocytes

To reveal the cardioprotective effects of IGFBP7, first to clarify the expression of IGFBP7 in the heart, high throughput analysis of the database (ProtomicsDB) showed that IGFBP7 is more stably expressed in many tissues in normal human (FIG. 2A, yellow is heart), and muscle tissue is only expressed in the heart. Immunofluorescent staining of adult mouse cardiomyocytes and fibroblasts revealed that IGFBP7 was distributed ubiquitously in the cardiomyocytes (fig. 2B), whereas in the fibroblasts, it was dotted and partially co-localized with vimentin (vimentin) (fig. 2B).

The Western blot results showed that IGFBP7 expression was significantly higher in cardiomyocytes than in fibroblasts (fig. 2C).

Example 4 expression of IGFBP7 in myocardial infarction

The antibody chip results show that IGFBP7 is a highly secreted factor from hCGCs (FIG. 3A), and Elisa measures the content of IGFBP7 in hCGCs conditioned medium (CdM) (FIG. 3B).

And (3) collecting a time course sample of myocardial infarction mouse heart tissue and serum by using a mouse myocardial infarction model, and detecting the change of IGFBP7 expression after myocardial infarction. As a result, it was found that the expression of IGFBP7 was continuously increased within one week after myocardial infarction (FIGS. 3C-3E), suggesting that it plays an important role in the development and subsequent progression of myocardial infarction.

Example 5 IGFBP7 myocardial-specific knockout aggravates post-myocardial infarction myocardial injury

Induced knock-out IGFBP7 ^CM-/- In mouse cardiomyocytes, IGFBP7 was knocked out (FIG. 4A), IGFBP7 ^CM-/- The surface area of the mouse myocardial cells is not different from that of a wild mouse, and the heart weight-body weight ratio and the heart function are not different.

However, after petiole surgery, IGFBP7 ^CM-/- The cardiac function of the mice (KO) was significantly reduced compared to the wild type mice (fig. 4B).

Example 6 myocardial injection of human recombinant protein IGFBP7 factor improves cardiac function and reduces scar area in myocardial infarction mice

A mouse myocardial infarction Model (MI) is utilized to carry out a myocardial injection experiment of the human recombinant protein IGFBP7 and evaluate the influence of the myocardial injection experiment on the cardiac function. The myocardial injection of IGFBP7 was performed simultaneously with myocardial infarction surgery using both 1ng/kg and 30ng/kg doses of factors, and mice were tested for cardiac function at 2, 7, 14 and 28 days following 2.4 ug/kg/day for 2, 7 and 28 days of subcutaneous injection over the following week, and myocardial infarction area was assessed for 4 weeks (FIGS. 5A, B).

The results show that myocardial injection of IGFBP7 did not affect survival in the mice following surgery (fig. 5B), significantly improved cardiac function in the mice 1 week post injection (fig. 5C), and significantly reduced fibrous scar area (fig. 5D).

Assessment of cardiomyocyte apoptosis by TUNEL staining it was found that myocardial injection of IGFBP7 significantly reduced the number of TUNEL-positive cells in the myocardial infarction region of mice (fig. 5E).

The a-SMA staining results showed an increase in the number of vessels in the myocardial infarction marginal zone of mice in the myocardial infarction group of IGFBP7 (FIG. 5F).

Example 7 neutralization of IGFBP7 antibodies impairs myocardial repair of hCGPCs

Using mouse myocardial infarction model, carrying out IGFBP7 antibody neutralization experiment (fig. 6A), detecting mouse cardiac function 2, 14 and 28 days after operation, and evaluating myocardial infarction area 28 days.

The results show that the IGFBP7 antibodies can impair the protective cardiac function of hCVPCs, limiting fibrous scarring (fig. 6B, fig. 6C).

Example 8 IGFBP7 protection of adult mouse cardiomyocytes against OGD injury

In order to further confirm that the myocardial protection of IGFBP7 is closely related to the protective effect on myocardial cells, adult mouse myocardial cells are separated, an in-vitro OGD damage model is established, and the capability of resisting damage of an IGFBP7 treatment group and a control group is compared. The results show that the proportion of rod cells in IGFBP7 treated group was significantly higher than in OGD control group (fig. 7A-B).

Meanwhile, it was found that the protective effect of IGFBP7 on cardiomyocytes was not affected after the addition of blocker NBI31772 of IGFBP7 binding to IGF, suggesting that IGFBP7 acts to protect myocardium through IGF receptor independent pathway.

The ability of the cardiomyocytes of WT and KO mice to resist OGD injury was further evaluated, and it was found that LDH release from the cardiomyocytes of KO mice was significantly increased after injury, and the proportion of rod-shaped cells was significantly decreased compared to the wild type (FIGS. 7C-E).

The above results indicate that IGFBP7 has the effect of protecting cardiomyocytes against injury.

Example 9 IGFBP7 inhibits post-myocardial infarction myocardial tissue RIP3 protein levels

Based on the above results, the role of IGFBP7 in protecting cardiomyocytes was clarified, and further it was revealed that the protective role was achieved through an IGF receptor-independent pathway. Necrosis is the main mode of myocardial cell death following myocardial infarction, where programmed necrosis accounts for an important proportion, and the classical RIP1/RIP3 pathway of programmed necrosis is detected in heart tissue samples 3 days after infarction.

The results showed that IGFBP7 treated histone necrosis significantly reduced the expression of the executive protein RIP3 (fig. 8), suggesting that IGFBP7 has an inhibitory effect on the occurrence of programmed necrosis after myocardial infarction.

Example 10 inhibition of RIP3 mRNA levels by IGFBP7

Further, QPCR assay was performed to analyze RIP3 mRNA expression following myocardial IGFBP7 injection.

The results show that myocardial injection of IGFBP7 significantly inhibited RIP3 mRNA levels. This result suggests that inhibition of RIP3 by IGFBP7 involves regulation of the transcriptional level (fig. 9).

Example 11 overexpression of IGFBP7 decreases RIP3 mRNA stability

To further determine how IGFBP7 regulates RIP3 mRNA levels, reduces transcription or promotes degradation, regulation of RIP3 mRNA degradation by IGFBP7 was evaluated by inhibiting transcription of adult mouse cardiomyocytes using actinomycin D (ActD, 10 ug/mL).

The results show that adenovirus (Ad) expressed IGFBP7 promoted its degradation by decreasing RIP3 mRNA stability (fig. 10), suggesting that IGFBP7 inhibited RIP3 expression by decreasing RIP3 mRNA stability, and thereby inhibited apoptotic necrosis of injured cardiomyocytes.

Example 12 screening method

(1) Screening based on IGFBP7 expression or activity

Cell: cardiomyocytes overexpressing IGFBP7.

Test group: culturing said cardiomyocytes overexpressing IGFBP7 and administering a candidate agent;

control group: culturing said cardiomyocytes overexpressing IGFBP7 without administration of a candidate substance.

The expression or activity of IGFBP7 in the test group and the control group is detected and compared. If the expression or activity of IGFBP7 in the test group is statistically higher (e.g., 30% or less higher) than that in the control group, it is indicated that the candidate is a potential substance for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, and protecting myocardial ischemic injury.

Further, the RIP1/RIP3 signal pathway is co-expressed in the cell, and the activity can be determined by observing: IGFBP7 was observed to reduce RIP3 mRNA stability in the test group. If the test group more remarkably promotes the reduction of RIP3 mRNA stability after the candidate substance is added, the candidate substance is a potential substance for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction and protecting myocardial ischemia injury.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims. Also, all references cited herein are incorporated by reference in this application as if each reference were individually incorporated by reference.

Sequence listing

<110> Shanghai Nutrition and health institute of Chinese academy of sciences

<120> cell secretion factor for promoting myocardial infarction repair and application thereof

<130> 216701

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 279

<212> PRT

<213> Homo sapiens

<400> 1

Met Glu Arg Pro Ser Leu Arg Ala Leu Leu Leu Gly Ala Ala Gly Leu

1 5 10 15

Leu Leu Leu Leu Leu Pro Leu Ser Ser Ser Ser Ser Ser Asp Thr Cys

20 25 30

Gly Pro Cys Glu Pro Ala Ser Cys Pro Pro Leu Pro Pro Leu Gly Cys

35 40 45

Leu Leu Gly Glu Thr Arg Asp Ala Cys Gly Cys Cys Pro Met Cys Ala

50 55 60

Arg Gly Glu Gly Glu Pro Cys Gly Gly Gly Gly Ala Gly Arg Gly Tyr

65 70 75 80

Cys Ala Pro Gly Met Glu Cys Val Lys Ser Arg Lys Arg Arg Lys Gly

85 90 95

Lys Ala Gly Ala Ala Ala Gly Gly Pro Gly Val Ser Gly Val Cys Val

100 105 110

Cys Lys Ser Arg Tyr Pro Val Cys Gly Ser Asp Gly Thr Thr Tyr Pro

115 120 125

Ser Gly Cys Gln Leu Arg Ala Ala Ser Gln Arg Ala Glu Ser Arg Gly

130 135 140

Glu Lys Ala Ile Thr Gln Val Ser Lys Gly Thr Cys Glu Gln Gly Pro

145 150 155 160

Ser Ile Val Thr Pro Pro Lys Asp Ile Trp Asn Val Thr Gly Ala Gln

165 170 175

Val Tyr Leu Ser Cys Glu Val Ile Gly Ile Pro Thr Pro Val Leu Ile

180 185 190

Trp Asn Lys Val Lys Arg Gly His Tyr Gly Val Gln Arg Thr Glu Leu

195 200 205

Leu Pro Gly Asp Arg Asp Asn Leu Ala Ile Gln Thr Arg Gly Gly Pro

210 215 220

Glu Lys His Glu Val Thr Gly Trp Val Leu Val Ser Pro Leu Ser Lys

225 230 235 240

Glu Asp Ala Gly Glu Tyr Glu Cys His Ala Ser Asn Ser Gln Gly Gln

245 250 255

Ala Ser Ala Ser Ala Lys Ile Thr Val Val Asp Ala Leu His Glu Ile

260 265 270

Pro Val Lys Lys Gly Thr Gln

275

<210> 2

<211> 840

<212> DNA

<213> Homo sapiens

<400> 2

atggagcggc cgtcgctgcg cgccctgctc ctcggcgccg ctgggctgct gctcctgctc 60

ctgcccctct cctcttcctc ctcttcggac acctgcggcc cctgcgagcc ggcctcctgc 120

ccgcccctgc ccccgctggg ctgcctgctg ggcgagaccc gcgacgcgtg cggctgctgc 180

cctatgtgcg cccgcggcga gggcgagccg tgcgggggtg gcggcgccgg cagggggtac 240

tgcgcgccgg gcatggagtg cgtgaagagc cgcaagaggc ggaagggtaa agccggggca 300

gcagccggcg gtccgggtgt aagcggcgtg tgcgtgtgca agagccgcta cccggtgtgc 360

ggcagcgacg gcaccaccta cccgagcggc tgccagctgc gcgccgccag ccagagggcc 420

gagagccgcg gggagaaggc catcacccag gtcagcaagg gcacctgcga gcaaggtcct 480

tccatagtga cgccccccaa ggacatctgg aatgtcactg gtgcccaggt gtacttgagc 540

tgtgaggtca tcggaatccc gacacctgtc ctcatctgga acaaggtaaa aaggggtcac 600

tatggagttc aaaggacaga actcctgcct ggtgaccggg acaacctggc cattcagacc 660

cggggtggcc cagaaaagca tgaagtaact ggctgggtgc tggtatctcc tctaagtaag 720

gaagatgctg gagaatatga gtgccatgca tccaattccc aaggacaggc ttcagcatca 780

gcaaaaatta cagtggttga tgccttacat gaaataccag tgaaaaaagg tacacaataa 840

Claims (11)

1. Use of insulin-like growth factor binding protein 7 or a upregulating agent thereof in the manufacture of a medicament or composition for the following function: promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, and relieving myocardial ischemia injury.

2. The use of claim 1, wherein promoting myocardial repair following myocardial infarction, improving cardiac function following myocardial infarction, and protecting against myocardial ischemic injury comprises:

reducing the area of the myocardial infarction area after myocardial infarction;

the area of a fibrous scar after myocardial infarction is reduced;

increasing the number of blood vessels in the myocardial infarction area and the myocardial infarction edge area after myocardial infarction;

promoting myocardial cells to resist damage;

the method can inhibit programmed necrosis of myocardial infarction, preferably inhibit activation of RIP1/RIP3 signal pathway of programmed necrosis, and further inhibit programmed necrosis of injured myocardial cell.

3. The use of claim 1, wherein the up-regulator of insulin-like growth factor binding protein 7 comprises an up-regulator selected from the group consisting of: (a) A substance that enhances the activity of insulin-like growth factor binding protein 7; (b) A substance that enhances the expression, stability or effective duration of action of insulin-like growth factor binding protein 7.

4. The use of claim 1, wherein the up-regulator of insulin-like growth factor binding protein 7 comprises a peptide selected from the group consisting of: an expression construct for recombinant expression of insulin-like growth factor binding protein 7, a polypeptide or compound that enhances the inhibitory effect of insulin-like growth factor binding protein 7 on the cellular programmed necrosis RIP1/RIP3 signal pathway, a chemical up-regulator of insulin-like growth factor binding protein 7, an up-regulator that promotes the driving ability of the insulin-like growth factor binding protein 7 gene promoter, a down-regulator of insulin-like growth factor binding protein 7 gene-specific microRNA, or a combination thereof; preferably, the expression construct comprises: viral vectors, non-viral vectors; more preferably, the expression vector comprises: adeno-associated virus vectors, lentiviral vectors, and adenoviral vectors.

5. Use of insulin-like growth factor binding protein 7 for screening a drug or compound having the following functions: promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, and protecting myocardial ischemia injury.

6. A method for screening a drug or a compound for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, and protecting myocardial ischemic injury, which comprises:

(1) Treating an expression system expressing insulin-like growth factor binding protein 7 with a candidate substance; and the combination of (a) and (b),

(2) Detecting the expression or activity of insulin-like growth factor binding protein 7 in said system; if the candidate substance statistically increases the expression or activity of insulin-like growth factor binding protein 7, it is indicative that the candidate substance is the desired drug or compound.

7. The method of claim 6, wherein step (1) comprises: in the test group, adding a candidate substance to the expression system; and/or the presence of a gas in the atmosphere,

the step (2) comprises the following steps: detecting the expression or activity of insulin-like growth factor binding protein 7 in said system and comparing said expression or activity to a control, wherein said control is an expression system without said candidate substance; if the candidate substance statistically increases the expression or activity of insulin-like growth factor binding protein 7, it is indicative that the candidate substance is the desired drug or compound.

8. The method of claim 6 or 7, wherein the system of step (1) further comprises a apoptosis RIP1/RIP3 signal pathway or pathway protein RIP3; and, step (2) also includes: and detecting the interaction condition of the insulin-like growth factor binding protein 7 and the programmed necrosis RIP1/RIP3 signal pathway or pathway protein RIP3 in the system, and if the insulin-like growth factor binding protein 7 inhibits the programmed necrosis RIP1/RIP3 signal pathway or pathway protein RIP3, indicating that the candidate substance is the required medicine or compound.

9. Use of a reagent specifically recognizing or amplifying insulin-like growth factor binding protein 7 for the preparation of a reagent or kit for assessing or prognosing disease progression after myocardial infarction.

10. The use according to claim 9, wherein a low expression of insulin-like growth factor binding protein 7 is indicative of a poor prognosis of the subject after myocardial infarction; if insulin-like growth factor binding protein 7 is normally expressed or highly expressed, it indicates that the prognosis of the subject after myocardial infarction is normal or good.

11. The use of claim 9, wherein said agent comprises:

a binding molecule that specifically binds to insulin-like growth factor binding protein 7;

primers for specifically amplifying the insulin-like growth factor binding protein 7 gene;

a probe that specifically recognizes insulin-like growth factor binding protein 7 gene; or

A chip for specifically recognizing the 7 gene of insulin-like growth factor.

Images