Application of allogenic liver precursor cells in preparation of medicine for treating chronic acute hepatic failure

Abstract

The invention relates to the technical field of medicines, in particular to application of allogenic liver precursor cells in preparation of a medicine for treating chronic acute hepatic failure. The allogenic liver precursor cells exert immunoregulation and promote regeneration and repair of liver cells through secreted active factors such as Kyn, PGE2, HGF and FGF7 so as to relieve and improve the chronic and acute hepatic failure.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of allogeneic liver progenitor cells in the preparation of drugs for treating acute-on-chronic liver failure. Background Technology

[0002] Acute-on-Chronic Liver Failure (ACLF) is a severe syndrome of liver decompensation caused by acute triggers (such as hepatitis virus infection, alcohol abuse, drug / toxin damage, or metabolic disorders) on the basis of chronic liver disease. It is characterized by high mortality and rapid progression. Its core pathogenesis involves excessive activation of the immune system, cytokine storm, massive hepatocyte necrosis, and impaired regeneration and repair in the context of chronic liver injury. For example, reactivation of viral hepatitis (HBV / HCV), alcoholic hepatitis, or drug-induced liver injury can trigger a systemic inflammatory response, leading to multiple organ failure. In addition, endotoxemia caused by gut microbiota dysbiosis is also considered an important driving factor for the exacerbation of ACLF.

[0003] Currently, clinical treatment for ACLF mainly involves comprehensive medical therapy, artificial liver support systems, and liver transplantation. Comprehensive medical therapy focuses on antiviral treatment, anti-inflammatory drugs, nutritional support, and complication management, but it cannot reverse hepatocyte necrosis or promote regeneration. Artificial liver support systems, including plasma exchange and molecular adsorption recirculation systems, can remove toxins and improve liver function in the short term, but they cannot repair liver tissue, resulting in limited improvement in long-term survival rates. Liver transplantation, as the only radical cure, is limited by donor shortages, surgical risks, and the side effects of lifelong immunosuppressive therapy (such as the risk of infection and tumors), and is only suitable for a small number of patients.

[0004] In recent years, cell therapy has attracted much attention due to its potential for multi-lineage differentiation, immunomodulation, and regeneration. For example, Chinese invention patent application CN116650530A discloses the pharmaceutical use of a CD146+ mesenchymal stem cell subset for ACLF, which for the first time demonstrated the role of umbilical cord mesenchymal stem cell subsets with specific surface markers in the treatment of acute-on-chronic liver failure, providing a scientific basis for future ACLF treatment and drug preparation. However, there is currently limited research on the application of hepatic progenitor cells in the preparation of drugs for treating acute-on-chronic liver failure, which limits its further research and application. Summary of the Invention

[0005] To address the problems in the prior art, this invention provides the application of allogeneic liver progenitor cells in the preparation of drugs for treating acute-on-chronic liver failure.

[0006] In one embodiment, the drug for treating acute-on-chronic liver failure is an immunomodulatory drug.

[0007] In one embodiment, the drug for treating acute-on-chronic liver failure is a drug that promotes hepatocyte regeneration and repair.

[0008] In one embodiment, the allogeneic liver progenitor cells are derived from human primary hepatocytes and are induced by small molecule compounds to produce liver progenitor cells derived from hepatocytes.

[0009] In one embodiment, the allogeneic liver progenitor cells are cultured according to the culture method disclosed in CN118995572A.

[0010] In one embodiment, the allogeneic liver progenitor cells secrete kynurenine (Kyn) in the process of immune regulation and promoting hepatocyte regeneration and repair.

[0011] Kynureic acid (Kyn) is a product of the conversion of tryptophan (Trp) catalyzed by IDO1 (indoleamine-2,3-dioxygenase 1), a rate-limiting step in tryptophan metabolism. T cell proliferation depends on tryptophan; therefore, tryptophan deficiency inhibits T cell proliferation and activity. Kynureic acid can activate aryl hydrocarbon receptors (AhR), thereby promoting the differentiation of regulatory T cells (Tregs) and suppressing inflammatory responses.

[0012] In one embodiment, the allogeneic liver progenitor cells secrete prostaglandin E2 (PGE2) in the process of immune regulation and promoting hepatocyte regeneration and repair.

[0013] PTGS2 is the rate-limiting enzyme in PGE2 biosynthesis, and the activity or expression level of PTGS2 directly affects the production of PGE2.

[0014] In one embodiment, the secreted growth factor family includes fibroblast growth factor (FGF) and hepatocyte growth factor (HGF).

[0015] In one embodiment, the fibroblast growth factor includes fibroblast growth factor 7 (FGF7).

[0016] In one embodiment, the dosage form of the drug includes an injection or an inhaler.

[0017] Beneficial effects

[0018] 1. The allogeneic liver progenitor cells obtained by the culture method disclosed in CN118995572A are used in drugs for the treatment of cirrhosis, and achieve high-efficiency treatment through the synergistic effect of the dual mechanism of "immunoregulation-liver regeneration".

[0019] 2. This invention uses transcriptomics to screen for genes related to the immune regulation and hepatocyte regeneration capabilities of hepatic progenitor cells, confirms this through the secretion levels in the supernatant of conditioned culture, and finally verifies the biological activity of factors in immune regulation and hepatocyte regeneration through in vitro pharmacological efficacy testing. It was found that Kyn, PGE2, HGF, FGF7, etc., secreted by allogeneic hepatic progenitor cells are key active factors in immune regulation and hepatocyte regeneration and repair.

[0020] 3. This invention is the first to demonstrate that allogeneic liver progenitor cells have a good therapeutic effect on chronic-onset liver failure, and can effectively promote hepatocyte proliferation, providing a new and effective method for the clinical treatment of chronic-onset liver failure. Attached Figure Description

[0021] Figure 1 A volcano diagram showing the transcriptomic expression of relevant genes in liver progenitor cells.

[0022] Figure 2 Analysis of the hepatic progenitor cell pathway.

[0023] Figure 3 The levels of immune-regulating factors secreted by hepatic progenitor cells and the inhibitory blocking effects are represented by A for Kyn and B for PGE2.

[0024] Figure 4 This refers to the secretion levels of HGF and FGF7 in the supernatant of liver progenitor cells.

[0025] Figure 5 This indicates the inhibitory effect of hepatic progenitor cells on the proliferation of PBMCs.

[0026] Figure 6 The effect of conditioned culture supernatant of liver progenitor cells on the inhibitory effect of THP-1 polarization to M1.

[0027] Figure 7 Cell counts were obtained by co-culturing primary hepatocytes with supernatant from different donors and with pathway inhibitors. (A) Cell counts were obtained by co-culturing primary hepatocytes with supernatant of XLV-23008 cells and with pathway inhibitors; (B) Cell counts were obtained by co-culturing primary hepatocytes with supernatant of XLV-23010 cells and with pathway inhibitors; (C) Cell counts were obtained by co-culturing primary hepatocytes with supernatant of XLV-23012 cells and with pathway inhibitors; (D) Cell counts were obtained by co-culturing primary hepatocytes with supernatant of XLV-23014 cells and with pathway inhibitors; **P<0.01, *P<0.05; MK2461: cMET and FGFR dual inhibitor; PD0325901: MEK inhibitor.

[0028] Figure 8 The effect of hepatic progenitor cell supernatant from different donors on the expression of cell cycle gene (CDK2) in primary hepatocytes. (A) qPCR assay of primary hepatocytes co-cultured with XLV-23008 cell conditioned culture supernatant and co-cultured with a pathway inhibitor to detect the expression of CDK2, a marker of the cell cycle G1 / S phase; (B) qPCR assay of primary hepatocytes co-cultured with XLV-23010 cell conditioned culture supernatant and co-cultured with a pathway inhibitor to detect the expression of CDK2, a marker of the cell cycle G1 / S phase; (C) qPCR assay of primary hepatocytes co-cultured with XLV-23012 cell conditioned culture supernatant and co-cultured with a pathway inhibitor to detect the expression of CDK2, a marker of the cell cycle G1 / S phase; The expression of CDK2, a marker of the cell cycle G1 / S phase, was detected by qPCR after co-culturing primary hepatocytes with (D)XLV-23014 cell supernatant and primary hepatocytes, and after co-culturing primary hepatocytes with pathway inhibitors; MK2461: cMET and FGFR dual inhibitor; PD0325901: MEK inhibitor. **P<0.01, *P<0.05, ***P<0.001. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents, biological materials, and detection kits described are commercially available unless otherwise specified.

[0030] Example 1

[0031] This embodiment provides the application of allogeneic liver progenitor cells in the preparation of drugs for treating acute-on-chronic liver failure.

[0032] The aforementioned medication for treating acute-on-chronic liver failure is an immunomodulatory agent that promotes hepatocyte regeneration and repair.

[0033] The allogeneic liver progenitor cells were cultured according to the culture method disclosed in CN118995572A.

[0034] The allogeneic hepatic progenitor cells secrete kynurenine (Kyn), prostaglandin E2 (PGE2), hepatocyte growth factor (HGF), and fibroblast growth factor 7 (FGF7) to exert immunomodulatory effects and promote hepatocyte regeneration and repair. Transcriptomic screening was used to identify genes related to the immunomodulatory and hepatocyte regeneration-promoting capabilities of hepatic progenitor cells. The secretion levels in the supernatant of conditioned cultured hepatic progenitor cells stimulated by inflammatory factors were used to confirm the relevant secreted factors. Finally, in vitro pharmacodynamic studies were conducted to verify the biological activity of the factors in immunomodulatory and hepatocyte regeneration-promoting effects of hepatic progenitor cells.

[0035] The transcriptomics screening method involved mailing allogeneic liver progenitor cells to Genewiz for transcriptome sequencing. RNA sample quality control was based on the quality control report from the sequencing department of a third-party company (Suzhou Genewiz Biotechnology Co., Ltd.). Genes related to immune regulation and cell regeneration were identified through differential gene expression analysis.

[0036] The drug is in the form of an injection.

[0037] Performance testing

[0038] 1. Study on transcriptional levels of allogeneic liver precursor cells:

[0039] Frozen liver progenitor cells (XLV23012) were mailed to Genewiz for transcriptome sequencing. RNA sample quality control was performed according to the quality control report from the sequencing department of a third-party company (Suzhou Genewiz Biotechnology Co., Ltd.). Gene expression differential analysis was used to identify genes related to immune regulation and cell regeneration. The results are as follows: Figure 1 , Figure 2 As shown:

[0040] Compared with primary hepatocytes (Pr), 7567 genes-specific genes (DEGs) were identified in hepatic progenitor cells, of which 3244 genes were upregulated and 4323 genes were downregulated. KEGG pathway analysis identified significantly enriched pathways in the DEGs, including extracellular matrix remodeling, cell proliferation and differentiation, tissue repair and regeneration, and inflammatory responses. Figure 2 Among them, genes involved in matrix degradation (MMP1, MMP2), cell proliferation (HGF, FGF7), and immune regulation (PTGS2) were significantly upregulated.

[0041] 2. Study on the levels of immune-regulating secreted factors in hepatic progenitor cells stimulated by inflammatory factors:

[0042] Preparation of supernatant from conditioned culture of hepatic progenitor cells stimulated by inflammatory factors: 3 × 10 5Two live cells per well were seeded into 6-well cell culture plates at a rate of 2 mL / well and incubated in a tri-gas incubator at 37℃±1℃, 5%±1% CO2, and 5%±0.5% O2. After 20–26 h, the culture medium was aspirated, and inflammatory factors or complete inhibitory medium were added to each well according to Table 1 at a rate of 1 mL / well. Unstimulated wells were incubated with 1 mL / well of normal culture medium. The 6-well cell culture plates were incubated for approximately 24 h in a tri-gas incubator at 37℃±1℃, 5%±1% CO2, and 5%±0.5% O2. After 24 h of stimulation, the culture medium was aspirated, and the cells were washed twice with 2 mL of PBS. Inhibitors were prepared using fresh DMEM / F12 basal medium (without serum). Continue adding inhibitors according to Table 1, then replace with fresh DMEM / F12 basal medium (without serum), 2 mL / well. Incubate 6-well cell culture plates at 37℃±1℃, 5%±1% CO2, and 5%±0.5% O2 for 24 h. Centrifuge the supernatant at 1000×g for 10 min to remove cell debris and collect the supernatant. Aliquot the supernatant and store at -80℃ for later use.

[0043] Table 1

[0044]

[0045] TSG-6 and PGE2 were detected using a commercially available kit (PGE2 ELISA Kit, Lianke, EK8103). Kynurenine was detected using a method established according to a literature published by the National Institutes for Food and Drug Control (Na Tao, Wang Pan, Liu Jing. Establishment and optimization of spectrophotometric method for evaluating human mesenchymal stem cell immune regulation function based on indoleamine 2,3-dioxygenase 1 activity. Chin J Biologicals September 2016, Vol.29 No.9.). Hepatic progenitor cell supernatant samples were analyzed.

[0046] The results are as follows Figure 3 As shown, compared with the resting state, the expression and secretion levels of immune regulation-related factors (Kyn, PGE2) were significantly increased after stimulation with a combination of inflammatory factors. This suggests that hepatic progenitor cells can secrete anti-inflammatory factors to exert immunomodulatory functions under inflammatory cytokine conditions. Furthermore, Kyn secretion can be blocked by the INCB024360 inhibitor, and PGE2 secretion can be blocked by NS-398.

[0047] 3. Study on the levels of secreted factors related to liver regeneration promoted by hepatic progenitor cells:

[0048] The liver progenitor cells (XLV23012) were removed from liquid nitrogen and revived. They were then seeded into 10cm culture dishes or T75 flasks at the appropriate density. Once the cell confluence reached 80% or higher, the cells were digested and plated at a density of 2 × 10⁶ cells / mL. 5 Cells were seeded per well in 6-well cell culture plates. After 24 hours of seeding, the medium was replaced with DMEM / F12 basal medium, and the culture supernatant was collected 72 hours after the DMEM / F12 medium replacement. The supernatant was centrifuged at 300×g for 10 min to remove cell debris, and the supernatant was aspirated. The supernatant was aliquoted and stored at -80℃ for later use.

[0049] Using commercially available kits (HGF ELISA Kit, Multi Sciences, EK1H01-96; FGF7 / KGF ELISA Kit, Sangon Biotech, D711444), liver progenitor cell supernatant samples were analyzed according to the instructions for each kit.

[0050] Test results as follows Figure 4 As shown, compared with primary hepatocytes, the secretion levels of both HGF and FGF7 in hepatic progenitor cells were significantly increased, with HGF secretion being the highest. This indicates that dedifferentiated hepatic progenitor cells have the potential to promote hepatocyte regeneration.

[0051] 4. Study on the mechanism of hepatic progenitor cells inhibiting the proliferation of peripheral blood mononuclear cells (PBMCs):

[0052] Take 1×10 5 Live hepatic progenitor cells (XLV-23012) per well were added to co-culture wells and cultured for 16–24 h under hepatic progenitor cell growth conditions. After labeling human PBMCs with CFSE, the labeled human PBMCs, hepatic progenitor cells, and various stimulants / inhibitors (see Table 2) were co-cultured in activated lymphocyte culture medium for 3–5 days. Under the stimulation of activated lymphocytes, the lymphocytes in the CFSE-labeled human PBMCs proliferated, and the CFSE signal weakened as the lymphocytes proliferated. Flow cytometry was used to detect changes in CFSE signal to express the inhibitory effect of the hepatic progenitor cell products on lymphocyte proliferation.

[0053] Table 2

[0054]

[0055] The results are as follows Figure 5As shown, hepatic progenitor cells can significantly inhibit lymphocyte proliferation in both resting and inflammatory factor-treated states, and this effect can be blocked by COX2 inhibitors (NS-398) and IDO1 inhibitors (INCB024360), revealing that PGE2 and IDO1 secreted by hepatic progenitor cells have an inhibitory effect on PBMC proliferation in vitro.

[0056] 5. Study on the mechanism of inhibition of THP-1 to M1 polarization by conditioned culture supernatant of liver progenitor cells:

[0057] Press THP-1 at 1×10 6 One live cell per well was seeded into a 12-well plate using THP-1 specific medium. After incubation with 200 ng / mL PMA for 12–18 h, the PMA was removed, and the cells were washed twice with PBS and replaced with fresh THP-1 cell-specific medium. After 6 h of incubation, one well of cells was digested to obtain M0 cells. Following M0 induction, cells were incubated with 1000 ng / mL LPS and 20 ng / mL IFN-γ in DMEM / F12 basal medium or conditioned medium for 24 h to induce M1 differentiation. Cells were then analyzed using TrypLE. TM The Express digested cell suspension was placed in a 1.5 mL centrifuge tube and centrifuged at 1000 × g for 5 min. After centrifugation, the supernatant was discarded, and the cell pellets were stored at -80°C for qPCR detection. Experimental groups are shown in Table 3. The following kits were used: RNA extraction kit (RNA Easy Fast Animal Tissue / Cell Total RNA Extraction Kit, Tiangen, DP451), RNA reverse transcription kit (HiScript III RT SuperMix for qPCR (+gDNA wiper), Novizan, R323-01), and qPCR Mix (BeyoFast). TM SYBR Green qPCR Mix (2X, High ROX), Beyotime, D7265) was used for qPCR detection of relevant genes.

[0058] Table 3

[0059]

[0060] The results are as follows Figure 6As shown, compared with M1, the XLV-23012 group significantly inhibited the expression of CD80, TNFα, and IL6 genes. Compared with the XLV-23012 group, the expression of related genes (CD80, TNFα, and IL6) in the group stimulated with inflammatory factors did not show a significant downward trend. Compared with the XLV-23012+(IL1β+TNFα+IFNγ) group, the COX2 inhibitor (NS-398) significantly upregulated the expression levels of CD80 and TNFα genes, while the addition of the IDO1 inhibitor (INCB024360) did not show an upregulation trend. This suggests that co-culturing the conditioned supernatant of hepatic progenitor cells with THP1 significantly reduced the expression of M1-type genes (CD80, TNFα, and IL6), and this effect could be blocked by the COX2 inhibitor (NS-398) that generates PGE2, revealing that PGE2 secreted by hepatic progenitor cells inhibits the polarization of THP-1 to M1 in vitro.

[0061] 6. Research on the mechanism of promoting primary hepatocyte regeneration:

[0062] According to 2.0×10 5 Cells were seeded in 6-well plates coated with rat tail collagen I and cultured for 12-16 h. Microscopic observation showed that most cells adhered to the plate, and the culture medium (including some dead cells) was removed. The experiment was divided into a control group and an experimental group. The control group was cultured in a 1:1 mixture of commercially available Livo hepatocyte maintenance medium and DMEM high-glucose medium. The experimental group was cultured in a 1:1 mixture of Livo hepatocyte maintenance medium and the supernatant of the tested cells, with 2 mL of medium per well. Inhibitors were added to the experimental group at the following concentrations: MK2461 (10 μM) and PD0325901 (10 μM). Cells were co-cultured for 48 h after medium change.

[0063] After co-culturing the cells, photographs were taken under a microscope, the supernatant culture medium was removed, and each well was washed once with 2.0 mL of PBS before cell digestion. AO / PI counts were performed. The remaining cells from each group were added to 1.5 mL centrifuge tubes, centrifuged at 1000×g for 3 min, the supernatant was removed, and 300 μL of RNA lysis buffer was added to the cell pellet. Subsequently, RNA was extracted from the cells using an RNA extraction kit, reverse transcribed, and analyzed by RT-PCR.

[0064] The results are as follows Figure 7-8 As stated above, after co-culturing the conditioned culture supernatant of liver progenitor cells from the four donors (XLV-23008, XLV-23010, XLV-23012, and XLV-23014) with primary hepatocytes, the number of primary hepatocytes was significantly increased in all cases. Figure 7 The expression of the gene CDK2 in the G1 / S phase of the cell cycle of primary hepatocytes was significantly upregulated. Figure 8This suggests that the conditioned culture supernatant from four donor cells promotes mitosis in primary hepatocytes. Furthermore, this proliferative effect can be blocked by inhibitors of corresponding pathways such as cMET, FGFR, and MEK.

Claims

1. Application of allogeneic liver progenitor cells in the preparation of drugs for the treatment of acute-on-chronic liver failure.

2. The use of allogeneic liver progenitor cells according to claim 1 in the preparation of drugs for treating acute-on-chronic liver failure, characterized in that, The aforementioned medication for treating acute-on-chronic liver failure is an immunomodulatory drug.

3. The use of allogeneic liver progenitor cells according to claim 1 in the preparation of drugs for treating acute-on-chronic liver failure, characterized in that, The aforementioned medication for treating acute-on-chronic liver failure is a drug that promotes the regeneration and repair of liver cells.

4. The use of allogeneic liver progenitor cells according to claim 2 or 3 in the preparation of a medicament for treating acute-on-chronic liver failure, characterized in that, The allogeneic liver progenitor cells are derived from human primary hepatocytes and are induced by small molecule compounds to produce liver progenitor cells derived from hepatocytes.

5. The use of allogeneic liver progenitor cells according to claim 4 in the preparation of drugs for treating acute-on-chronic liver failure, characterized in that, The allogeneic liver progenitor cells secrete kynurenine in the process of immune regulation and promoting hepatocyte regeneration and repair.

6. The use of allogeneic liver progenitor cells according to claim 5 in the preparation of drugs for treating acute-on-chronic liver failure, characterized in that, The allogeneic liver progenitor cells secrete prostaglandin E2 in the process of immune regulation and promoting hepatocyte regeneration and repair.

7. The use of allogeneic liver progenitor cells according to claim 5 in the preparation of drugs for treating acute-on-chronic liver failure, characterized in that, The active factors secreted by the allogeneic liver progenitor cells in their role of immune regulation and promoting hepatocyte regeneration and repair also include the secretory growth factor family.

8. The use of allogeneic liver progenitor cells according to claim 6 in the preparation of drugs for treating acute-on-chronic liver failure, characterized in that, The secreted growth factor family includes fibroblast growth factor and hepatocyte growth factor.

9. The use of allogeneic liver progenitor cells according to claim 8 in the preparation of drugs for treating acute-on-chronic liver failure, characterized in that, The fibroblast growth factor includes fibroblast growth factor 7.

10. The use of allogeneic liver progenitor cells according to claim 1 in the preparation of drugs for treating acute-on-chronic liver failure, characterized in that, The dosage form of the drug includes injections or inhalers.

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