Application of CD177 positive neutrophil in liver regeneration

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of CD177 positive neutrophil in liver regeneration. A series of experiments reveal that neutrophil is an immune cell necessary for liver regeneration after a liver resection operation, especially after a two-step liver resection operation (ALPPS) combining liver separation and portal vein ligation, and after the neutrophil is depleted by an antibody, liver cell proliferation and liver volume increase are remarkably inhibited. The invention further finds that the CD177 positive neutrophil is a specific functional subgroup which plays a key role in the CD177 positive neutrophil. Liver regeneration can be obviously inhibited by clearing the CD177 positive neutrophil in the mouse body, and inhibited liver regeneration can be recovered by in-vivo feedback of the CD177 positive neutrophil. The invention discloses an unknown effect of the CD177 positive neutrophil in promotion of ALPPS induced liver regeneration previously. The research lays a foundation for a strategy taking the neutrophil as a target spot, and a regenerative liver microenvironment is expected to be established and is used for preventing and treating hepatic failure.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to the application of CD177 positive neutrophils in liver regeneration. Background Technology

[0002] Primary liver cancer is the fourth most common malignant tumor in China and the second leading cause of cancer-related deaths. The liver is also a common site of metastasis, accounting for approximately 25% of all metastatic cases. The liver possesses a unique capacity for regeneration after injury, a process dependent on the precise regulation of immune cells. When the liver is damaged by toxins (such as CCl4), viral infections, or surgical resection (such as PHx), hepatocytes repair liver tissue through compensatory hypertrophy and proliferation. However, late diagnosis often results in insufficient Future Liver Remnant (FLR) in most patients, making radical surgical treatment difficult.

[0003] ALPPS (Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy) has been reported to induce rapid liver regeneration, increasing FLR volume by 49%-84% within 1-2 weeks. This characteristic is specifically designed to address the challenge of insufficient remaining liver volume for conventional tumor resection, facilitating radical resection of large liver tumors or multiple metastatic colorectal cancer tumors, and reducing the risk of postoperative liver failure. Reports indicate that long-term survival rates after ALPPS are significantly better than those after transcatheter arterial chemoembolization (TACE) in similar patients.

[0004] ALPPS surgery comprises two surgical elements: partial portal vein ligation and hepatic dissection. However, partial portal vein ligation alone is insufficient to induce liver regeneration, making the acceleration of liver regeneration a challenging issue. Even combining ALPPS with partial portal vein ligation and hepatic dissection carries certain surgical risks; approximately 10% of patients may experience liver failure due to insufficient liver volume increase, leading to surgical failure. Therefore, clinical induction of liver regeneration is a balance between surgical procedures and patient tolerance. ALPPS surgery triggers key physiological changes, including increased portal vein blood flow and pressure, vascular shear stress, FLR hypoxia, and systemic inflammation, all of which collectively drive rapid liver regeneration. This regeneration process relies on complex interactions between parenchymal and non-parenchymal cells. Changes in the immune microenvironment activate key mediators in the regeneration cascade, such as hepatocyte growth factor (HGF) and interleukin-6 (IL-6). Understanding these cellular dynamics and signaling events is crucial for elucidating regeneration mechanisms and improving treatment outcomes. However, the mechanisms by which ALPPS modulates the hepatic immune microenvironment to promote regeneration remain poorly understood. Previous studies have focused primarily on the immune microenvironment of liver regeneration after partial hepatectomy, rather than the immune microenvironment in the context of ALPPS surgery.

[0005] Neutrophils infiltrate various organ tissues under both normal and pathological conditions, and their migration to sites of injury and inflammation is a key host defense mechanism. In liver-related diseases such as ischemia-reperfusion injury, fibrosis, and cirrhosis, neutrophils participate in both tissue damage and repair. These cells can differentiate into functionally distinct and specifically pre-programmed subsets. Local microenvironment signals can reprogram neutrophil behavior, thereby modulating their functional characteristics. Recent studies have shown that neutrophils promote liver regeneration by inducing a regenerative phenotype of monocytes / macrophages, promoting the burial of apoptotic extracellular vesicles, and secreting HGF. Despite these advances, the functional heterogeneity of neutrophils and the specific subtypes involved in liver regeneration remain unclear and warrant further investigation.

[0006] To address the aforementioned issues, those skilled in the art urgently need to develop novel methods to promote liver regeneration after hepatectomy to benefit liver disease patients in need. Summary of the Invention

[0007] The purpose of this invention is to disclose CD177 + The role of neutrophils in promoting liver regeneration induced by hepatectomy (especially ALPPS) was previously unknown. This study lays the foundation for neutrophil-targeted strategies that may establish a pro-regenerative liver microenvironment for the treatment of liver failure.

[0008] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides the application of CD177-positive neutrophils in liver regeneration, said application being at least one of the following: 1) Application in the preparation of products for indicating or evaluating the rate and effectiveness of liver regeneration after hepatectomy; 2) Application in the preparation of products for diagnosing or assisting in the diagnosis of liver regeneration rate and efficacy after hepatectomy; 3) Application in the preparation of products for the treatment or adjuvant treatment of liver regeneration speed and efficacy after hepatectomy; 4) Application in the preparation of products for predicting the risk of liver failure after liver resection surgery.

[0009] In some specific embodiments of the present invention, the liver resection surgery induces liver regeneration. Further, the liver resection surgery includes: left hemihepatectomy, right hemihepatectomy, right anterior resection, right posterior resection, left medial resection, left lateral resection, right tri-region resection, left tri-region resection, and hepatic segment resection (1-9 segments or overlapping), combined with a two-step hepatectomy (ALPPS) involving liver septation and portal vein ligation. Further, the liver resection surgery is a two-step hepatectomy (ALPPS) involving liver septation and portal vein ligation.

[0010] In some specific embodiments of the present invention, the CD177 positive neutrophils are derived from whole blood or liver. Further, the whole blood or liver is derived from the human body or a mammal. Even further, the mammal is selected from mice, rats, guinea pigs, rabbits, sheep, pigs, dogs, or non-human primates. Even further, the non-human primate is a monkey.

[0011] In some specific embodiments of the present invention, an increase in the number of CD177-positive neutrophils in the body promotes liver regeneration; a decrease in the number of CD177-positive neutrophils in the body inhibits liver regeneration.

[0012] In some specific embodiments of the present invention, the increase in the content of CD177-positive neutrophils in vivo is achieved through at least one of the following methods: 1) By injecting exogenous CD177-positive neutrophils, the proliferation rate of hepatocytes or FLR growth can be increased, thereby promoting liver regeneration; 2) By injecting agonists of CD177-positive neutrophils or their pharmaceutically acceptable salts or bioequivalents, or inducers of CD177-positive neutrophils or their pharmaceutically acceptable salts or bioequivalents, the proliferation rate of hepatocytes or FLR growth can be increased, thereby promoting liver regeneration. The decrease in the level of CD177-positive neutrophils in vivo was addressed by targeting CD177. +Inhibitors of neutrophils reduce the proliferation rate of hepatocytes or the growth of FLR, thus hindering liver regeneration.

[0013] In some specific embodiments of the present invention, the agonists or inducers of CD177-positive neutrophils include: granulocyte colony-stimulating factor (G-CSF), sildenafil, vardenafil, auranofin, forskolin, rolipram, rolipram, and curcumin; the inhibitors of CD177-positive neutrophils include: azathioprine, sulfasalazine, dexamethasone, aspirin, ibuprofen, naproxen, indomethacin, methotrexate, hydroxychloroquine, anakinra, and andecaliximab.

[0014] In some specific embodiments of the present invention, the product includes one or more of the following: pharmaceuticals, reagents, reagent kits, chips, test strips, membrane strips, and detection platforms.

[0015] In a second aspect, the present invention provides a medicament for promoting liver regeneration, the medicament comprising at least one of the following: (1) CD177 positive neutrophils; (2) An agonist of CD177-positive neutrophils or a pharmaceutically acceptable salt or bioequivalent thereof; (3) Inducers of CD177 positive neutrophils or their pharmaceutically acceptable salts or bioequivalents.

[0016] In some specific embodiments of the present invention, the medicament is a medicament or pharmaceutical composition for treating liver regeneration, and further, the medicament also includes pharmaceutically acceptable excipients.

[0017] In some specific embodiments of the present invention, the dosage form of the drug includes liquid preparations, solid preparations, sprays, or aerosols; the liquid preparations include injections, suspensions, emulsions, solutions, or syrups; the solid preparations include tablets, capsules, granules, or powders; the administration method of the drug includes injection, oral administration, sublingual administration, or mucosal dialysis; the injection includes intravenous injection, intravenous drip, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

[0018] In a third aspect, the present invention provides a method for using CD177-positive neutrophils in liver regeneration, wherein CD177-positive neutrophils are targeted and introduced into the body requiring induction of liver regeneration at a recommended dose. Further, the method of use can be performed independently of or in conjunction with the liver resection surgery. Further, the method of use is applicable to mice, rats, guinea pigs, rabbits, sheep, pigs, dogs, monkeys, or humans. Even further, the method of use can be used continuously as recommended. Further, the recommended method is the number of consecutive days of use adjusted according to the remaining liver volume.

[0019] In a fourth aspect, the present invention provides a method for predicting the risk of liver regeneration in a patient after hepatectomy, the method comprising detecting the number of CD177-positive neutrophils in a whole blood sample from the patient, the method comprising: 1) Bring the sample into contact with a reagent or instrument capable of detecting the number of CD177-positive neutrophils; 2) Obtain the detection results of the number of CD177 positive neutrophils; 3) The number of CD177-positive neutrophils in the test results can be used to predict the rate and effect of liver regeneration after hepatectomy.

[0020] The technical solution provided by this invention has the following technical contributions: (1) This invention reveals for the first time through a series of experiments that neutrophils are immune cells necessary for liver regeneration after liver resection surgery, especially after ALPPS surgery. When neutrophils are cleared by antibodies, hepatocyte proliferation and liver volume increase are significantly inhibited.

[0021] (2) The present invention further discovers CD177 + Neutrophils are a specific functional subset that plays a key role. After hepatectomy, CD177-positive neutrophils infiltrate the liver. In the ALPPS mouse model, neutrophil depletion significantly reduced the proportion of proliferating hepatocytes and the FLR / BW ratio; the same principle applies to the 2 / 3 hepatectomy mouse model.

[0022] (3) This invention found that liver regeneration induced by 2 / 3 hepatectomy was severely impaired in Cd177 gene knockout mice. The same principle also applies to ALPPS-induced Cd177 gene knockout mice. Specific reinfusion of CD177... + Neutrophils (not CD177-negative cells) effectively reversed this defect, significantly restoring hepatocyte proliferation rate and liver weight / body weight ratio. This demonstrates that CD177... + Neutrophils possess unique therapeutic functions that can be applied clinically to promote liver regeneration.

[0023] (4) Based on the above biological findings, this invention creatively proposes a cell reinfusion technique that can be used independently or as an adjunct to ALPPS surgery to promote liver regeneration. This technique involves enriching and reinfusing activated CD177 cells in vitro. + Neutrophils directly replenish the body with key cellular components that promote regeneration, thereby precisely and controllably enhancing regenerative capacity. Compared to simply relying on surgical trauma to stimulate regeneration, this method offers flexibility in intervention timing and dosage, providing clinicians with a novel "accelerator" that promises to ensure liver regeneration speed, reduce the risk of liver failure, and improve surgical success rates without increasing surgical trauma. Attached Figure Description

[0024] Figure 1 This study investigated the effect of neutrophil depletion on liver regeneration in an ALPPS mouse model. Figure 1 A is an experimental design diagram for depleting neutrophils in an ALPPS mouse model using anti-Ly6G antibody or isotype control antibody (rat IgG2a). Figure 1 B is a Kaplan-Meier survival curve comparing the 7-day survival rates of the anti-Ly6G group (N=29) and the control antibody group (N=26) after ALPPS surgery. Figure 1 C represents representative images of hematoxylin-eosin (H&E) staining, Ly6G immunohistochemical staining, Ki67 immunohistochemical staining, and gross liver images, demonstrating the impact of neutrophil depletion on the regeneration of remaining liver tissue. Figure 1 D is a scatter plot showing the number of Ly6G-positive cells and the proportion of Ki67-positive hepatocytes per high-power field (HPF, 400x) in the remaining liver tissue on the 3rd postoperative day after anti-Ly6G antibody treatment. Figure 1 E is a scatter plot of the ratio of remaining liver tissue weight to body weight on day 7 after surgery in neutrophil-depleted mice and control mice.

[0025] Figure 2 The effect of neutrophil depletion on liver regeneration in a mouse model with 2 / 3 hepatectomy. Figure 2 A is an experimental design diagram for in vivo neutrophil depletion using anti-Ly6G antibody or isotype control (rat IgG2a) in a 2 / 3 hepatectomy mouse model. Figure 2 Representative H&E, Ly6G, and Ki67 immunohistochemical staining images taken on postoperative day 2 (POD2), and gross liver images taken on postoperative day 2 and day 7 (POD7), show the effect of neutrophil depletion on FLR regeneration. Figure 2 C is a scatter plot showing the number of Ly6G cells per high-power field (HPF, 400×) in FLR on day 2 post-surgery after anti-Ly6G antibody treatment. + Cell count and Ki67 + Hepatocyte ratio. Figure 2 D is a scatter plot showing the ratio of remaining liver tissue weight to body weight on day 7 post-surgery in mice of the neutrophil depletion group and the control group.

[0026] Figure 3 It is Cd177 - / - Effects on mice in a 2 / 3 hepatectomy mouse model. Figure 3 A is to construct a Cd177 gene knockout (Cd177) gene knockout mechanism in C57BL / 6 background mice using CRISPR / Cas9 technology. - / - (Illustration of the mouse's strategy) Figure 3 B is in wild-type (WT) mice, Cd177 - / - A schematic diagram of establishing a 2 / 3 hepatectomy model in mice. Figure 3 C is the ratio of the remaining liver tissue weight to the body weight on the 7th day after surgery, quantified by scatter plot.

[0027] Figure 4 It is Cd177 - / - Effects of mice on the ALPPS model. Figure 4 A is in wild-type (WT) mice, Cd177 - / - Cd177 in mice and CD177-positive neutrophils injected via tail vein - / - A schematic diagram of establishing an ALPPS (10% remaining liver tissue) model in mice. Figure 4 B is a wild-type mouse, Cd177 - / - Mice and Cd177 infused via CD177-positive neutrophil infusion - / - Representative H&E staining and Ki67 immunohistochemical staining images of the remaining liver tissue of mice on postoperative day 3, and gross images of the liver on postoperative day 3 and day 7. Figure 4 C-4D is a scatter plot that quantifies wild-type mice and Cd177 in the ALPPS model. - / - Mice and Cd177 infused via CD177-positive neutrophil infusion - / - The percentage of Ki67-positive hepatocytes in the remaining liver tissue of mice on postoperative day 3 (4C), and the ratio of remaining liver tissue to body weight on postoperative day 7 (4D).

[0028] Abbreviations: ALPPS: Two-step hepatectomy combining liver transection and portal vein ligation; BW: Body weight; DMEM: Duchenne modified Eagle medium; ECM: Extracellular matrix; ELISA: Enzyme-linked immunosorbent assay; FFPE: Formalin-fixed paraffin-embedded material; FLR: Future remaining liver volume; H&E: Hematoxylin-eosin; HGF: Hepatocyte growth factor; IHC: Immunohistochemistry; IF: Immunofluorescence; MMP9: Matrix metalloproteinase 9; PBS: Phosphate-buffered saline; PCNA: Proliferating cell nuclear antigen; POD: Postoperative days; scRNA-seq: Single-cell RNA sequencing; VEGF: Vascular endothelial growth factor. Detailed Implementation

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] In the following embodiments, unless otherwise specified, the terms used are explained as follows: In this disclosure, unless otherwise specified, the term "Liver Regeneration" refers to the process by which the liver, after being damaged (such as by surgical resection, trauma, etc.), restores its original volume and function through mechanisms such as hepatocyte proliferation and extracellular matrix remodeling.

[0031] In this disclosure, unless otherwise specified, the term "ALPPS" stands for Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy, a surgical procedure used to treat liver tumors that promotes rapid regeneration of remaining liver tissue.

[0032] In this disclosure, unless otherwise specified, the term "FLR" stands for Future Liver Remnant, referring to the liver tissue remaining after partial liver resection, whose regenerative capacity is crucial for surgical success. FLR is usually expressed as a percentage of the preoperative total liver volume (%FLR), or it can be calculated as a relative value in conjunction with standard liver volume (SLV).

[0033] In this disclosure, unless otherwise specified, the terms "IHC / IF" refer to Immunohistochemistry and Immunofluorescence, respectively, both of which are experimental techniques for detecting the expression of specific proteins in tissues.

[0034] In this disclosure, unless otherwise specified, the term "MMP9" stands for Matrix Metalloproteinase 9, also known as gelatinase B. It is a zinc ion-dependent endopeptidase that is mainly synthesized and secreted by immune cells, endothelial cells, etc. It can degrade the extracellular matrix and plays an important role in tissue repair and regeneration.

[0035] In this disclosure, unless otherwise specified, the term "HGF" stands for Hepatocyte Growth Factor, which promotes hepatocyte proliferation and is a key regulator of liver regeneration.

[0036] In this disclosure, unless otherwise specified, the terms "HGF-alpha" and "HGF-α" refer to hepatocyte growth factor-alpha, the active α-chain of hepatocyte growth factor (HGF). Its core function is to bind to receptors and activate signaling pathways, regulating cell growth, migration, and tissue repair, playing a crucial role in embryonic development, organ regeneration, and tumor progression. HGF initially exists as an inactive single-chain precursor (pro-HGF). After cleavage by proteases, it generates a heterodimer linked by disulfide bonds, namely the α-chain (HGF-α) and the β-chain (HGF-β). HGF-α contains four kringle domains (kringle 1-4), which are key regions for its receptor binding and specific recognition function; HGF-β contains a serine protease-like domain responsible for subsequent signal activation. The function of HGF-α is initiated by binding to specific receptors, with the core being the activation of the HGF / c-MET signaling pathway.

[0037] In this disclosure, unless otherwise specified, the term "Neutrophils" refers to neutrophils, which are the most numerous white blood cells in human peripheral blood. They are mainly involved in inflammatory responses and tissue repair processes, and play an important role in the body's fight against infection, removal of necrotic tissue, and promotion of tissue regeneration.

[0038] In this disclosure, unless otherwise specified, the term "neutrophil infiltration" refers to the process by which neutrophils aggregate towards the site of lesion in an inflammatory response, typically associated with infection and tissue damage. The neutrophil infiltration process generally involves the following steps: 1. Rolling: As neutrophils flow in the bloodstream, they are influenced by molecules released from inflamed tissue (such as histamine and leukotrienes), initiating a rolling and adhesion process on the vascular endothelium. 2. Adhesion: Neutrophils firmly adhere to the endothelium by binding to adhesion molecules on the surface of vascular endothelial cells. 3. Crawling: Neutrophils move along the surface of endothelial cells, crawling towards areas of high chemokine concentration at the site of inflammation. 4. Transmigration: Neutrophils penetrate the vascular endothelial cells, enter the tissue, and ultimately reach the site of inflammation or infection.

[0039] In this disclosure, unless otherwise specified, the term "CD177" refers to a glycosylphosphatidylinositol-anchored glycoprotein with a molecular weight of approximately 58-64 kDa, also known as human neutrophil antigen NB1 or polycythemia vera-1 antigen. It is primarily expressed on neutrophils, neutrophils, and metamyelocytes, and is anchored to the cell membrane via GPI linkages. CD177 regulates neutrophil function by binding to β2-integrin (ITGAM / CD11b and ITGB2 / CD18), platelet endothelial cell adhesion molecule-1 (PECAM-1 / CD31), and proteinase 3 (PR3). Its interaction with β2 integrin mediates tumor necrosis factor-α (TNF-α)-induced neutrophil degranulation and superoxide generation; its binding with PECAM-1 promotes transendothelial migration of CD177-positive neutrophils; and it forms a complex with membrane-bound PR3 (mPR3) (requiring GPR97 participation), triggering neutrophil activation by activating the G protein-coupled receptor PAR2.

[0040] In this disclosure, unless otherwise specified, the term "CD177 gene" belongs to the Ly-6 superfamily, located on chromosome 19q13.31, and has HNA-2a and NB1 alleles. The glycoprotein it encodes contains 437 amino acids and is primarily expressed in granulocytes, with lower expression in monocytes and lymphocytes.

[0041] In this disclosure, unless otherwise specified, the term "CD177" refers to... + Neutrophils, CD177-positive neutrophils, CD177 +Neutrophils are a key population in NETosis, participating in processes such as bile duct epithelial cell damage, intestinal protection in IBD, and acute lung injury (ALI) by releasing NETs (containing DNA, histones, myeloperoxidase MPO, etc.). Exogenous CD177 protein can inhibit NETosis, and its mechanism may be related to CD177-CD31 binding inducing SHP-1 phosphorylation. In addition, CD177 also regulates the production of reactive oxygen species (ROS), activation of inflammasomes (such as NLRP3 and AIM2), and the release of inflammatory factors such as IL-1β.

[0042] In this disclosure, unless otherwise specified, the term "ECM" refers to the extracellular matrix, a complex network surrounding cells composed of proteins, polysaccharides, and other substances. The term "ECM remodeling" refers to the dynamic changes in the structure, composition, and function of this network through processes such as synthesis, degradation, and modification, which are regulated by various enzymes and cytokines and are crucial for tissue regeneration.

[0043] In this disclosure, unless otherwise specified, the term "Ly-6G antibody (RB6-8C5)" is a mouse monoclonal IgG2b antibody that can be detected by Western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), immunohistochemistry of paraffin-embedded sections (IHCP), and flow cytometry (FCM) of mouse Ly-6G protein. Mouse Ly6G is a glycosylated phosphatidylinositol-anchored cell surface protein that is typically transiently expressed during monocyte development and persistently expressed in mature granulocytes and peripheral blood neutrophils. Its 1A8 monoclonal antibody specifically reacts with mouse Ly6G and is widely used for in vivo neutrophil clearance.

[0044] In this disclosure, unless otherwise specified, the terms “treating” or “treatment” mean alleviating, reducing, ameliorating, relieving, or controlling one or more clinical signs of a disease or disorder, and lowering, stopping, or reversing the progression of the severity of a condition or symptom that is being treated.

[0045] In this disclosure, unless otherwise specified, pharmaceutical products may be manufactured using techniques known to those skilled in the art into a dosage form suitable for parenterally administration, including, but not limited to: injections [e.g., sterile aqueous solutions or dispersions], sterile powders, tablets, troche, lozenges, pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, and the like.

[0046] In this disclosure, unless otherwise specified, pharmaceutical products according to the present invention can be administered via a parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intraepidermal injection, intradermal injection, intramuscular injection, intravenous injection, and intralesional injection. In this disclosure, unless otherwise specified, pharmaceutically acceptable excipients preferably include one or more of fillers, disintegrants, binders, lubricants, flavoring agents, coating agents, surfactants, preservatives, antioxidants, sustained-release materials, colorants, solvents, and suspending agents.

[0047] In this invention, the filler preferably includes one or more of the following: lactose, microcrystalline cellulose, pregelatinized starch, starch, dextrin, mannitol, dicalcium phosphate, cellulose, sucrose, glucose, sorbitol, and xylitol.

[0048] In this invention, the disintegrant preferably includes one or more of the following: crospovidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and ethyl cellulose.

[0049] In this invention, the adhesive preferably includes one or more of the following: povidone, hydroxypropyl cellulose, methylcellulose, sodium hydroxymethylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, and gelatin.

[0050] In this invention, the lubricant preferably includes one or more of the following: micronized silica gel, magnesium stearate, zinc stearate, calcium stearate, stearic acid, hydrogenated vegetable oil, palmitic acid, talc, polyethylene glycol, sodium dodecyl sulfate, and magnesium dodecyl sulfate.

[0051] In this invention, the flavoring agent preferably includes flavorings, sucrose, aspartame, sucralose, simple syrup, glycerin, etc. It contains one or more of the following: sorbitol, mannitol, steviol glycoside, sodium saccharin, and citric acid.

[0052] In this invention, the coating agent preferably includes one or more of hydroxypropyl methylcellulose, polyvinyl alcohol, cellulose acetate phthalate, acrylic resin, sucrose, talc, and ethyl cellulose.

[0053] In this invention, the surfactant preferably includes one or more of polysorbate, poloxamer, sodium dodecyl sulfate, and benzalkonium chloride.

[0054] In this invention, the preservative preferably includes one or more of sodium benzoate, benzoic acid, methylparaben, ethylparaben, propylparaben, benzalkonium chloride, benzalkonium bromide, ethanol, phenethyl alcohol, and potassium sorbate.

[0055] In this invention, the antioxidant preferably includes one or more of sodium sulfite, sodium bisulfite, ascorbic acid, cysteine, vitamin E, tert-butyl-p-hydroxyanisole, tert-butyl-hydroxytoluene, disodium EDTA and citric acid.

[0056] In this invention, the sustained-release material preferably includes hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, ethyl cellulose, acrylic resin, cellulose acetate phthalate, polylactic acid, and polylactic acid. One or more of the glycolic acid copolymers.

[0057] In this invention, the colorant preferably includes natural pigments and / or synthetic pigments; the natural pigments preferably include sodium copper chlorophyllin and / or caramel color; the synthetic pigments preferably include one or more of titanium dioxide, iron oxide red, iron oxide yellow, iron oxide black, indigo and lemon yellow.

[0058] In this invention, the solvent preferably includes one or more of purified water, water for injection, ethanol, propylene glycol, and glycerol.

[0059] In this invention, the suspending agent preferably includes one or more of xanthan gum, gum arabic, carbomer, and sodium carboxymethyl cellulose.

[0060] The selection and quantity of these reagents fall within the scope of the professional competence and routine techniques of those familiar with this technology.

[0061] The following description is based on specific embodiments.

[0062] I. Research Methods 1.1 Material Preparation All animal experiments were approved by the Animal Management Committee of Zhongshan Hospital Affiliated to Fudan University. The specific procedures are as follows: 1.1.1. Animal husbandry Male C57BL / 6 mice aged 8-10 weeks were housed under a 12-hour light-12-hour dark cycle with free access to food and water. The mice were purchased from Vital River.

[0063] 1.1.2. Donor pretreatment: Healthy C57BL / 6 mice were selected as donors. Clinically, the mouse homologous donors underwent "short-term pretreatment": 12 hours before sacrifice, an approximately 2cm abdominal incision was made under anesthesia, and lipopolysaccharide derived from *E. coli* (LPS, catalog number: HY-D1056, Med Chem Express) was injected intraperitoneally at a dose of 40μg / 25g body weight. The abdominal wound was then sutured. The combination of LPS and abdominal injury induced neutrophil activation, further upregulating CD177 expression and increasing CD177 levels in peripheral blood. + Neutrophil percentage.

[0064] 1.1.3. Peripheral blood collection and erythrocyte removal Twelve hours after pretreatment, donor mice were anesthetized by intraperitoneal injection of tribromoethanol, and final blood collection was performed via enucleation. Fresh peripheral blood was mixed with erythrocyte lysis buffer at a volume ratio of 1:9 and treated under ice bath conditions to lyse erythrocytes. Subsequently, the mixture was centrifuged at 300g for 10 minutes, the cell pellet was collected, and washed twice with PBS buffer to obtain a cell suspension containing neutrophils.

[0065] 1.1.4. Neutrophil enrichment and CD177 + Subtype sorting (1) Preliminary enrichment Using a mouse neutrophil enrichment kit (catalog number: 19762, Stem Cell Technologies), neutrophils were initially isolated from the cell suspension according to the kit instructions, and impurity cells such as monocytes and lymphocytes were removed.

[0066] (2) Flow cytometry purification Preliminarily enriched neutrophils were resuspended in PBS, and a mixture of fluorescently labeled antibodies (containing CD45 antibody, CD11B antibody, LY6G antibody, and CD177-AF647 antibody, catalog number: 566599, BD Biosciences) was added. The cells were incubated at 4°C in the dark for 30 minutes. After incubation, the cells were centrifuged at 300×g for 5 minutes, the supernatant was discarded, and the cells were resuspended in PBS. CD45 antibody was screened using flow cytometry (FACS). + CD11B + LY6G + CD177 + Positive cells are CD177-positive neutrophils.

[0067] (3) Staining antibodies CD45 (BD Biosciences, #550994), CD11B (Bio Legend, #101216), LY6G (BD Biosciences, #551460), CD177 (AF647, BD Biosciences, #566599).

[0068] 1.2. Constructing various mouse models 1.2.1. ALPPS mouse model A mouse ALPPS model was established, preserving 10% of the flaccid respiration rate (FLR). The specific steps for constructing the ALPPS model, simulating clinical ALPPS surgery, are as follows: (1) Anesthesia: Mice were anesthetized by intraperitoneal injection of 1% (w / v) sodium pentobarbital (0.125 mL per 25 g body weight) and 30 μL of atropine was injected intraperitoneally at the same time. After the anesthesia was stable for 5-10 minutes, a 15 mL centrifuge tube containing ether-soaked gauze was placed near the nose of the mouse for short-term additional sedation.

[0069] (2) Construction of 10% FLR mouse ALPPS model (operation under surgical microscope): Gallbladder treatment: The gallbladder was ligated with 5-0 silk suture and removed.

[0070] Portal vein branch ligation: Free the ligaments surrounding the left lateral lobe, separate and ligate the portal vein branches supplying the left lateral lobe (using 7-0 absorbable sutures), ensuring the bile duct and hepatic artery remain intact. Darkening of the left lateral lobe indicates successful ligation.

[0071] Ligation of other lobes' portal vein branches: The portal vein branches of the caudate lobe, right lobe, and right middle lobe were ligated sequentially with 7-0 absorbable sutures. The appearance of an ischemic boundary between the left and right middle lobes indicates successful vascular occlusion.

[0072] Hepatic lobe separation: The left and right middle lobes were separated using bipolar electrocoagulation.

[0073] The muscle layer was closed with continuous sutures using 5-0 nylon suture knots, and the skin layer was closed with intermittent sutures.

[0074] This baseline control group (ALPPS mouse model) consists only of male C57BL / 6 wild-type mice around 8 weeks old. The mice underwent a 10% FLRAPPS surgery (i.e., by ligating the portal veins corresponding to the left lateral lobe, caudate lobe, right lobe, and right middle lobe, and splitting the left and right middle lobes of the liver, while preserving approximately 10% of the liver tissue corresponding to the left middle lobe) to construct the mouse ALPPS model. The core objective is to establish a "natural regeneration baseline" for the mouse ALPPS model and verify whether the model can normally initiate the liver regeneration process.

[0075] 1.2.2. 2 / 3 Hepatectomy Mouse Model The specific steps for constructing a 2 / 3 hepatectomy mouse model are as follows: (1) Anesthesia treatment, same as ALPPS surgery; (2) Construction of a 2 / 3 liver resection mouse model (operation under a surgical microscope): Gallbladder treatment: The gallbladder was ligated with 5-0 silk suture and removed.

[0076] Resection of the left lateral lobe: Free the ligaments around the left lateral lobe, separate and ligate the portal vein branches, hepatic artery branches and bile duct branches supplying the left lateral lobe (using 7-0 absorbable sutures), remove the left lateral lobe with a blade, and perform bipolar electrocoagulation to stop bleeding, ensuring that there is no bleeding or bile leakage at the cut surface.

[0077] The right and left middle lobes were removed, and the portal vein branches, hepatic artery branches, and bile duct branches supplying the right and left middle lobes were separated and ligated (using 7-0 absorbable sutures). The right lobe was removed with a blade, and bipolar electrocoagulation was used to stop the bleeding, ensuring that there was no bleeding or bile leakage at the cut surface.

[0078] The left lateral lobe and right lobe (including the left and right middle lobes) were removed, accounting for approximately 68% of the total liver weight, or about two-thirds of the liver. The muscle layer was closed with continuous sutures using 5-0 nylon sutures, and the skin layer was closed with interrupted sutures.

[0079] 1.3. Constructing Cd177 - / - mice Cd177 knockout mouse models were generated using CRISPR / Cas9 technology, targeting exons 3-17 (…). Figure 3 A).

[0080] F0 generation mice were obtained by microinjecting Cas9 mRNA and guide RNA (gRNA) into fertilized eggs of C57BL / 6J mice. F0 generation mice were bred to obtain F1 generation mice, and seven F1 generation mice carrying the target mutation were selected and cross-crossed to establish a Cd177 homozygous knockout mouse strain. Genotyping was performed using primer pairs P1+P2 and P3+P4, confirming successful Cd177 gene knockout, and these mice were designated Cd177. - / - Mice.

[0081] Other genes involved in this experiment are shown in the table below: 1.4. Effects of neutrophil depletion on liver regeneration induced by hepatectomy 1.4.1. Neutrophil depletion in the ALPPS mouse model In the ALPPS mouse model, neutrophils were depleted using anti-Ly6G antibody or isotype control antibody (rat IgG2a). Figure 1 A), the specific steps are as follows: (1) Neutrophil depletion group One day before surgery, mice were injected intraperitoneally with 50 μg of anti-Ly6G antibody to antagonize neutrophils. During and after surgery, mice were injected intraperitoneally daily with 100 μg of anti-Ly6G antibody at a dose of 25 g body weight, and simultaneously underwent ALPPS surgery (10% FLR).

[0082] (2) Depletion control (IgG2a group) The anti-Ly6G antibody was replaced with the same dose of rat IgG2a (an isotype control of the anti-Ly6G antibody), and the remaining treatments (surgical procedure, injection timing and dosage) were completely consistent with the exhaustion group.

[0083] Liver tissue was collected on postoperative days 3 and 7 for verification of liver regeneration function: ① Hepatocyte proliferation: Ki67 and Ly6G expression were detected by IHC; ② FLR volume: the FLR / body weight (BW) ratio was calculated on postoperative day 7; ③ Immunohistochemical staining of the collected liver tissue was performed with H&E, Ly6G, and Ki67.

[0084] 1.4.2 Neutrophil depletion in a 2 / 3 hepatectomy mouse model In a 2 / 3 hepatectomy mouse model, neutrophils were depleted using anti-Ly6G antibody or isotype control antibody (rat IgG2a). Figure 2 A), the specific steps are as follows: (1) Neutrophil depletion group Mice were injected intraperitoneally with 50 μg of anti-Ly6G antibody one day before surgery to antagonize neutrophils. During and after surgery, mice were injected intraperitoneally with anti-Ly6G antibody at a dose of 100 μg / 25g body weight daily, and underwent 2 / 3 hepatectomy.

[0085] (2) Depletion control (IgG2a group) The anti-Ly6G antibody was replaced with the same dose of rat IgG2a (an isotype control of the anti-Ly6G antibody), and the remaining treatments (surgical procedure, injection timing and dosage) were completely consistent with the exhaustion group.

[0086] Liver tissue was collected on postoperative days 2 and 7 for verification of liver regeneration function: ① Hepatocyte proliferation: H&E and IHC were used to detect Ki67 and Ly6G expression; ② FLR volume: the FLR / body weight (BW) ratio was calculated on postoperative day 7.

[0087] 1.5Cd177 - / - The role of mice in a 2 / 3 hepatectomy mouse model In wild-type (WT) mice, Cd177 - / - Establish a 2 / 3 hepatectomy mouse model in mice ( Figure 3 (A-3B) The FLR / body weight (BW) ratio was calculated on the 7th day after surgery.

[0088] 1.6 Injection of CD177-positive neutrophils to counteract Cd177 - / - The role of liver regeneration in a mouse ALPPS model In wild-type (WT) mice, Cd177 - / - Establish an ALPPS (10% remaining liver tissue) model in mice. Figure 4 A), then prepare Cd177 - / - The specific steps for ALPPS combined with CD177-positive neutrophil salvage therapy in mice are as follows: (1) ALPPS group of wild-type (WT) mice WT mice were prepared using the ALPPS mouse model.

[0089] (2) Cd177 - / - ALPPS group of mice C57BL / 6 mice conditionally knocked out of Cd177 were designated as Cd177 mice. - / - Mice, Cd177 - / - Mice underwent only ALPPS surgery (10% FLR) without any additional intervention.

[0090] (3) Cd177 - / - ALPPS combined with CD177-positive neutrophil salvage therapy in mice Cd177 - / -Mice underwent ALPPS surgery (10% FLR), and CD177-positive neutrophils were isolated from lipopolysaccharide (LPS) and surgically treated mice during the operation and for 3 days post-operation. 1×10⁻⁶ neutrophils were then infused daily via the tail vein. 5 CD177 per 25g of body weight + Neutrophils. The core objective is to validate the efficacy of exogenous CD177 supplementation. + Can neutrophils rescue regenerative defects in knockout mice?

[0091] Liver tissue was collected on postoperative days 3 and 7 for verification of liver regeneration function: ① Hepatocyte proliferation: Ki67 and Ly6G expression were detected by IHC; ② FLR volume: the FLR / body weight (BW) ratio was calculated on postoperative day 7; ③ The collected liver tissue was subjected to H&E and Ki67 immunohistochemical staining.

[0092] Timing and method of reinfusion: Timing of reinfusion: The drug is administered in two doses. The first intraoperative reinfusion is given immediately after the ALPPS procedure (hepatic transection and portal vein ligation). The second postoperative supplementary reinfusion is given once a day after the procedure until 6 days postoperatively (covering the peak period of hepatocyte proliferation).

[0093] Infusion method: The tail vein is used for injection to ensure that the cells migrate directionally to the FLR tissue via blood circulation.

[0094] Infusion Dosage and Control Design Infusion dose: 100,000 CD177 + The single infusion volume was calculated using the ratio of neutrophils to 25g mouse body weight. Each model mouse received a single infusion of 1×10⁻⁶ cells / 25g mouse body weight. 5 CD177 + Neutrophils, specifically adjusted based on FLR volume.

[0095] Control setting: A normal saline control group (infused with an equal volume of normal saline) was set up to exclude non-specific interference and verify the specific role of the CD177 positive subtype.

[0096] II. Research Results 2.1 Neutrophils are a necessary condition for inducing liver regeneration in the ALPPS mouse model. In such Figure 1 In the ALPPS mouse model constructed by A, neutrophils were depleted by anti-Ly6G antibody before, during and after surgery. The results showed that the 7-day survival rate of the anti-Ly6G group (57.2%) was lower than that of the control group (87.1%; P<0.01). Figure 1 B). The number of Ly6G-positive cells per high-power field (HPF, 400×) decreased from 13.9 to 2.4 (P<0.001). Figure 1C-1D). Hepatocyte proliferation was also significantly impaired in the neutrophil depletion group, and the Ki67 positivity rate on postoperative day 3 (13.4%) was significantly lower than that in the control group (71.4%) (P<0.001); Figure 1 C-1D). On postoperative day 7, the FLR / BW ratio in the anti-Ly6G group decreased to 1.7%, while it was 2.6% in the control group (P<0.01; Figure 1 C, 1E).

[0097] 2.2 Neutrophils are a necessary condition for inducing liver regeneration in a 2 / 3 hepatectomy mouse model. Similarly, in such Figure 2 In the 2 / 3 hepatectomy mouse model constructed by A, neutrophil depletion significantly reduced the proportion of proliferating hepatocytes (6.6% vs. 22.7%, P<0.001). Figure 2 B-2C) and FLR / BW ratio (4.6% vs. 5.3%, P<0.05; Figure 2 D).

[0098] Therefore, although hepatectomy induces FLR proliferation to some extent in various mouse models, neutrophil depletion significantly impairs hepatocyte proliferation and FLR growth. These results collectively highlight the crucial role of neutrophils in hepatectomy-induced liver regeneration.

[0099] 2.3CD177 + Subtypes play an important role in liver regeneration in a 2 / 3 hepatectomy mouse model. On day 7 post-2 / 3 hepatectomy (POD7), compared to wild-type (WT) mice, Cd177 knockout mice (e.g., Figure 3 As shown in A-3B, Cd177 gene knockout mice were constructed, denoted as Cd177. - / - The FLR / BW ratio in mice was significantly decreased (4.8% vs. 3.4%, P<0.01). Figure 3 C).

[0100] In summary, these findings indicate that neutrophils (especially CD177) are... + (Subtype) plays a key role in promoting liver regeneration after 2 / 3 hepatectomy.

[0101] 2.4 Injection of CD177-positive neutrophils can promote Cd177... - / - Liver regeneration in a mouse ALPPS model like Figure 4 As shown in A, in wild-type (WT) mice, Cd177 - / - An ALPPS (10% remaining liver tissue) model was established in mice, and Cd177 was then prepared. - / -Mice were treated with ALPPS combined with CD177-positive neutrophil salvage therapy. Results showed that on day 7 after ALPPS surgery, CD177 levels were significantly higher in mice compared to wild-type mice. - / - The hepatocyte proliferation rate in mice decreased from 81.6% to 32.4% (P<0.001). Figure 4 B-4C), the FLR / body weight ratio decreased from 2.2% to 1.6% (P<0.05, Figure 4 B, 4D), while injecting CD177 + Neutrophils significantly restored the hepatocyte proliferation rate in gene knockout mice to 68.2% (P<0.01). Figure 4 B-4C), and increased the FLR / BW ratio from 1.6% to 2.0% (P<0.05, Figure 4 B, 4D).

[0102] These findings suggest that CD177-positive neutrophils may serve as a potential therapeutic target for liver regeneration, and their in vivo administration could promote liver regeneration and potentially have therapeutic applications in liver failure.

[0103] In summary, this invention is the first to develop CD177-positive neutrophils into a cell therapy product that can precisely and efficiently promote liver regeneration. This approach, through the specific reinfusion of key functional cells (CD177-positive neutrophils), can significantly address the challenge of liver failure caused by insufficient liver regeneration in approximately 10% of patients after ALPPS surgery, effectively increasing hepatocyte proliferation rate and liver volume, creating surgical opportunities for high-risk patients, and offering significant advantages such as flexible intervention and high safety.

[0104] The above specific embodiments are merely illustrative of the invention and do not represent a limitation thereof. Those skilled in the art will recognize that other variations of the specific structure of this invention are possible.

Claims

1. The application of CD177-positive neutrophils in liver regeneration, characterized in that, The application is at least one of the following: 1) Use in the preparation of products for indicating or evaluating the effects of liver regeneration after hepatectomy; 2) Application in the preparation of products for diagnosing or assisting in the diagnosis of liver regeneration effects after hepatectomy; 3) Application in the preparation of products for treating or adjuvant treatment of liver regeneration after hepatectomy; 4) Application in the preparation of products for predicting the risk of liver failure after liver resection surgery.

2. The application according to claim 1, characterized in that, The liver resection surgery induces liver regeneration. Further, the liver resection surgery includes: left hemihepatectomy, right hemihepatectomy, right anterior resection, right posterior resection, left medial resection, left lateral resection, right tri-region resection, left tri-region resection, and hepatic segment resection (1-9 segments or overlapping), combined with a two-step hepatectomy (ALPPS) involving liver septation and portal vein ligation. Further, the liver resection surgery is a two-step hepatectomy (ALPPS) involving liver septation and portal vein ligation.

3. The application according to claim 1, characterized in that, The CD177-positive neutrophils are derived from whole blood or liver. Further, the whole blood or liver is derived from the human body or a mammal. Even further, the mammal is selected from mice, rats, guinea pigs, rabbits, sheep, pigs, dogs, or non-human primates. Even further, the non-human primate is a monkey.

4. The application according to claim 1, characterized in that, An increase in the number of CD177-positive neutrophils in the body promotes liver regeneration; a decrease in the number of CD177-positive neutrophils in the body inhibits liver regeneration.

5. The application according to claim 4, characterized in that, The increase in the level of CD177-positive neutrophils in vivo occurs in at least one of the following ways: 1) By injecting exogenous CD177-positive neutrophils, the proliferation rate of hepatocytes or FLR growth can be increased, thereby promoting liver regeneration; 2) By injecting agonists of CD177 positive neutrophils or their pharmaceutically acceptable salts or bioequivalents, or inducers of CD177 neutrophils or their pharmaceutically acceptable salts or bioequivalents, the proliferation rate of hepatocytes or FLR growth can be increased, thereby promoting liver regeneration. The reduction in the number of CD177-positive neutrophils in vivo is achieved by reducing the proliferation rate of hepatocytes or FLR growth and inhibiting liver regeneration through inhibitors targeting CD177-positive neutrophils or their pharmaceutically acceptable salts or bioequivalents.

6. The application according to claim 5, characterized in that, The agonist or inducer of CD177 positive neutrophils is one or more of the following: granulocyte colony-stimulating factor (G-CSF), sildenafil, vardenafil, auranofin, forskolin, rolipram, and curcumin. The inhibitor of CD177-positive neutrophils is selected from one or more of the following: azathioprine, sulfasalazine, dexamethasone, aspirin, ibuprofen, naproxen, indomethacin, methotrexate, hydroxychloroquine, Anakinra, and andecaliximab.

7. The application according to any one of claims 1-6, characterized in that, The products include one or more of the following: drugs, reagents, reagent kits, chips, test strips, membrane strips, and detection platforms.

8. A drug for promoting liver regeneration, characterized in that, The drug comprises at least one of the following: (1) CD177 positive neutrophils; (2) An agonist of CD177-positive neutrophils or a pharmaceutically acceptable salt or bioequivalent thereof; (3) Inducers of CD177 positive neutrophils or their pharmaceutically acceptable salts or bioequivalents.

9. The medicament according to claim 8, characterized in that, The drug is a drug or drug composition for treating liver regeneration, and further, the drug also includes pharmaceutically acceptable excipients.

10. The medicament according to claim 9, characterized in that, The dosage forms of the drug include liquid preparations, solid preparations, sprays, or aerosols; the liquid preparations include injections, suspensions, emulsions, solutions, or syrups; the solid preparations include tablets, capsules, granules, or powders; the administration methods of the drug include injection, oral administration, sublingual administration, or mucosal dialysis; the injections include intravenous injection, intravenous drip, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

11. A method for using CD177-positive neutrophils in liver regeneration, characterized in that, CD177-positive neutrophils are targeted and introduced into the body requiring liver regeneration at a recommended dose. Further, the recommended dose is a single-use dose adjusted proportionally to the remaining liver volume. Further, the method of administration can be performed independently of or in conjunction with the liver resection surgery. Further, the method of administration is applicable to mice, rats, guinea pigs, rabbits, sheep, pigs, dogs, monkeys, or humans. Further, the method of administration can be used continuously as recommended. Further, the recommended method is the number of consecutive days of administration adjusted according to the remaining liver volume.

12. A method for predicting the risk of liver regeneration in patients after hepatectomy, characterized in that, The method for detecting the number of CD177-positive neutrophils in whole blood or liver tissue samples from a patient includes: 1) Bring the sample into contact with a reagent or instrument capable of detecting the number of CD177-positive neutrophils; 2) Obtain the detection results of the number of CD177 positive neutrophils; 3) The number of CD177-positive neutrophils in the test results can be used to predict the rate and effect of liver regeneration after hepatectomy.

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