CN115381949A

CN115381949A - Application of targeted inhibition of pigment epithelium derived factor in promotion of liver regeneration and improvement of liver injury

Info

Publication number: CN115381949A
Application number: CN202110572595.8A
Authority: CN
Inventors: 王红阳; 陈瑶; 沈皓; 于晗; 付静; 董慧; 魏雅婷; 王碧波; 任一彬
Original assignee: Third Affiliated Hospital Of Chinese People's Liberation Army Naval Medical University
Current assignee: Third Affiliated Hospital Of Chinese People's Liberation Army Naval Medical University
Priority date: 2021-05-25
Filing date: 2021-05-25
Publication date: 2022-11-25

Abstract

The invention provides an application of targeted inhibition of Pigment Epithelium Derived Factor (PEDF) in promoting liver regeneration and improving liver injury. The invention discloses that PEDF plays a negative regulation role in the liver regeneration process for the first time, and the PEDF can be used as a novel target spot for diagnosis or treatment. The compound can be used as a target to develop a medicament for promoting liver regeneration, preventing, relieving and/or treating liver injury; the molecular marker can be used for diagnosing and prognostically evaluating the liver regeneration capability or the liver injury condition. Meanwhile, the inventor also screens and optimizes a targeted drug which can be targeted to PEDF and has particularly excellent in-vivo and in-vitro action effects.

Description

Application of targeted inhibition pigment epithelium derived factor in promoting liver regeneration and improving liver injury

Technical Field

The invention belongs to the field of application of biotechnology medicines; more specifically, the invention relates to an application of targeted inhibition of Pigment epithelium-derived factor (PEDF, also known as Serpinf 1) in promoting liver regeneration and improving liver injury.

Background

The liver is one of the most functional and complex parenchymal organs in the body, and has the main functions of regulating and controlling the balance of carbohydrates and lipids, participating in the metabolism and biotransformation of exogenous substances, generating and excreting bile, storing vitamins, synthesizing secretory proteins and generating and eliminating blood coagulation substances, and plays an important role in specific and non-specific immunity. The liver, as the center of human metabolism, plays an important role in various physiological and pathological processes, and many diseases also affect the liver.

Liver resection is currently the most common and effective method for treating liver tumors, and the strong regenerative capacity of normal liver tissue is the basis for effective liver resection. However, the method is limited by the bottleneck of early diagnosis technology of liver tumors, most of liver tumors are developed to the middle and late stages when being diagnosed for the first time, and because the tumors have large volumes and more involved liver segments, the volume of residual liver after resection cannot meet the metabolic demand of a human body, so that life-threatening complications such as liver failure can be caused. To solve the problem of insufficient volume of the residual Liver, various surgical methods for stimulating rapid regeneration of the residual Liver, such as Portal Vein Ligation (PVL)/Portal Vein Embolism (PVE), and two-step Hepatectomy (Associating Liver Partition and Portal Vein Ligation for Staged hepatology, ALPPS) combined with Liver Partition and Portal Vein Ligation, have been developed clinically.

However, the effectiveness of such procedures is still controversial, and the risk of liver failure in postoperative patients due to insufficient residual liver volume remains high. Researches related liver regeneration pathways, explores targets for regulating and controlling liver regeneration, develops safe and effective medicaments, accelerates the regeneration of residual liver in perioperative period, reduces the incidence rate of liver failure, and has important significance for the operative treatment of liver cancer.

Liver regeneration is a complex process which is commonly regulated and controlled by different types of Cells, and Liver Cells, immune Cells, bile duct Cells, stellate Cells and Liver Sinus Endothelial Cells (LSEC) are all involved in the regulation and control of the Liver regeneration process.

Therefore, there is a need in the art to further study and develop new technical schemes for promoting liver regeneration and improving liver damage, so as to provide new treatment approaches for clinical treatment.

Disclosure of Invention

The invention aims to provide an application of targeted inhibition of pigment epithelium derived factor in promoting liver regeneration and improving liver injury.

In a first aspect of the invention there is provided the use of an inhibitor of Pigment Epithelium Derived Factor (PEDF) for: preparing a composition for promoting liver regeneration; or for the preparation of a composition for the prevention, alleviation and/or treatment of liver damage.

In a preferred embodiment, the composition is also used for promoting proliferation of endothelial cells.

In a preferred embodiment, the composition is also used for promoting the looping ability of endothelial cells;

in a preferred embodiment, the composition is also used to stimulate the production of new blood vessels in the residual liver or in the liver after injury; and/or

In a preferred embodiment, the composition is also used for accelerating the recovery of liver function and liver size after hepatectomy.

In another preferred embodiment, the endothelial cells include (but are not limited to): hepatic sinus endothelial cells (LSEC), vascular endothelial cells (e.g., venous endothelial cells), lymphatic endothelial cells.

In another preferred embodiment, the inhibitor of pigment epithelium derived factor comprises a compound selected from (but not limited to): an agent that knocks out or silences a pigment epithelium-derived factor; binding molecules such as antibodies that specifically bind to pigment epithelium-derived factors; a chemical small molecule antagonist or inhibitor against pigment epithelium-derived factor; or agents that interfere with the interaction of the pigment epithelium-derived factor with an effector molecule or its receptor.

In another preferred embodiment, the agent that knocks out or silences pigment epithelium-derived factor comprises (but is not limited to): the gene coding sequence of the pigment epithelium derived factor is expressed by the interference molecule specifically interfering the coding gene of the pigment epithelium derived factor, the CRISPR gene editing reagent aiming at the pigment epithelium derived factor, the homologous recombination reagent aiming at the pigment epithelium derived factor or the site-directed mutation reagent, and the homologous recombination reagent or the site-directed mutation reagent carries out the function-loss mutation on the pigment epithelium derived factor.

In another preferred embodiment, the interfering molecule includes, but is not limited to, an shRNA, siRNA, miRNA, antisense nucleic acid, or a construct capable of forming the shRNA, siRNA, miRNA, antisense nucleic acid;

in another preferred embodiment, the agent for knocking out or silencing the pigment epithelium derived factor is an interfering molecule, and targets 382 to 402 th, 323 to 343 th, 246 to 266 th, 447 to 467 th or a combination thereof in the nucleotide sequence shown in SEQ ID NO. 1; preferably at positions 382 to 402, 323 to 343 or combinations thereof in the nucleotide sequence shown in SEQ ID NO. 1.

In another preferred embodiment, the inhibitor (e.g., an interfering molecule or sgRNA, etc.) is introduced to the target site (the lesion, such as post-operative liver) via an expression construct (expression vector); the expression construct comprises: viral vectors, non-viral vectors; preferably the viral vectors include (but are not limited to): adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors.

In another preferred embodiment, the liver injury comprises liver dysfunction after liver surgery, liver injury caused by hepatitis, liver fibrosis, liver cirrhosis, end-stage liver disease, liver cancer, alcoholic liver disease, metabolic liver disease or liver failure;

in another preferred embodiment, the liver operation comprises a treatment method based on the regeneration capacity of normal liver, which is used for destroying the liver tissue of a lesion part, preserving the normal liver tissue and enabling the normal liver tissue to compensate and proliferate to play the normal liver function; more preferably, it includes but is not limited to: traditional Hepatectomy, portal Vein Ligation secondary Hepatectomy (PVL), association Liver segmentation and Portal Vein Ligation secondary Hepatectomy (ALPPS), radiofrequency ablation, microwave ablation, cryoablation, hepatic artery interventional embolization chemotherapy, hepatic artery interventional embolization radiotherapy, and stereotactic radiotherapy.

In another aspect of the present invention, there is provided a use of a reagent specifically recognizing or amplifying a pigment epithelium-derived factor for the preparation of a diagnostic reagent or a kit for the diagnosis or prognostic evaluation of liver regeneration ability or liver damage; preferably, the agents include (but are not limited to): a binding molecule (e.g., an antibody or ligand) that specifically binds to a pigment epithelium-derived factor protein; primers for specifically amplifying pigment epithelium derived factor genes; a probe that specifically recognizes a pigment epithelium-derived factor gene; or a chip which specifically recognizes the pigment epithelium derived factor gene.

In another aspect of the present invention, there is provided a pharmaceutical composition or kit for promoting liver regeneration or preventing, alleviating and/or treating liver injury, comprising an inhibitor of pigment epithelium-derived factor, the inhibitor comprising a compound selected from the group consisting of: interference molecules specifically interfering the expression of coding genes of the pigment epithelium derived factor, a CRISPR gene editing reagent aiming at the pigment epithelium derived factor, a homologous recombination reagent or a site-directed mutation reagent aiming at the pigment epithelium derived factor, wherein the homologous recombination reagent or the site-directed mutation reagent performs function-loss mutation on the pigment epithelium derived factor; preferably, the interfering molecule includes (but is not limited to) shRNA, siRNA, miRNA, antisense nucleic acid, or a construct capable of forming the shRNA, siRNA, miRNA, antisense nucleic acid; more preferably, the interfering molecule targets positions 382-402, 323-343, 246-266, 447-467 or a combination thereof in the nucleotide sequence shown in SEQ ID NO. 1; preferably at positions 382 to 402, 323 to 343 or combinations thereof in the nucleotide sequence shown in SEQ ID NO. 1.

In another preferred embodiment, the composition is a pharmaceutical composition, a bioactive agent composition, a nutraceutical composition or a food composition.

In another preferred embodiment, the composition is prepared by mixing one or more of an inhibitor targeting PEDF gene (including bioactive agents, PEDF-related inhibitors, and PEDF-neutralizing antibodies) with a pharmaceutically acceptable carrier to obtain a composition for stimulating liver regeneration after liver surgery and preventing, alleviating, and/or treating liver dysfunction after hepatectomy.

In another aspect of the present invention, there is provided a method of screening for a potential substance for promoting liver regeneration or preventing, alleviating and/or treating liver damage, the method comprising: (1) Treating an expression system with a candidate substance, the expression system expressing pigment epithelium-derived factor; and, (2) detecting the expression or activity of a pigment epithelium-derived factor in said system; a candidate substance is a potential substance for promoting liver regeneration or preventing, alleviating and/or treating liver damage if the candidate substance statistically downregulates (significantly downregulates, e.g., downregulates by more than 10%, more than 20%, more than 50%, more than 80%, etc., or renders it non-expressed or inactive) the expression or activity of pigment epithelium-derived factors.

In another preferred embodiment, the system in step (1) is an endothelial cell (culture) system; preferably, the endothelial cells include (but are not limited to): hepatic sinus endothelial cells (LSEC), vascular endothelial cells (e.g., venous endothelial cells), lymphatic endothelial cells; the step (2) further comprises the following steps: detecting the proliferation capacity or the cyclization capacity of endothelial cells in the system; if the proliferation ability or the cyclization ability is promoted (significantly promoted, such as increased by more than 10%, more than 20%, more than 50%, more than 80% or more), the candidate substance is a potential substance for promoting liver regeneration or preventing, alleviating and/or treating liver injury.

In another preferred example, step (1) includes: in the test group, adding a candidate substance to the expression system; and/or, the step (2) comprises: detecting the expression or activity of the pigment epithelium derived factor in the system, or detecting the proliferation capacity or cyclization capacity of endothelial cells; and comparing the expression system with a control group, wherein the control group is an expression system without the candidate substance; if the candidate substance statistically reduces the expression or activity of the pigment epithelium derived factor or the proliferation capacity or the cyclization capacity of endothelial cells, the candidate substance is a potential substance for promoting liver regeneration or preventing, relieving and/or treating liver injury.

In another preferred embodiment, the candidate substance includes (but is not limited to): a regulatory molecule designed aiming at the pigment epithelium derived factor, a fragment or a variant thereof, a coding gene thereof or upstream and downstream molecules thereof or a signal path or a construct thereof (such as shRNA, siRNA, gene editing actual, an expression vector, a recombinant virus or non-virus construct and the like), a chemical small molecule (such as a specific inhibitor or antagonist), an interactive molecule and the like.

In another preferred embodiment, the system is selected from: a cell system (e.g., a cell or cell culture expressing pigment epithelium-derived factor), a subcellular (culture) system, a solution system, a tissue system, an organ system, or an animal system.

In another preferred example, the method further comprises: the obtained potential substances are subjected to further cell experiments and/or animal experiments to further select and identify substances useful for promoting liver regeneration and preventing, alleviating and/or treating liver damage from the candidate substances.

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

Drawings

Fig. 1A, perioperative survival curves of littermate C57 wild-type mice in ALPPS after administration of anti-PEDF antibody and placebo, respectively.

Fig. 1B is a graph of liver-to-body recovery rate of littermate C57 wild-type mice perioperative with ALPPS after administration of anti-PEDF antibody and placebo, respectively.

Fig. 2, ALPPS perioperative liver function test serum ALT and AST profiles of littermate C57 wild-type mice after administration of anti-PEDF antibody and placebo, respectively.

Figure 3, multiplex immunofluorescent staining profiles of liver tissue 2 days and 6 days post ALLPS I for the PEDF antibody group and the placebo group.

Fig. 4, western immunoblots of LSEC-associated markers LYVE and CD146 in liver tissue at 2 and 6 days post ALLPS I phase for the PEDF antibody group and placebo group.

Fig. 5 and the graphs for the detection of EDU proliferation assay after 48-hour incubation of LSEC in media supplemented with PEDF antibody and antibody diluent, respectively.

Fig. 6 and a microscopic brightfield image of the cyclization ability of LSEC after 6 hours of culture in a medium to which PEDF antibody and an antibody diluent were added, respectively.

FIG. 7 correlation of liver tissue PEDF transcript levels and post-operative ALT levels in hepatectomized patients.

FIG. 8, conditioned medium of hepatocytes with low transcription levels of PEDF promotes the cyclization ability of HUVEC cells.

FIG. 9, CCK8 proliferation assay of HUVEC cells stimulated by conditioned medium from primary hepatocytes from adenovirally mediated targeted interfering hepatoablation patients.

FIG. 10, microscopic brightfield image of HUVEC cell looping ability under conditioned medium stimulation of hepatocytes after adenovirus-mediated targeting interferes with the different sequences of PEDF in primary hepatocytes from a hepatectomy patient.

Detailed Description

Through intensive research and analysis, the inventor firstly reveals that Pigment Epithelium Derived Factor (PEDF) plays a negative regulation role in the liver regeneration process, and the PEDF can be used as a novel target point for diagnosis or treatment. By taking the compound as a target, a medicine for promoting liver regeneration, preventing, relieving and/or treating liver injury can be developed; the molecular marker can be used for diagnosing and prognostically evaluating the liver regeneration capability or the liver injury condition. Meanwhile, the inventor also screens and optimizes a targeted drug which can be targeted to PEDF and has particularly excellent in-vivo and in-vitro action effects.

PEDF

PEDF, also known as Serpin F1, gene ID:5176, is a secreted glycoprotein that is a member of the serine protease inhibitor superfamily and is widely distributed in human tissues, particularly in the liver and fat. Its main physiological functions include inhibiting angiogenesis, regulating lipid metabolism, resisting oxidation, resisting inflammation, resisting tumor, nourishing nerve, etc. It has been found that PEDF can inhibit the growth of transplanted tumor of human liver cancer nude mice by inhibiting the generation of tumor blood vessels (Gaoyun, etc.; pigment epithelium derived factor inhibits the growth of transplanted tumor of human liver cancer nude mice; national artificial liver experts forum; 2009). PEDF can also inhibit the invasion and metastasis of breast cancer cells by regulating epithelial-mesenchymal transition (Zhoudan et al; pigment epithelium derived factor inhibits breast cancer cell invasion and metastasis by regulating epithelial-mesenchymal transition; southern medical university proceedings; 2018). In terms of regulating normal cellular physiological functions, studies have shown that PEDF is involved in regulating lipid metabolism processes of human HepG2 hepatocytes (Stadium et al; influence of pigment epithelium-derived factors on lipid metabolism of human HepG2 hepatocytes. Proceedings of Chongqing university of medicine 2012), and that oxidative low density lipoprotein-induced human umbilical vein endothelial cell damage can be mitigated by antioxidant action (Madao et al; pigment epithelium-derived factors mitigate oxidative low density lipoprotein-induced human umbilical vein endothelial cell damage experiments; release military medical school proceedings 2017. In addition, studies suggest that PEDF has a function in the nourishment and repair of nerves. Thus, studies in the art on PEDF have shown that it has multiple functions, however, to date no researchers have correlated it with liver regeneration and the promotion of recovery from liver injury. After intensive in vitro and in vivo studies, the inventors have determined that PEDF is a target for liver regeneration.

PEDF according to the invention may be naturally occurring, e.g. it may be isolated or purified from a mammal. In addition, the PEDF may be artificially prepared, for example, recombinant PEDF may be produced according to a conventional recombinant technology of genetic engineering for experimental or clinical applications. In use, recombinant PEDF may be employed. The PEDF comprises full-length PEDF or a biologically active fragment thereof. Preferably, the amino acid sequence of PEDF can be substantially identical to the sequence shown in SEQ ID NO. 2; the nucleotide sequence thereof may be substantially the same as that shown in SEQ ID NO. 1.

The amino acid sequence of PEDF formed by substitution, deletion or addition of one or more amino acid residues is also included in the present invention. PEDF or a biologically active fragment thereof includes a portion of a conservative amino acid substitution sequence 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 ensure that the biological activity of the resulting molecule is not altered. It is contemplated that useful biologically active fragments of PEDF may be used in the present invention. Herein, a biologically active fragment of PEDF is meant to be a polypeptide that still retains all or part of the function of full-length PEDF. Typically, the biologically active fragment retains at least 50% of the activity of full-length PEDF. More preferably, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of full-length PEDF.

Around this PEDF target, the present inventors performed extensive experimental demonstrations, including demonstration at the cellular level as well as at the animal level, to determine the relationship of PEDF gene and liver regeneration. According to the embodiment of the present invention, overexpression of PEDF gene can inhibit liver regeneration after ALPPS operation, and delay recovery of liver function after ALPPS operation. The targeted inhibition of PEDF expression, such as interference of PEDF gene expression, the use of PEDF neutralizing antibodies and/or related preparations for inhibiting the PEDF neutralizing antibodies, and the like, can stimulate liver regeneration after ALPPS operation and accelerate the recovery of liver function after ALPPS operation.

In a more specific embodiment of the present invention, the present inventors determined the effect of PEDF gene in liver regeneration by using various experimental means such as primary cell in vitro proliferation assay, primary sinus hepatomere endothelial cell in vitro cyclization function assay, animal in vivo liver function assay, liver tissue immunofluorescence, liver tissue immunowestern blot, and the like, specifically as follows: 1. the survival rate of animals after ALPPS surgery can be remarkably improved by using a PEDF neutralizing antibody; 2. the use of PEDF neutralizing antibodies significantly increased the rate of liver-to-body recovery in animals following ALPPS surgery; 3. the use of the PEDF neutralizing antibody can significantly reduce serum alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) levels in animals after ALPP surgery; 4. the PEDF neutralizing antibody can obviously stimulate the proliferation of hepatic sinus endothelial cells in the later period of liver regeneration after ALPPS operation; 5. the primary hepatocytes sorted after hepatectomy are incubated with endothelial cells, and the effect of the hepatocytes on promoting proliferation and cyclization of the endothelial cells can be remarkably enhanced by PEDF neutralizing antibodies; 6. dividing patients who receive hepatectomy into low and medium high groups according to the PEDF transcription level of liver tissues, and detecting the ALT level after 3 days of operation to find that the PEDF transcription level is higher and the ALT level after 3 days of operation is relatively higher; 7. after primary hepatocytes were isolated from liver tissues of the above patients and cultured in vitro, HUVEC cells were stimulated using conditioned medium of the hepatocytes. Finding that the hepatocyte conditioned medium with low PEDF transcription level can promote the cyclization capability of the HUVEC cells better than the hepatocyte conditioned medium with high PEDF transcription level; 8. after human primary hepatocytes are sorted and targeted to interfere with PEDF through adenovirus mediation, the conditioned medium of the hepatocytes can promote the proliferation and cyclization capabilities of HUVEC cells.

From the above results, it was found that when PEDF-neutralizing antibody was used to inhibit PEDF during ALPPS operation, the proliferation of hepatic sinus endothelial cells was stimulated, the recovery rate of liver-body ratio was accelerated, the recovery of liver function was promoted, and the survival rate of animals after ALPPS operation was improved. Moreover, PEDF transcript levels in liver tissues of patients undergoing hepatectomy are closely associated with post-operative recovery of liver function, and patients with low PEDF transcript levels, which recover relatively quickly from post-operative liver function, may be associated with improved function of vascular endothelial cells. After the interference of the PEDF to the liver cells of patients with liver resection to inhibit the expression of the PEDF, the conditioned medium of the liver cells can promote the proliferation and the cyclization capacity of HUVEC cells of a human umbilical cord endothelial cell line (serving as a cell model of endothelial cells). Therefore, the targeted inhibition of PEDF has the effects of promoting liver regeneration and accelerating the functional recovery of the liver after hepatectomy, and provides theoretical basis and clinical basis for preventing, relieving and/or treating the liver dysfunction after hepatectomy.

Aiming at the functions of the PEDF gene, the bioactive preparation for targeting and interfering the PEDF gene, the related inhibitor of the PEDF and the neutralizing antibody thereof can be used as medicaments for promoting liver regeneration and preventing, relieving and/or treating liver dysfunction after liver surgery. PEDF can be used as a drug target for screening drugs for promoting liver regeneration and preventing, relieving and/or treating liver dysfunction after liver surgery; PEDF can also be used as a target gene in gene therapy, and is used for designing and preparing medicaments and/or biological preparations for promoting liver regeneration and preventing, relieving and/or treating liver dysfunction after liver surgery.

PEDF inhibitors and uses thereof

Based on the above new findings of the present inventors, the present invention provides a use of an inhibitor of PEDF or a gene encoding the same for preparing a composition for inhibiting and promoting liver regeneration and preventing, alleviating and/or treating liver injury (e.g., liver dysfunction after hepatectomy).

As used herein, the inhibitor of PEDF or a gene encoding the same includes down-regulators (e.g., down-regulators of expression, down-regulators of activity), antagonists, blockers, degradants, and the like, and these terms are used interchangeably.

The inhibitor of PEDF or the gene encoding the PEDF is any substance which can reduce the activity of PEDF, reduce the stability of PEDF or the gene encoding the PEDF, down-regulate the expression of PEDF, reduce the effective action time of PEDF, or inhibit the transcription and translation of the PEDF gene, and can be used in the invention as a substance which is useful for down-regulating PEDF, thereby being used for promoting liver regeneration or preventing, relieving or treating liver injury (such as liver dysfunction after hepatectomy). For example, the inhibitor includes an interfering RNA molecule or antisense nucleotide that specifically interferes with the expression of the PEDF gene; an antibody or ligand that specifically binds to a protein encoded by the PEDF gene; and so on.

In a particularly preferred embodiment of the present invention, the inhibitor is an interfering RNA molecule (e.g., siRNA, shRNA, miRNA, etc.) specific to PEDF, and such interfering RNA molecule can be prepared according to the sequence information of PEDF provided in the present invention. The method for preparing the interfering RNA molecules is not particularly limited, and includes, but is not limited to: chemical synthesis, in vitro transcription, and the like. The interfering RNA may be delivered into the cell by using an appropriate transfection reagent, or may also be delivered into the cell using a variety of techniques known in the art. The research results of the inventor find that, although molecules with certain interference capacity can be obtained for different sections of the PEDF gene, the interference sequence targeting certain specific sections has good specificity and particularly excellent target inhibition effect. Therefore, as the most preferred mode of the present invention, interfering molecules targeting positions 382 to 402 and 323 to 343 in the nucleotide sequence shown in SEQ ID NO.1 are used as inhibitors. The preferred inhibitors achieve just as good a down-regulation of PEDF (without affecting other in vivo mechanistic mechanisms of its production) without producing visible side effects, effectively promoting liver regeneration.

As a particularly preferred mode of the invention, the inhibitor is a neutralizing antibody (e.g. polyclonal antibody in the present example) specifically targeting PEDF, which inhibits PEDF function at the protein level. Polyclonal antibodies against PEDF can be prepared by conventional methods, for example, by introducing the PEDF protein into an animal, for example, by immunizing an animal after mixing the PEDF protein with freund's adjuvant at an appropriate ratio (e.g., 1. The immunization method may use subcutaneous injection into the animal. The animal can be selected from rabbit, sheep, cattle, etc. For example, a rabbit may be used to produce the polyclonal antibody: after 2-3 months of rabbit immunization, antiserum was harvested from its venous blood and purified to obtain specific polyclonal antibodies.

According to the novel findings of the present invention, monoclonal antibodies against PEDF can also be prepared using hybridoma technology. When the hybridoma is obtained, the hybridoma cells may be cultured and expanded in vitro according to a conventional animal cell culture method to secrete the anti-PEDF monoclonal antibody. As an embodiment, the anti-PEDF mab may be prepared by the following preparation method: (1) providing an adjuvant-pretreated mouse; (2) Inoculating the hybridoma cells in the abdominal cavity of a mouse and secreting a monoclonal antibody; (3) Ascites is extracted and separated to obtain the monoclonal antibody. The monoclonal antibody is separated from the ascites fluid and further purified, so that the antibody with high purity can be obtained. The monoclonal antibodies of the invention can also be prepared by recombinant methods or synthesized using a polypeptide synthesizer. It is well known to those skilled in the art that the monoclonal antibody can be easily obtained after obtaining the hybridoma cell line from which the monoclonal antibody is obtained or by means of sequencing or the like.

As a preferred embodiment of the present invention, there is provided an anti-PEDF neutralizing antibody having excellent properties, which has high specificity for PEDF and does not bind to proteins other than PEDF. And the effect of effectively blocking PEDF to inhibit angiogenesis (not influencing other in vivo mechanism mechanisms generated by the PEDF) can be realized, and the liver regeneration can be effectively promoted.

As an alternative of the invention, the inhibitor may be a small molecule compound directed against PEDF. Screening of such small molecule compounds can be performed by one skilled in the art using routine screening methods in the art. For example, in the examples of the present invention, several alternative screening methods are provided in conjunction with the regulatory mechanisms disclosed herein.

As a preferred mode of the invention, the CRISPR/Cas (e.g. Cas 9) system can be used for targeted gene editing, thereby knocking out PEDF gene in the region of targeted disease. Common methods for knocking out the PEDF gene include: co-transferring the sgRNA or a nucleic acid capable of forming the sgRNA, cas9 mRNA or a nucleic acid capable of forming the Cas9 mRNA into a targeted region or targeted cell. After the target site is determined, known methods can be employed to cause the sgRNA and Cas9 to be introduced into the cell. The nucleic acid capable of forming the sgRNA is a nucleic acid construct or an expression vector, or the nucleic acid capable of forming the Cas9 mRNA is a nucleic acid construct or an expression vector, and these expression vectors are introduced into cells, so that active sgrnas and Cas9 mrnas are formed in the cells.

As an alternative to the present invention, PEDF can be specifically targeted by homologous recombination to be defective or absent in expression. The Cre and loxp methods can also be used to selectively knock-out, reduce expression or inactivate a gene of interest in the genome of a cell.

The above are some representative or preferred ways to down-regulate PEDF. Other methods known in the art for modulating PEDF may also be used as would be known to one of skill in the art after understanding the general protocol of the present invention and are also encompassed by the present invention.

Applications related to diagnosis and prognosis evaluation

In the present invention, a target having an important regulatory effect on liver regeneration is disclosed. Based on this novel discovery by the present inventors, PEDF can be used as a target for the diagnostic or prognostic evaluation of liver regeneration ability or recovery from liver injury: (i) typing and differential diagnosis after liver injury; (ii) Evaluating the treatment, drug efficacy, prognosis evaluation, and selecting appropriate treatment for the relevant population (e.g., liver operated population). For example, people with abnormal gene expression of PEDF can be isolated, and targeted therapy can be performed more specifically.

The prognosis of a subject providing a sample to be assessed can be predicted by determining the expression or activity of PEDF in the sample to be assessed, and selecting an appropriate drug for treatment. Typically, a threshold level of PEDF expression is defined and when PEDF expression is above the defined threshold level, treatment with a regimen that inhibits PEDF is contemplated. Such a threshold value is readily determinable by one skilled in the art, and can be obtained, for example, by comparing the expression of PEDF in normal human cells or tissues to the expression of PEDF in cells or tissues of the subject. The specific value of the threshold may be different according to the measurement parameters, measurement instruments, and the like.

The presence or absence and expression of PEDF can 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 sequence analysis, PCR and the like can be used, and these methods can be used in combination.

The invention also provides reagents for detecting the presence or absence and expression of PEDF or a gene encoding it in an analyte. Preferably, when the detection at the gene level is performed, primers that specifically amplify PEDF can be used; or a probe that specifically recognizes PEDF to determine the presence or absence of the PEDF gene; when detecting protein levels, antibodies or ligands that specifically bind to proteins encoded by PEDF can be used to determine PEDF expression.

Methods for detecting PEDF expression in an analyte using antibodies that specifically bind to PEDF are well known to those skilled in the art.

The design of a specific probe for the PEDF 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 the PEDF gene but not to genes other than the PEDF gene, and which carries a detectable signal.

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

In addition, the kit may further include 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.

Drug screening

After close correlation between PEDF and liver regeneration is known, substances that inhibit the expression or activity of PEDF or its encoding gene can be screened based on this feature. From the substances, a drug which is truly useful for promoting liver regeneration or preventing, alleviating or treating liver damage can be found.

Accordingly, the present invention provides a method for screening a potential substance (candidate substance or candidate drug) for promoting liver regeneration or preventing, alleviating or treating liver damage, the method comprising: treating the system expressing PEDF with a candidate substance; and detecting the expression or activity of PEDF in the system; if the candidate substance inhibits the expression or activity of PEDF, it is indicated that the candidate substance is a potential substance for promoting liver regeneration or preventing, alleviating or treating liver damage. The system expressing PEDF is preferably a cell (or cell culture) system, and the cell may be a cell endogenously expressing PEDF; or may be a cell recombinantly expressing PEDF. Furthermore, it is also possible to assess whether the potential substance is useful by observing the interaction of PEDF with its upstream and downstream proteins.

In combination with the results of the studies of the present inventors, as a preferred mode of the screening method of the present invention, the effectiveness of the potential substance (candidate substance or drug candidate) can be further determined by analyzing the proliferative capacity or the cyclization capacity of liver tissue-derived endothelial cells. This can be assayed by culturing PEDF-expressing hepatocytes, or a screening system in which they are co-cultured with endothelial cells. An observable increase in proliferative capacity or looping capacity is indicative of the effectiveness of the potential substance.

In a preferred embodiment of the present invention, a Control group (Control) may be provided in order to more easily observe the change in the expression or activity of PEDF during screening, and the Control group may be a system expressing PEDF without adding the candidate substance. The control group includes but is not limited to: blank control without candidate substance, blank plasmid control, etc.

As a preferred embodiment of the present invention, the method further comprises: the obtained potential substances are subjected to further cell experiments and/or animal experiments to further select and identify substances which are truly useful for promoting liver regeneration or preventing, alleviating or treating liver damage.

In another aspect, the invention also provides a potential substance obtained by the screening method for promoting liver regeneration or preventing, relieving or treating liver injury. These preliminarily selected substances may constitute a screening library so that one may finally select therefrom substances useful for inhibiting the expression and activity of PEDF, thereby promoting liver regeneration or preventing, alleviating or treating liver damage.

Pharmaceutical composition

The invention also provides a pharmaceutical composition, which comprises an effective amount (such as 0.000001-50wt%, preferably 0.00001-20wt%, more preferably 0.0001-10 wt%) of the inhibitor of PEDF or the encoding gene thereof, and a pharmaceutically acceptable carrier.

As a preferred mode of the present invention, there is provided a composition for promoting liver regeneration or preventing, alleviating or treating liver damage, which comprises an effective amount of an inhibitor of PEDF or a gene encoding the same, and a pharmaceutically acceptable carrier.

In a preferred embodiment of the present invention, the inhibitor includes, but is not limited to: agents that knock-out or silence PEDF, binding molecules (e.g., antibodies or ligands) that specifically bind to PEDF, small chemical molecule antagonists or inhibitors against PEDF, and the like. In a more specific manner, the inhibitors include, but are not limited to: an interfering molecule that specifically interferes with the expression of a gene encoding PEDF, a CRISPR gene editing reagent directed to PEDF, a homologous recombination reagent or a site-directed mutagenesis reagent directed to PEDF, which carries out loss-of-function mutagenesis of PEDF. Particularly preferably, the composition comprises an antibody that specifically neutralizes PEDF; or an interfering molecule targeted to positions 382 to 402, 323 to 343 or a combination thereof in the nucleotide sequence shown in SEQ ID NO. 1.

In the present invention, the term "comprising" means that various ingredients can be used together in the mixture or composition of the present invention. Thus, the terms "consisting essentially of 8230and" consisting of 8230are encompassed by the term "comprising".

As used herein, the "effective amount" refers to an amount that produces a function or activity in and is acceptable to humans and/or animals. The term "pharmaceutically acceptable carrier" refers to a vehicle for the administration of a therapeutic agent, including various excipients, diluents, solvents, suspending agents, and the like, which may be liquid or solid, and which is a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in the composition may comprise liquids such as water, saline, buffers. In addition, auxiliary substances, such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. The vector may also contain a cell transfection reagent.

The dosage form of the pharmaceutical composition of the present invention may be various available dosage forms as long as the active ingredient can be efficiently delivered into the body of a mammal. In a preferred form of the invention, the pharmaceutical composition is in the form of a solution in which the antibody to PEDF is present in the form of a sol in a suitable liquid carrier or diluent.

Once the use of the PEDF or gene encoding inhibitor is known, a variety of methods well known in the art can be used to administer the inhibitor agent or gene encoding, or pharmaceutical composition thereof, to a mammal or human.

Preferably, it can be carried out by means of gene therapy. For example, an inhibitor of PEDF can be directly administered to a subject by a method such as injection; alternatively, an expression unit (e.g., an expression vector or virus, etc., or siRNA) carrying the inhibitor of PEDF can be delivered to the target site in a manner that allows expression of the active PEDF inhibitor, depending on the type of inhibitor. Preferably, the inhibitor is introduced into the target site (lesion, such as post-operative liver) via an expression construct (expression vector); the expression construct comprises: viral vectors, non-viral vectors; preferably the viral vectors include, but are not limited to: adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, retroviral vectors.

The effective amount of the PEDF or the gene encoding inhibitor thereof according to the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. 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 such as bioavailability, metabolism, half-life, etc. of said inhibitor of PEDF or its encoding gene; 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 the specific examples of the present invention, some dosing regimens for animals such as mice are given. Conversion from an animal, e.g. murine, dose to a dose suitable for human administration is readily made by those skilled in the art, and can be calculated, for example, according to the Meeh-Rubner equation: meeh-Rubner formula: a = kx (W) ^2/ 3)/10,000. Wherein A is the body surface area in m ² Calculating; w is body weight, calculated as g; k is constant and varies with species of animal, in general, mouse and rat 9.1, guinea pig 9.8, rabbit 10.1, cat 9.9, dog 11.2, monkey 11.8, human 10.6. It will be appreciated that the conversion of the dosage administered may vary according to the drug and clinical situation, as assessed by the skilled pharmacist.

In a preferred embodiment, the MOI value of the viral vector as an active ingredient in the composition may be 1 to 100, more preferably 5 to 50; further more preferably 8 to 20. The dose of PEDF antibody or PEDF-related inhibitor is in the range of 0.1mg to 1g/kg (more preferably 1mg to 0.5g/kg; still more preferably 10mg to 0.1 g/kg). It is understood that the amount may be above or below these ranges depending on the actual clinical needs.

In a preferred embodiment, the pharmaceutical composition is an oral formulation or an injectable formulation.

In a preferred embodiment, the composition further comprises an effective drug which can stimulate liver regeneration besides the PEDF antibody or the PEDF-related inhibitor.

The invention also provides a kit containing the pharmaceutical composition or directly containing the PEDF or the inhibitor of the coding gene thereof. In addition, the kit can also comprise instructions for the use of the drugs in the kit.

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

PEDF gene sequence (SEQ ID NO: 1:

atgaaagggaagctcgccaggtccacaaaggaaattcccgatgagatcagcattctccttctcggtgtggcgcacttcaaggggcagtgggt

aacaaagtttgactccagaaagacttccctcgaggatttctacttggatgaagagaggaccgtgagggtccccatgatgtcggaccctaaggc

tgttttacgctatggcttggattcagatctcagctgcaagattgcccagctgcccttgaccggaagcatgagtatcatcttcttcctgcccctga

aagtgacccagaatttgaccttgatagaggagagcctcacctccgagttcattcatgacatagaccgagaactgaagaccgtgcaggcggt

cctcactgtccccaagctgaagctgagttatgaaggcgaagtcaccaagtccctgcaggagatgaagctgcaatccttgtttgattcacca

gactttagcaagatcacaggcaaacccatcaagctgactcaggtggaacaccgggctggctttgagtggaacgaggatggggcgggaacc

acccccagcccagggctgcagcctgcccacctcaccttcccgctggactatcaccttaaccagcctttcatcttcgtactgagggacacagac

acaggggcccttctcttcattggcaagattctggaccccaggggcccctaa

PEDF amino acid sequence (SEQ ID NO: 2:

MKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDS

DLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQE

MKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTD

TGALLFIGKILDPRGP

example 1 Targeted inhibition of the Effect of PEDF on post-ALPPS liver regeneration

In this example, an animal (mouse) model of stepwise hepatectomy (ALPPS) combined with liver segmentation and portal vein branch ligation was prepared, and liver recovery was observed by administering PEDF antibody (purchased from R & D systems, AF 1177). The main operation steps are as follows:

(1) 50C 57BL/6J male mice (8 weeks old) were randomized into two groups, and both received the same ALPPS procedure, 48 hours post-first surgery, with second phase surgery.

(2) From 12 hours before the first-stage operation to 12 hours after the second-stage operation, the PEDF group is injected with 10mg/kg (R & D Systems, AF 1177) of PEDF antibody by the abdominal cavity every 12 hours, and the control group is injected with normal saline with the same volume for 7 times; two groups of mice were recorded for mortality.

(3) Killing 5 mice daily from the stage I operation according to a plan, weighing liver weight and body weight before killing, taking 300ul blood from tail vein, taking serum after centrifugation, and detecting alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST); freezing half of the liver, and storing in a-80 deg.C ultra-low temperature refrigerator; the other half was fixed with formalin and embedded in paraffin for use.

The postoperative measurement results are shown in fig. 1, the survival rate of mice with the PEDF antibody group after the ALPPS I phase operation is not obviously different, but the survival rate after the II phase operation (the second day after the I phase operation) is higher (left); liver bodies recovered more rapidly than the rate of recovery (right).

The results of liver function measurements are shown in FIG. 2, where serum ALT levels (left) and AST levels (right) were lower after phase II surgery (second day after phase I surgery) in mice of the PEDF antibody group.

The above results indicate that targeted inhibition of PEDF can accelerate the regeneration of residual liver after ALPPS surgery, accelerate the recovery of liver function, and reduce the mortality associated with hepatic failure after ALPPS surgery.

Example 2 immunofluorescence histochemical staining to examine the effect of targeted inhibition of PEDF on post-ALPPS liver regeneration

An ALPPS procedure was performed as described in example 1 to prepare a mouse ALPPS model, which was treated with PEDF antibody and control, and liver tissues of PEDF antibody-group mice and control-group mice were obtained 2 days and 6 days after phase I surgery, respectively. After slicing, immunofluorescence staining is carried out on specific markers HNF4 alpha of liver cells, specific markers LYVE1 of Liver Sinus Endothelial Cells (LSEC) and proliferation cell specific nuclear antigen Ki-67, and observation and counting are carried out through a confocal microscope. Ki-67 is a proliferation cell-associated nuclear antigen, the function of which is closely related to mitosis and is essential in cell proliferation, and positive staining indicates that the cell proliferation is active.

The immunofluorescence staining method mainly comprises the following steps:

(1) Carrying out paraffin embedding on the liver tissue to obtain a paraffin section, and dewaxing to water;

(2)3％H ₂ O ₂ washing with water at room temperature for 10 min;

(3) Acid antigen retrieval;

(4) 1% BSA blocking for 30min;

(5) Ki-67 antibody (1, cell Signaling Technology) was added dropwise overnight at 4 ℃;

(6) Dripping horseradish peroxidase-labeled secondary antibody (Shanghai Changshai biology) for 30min at 37 ℃;

(7) TSA fluorescent staining solution (Perkin Elmer) is added dropwise for 30min at room temperature;

(8) Washing the film;

(9) Antibodies HNF4 α (1, 200, abcam) and LYVE1 (1;

(10) DAPI staining, glycerol mounting, leica confocal microscopy.

As shown in the left panel of fig. 3, the PEDF antibody group and the control group proliferated hepatocytes at a similar ratio 2 days after phase I (hepatocyte proliferation peak period). As shown in the right panel of fig. 3, the proportion of LSEC proliferating cells in PEDF antibody group was much higher than that in control group 6 days after phase I operation (LSEC proliferating peak period).

This result demonstrates that targeted inhibition of PEDF promotes liver regeneration primarily by stimulating LSEC proliferation.

Example 3 immunoblotting to examine the Effect of Targeted inhibition of PEDF on post-operative liver regeneration by ALPPS

ALPPS procedures were performed as described in example 1 to prepare a mouse ALPPS model, which was treated with PEDF antibody and control, and the LSEC-specific marker molecules of the PEDF antibody group and control group were detected by immunoblotting. CD146, LYVE1, is a specific marker molecule for LSEC, and its abundance in liver tissue represents the proportion of LSEC in all cells of the liver.

The immunoblot detection method mainly comprises the following steps:

(1) Liver tissue was ultrasonically lysed in IP lysate (Shanghai Biyun), centrifuged at 12000rpm/min and 4 ℃.

(2) After centrifugation, the supernatant was subjected to BCA (Shanghai Saimeishi fly) measurement, and SDS denatured liquid was added thereto and heated at 95 ℃ for 5min at intervals.

(3) The prepared denatured sample is added to polyacrylamide gel to start electrophoresis, and the separated bands are transferred to nitrocellulose membrane.

(4) Blocking with 5% skim milk for 30min, adding diluted PEDF (1, 200, santa cruz TECHNOLOGY), CD146 (1, cell SIGNALING techrology), LYVE1 (1, 500, abcam), reference molecule GAPDH (1, 5000, cell SIGNALING techrology), and incubating overnight at 4 ℃.

(5) After the next day of washing, a fluorescent protein-conjugated secondary antibody (1.

(6) After washing, the relevant molecular expression intensity was read using an Odyssey membrane scanner.

As shown in fig. 4, compared with the control group, the expression level of the LSEC-specific marker of the PEDF antibody group after 2 days after the phase I operation was not changed much, and the expression level of the LSEC-specific marker of the phase I after 6 days after the phase I operation was significantly increased.

This result demonstrates that targeted inhibition of PEDF accelerates liver regeneration mainly by promoting proliferation of LSEC during the late stage of regeneration.

Example 4 Targeted inhibition of the Effect of PEDF on the in vitro proliferation potency of LSEC

In this example, ALPPS surgery was performed as described in example 1 to prepare a mouse ALPPS model, which was treated with PEDF antibody and control to examine the effect of targeted inhibition of PEDF on the proliferation capacity of LSEC in vitro. EDU, a thymidine analog that inserts into replicating DNA molecules during cell proliferation, can be analyzed by detecting the presence of EDU-positive cells, which can effectively detect the percentage of cells in S phase.

The main operation steps are as follows:

(1) 8-week-old C57BL/6J male mice were selected for ALPPS surgery, anesthetized on day 2 after the second phase surgery, and then subjected to in situ perfusion digestion of the liver.

(2) And obtaining the primary mouse hepatocytes and the primary LSEC by density gradient centrifugation.

(3) The obtained primary hepatocytes were cultured at 4X 10 ⁵ The cells were inoculated at a density of one ml in a 3D culture system using Matrigel (Corning) as a substrate, and the cells were changed once every 48 hours for three to four weeks.

(4) When the number of liver organoids exceeds 50/well, the old medium is collected during fluid exchange, centrifuged and the supernatant is removed.

(5) Mouse LSEC from in situ perfusion digestion were expressed at 1X 10 ⁶ The cells are inoculated on a 6-well plate at a density of one well and the cells are stable in adherence.

(6) Preparing a mixed culture medium by using the hepatocyte culture medium supernatant collected in the step 4) and the ECM culture medium 1 in a ratio, adding the PEDF antibody into the mixed culture medium at a concentration of 100ng/ml, and adding an equal volume of antibody diluent into a control group.

(7) After culturing LSEC in the mixed medium for 48 hours, the proliferation ratio of LSEC was measured using an EDU kit (shanghai keebo).

As a result, as shown in FIG. 5, the number of EDU-positive cells was significantly greater in the PEDF antibody group than in the control group.

This result demonstrates that targeted inhibition of PEDF promotes proliferation of sinusoidal endothelial cells (LSEC).

Example 5 Targeted inhibition of the Effect of PEDF on the in vitro cyclization Capacity of LSEC

In this example, ALPPS surgery was performed as described in example 1 to prepare a mouse ALPPS model, which was treated with PEDF antibody and control to examine the effect of targeted inhibition of PEDF on the ability of in vitro cyclization of mouse LSEC.

The main operation steps are as follows:

steps (1) to (4) were the same as in example 4.

(5) LSEC at 5X 10 ³ The density per well was plated on u-slide angiogenic slides using 10. Mu.l Matrigel plated prior to plating.

(6) Same as example 4, step (6).

(7) After culturing the LSEC in the mixed culture medium for 6 hours, the cyclization condition of the LSEC is observed under a microscope bright field.

The result is shown in fig. 6, the ring formation number of LSEC in the PEDF antibody group is significantly greater than that in the control group, which indicates that the PEDF antibody has a very significant promoting effect on the ring formation ability of the endothelial cells in the liver sinusoidal.

Example 6 correlation of liver tissue PEDF transcript levels and post-operative ALT levels in Supractomized patients

In this example, liver tissue from patients with major hepatectomy was analyzed for PEDF transcript levels and post-operative ALT levels.

The main operation steps are as follows:

(1) After the approval of the ethical committee of the hospital is obtained and the patient signs an informed consent, taking fresh cancer tissues beside the patient in the operation, fully shaking and cracking a part of the fresh cancer tissues by Trizol, and extracting RNA in the fresh cancer tissues by an ethanol extraction method; another part was stored in 4 ℃ medium and used in examples 7, 8, 9 within 8 hours.

(2) The cDNA was obtained by M-MLV (Invitrogen, 28025013) reverse transcription system.

(3) The transcriptional level of PEDF in liver tissue was detected by qRT-PCR system.

(4) PEDF transcript levels were correlated with serum ALT levels 3 days post-surgery.

As shown in FIG. 7, the patients were classified into three groups of PEDF low and high (high: nos. 1-7; medium: nos. 8-15; low: nos. 16-22) according to the level of PEDF transcript in liver tissue, and the ALT level after 3 days of operation was higher than that of the low PEDF transcript.

Thus, the level of PEDF transcript in liver tissue of most hepatectomies correlates with post-operative ALT levels, which are higher than those of PEDF transcripts, which are also relatively higher than those of post-operative ALT.

Example 7 promotion of cyclization Capacity of HUVEC cells by cultures of hepatocytes of PEDF Low transcript level group

In this example, the effect of cultures of hepatocytes from the PEDF low transcription level group on the cyclization ability of HUVEC cells was analyzed.

The main operation steps are as follows:

(1) Fresh para-cancer tissue of the patient in the operation of example 6 is taken, cut into pieces of about 1-2mm, digested with 1mg/ml collagenase at 37 ℃ for 1-1.5 hours, and digested until no visible tissue fragments are observed, by adding an equal volume of complete medium to stop the digestion.

(2) Filtering the cell suspension by a 70-micron filter screen, centrifuging for 50g multiplied by 3min, centrifuging twice, and collecting the hepatic cells at the bottom of the tube.

(3) After the culture medium is resuspended, the liver cells are planted into a 6-well plate coated by type I collagen, and the planting density is 1.2-1.5 multiplied by 10 ⁶ Each patient's hepatocytes were seeded in 5 wells per well.

(4) After 6 hours the solution was changed to remove non-adherent cells.

(5) Culturing for 48 hours after changing the liquid, and collecting the hepatocyte conditioned medium for later use.

(6) HUVEC according to 5X 10 ³ The density per well was plated on u-slide angiogenic slides using 10. Mu.l Matrigel plated prior to plating.

(7) The hepatocyte culture medium was collected, and HUVEC cells were cultured after mixing this conditioned medium with complete medium 1.

(8) After culturing HUVEC in the mixed culture medium for 24 hours, the cyclization condition is observed under a microscope under a bright field.

As a result, as shown in FIG. 8, the medium conditioned by the liver cells of the PEDF low transcription level group stimulated the cyclization ability of HUVEC cells more significantly. Suggesting that PEDF in hepatocytes may influence the recovery of liver function after hepatectomy by modulating the function of vascular endothelium.

Therefore, the conditioned medium of hepatocytes of the PEDF low transcription level group can promote the cyclization ability of HUVEC cells.

Example 8 Effect of PEDF on HUVEC cell proliferation in hepatocytes from Targeted interfering Subtraction patients

In this example, the effect on the proliferation of HUVEC cells was analyzed using PEDF in hepatocytes from an adenovirus-mediated targeted interfering hepatotomy patient.

The adenovirus vector is: h17692 (obtained from Shanghai and Yuanbiol Ltd.).

Sequence 1: cccaagctgaagctgagttat (SEQ ID NO:3; corresponding to positions 382 to 402 in SEQ ID NO: 1);

sequence 2: ccgagttcatttcatgacacag (SEQ ID NO:4; corresponding to positions 323 to 343 in SEQ ID NO: 1);

and (3) sequence: cggaagcatgagtatctactt (SEQ ID NO:5; corresponding to positions 246-266 in SEQ ID NO: 1);

and (4) sequence: cttgttgattcaccagactt (SEQ ID NO:6; corresponding to positions 447-467 in SEQ ID NO: 1).

The above sequences are introduced into an adenovirus vector.

The main operation steps are as follows:

(2) After filtering the cell suspension by a 70-micron filter screen, centrifuging for 50g multiplied by 3min, centrifuging twice, and collecting the hepatic cells at the bottom of the tube.

(3) After the culture medium is resuspended, the liver cells are planted into a 6-well plate coated by type I collagen, and the planting density is 1.2-1.5 multiplied by 10 ⁶ Each patient's hepatocytes were seeded with 5 wells per well.

(4) After 6 hours the solution was changed to remove non-adherent cells.

(5) Hepatocytes were infected with an empty vector and adenovirus loaded with different sequences (MOI = 8) and replaced 48 hours later.

(6) After another 48 hours of culture after the medium change, the hepatocyte culture medium was collected, and HUVEC cells were cultured after mixing this conditioned medium with-complete medium 1, and CCK8 assay was performed.

As shown in FIG. 9, conditioned medium of adenovirus targeting different sequences that interfere with PEDF did not stimulate HUVEC cells differently after infection of hepatocytes. Wherein, the

sequences

1 and 2 can remarkably improve the proliferation level of HUVEC cells, and the effect is remarkably superior to that of other groups.

Therefore, after the different sequences of PEDF in the liver cells of the patients with the high transcription level group of PEDF are targeted and interfered by the mediation of adenovirus, the conditioned medium of the primary liver cells can obviously promote the proliferation of HUVEC cells compared with the empty vector control group.

Example 9 targeting of PEDF in hepatocytes from patients with interfering hepatoablation promotes the ability of HUVEC cell lines to form loops

In this example, the effect on the cycling ability of HUVEC cells was analyzed using PEDF in hepatocytes from an adenovirus-mediated targeted interfering hepatotomy patient.

The main operation steps are as follows:

(1) - (5) same as in example 8.

(6) HUVEC according to 5X 10 ³ Perwell density was plated on u-slide angiogenic slides using 10. Mu.l Matrigel plates prior to plating.

(7) The hepatocyte medium was collected, and HUVEC cells were cultured after mixing this conditioned medium with complete medium 1.

(8) After culturing HUVEC in the mixed culture medium for 24 hours, the cyclization was observed under a microscope under a bright field.

As shown in FIG. 10, conditioned medium of different adeno-associated viruses targeted to interfere with the PEDF sequence stimulated the loop formation ability of HUVEC cells differently after infection of hepatocytes. Wherein, the loop formation number of the

groups

1 and 2 is obviously more than that of the control group, which shows that the targeted

interference sequences

1 and 2 can obviously improve the loop formation capability of HUVEC cells. The inventors observed that it was even superior to the AF1177 antibody.

Therefore, after adenovirus-mediated targeting to interfere with PEDF different sequences in hepatocytes of patients with liver resection in the high transcription level group of PEDF, the conditioned medium of the primary hepatocytes can promote the cyclization ability of HUVEC cells compared with the empty vector control group.

Example 10 screening method

(1) Screening based on PEDF expression or activity

And (3) screening system: a human liver cell line overexpressing PEDF (L-02).

Test group: culturing said liver cell line overexpressing PEDF and administering a candidate agent;

control group: culturing said liver cell line overexpressing PEDF without administration of a candidate substance.

The expression or activity of PEDF in the culture medium of the test group and the control group is detected and compared. If the expression or activity of PEDF in the test group is statistically lower (e.g., 30% or less lower) than that in the control group, the candidate is a potential agent for promoting liver regeneration, alleviating or treating liver damage.

(2) Screening based on endothelial cell cultures

Screening system: human liver cell line overexpressing PEDF (L-02) and human umbilical vein endothelial cell line (HUVEC).

Test group: stimulating HUVEC cells with L-02 conditioned medium over-expressing PEDF, and administering the candidate agent;

control group: HUVEC cells were stimulated with L-02 conditioned medium overexpressing PEDF, and no candidate substance was administered.

The proliferation ability or cyclization ability of HUVEC cells in the test group and the control group were detected and compared, respectively. If the proliferation or cyclization of HUVEC cells in the test group is significantly higher (e.g., 10% or more higher) than that in the control group, the candidate is a potential substance for promoting liver regeneration, alleviating or treating liver injury.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> third subsidiary Hospital of China's liberation army, navy, military and college

<120> application of targeted inhibition of pigment epithelium derived factor in promotion of liver regeneration and improvement of liver injury

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ctcggtgtgg cgcacttcaa ggggcagtgg gtaacaaagt ttgactccag aaagacttcc 120

ctcgaggatt tctacttgga tgaagagagg accgtgaggg tccccatgat gtcggaccct 180

aaggctgttt tacgctatgg cttggattca gatctcagct gcaagattgc ccagctgccc 240

ttgaccggaa gcatgagtat catcttcttc ctgcccctga aagtgaccca gaatttgacc 300

ttgatagagg agagcctcac ctccgagttc attcatgaca tagaccgaga actgaagacc 360

gtgcaggcgg tcctcactgt ccccaagctg aagctgagtt atgaaggcga agtcaccaag 420

tccctgcagg agatgaagct gcaatccttg tttgattcac cagactttag caagatcaca 480

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ggggcgggaa ccacccccag cccagggctg cagcctgccc acctcacctt cccgctggac 600

tatcacctta accagccttt catcttcgta ctgagggaca cagacacagg ggcccttctc 660

ttcattggca agattctgga ccccaggggc ccctaa 696

<210> 2

<211> 231

<212> PRT

<213> Homo sapiens

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Met Lys Gly Lys Leu Ala Arg Ser Thr Lys Glu Ile Pro Asp Glu Ile

1 5 10 15

Ser Ile Leu Leu Leu Gly Val Ala His Phe Lys Gly Gln Trp Val Thr

20 25 30

Lys Phe Asp Ser Arg Lys Thr Ser Leu Glu Asp Phe Tyr Leu Asp Glu

35 40 45

Glu Arg Thr Val Arg Val Pro Met Met Ser Asp Pro Lys Ala Val Leu

50 55 60

Arg Tyr Gly Leu Asp Ser Asp Leu Ser Cys Lys Ile Ala Gln Leu Pro

65 70 75 80

Leu Thr Gly Ser Met Ser Ile Ile Phe Phe Leu Pro Leu Lys Val Thr

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100 105 110

Asp Ile Asp Arg Glu Leu Lys Thr Val Gln Ala Val Leu Thr Val Pro

115 120 125

Lys Leu Lys Leu Ser Tyr Glu Gly Glu Val Thr Lys Ser Leu Gln Glu

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Met Lys Leu Gln Ser Leu Phe Asp Ser Pro Asp Phe Ser Lys Ile Thr

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165 170 175

Trp Asn Glu Asp Gly Ala Gly Thr Thr Pro Ser Pro Gly Leu Gln Pro

180 185 190

Ala His Leu Thr Phe Pro Leu Asp Tyr His Leu Asn Gln Pro Phe Ile

195 200 205

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ccgagttcat tcatgacata g 21

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cggaagcatg agtatcatct t 21

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cttgtttgat tcaccagact t 21

Claims

1. Use of an inhibitor of a pigment epithelium-derived factor for:

preparing a composition for promoting liver regeneration; or

Preparing a composition for preventing, alleviating and/or treating liver injury.

2. The use of claim 1, wherein said composition is further used for:

promoting proliferation of endothelial cells;

promoting the cyclization ability of endothelial cells;

stimulating the production of new blood vessels in the residual liver or damaged liver; and/or

Accelerate the recovery of liver body ratio and liver function after hepatectomy.

3. The use of claim 1, wherein the inhibitor of a pigment epithelium-derived factor comprises an inhibitor selected from the group consisting of: an agent that knocks out or silences a pigment epithelium-derived factor; binding molecules such as antibodies that specifically bind to pigment epithelium-derived factors; a chemical small molecule antagonist or inhibitor against pigment epithelium derived factor; or agents that interfere with the interaction of the pigment epithelium-derived factor with an effector molecule or its receptor.

4. The use of claim 3, wherein the agent that knocks out or silences a pigment epithelium-derived factor comprises: interference molecules specifically interfering the expression of coding genes of the pigment epithelium derived factor, CRISPR gene editing reagent aiming at the pigment epithelium derived factor, homologous recombination reagent or site-directed mutation reagent aiming at the pigment epithelium derived factor, wherein the homologous recombination reagent or the site-directed mutation reagent performs loss-of-function mutation on the pigment epithelium derived factor;

preferably, the interfering molecule comprises an shRNA, siRNA, miRNA, antisense nucleic acid, or a construct capable of forming the shRNA, siRNA, miRNA, antisense nucleic acid;

more preferably, the agent for knocking out or silencing pigment epithelium derived factor is an interference molecule, and targets 382-402 th, 323-343 th, 246-266 th, 447-467 th or the combination thereof in the nucleotide sequence shown in SEQ ID NO. 1; preferably at positions 382 to 402, 323 to 343 or combinations thereof in the nucleotide sequence shown in SEQ ID NO. 1.

5. The use of claim 4, wherein the inhibitor is introduced to the target site via an expression construct; the expression construct comprises: viral vectors, non-viral vectors; preferably the viral vector comprises: adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, retroviral vectors.

6. The use of claim 1, wherein the liver injury comprises liver dysfunction following liver surgery, liver damage resulting from hepatitis, liver fibrosis, cirrhosis, end-stage liver disease, liver cancer, alcoholic liver disease, metabolic liver disease, or liver failure;

preferably, the liver operation comprises a treatment method based on the regeneration capacity of the normal liver, which is used for preserving the normal liver tissue and enabling the normal liver tissue to compensate and proliferate to play the normal liver function by destroying the liver tissue of the lesion part; more preferably, the method comprises the following steps: traditional hepatectomy, secondary hepatectomy by portal vein ligation, secondary hepatectomy by combining liver segmentation and portal vein ligation, radiofrequency ablation, microwave ablation, cryoablation, hepatic artery interventional embolization chemotherapy, hepatic artery interventional embolization radiotherapy and stereotactic radiotherapy.

7. The application of a reagent for specifically identifying or amplifying pigment epithelium derived factors, which is used for preparing a diagnostic reagent or a kit for diagnosing or prognostically evaluating the liver regeneration capability or liver injury; preferably, the reagent comprises: a binding molecule that specifically binds to a pigment epithelium-derived factor protein; primers for specifically amplifying pigment epithelium derived factor genes; a probe that specifically recognizes a pigment epithelium-derived factor gene; or a chip which specifically recognizes the pigment epithelium derived factor gene.

8. A pharmaceutical composition or kit for promoting liver regeneration or preventing, ameliorating and/or treating liver injury comprising an inhibitor of pigment epithelium-derived factor, said inhibitor comprising a compound selected from the group consisting of: interference molecules specifically interfering the expression of coding genes of the pigment epithelium derived factor, CRISPR gene editing reagent aiming at the pigment epithelium derived factor, homologous recombination reagent or site-directed mutation reagent aiming at the pigment epithelium derived factor, wherein the homologous recombination reagent or the site-directed mutation reagent performs loss-of-function mutation on the pigment epithelium derived factor; preferably, the interfering molecule comprises an shRNA, siRNA, miRNA, antisense nucleic acid, or a construct capable of forming the shRNA, siRNA, miRNA, antisense nucleic acid; more preferably, the interfering molecule targets positions 382-402, 323-343, 246-266, 447-467 or a combination thereof in the nucleotide sequence shown in SEQ ID NO 1; preferably at positions 382 to 402, 323 to 343 or combinations thereof in the nucleotide sequence shown in SEQ ID NO. 1.

9. A method of screening for potential agents for promoting liver regeneration or preventing, ameliorating and/or treating liver damage, the method comprising:

(1) Treating an expression system with a candidate substance, the expression system expressing pigment epithelium-derived factor; and the combination of (a) and (b),

(2) Detecting the expression or activity of pigment epithelium derived factor in said system; the candidate substance is a potential substance for promoting liver regeneration or preventing, alleviating and/or treating liver damage if the candidate substance statistically modulates the expression or activity of the pigment epithelium-derived factor.

10. The method of claim 9, wherein the system of step (1) is an endothelial cell system; preferably, the endothelial cells comprise: hepatic sinus endothelial cells, vascular endothelial cells, lymphatic endothelial cells;

the step (2) further comprises: detecting the proliferation capacity or the cyclization capacity of endothelial cells in the system; if the proliferative capacity or the cyclization capacity is promoted, the candidate substance is a potential substance for promoting liver regeneration or preventing, alleviating and/or treating liver injury.