WO2022188788A1 - 肝病调控制剂及其应用 - Google Patents

肝病调控制剂及其应用 Download PDF

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WO2022188788A1
WO2022188788A1 PCT/CN2022/079802 CN2022079802W WO2022188788A1 WO 2022188788 A1 WO2022188788 A1 WO 2022188788A1 CN 2022079802 W CN2022079802 W CN 2022079802W WO 2022188788 A1 WO2022188788 A1 WO 2022188788A1
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liver
cells
preparation
cell
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French (fr)
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张洪丹
朱雪晶
黄仁杰
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上海赛立维生物科技有限公司
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Priority to US18/463,267 priority Critical patent/US20230414676A1/en

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Definitions

  • the invention relates to the field of biotechnology, in particular to an anti-hepatic fibrosis preparation and a preparation method and application thereof.
  • liver regeneration microenvironment is an important mechanism to maintain the physiological function of the liver, and remodeling the liver regeneration microenvironment is of great significance to prevent the occurrence and development of chronic liver diseases.
  • the purpose of the present invention is to provide a liver disease regulating preparation, which is beneficial to prevent the occurrence and development of chronic liver disease by remodeling the liver regeneration microenvironment.
  • the preparation for regulating liver disease of the present invention comprises liver-derived precursor cells or a secretory supernatant of the liver-derived precursor cells.
  • the liver-derived precursor cells are liver precursor cells.
  • the liver-derived precursor cells are liver precursor-like cells.
  • the liver-derived precursor cells are human liver-derived precursor cells.
  • the human liver-derived precursor cells are human liver precursor cells.
  • the human liver-derived precursor cells are human liver precursor-like cells.
  • the secretory supernatant of the liver-derived precursor cells includes at least one miRNA, and the at least one miRNA is at least one of miRNA-182, miRNA-183 and miRNA-574, which can effectively promote hepatocytes. proliferation.
  • the secretory supernatant of the liver-derived precursor cells comprises an active ingredient acting on the JAK-STAT pathway to inhibit activation of hepatic stellate cells or induce death of the hepatic stellate cells.
  • the secretory supernatant of the liver-derived precursor cells comprises at least one of leukemia inhibitory factor, endothelin, colony-stimulating factor, amphiregulin, and fibroblast growth factor to inhibit hepatic stellate cells. Activation or induction of death of the hepatic stellate cells.
  • the fibroblast growth factor is FGF19.
  • the secretory supernatant of the liver-derived precursor cells comprises secretory components capable of inducing the recipient to establish effective immune tolerance by inhibiting the proliferation of the immune cells.
  • the immune cells are any one of macrophages, B cells, T cells, NK cells and NKT cells.
  • the liver disease regulating preparation further includes a resuspending component, and the resuspending component includes at least one of physiological saline, compound electrolyte solution, buffer solution and basal medium.
  • the liver disease regulating preparation further includes auxiliary components, the auxiliary components include at least one of immunosuppressive components, serum, antibiotics and synergistic active components.
  • liver-derived precursor cells of the embodiments of the present invention are cultured in an in vitro medium to obtain the secretory supernatant of the liver-derived precursor cells.
  • the in vitro culture medium is a basal medium
  • the basal medium is HepX medium, DMEM/F12 cell culture medium, William's E cell culture medium, Neurobasal Medium cell culture medium, MEM cell culture medium, DMEM At least one of cell culture medium, 1640RPMI cell culture medium, and F12 cell culture medium.
  • the in vitro culture medium includes the basal medium and serum-like substances.
  • the in vitro culture medium consists of the basal medium and serum-like substances. Wherein, based on the volume content of the basal medium, the content of serum-like substances does not exceed 20%.
  • the in vitro culture medium is further supplemented with a double antibody, and the content of the double antibody does not exceed 2%.
  • the in vitro culture medium further comprises N2, B27, growth factors, ROCK kinase inhibitors, Wnt signaling pathway agonists, TGF- ⁇ signaling inhibitors, N-acetyl-L-cysteine and ascorbic acid. at least one.
  • the liver-derived precursor cells are obtained by culturing primary hepatocytes in vitro through hepatocyte expansion transformation medium (TEM medium).
  • TEM medium hepatocyte expansion transformation medium
  • the TEM medium includes basal medium, serum-free supplements, serum-like substances, growth factors, TGF-beta signaling inhibitors, Wnt signaling pathway agonists, and ROCK kinase inhibitors.
  • the content of the growth factor is 0.1-100 ng/ml
  • the content of the ROCK kinase inhibitor is 0.1-100 ⁇ mol
  • the content of the Wnt signaling pathway agonist The content of the TGF- ⁇ signal inhibitor is 0.1-100 micromol
  • the content of the serum-like substance is not more than 20%
  • the volume content of the serum-free additive is not more than 2%.
  • the TEM medium further comprises at least one of N-acetyl-L-cysteine and ascorbic acid.
  • the growth factor is at least one of epidermal growth factor, fibroblast growth factor 2, vascular endothelial growth factor, platelet-derived growth factor, hepatocyte growth factor, interleukin-6, and oncostatin.
  • the ROCK kinase inhibitor is at least one of Fasudil, Y-27632, Thiazovivin, and SB-772077-B.
  • the Wnt signaling pathway agonist is at least one of recombinant Wnt protein, recombinant R-spondin protein and glycogen synthase kinase 3 ⁇ inhibitor.
  • the TGF-beta signaling inhibitor is at least one of RepSox, SB431542, and A83-01.
  • the serum-like substance in any one of the in vitro medium and the TEM medium is animal-derived serum.
  • the animal-derived serum in any of the in vitro medium and the TEM medium can be replaced with a serum replacement.
  • the serum replacement is animal-derived component-free platelets and derivatives thereof.
  • the serum replacement is sphingosine monophosphate and indoleacetic acid.
  • the animal-derived serum is fetal bovine serum.
  • the content of the sodium pyruvate is 0.5-1.5 mmol/L
  • the content of the ascorbic acid is 5-50 ⁇ g/mL
  • the epidermis is The content of the cell growth factor is 5-25 ng/ml
  • the content of the hepatocyte growth factor is 5-25 ng/ml
  • the content of the ROCK kinase inhibitor is 5-20 ⁇ mol/L
  • the The content of the Wnt signaling pathway agonist is 1-5 ⁇ mol/L
  • the content of the TGF- ⁇ signaling inhibitor is 0.5-2 ⁇ mol/L
  • the content of the sphingosine monophosphate is 0.5-2 ⁇ mol/L /L
  • the content of the indoleacetic acid is 2-10 ⁇ mol/L
  • the volume percentage of the N2 additive and the B27 additive is not more than 1%
  • the content of N-acetyl-L-cysteine is 0.5 -10 ⁇ mol/L.
  • the embodiments of the present invention provide the in vitro application of the liver disease regulating preparation, including co-culturing the liver disease regulating preparation with target cells.
  • the target cells are any one of primary hepatocytes, hepatic stellate cells, macrophages and immune-related cells.
  • the step of co-culturing the liver disease-modulating preparation with the target cells includes co-culturing the liver disease-modulating preparation and the hepatic stellate cells with a co-culture medium to account for the volume of the co-culture medium.
  • the content of the liver disease regulating preparation is not less than 1%.
  • the plating density of the target cells is 1 ⁇ 10 4 /cm 2 .
  • the co-culture medium consists of a basal medium and the serum-like substance.
  • the content of the serum-like substance does not exceed 20% based on the volume percentage of the basal medium.
  • the co-culture medium further contains a hepatic stellate cell activator.
  • the hepatic stellate cell activating agent is hepatic stellate cell activating factor.
  • the hepatic stellate cell activating factor is TGF- ⁇ 1.
  • the step of co-culturing the liver disease regulating preparation with the target cells includes co-culturing the liver disease regulating preparation and a liver macrophage model, and the liver macrophage model is an inflammatory cell model or a repair type. cell model.
  • the step of co-culturing the liver disease modulating agent with target cells includes co-culturing the liver disease modulating agent with the immune-related cells, and using a stimulator to induce proliferation of the immune-related cells.
  • the immune-related cells are any one of peripheral blood mononuclear cells and spleen cells.
  • the step of co-culturing the liver disease regulating preparation with the immune-related cells includes resuspending the liver disease regulating preparation using a co-culture medium, and controlling the liver disease regulating preparation to occupy the co-culture medium.
  • the volume concentration is not less than 5%, so that the proliferation inhibition rate of the immune-related cells by the liver disease regulating preparation is not less than 30%.
  • the step of co-culturing the liver disease-modulating preparation with the immune-related cells includes co-culturing the immune-related cells with different liver disease-modulating preparations, wherein the liver-derived pre-existing liver disease regulating preparations
  • the culture supernatants of somatic cells are derived from different donors.
  • the application of the liver disease regulating preparation in the embodiment of the present invention in the treatment of liver disease includes: examining the effect on liver regeneration after intervening the liver disease regulating preparation in an in vivo animal model.
  • the animal in vivo model is carbon tetrachloride-induced mouse acute liver failure model, acetaminophen-induced mouse acute liver failure model, thioacetamide-induced mammalian liver cirrhosis model, tetrachloride Carbon-induced mammalian liver cirrhosis model, mammalian nonalcoholic steatohepatitis model, ConA-induced T cell and NKT cell-mediated mouse autoimmune hepatitis model, immune rejection after hepatocyte or liver tissue transplantation Any one of the mouse model and the acute host-versus-graft reaction model after liver transplantation.
  • Fig. 1 is the transmission electron microscope photograph of the PHH Exo sample of embodiment 1-1;
  • Fig. 2 is the transmission electron microscope photograph of the Hep Exo sample of embodiment 1-1;
  • Fig. 3 is a comparison diagram of the average particle size of exosomes in the PHH Exo sample of Example 1-1 and the Hep Exo sample;
  • Figure 4 is a comparison diagram of the expression of CD63 and CD81 in different cell-derived exosomes detected by flow analysis in Example 1-1;
  • Figure 5 is a photo of the expression of CD63 and TSG101 in different cells and exosomes derived from different cells examined by Western blotting in Example 1-1;
  • Figure 7 is a comparison diagram of BrdU incorporation in cells obtained after co-culture of PHH Exo samples with different exosome concentrations and Hep Exo samples of Examples 1-3;
  • Fig. 8 is the immunofluorescence photograph obtained after the cells obtained by co-culturing the PHH Exo sample and the Hep Exo sample with PHHs respectively in Example 1-3 are subjected to EdU fluorescent staining;
  • FIG. 9 is an immunofluorescence photograph obtained by performing Ki67 immunofluorescence staining on cells obtained after PHH Exo samples and Hep Exo samples were co-cultured with PHHs in Example 1-3;
  • Fig. 10 is a flow cytometric analysis result comparison diagram obtained by carrying out flow cytometry cycle analysis to PHH Exo-cell and Hep Exo-cell in Example 1-4;
  • Figure 11 is a comparison diagram of the expression levels of cell cycle-related molecules miRNA obtained by real-time quantitative PCR analysis of the control group, PHH Exo-cell and Hep Exo-cell in Examples 1-4;
  • Figure 12 is a comparison diagram of imprinted photos obtained by using Western blotting to investigate the expression of cell cycle-related molecules in control group, PHH Exo-cell and Hep Exo-cell in Example 1-4;
  • Figure 13 is a clustering diagram of the sequencing and expression of different cell-derived exosomal miRNAs of Examples 1-5;
  • Figure 14 shows the primary hepatocytes of Examples 1-5 transfected in vitro with hsa-miR-182, hsa-miR-183, hsa-miR-149, hsa-miR-215, hsa-miR-574, hsa-miR- Comparison of BrdU incorporation of 654 and hsa-miR-675 in each transfected cell detected by ELISA after 48 hours;
  • Figure 15 shows EdU fluorescence micrographs obtained after 48 hours after primary hepatocytes of Examples 1-5 were transfected with hsa-miR-182, hsa-miR-183 and hsa-miR-574, respectively, after EdU staining;
  • Figure 16 shows the EdU of each transfected cell detected by EdU fluorescence method 48 hours after the primary hepatocytes of Examples 1-5 were transfected with hsa-miR-182, hsa-miR-183 and hsa-miR-574 respectively Incorporation rate comparison.
  • Fig. 17 is the change situation of survival rate with time after the completion of the modeling of each mouse in the Hep Exo treatment group and the PBS control group of Example 1-6;
  • Figure 18 is the HE staining photos of the liver tissue sections of the mice in the normal control group, the PBS control group and the Hep Exo treatment group of Examples 1-6 at 24 hours and 48 hours after modeling;
  • Figure 19 is a comparison diagram of immunohistochemical Ki67 staining of liver tissue sections of mice in the PBS control group and Hep Exo treatment group of Examples 1-6;
  • Figure 20 is a comparison diagram of the percentage of Ki67 positive cells in the paraffin sections of each mouse liver tissue in the normal group, the PBS control group and the Hep Exo treatment group of Examples 1-6;
  • Figure 21 shows the results of comparing the ALT levels of each mouse in the normal group, the PBS control group and the Hep Exo treatment group of Examples 1-6 at different times;
  • Fig. 22 is the AST level comparison result of each mouse in the normal group of embodiment 1-6, PBS control group and Hep Exo treatment group at different time;
  • Figure 23 is the change of survival rate of mice in each group of CCl4 models of Examples 1-7 over time;
  • Figure 24 shows the changes in the survival rate of mice in each group of APAP models of Examples 1-7 over time
  • Figure 25 is a graph showing the comparison of AST levels at different times after each mouse of the CCl4 model shown in Examples 1-8;
  • Figure 26 is a graph showing the comparison of ALT levels at different times after the CCl4 model of each mouse model shown in Examples 1-8;
  • Figure 27 is a graph showing the comparison of AST levels and ALT levels in each mouse of the APAP model shown in Examples 1-8 24 hours after modeling;
  • Figure 28 is a comparison diagram of the pathological tissue sections of each mouse of the CCl4 model of Examples 1-8 at different times after modeling;
  • Figure 29 is a comparison diagram of pathological tissue sections 24 hours after the APAP model of each mouse of Examples 1-8;
  • Figure 30 is a comparison diagram of immunohistochemical Ki67 staining of liver tissue sections at different times after the modeling of CCl4-NC agomir group mice and CCl4-miRNA 183-5p agomir group mice of Example 1-8;
  • Figure 31 is a comparison chart of immunohistochemical Ki67 staining of liver tissue sections of the mice in the APAP-NC agomir group and the APAP-miRNA 183-5p agomir group 24 hours after the modeling of Examples 1-8;
  • Figure 32 is a schematic diagram showing the comparison of the percentage of Ki67 positive cells in the liver tissue sections of the mice in the CCl4-NC agomir group and the mice in the CCl4-miRNA 183-5p agomir group at different times after the modeling in Example 1-8;
  • Figure 33 is a graph showing the comparison of the expression of cell cycle-related proteins in liver tissue of mice in the CCl4-miRNA 183-5p agomir group of Examples 1-8 at different times after modeling.
  • Figure 34 is a schematic diagram of the gene expression of primary human hepatocytes of Example 2-1;
  • Figure 35 is a schematic diagram of the gene expression of the human liver precursor-like cells of Example 2-1;
  • Fig. 36 is a photograph comparing the microscopic morphology of each hepatic stellate cell in the experimental group, the control group and the control group of Example 2-2;
  • Figure 37 is a comparison chart of the relative mRNA expression of genes related to HSCs activation in the experimental group, the control group and the control group of Example 2-2;
  • Figure 38 is a microscopic topography photo of the cell aggregates in the experimental group of Example 2-2 under transmission microscope observation;
  • Figure 39 is the analysis result obtained by flow cytometry on the cell aggregates of the control group of Example 2-2;
  • Figure 40 is an analysis result obtained by flow cytometry assay for the cell aggregates of the experimental group of Example 2-2;
  • Figure 41 is a comparison diagram of the expression of fibrosis-related proteins and key fibrosis signals in cells of each group obtained by Western blot analysis on the cell aggregates of the control group, the control group, and the experimental group of Example 2-2;
  • Figure 42 is a graph showing the comparison of the expression of hepatic progenitor gene and hepatic parenchymal cell markers in primary murine hepatic cells and murine hepatic precursor-like cells of Example 2-3;
  • Figure 43 is a comparison diagram of the micro-morphological photos of the HSCs-T6 cells in each group after 48 hours of co-culture in the control group, the control group and the experimental group of Example 2-4;
  • Figure 44 is a comparison chart of the relative mRNA expression of genes related to HSCs activation in the HSCs-T6 of the control group, the control group and the experimental group of Example 2-4;
  • Figure 45 is a photo of the microscopic morphology of cells in the experimental group of Example 2-4;
  • Figure 46 is an analysis result obtained by flow cytometry on the cells of the control group of Examples 2-4;
  • Figure 47 shows the analysis results obtained by flow cytometry on the cells of the experimental groups of Examples 2-4;
  • Figure 48 shows the JAK-STAT pathway and the proteins involved in growth factor activity, cytokine activity and receptor-ligand activity in the first anti-hepatic fibrosis preparation of Examples 2-5 A schematic diagram of the visual network between;
  • Figure 49 is a comparison diagram of the expression of the pSTAT1 signal in the control group, the control group and the experimental group of Example 2-6 obtained by Western blot analysis shown in Figure 16 in each group of cells;
  • Figure 50 is a comparison chart of the analysis results of the flow analysis of the cell aggregates in the control group, the control group and the experimental group of Examples 2-7;
  • Figure 51 is a comparison chart of the percentage of apoptotic cells in each group obtained according to the statistics of Figure 51;
  • Figure 52 is a comparison diagram of the microscopic topography of the liver tissue slices of the mice in the normal group, the PBS injection group and the anti-hepatic fibrosis preparation intervention group of Examples 2-8;
  • Figure 53 is a comparison photo of the distribution of activated hepatic stellate cells in the liver of the normal group, the PBS injection group and the anti-hepatic fibrosis preparation intervention group of Example 2-8.
  • Figure 54 is a graph showing the comparison of collagen-related genes and fibrosis-related gene expressions of LX-2 cells obtained after 48 hours of co-cultivation in the LX-2 group, the LX-2 addition group and the co-culture group of Example 2-9;
  • Figure 55 is a photograph of liver tissue obtained from the normal group, sham-operated group and cell transplantation group of Example 2-10 after H&E staining, picro-Sirius staining, Masson's trichrome staining and fibronectin (FN) immunostaining comparison chart;
  • Figure 56 shows the statistical hepatic fibrosis area after liver tissue was taken from the normal group, sham-operated group and cell transplantation group of Example 2-10 and subjected to picro-Sirius staining, Masson's trichrome staining and fibronectin (FN) immunostaining Comparison of relative quantification with fibronectin-positive staining areas;
  • Figure 57 is the analysis result of the determination of hydroxyproline content in the normal group, the sham operation group and the cell transplantation group of Example 2-10;
  • Figure 58 is the analysis results of liver fibrosis scoring from the sham operation group and the cell transplantation group of Example 2-10;
  • Figure 59 is a comparison diagram of photos obtained by taking liver tissue from the normal group, the sham-operated group and the cell transplantation group of Example 2-10 for Ki67 immunohistochemical staining;
  • Figure 60 is the quantification result of counting positive stained cells according to the photo shown in Figure 9;
  • Figure 61 is a photograph of liver tissue obtained from the normal group, sham-operated group and cell transplantation group of Example 2-11 after H&E staining, picro-Sirius staining, Masson's trichrome staining and fibronectin (FN) immunostaining comparison chart;
  • Figure 62 shows the statistical hepatic fibrosis area after liver tissue was taken from the normal group, sham-operated group and cell transplantation group of Example 2-11 and subjected to picro-Sirius staining, Masson's trichrome staining and fibronectin (FN) immunostaining Comparison of relative quantification with fibronectin-positive staining areas;
  • Figure 63 shows the analysis results of liver tissue taken from the normal group, the sham-operated group and the cell transplantation group of Example 2-11 for hydroxyproline content determination;
  • Figure 64 shows the analysis results of liver fibrosis scoring in the sham-operated group and the cell transplantation group of Example 2-11;
  • Figure 65 is a heat map obtained by analyzing the gene expression levels related to fibrosis, extracellular matrix, signal transduction-related liver fibrosis in the blood of the normal group, the sham-operated group and the cell transplantation group of Example 2-11 ;
  • Figure 66 is a bright-field photograph of HepLPCs obtained by taking a bright-field photograph of cells obtained after culturing in a TEM medium for 10 days in Example 3-1;
  • Figure 67 is the analysis result of the proportion of total macrophages obtained by the cell identification of BMDMs obtained after induction with mouse GM-CSF by flow cytometry in Example 3-2;
  • Figure 68 shows the analysis results of the proportion of M1 macrophages obtained by the cell identification of BMDMs induced by LPS in vitro directed polarization by flow cytometry in Example 3-2;
  • Figure 69 is a comparison chart of the expression of M1-related inflammatory genes obtained by cell identification of the Control group and the DM+LPS group by flow cytometry in Example 3-2;
  • Figure 70 is a comparison diagram of M1-related inflammatory gene expression in each group obtained after RNA extraction and gene expression analysis of HepLPCs-CM+LPS group, Control group and DM+LPS group in Example 3-2;
  • Figure 71 is a comparison chart of the expression levels of M1-related inflammatory genes obtained by detecting cytokine concentrations in the cell culture supernatants of the HepLPCs-CM+LPS group, the Control group and the DM+LPS group in Example 3-2;
  • Figure 72 is the analysis results of the proportion of M2 macrophages obtained by the cell identification of BMDMs induced by IL-4 in vitro directed polarization by flow cytometry in Example 3-3;
  • Figure 73 is a comparison chart of M2-related inflammatory gene expression obtained by cell identification of Control group and DM+IL-4 group by flow cytometry in Example 3-3;
  • Figure 74 is a comparison chart of the secretion of M2-related inflammatory factor IL10 in each group obtained by RNA extraction and gene expression analysis of HepLPCs-CM+IL-4, Control group and DM+IL-4 group in Example 3-3;
  • Figure 75 is the fusion diagram after DAPI nuclear staining of exosomes with different concentrations of Example 3-4 and macrophage BMDMs for 6 hours;
  • Figure 76 is the control group of embodiment 3-4, DM+LPS group, CM+LPS group and EV+LPS group M1-related inflammatory gene expression comparison chart;
  • Figure 77 is a graph showing the comparison of M2-related inflammatory gene expression in the Control group, the DM+IL4 group, the CM+IL4 group and the EV+IL4 group in Example 3-5.
  • Figure 78 is a stained photograph obtained after the liver tissue of the early NASH mouse model of Example 3-6 was embedded in paraffin, sectioned, and stained with H&E;
  • Figure 79 is a staining photograph obtained after paraffin embedding, section preparation and H&E staining of the liver tissue of the mice in the control group of Examples 3-6;
  • Figure 80 is a stained photograph obtained after the liver tissue of the early NASH mouse model of Example 3-6 is paraffin-embedded and sectioned and subjected to Masson staining;
  • Figure 81 is a stained photograph obtained after the liver tissue of the early NASH mouse model of Example 3-6 was paraffin-embedded and sectioned and stained with Oil Red O;
  • Figure 82 shows the staining photos obtained by immunohistochemical staining of the M1 macrophage-specific marker CD68 after paraffin-embedding and sectioning the liver tissue of the early NASH mouse model in Example 3-6;
  • Figure 83 is a staining photograph obtained by immunohistochemical staining of the M2 macrophage-specific marker CD163 after paraffin-embedding and sectioning the liver tissue of the early NASH mouse model in Example 3-6;
  • Figure 84 is a comparison chart of blood biochemistry and TC, TG content of the early NASH mouse model of Example 3-6 and the control group;
  • Figure 85 is a comparison diagram of the liver H&E staining photos and Oil Red O staining photos of the mice in the sham-operated group, the low-dose cell treatment group, and the high-dose cell treatment group of Example 3-7;
  • Figure 86 is a comparison chart of the NAS scores of Example 3-7 for the sham operation group, the cell low-dose treatment group, and the cell high-dose treatment group;
  • Figure 87 is a Masson staining photograph of the liver sections of the mice in the sham-operated group, the low-dose cell treatment group and the high-dose cell treatment group in Example 3-7;
  • Figure 88 is a comparison chart of the statistical results of the liver fibrosis areas of Examples 3-7 for each group of staining photos in Figure 11;
  • Figure 89 is the staining photos obtained by Ki67 staining of liver sections of mice in the sham-operated group, the low-dose cell treatment group and the high-dose cell treatment group in Example 3-7;
  • Figure 90 shows the levels of ALT, AST and LDH indexes in the blood of the mice in the normal control group, the immunosuppressive group, the model group with oral saline, the sham-operated group, the low-dose cell treatment group and the high-dose cell treatment group in Example 3-7 comparison chart;
  • Figure 91 is a graph showing the comparison of TC and TG content in the livers of the mice in the normal control group, the sham-operated group, the mice in the low-dose cell treatment group and the mice in the high-dose cell treatment group in Example 3-7;
  • Figure 92 is a comparison diagram of staining photos obtained after immunohistochemical staining of anti-CD163 and anti-CD68 was performed on mice in the sham-operated group, the low-dose cell treatment group, and the mice in the high-dose cell treatment group in Example 3-7;
  • Figure 93 is a graph comparing the numbers of CD163 + macrophages and CD68 + macrophages in the liver of each group obtained according to the statistics of Figure 93;
  • Figure 94 is a comparison diagram of the H&E staining photos of the liver sections of the mice in the sham-operated group, the low-dose cell treatment group, and the high-dose cell treatment group of the middle-late NASH mouse model of Example 3-8;
  • Figure 95 is a comparison chart of Masson staining photos of liver sections of mice in the sham-operated group, the low-dose cell treatment group, and the high-dose cell treatment group of the middle-late NASH mouse model of Example 3-8;
  • Figure 96 is a comparison chart of the statistical results of the hepatic fibrosis regions of each group obtained according to the statistics of Figure 96;
  • Figure 97 shows the analysis results of the proportion of total macrophages obtained by the cell identification of BMDMs obtained after induction with mouse GM-CSF by flow cytometry in Examples 3-9;
  • Figure 98 is the analysis results of the proportion of M1 macrophages obtained by the cell identification of BMDMs induced by LPS in vitro directed polarization by flow cytometry in Examples 3-9;
  • Figure 99 is a comparison chart of the expression of M1-related inflammatory genes obtained by cell identification of the Control group and the DM+LPS group by flow cytometry in Example 3-9;
  • Figure 100 shows the analysis results of the proportion of M2 macrophages obtained by the cell identification of BMDMs induced by IL-4 in vitro directed polarization by flow cytometry in Examples 3-10;
  • Figure 101 is a comparison diagram of M2-related inflammatory gene expression obtained by cell identification of Control group and DM+IL-4 group by flow cytometry in Example 3-10;
  • Figure 102 is the control group of embodiment 3-11, DM+LPS group, CM+LPS group and EV+LPS group M1-related inflammatory gene expression comparison chart;
  • Figure 103 is a graph showing the comparison of M2-related inflammatory gene expression in the Control group, the DM+IL4 group, the CM+IL4 group and the EV+IL4 group in Examples 3-12.
  • Figure 104 is a graph showing the comparison of the expression of various liver precursor-related markers in HepLPCs cells of Example 4-1;
  • Figure 105 is a brightfield photograph of HepLPCs obtained by brightfield photographing of cells obtained after culturing in TEM medium for 10 days in Example 4-1;
  • Figure 106 is a comparison diagram obtained by using flow cytometry to investigate the proliferation inhibition of spleen cells by HepLPC-CM in the positive control group and each co-culture group in Example 4-1;
  • Figure 107 is a comparison diagram obtained in Example 4-2 using flow cytometry to investigate the inhibition of spleen cell proliferation by HepLPC-CM derived from Donor 1 and the spleen cell proliferation in the corresponding positive control group;
  • Figure 108 is a comparison diagram obtained by using flow cytometry in Example 4-2 to investigate the inhibition of spleen cell proliferation by HepLPC-CM derived from Donor 2 and the spleen cell proliferation in the corresponding positive control group;
  • Figure 109 is a comparison diagram obtained by using flow cytometry in Example 4-2 to investigate the inhibition of spleen cell proliferation by HepLPC-CM derived from Donor 3 and the spleen cell proliferation in the corresponding positive control group;
  • Figure 110 is a comparison diagram obtained by using flow cytometry in Example 4-2 to investigate the inhibition of spleen cell proliferation by HepLPC-CM derived from Donor 4 and the spleen cell proliferation in the corresponding positive control group.
  • Figure 111 is a comparison diagram of the proliferation inhibition of spleen cells by HepLPC in the positive control group, the FK506 control group and each co-culture group by flow cytometry in Example 4-3;
  • Figure 112 is a comparison diagram obtained by using flow cytometry to investigate the inhibition of spleen cell proliferation by HepLPC derived from Donor 1 and the spleen cell proliferation in the corresponding positive control group;
  • Figure 113 is a comparison diagram obtained by using flow cytometry to investigate the inhibition of spleen cell proliferation by HepLPC derived from Donor 2 and the corresponding positive control group of spleen cell proliferation;
  • Figure 114 is a comparison diagram obtained by using flow cytometry to investigate the inhibition of spleen cell proliferation by HepLPC derived from Donor 3 and the corresponding positive control group of spleen cell proliferation;
  • Figure 115 is a comparison diagram obtained by using flow cytometry in Example 4-4 to investigate the inhibition of spleen cell proliferation by HepLPC derived from Donor 4 and the spleen cell proliferation in the corresponding positive control group;
  • Figure 116 is a comparison diagram obtained by using flow cytometry to investigate the proliferation inhibition of PBMCs in the PBMC co-culture group of each donor-derived HepLPC and the corresponding positive control group;
  • Figure 117 is the relationship curve of different ConA injection doses and mouse survival rate of Examples 4-6;
  • Figure 118 is a comparison chart of the ALT, AST and LDH index levels of each group obtained by taking blood from the orbit of the mice in the 8 mg/kg injection dose group at different times in Example 4-7 and analyzing and investigating the blood biochemical levels;
  • Figure 119 is a comparison chart of ALT, AST, LDH and ALP index levels of each group obtained by taking blood from the orbit 6 hours after the injection of each mouse in the experimental group 1 and the control group in Example 4-7 and investigating the blood biochemical level analysis. ;
  • Figure 120 is a comparison chart of ALT, AST and LDH index levels of each group obtained by taking blood from the orbit 6 hours after the injection of each mouse in experimental group 2 and control group in Example 4 and investigating and investigating through blood biochemical level analysis.
  • cell culture is carried out in a cell culture incubator with a carbon dioxide concentration of 5% under an environment of 37 degrees Celsius.
  • the media used in cell culture and various reagents used in cell processing, such as buffers, are sterilized and filtered through a 0.22-micron filter to remove impurities before use.
  • liver disease-modulating preparations prepared hepatocyte-modulating preparations containing at least one miRNA as liver disease-modulating preparations, and examine the application of such liver-disease-modulating preparations. details as follows:
  • human primary hepatocytes (abbreviated as PHHs) and human hepatic precursor-like cells (abbreviated as HepLPCs) were used as seed cells, and were successfully cultured in serum-containing medium and serum-free medium.
  • the PHHs in this example were purchased from Guangzhou Shenzhen Liwo Technology Co., Ltd., with the batch number of Lot#201904001; HepLPCs were from Sailiwei Biotechnology Co., Ltd. with the batch number of XLV-19006; Hep-X basal medium was from Shanghai Yuanpei Biology Technology Co., Ltd.; fetal bovine serum, 1% penicillin-streptomycin solution and rat tail collagen are all derived from Gibco; hepatocyte growth factor HGF is derived from inshore organisms; epithelial cell growth factor EGF is derived from inshore organisms; ROCK kinase inhibitor Agent Y-27632 was from Taozhu Bio; Wnt signaling pathway agonist CHIR-99021 was from Taozhu Bio; TGF- ⁇ signaling inhibitor A-8301 was from Taozhu Bio; CD63-FITC and CD81-PE flow-through antibodies were From the United States BD bioscience.
  • the serum-containing medium used was composed of the following: Hep-X basal medium, and 1% N2 nutrient supplement (100X), 1% B27 nutrient based on the volume of Hep-X basal medium Supplement (50X), 10% Fetal Bovine Serum FBS, 1% Penicillin-Streptomycin Solution; 20ng/mL Hepatocyte Growth Factor HGF, 50ng/mL Epithelial Growth Factor EGF, 10 ⁇ M ROCK kinase inhibitor Y-27632, 3 ⁇ M Wnt signaling pathway agonist CHIR-99021, 1 ⁇ M TGF- ⁇ signaling inhibitor A-8301.
  • composition of the serum-like substance-free medium used is the composition of the serum-like substance-containing medium after the fetal bovine serum is removed.
  • the above serum-containing medium and serum-free medium were filtered through a 0.22 micron filter to remove impurities before use.
  • the present embodiment provides the process of separately obtaining the precipitated material containing exosomes from two kinds of seed cells, specifically:
  • Seed cells were inoculated into a 15cm petri dish at a seeding density of 1 ⁇ 10 5 cells/square centimeter, and 2 ml of serum-containing medium was added to each well to culture until the cell confluence was not less than 95% and the growth state was good, and the expansion was completed. Increase cultivation. During the expansion culture, the medium containing serum-like substances is replaced every 2-3 days.
  • the medium in the 15 cm culture dish was replaced with a serum-free medium for continuing culture for 48 hours, and then the culture supernatant was collected.
  • the exosome isolation kit from System Biosciences, USA ULTRA EV Isolation separates PHHs-derived precipitates and HepLPCs-derived precipitates from the culture supernatant. The specific operation steps are described in the instructions attached to the exosome isolation kit, and will not be repeated here.
  • PHH Exo sample The precipitation material derived from PHHs is abbreviated as PHH Exo sample, and the precipitation material derived from HepLPCs is abbreviated as Hep Exo sample.
  • PHH Exo samples and Hep Exo samples were diluted and fixed with phosphate buffer containing 1% glutaraldehyde at a concentration of 0.1M, and then dropped onto a copper grid, then negatively stained with 1% uranyl acetate, and dried at room temperature. Then observe and take pictures with transmission electron microscope, and obtain the comparison photos of transmission electron microscope of PHH Exo and Hep Exo samples shown in Fig. 1 and Fig. 2 respectively, and the pH of phosphate buffer is 7.4.
  • the PHH Exo sample and the Hep Exo sample were analyzed using the PMX110 nanoparticle tracking analyzer from Particle Metrix, Germany, respectively, and the comparison chart of the average particle size of the particles in the two samples shown in Figure 3 was obtained.
  • the specific detection and analysis methods are conventional technical means of those skilled in the art, and will not be repeated here.
  • the diameter of the particles in the PHH Exo sample and the Hep Exo sample is about 100 nanometers, and the shape is regular like a circle. Further referring to Figure 3, the average particle size of the particles in the PHH Exo sample is 135 ⁇ 9.103nm , the average particle size of Hep Exo samples was 136.4 ⁇ 4.323 nm, which was in line with the morphological characteristics of exosomes.
  • PHH Exo samples and Hep Exo samples were diluted and mixed with PBS solution respectively, and one part was stained with CD63-FITC and CD81-PE flow antibody, and the other part of unstained PHH Exo samples and Hep Exo samples were used as negative controls.
  • the above-mentioned samples were tested on the Accuri C6flow cytomenter of BD bioscience in the United States, and the flow analysis results described in Figure 4 were obtained. The specific operations and analysis steps are conventional technical means of those skilled in the art, and will not be repeated here.
  • the supernatant was collected by centrifugation at 12000 rpm for 10 min as a test sample.
  • protein quantification used SDS-PAGE protein loading buffer (5 ⁇ ) of Biyuntian Biotechnology Co., Ltd. and PierceTM BCA Protein Assay Kit from Thermo Fisher of the United States; high-sensitivity ECL of Nanjing Novizan Biotechnology Co., Ltd. was used.
  • Chemiluminescence detection kit and ChemiDoc chemiluminescence imager from BIO-RAD, USA were used for WB test. The specific operations and analysis steps are conventional technical means of those skilled in the art, and will not be repeated here.
  • both PHH Exo samples and Hep Exo samples positively expressed exosome marker proteins CD63 and CD81.
  • the positive rates of CD63 in PHH Exo samples and Hep Exo samples were 61.85 ⁇ 3.465% and 90.85 ⁇ 2.475%, respectively.
  • the CD81 positive rates of the samples and Hep Exo samples were 69.90 ⁇ 4.95% and 89.40 ⁇ 1.273%, respectively.
  • the PHH Exo samples and Hep Exo samples of the Exosome group both positively expressed exosome marker proteins CD63 and TSG101.
  • Example 1-1 the PHH Exo samples and Hep Exo samples of Example 1-1 were labeled, and then co-cultured with PHHs to investigate the expression of exosomes in the cytoplasm of hepatocytes, proving that exosomes derived from PHHs and HepLPCs Can be successfully taken up by hepatocytes.
  • PHH Exo samples and Hep Exo samples were diluted with PBS buffer solution, and the diluted PHH Exo samples and Hep Exo samples with different concentrations were labeled with PKH26Red Fluorescent Cell Linker Kit from Sigma in the United States. The samples were then separately incubated with PHHs for 24 hours to complete the co-culture. After the co-cultivation, the culture containing cells was fixed with phosphate buffer containing 1% glutaraldehyde and the concentration of 0.1M, and then stained with DAPI, and then observed under a fluorescence microscope to obtain the PHH Exo sample shown in Figure 6.
  • This example provides the application of the hepatocyte regulation preparation containing exosomes in in vitro culture.
  • the PHH Exo samples and Hep Exo samples of Example 1-1 were diluted with buffer solution and then co-cultured with PHHs, and the obtained cells were subjected to proliferation analysis by BrdU ELISA detection and EdU fluorescence method, and Ki67 positive by immunofluorescence detection
  • the expression of expressing cells proved that both PHHs-derived and HepLPCs-derived exosomes could promote hepatocyte proliferation, and HepLPCs-derived exosomes had a more significant effect on promoting hepatocyte proliferation.
  • PHH Exo samples and Hep Exo samples were diluted with PBS buffer to different concentrations, 0, 1, 10, and 100 ⁇ g/ml, respectively, and were co-cultured with PHHs for 24 hours, and the PHHs were seeded at a density of 1 ⁇ 10 5 cells. /cm2, use a 12-well plate for the culture vessel.
  • the EdU Apollo 567 In Vitro Imaging Kit kit from Guangzhou Ribo Biotechnology Co., Ltd. was used to carry out EdU labeling, cell fixation, Apollo staining and DNA on the cells obtained by co-cultivation of samples with a concentration of 100 ⁇ g/ml exosomes. After staining, the DNA was stained and observed with a fluorescence microscope, and the PBS buffer was used as a negative control to obtain the immunofluorescence photo shown in FIG. 8 .
  • the specific EdU labeling, cell fixation, Apollo staining and DNA staining steps are provided by the kit.
  • the obtained cells were subjected to Ki67 immunofluorescence staining to obtain the immunofluorescence photograph shown in FIG. 9 .
  • EdU and BrdU are thymidine analogs, which replace thymidine (T) and infiltrate into DNA molecules being synthesized during DNA replication, and are used to detect DNA replication activity.
  • Ki67 is an antigen associated with proliferating cells and is mainly used to label cells in the proliferative cycle. It can be seen from this example that since both PHHs-derived and HepLPCs-derived exosomes can promote the proliferation of hepatocytes, the hepatocyte-regulating preparation formed by combining any of the above exosomes with a diluent can be used to promote hepatocyte proliferation. Proliferation medium is used.
  • the diluent is a resuspension
  • the concentration of exosomes is 10-200 ⁇ g/ml.
  • the resuspension solution is any one of PBS buffer solution, physiological saline and compound electrolyte solution.
  • Example 1-3 the cells obtained by co-culturing HepLPCs-derived exosomes with PHHs in Example 1-3 were used as examples to analyze the cell cycle and the expression of cell cycle-related molecules.
  • Cell cycle progression promotes hepatocyte proliferation.
  • PHH Exo-cell and Hep Exo-cell Use trypsin to digest the PHH Exo samples and Hep Exo samples co-cultured with PHHs in Examples 1-3 (respectively abbreviated as PHH Exo-cell and Hep Exo-cell), using Propidium Iodide Flow Cytometry Kit from Abcam, USA
  • the kit and the BD FACS Verse flow cytometer of BD bioscience in the United States were used for cell cycle analysis, and the red fluorescence was detected at the excitation wavelength of 488 nm, and the light scattering was detected at the same time.
  • the specific pre-operation steps are provided by the kit.
  • control group PHH Exo-cell and Hep Exo-cell were subjected to real-time fluorescence quantitative PCR using HiScript III 1st Strand cDNA Synthesis Kit from Nanjing Novizan Biotechnology Co., Ltd. and ChamQ SYBR Color qPCR Master Mix in Roche, Germany.
  • Quantitative PCR analysis the comparison chart of miRNA expression levels of cell cycle-related molecules shown in Figure 11, in which: three histograms corresponding to each cell cycle-related molecule, from left to right are the control group, PHH Exo samples and Hep Exo samples sample.
  • the exosomal miRNAs in the PHH Exo and Hep Exo samples of Example 1-1 were extracted, and the exosomal miRNA high-throughput sequencing analysis, sequencing bioinformatics analysis and miRNA mimic in vitro transfection into primary liver Post-cell BrdU ELISA test and EdU proliferation analysis showed that the miRNAs that were significantly increased in exosome expression and could effectively promote hepatocyte proliferation were hsa-miR-182, hsa-miR-183 and hsa-miR-574.
  • the Total Exosome RNA and Protein Isolation Kit from Invitrogen, USA was used to extract miRNA to obtain PHH Exo-derived analysis samples PHH-Exo-mi and Hep Exo-derived analysis samples Hep Exo-mi, and then use Nanjing Novizan Biotechnology Co., Ltd.
  • the miRNA 1st Strand cDNA Synthesis Kit, miRNA Universal SYBR qPCR Master Mix and HiScript III 1st Strand cDNA Synthesis Kit and ChamQ SYBR Color qPCR Master Mix connect the 3' and 5' connectors successively, reverse transcribed into cDNA, and then carry out PCR amplification. increase.
  • the target fragment library was recovered by cutting the gel, and the library that passed the quality inspection was sequenced and analyzed by the Illumina HiSeqTM 2500 high-throughput sequencer of Illumina in the United States. Top 15 miRNAs.
  • the primary hepatocytes transfected with hsa-miR-182, hsa-miR-183 and hsa-miR-574 were further stained with EdU, and the EdU incorporation rate was detected by EdU fluorescence method, as shown in Figure 15.
  • Examples 1-3 For specific detection kits, please refer to Examples 1-3.
  • hsa-miR-182 hsa-miR-183 and hsa-miR-574 significantly promoted the proliferation of primary hepatocytes (p ⁇ 0.05).
  • the EdU incorporation rate of hsa-miR-183 mimic transfection in vitro was significantly increased, and was significantly higher than that of hsa-miR-182 and hsa-miR-574 mimic transfection groups
  • the EdU incorporation rates of NC mimic, hsa-miR-182, hsa-miR-183 and hsa-miR-574 mimic transfection groups were 10.04 ⁇ 2.946%, 18.22 ⁇ 2.67%, 29.46 ⁇ 4.799% and 14.6 ⁇ 3.173%, respectively .
  • the injection containing Hep Exo samples was injected into the tail vein as an exosome preparation to intervene in the in vivo animal model of liver failure to promote liver tissue regeneration.
  • a carbon tetrachloride-induced mouse acute liver failure model and a control model were constructed using several C57BL/6 mice aged 6-8 weeks and weighing 22-25 g. Specifically, the induction injection of carbon tetrachloride and olive oil diluted by 1:4 was injected intraperitoneally to construct the acute liver failure model in mice, and the same volume of olive oil was injected intraperitoneally to construct the normal group. The intraperitoneal injection doses of the two models were both is 1 mL/kg.
  • the treatment injection mixed with PBS buffer and Hep Exo samples was injected into the tail vein of some mice with acute liver failure to form a Hep Exo treatment group; for some mice with acute liver failure
  • the model was injected with the same volume of PBS buffer as the treatment injection into the tail vein to form a PBS control group.
  • the Hep Exo sample concentration is 2 ⁇ g/ ⁇ L.
  • the injection dose of Hep Exo treatment group and PBS control group was 15 mg/kg.
  • the injection dose of Hep Exo treatment group and PBS control group is 1-100 mg/kg.
  • the survival of each mouse in the Hep Exo treatment group and the PBS control group within 7 days after modeling was investigated. Specifically, the survival curve was drawn using the Kaplan-meier method for survival analysis and the log-rank test was performed. p ⁇ 0.05 was considered to be statistically different, and the results shown in Figure 17 were obtained.
  • the PBS control group had 25% mortality at 24 hours and more than 50% mortality within 48 hours, while the Hep Exo treated mice had a 20% mortality rate at 24 hours and only a 48-hour mortality rate. 30%, and no death after 72 hours (p ⁇ 0.05), indicating that human liver precursor-like cell-derived exosomes can improve the 7-day survival rate of mice and have a significant therapeutic effect on acute liver failure.
  • the liver tissue of the mice in the normal group was in the form of normal hepatocytes, the hepatic lobule structure was complete and clear, and there was no inflammatory cell infiltration.
  • the mice in the PBS control group had obvious hepatocyte swelling, nuclear fragmentation, increased vacuoles, local infiltration of inflammatory cells, damage to the normal connection structure of the hepatic cord, and severe hepatic sinusoidal congestion.
  • the mice in the Hep Exo treatment group showed milder hepatocyte damage at 24h and 48h after modeling, with less vacuolization and inflammatory cell infiltration than the control group, and the liver damage was alleviated to a certain extent.
  • mice were anesthetized by sevoflurane inhalation 24 hours and 48 hours after modeling, and the eyeballs of the mice were removed to collect blood, allowing the blood to flow out naturally. Centrifuge at 4°C, 3000 rpm for 10 min, and then slowly aspirate the supernatant as mouse serum.
  • the alanine aminotransferase (ALT) kit and aspartate aminotransferase (AST) detection kit from Beckman Coulter were used to detect the biochemical indexes of ALT and AST, and the AST level and AST were calculated according to the average absorbance ⁇ A per minute.
  • ALT levels the comparison results of AST levels and ALT levels of each mouse in the normal group, the PBS control group and the Hep Exo treatment group at different times shown in Figures 21 and 22 were obtained, and the number of mice used for statistics in each group was 8.
  • the three bar graphs corresponding to each modeling time in Figure 21 and Figure 22 are the normal group, the PBS control group and the Hep Exo treatment group from left to right.
  • the levels of serum AST and ALT in the PBS control group and the Hep Exo treatment group gradually increased after modeling, and reached a peak at 48h.
  • the serum AST and ALT levels of the mice in the Hep Exo treatment group were significantly reduced. It can be seen that HepLPC-derived exosomes can effectively reduce the serum ALT and AST levels, and play a role in the acute liver injury in mice. Protective effects.
  • This example takes miRNA-183 as an example, and provides the application of exosome preparations containing miRNA-183 in the preparation of drugs for the treatment of liver failure.
  • mice models of acute liver failure induced by carbon tetrachloride and acetaminophen were constructed to simulate different mechanisms of liver injury, respectively.
  • the injection containing miRNA-183 was injected into the tail vein as an exosome preparation to interfere with liver failure in vivo. animal model to promote liver tissue regeneration.
  • the model of acute liver failure induced by carbon tetrachloride (abbreviated as CCl4 model) is constructed as follows: C57BL/6 mice weighing 22-25 g for 6-8 weeks were intraperitoneally injected with carbon tetrachloride and olive oil at a ratio of 1:4 Diluted carbon tetrachloride induction injection, the injection dose is 1mL/kg. The corresponding normal control group was intraperitoneally injected with an equal volume of olive oil (abbreviated as CCl4 normal group). The carbon tetrachloride induction injection was sterile filtered using a 0.22 micron filter prior to injection.
  • Acetaminophen-induced acute liver failure model (abbreviated as APAP model) is constructed as follows: C57BL/6 mice weighing 22-25 g at 6-8 weeks are intraperitoneally injected with acetaminophen mixed with PBS buffer. Acetaminophen induction injection, the injection dose is 1mL/kg. The corresponding normal control group was intraperitoneally injected with an equal volume of PBS buffer (abbreviated as APAP normal group). The acetaminophen-induced injection was sterile filtered using a 0.22 micron filter prior to injection.
  • the tail vein injection of part of the CCl4 model and part of the APAP model 6 hours after modeling was diluted with CCl4 and PBS buffer, respectively, and the negative control injection containing 15 nanomolar miRNA-183 agomir was 200 ⁇ l each (respectively abbreviated as CCl4 -NC agomir group and APAP-NC agomir group).
  • Cy5-labeled miRNA-183 agomir was encapsulated with Entranster TM -in vivo RNA transfection reagent, and diluted with CCl4 and PBS buffers, respectively, and then tail vein injection was performed on part of the CCl4 model and part of the APAP model 6 hours after modeling (respectively simplified).
  • CCl4-miRNA 183-5p agomir group and APAP-miRNA 183-5p agomir group.
  • each microliter of transfection reagent in the injection contains 2 micrograms of nucleic acid
  • the injection volume of each injection is 200 microliters
  • the content of miRNA-183 agomir is 15 nanomolar.
  • Entranster TM -in vivo RNA transfection reagent was from Beijing Ingen Biotechnology Co., Ltd.
  • Cy5-labeled miRNA-183 agomir was from Guangzhou Ribo Biotechnology Co., Ltd.
  • liver tissue and blood samples were taken from each group at different time points after modeling for detection of liver injury and regeneration-related indicators.
  • the number of mice used for testing in each group was 8. details as follows:
  • the mortality rate of mice in the CCl4-NC agomir group was 20% at 24 hours and more than 70% within 48 hours, while the mice in the CCl4-miRNA 183-5p agomir group did not die within 24 hours and died within 48 hours The mortality rate was only 20% (p ⁇ 0.05); referring to Figure 24, the mortality rate of mice in the APAP-NC agomir group was as high as 80% within 24 hours, while the mortality rate of mice in the APAP-miRNA 183-5p agomir group was only 50% within 24 hours. Both Figure 23 and Figure 24 illustrate that miRNA 183-5p can significantly improve the 7-day survival rate of mice, and has a significant therapeutic effect on acute liver failure.
  • the peripheral blood of each mouse of the CCl4 model was taken at different time points within 72 hours after modeling for ALT and AST detection, and the peripheral blood of each mouse of APAP model was taken for ALT and AST detection at 24 hours after modeling.
  • the three bar graphs corresponding to each modeling time in Figures 25 and 26 are the normal group, the CCl4-NC agomir group and the CCl4-miRNA 183-5p agomir group from left to right.
  • the three bar graphs corresponding to each factor (ALT or AST) in Figure 27 are the normal group, the APAP-NC agomir group and the APAP-miRNA 183-5p agomir group from left to right.
  • the levels of AST and ALT in the mice in the CCl4-NC agomir group and the mice in the CCl4-miRNA 183-5p agomir group gradually increased after modeling, and reached a peak at 48h, but decreased at 72h.
  • the levels of AST and ALT in the CCl4-miRNA 183-5p agomir group were significantly lower than those in the CCl4-NC agomir group.
  • the AST and ALT levels of mice in APAP-miRNA 183-5p agomir group were significantly lower than those in APAP-NC agomir group. All of the above indicated that miRNA 183-5p could effectively reduce the levels of ALT and AST in serum.
  • Figure 32 shows the percentage of Ki67-positive cells in liver tissue sections of mice in the CCl4-NC agomir group and mice in the CCl4-miRNA183-5p agomir group at different times after modeling. Among them: the two histograms corresponding to the completion time of each modeling are CCl4-NC agomir group and CCl4-miRNA183-5p agomir group from left to right.
  • liver tissues of the mice in the CCl4 normal group and the APAP normal group showed normal hepatocyte morphology, the hepatic lobule structure was complete and clear, and there was no inflammatory cell infiltration.
  • the mice in the CCl4-NC agomir group and APAP-NC agomir group had obvious liver damage at 24h, and at 48h, severe hepatocyte necrosis, nuclear fragmentation, increased vacuoles, a large number of inflammatory cell infiltration, and severe liver sinus congestion occurred.
  • mice in the CCl4-miRNA 183-5p agomir group and APAP-miRNA 183-5p agomir group also had liver damage after 24 hours of modeling, but the degree of liver damage was significantly alleviated after 48 hours of modeling, with both vacuolization and inflammatory cell infiltration. less than the corresponding NC agomir group.
  • mice in the CCl4-miRNA 183-5p agomir group had significantly more Ki67-expressing cells in the 48-hour section (p ⁇ 0.05); compared with the APAP-NC agomir group, the APAP-miRNA 183 The number of Ki67-positive cells in the -5p agomir group was significantly increased (p ⁇ 0.05) in the 24-hour section, indicating that liver regeneration was effectively initiated.
  • Example 1 the protein expression of cell cycle-related molecules in each mouse in the CCl4-miRNA 183-5p agomir group at different times after modeling was investigated by Western blotting, and the schematic diagram shown in FIG. 33 was obtained.
  • the specific operation method please refer to Example 1.
  • liver disease regulating preparations prepared anti-hepatic fibrosis preparations as liver disease regulating preparations, and investigated the application of such liver disease regulating preparations. details as follows:
  • This embodiment provides the first anti-hepatic fibrosis preparation, and its preparation method is as follows:
  • step S2 Collect the in vitro culture supernatant, remove the cell debris in the in vitro culture supernatant, and perform 25-fold filtration and concentration, and the obtained secretory supernatant is used as an anti-hepatic fibrosis preparation. Specifically, in the step S2, the cell debris in the culture supernatant is removed under a centrifugal force of 3000 g; the 10 kDa Amicon Ultra filter is used for filtration and concentration.
  • BCA protein quantitative kit (from Shanghai Biyuntian Biotechnology Co., Ltd.) was used to detect the total protein content in the secretion supernatant as 2.2 mg/ml according to the detection method provided in the instructions.
  • the Human-HepLPCs in step S0 described in this example were transformed and expanded by TEM medium from Human-primary hepatocytes for 7-9 days, and then subcultured at 1:(3-6) to Generations 2-5 are obtained.
  • the TEM medium consists of the following components: DMEM/F12 basal medium, and based on the content of the DMEM/F12 basal medium: N2 nutritional supplement (100X) with a content of 1%, B27 with a content of 1% Nutrient supplement (50X), 1 mM sodium pyruvate, 10 ⁇ g/mL ascorbic acid, 20 ng/mL hepatocyte growth factor HGF, 20 ng/mL epithelial cell growth factor EGF, 10 ⁇ M ROCK kinase inhibitor Y27632, 3 ⁇ M Wnt Signaling pathway agonist CHIR99021, 1 ⁇ M TGF- ⁇ signaling inhibitor A8301, 1 ⁇ M sphingosine-1-phosphate S1P and 5
  • DMEM/F12, N2 nutritional supplement, B27 nutritional supplement and sodium pyruvate are from Invitrogen; ascorbic acid is from Sigma-Aldrich; HGF and EGF are from Novoprotein; Y27632, CHIR99021, A8301, s1p and LPA are all from TargetMol .
  • the hepatocyte marker HNF4 ⁇ of human hepatic precursor-like cells and the hepatic stem cell/liver progenitor cell marker CD24 and CK19 were significantly expressed, and the expression levels of hematopoietic stem cell antigen CD34, leukocyte common antigen CD45 and liver fetal cell marker AFP were all expressed. less than 2%.
  • Human hepatic precursor-like cells do not express the MHC class II antigens HLA-DP, HLA-DQ and HLA-DR and exhibit low immunogenicity.
  • Example 2-1 the first anti-hepatic fibrosis preparation of Example 2-1 was co-cultured with the human immortalized hepatic stellate cell line LX-2, and the pro-apoptotic effect of the anti-hepatic fibrosis preparation on LX-2 was investigated.
  • LX-2 of this example was purchased from Procell.
  • the first anti-hepatic fibrosis preparation of Example 1 was co-cultured with LX-2 as follows: LX-2 was fixed in DMEM medium containing 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin , add 2.5ng/mL of TGF- ⁇ 1 to activate LX-2; then add the anti-hepatic fibrosis preparation of Example 1, mix well, and let stand for 48 hours. In the co-culture mixture, the volume percentage of the first anti-hepatic fibrosis preparation was 1%, 2.5% and 5%.
  • the first anti-hepatic fibrosis preparation was added with 10 ⁇ g/mL of anti-FGF19 antibody (rabbit monoclonal antibody, from R&D Systems) and 10 ⁇ g/mL of anti-AREG antibody (rabbit polyclonal antibody, from R&D) before use Systems) for 2 hours.
  • RT-PCR real-time polymerase chain reaction
  • TGF- ⁇ 1 activated LX-2 cells, which up-regulated the expression levels of the above-mentioned HSCs activation-related genes; while the introduction of the first anti-hepatic fibrosis agent significantly inhibited the above-mentioned HSCs activation-related gene expression levels , it can be seen that the first anti-hepatic fibrosis agent has a significant inhibitory effect on the activation of HSCs.
  • the cells of the experimental group (the volume percentage of the first anti-hepatic fibrosis preparation is 1%) were prepared for transmission electron microscope observation, and the microscopic photo shown in FIG. 38 was obtained.
  • apoptotic bodies indicated by arrows were observed in the cell aggregates obtained by co-culture of the first anti-hepatic fibrosis preparation with LX-2.
  • the cells obtained from the experimental group and the TGF- ⁇ 1 activation group were further stained with Annexin V/PI and then measured by flow cytometry.
  • the flow cytometry chart of the control group shown in Figure 39 and the experimental group shown in Figure 40 were obtained. flow cytometry plot. Referring to Figure 39 and Figure 40, the first anti-hepatic fibrosis agent induced HSC apoptosis.
  • This example provides a second anti-hepatic fibrosis preparation, the preparation method of which uses Rat-HepLPCs, which have a confluence of not less than 60%, as seed cells.
  • Rat-HepLPCs which have a confluence of not less than 60%, as seed cells.
  • Rat-HepLPCs were obtained by using Rat primary hepatocytes of rat liver as seed cells and cultured in TEM medium.
  • the second anti-hepatic fibrosis preparation of Example 2-3 was co-cultured with the murine hepatic stellate cell line HSCs-T6, and the pro-apoptotic effect of the anti-hepatic fibrosis preparation on HSCs-T6 was investigated.
  • the HSCs-T6 of this example were purchased from Procell.
  • Example 2-2 For the process of co-culturing the second anti-hepatic fibrosis preparation of Example 2-3 with HSCs-T6, please refer to Example 2-2.
  • the volume percentage of the second anti-hepatic fibrosis preparation in the co-culture mixture is 1%.
  • HSCs-T6 cells by TGF- ⁇ 1 resulted in a change in the morphology of HSCs-T6 cells into elongated dendritic cells, and the addition of the first anti-hepatic fibrosis agent for co-culture could reverse this change.
  • HSCs activation-related genes Col1a1, Col3a1, TGF- ⁇ 1, Desmin, ⁇ -SMA in the HSCs-T6 of the control group, the TGF- ⁇ 1 activation group and the experimental group were investigated by real-time polymerase chain reaction (RT-PCR).
  • RT-PCR real-time polymerase chain reaction
  • TGF- ⁇ 1 activated HSCs-T6 cells, which increased the expression levels of the above-mentioned HSCs activation-related genes; while the introduction of the second anti-hepatic fibrosis agent significantly inhibited the above-mentioned HSCs activation-related gene expression levels, It can be seen that the second anti-hepatic fibrosis agent has a significant inhibitory effect on the activation of HSCs.
  • tandem mass spectrometry tag (TMT) was used to analyze the proteomic composition of the first anti-hepatic fibrosis preparation obtained by in vitro culture of human liver precursor-like cells Human-HepLPCs in Example 1.
  • PPI protein-protein interaction
  • LIF leukemia inhibitory factor
  • EDN1 endothelin 1
  • CSF1 colony stimulating factor 1
  • AVG amphiregulin
  • FGF19 fibroblast growth factor 19
  • This example provides a third anti-hepatic fibrosis preparation, comprising recombinant human FGF19 (rhFGF19) and recombinant human AREG (rhAREG).
  • rhFGF19 recombinant human FGF19
  • rhAREG recombinant human AREG
  • the third anti-hepatic fibrosis preparation was co-cultured with LX-2 to form a TGF- ⁇ 1+rhFGF19+rhAREG group, and its effect on LX-2 was investigated.
  • the difference is that :
  • the concentrations of rhFGF19 were 0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL and 1000ng/mL, and the concentrations of rhAREG were 0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL and 1000ng, respectively /mL.
  • the concentrations of rhFGF19 and rhAREG were different in different experimental groups.
  • p-STAT1 plays an important role in the process of liver fibrosis, and mainly achieves anti-fibrotic effect by inhibiting the function of hepatic stellate cells.
  • the control group, the control group and the different experimental groups were subjected to Western blot analysis, and the comparison chart of the expression of pSTAT1 signal in the cells of each group as shown in FIG. 49 was obtained. Referring to Fig. 49, it can be seen that when the concentration of each recombinant protein is not less than 10 ng/mL, the level of p-STAT1 increases.
  • the neutralizing antibody FGF19Ab of FGF19 and the neutralizing antibody AREG Ab of AREG were added to the co-culture system of the first anti-hepatic fibrosis preparation of Example 2-2 and activated LX-2 to form TGF- ⁇ 1+
  • the synergistic effect of rhFGF19 and rhAREG was investigated together with the TGF- ⁇ 1+rhFGF19+rhAREG group in Example 2-6, the TGF- ⁇ 1 activation group and the experimental group in Example 2-2.
  • concentrations of rhFGF19 and rhFGF19 in the co-culture system were both 100 ng/ml, and the concentrations of FGF19 neutralizing antibody FGF19Ab and AREG neutralizing antibody AREG Ab in the co-culture system were both 10 ⁇ g/ml.
  • This example provides the application of the anti-hepatic fibrosis preparation in preparing the anti-hepatic fibrosis drug.
  • thioacetamide was used to induce liver fibrosis.
  • 5-6 week old female mice C57BL/C were induced by thioacetamide (TAA) to form a liver fibrosis model.
  • TAA was diluted in normal saline and injected intraperitoneally at a dose of 200 mg/kg, 3 times a week. 7 weeks in total.
  • Example 1 The secretory supernatant of Example 1 as an anti-hepatic fibrosis preparation was diluted with PBS to a total protein concentration of 2 mg/ml to obtain an anti-hepatic fibrosis preparation injection.
  • the normal control group was left untreated, the sham operation group was injected with 250ul PBS solution through the spleen, and the anti-hepatic fibrosis preparation intervention group was injected with 250ul of the anti-hepatic fibrosis preparation injection through the spleen.
  • the livers of mice were soaked in formalin solution for fixation, embedded and sliced for HE staining, Masson's trichrome detection and Sirius detection to comprehensively analyze the degree of liver fibrosis in mice. .
  • liver fibrosis preparations significantly improved the degree of liver fibrosis induced by TAA in mice.
  • the qPCR test process in the embodiment of the present invention is as follows: the total mRNA is extracted using the Eastep Super RNA extraction kit (Item No. LS1040, derived from Promega). use First-strand cDNA synthesis kit (Cat. No. R211-01, from Vazyme) was used for reverse transcription. Real-time PCR was then developed using the AceQ qPCR SYBR Green Master Mix (Cat. No. Q131-02 from Vazyme) and the Life Technology ABI 7500 system. GAPDH expression was used as an internal control, threshold cycle (CT) was determined, and relative changes in gene expression were calculated using the delta ( ⁇ CT) method.
  • CT threshold cycle
  • a transmission electron microscope (model Jem 1200ex II, derived from JEOL) was used to observe the morphology of cells.
  • Cells were fixed with 2.5% glutaraldehyde and 2% osmic acid, then dehydrated and embedded in epoxy resin, cut into 80 nm thick sections, and then visualized after double staining with uranyl acetate and lead acetate.
  • RNA sequencing and bioinformatics analysis of the embodiments of the present invention use Reagents Total RNA was isolated from liver tissue; genomic DNA was removed using DNase I (from TaKara); the concentration and purity of RNA samples were determined by a 2100 Bioanalyzer (from Agilent) and quantified using an ND-2000.
  • RNA seq transcriptome libraries were prepared according to the TruSeqTM RNA sample preparation kit from Illumina, using 1 ⁇ g of total RNA; according to Illumina's library construction protocol, fragment RNA was first-strand and first-strand Double-stranded cDNA was synthesized, followed by adaptor ligation and enrichment at low cycles; after quantification, paired-end RNA seq sequencing libraries were sequenced using an Illumina HiSeq xten/NovaSeq 6000 sequencer at Guangzhou RiboBio Co., Ltd.
  • Gene expression of the examples of the present invention was normalized by EDASEQ. Differentially expressed genes were obtained using version 1.10.1 of DESeq2 with a cutoff of Q-value ⁇ 0.05 and log2 (fold change) > 1 for identifying differentially expressed genes. All differentially expressed mRNAs were selected for GO analysis with clusterProfiler. Use glbase for additional analysis.
  • RIPA buffer P0013B, derived from Beyotime
  • protease inhibitor cocktail P1010, derived from Beyotime
  • P1010 protease inhibitor cocktail
  • BCA protein assay kit ZJ101, derived from Epizyme
  • Quantitative protein samples were resolved by 5x SDS-PAGE (P0015, from Beyotime) and transferred to a hydrophobic PVDF transfer membrane (IPVH00010, from Merck Millipore).
  • Membranes were blocked in 5% BSA in TBST for 1.5 h and incubated with primary antibodies overnight at 4 °C. Membranes were then washed three times with TBST and incubated with secondary antibodies for 2 h at room temperature. Blots were detected using the Enhanced ECL Chemiluminescence Detection Kit (E411-04 from Vazyme and a digital luminescence image analyzer (BioRad). Densitometric analysis of each band was determined using ImageJ software or QingXiang software. Primary and secondary antibodies used See Table 1.
  • the annexin V-FITC apoptosis detection kit derived from Beyotime and the annexin V-APC apoptosis detection kit derived from Biogems were used to pass the annexin V/propidium iodide (PI) or Annexin V/7-AAD assay detects apoptosis in LX-2. Specifically, cells were harvested and resuspended in binding buffer, then stained with Annexin V and PI or 7-AAD according to the kit instructions. Staurosporine, a protein kinase inhibitor at 0.5 ⁇ M, was used as a pro-apoptotic control (positive control). Fluorescence was detected by a BD-facverse flow cytometer, and data analysis was performed with FlowJo software.
  • PI annexin V/propidium iodide
  • Annexin V/7-AAD Annexin V/7-AAD
  • Cytokine antibody arrays (AAH-INF-G3, series) to measure the expression of 40 cytokines in the culture supernatant. Positive signals were detected with a laser scanner. The basic statistic used for significance analysis is fold change. Differentially expressed proteins (DEPs) were defined as proteins with a fold change greater than 1.2 or less than 0.83 (absolute log fc > 0.263). The functions of cytokines were annotated by Gene Ontology (GO) annotations.
  • GO Gene Ontology
  • MS mass spectrometry
  • PD Proteome Discovery
  • Sequest HT search engine A list of MS spectra was searched against the Homo sapiens UniProt FASTA database (UniProt-Human-9606-2020-10.FASTA) with aminomethyl [C], TMT 6 complex (K) and TMT 6 complex (N-term) as Fixed modifications, oxidation (M) and acetyl (protein N-term) as variable modifications. Parameters used to identify peptides were: 10 ppm precursor ion mass tolerance, 0.02 Da fragment mass tolerance, up to 2 deletion cleavage.
  • the false discovery rate (FDR) at both the PSM and peptide levels was set to 0.01.
  • the functions of the proteins were annotated by Gene Ontology (GO) annotation (http://www.geneontology.org/).
  • the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to analyze enrichment pathways. A two-tailed Fisher's exact test was used in the GO-KEGG enrichment analysis. P ⁇ 0.05 was considered significant.
  • Cytoscape version 2.6 www.Cytoscape.org was used to visualize and analyze molecular and protein interaction networks. Differentially expressed proteins were arranged by hierarchical clustering and represented as a heatmap. Heatmaps were generated by R software (http://www.r-project.org).
  • the HepLPCs obtained in Example 2-1 were used as the main components of the first cell preparation, and the first cell preparation was co-cultured with the human immortalized hepatic stellate cell line LX-2. 2's pro-death effect.
  • LX-2 of this example was purchased from Procell.
  • Human-HepLPCs and LX-2 were first stained with lipophilic membrane dyes DiO and DiI, respectively, and then the stained cells were resuspended in DMEM complete medium to obtain different cell preparations. Specifically, after digesting and washing the Human-HepLPCs obtained by in vitro culture in TEM medium in Example 2-1, they were resuspended in serum-depleted LPCs containing 5uM DiO (derived from Beyotime) or DiI (derived from Beyotime), respectively.
  • DiO lipophilic membrane dyes DiO and DiI
  • DMEM complete medium In DMEM complete medium, incubate for 10 minutes in an incubator, then wash with PBS buffer to remove unbound staining, and finally resuspend the stained cells in DMEM complete medium to obtain the markers used in Example 3 cell preparation. After DiO enters the cell membrane, it can diffuse laterally and gradually make the cell membrane of the entire cell stained green; after DiI enters the cell membrane, it can diffuse laterally and gradually make the cell membrane of the entire cell stained red.
  • Example 2-9 The cell preparation containing DiI-labeled LX-2 and the cell preparation containing DiO-labeled HepLPCs were mixed in a ratio of 1:1, and then 2.5ng/ml TGF- ⁇ was added, and then 3000 cells/well were added. Inoculated into a low-adherence 96-well plate as a co-culture group; inoculated the cell preparation containing DiI-labeled LX-2 into a low-adherence 96-well plate at 3000 cells/well as the LX-2 group; DiI-labeled LX-2 The LX-2 cell preparation was added with 2.5ng/ml of TGF- ⁇ and then seeded into a low-adherence 96-well plate at 3000 cells/well as the LX-2 supplemented group.
  • the medium was aspirated and discarded, and the RNA of the LX-2 group and the LX-2 supplemented group were collected respectively after observing and taking pictures under the microscope;
  • LX2 cells labeled with red DiI fluorescence were sorted by flow cytometry, and then the RNA of the cells was collected.
  • RNA extraction kit (purchased from Promaga company) was used to extract the total RNA of LX2 in 3 groups; SYBR Green PCR kit (purchased from Novizan company) was used to carry out on PCR instrument (purchased from Roche company).
  • Validated genes include collagen-related genes COL3A1, COL1A1; fibrosis-related genes a-SMA, Vimentin, Timp1.
  • the levels of fibrosis-related genes COL3A1, COL1A1, a-SMA, Vimentin, Timp1 and Desimin were significantly up-regulated in hepatic stellate cells activated by TGF- ⁇ 1, while hepatic stellate cells activated by TGF- ⁇ 1 were significantly associated with liver stellate cells.
  • the above genes were significantly down-regulated, which proved that hepatic precursor cells could significantly inhibit the activation of hepatic stellate cells in vitro.
  • This example provides the application of the second cell preparation prepared from the Human-HepLPCs of Example 2-1 as hepatic-derived cells with precursor characteristics in the preparation of anti-hepatic fibrosis drugs.
  • the second cell preparation is a cell suspension obtained by resuspending Human-HepLPCs in PBS. Specifically, 5 ⁇ 10 6 Human-HepLPCs were resuspended in 500 ⁇ l PBS.
  • the in vitro organoid model of liver fibrosis is a mammalian liver cirrhosis model induced by thioacetamide (TAA).
  • TAA thioacetamide
  • the modeling method is as follows: 5-6 week old female Sprague-Dawley rats (derived from Vitalriver) were induced with TAA diluted in normal saline at an injection dose of 200 mg/kg every week, twice a week, and the induction time was 13 weeks to complete modeling.
  • 8 rats that were successfully modeled were treated with any treatment, as the normal group; 500 ⁇ l PBS was injected into the spleen of the 8 rats that were successfully modeled, as the sham-operated group; the first induction was used as the initial time node, the The 8 successfully modeled rats were injected with the immunosuppressive drug tacrolimus (FK506) in the spleen at an injection dose of 0.2 mg/kg daily at weeks 13 and 15, and weekly starting the day after the first injection of FK506. Two splenic injections of the second cell preparation were performed and the rats were sacrificed on the third day of week 17.
  • FK506 immunosuppressive drug tacrolimus
  • livers of rats in each experimental group were used for further staining analysis, hydroxyproline content analysis and liver fibrosis scoring.
  • the injection of the second cell preparation can reduce the extracellular matrix (ECM) Accumulation, reducing the level of hydroxyproline in rats, and alleviating liver cirrhosis in rats.
  • ECM extracellular matrix
  • Figure 55 is a comparison of photos obtained after liver tissue from the normal group, the sham-operated group and the cell transplantation group was stained with H&E, picro-Sirius red, Masson's trichrome and fibronectin (FN) immunostaining, the scale bar in the figure is 100 ⁇ m.
  • Figure 56 shows the statistical hepatic fibrosis area and fibronectin-positive staining of liver tissue from normal group, sham-operated group and cell transplantation group after picro-Sirius staining, Masson's trichrome staining and fibronectin (FN) immunostaining Comparison of relative quantification of regions.
  • Fig. 57 shows the analysis results of the determination of hydroxyproline content in the normal group, the sham-operated group and the cell transplantation group.
  • Figure 58 shows the analysis results of liver fibrosis scoring in the sham operation group and the cell transplantation group.
  • Fig. 59 and Fig. 60 are the comparison pictures of photos obtained by taking liver tissue for Ki67 immunohistochemical staining in the normal group, the sham-operated group and the cell transplantation group, respectively, and the quantification results of positive staining cells according to the photos shown in Fig. 59 .
  • This example provides the application of the third cell preparation prepared from the Rat-HepLPCs of Example 2-2 as hepatic-derived cells with precursor characteristics in the preparation of anti-hepatic fibrosis drugs.
  • liver-derived cells are Rat-HepLPCs.
  • the in vitro organoid model of liver fibrosis is a carbon tetrachloride-induced mammalian liver cirrhosis model.
  • the modeling method is as follows: 5-6 week-old female Sprague-Dawley rats (from Vitalriver) were induced weekly with a mixed injection of CCl4 and olive oil with a content of 40% CCl4 at a dose of 1 mL/kg. The time period was 13 weeks to complete the modeling.
  • the treatment conditions of the normal group and the sham operation group in this example are the same as those of Example 4.
  • the difference between the treatment conditions of the cell transplantation group and Examples 2-4 is that the immunosuppressive drug tacrolimus was not used for intervention, and the 13th After weekly injection of the third cell preparation, the rats were sacrificed at week 17.
  • the livers and blood of each group of rats were taken for further staining analysis, hydroxyproline content analysis and liver fibrosis scoring.
  • the injection of the second cell preparation can reduce the amount of extracellular matrix (ECM) ) accumulation, reduced the level of hydroxyproline in rats, inhibited the production of fibrotic cytokine TGF- ⁇ and its intracellular signaling molecules in fibrotic tissues, and alleviated liver cirrhosis in rats.
  • ECM extracellular matrix
  • Figure 61 is a comparison chart of photos obtained after liver tissue from the normal group, the sham-operated group and the cell transplantation group was stained with H&E, picro-Sirius red, Masson's trichrome and fibronectin (FN) immunostaining, the scale bar in the figure is 100 ⁇ m.
  • Figure 62 shows the statistical hepatic fibrosis area and fibronectin-positive staining of liver tissue from the normal group, the sham-operated group and the cell transplantation group after picro-Sirius staining, Masson's trichrome staining and fibronectin (FN) immunostaining Comparison of relative quantification of regions.
  • Figure 63 shows the analysis results of liver tissue taken from the normal group, the sham-operated group and the cell transplantation group for the determination of hydroxyproline content.
  • Figure 64 shows the analysis results of liver fibrosis scoring in the sham operation group and the cell transplantation group.
  • Figure 65 is a heat map obtained by analyzing the gene expression levels related to the occurrence of hepatic fibrosis related to pro-fibrosis, extracellular matrix, and signal transduction in the blood of the normal group, the sham-operated group and the cell transplantation group.
  • Liver tissue was fixed in 4% paraformaldehyde (PFA), embedded in paraffin, and then cut into 4 ⁇ m thick sections. Liver sections were routinely stained for collagen deposition using hematoxylin and eosin (H&E) staining, picro-Sirius red staining, and Masson's trichrome (MT) staining. Collagen type I and III quantification were analyzed using a microscope (Olympus BX50) under polarized light. Using a polarizing filter, type I collagen fibers in Sirius red stained sections will appear orange to red, and type III collagen fibers will appear yellow to green. Sections stained with Sirius Red were used for liver fibrosis scoring under the Ishak scoring system.
  • PFA paraformaldehyde
  • tissue sections were stained with primary antibodies against fibronectin, GFP, KI67, HLA class I, CK18, ALB. Liver fibrosis scores and Ki67+ cells were calculated in a double-blind manner from two independent individuals, and the results were averaged for analysis. Representative images of H&E, picro Sirius red, MT staining and IHC were taken at Turbo by Leica Aperio. Quantification was performed by dividing the area of positive staining by the total sampling area using Image J software.
  • liver sections or cell spheroids were fixed in 4% paraformaldehyde (PFA), then permeabilized with 0.3% Triton X-100 and blocked with 3% bovine serum albumin (BSA), then the sections were incubated to obtain Primary antibody against ⁇ -SMA, cleaved caspase 3 overnight at 4°C (see Supplementary Table 1 for details), and cultured cell spheroids at 4°C overnight for ALB, CYP3A4, TTR, CK19, SOX9 , AFP (see Supplementary Table 1 for details).
  • PFA paraformaldehyde
  • BSA bovine serum albumin
  • Hydroxyproline assays were performed according to the manufacturer's protocol (Solarbio, BC0250). Briefly, liver tissue (200 mg) was homogenized in extract, boiled in an oven at 110°C for 2 to 6 hours until no visible bulk was observed, then centrifuged at 16,000 rpm for 20 min and washed with 10 mol/L NaOH (approximately 1 ml) to adjust the pH to 6 to 8. Set the volume of the hydrolyzed sample to 4 ml with distilled water, transfer the supernatant to a 96-well plate, and measure according to the manufacturer's protocol.
  • liver disease regulating preparation prepared a hepatic macrophage regulator as a liver disease regulating preparation, and investigated the application of this liver disease regulating preparation. details as follows:
  • This embodiment provides the first hepatic macrophage regulator, and its preparation method is as follows:
  • S0 provide primary human hepatocytes
  • hepatocyte proliferation medium (abbreviated as TEM medium) to culture the primary hepatocytes until the confluence is not less than 80%;
  • S4 Collect the cell supernatant after the in vitro culture is completed, perform centrifugation on the cell supernatant to remove cell debris to obtain a culture supernatant, and use the culture supernatant as the first hepatic macrophage regulator.
  • the primary human hepatocytes are from Shanghai Ruide Liver Co., Ltd. Specifically, the human primary hepatocytes were subjected to Percoll density gradient centrifugation combined with flow sorting to exclude CD24-positive and EpCAM-positive precursor cells before being cultured by TEM.
  • the TEM medium of step S1 in this example contains 1% serum-free additive N2 (100 ⁇ ), 1% serum-free additive B27 (50 ⁇ ), 20 ng based on the content of HepX Basal medium.
  • the step of culturing the primary hepatocytes includes: resuspending the primary human hepatocytes in a TEM medium and inoculating them with Vitronectin at a density of 0.5-1 ⁇ 10 5 /cm 2 .
  • Cultures were performed in 6-well plates coated with XF TM (from Canada Stem Cell Technology, Inc.). During the culture, the TEM medium was changed every 2-3 days.
  • the step of subculturing the cells obtained after the step S1 after digestion at a ratio of 1:3 includes: using 0.25% Trypsin-EDTA (from Gibco, USA) for digestion, and according to the following steps: The ratio of 1:3 was inoculated into a new culture dish to expand the culture to the third passage and until the cell confluence rate reached 80%.
  • the cell supernatant was centrifuged at 300 g for 10 minutes to remove cell debris to obtain the first hepatic macrophage regulator.
  • HepLPCs human liver precursor-like cells obtained in the step S2 were identified and analyzed by flow cytometry, and the expression comparison of the obtained liver precursor-related markers in the HepLPCs cells is shown in Fig. 34.
  • FIG. 34 it was found that HepLPCs expressed the liver precursor-related markers CK19 and CD24 and the liver marker ALB, and exhibited the characteristics of liver precursor cells.
  • the expression levels of hematopoietic stem cell antigen CD34 and leukocyte common antigen CD45 were less than 2%, showing low immunogenicity.
  • This example provides a modeling method for an inflammatory cell model, and co-cultures the first hepatic macrophage regulator (referred to as HepLPCs-CM) in Example 1 with an inflammatory cell model, and investigates the first hepatic macrophage regulator. Effects of phagocytic modulators on M1-type macrophages.
  • lipopolysaccharide LPS stimulates macrophages to establish an inflammatory cell model.
  • the specific process includes: Obtaining mouse primary bone marrow-derived macrophages (BMDMs), resuspending and inoculating in BMDM medium, and adding Mouse granulocyte-macrophage colony-stimulating factor GM-CSF was used to induce differentiation of BMDMs for 7 days until the cells matured.
  • the obtained mature primary macrophages were induced by LPS for 6 hours in vitro after directional polarization induction. Digest the macrophages to obtain the inflammatory cell model. Among them, the concentration of control LPS in the process of LPS stimulation was 100 ng/mL.
  • the purified cells were resuspended in BMDM medium and then inoculated into 12-well plates at (8-10) ⁇ 10 5 /well, and mouse GM-CSF was added to each well and the concentration was controlled at 40ng/mL. After 6-8 hours after inoculation, the cells were transferred to a new culture dish for culture, and the BMDM medium supplemented with 40 ng/mL mouse GM-CSF was replaced every 3 days until the 7th day of culture, to achieve a large amount of cells. Differentiation and maturation of phagocytes.
  • the BMDM medium consists of 500 mL of 1640 medium (from Shanghai Yuanpei Biotechnology Co., Ltd.), 5% penicillin-streptomycin double antibody and 10% FBS.
  • the medium was replaced with BMDM medium containing 100 ng/mL LPS and then cultured for 6 hours to complete the induction of directed polarization in vitro.
  • the expression level of IL6 was up-regulated to 823.200 ⁇ 174.500
  • the expression level of IL1 ⁇ was up-regulated to 8.389 ⁇ 0.029
  • the expression level of iNOS was up-regulated to 24.650 ⁇ 1.196.
  • HepLPCs-CM was co-cultured with the aforementioned BMDMs induced by LPS in vitro directional polarization for 6 hours to obtain the HepLPCs-CM+LPS group.
  • Gene expression analysis, and the comparison chart of M1-related inflammatory gene expression in each group shown in FIG. 70 was obtained.
  • the expression of M1-related genes was significantly down-regulated after co-culture of HepLPCs-CM BMDMs induced by directional polarization in vitro with LPS.
  • the expression level of IL6 was down-regulated to 346.300 ⁇ 20.810
  • the expression level of IL1 ⁇ was down-regulated to 11.290 ⁇ 0.10
  • the expression level of iNOS was down-regulated to 169.800 ⁇ 9.711.
  • the secretion of inflammatory factors in the supernatant of BMDMs induced by directional polarization in vitro with LPS was reduced after treatment with HepLPCs-CM.
  • the secretion of IL6 was 138.700 ⁇ 32.130pg/(mL*105cell)
  • the secretion of IL1 ⁇ was 0.710 ⁇ 0.019pg/(mL*105cell)
  • the secretion of iNOS was 0.095 ⁇ 0.001pg/(mL*105cell).
  • Macrophages can appear two classical cell subsets under the induction of different factors, namely M1 macrophages and M2 macrophages. The two functions are completely different. It is generally believed in the industry that M1 macrophages promote inflammatory responses by producing a large number of pro-inflammatory cytokines such as interleukin-1 ⁇ (IL-1 ⁇ ), TNF- ⁇ , interleukin-6 (interleukin-6, IL-6). ), as well as nitric oxide (NO) and reactive oxygen species (ROS) mediate the body's inflammatory response.
  • IL-1 ⁇ interleukin-1 ⁇
  • TNF- ⁇ interleukin-6
  • IL-6 interleukin-6
  • ROS reactive oxygen species
  • HepLPCs-CM can inhibit LPS-induced inflammatory activation of macrophages, and significantly reduce the gene and secreted protein expressions of inflammation-related factors.
  • This example provides a modeling method for a repair cell model, and co-cultures the first hepatic macrophage regulator (referred to as HepLPCs-CM) in Example 1 with a repair cell model, and investigates the first Effects of hepatic macrophage modulators on M2-type macrophages.
  • HepLPCs-CM first hepatic macrophage regulator
  • a repair cell model is established by stimulating macrophages with interleukin 4 (IL-4) derived from near-shore organisms.
  • the specific process includes: the acquisition method of mouse primary bone marrow-derived macrophages (BMDMs) and the use of mouse Please refer to Example 2 for the method of inducing differentiation of BMDMs by GM-CSF for 7 days.
  • the obtained mature primary macrophages were induced to polarize in vitro for 6 hours with IL-4, and then the macrophages were digested to obtain a repair cell model.
  • the concentration of IL-4 was controlled at 40 ng/mL during the stimulation of IL-4.
  • Example 3-2 For other specific experimental steps, please refer to Example 3-2.
  • HepLPCs-CM and the aforementioned BMDMs induced by IL-4 in vitro directional polarization were co-cultured in BMDM medium for 6 hours to obtain the HepLPCs-CM+IL-4 group.
  • RNA extraction and gene expression analysis were performed with the DM+IL-4 group, and the comparison chart of the secretion of M2-related inflammatory factor IL10 in each group shown in FIG. 74 was obtained.
  • BMDMs induced by IL-4 in vitro directed polarization were co-cultured with HepLPCs-CM, and the secretion of inflammatory factor IL10 in the supernatant was increased.
  • IL10 secretion was 108.052 ⁇ 0.472pg/(mL*105cell).
  • M2 macrophages mainly produce immunomodulatory factors such as interleukin-10 (interleukin-10, IL-10), participate in Th2 cell-type immune responses, inhibit inflammation and fibrosis, and play an important role in tissue repair. effect. It can be seen from Figure 73 and Figure 74 and the respective analysis results that HepLPCs-CM can promote IL-4-induced gene expression of repaired M2 macrophages and a small increase in the anti-inflammatory factor IL-10.
  • interleukin-10 interleukin-10
  • HepLPCs-CM can inhibit the inflammatory response and promote tissue repair by affecting the changes of macrophage subsets.
  • Example 3-1 the exosome component (abbreviated as HepLPCs-Ex) in the HepLPCs-CM of Example 3-1 was extracted, and its effect on the inflammatory cell model of Example 3-2 was investigated.
  • HepLPCs-Ex exosome component in the HepLPCs-CM of Example 3-1
  • the ExoQuick-TC exosome extraction kit was used to extract the exosome components in the HepLPCs-CM of Example 3-1 to obtain HepLPCs-Ex.
  • the extracted HepLPCs-Ex was entrusted to Xiaopeng Bio for NTA detection. The results showed that the diameter of the extracted sample particles was mostly between 90-110nm, and the peak value was 96nm.
  • the exosomal HepLPCs-Ex in HepLPCs-CM can inhibit the inflammatory activation of M1 macrophages.
  • This example examines the effect of the exosome component HepLPCs-Ex of Example 3-4 on the repair cell model of Example 3-3.
  • the exosomal HepLPCs-Ex in HepLPCs-CM can promote the generation of repairing macrophages.
  • This example provides a modeling method for the NASH mouse model.
  • mice This example uses healthy male 5-week-old wild-type (WT) C57BL/6 mice purchased from Shanghai Lingchang Biotechnology Co., Ltd. for modeling. The weight of the mice reached an average of 20 g. There are 7 mice in each group, and the grouping is as follows:
  • Model group feed CDAHFD high-fat diet at a feeding amount of 2.5-3g/bird, 2-3 times a week.
  • Control group The feeding amount of 2.5-3g/bird was fed with standard diet.
  • mice in each group were reared in a specific pathogen-free environment (SPF grade) rearing environment, controlled at room temperature of 20-26 °C, humidity of 40-70%, and alternating light and dark for 12 hours. No more than 5 animals.
  • SPF grade pathogen-free environment
  • mice in the model group were reared for 3 weeks to obtain an early NASH mouse model; after 6 weeks of rearing, a middle-late NASH mouse model was obtained.
  • liver tissues of the early NASH mouse model and the control group were used for paraffin embedding, section preparation and H&E staining to obtain the H&E staining photos shown in Figure 78 and Figure 79 respectively.
  • hepatocytes appeared balloon-like degeneration, showing obvious diffuse fatty degeneration.
  • the liver tissue of the early NASH mouse model was paraffin-embedded and sliced, and then the immunohistochemical staining of the M1 macrophage specific marker CD68 and the M2 macrophage specific marker CD163 were performed respectively.
  • the immunohistochemical staining of , and the staining photos shown in Figure 82 and Figure 83 were obtained, respectively. It can be seen that the mouse liver tissue of the early NASH mouse model was accompanied by inflammatory cell infiltration.
  • orbital blood was used to detect blood samples related to liver function indicators AST, ALT, LDH, and to detect the content of total cholesterol TC and hepatic triglyceride TG in isolated liver tissue, and to evaluate the liver function of early NASH mouse model and control group mice.
  • the HepLPCs of Example 3-1 were used to intervene in the early NASH mouse model, and the effect of HepLPCs on NASH disease was investigated.
  • the early NASH mouse models were grouped as follows, with 7 mice in each group:
  • Model group Oral saline.
  • Immunosuppression group oral immunosuppressant FK506 at a dose of 0.2 mg/kg;
  • Cell low-dose treatment group 0.2 mg/kg of immunosuppressant was orally administered one day before surgery; intraperitoneal injection of 200 microliters of cell injection solution was performed on each animal during surgery, and 0.5 ⁇ 10 6 HepLPCs were resuspended in normal saline to obtain cell injection solution;
  • Cell high-dose treatment group The difference from cell low-dose treatment group is that each injected cell injection contains 1 ⁇ 10 6 HepLPCs;
  • Sham operation group 200 microliters of normal saline was injected into each spleen;
  • mice in the above groups were killed on the 10th day after the injection or oral administration, and the liver tissue was taken for paraffin embedding and sectioning, followed by H&E staining and Oil Red O staining, and the liver pathological changes and lipid deposition were observed, and the results shown in Figure 85 were obtained.
  • FIG. 86 compared with the sham-operated group, the cell low-dose treatment group and the cell high-dose treatment group showed that the lipid deposition in the liver was significantly reduced.
  • the NAFLD activity score was used to analyze the pathological changes under the liver microscope, and the NAS score comparison chart of the sham operation group, the cell low-dose treatment group and the cell high-dose treatment group shown in FIG. 86 was obtained.
  • the NAS score of the sham operation group was between 4 and 6 points, which met the pathological evaluation criteria of NASH, while each cell treatment group was lower than 2 points, indicating that NASH could be excluded. showed that HepLPCs played a positive intervention role in early NASH mouse model.
  • the normal control group NC the immunosuppression group CDA+FK506, the oral normal saline model group CDA+saline, the sham operation group CDA+sham, the cell low dose treatment group CDA+HepLPCs (low dose) and the cell high dose treatment Orbital blood was collected from the CDA+HepLPCs (high dose) mice in the group, and the ALT, AST, and LDH indexes of each group were examined through the analysis of blood biochemical levels. Compared with the sham operation group, the three indexes of ALT, AST and LDH in the two cell treatment groups decreased to a certain extent.
  • the treatment window of liver function index ALT in the low-dose cell treatment group decreased by 16%; the treatment window of liver function index ALT in the high-dose cell treatment group decreased by 31.5%; compared with the sham operation group, the treatment window of liver function index AST in the low-dose cell treatment group decreased by 15% %; the treatment window of liver function index AST in the high-dose cell treatment group decreased by 22%; compared with the sham operation group, the treatment window of liver function index LDH in the low-dose cell treatment group decreased by 13.9%; the treatment window of liver function index LDH in the high-dose cell treatment group down 17%.
  • HepLPCs can improve liver function in an early NASH mouse model.
  • the liver hepatic sterol TC content in the high-dose cell treatment group decreased by 23.4%; compared with the sham-operation group, the liver triglyceride TG content in the cell low-dose treatment group decreased by 17.6%; There was no significant difference in triglyceride TG; from the detection of TC and TG, the contents of TC and TG in the sham operation group were higher than those in the normal group.
  • HepLPCs were shown to significantly improve NASH hepatic lipid metabolism.
  • Example 3 the HepLPCs of Example 3-1 were used to intervene in the middle-late NASH mouse model, and the effect of HepLPCs on NASH disease was investigated.
  • Example 3 the grouping treatment of the NASH mouse model in the middle and late stages. The difference is: the cell injection infused into the spleen of each mouse in the low-dose treatment group contains 1 ⁇ 10 6 cells, and the cell injection in the high-dose treatment group contains 1 ⁇ 10 6 cells.
  • the mouse spleen infused cell injection contained 2 x 10< 6 > cells.
  • This example provides a modeling method for an inflammatory cell model.
  • Macrophages can appear two classical cell subsets under the induction of different factors, namely M1 type macrophages and M2 type macrophages. The two functions are completely different. It is generally believed in the industry that M1 macrophages promote inflammatory responses by producing a large number of pro-inflammatory cytokines such as interleukin-1 ⁇ (IL-1 ⁇ ), TNF- ⁇ , interleukin-6 (interleukin-6, IL-6). ), as well as nitric oxide (NO) and reactive oxygen species (ROS) mediate the body's inflammatory response.
  • IL-1 ⁇ interleukin-1 ⁇
  • TNF- ⁇ interleukin-6
  • IL-6 interleukin-6
  • ROS reactive oxygen species
  • the purified cells were resuspended in BMDM medium and then inoculated into 12-well plates at (8-10) ⁇ 10 5 /well, and mouse GM-CSF was added to each well and the concentration was controlled at 40ng/mL. After 6-8 hours after inoculation, the cells were transferred to a new culture dish for culture, and the BMDM medium supplemented with 40 ng/mL mouse GM-CSF was replaced every 3 days until the 7th day of culture, to achieve a large amount of cells. Differentiation and maturation of phagocytes.
  • the BMDM medium consists of 500 mL of 1640 medium (from Shanghai Yuanpei Biotechnology Co., Ltd.), 5% penicillin-streptomycin double antibody and 10% FBS.
  • the medium was replaced with BMDM medium containing 100 ng/mL LPS and then cultured for 6 hours to complete the induction of directed polarization in vitro.
  • the expression level of IL6 was up-regulated to 823.200 ⁇ 174.500
  • the expression level of IL1 ⁇ was up-regulated to 8.389 ⁇ 0.029
  • the expression level of iNOS was up-regulated to 24.650 ⁇ 1.196.
  • This example provides a modeling method for a repaired macrophage model.
  • M2 macrophages mainly produce immunomodulatory factors such as interleukin-10 (IL-10), participate in Th2 cell-type immune responses, inhibit inflammation and fibrosis, and play an important role in tissue repair. effect.
  • IL-10 interleukin-10
  • a repairing macrophage model is established by stimulating macrophages with IL-4.
  • the specific process includes: the acquisition method of mouse primary bone marrow-derived macrophages (BMDMs) and the use of mouse GM-CSF to conduct a 7-year study on BMDMs.
  • BMDMs mouse primary bone marrow-derived macrophages
  • GM-CSF mouse GM-CSF
  • Example 3-2 for the method of inducing differentiation for one day.
  • the obtained mature primary macrophages were induced to polarize in vitro for 6 hours with IL-4, and then the macrophages were digested to obtain a repaired macrophage model.
  • the concentration of IL-4 was controlled at 40 ng/mL during the stimulation of IL-4.
  • Example 5 For other specific experimental steps, please refer to Example 5.
  • the HepLPCs of Example 3-1 were cultured in vitro, and exosome components (abbreviated as HepLPCs-Ex) were extracted from the obtained in vitro culture supernatant, and its effect on the inflammatory cell model of Example 3-5 was investigated. effect.
  • the in vitro culture of HepLPCs of Example 3-1 and the process of obtaining the in vitro culture supernatant include: after the subculture of Example 1 is completed, the TEM medium is replaced with serum-free high-glucose DMEM medium. , continued in vitro culture for 24 hours; after the in vitro culture was completed, the cell supernatant was collected, and the cell supernatant was centrifuged at 300 g for 10 minutes to remove cell debris to obtain the in vitro culture supernatant.
  • the ExoQuick-TC exosome extraction kit was used to extract the exosome components in HepLPCs-CM to obtain HepLPCs-Ex.
  • the extracted HepLPCs-Ex were commissioned to Xiaopeng Bio for NTA detection. The results showed that the diameter of the extracted sample particles was mostly between 90-110nm, and the peak value was 96nm.
  • the number of BMDMs cells was controlled to be 5 ⁇ 10 5 , and the exosome concentration and HepLPCs-CM concentration were both 1.3ug/uL.
  • HepLPCs-CM and HepLPCs-Ex were added to the inflammatory cell model and mixed for 6 hours to form CM+LPS group and EV+LPS group.
  • Control group, DM+LPS group, CM+LPS group and EV+LPS group were examined by qPCR.
  • the expression of M1-related inflammatory genes in each group is obtained, and the comparison chart of M1-related inflammatory gene levels in each group shown in FIG. 102 is obtained.
  • exosomal HepLPCs-Ex released from HepLPCs induced by serum-free basal medium can inhibit the inflammatory activation of M1 macrophages.
  • This example examines the effect of the exosome component HepLPCs-Ex of Examples 3-10 on the aforementioned repaired macrophage model.
  • the number of BMDMs cells was controlled to be 5 ⁇ 10 5 , and the exosome concentration and HepLPCs-CM concentration were both 1.3ug/uL.
  • HepLPCs-CM and HepLPCs-Ex were added to the repaired macrophage model of Example 6, respectively, and cultured for 6 hours to form a CM+IL4 group and an EV+IL4 group.
  • the Control group, DM+IL4 group, CM group were investigated by qPCR.
  • the expression of M2-related inflammatory genes in the +IL4 group and the EV+IL4 group, the comparison chart of M2-related inflammatory gene levels in each group shown in Figure 103 was obtained.
  • the exosomal HepLPCs-Ex released by HepLPCs under the induction of serum-free basal medium can promote the generation of repairing macrophages, namely M2 macrophages.
  • liver disease regulating preparations prepared immune cell proliferation inhibitor and proliferation inhibitory cell preparation as liver disease regulating preparations respectively, and investigated the application of such liver disease regulating preparations. details as follows:
  • HepLPC-CM conditioned culture supernatant
  • the in vitro culture method of HepLPC includes: firstly inoculate human primary hepatocytes at a density of 2 ⁇ 10 4 cells/cm 2 after removing CD24-positive and EpCAM-positive precursor cells by Percoll density gradient centrifugation combined with flow sorting Cells were cultured in Matrigel (Corning)-coated 6-well plates in WE medium (Invitrogen) containing 10% serum until adherent, and then transferred to TEM medium at a seeding density of 2 ⁇ 10 4 /cm 2 Cultures were carried out for 10 days, and the TEM medium was changed every other day.
  • composition of the TEM medium in this example and the sources of the components are as follows: DMEM/F12 basal medium (Invitrogen), and based on the content of the DMEM/F12 basal medium: 1% by volume N2 additive and volume content 1% B27 additive (Invitrogen company), 1mmol/L sodium pyruvate (Invitrogen company), 10 ⁇ g/mL ascorbic acid (Sigma-Aldrich company), 20ng/mL hepatocyte growth factor HGF (Peprotech company), 20ng/mL epidermal cells Growth factor EGF (Peprotech Company), 10 ⁇ mol/L ROCK kinase inhibitor Y27632 (TargetMol Company), 3 ⁇ mol/L Wnt signaling pathway agonist CHIR99021 (TargetMol Company), 1 ⁇ mol/L TGF- ⁇ signaling inhibitor A83-01 (TargetMol Company) ), 1 ⁇ mol/L sphingosine monophosphate S1P (Santa Cruz
  • the preparation method of HepLPC-CM includes: digesting the precursor-like cells obtained after 10 days of TEM culture with 0.25% Trypsin-EDTA (from Gibco, USA), and inoculating them into new culture at a ratio of 1:3
  • the cells were expanded and cultured in the dish to the third passage until the cell confluence rate reached 80%, and then the TEM medium was replaced with serum-free DMEM medium for 24 hours of in vitro culture; after the in vitro culture, the cell supernatant was collected, and the described The cell supernatant was centrifuged at 300 g for 10 minutes to remove cell debris to obtain the conditioned culture supernatant HepLPC-CM.
  • mice are sacrificed by cervical dislocation, the spleen is taken out and ground with a 200-mesh sieve, and then washed with lymphatic separation liquid, and the separation liquid containing lymphocytes is collected. and resuspended in RPMI-1640 complete medium containing 10% fetal bovine serum and centrifuged at 800g for 30 minutes at low temperature; after centrifugation, the lymphocyte layer was aspirated and resuspended in RPMI-1640 complete medium containing 10% fetal bovine serum.
  • the co-cultivation steps include: seeding the aforementioned mouse spleen cell suspension with stimulators at a density of 2 ⁇ 10 4 cells/cm 2 in 6-well plates; using HepLPC-CM and using RPMI-10% fetal bovine serum.
  • the HepLPC-CM from Donor 1 was resuspended in 1640 complete medium to obtain HepLPC-CM suspensions with HepLPC-CM concentrations of 100%, 50%, 25%, 12.5% and 6.25%, respectively.
  • Human-HepLPCs In Human-HepLPCs, the hepatocyte marker HNF4 ⁇ and the hepatic stem/liver progenitor cell markers CD24 and CK19 were significantly expressed, and the expression levels of hematopoietic stem cell antigen CD34, leukocyte common antigen CD45 and liver fetal cell marker AFP were all less than 2%. Human-HepLPCs do not express the MHC class II antigens HLA-DP, HLA-DQ and HLA-DR, showing low immunogenicity.
  • human primary hepatocytes from different donor sources from Invitrogen were cultured in vitro as liver precursor-like cells HepLPC, and the conditioned culture supernatants from different donor sources were obtained, and then the conditioned culture supernatants from different donor sources were cultured in vitro.
  • the supernatant was co-cultured with ConA-stimulated mouse spleen cells, and the inhibitory effect of HepLPC-CM on the proliferation of spleen cells was investigated.
  • Example 4-1 for the in vitro culture method of human primary hepatocytes, the preparation method of the conditioned culture supernatant, and the preparation method of the mouse spleen cell suspension added with stimulators.
  • the difference between the co-cultivation step and Example 1 is that each donor-derived HepLPC is not resuspended in RPMI-1640 complete medium containing 10% fetal bovine serum, but is directly co-cultured with mouse spleen cells added with stimulators .
  • HepLPC human primary hepatocytes from donor 1 from Invitrogen were cultured in vitro into HepLPC, which were hepatic precursor-like cells, and then HepLPC was co-cultured with ConA-stimulated mouse spleen cells to investigate the effect of HepLPC-CM on spleen cells. proliferation inhibitory effect.
  • the composition of the TEM medium and its components, and the preparation method of the mouse spleen cell suspension added with stimulators please refer to the foregoing examples.
  • the co-cultivation step includes: after the aforementioned TEM culture is completed, the cell fusion rate reaches 90%, and then the obtained cells are continuously subcultured in the TEM medium at a ratio of 1:3 at a seeding density of 1 ⁇ 10 4 cells/square centimeter.
  • the third passage using 0.05% trypsin/EDTA solution to digest the HepLPC subcultured to the third passage and the cell confluence rate reached 80%, the digestion was terminated with RPMI-1640 complete medium containing 10% fetal bovine serum and resuspended as Single cell suspension; repeated washing of single cell suspension with PBS to ensure removal of digestion solution; resuspend and adjust cell concentration in complete RPMI-1640 medium containing 10% fetal bovine serum; mouse spleen cells previously added with stimulant
  • the suspension was inoculated in a 6-well plate, and the number ratio of HepLPC and mouse spleen cells was adjusted to 1:1, 1:2, 1:5, and 1:10 by controlling the amount of mouse spleen cell suspension added with stimulators.
  • the co-culture group without HepLPC was used as the positive control group
  • the co-culture group with the addition of the immunosuppressive drug tacrolimus FK506 at a final concentration of 10 nM was used as the FK506 control group.
  • qPCR and flow cytometry were used to analyze the gene expression of primary human hepatocytes of donor 1 and HepLPC cultured by TEM. Please refer to the foregoing examples.
  • the positive control group, the FK506 control group and each co-cultivation group were treated with a CFSE cell proliferation detection kit and detected by flow cytometry.
  • the results of inhibition of cell proliferation are shown in FIG. 111 .
  • the ratios of HepLPC and mouse spleen cells are 1:1, 1:2, 1:5, 1, respectively.
  • the inhibition rates of HepLPC on spleen cell proliferation were 90%, 87%, 85%, 80% and 50%, respectively.
  • human primary hepatocytes from different donor sources from Invitrogen were cultured in vitro as liver precursor-like cells HepLPC, and HepLPC from different donor sources were co-cultured with ConA-stimulated mouse spleen cells to investigate HepLPC. Proliferation inhibitory effect on spleen cells.
  • Example 4-1 for the in vitro culture method of human primary hepatocytes, the preparation method of the conditioned culture supernatant, the preparation method of the mouse spleen cell suspension added with stimulators, and the co-cultivation method.
  • the number ratio of each donor-derived HepLPC to mouse spleen cells was controlled to be 1:5, respectively.
  • the four donor-derived HepLPCs obtained in Example 2 were used to co-culture with human peripheral blood mononuclear cells (PBMCs) respectively, and the phytohemagglutinin PHA was used to stimulate the proliferation of T cells in PBMCs to investigate the effect of HepLPCs on PBMCs. Proliferation inhibition.
  • the peripheral blood of healthy males was diluted with an appropriate amount of D-PBS and Histopaque-1077, then centrifuged at 2000 rpm for 25 minutes, and the buffy coat layer in the middle was added to D-PBS for repeated centrifugation and washing, and the supernatant was discarded to obtain cell pellets;
  • the cell pellet was resuspended in RPMI-1640 medium of ⁇ g/ml PHA, 10% PBS, 2mM GlutaMAX to obtain a PBMC suspension.
  • the co-cultivation process of this example is as follows: take the HepLPC obtained after subculture and digestion in Example 4-1, and resuspend it into a single-cell suspension with PBS; use PBS to repeatedly wash the single-cell suspension to ensure that the digestion solution is removed; Use RPMI-1640 complete medium containing 10% fetal bovine serum to resuspend and adjust the cell concentration; inoculate HepLPC resuspended in RPMI-1640 complete medium containing 10% fetal bovine serum in a 6-well plate, add PBMC suspension, By controlling the amount of PBMC suspension and HepLPC suspension and the concentration of each suspension, the number ratio of HepLPC and PBMC was 1:5, and the final concentration of PHA was 5 ⁇ g/ml. The PBMC culture group without HepLPC suspension was used as the positive control group.
  • HepLPC of Example 1 was used for cell transplantation, and the effect of HepLPC on ConA-induced, T cell and NKT cell-mediated mouse autoimmune hepatitis model was investigated.
  • mice in the 8 mg/kg injection dose group were collected blood from the orbits at different times, and the ALT, AST and LDH indexes of each group were investigated through the analysis of blood biochemical levels, and the ALT, AST, and LDH indexes shown in Figure 118 were obtained. Horizontal comparison chart. Referring to Figure 118, for non-lethal dose ConA-treated mice, blood ALT, AST, LDH were highest at 6 h after injection. Therefore, mice injected at the 8 mg/kg tail vein were successfully modeled at 6 hours after the end of the injection. The 8 mice in the control group NC of this example were injected with the same dose of PBS through the tail vein.
  • HepLPC obtained after subculture and digestion in Example 4-1 was used, and after repeated resuspending and washing with PBS to ensure the purification effect, HepLPC was resuspended in PBS and grouped as follows:
  • Experimental group 1 8 C57BL/6 male mice were injected with PBS-resuspended HepLPC suspension and PBS-resuspended ConA into 8 C57BL/ 6 male mice, respectively.
  • the injection dose of control HepLPC was 106/mice
  • the injection dose of ConA was 8 mg/mice. non-lethal dose in kilograms.
  • Experimental group 2 The difference from experimental group 1 is that the injected dose of ConA is a lethal dose of 20 mg/kg.
  • Control group each C57BL/6 male mouse was injected with the same volume of PBS through the tail vein.
  • mice in each group were still alive 6 hours after the injection.

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Abstract

提供一种肝病调控制剂，以有利于通过重塑肝脏再生微环境阻止慢性肝病发生发展。

Description

肝病调控制剂及其应用
本申请要求2021年03月08日提交的申请号为2021102495437的中国专利申请的优先权。上述申请的内容以引用方式被包含于此。
技术领域
本发明涉及生物技术领域，尤其涉及抗肝纤维化制剂及其制备方法和应用。
背景技术
寻找有效方法促进肝再生，增加有效肝体积和肝储备，进而改善患者肝功能状况，最大限度减少肝硬化等肝病相关严重并发症的发生率并为后续有效治疗创造条件，成为亟待解决的临床问题。肝脏再生微环境的有序调控是维持肝脏生理功能的重要机制，重塑肝脏再生微环境对于阻止慢性肝病发生发展具有重要意义。
因此，有必要开发新型的肝病调控制剂以解决现有技术中存在的上问题。
发明内容
本发明的目的在于提供一种肝病调控制剂，以有利于通过重塑肝脏再生微环境阻止慢性肝病发生发展。
本发明的肝病调控制剂包含肝脏来源前体细胞或所述肝脏来源前体细胞的分泌上清。
一些实施例中，所述肝脏来源前体细胞为肝前体细胞。
一些实施例中，所述肝脏来源前体细胞为肝前体样细胞。
一些实施例中，所述肝脏来源前体细胞为人肝来源前体细胞。
一些实施例中，所述人肝来源前体细胞为人肝前体细胞。
一些实施例中，所述人肝来源前体细胞为人肝前体样细胞。
一些实施例中，所述肝脏来源前体细胞的分泌上清包括至少一种miRNA，所述至少一种miRNA为miRNA-182、miRNA-183和miRNA-574的至少一种，能够有效促进肝细胞增殖。
一些实施例中，所述肝脏来源前体细胞的分泌上清包含作用于JAK-STAT通路的起效成分，以抑制肝星状细胞活化或诱导所述肝星状细胞死亡。
一些实施例中，所述肝脏来源前体细胞的分泌上清包含白血病抑制因子、内皮素、集落刺激因子、双调蛋白和成纤维细胞生长因子的至少一种，以起到抑制肝星状细胞活化或诱导所述肝星状细胞死亡的作用。
一些实施例中，所述成纤维生长因子为FGF19。
一些实施例中，所述肝脏来源前体细胞的分泌上清包含能够通过抑制所述免疫细胞的增殖来诱导受体建立有效免疫耐受的分泌成分。
一些实施例中，所述免疫细胞为巨噬细胞、B细胞、T细胞、NK细胞和NKT细胞的任意一种。
一些实施例中，所述肝病调控制剂还包括重悬成分，所述重悬成分包括生理盐水、复方电解质溶液、缓冲溶液和基础培养基的至少一种。
一些实施例中，所述肝病调控制剂还包括辅助成分，所述辅助成分包括免疫抑制成分、血清、抗生素和协同起效成分的至少一种。
本发明实施例的所述肝脏来源前体细胞经体外培养基的培养得到所述肝脏来源前体细胞的分泌上清。
一些实施例中，所述体外培养基为基础培养基，所述基础培养基为HepX培 养基、DMEM/F12细胞培养基、William’s E细胞培养基、Neurobasal Medium细胞培养基、MEM细胞培养基、DMEM细胞培养基、1640RPMI细胞培养基和F12细胞培养基的至少一种。
一些实施例中，所述体外培养基包括所述基础培养基和血清类物质。
一些实施例中，所述体外培养基由所述基础培养基和血清类物质组成。其中，以占所述基础培养基的体积含量计，血清类物质的含量不超过20％。
一些实施例中，所述体外培养基还添加有双抗，所述双抗的含量不超过2％。
一些实施例中，所述体外培养基还包括N2、B27、生长因子、ROCK激酶抑制剂、Wnt信号通路激动剂、TGF-β信号抑制剂、N-乙酰-L-半胱氨酸和抗坏血酸的至少一种。
一些实施例中，所述肝脏来源前体细胞经肝细胞扩增转化培养基(TEM培养基)对原代肝细胞进行体外培养得到。
一些实施例中，TEM培养基包括基础培养基、无血清添加物、血清类物质、生长因子、TGF-β信号抑制剂、Wnt信号通路激动剂和ROCK激酶抑制剂。
以占所述基础培养基的含量计，所述生长因子的含量为0.1-100纳克/毫升，所述ROCK激酶抑制剂的含量为0.1-100微摩尔，所述Wnt信号通路激动剂的含量为0.1-50微摩尔，所述TGF-β信号抑制剂的含量为0.1-100微摩尔，所述血清类物质的含量不超过20％，所述无血清添加物的体积含量不超过2％。
一些实施例中，TEM培养基还包括N-乙酰-L-半胱氨酸、抗坏血酸的至少一种。
一些实施例中，所述生长因子为表皮生长因子、成纤维细胞生长因子2、血管内皮生长因子、血小板衍生生长因子、肝细胞生长因子、白介素-6和抑瘤素的至少一种。
一些实施例中，所述ROCK激酶抑制剂为Fasudil、Y-27632、Thiazovivin和SB-772077-B的至少一种。
一些实施例中，所述Wnt信号通路激动剂为重组Wnt蛋白、重组R-spondin蛋白和糖原合成酶激酶3β抑制剂的至少一种。
一些实施例中，所述TGF-β信号抑制剂为RepSox、SB431542和A83-01的至少一种。
一些实施例中，所述体外培养基和所述TEM培养基中任意一种的血清类物质为动物源血清。
一些实施例中，所述体外培养基和所述TEM培养基中任意一种中的动物源血清可以用血清替代物替换。
一些实施例中，所述血清替代物为无动物源成份的血小板及其衍生物。
一些实施例中，所述血清替代物为一磷酸鞘氨酸和吲哚乙酸。
一些实施例中，所述动物源血清为胎牛血清。
一些实施例的TEM培养基中，以占基础培养基的含量计，所述丙酮酸钠的含量为0.5-1.5毫摩尔/升，所述抗坏血酸的含量为5-50微克/毫升，所述表皮细胞生长因子的含量为5-25纳克/毫升，所述肝细胞生长因子的含量为5-25纳克/毫升，所述ROCK激酶抑制剂的含量为5-20微摩尔/升，所述Wnt信号通路激动剂的含量为1-5微摩尔/升，所述TGF-β信号抑制剂的含量为0.5-2微摩尔/升，所述一磷酸鞘氨酸的含量为0.5-2微摩尔/升，所述吲哚乙酸的含量为2-10微摩尔/升，所述N2添加剂和所述B27添加剂的体积百分比不超过1％，N-乙酰-L-半胱氨酸的含量为0.5-10微摩尔/升。
本发明实施例提供了所述肝病调控制剂的体外应用，包括将所述肝病调控制剂与目标细胞共培养。所述目标细胞为原代肝细胞、肝星状细胞、巨噬细胞和 免疫相关细胞的任意一种。
一些实施例中，将所述肝病调控制剂与目标细胞共培养的步骤包括，使用共培养基对所述肝病调控制剂与所述肝星状细胞进行共培养，以占所述共培养基的体积百分比计，所述肝病调控制剂的含量不低于1％。
一些实施例的共培养过程中，所述目标细胞的铺板密度为1×10 4个/平方厘米。
一些实施例中，所述共培养基由基础培养基和所述血清类物质组成。
一些实施例中，所述共培养基中，以占所述基础培养基的体积百分比计，所述血清类物质的含量不超过20％。
一些实施例中，所述共培养基还含有肝星状细胞活化剂。
一些实施例中，所述肝星状细胞活化剂为肝星状细胞活化因子。
一些实施例中，所述肝星状细胞活化因子为TGF-β1。
一些实施例中，将所述肝病调控制剂与目标细胞共培养的步骤包括，将所述肝病调控制剂与肝巨噬细胞模型进行共培养，所述肝巨噬细胞模型为炎症细胞模型或修复型细胞模型。
一些实施例中，将所述肝病调控制剂与目标细胞共培养的步骤包括，将所述肝病调控制剂与所述免疫相关细胞共培养，并使用刺激剂诱导所述免疫相关细胞的增殖。
一些实施例中，所述免疫相关细胞为外周血单个核细胞和脾脏细胞的任意一种。
一些实施例中，将所述肝病调控制剂与所述免疫相关细胞共培养的步骤包括，使用共培养基对所述肝病调控制剂进行重悬，并控制所述肝病调控制剂占所述共培养基的体积浓度不低于5％，以使所述肝病调控制剂对所述免疫相关细胞的 增殖抑制率不低于30％。
一些实施例中，将所述肝病调控制剂与所述免疫相关细胞共培养的步骤包括，使用不同肝病调控制剂与所述免疫相关细胞共培养，所述不同肝病调控制剂中所含有的肝脏来源前体细胞的培养上清来源于不同供体。
本发明实施例的所述肝病调控制剂在肝病治疗方面的应用包括，将所述肝病调控制剂干预体内动物模型后考察对肝脏再生的影响。
一些实施例中，所述动物体内模型为四氯化碳诱导的小鼠急性肝衰竭模型、乙酰氨基酚诱导的小鼠急性肝衰竭模型、硫代乙酰胺诱导的哺乳动物肝硬化模型、四氯化碳诱导的哺乳动物肝硬化模型、哺乳动物非酒精性脂肪性肝炎模型、ConA诱导且T细胞和NKT细胞介导的小鼠自身免疫性肝炎模型、肝细胞或肝组织移植后的免疫排斥大鼠模型以及肝移植后急性宿主抗移植物反应模型中的任意一种。
附图说明
图1为实施例1-1的PHH Exo样本的透射电镜照片；
图2为实施例1-1的Hep Exo样本的透射电镜照片；
图3为实施例1-1的PHH Exo样本和Hep Exo样本中外泌体平均粒径对比图；
图4为实施例1-1通过流式分析检测不同细胞来源外泌体的CD63和CD81表达情况对比图；
图5为实施例1-1通过蛋白质印迹测试考察的不同细胞以及不同细胞来源外泌体的CD63和TSG101的表达情况照片；
图6为实施例1-2的PHH Exo样本和Hep Exo样本在标记后、染色后以及共培养后的免疫荧光共聚焦检测结果照片；
图7为实施例1-3的不同外泌体浓度的PHH Exo样本和Hep Exo样本共培养后得到的细胞的BrdU掺入作用对比图；
图8为实施例1-3对PHH Exo样本和Hep Exo样本分别与PHHs进行共培养后得到的细胞进行EdU荧光染色后得到的免疫荧光照片；
图9为实施例1-3对PHH Exo样本和Hep Exo样本分别与PHHs进行共培养后得到的细胞进行Ki67免疫荧光染色得到的免疫荧光照片；
图10为实施例1-4对PHH Exo-cell和Hep Exo-cell进行流式细胞周期分析得到的流式分析结果对比图；
图11为实施例1-4通过对对照组、PHH Exo-cell和Hep Exo-cell进行实时荧光定量PCR分析得到的细胞周期相关分子miRNA表达水平对比图；
图12为实施例1-4使用蛋白质印迹测试考察对照组、PHH Exo-cell和Hep Exo-cell表达细胞周期相关分子得到的印记照片对比图；
图13为实施例1-5的不同细胞来源外泌体miRNA测序表达聚类图；
图14为实施例1-5的原代肝细胞体外转染hsa-miR-182，hsa-miR-183，hsa-miR-149，hsa-miR-215，hsa-miR-574，hsa-miR-654和hsa-miR-675的48小时后使用ELISA检测各转染细胞的BrdU掺入作用对比图；
图15为实施例1-5的原代肝细胞分别转染hsa-miR-182，hsa-miR-183和hsa-miR-574的48小时后分别进行EdU染色后得到的EdU荧光显微照片；
图16为实施例1-5的原代肝细胞分别转染hsa-miR-182，hsa-miR-183和hsa-miR-574的48小时后，使用EdU荧光法检测得到的各转染细胞EdU掺入率对比情况。
图17为实施例1-6的Hep Exo治疗组和PBS对照组各小鼠造模完毕后生存率随 时间的变化情况；
图18为实施例1-6的正常对照组、PBS对照组和Hep Exo治疗组小鼠造模后24小时和48小时的肝组织切片HE染色照片；
图19为实施例1-6的PBS对照组和Hep Exo治疗组小鼠肝组织切片免疫组化Ki67染色对比图；
图20为实施例1-6的正常组、PBS对照组和Hep Exo治疗组各小鼠肝脏组织石蜡切片中Ki67阳性细胞百分比对比图；
图21为实施例1-6的正常组、PBS对照组和Hep Exo治疗组各小鼠在不同时间的ALT水平对比结果；
图22为实施例1-6的正常组、PBS对照组和Hep Exo治疗组各小鼠在不同时间的AST水平对比结果；
图23为实施例1-7的CCl4模型各组小鼠的生存率随时间的变化情况；
图24为实施例1-7的APAP模型各组小鼠的生存率随时间的变化情况；
图25为实施例1-8所示的CCl4模型各小鼠造模后不同时间的AST水平对比图；
图26为实施例1-8所示的CCl4模型各小鼠造模后不同时间的ALT水平对比图；
图27为实施例1-8所示的APAP模型各小鼠造模后24小时的AST水平和ALT水平对比图；
[根据细则91更正 30.03.2022]　
[根据细则91更正 30.03.2022]　
图28为实施例1-8的CCl4模型各小鼠造模后不同时间的病理组织切片对比图；
[根据细则91更正 30.03.2022]　
图29为实施例1-8的APAP模型各小鼠造模后24小时的病理组织切片对比图；
[根据细则91更正 30.03.2022]　
图30为实施例1-8的CCl4-NC agomir组小鼠和CCl4-miRNA 183-5p agomir组小鼠造模完成后不同时间肝组织切片免疫组化Ki67染色对比图；
[根据细则91更正 30.03.2022]　
图31为实施例1-8的APAP-NC agomir组和APAP-miRNA 183-5p agomir组小鼠造模完成后24小时的肝组织切片免疫组化Ki67染色对比图；
[根据细则91更正 30.03.2022]　
图32为实施例1-8的造模完成后不同时间CCl4-NC agomir组小鼠和CCl4-miRNA 183-5p agomir组小鼠肝组织切片的Ki67阳性细胞百分比对比情况示意图；
[根据细则91更正 30.03.2022]　
图33为实施例1-8的CCl4-miRNA 183-5p agomir组小鼠在造模后不同时间的肝组织细胞周期相关蛋白表达情况对比图。
[根据细则91更正 30.03.2022]　
图34为实施例2-1的人原代肝细胞的基因表达情况示意图；
[根据细则91更正 30.03.2022]　
图35为实施例2-1的人肝前体样细胞的基因表达情况示意图；
[根据细则91更正 30.03.2022]　
图36为实施例2-2的实验组、控制组和对照组中各肝星状细胞的微观形貌对比照片；
[根据细则91更正 30.03.2022]　
图37为实施例2-2的实验组、控制组和对照组中与HSCs活化相关基因的相对mRNA表达情况对比图；
[根据细则91更正 30.03.2022]　
图38为实施例2-2的实验组细胞聚集体在透射显微镜观察下的微观形貌照片；
[根据细则91更正 30.03.2022]　
图39为针对实施例2-2的对照组细胞聚集体进行流式细胞术测定得到的分析结果；
[根据细则91更正 30.03.2022]　
图40为针对实施例2-2的实验组细胞聚集体进行流式细胞术测定得到的分析结果；
[根据细则91更正 30.03.2022]　
图41为针对实施例2-2的控制组、对照组和实验组的细胞聚集体进行蛋白质印记分析得到的各组细胞的纤维化相关蛋白表达和关键纤维化信号的表达情况对 比图；
[根据细则91更正 30.03.2022]　
图42为实施例2-3的鼠肝原代细胞和鼠肝前体样细胞的肝祖细胞基因和肝实质细胞标志物的表达情况对比图；
[根据细则91更正 30.03.2022]　
图43为实施例2-4的控制组、对照组和实验组共培养48小时后，各组HSCs-T6细胞的微观形貌照片对比图；
[根据细则91更正 30.03.2022]　
图44为实施例2-4的控制组、对照组和实验组的HSCs-T6中与HSCs活化相关基因的相对mRNA表达情况对比图；
[根据细则91更正 30.03.2022]　
图45为实施例2-4的实验组中细胞微观形貌照片；
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图46为针对实施例2-4的对照组的细胞进行流式细胞术测定得到的分析结果；
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图47为针对实施例2-4的实验组的细胞进行流式细胞术测定得到的分析结果；
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图48为实施例2-5的第一种抗肝纤维化制剂中，JAK-STAT通路与第一种抗肝纤维化制剂中参与生长因子活性、细胞因子活性和受体-配体活性的蛋白质之间的可视化网络示意图；
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图49为实施例2-6的控制组、对照组和实验组进行蛋白质印记分析后得到图16所示的各组细胞的pSTAT1信号的表达情况对比图；
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图50为针对实施例2-7的控制组、对照组和实验组各组的细胞聚集体进行流式分析的分析结果对比图；
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图51为根据图51统计得到的各组凋亡细胞数量百分比对比图；
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图52为实施例2-8的正常组、PBS注射组和抗肝纤维化制剂干预组小鼠的肝组织切片微观形貌照片对比图；
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图53为实施例2-8的正常组、PBS注射组和抗肝纤维化制剂干预组小鼠肝脏内 活化的肝星状细胞分布状态对比照片。
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图54为实施例2-9的LX-2组、LX-2添加组和共培养组共培养48小时后得到的LX-2细胞的胶原相关基因和纤维化相关基因表达情况对比图；
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图55为实施例2-10的正常组、假手术组和细胞移植组取肝组织进行H&E染色、picro-Sirius天狼星红染色、Masson三色染色和纤维粘连蛋白(FN)免疫染色后得到的照片对比图；
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图56为实施例2-10的正常组、假手术组和细胞移植组取肝组织进行picro-Sirius天狼星红染色、Masson三色染色和纤维粘连蛋白(FN)免疫染色后统计的肝纤维化区域和纤维连接蛋白阳性染色区域的相对定量情况对比图；
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图57为实施例2-10的正常组、假手术组和细胞移植组进行羟脯氨酸含量测定的分析结果；
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图58为实施例2-10的假手术组和细胞移植组取肝组织进行肝纤维化评分的分析结果；
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图59为实施例2-10的正常组、假手术组和细胞移植组取肝组织进行Ki67免疫组化染色得到的照片对比图；
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图60为根据图9所示的照片统计阳性染色细胞的量化结果；
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图61为实施例2-11的正常组、假手术组和细胞移植组取肝组织进行H&E染色、picro-Sirius天狼星红染色、Masson三色染色和纤维粘连蛋白(FN)免疫染色后得到的照片对比图；
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图62为实施例2-11的正常组、假手术组和细胞移植组取肝组织进行picro-Sirius天狼星红染色、Masson三色染色和纤维粘连蛋白(FN)免疫染色后统计的肝纤维化区域和纤维连接蛋白阳性染色区域的相对定量情况对比图；
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图63为实施例2-11的正常组、假手术组和细胞移植组取肝组织进行羟脯氨酸含量测定的分析结果；
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图64为实施例2-11的假手术组和细胞移植组取肝组织进行肝纤维化评分的分析结果；
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图65为实施例2-11的正常组、假手术组和细胞移植组血液的促纤维化、细胞外基质、信号转导相关的肝纤维化发生相关的基因表达水平进行分析后得到的热图；
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图66为实施例3-1的经TEM培养基培养10天后得到的细胞进行明场拍照得到的HepLPCs的明场照片；
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图67为实施例3-2采用流式细胞术对经小鼠GM-CSF诱导后得到的BMDMs进行细胞鉴定得到的总巨噬细胞占比分析结果；
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图68为实施例3-2采用流式细胞术对经LPS体外定向极化诱导得到的BMDMs进行细胞鉴定得到的M1型巨噬细胞占比分析结果；
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图69为实施例3-2采用流式细胞术对Control组和DM+LPS组进行细胞鉴定得到的M1相关炎性基因表达情况对比图；
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图70为实施例3-2对HepLPCs-CM+LPS组、Control组和DM+LPS组进行RNA提取和基因表达分析后得到的各组M1相关炎性基因表达情况对比图；
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图71为实施例3-2对HepLPCs-CM+LPS组、Control组和DM+LPS组的细胞培养上清进行细胞因子浓度检测得到的M1相关炎性基因表达水平对比图；
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图72为实施例3-3采用流式细胞术对经IL-4体外定向极化诱导得到的BMDMs进行细胞鉴定得到的M2型巨噬细胞占比分析结果；
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图73为实施例3-3采用流式细胞术对Control组和DM+IL-4组进行细胞鉴定得 到的M2相关炎性基因表达情况对比图；
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图74为实施例3-3对HepLPCs-CM+IL-4、Control组和DM+IL-4组进行RNA提取和基因表达分析得到的各组M2相关炎性因子IL10的分泌情况对比图；
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图75为实施例3-4的不同浓度外泌体与巨噬细胞BMDMs作用6小时后的DAPI核染后融合图；
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图76为实施例3-4的Control组，DM+LPS组、CM+LPS组和EV+LPS组的M1相关炎性基因表达情况对比图；
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图77为实施例3-5的Control组，DM+IL4组、CM+IL4组和EV+IL4组的M2相关炎性基因表达情况对比图。
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图78为实施例3-6的早期NASH小鼠模型的肝脏组织进行石蜡包埋、切片制样和H&E染色后得到的染色照片；
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图79为实施例3-6的对照组小鼠的肝脏组织进行石蜡包埋、切片制样和H&E染色后得到的染色照片；
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图80为实施例3-6的早期NASH小鼠模型的肝脏组织进行石蜡包埋和切片制样后进行Masson染色后得到的染色照片；
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图81为实施例3-6的早期NASH小鼠模型的肝脏组织进行石蜡包埋和切片制样后进行油红O染色后得到的染色照片；
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图82为实施例3-6对对早期NASH小鼠模型的肝脏组织进行石蜡包埋和切片制样后进行M1型巨噬细胞特异性标志物CD68的免疫组化染色得到的染色照片；
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图83为实施例3-6对早期NASH小鼠模型的肝脏组织进行石蜡包埋和切片制样后进行M2型巨噬细胞特异性标志物CD163的免疫组化染色后得到的染色照片；
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图84为实施例3-6的早期NASH小鼠模型和对照组的血生化及TC、TG含量对 比图；
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图85为实施例3-7的假手术组、细胞低剂量治疗组和细胞高剂量治疗组小鼠的肝脏H&E染色照片和油红O染色照片对比图；
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图86为实施例3-7对假手术组、细胞低剂量治疗组和细胞高剂量治疗组的NAS评分对比图；
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图87为实施例3-7对假手术组、细胞低剂量治疗组和细胞高剂量治疗组小鼠的肝脏切片的Masson染色照片；
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图88为实施例3-7对图11的各组染色照片的肝纤维化区域统计结果对比图；
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图89为实施例3-7对假手术组、细胞低剂量治疗组和细胞高剂量治疗组小鼠的肝脏切片进行Ki67染色得到的染色照片；
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图90为实施例3-7的正常对照组、免疫抑制组、口服生理盐水的模型组、假手术组、细胞低剂量治疗组和细胞高剂量治疗组小鼠血液中ALT、AST、LDH指标水平对比图；
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图91为实施例3-7的正常对照组、假手术组、细胞低剂量治疗组小鼠和细胞高剂量治疗组小鼠肝脏的TC和TG含量对比图；
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图92为实施例3-7对假手术组、细胞低剂量治疗组小鼠和细胞高剂量治疗组小鼠进行抗CD163和抗CD68的免疫组化染色后得到的染色照片对比图；
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图93为根据图93统计得到的各组肝脏中CD163 +巨噬细胞和CD68 +巨噬细胞数量对比图；
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图94为实施例3-8的中晚期NASH小鼠模型的假手术组、细胞低剂量治疗组和细胞高剂量治疗组小鼠肝脏切片的H&E染色照片对比图；
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图95为实施例3-8的中晚期NASH小鼠模型的假手术组、细胞低剂量治疗组和 细胞高剂量治疗组小鼠肝脏切片的Masson染色照片对比图；
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图96为根据图96统计得到的各组肝纤维化区域统计结果对比图；
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图97为实施例3-9采用流式细胞术对经小鼠GM-CSF诱导后得到的BMDMs进行细胞鉴定得到的总巨噬细胞占比分析结果；
[根据细则91更正 30.03.2022]　
图98为实施例3-9采用流式细胞术对经LPS体外定向极化诱导得到的BMDMs进行细胞鉴定得到的M1型巨噬细胞占比分析结果；
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图99为实施例3-9采用流式细胞术对Control组和DM+LPS组进行细胞鉴定得到的M1相关炎性基因表达情况对比图；
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图100为实施例3-10采用流式细胞术对经IL-4体外定向极化诱导得到的BMDMs进行细胞鉴定得到的M2型巨噬细胞占比分析结果；
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图101为实施例3-10采用流式细胞术对Control组和DM+IL-4组进行细胞鉴定得到的M2相关炎性基因表达情况对比图；
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图102为实施例3-11的Control组，DM+LPS组、CM+LPS组和EV+LPS组的M1相关炎性基因表达情况对比图；
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图103为实施例3-12的Control组，DM+IL4组、CM+IL4组和EV+IL4组的M2相关炎性基因表达情况对比图。
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图104为实施例4-1的各肝前体相关标志物在HepLPCs细胞中的表达情况对比图；
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图105为实施例4-1的经TEM培养基培养10天后得到的细胞进行明场拍照得到的HepLPCs的明场照片；
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图106为实施例4-1采用流式细胞术考察阳性对照组以及各共培养组中HepLPC-CM对脾脏细胞的增殖抑制情况得到的对比图；
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图107为实施例4-2采用流式细胞术考察供体1来源HepLPC-CM对脾脏细胞增殖的抑制情况以及对应的阳性对照组中脾脏细胞增殖情况得到的对比图；
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图108为实施例4-2采用流式细胞术考察供体2来源HepLPC-CM对脾脏细胞增殖的抑制情况以及对应的阳性对照组中脾脏细胞增殖情况得到的对比图；
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图109为实施例4-2采用流式细胞术考察供体3来源HepLPC-CM对脾脏细胞增殖的抑制情况以及对应的阳性对照组中脾脏细胞增殖情况得到的对比图；
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图110为实施例4-2采用流式细胞术考察供体4来源HepLPC-CM对脾脏细胞增殖的抑制情况以及对应的阳性对照组中脾脏细胞增殖情况得到的对比图。
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图111为实施例4-3采用流式细胞术考察阳性对照组、FK506对照组以及各共培养组中HepLPC对脾脏细胞的增殖抑制情况得到的对比图；
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图112为实施例4-4采用流式细胞术考察供体1来源HepLPC对脾脏细胞增殖的抑制情况以及对应的阳性对照组中脾脏细胞增殖情况得到的对比图；
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图113为实施例4-4采用流式细胞术考察供体2来源HepLPC对脾脏细胞增殖的抑制情况以及对应的阳性对照组中脾脏细胞增殖情况得到的对比图；
[根据细则91更正 30.03.2022]　
图114为实施例4-4采用流式细胞术考察供体3来源HepLPC对脾脏细胞增殖的抑制情况以及对应的阳性对照组中脾脏细胞增殖情况得到的对比图；
[根据细则91更正 30.03.2022]　
图115为实施例4-4采用流式细胞术考察供体4来源HepLPC对脾脏细胞增殖的抑制情况以及对应的阳性对照组中脾脏细胞增殖情况得到的对比图；
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图116为实施例4-5采用流式细胞术考察各供体来源HepLPC的PBMC共培养组对PBMC的增殖抑制情况与对应的阳性对照组中PBMC的增殖情况得到的对比图；
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图117为实施例4-6的不同ConA注射剂量与小鼠生存率的关系曲线；
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图118为实施例4-7对8毫克/公斤注射剂量组的小鼠在不同时间眼眶取血并通过血生化水平分析考察得到的各组ALT、AST和LDH指标水平对比图；
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图119为实施例4-7对实验组1和对照组的各小鼠注射完毕的6小时后眼眶取血并通过血生化水平分析考察得到的各组ALT、AST、LDH和ALP指标水平对比图；
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图120为实施例4对实验组2和对照组的各小鼠注射完毕的6小时后眼眶取血并通过血生化水平分析考察得到的各组ALT、AST和LDH指标水平对比图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。除非另外定义，此处使用的技术术语或者科学术语应当为本发明所属领域内具有一般技能的人士所理解的通常意义。本文中使用的“包括”等类似的词语意指出现该词前面的元件或者物件涵盖出现在该词后面列举的元件或者物件及其等同，而不排除其他元件或者物件。
本发明各实施例中，如无特别说明，细胞培养均在37摄氏度环境下且二氧化碳浓度为5％的细胞培养箱中进行。细胞培养使用的培养基以及处理细胞所使用的各类试剂，例如缓冲液使用前均经无菌化处理以及0.22微米滤器过滤以去除杂质。
本发明各实施例中涉及统计学分析的数据，每组实验至少重复3次，实验结果数据利用GraphPad Prism 8.0软件进行统计学分析。两组数据间比较使用双尾非配对t检验来计算统计学差异，多组数据间差异的比较使用用ANOVA方差分析计算统计学差异。p<0.05被认为具有统计学差异，说明书附图中：*代表P<0.05； **代表P<0.005；***代表P<0.001；****代表P<0.0001。
以下通过具体的实施例进行详细说明：
以下实施例制备了包含至少一种miRNA的肝细胞调控制剂作为肝病调控制剂，并考察了这种肝病调控制剂的应用。具体如下：
实施例1-1
本实施例以人原代肝细胞(简记为PHHs)和人肝前体样细胞(简记为HepLPCs)作为种子细胞，采用含血清类物质培养基和无血清类物质培养基进行培养后成功分离出粒径100纳米左右，并表达外泌体标志蛋白TSG101、CD63和CD81的外泌体。
本实施例的PHHs购自广州深圳立沃科技有限公司，批号为Lot#201904001；HepLPCs来源于赛立维生物科技有限公司，批号为XLV-19006；Hep-X基础培养基来源于上海源培生物科技有限公司；胎牛血清、1％青霉素-链霉素溶液以及鼠尾胶原均来源于Gibco；肝细胞生长因子HGF来源于近岸生物；上皮细胞生长因子EGF来源于近岸生物；ROCK激酶抑制剂Y-27632来源于陶术生物；Wnt信号通路激动剂CHIR-99021来源于陶术生物；TGF-β信号抑制剂A-8301来源于陶术生物；CD63-FITC和CD81-PE流式抗体均来源于美国BD bioscience。
使用的含血清类物质培养基组成如下：Hep-X基础培养基，以及以占Hep-X基础培养基体积计，含量为1％的N2营养补充剂(100X)，含量为1％的B27营养补充剂(50X)、10％胎牛血清FBS、含量为1％青霉素-链霉素溶液；含量为20ng/mL的肝细胞生长因子HGF，含量为50ng/mL的上皮细胞生长因子EGF，含量为10μM的ROCK激酶抑制剂Y-27632，含量为3μM的Wnt信号通路激动剂CHIR-99021，含量为1μM的TGF-β信号抑制剂A-8301。
使用的无血清类物质培养基组成为所述含血清类物质培养基去除胎牛血清 后的组成成分。
上述含血清类物质培养基和无血清类物质培养基使用前均经0.22微米滤器过滤以去除杂质。
本实施例提供了从两种种子细胞中分别获取包含外泌体的沉淀物质的过程，具体为：
以1×10 5个/平方厘米的接种密度将种子细胞接种于15cm培养皿中，每孔加2毫升含血清类物质培养基培养至细胞融合度不低于95％且生长状态良好，完成扩增培养。所述扩增培养的过程中，每2-3天更换一次含血清类物质培养基。
所述扩增培养完成后，将15cm培养皿中的培养基更换为无血清类物质培养基继续培养48小时后收取培养上清。使用美国System Biosciences公司的外泌体分离试剂盒
Figure PCTCN2022079802-appb-000001
ULTRA EV Isolation从培养上清中分离出来源于的PHHs的沉淀物质以及来源于HepLPCs的沉淀物质。具体的操作步骤记载于外泌体分离试剂盒附带的说明中，在此不做赘述。
本实施例利用透射电镜、纳米颗粒追踪检测和流式分析对上述两种沉淀物质进行分析。来源于的PHHs的沉淀物质简记为PHH Exo样本，来源于HepLPCs的沉淀物质简记为Hep Exo样本。
将PHH Exo样本和Hep Exo样本稀释后使用含1％戊二醛，浓度为0.1M的磷酸盐缓冲液固定后滴加在铜网格上，然后使用1％的醋酸铀酰负染，室温干燥后用透射电镜观察拍照，得到图1和图2分别所示的PHH Exo和Hep Exo样本的透射电镜对比照片，磷酸盐缓冲液的pH为7.4。
利用德国Particle Metrix的PMX110纳米颗粒跟踪分析仪分别对PHH Exo样本和Hep Exo样本进行分析，得到图3所示的两种样本中颗粒平均粒径对比图。具体的检测和分析方法为本领域技术人员的常规技术手段，在此不做赘述。
参照图1和图2，PHH Exo样本和Hep Exo样本中颗粒直径大小约100纳米，且呈现形态规则的类圆形，进一步的参照图3，PHH Exo样本中颗粒平均粒径为135±9.103nm，Hep Exo样本中颗粒平均粒径为136.4±4.323nm，符合外泌体的形态特征。
使用PBS溶液对PHH Exo样本和Hep Exo样本分别稀释并混匀后，对一部分用CD63-FITC和CD81-PE流式抗体染色，另一部分未染色的PHH Exo样本和Hep Exo样本作为阴性对照，将上述样本在美国BD bioscience的Accuri C6flow cytomenter进行上机检测，得到图4所述的流式分析结果。具体操作和分析步骤为本领域技术人员的常规技术手段，在此不做赘述。
本实施例对经体外培养后得到的两种细胞产物(简记为PHH和Hep)、PHH Exo样本以及Hep Exo样本和培养上清产物通过BCA法进行蛋白定量分析，并进行蛋白质印迹(Western Blotting，WB)测试，得到图5所示的各样品的CD63、CD81、TSG101、EEA1、GRP78和β-actin的表达情况对比照片。其中，对细胞产物进行裂解的方法为：吸除培养上清后对细胞沉淀物使用PBS缓冲液洗涤入12孔板并加入适量RIPA裂解液后收集细胞，并在冰上裂解后于4摄氏度下12000rpm离心10min以收集上清作为测试样品。其中，蛋白定量使用碧云天生物技术有限公司的SDS-PAGE蛋白上样缓冲液(5×)以及美国Thermo Fisher的PierceTM BCA Protein Assay Kit试剂盒；使用南京诺唯赞生物科技有限公司的高敏型ECL化学发光检测试剂盒和美国BIO-RAD的ChemiDoc化学发光成像仪进行WB测试。具体操作和分析步骤为本领域技术人员的常规技术手段，在此不做赘述。
参照图4，PHH Exo样本和Hep Exo样本均阳性表达外泌体标志蛋白CD63和CD81，经统计PHH Exo样本和Hep Exo样本的CD63阳性率分别为61.85±3.465％和90.85±2.475％，PHH Exo样本和Hep Exo样本的CD81阳性率分别为69.90±4.95％和89.40±1.273％。参照图5，相较于细胞裂解产物Cell Iysate 组的各样品，Exosome组的PHH Exo样本和Hep Exo样本均阳性表达了外泌体标志蛋白CD63和TSG101。
实施例1-2
本实施例对实施例1-1的PHH Exo样本和Hep Exo样本进行标记，然后与PHHs共培养，考察外泌体在肝细胞胞质内的表达情况，证明来源于PHHs和HepLPCs的外泌体可成功被肝细胞摄取。
以加入PBS缓冲溶液的PHHs为阴性对照，PHH Exo样本和Hep Exo样本使用PBS缓冲溶液进行稀释，使用美国Sigma的PKH26Red Fluorescent Cell Linker Kit试剂盒分别标记稀释后的PHH Exo样本和不同浓度的Hep Exo样本，然后分别与PHHs共孵育24小时以完成共培养。共培养完毕后，取含细胞的培养物使用含1％戊二醛，浓度为0.1M的磷酸盐缓冲液固定后进行DAPI染色，然后在荧光显微镜下观察，得到图6所示的PHH Exo样本和Hep Exo样本在标记后、染色后以及共培养后的免疫荧光共聚焦检测结果。具体的标记步骤由试剂盒提供。图6显示，无论是PHHs来源还是HepLPCs来源的外泌体在肝细胞胞质内都有明显表达，即可以成功被肝细胞摄取。
实施例1-3
本实施例提供了包含外泌体的肝细胞调控制剂在体外培养方面的应用。具体的，将实施例1-1的PHH Exo样本和Hep Exo样本使用缓冲溶液稀释后与PHHs共培养，对得到的细胞通过BrdU ELISA检测和EdU荧光法进行增殖分析，以及通过免疫荧光检测Ki67阳性表达细胞的表达情况，证明PHHs来源和HepLPCs来源的外泌体均能够促进肝细胞增殖，且HepLPCs来源的外泌体促进肝细胞增殖的效果更为显著。
首先，将PHH Exo样本和Hep Exo样本分别用PBS缓冲液稀释为不同浓度，分别为0、1、10和100微克/毫升，分别与PHHs共培养24小时，PHHs接种密 度为1×10 5个/平方厘米，培养容器使用12孔板。
共培养结束后，使用香港Abcam的BrdU Cell Proliferation ELISA Kit试剂盒通过ELISA检测获取波长为450纳米的OD值，进一步统计BrdU掺入作用，得到图7所示的不同外泌体浓度的PHH Exo样本和Hep Exo样本共培养后得到的细胞的BrdU掺入作用对比图，其中：每组浓度所对应的两个柱形图中，左侧为PHH Exo样本，右侧为Hep Exo样本。具体的检测步骤由试剂盒提供。ELISA检测和结果统计方法为本领域技术人员的常规技术手段。
共培养结束后，使用广州锐博生物技术公司的EdU Apollo 567 In Vitro Imaging Kit试剂盒对100微克/毫升外泌体浓度的样本经共培养得到的细胞进行EdU标记、细胞固定、Apollo染色以及DNA染色，DNA染色后使用荧光显微镜观察，并以PBS缓冲液作为阴性对照，得到图8所示的免疫荧光照片。具体的EdU标记、细胞固定、Apollo染色以及DNA染色步骤由试剂盒提供。
共培养结束后，对得到的各细胞进行Ki67免疫荧光染色得到图9所示的免疫荧光照片。
参照图7，浓度为100微克/毫升外泌体浓度的PHH Exo样本和Hep Exo样本对PHHs的增殖作用显著。进一步参照图8和图9，与阴性对照组相比，PHH Exo样本和Hep Exo样本均促进了肝细胞的增殖，从图8和图9中统计得到，Hep Exo样本和PHH Exo样本的EdU掺入率分别为19.89±1.049％和27.09±3.308％，Ki67阳性细胞百分比分别为38.7±2.406％和55.75±6.014％。
EdU和BrdU是胸腺嘧啶核苷类似物，在DNA复制时期代替胸腺嘧啶(T)渗入正在合成的DNA分子中，用来检测DNA复制活性。Ki67是增殖细胞的相关抗原，主要用于标记增殖周期中的细胞。从本实施例中可以看到，由于PHHs来源和HepLPCs来源的外泌体均能够促进肝细胞的增殖，由上述任意一种外泌体与稀释剂组合形成的肝细胞调控制剂能够作为促进肝细胞增殖的培养基使用。
一些实施例中，稀释剂为重悬液，外泌体的浓度为10-200微克/毫升。一些具体的实施例中，重悬液为PBS缓冲溶液、生理盐水和复方电解质溶液的任意一种。
实施例1-4
本实施例以实施例1-3中HepLPCs来源的外泌体经与PHHs共培养得到的细胞为例进行细胞周期分析和细胞周期相关分子的表达情况分析，证明了HepLPCs来源的外泌体通过加快细胞周期进程促进了肝细胞的增殖。
使用胰酶分别消化实施例1-3中PHH Exo样本和Hep Exo样本与PHHs共培养得到的细胞(分别简记为PHH Exo-cell和Hep Exo-cell)，采用美国Abcam的Propidium Iodide Flow Cytometry Kit试剂盒和美国BD bioscience的BD FACS Verse流式细胞仪进行细胞周期分析，检测激发波长488nm波长处检测红色荧光，同时检测光散射情况，用Flowjo软件进行细胞DNA含量和光散射分析，得到图10所示的细胞周期分析结果，其中的对照组为PHHs和等体积PBS缓冲液。具体的上机前操作步骤由试剂盒提供。
使用南京诺唯赞生物科技有限公司的HiScript III 1st Strand cDNA Synthesis Kit以及ChamQ SYBR Color qPCR Master Mix在德国Roche的LightCycler480I实时荧光定量PCR仪对对照组、PHH Exo-cell和Hep Exo-cell进行实时荧光定量PCR分析，得到图11所示的细胞周期相关分子miRNA表达水平对比图，其中：每种细胞周期相关分子对应的三个柱状图，自左向右依次为对照组、PHH Exo样本和Hep Exo样本。
使用蛋白质印迹(Western Blotting，WB)测试考察对照组、PHH Exo-cell和Hep Exo-cell表达细胞周期相关分子的情况，得到图12所示的细胞周期相关分子miRNA表达情况对比图。具体操作步骤请参见实施例1-1论述的WB测试步骤。
参照图10，与对照组以及PHH Exo-cell相比，Hep Exo-cell的G0期和G1期细胞比例分别下降15.6±1.353％和10.733±0.874％(P<0.01)，S期细胞比例分别增加6.47±0.97％和4.17±1.527％(P<0.01)，G2/M期细胞比例分别增加14.9±1.413％和9.133±2.101％，表明Hep Exo样本加快了原代肝细胞G1期向S期及G2/M期进程，从而促进了细胞增殖。
参照图11和图12，细胞周期相关蛋白Cyclin A2，Cyclin D1，Cyclin E表达明显上调，而p27kip1表达明显下调，说明Hep Exo样本可能通过增加cyclin家族蛋白同时抑制p27蛋白表达，促进细胞周期进程，从而促进肝细胞增殖。
实施例1-5
本实施例对实施例1-1的PHH Exo和Hep Exo样本中外泌体的miRNA进行了提取，并通过外泌体miRNA高通量测序分析、测序生信分析以及miRNA mimic体外转染原代肝细胞后的BrdU ELISA测试和EdU增殖分析证明，外泌体中表达显著升高且能够有效促进肝细胞增殖的miRNA为hsa-miR-182、hsa-miR-183以及hsa-miR-574。
使用美国Invitrogen的Total Exosome RNA and Protein Isolation Kit进行miRNA的提取，得到PHH Exo来源的分析样本PHH-Exo-mi和Hep Exo来源的分析样本Hep Exo-mi，然后使用南京诺唯赞生物科技有限公司的miRNA 1st Strand cDNA Synthesis Kit、miRNA Universal SYBR qPCR Master Mix以及HiScript III 1st Strand cDNA Synthesis Kit和ChamQ SYBR Color qPCR Master Mix先后连接3’端和5’的接头，反转录成cDNA，再进行PCR扩增。PCR扩增后切胶回收目的片段文库，将质检合格的文库通过美国Illumina的Illumina HiSeqTM 2500高通量测序仪测序分析，得到图13所示的聚类热图，提供了上调或下调差异表达前15个miRNA。
本实施例选取了7个表达明显上调的miRNA，分别记为hsa-miR-182， hsa-miR-183，hsa-miR-149，hsa-miR-215，hsa-miR-574，hsa-miR-654和hsa-miR-675，通过原代肝细胞体外转染miRNA mimic增强miRNA表达，然后进行BrdU ELISA检测BrdU掺入作用，得到图14所示的转染各miRNA细胞的BrdU掺入作用对比图，其中NC为空白转染组。体外转染使用广州锐博生物技术公司的miRNA mimic/inhibitor)，BrdU ELISA检测BrdU掺入作用的过程以及使用的试剂盒请参见实施例1-3。
本实施例进一步对转染hsa-miR-182，hsa-miR-183和hsa-miR-574的原代肝细胞分别进行EdU染色后，通过EdU荧光法检测EdU掺入率，得到图15所示的EdU荧光显微照片以及图16所示的EdU掺入率对比图。具体的检测用试剂盒请参见实施例1-3。
参照图14，7个表达明显上调的miRNA中，hsa-miR-182，hsa-miR-183和hsa-miR-574对原代肝细胞增殖具有明显的促进作用(p<0.05)。参照图15和图16，与NC mimic转染组相比，hsa-miR-183mimic体外转染的EdU掺入率明显增多，且明显高于hsa-miR-182和hsa-miR-574mimic转染组，NC mimic、hsa-miR-182、hsa-miR-183以及hsa-miR-574mimic转染组的EdU掺入率分别为10.04±2.946％，18.22±2.67％，29.46±4.799％和14.6±3.173％。
实施例1-6
本实施例构建四氯化碳诱导的小鼠急性肝衰竭模型后，尾静脉注射包含Hep Exo样本的注射液作为外泌体制剂干预肝衰竭体内动物模型，以促进肝组织再生。
首先，使用若干6-8周龄，体重22-25g的C57BL/6小鼠构建四氯化碳诱导的小鼠急性肝衰竭模型以及对照模型。具体的，腹腔注射四氯化碳与橄榄油按照1：4稀释的诱导注射液以构建小鼠急性肝衰竭模型，腹腔注射同等体积的橄榄油以构建正常组，两种模型的腹腔注射剂量均为1mL/kg。
然后，诱导注射液注射完毕的6小时后，对部分小鼠急性肝衰竭模型尾静脉 注射PBS缓冲液和Hep Exo样本混匀的治疗注射液，形成Hep Exo治疗组；对部分小鼠急性肝衰竭模型尾静脉注射与治疗注射液等体积的PBS缓冲液，形成PBS对照组。治疗注射液中，Hep Exo样本浓度为2微克/微升。Hep Exo治疗组和PBS对照组的注射剂量均为15毫克/kg。
一些其他的实施例中，Hep Exo治疗组和PBS对照组的注射剂量为1-100毫克/公斤。
以上所有注射液以及缓冲液注射前均灭菌并经0.22μm滤器过滤。
本实施例考察了造模完毕后7天内Hep Exo治疗组和PBS对照组各小鼠的生存情况。具体的，使用卡普兰-迈尔(Kaplan-meier)法进行生存分析绘制生存曲线并进行行对数秩检验(Log-Rank)，p<0.05被认为具有统计学差异，得到图17所示的Hep Exo治疗组和PBS对照组各小鼠的生存情况对比图，其中各组用于分析的小鼠数目为15。具体的分析方法为本领域技术人员的常规技术手段，在此不做赘述。
参照图17，PBS对照组小鼠24小时死亡率为25％，并且在48小时内死亡率超过50％，而Hep Exo治疗组小鼠24小时死亡率为20％，48小时内死亡率仅为30％，并且72小时后再无死亡(p<0.05)，说明人肝前体样细胞来源外泌体可以改善小鼠7天生存率，对急性肝衰竭具有明显的治疗作用。
本实施例在造模后的24小时和48小时取正常对照组、PBS对照组和Hep Exo治疗组小鼠的肝脏组织进行H&E(hematoxylin-eosin staining)染色后制片，于显微镜下观察，得到图18所示的各组小鼠病理损伤情况对比图。具体的制片和观察步骤为本领域技术人员的常规技术手段，在此不做赘述。
参照图18，正常组小鼠肝组织呈正常肝细胞形态，肝小叶结构完整清晰，无炎性细胞浸润。PBS对照组小鼠造模24h和48h均发生明显的肝细胞肿胀，细胞核碎裂，空泡增多，局部有炎症细胞浸润，肝索正常接结构破坏，肝窦淤血 严重。然而，Hep Exo治疗组小鼠造模24h和48h均变现出程度较轻的肝细胞损伤，空泡形成及炎症细胞浸润都较对照组少，肝损伤在一定程度上得到减轻。
本实施例在造模后的24小时和48小时取正常组、PBS对照组和Hep Exo治疗组小鼠的肝脏组织进行石蜡切片后进行Ki67免疫组化染色，考察肝组织的再生情况，得到图19所示的PBS对照组和Hep Exo治疗组小鼠肝组织切片免疫组化Ki67染色对比图以及图10所示的各组小鼠肝脏组织石蜡切片中Ki67阳性细胞百分比，每组用于统计的小鼠数目为8。具体的Ki67免疫组化染色步骤请参见实施例1-3。图20每个造模时间对应的两个柱形图从左至右依次为PBS对照组和Hep Exo治疗组。
参照图19和图20，Hep Exo治疗组小鼠在48小时切片中，Ki67阳性表达细胞与PBS对照组相比明显增多，经统计Hep Exo治疗组和PBS对照组的Ki67阳性表达细胞百分比分别为16.587±3.381％和7.021±2.415％，说明肝再生被有效启动。可见，Hep Exo样本在急性肝衰竭中具有重要的治疗作用，能够提高小鼠生存率，减轻肝脏损伤，有效促进肝脏组织再生。
本实施例在造模后的24小时和48小时利用七氟烷吸入麻醉小鼠，摘掉小鼠眼球取血，让血液自然流出，采集的血液收集到1.5mL EP管中，常温放置30min后在4℃，3000rpm下离心10min，然后缓慢吸取上清液为小鼠的血清。采用美国Beckman Coulter的丙氨酸氨基转移酶(ALT)试剂盒和天冬氨酸氨基转移酶(AST)检测试剂盒进行ALT和AST的生化指标检测，根据每分钟的平均吸光度ΔA计算AST水平和ALT水平，得到图21和22所示的正常组、PBS对照组和Hep Exo治疗组各小鼠在不同时间的AST水平和ALT水平对比结果，每组用于统计的小鼠数目为8。图21和图22的每个造模时间对应的三个柱形图从左至右依次为正常组、PBS对照组和Hep Exo治疗组。
参照图21和图22，PBS对照组和Hep Exo治疗组的血清AST和ALT的水 平均在造模后逐渐上升，并在48h达到峰值。Hep Exo治疗组小鼠血清的AST和ALT水平与PBS对照组相比，均出现明显降低，可见HepLPC来源的外泌体可以有效降低血清中ALT和AST的水平，对小鼠急性肝脏损伤起到保护作用。
实施例1-7
本实施例以miRNA-183为例，提供了包含miRNA-183的外泌体制剂在制备肝衰竭治疗类药物方面的应用。
具体的，构建四氯化碳以及乙酰氨基酚诱导的两种小鼠急性肝衰竭模型以分别模拟不同的肝损伤机制，尾静脉注射包含miRNA-183的注射液作为外泌体制剂干预肝衰竭体内动物模型，以促进肝组织再生。
四氯化碳诱导的急性肝衰竭模型(简记为CCl4模型)构建方法为：对6-8周，体重22-25g的C57BL/6小鼠腹腔注射四氯化碳与橄榄油按照1：4稀释的四氯化碳诱导注射液，注射剂量为1mL/kg。相应正常对照组腹腔注射等体积的橄榄油(简记为CCl4正常组)。四氯化碳诱导注射液注射前使用0.22微米滤器进行除菌过滤。
对乙酰氨基酚诱导的急性肝衰竭模型(简记为APAP模型)构建方法为：对6-8周，体重22-25g的C57BL/6小鼠腹腔注射乙酰氨基酚与PBS缓冲液混合形成的对乙酰氨基酚诱导注射液，注射剂量为1mL/kg。相应正常对照组腹腔注射等体积的PBS缓冲液(简记为APAP正常组)。乙酰氨基酚诱导注射液注射前使用0.22微米滤器进行除菌过滤。
对造模6小时后的部分CCl4模型和部分APAP模型尾静脉注射分别用CCl4和PBS缓冲液稀释，含15纳摩尔的miRNA-183 agomir的阴性对照注射液各200微升(分别简记为CCl4-NC agomir组和APAP-NC agomir组)。
使用Entranster TM-in vivo RNA转染试剂包裹Cy5标记miRNA-183 agomir， 并分别用CCl4和PBS缓冲液稀释，然后对造模6小时后的部分CCl4模型和部分APAP模型进行尾静脉注射(分别简记为CCl4-miRNA 183-5p agomir组和APAP-miRNA 183-5p agomir组)。其中，注射液中每微升转染试剂含2微克核酸，注射液每只注射量为200微升，miRNA-183 agomir的含量为15纳摩尔。Entranster TM-in vivo RNA转染试剂来源于北京英格恩生物技术公司，Cy5标记miRNA-183 agomir来源于广州锐博生物技术公司。
造模后的不同时间点取各组肝组织和血样样本进行肝损伤和再生相关指标检测。每组用于检测的小鼠数目为8。具体如下：
本实施例考察了不同处理小鼠造模后的7天生存率，得到图23和图24分别所示的CCl4模型各组小鼠的7天生存率情况以及APAP模型各组小鼠的7天生存率情况。具体操作和分析过程请参见实施例1-3。
参照图23，CCl4-NC agomir组小鼠24小时死亡率为20％，并且在48小时内死亡率超过70％，而CCl4-miRNA 183-5p agomir组小鼠24小时无死亡，48小时内死亡率仅为20％(p<0.05)；参照图24，APAP-NC agomir组小鼠24小时死亡率高达80％，而APAP-miRNA 183-5p agomir组小鼠24小时内死亡率仅50％。图23和图24均说明miRNA 183-5p可以明显改善小鼠7天生存率，对急性肝衰竭具有明显的治疗作用。
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本实施例对造模后的72小时内的不同时间点取CCl4模型各小鼠外周血进行ALT和AST检测，造模后的第24小时取APAP模型各小鼠外周血进行ALT和AST检测，得到图25至图27分别所示的AST水平和ALT水平对比图。具体的ALT和AST检测过程请参见实施例1-3。图25和图26的每个造模时间对应的三个柱形图从左至右依次为正常组、CCl4-NC agomir组和CCl4-miRNA 183-5p agomir组。图27的每个因子(ALT或AST)对应的三个柱形图从左至右依次为正常组、APAP-NC agomir组和APAP-miRNA 183-5p agomir组。
参照图25和图26，CCl4-NC agomir组小鼠和CCl4-miRNA 183-5p agomir组小鼠的AST和ALT的水平均在造模后逐渐上升，并在48h达到峰值，而在72h有所降低，且CCl4-miRNA 183-5p agomir组小鼠的AST和ALT的水平相较CCl4-NC agomir组小鼠有明显降低。参照图27，APAP-miRNA 183-5p agomir组小鼠的AST和ALT水平显著低于APAP-NC agomir组各小鼠。以上均说明miRNA 183-5p能够有效降低血清中ALT和AST的水平。
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本实施例在造模后的不同时间点取CCl4模型各小鼠肝脏组织进行病理HE染色考察肝损伤情况，并在造模后的第24小时取APAP模型各小鼠肝脏组织进行病理HE染色考察肝损伤情况，得到图28至图29分别所示的CCl4模型各小鼠和APAP模型各小鼠造模后的病理组织切片对比图。具体的操作过程请参见实施例1-3。
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本实施例进一步通过免疫组化检测Ki67的表达，对不同处理的小鼠肝组织再生情况进行评价，得到图30所示的造模完成后不同时间CCl4-NC agomir组小鼠和CCl4-miRNA 183-5p agomir组小鼠肝组织切片免疫组化Ki67染色对比图，以及图31所示的造模完成的24小时后APAP-NC agomir组和APAP-miRNA183-5p agomir组小鼠肝组织切片免疫组化Ki67染色对比图。具体操作过程请参见实施例1-3。
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图32统计了造模完成后不同时间CCl4-NC agomir组小鼠和CCl4-miRNA183-5p agomir组小鼠肝组织切片的Ki67阳性细胞百分比。其中：每个造模完成时间对应的两个柱状图从左自右依次为CCl4-NC agomir组和CCl4-miRNA183-5p agomir组。
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参照图28至图32，CCl4正常组和APAP正常组小鼠肝组织均呈正常肝细胞形态，肝小叶结构完整清晰，无炎性细胞浸润。CCl4-NC agomir组和APAP-NC agomir组小鼠造模24h出现明显肝损伤，在48h出现严重的肝细胞坏死，细胞核碎裂，空泡增多，大量炎症细胞浸润，肝窦淤血严重。然而，CCl4-miRNA 183-5p  agomir组和APAP-miRNA 183-5p agomir组小鼠造模24小时也出现肝损伤，但造模48小时后肝脏损伤程度明显减轻，空泡形成及炎症细胞浸润都较对应的NC agomir组少。
与CCl4-NC agomir组相比，CCl4-miRNA 183-5p agomir组小鼠在48小时切片中，Ki67阳性表达细胞明显增多(p<0.05)；与APAP-NC agomir组相比，APAP-miRNA 183-5p agomir组小鼠在24小时切片中，Ki67阳性表达细胞明显增多(p<0.05)，说明肝再生被有效启动。
本实施例通过Western blotting法考察了CCl4-miRNA 183-5p agomir组各小鼠在完成造模后的不同时间的细胞周期相关分子的蛋白表达情况，得到图33所示的示意图。具体的操作方法请参见实施例1。
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参照图33，CCl4-miRNA 183-5p agomir组的肝组织细胞周期相关蛋白Cyclin A2，Cyclin D，Cyclin E表达上调,而p27kip1表达下调，说明miRNA-183-5p可能通过增加cyclin家族蛋白同时抑制p27蛋白表达，促进细胞周期进程，从而促进肝细胞增殖。
以下实施例制备了抗肝纤维化制剂作为肝病调控制剂，并考察了这种肝病调控制剂的应用。具体如下：
实施例2-1
本实施例提供了第一种抗肝纤维化制剂，其制备方法如下：
S0：提供汇合度不低于60％的人肝前体样细胞Human-HepLPCs作为种子细胞；
S1：使用无血清的DMEM培养基对Human-HepLPCs进行24小时的体外培养；
S2：收集体外培养上清，去除所述体外培养上清中的细胞碎片后进行25倍 的过滤浓缩，得到的分泌上清作为抗肝纤维化制剂。具体的，所述步骤S2中，在3000g的离心力下去除培养上清中的细胞碎片；使用10kDa的Amicon Ultra超滤器进行过滤浓缩。
本实施例采用BCA蛋白定量试剂盒(来源于上海碧云天生物技术有限公司)按说明书提供的检测方法检测上述分泌上清中的总蛋白含量为2.2毫克/毫升。
本实施例所述步骤S0的Human-HepLPCs由人原代肝细胞Human-primary hepatocytes经TEM培养基进行7-9天的转化扩增培养后，再进行1:(3-6)的传代培养至第2-5代得到。具体的，TEM培养基由以下成分组成：DMEM/F12基础培养基，以及以占DMEM/F12基础培养基的含量计：含量为1％的N2营养补充剂(100X)，含量为1％的B27营养补充剂(50X)，1mM的丙酮酸钠，10μg/mL的抗坏血酸，20ng/mL的肝细胞生长因子HGF,20ng/mL的上皮细胞生长因子EGF,10μM的ROCK激酶抑制剂Y27632，3μM的Wnt信号通路激动剂CHIR99021,1μM的TGF-β信号抑制剂A8301,1μM的鞘氨醇-1-磷酸S1P and 5μM的吲哚乙酸LPA。其中的DMEM/F12、N2营养补充剂、B27营养补充剂和丙酮酸钠来源于Invitrogen；抗坏血酸来源于Sigma-Aldrich；HGF和EGF来源于Novoprotein；Y27632、CHIR99021、A8301、s1p和LPA均来源于TargetMol。
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本实施例分别使用qPCR和流式细胞术分析了人原代肝细胞和人肝前体样细胞的基因表达情况，结果如图34和图35所示。参照图34，箭头所指的柱状图代表人肝前体样细胞的基因相对表达情况。人原代肝细胞在TEM培养基作用下，肝祖细胞基因Ck7、Ck19和Sox9的表达显著增加，而Alb、Cyp3a4和Hnf4α这类肝实质细胞标志物表达显著下降。参照图35，人肝前体样细胞的肝细胞标志物HNF4α以及肝干细胞/肝祖细胞标志物CD24and CK19显著表达，造血干细胞抗原CD34、白细胞共同抗原CD45以及肝胎儿细胞标志物AFP的表达水平均小于2％。人肝前体样细胞不表达MHC II类抗原HLA-DP，HLA-DQ和HLA-DR，表现出低免疫原性。
实施例2-2
本实施例对实施例2-1的第一种抗肝纤维化制剂与人永生化肝星状细胞系LX-2进行共培养，考察抗肝纤维化制剂对LX-2的促凋亡作用。本实施例的LX-2购自Procell。
实施例1的第一种抗肝纤维化制剂与LX-2进行共培养的过程如下：LX-2固定于含10％FBS、100U/mL的青霉素和100mg/mL的链霉素的DMEM培养基中，加入2.5ng/mL的TGF-β1活化LX-2；再加入实施例1的抗肝纤维化制剂混匀后静置48小时。共培养混合物中，第一种抗肝纤维化制剂的体积百分比含量为1％、2.5％和5％。其中，第一种抗肝纤维化制剂在使用前先加入10μg/mL的抗FGF19抗体(兔单克隆抗体，来源于R&D Systems)和10μg/mL的抗AREG抗体(兔多克隆抗体，来源于R&D Systems)孵育2小时。
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本实施例将上述共培养得到的细胞作为实验组，LX-2和DMEM培养基共培养48小时得到的细胞作为控制组，加入TGF-β1活化LX-2后与DMEM培养基共培养48小时得到的细胞聚集体作为TGF-β1活化组，三组制样后使用透射电子显微镜观察形态，得到图36所示的各组LX-2细胞的微观形貌照片。参照图3，TGF-β1激活LX-2细胞导致LX-2细胞形态改变为细长的树突状，而加入第一种抗肝纤维化制剂共培养则能够逆转这种变化。
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本实施例通过实时聚合酶链反应(RT-PCR)考察了TGF-β1活化组和实验组(第一种抗肝纤维化制剂的体积百分比含量为1％)的LX-2中与HSCs活化相关基因Col1a1、Col3a1、TGF-β1、Desmin、α-SMA和Pdgfb的相对mRNA表达情况，将数据标准化为GAPDH表达，并与对照组相比统计差异化程度，得到图37所示的各组与HSCs活化相关基因的相对mRNA表达情况对比图。从图37可以看到，TGF-β1激活了LX-2细胞，使得上述HSCs活化相关基因的表达水平上调；而第一种抗肝纤维化制剂的引入显著抑制了上述HSCs活化相关基因的表达水平，可见第一种抗肝纤维化制剂对HSCs活化具有显著的抑制作用。
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本实施例对实验组(第一种抗肝纤维化制剂的体积百分比含量为1％)的细胞制样后进行透射电子显微镜观察，得到图38所示的微观照片。参见图38所示，第一种抗肝纤维化制剂与LX-2共培养得到的细胞聚集体中观察到箭头所示的凋亡小体。进一步对实验组和TGF-β1活化组得到的细胞进行膜联蛋白V/PI染色后进行流式细胞术测定，得到图39所示的对照组流式细胞术图和图40所示的实验组的流式细胞术图。参照图39和图40，第一种抗肝纤维化制剂诱导了HSC凋亡。
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本实施例分别对控制组、TGF-β1活化组以及不同抗肝纤维化制剂的质量百分比含量的实验组的细胞聚集体进行蛋白质印记分析(Western blot)，得到图41所示的各组细胞的纤维化相关蛋白表达和关键纤维化信号的表达情况对比图。可见，在存在TGF-β1的情况下，第一种抗肝纤维化制剂诱导纤维化相关蛋白表达和关键纤维化信号TGF-β-SMAD途径的剂量依赖性降低。
实施例2-3
本实施例提供了第二种抗肝纤维化制剂，其制备方法以汇合度不低于60％的鼠肝前体样细胞Rat-HepLPCs作为种子细胞，其余制备过程请参见实施例1。
Rat-HepLPCs以鼠肝原代细胞Rat primary hepatocytes为种子细胞，通过TEM培养基培养得到。具体的培养过程请参见实施例2-1。
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本实施例分别使用qPCR和流式细胞术分析了Rat primary hepatocytes和Rat-HepLPCs的基因表达情况，结果如图42所示。参照图42，鼠肝前体样细胞在TEM培养基作用下，肝祖细胞基因Ck7,Ck19和Sox9的表达显著增加，而Alb、Cyp3a4和Hnf4α这类肝实质细胞标志物表达显著下降。
实施例2-4
本实施例对实施例2-3的第二种抗肝纤维化制剂与鼠肝星状细胞系HSCs-T6进行共培养，考察抗肝纤维化制剂对HSCs-T6的促凋亡作用。本实施例的 HSCs-T6购自Procell。
实施例2-3的第二种抗肝纤维化制剂与HSCs-T6进行共培养的过程请参见实施例2-2。其中共培养混合物中第二种抗肝纤维化制剂的体积百分比含量为1％。
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本实施例将第二种抗肝纤维化制剂与HSCs-T6进行共培养得到的细胞作为实验组，HSCs-T6和DMEM培养基共培养48小时得到的细胞作为控制组，加入TGF-β1活化HSCs-T6后与DMEM培养基共培养48小时得到的细胞作为TGF-β1活化组，三组制样后使用透射电子显微镜观察形态，得到图43所示的各组HSCs-T6细胞的微观形貌照片。参照图43，TGF-β1激活HSCs-T6细胞导致HSCs-T6细胞形态改变为细长的树突状，而加入第一种抗肝纤维化制剂共培养则能够逆转这种变化。
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本实施例通过实时聚合酶链反应(RT-PCR)考察了控制组、TGF-β1活化组和实验组的HSCs-T6中与HSCs活化相关基因Col1a1、Col3a1、TGF-β1、Desmin、α-SMA和Pdgfb的相对mRNA表达情况，将数据标准化为GAPDH表达，并与对照组相比统计差异化程度，得到图44所示的各组与HSCs活化相关基因的相对mRNA表达情况对比图。从图44可以看到TGF-β1激活了HSCs-T6细胞，使得上述HSCs活化相关基因的表达水平上调；而第二种抗肝纤维化制剂的引入显著抑制了上述HSCs活化相关基因的表达水平，可见第二种抗肝纤维化制剂对HSCs活化具有显著的抑制作用。
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本实施例对实验组的细胞制样后进行透射电子显微镜观察，得到图45所示的微观照片。参见图45所示，第二种抗肝纤维化制剂与HSCs-T6共培养得到的细胞中也观察到了箭头所示的凋亡小体。进一步对实验组和TGF-β1活化组得到的细胞进行膜联蛋白V/PI染色后进行流式细胞术测定，得到图46所示的TGF-β1活化组流式细胞术图和图47所示的实验组的流式细胞术图。参照图46和图47，第二种抗肝纤维化制剂诱导了HSC凋亡。
实施例2-5
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JAK/STAT通路在肝纤维化的形成进程中具有重要的调控作用。本实施例对实施例1的由人肝前体样细胞Human-HepLPCs体外培养得到的第一种抗肝纤维化制剂使用串联质谱标签(TMT)分析蛋白质组学组成，通过蛋白质-蛋白质相互作用(PPI)分析构建得到图48所示的第一种抗肝纤维化制剂中JAK-STAT通路与第一种抗肝纤维化制剂中参与生长因子活性、细胞因子活性和受体-配体活性的蛋白质之间的可视化网络示意图。
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参照图48，白血病抑制因子(LIF)、内皮素1(EDN1)、集落刺激因子1(CSF1)、双调蛋白(AREG)、成纤维细胞生长因子19(FGF19)直接或间接与JAK-STAT通路的中间分子相互作用。
实施例2-6
本实施例提供了第三种抗肝纤维化制剂，包含重组人FGF19(rhFGF19)和重组人AREG(rhAREG)。本实施例参照实施例2-2的方法将所述第三种抗肝纤维化制剂与LX-2共培养，形成TGF-β1+rhFGF19+rhAREG组，考察其对LX-2的作用，区别在于：rhFGF19的浓度分别为0.1ng/mL、1ng/mL、10ng/mL、100ng/mL和1000ng/mL，rhAREG的浓度分别为0.1ng/mL、1ng/mL、10ng/mL、100ng/mL和1000ng/mL。不同实验组的rhFGF19浓度和rhAREG浓度不同。
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作为公知的是，p-STAT1在肝纤维化过程中发挥重要作用，主要通过抑制肝星状细胞的功能实现抗纤维化作用。本实施例控制组、对照组和不同实验组进行蛋白质印记分析(Western blot)，得到图49所示的各组细胞的pSTAT1信号的表达情况对比图。参照图49可知，每种重组蛋白的浓度不低于10ng/mL时，p-STAT1水平增加。
实施例2-7
本实施例将FGF19的中和抗体FGF19Ab和AREG的中和抗体AREG Ab加入到实施例2-2的第一种抗肝纤维化制剂与活化的LX-2的共培养体系中形成TGF-β1+scrtms+FGF19Ab+AREG Ab组，与实施例2-6的TGF-β1+rhFGF19+rhAREG组，以及实施例2-2的TGF-β1活化组以及实验组一同考察rhFGF19和rhAREG的协同作用。rhFGF19和rhFGF19在共培养体系中的浓度均为100纳克/毫升，FGF19的中和抗体FGF19Ab和AREG的中和抗体AREG Ab在共培养体系中的浓度均为10微克/毫升。
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共培养48小时后，对各组细胞进行流式分析，根据图50所示的各组细胞计数情况对比图。根据图50统计凋亡细胞的数量百分比，得到图51所示的各种各组凋亡细胞数量百分比对比图。参照图50和图51，在TGF-β1存在的情况下，相较于TGF-β1和LX-2的共培养体系，rhFGF19和rhAREG的联合使用在一定程度上诱导了LX-2的凋亡；而rhFGF19Ab和rhAREG Ab的联合使用则降低了上述的促LX-2凋亡作用，说明rhFGF19和rhAREG的联合使用有助于诱导STAT1介导的HSC凋亡。
实施例2-8
本实施例提供了抗肝纤维化制剂在制备抗肝纤维化药物方面的应用。
首先，为了诱发肝纤维化，使用硫代乙酰胺(Thioacetamide，TAA)诱导肝纤维化。具体的，由硫代乙酰胺(TAA)诱导5～6周龄雌性老鼠(C57BL/C)造成肝纤维化模型，TAA稀释于生理盐水中，采用200mg/kg剂量腹腔注射，1周3次，共7周。动物共三个组，正常对照组、假手术组(PBS注射组)、抗肝纤维化制剂干预组、动物数量分别为：8只、8只、8只。
采用PBS将实施例1的作为抗肝纤维化制剂的分泌上清稀释至总蛋白浓度为2mg/ml，得到抗肝纤维化制剂注射液。TAA注射7周后，正常对照组不做处理，假手术组通过脾脏注射予以250ulPBS溶液，抗肝纤维化制剂干预组通过脾脏注 射250ul的抗肝纤维化制剂注射液。注射完毕的7日后，取小鼠肝脏用福尔马林溶液浸泡进行固定后包埋和切片后用于HE染色检测，马森三色检测和天狼星检测，对小鼠肝纤维化程度进行综合分析。
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如图52所示，经过7周TAA药物的诱导，PBS注射组小鼠肝脏表面不平整，呈粗糙质地，免疫形态学显示胶原广泛存在，纤维之间相互连接将正常肝组织间隔。而经过抗肝纤维化制剂治疗后，抗肝纤维化制剂干预组小鼠肝脏质地更接近正常组，免疫形态学显示纤维组织明显在肝脏中减少，纤维组织呈现更为纤细的结构，这提示抗肝纤维化制剂干预明显改善了TAA诱导的小鼠肝纤维化程度。
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如图53所示，经过7周TAA药物的诱导，PBS注射组大鼠肝脏内活化的肝星状细胞(α-SMA阳性细胞)数量明显增多，而抗肝纤维化制剂干预组中，活化肝星状细胞(α-SMA阳性细胞)数量较假手术组明显减少，这说明抗肝纤维化制剂可抑制体内肝星状细胞的活化。
本发明实施例的qPCR测试过程如下：使用Eastep Super RNA提取试剂盒(货号为LS1040，来源于Promega)提取总mRNA。使用
Figure PCTCN2022079802-appb-000002
第一链cDNA合成试剂盒(货号为R211-01，来源于Vazyme)进行反转录。然后，使用AceQ qPCR SYBR Green Master Mix(货号为Q131-02，来源于Vazyme)和Life Technology ABI 7500系统开发实时PCR。GAPDH表达用作内部对照，确定阈值周期(CT)，并使用Δ(ΔCT)方法计算基因表达的相对变化。本发明实施例使用透射电子显微镜(型号为Jem 1200ex II，来源于JEOL)观察细胞的形态。将细胞用2.5％戊二醛和2％锇酸固定，然后脱水并包埋在环氧树脂中，切成80nm厚的切片，然后用醋酸铀酰和醋酸铅双重染色后观察。
本发明实施例的RNA测序和生物信息学分析使用
Figure PCTCN2022079802-appb-000003
试剂从肝组织中分离总RNA；使用DNA酶I(来源于TaKara)去除基因组DNA；RNA样品的浓度和纯度由2100生物分析仪(来源于安捷伦)测定，并使用ND-2000进行定 量。文库制备和Illumina Hiseq xten/Nova seq 6000测序RNA seq转录组文库按照来自Illumina的TruSeqTM RNA样品制备试剂盒制备，使用1μg总RNA；根据Illumina的文库构建方案的说明，片段RNA经过第一链和第二链cDNA合成，然后以低周期进行接头连接和富集；量化后，使用Illumina HiSeq xten/NovaSeq 6000测序仪在广州RiboBio有限公司对配对末端RNA seq测序文库进行测序。
本发明实施例的基因表达通过EDASEQ标准化。使用版本1.10.1的DESeq2获得差异表达基因，Q值的截止值<0.05和log2(折叠变化)>1用于识别差异表达基因。选择所有差异表达的mRNA进行GO分析clusterProfiler。使用glbase进行其他分析。
本发明实施例使用含有蛋白酶抑制剂混合物(P1010，来源于Beyotime)的RIPA缓冲液(P0013B，来源于Beyotime)提取细胞或分泌体的总蛋白。样品在冰上超声处理30秒，然后在4℃下以12000xg离心15分钟。收集上清液并用BCA蛋白质分析试剂盒(ZJ101，来源于Epizyme)定量。定量蛋白质样品通过5x SDS-PAGE(P0015，来源于Beyotime)进行解析，并转移至疏水性PVDF转移膜(IPVH00010，来源于默克密理博)。将膜在TBST中的5％BSA中封闭1.5小时，并在4℃下与一级抗体孵育过夜。然后用TBST洗涤膜三次，并在室温下与二级抗体孵育2小时。使用增强ECL化学发光检测试剂盒(E411-04，来源于Vazyme和数字发光图像分析仪(BioRad)检测印迹。使用ImageJ软件或QingXiang软件确定每个波段的密度分析。使用的一级和二级抗体见表1。
表1
Figure PCTCN2022079802-appb-000004
Figure PCTCN2022079802-appb-000005
本发明实施例使用来源于Beyotime的膜联蛋白V-FITC凋亡检测试剂盒以及来源于Biogems的膜联蛋白V-APC凋亡检测试剂盒通过膜联蛋白V/碘化丙啶(PI)或膜联蛋白V/7-AAD分析检测LX-2中的凋亡。具体的，收集细胞并重新悬浮在结合缓冲液中，然后根据试剂盒说明用膜联蛋白V和PI或7-AAD染色。0.5μM的蛋白激酶抑制剂Staurosporine用作促凋亡对照(阳性对照)。采用BD-facverse流式细胞仪检测荧光，并用FlowJo软件进行数据分析。
本发明实施例使用细胞因子抗体阵列(AAH-INF-G3，
Figure PCTCN2022079802-appb-000006
系列)测量培养上清液中40种细胞因子的表达。用激光扫描仪检测阳性信号。用于显著性分析的基本统计数据是折叠变化。差异表达蛋白(DEP)被定义为折叠变化超过1.2或小于0.83(绝对对数fc>0.263)的蛋白质。细胞因子的功能通过基因本体(GO)注释进行注释。
原始质谱(MS)数据文件使用蛋白质组发现(PD)软件(版本2.4.0.305)和内置的Sequest HT搜索引擎进行处理。根据智人UniProt FASTA数据库(UniProt-Human-9606-2020-10.FASTA)搜索MS光谱列表，其中氨基甲基[C]、TMT 6复合物(K)和TMT 6复合物(N-术语)作为固定修饰，氧化(M)和乙酰基(蛋白质N-术语)作为可变修饰。用于鉴定肽的参数为：10ppm前体离子质量耐受性，0.02Da片段质量耐受性，最多2次缺失裂解。PSM和肽水平的错误发现率(FDR)均设置为0.01。通过基因本体(GO)注释对蛋白质的功能进行了注释(http://www.geneontology.org/).京都基因和基因组百科全书(KEGG) 数据库用于分析富集途径。GO-KEGG富集分析中使用了双尾Fisher精确检验。P<0.05被认为是显著的。Cytoscape 2.6版(www.Cytoscape.org)用于可视化和分析分子和蛋白质相互作用网络。差异表达的蛋白质通过层次聚类进行排列，并以热图表示。热图由R软件生成(http://www.r-project.org).。
实施例2-9
本实施例使用实施例2-1得到的HepLPCs作为第一种细胞制剂的主要成分，将第一种细胞制剂与人永生化肝星状细胞系LX-2进行共培养，考察细胞制剂对LX-2的促死亡作用。本实施例的LX-2购自Procell。
为实验观察方便起见，首先使用亲脂性膜染料DiO及DiI分别对Human-HepLPCs和LX-2进行染色，然后再将染色后的细胞重悬于DMEM完全培养基中得到不同的细胞制剂。具体的，将实施例2-1中经TEM培养基体外培养得到的Human-HepLPCs进行消化和洗涤后，分别重悬于含5uM DiO(来源于Beyotime)或DiI(来源于Beyotime)的去血清的DMEM完全培养基中，放培养箱中孵育10分钟，之后采用PBS缓冲液进行洗涤，去除未结合的染色，最后将染色细胞重新重悬于DMEM完全培养基中，得到实施例3所使用的标记细胞制剂。DiO进入细胞膜后可以侧向扩散逐渐使整个细胞的细胞膜被染成绿色；DiI进入细胞膜后可以侧向扩散逐渐使整个细胞的细胞膜被染成红色。
实施例2-9将含DiI标记的LX-2的细胞制剂和含DiO标记的HepLPCs的细胞制剂按照1：1的比例混合后添加2.5ng/ml的TGF-β，然后按3000个细胞/孔接种至低黏附的96孔板中作为共培养组；将含DiI标记的LX-2的细胞制剂按3000个细胞/孔接种至低黏附的96孔板中作为LX-2组；将含DiI标记的LX-2的细胞制剂添加2.5ng/ml的TGF-β后按3000个细胞/孔接种至低黏附的96孔板中作为LX-2添加组。对LX-2组、LX-2添加组和共培养组共培养48小时后，吸弃培养基，在镜下进行观察拍照后，分别收集LX-2组和LX-2添加组的RNA；对于共培养组，采用流式细胞仪将带有红色DiI荧光标记的LX2细胞分选出， 之后收集该细胞的RNA。
采用RNA提取试剂盒(购自Promaga公司)分别提取3组中LX2的总RNA；使用SYBR Green PCR kit(购自诺维赞公司)在PCR仪器上(购自Roche公司)中进行。验证基因包括胶原相关基因COL3A1、COL1A1；纤维化相关基因a-SMA、Vimentin、Timp1。
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本实施例对LX-2组、LX-2添加组和共培养组共培养48小时后使用定量荧光PCR(Real-Time PCR)对各组LX-2细胞的胶原相关基因和纤维化相关基因进行测定，得到图54所示的各组LX-2细胞的胶原相关基因和纤维化相关基因表达情况对比图。参照图54，经过TGF-β1活化的肝星状细胞，纤维化相关基因COL3A1、COL1A1、a-SMA、Vimentin、Timp1和Desimin水平均明显上调，而经TGF-β1活化的肝星状细胞与肝前体细胞共培养后，上述基因均明显下调，证明肝前体细胞能显著抑制肝星状细胞在体外的活化。
实施例2-10
本实施例提供了由实施例2-1的Human-HepLPCs作为具有前体特征的肝源细胞制备的第二细胞制剂在制备抗肝纤维化药物方面的应用。
其中，所述第二细胞制剂为Human-HepLPCs经PBS重悬得到的细胞悬液。具体的，在500μl PBS中重悬5x10 6个Human-HepLPCs。
所述肝纤维化体外类器官模型为硫代乙酰胺(TAA)诱导的哺乳动物肝硬化模型。建模方法如下：对5～6周龄的雌性Sprague-Dawley大鼠(来源于Vitalriver)每周用生理盐水稀释的TAA以200mg/kg的注射剂量进行诱导，每周注射两次，诱导时间为13周以完成建模。
本实施例中：对建模成功的8只大鼠进行任何处理，作为正常组；对建模成功的8只大鼠脾脏注射500μl PBS，作为假手术组；以首次诱导作为初始时间 节点，对建模成功的8只大鼠在第13周和第15周按0.2mg/kg的注射剂量每天脾脏注射免疫抑制药物他克莫司(FK506)，在首次注射FK506后的第二天开始每周两次脾脏注射第二细胞制剂，并在第17周的第三天处死大鼠。
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本实施例取各实验组大鼠的肝脏进行进一步染色分析、羟脯氨酸含量分析和肝纤维化评分，具体参照图55至图60可知，注射第二细胞制剂可减少细胞外基质(ECM)的积累，降低大鼠的羟脯氨酸水平，对大鼠的肝硬化有所缓解。
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图55为正常组、假手术组和细胞移植组取肝组织进行H&E染色、picro-Sirius天狼星红染色、Masson三色染色和纤维粘连蛋白(FN)免疫染色后得到的照片对比图，图中比例尺为100μm。
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图56为正常组、假手术组和细胞移植组取肝组织进行picro-Sirius天狼星红染色、Masson三色染色和纤维粘连蛋白(FN)免疫染色后统计的肝纤维化区域和纤维连接蛋白阳性染色区域的相对定量情况对比图。
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图57为正常组、假手术组和细胞移植组进行羟脯氨酸含量测定的分析结果。图58为假手术组和细胞移植组取肝组织进行肝纤维化评分的分析结果。
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图59和图60分别为正常组、假手术组和细胞移植组取肝组织进行Ki67免疫组化染色得到的照片对比图以及根据图59所示的照片统计阳性染色细胞的量化结果。
实施例2-11
本实施例提供了由实施例2-2的Rat-HepLPCs作为具有前体特征的肝源细胞制备的第三细胞制剂在制备抗肝纤维化药物方面的应用。
本实施例的第三细胞制剂与实施例2-4的第二细胞制剂的区别在于：肝源细胞为Rat-HepLPCs。
所述肝纤维化体外类器官模型为四氯化碳诱导的哺乳动物肝硬化模型。建模 方法如下：对5～6周龄的雌性Sprague-Dawley大鼠(来源于Vitalriver)每周用含量为40％CCl4的CCl4和橄榄油混合注射液以1mL/kg的注射剂量进行诱导，诱导时间为13周以完成建模。
本实施例的正常组和假手术组与实施例4的处理情况一致，细胞移植组的处理情况与实施例2-4的区别在于：不使用免疫抑制药物他克莫司进行干预，且第13周注射第三细胞制剂后，于第17周处死大鼠。
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本实施例对各组大鼠取肝脏和血液进行进一步染色分析、羟脯氨酸含量分析和肝纤维化评分，具体参照图61至图65可知，注射第二细胞制剂可减少细胞外基质(ECM)的积累，降低大鼠的羟脯氨酸水平，使纤维化组织中纤维化细胞因子TGF-β及其细胞内信号分子的产生受到抑制，对大鼠的肝硬化有所缓解。
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图61为正常组、假手术组和细胞移植组取肝组织进行H&E染色、picro-Sirius天狼星红染色、Masson三色染色和纤维粘连蛋白(FN)免疫染色后得到的照片对比图，图中比例尺为100μm。
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图62为正常组、假手术组和细胞移植组取肝组织进行picro-Sirius天狼星红染色、Masson三色染色和纤维粘连蛋白(FN)免疫染色后统计的肝纤维化区域和纤维连接蛋白阳性染色区域的相对定量情况对比图。
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图63为正常组、假手术组和细胞移植组取肝组织进行羟脯氨酸含量测定的分析结果。图64为假手术组和细胞移植组取肝组织进行肝纤维化评分的分析结果。
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图65为正常组、假手术组和细胞移植组血液的促纤维化、细胞外基质、信号转导相关的肝纤维化发生相关的基因表达水平进行分析后得到的热图。
本发明实施例中：
将肝组织固定在4％多聚甲醛(PFA)中，并包埋在石蜡中，随后切割成4μm 厚的切片。肝脏切片常规用苏木精和伊红(H&E)染色、picro-Sirius天狼星红染色和Masson三色(MT)染色进行胶原沉积染色。在偏振光下使用显微镜(Olympus BX50)分析I型和III型胶原定量。使用极化过滤器，天狼星红染色切片中的I型胶原纤维将呈现橙色到红色，III型胶原纤维将呈现黄色到绿色。用天狼星红染色的切片用于Ishak评分系统下的肝纤维化评分。对于免疫组织化学(IHC)，组织切片用抗纤维连接蛋白、GFP、KI67、HLA I类、CK18、ALB的一级抗体染色。两名独立个体采用双盲法计算肝纤维化评分和Ki67+细胞，并将结果平均值用于分析。H&E、picro天狼星红、MT染色和IHC的代表性图像由徕卡Aperio在Turbo拍摄。通过使用Image J软件将阳性染色面积除以总采样面积进行量化。
在低温恒温器肝脏切片(5μm厚)或细胞球体上进行免疫荧光。在抗体染色之前，将肝切片或细胞球固定在4％多聚甲醛(PFA)中，然后用0.3％Triton X-100渗透并用3％牛血清白蛋白(BSA)封闭，然后将切片孵育以获得针对α-SMA的一级抗体，在4℃下过夜切割半胱天冬酶3(详见补充表1)，并在4℃下过夜培养细胞球体以获得针对ALB、CYP3A4、TTR、CK19、SOX9、AFP(详见补充表1)的一级抗体。然后用PBS清洗样本，并用荧光标记的二级抗体染色。对照样品在未与第一抗体孵育的情况下进行类似处理。用共焦显微镜(徕卡TCS SP8)拍摄免疫荧光染色的代表性图像。
根据制造商的方案(Solarbio，BC0250)进行羟脯氨酸测定。简单地说，将肝组织(200mg)在提取液中均质化，在110℃的烘箱中煮沸2至6小时，直到没有可见的大质量，然后在16000rpm下离心20分钟，并用10mol/L NaOH(约1ml)将pH值调节至6至8。用蒸馏水将水解样品的体积设置为4ml，将上清液转移到96孔板上，并按照制造商的方案进行测量。
以下实施例制备了肝巨噬细胞调节剂作为肝病调控制剂，并考察了这种肝病调控制剂的应用。具体如下：
实施例3-1
本实施例提供了第一种肝巨噬细胞调节剂，其制备方法如下：
S0：提供人原代肝细胞；
S1：使用肝细胞增殖培养基(简记为TEM培养基)对所述原代肝细胞进行培养直至汇合度不低于80％；
S2：将经所述步骤S1后得到的细胞消化后按1：3的比例进行传代培养；
S3：将TEM培养基更换为使用无血清的高糖DMEM培养基后，继续进行24小时的体外培养；
S4：所述体外培养结束后收集细胞上清，对所述细胞上清进行离心处理以去除细胞碎片得到培养上清，将所述培养上清作为所述第一肝巨噬细胞调节剂。
本实施例的所述步骤S0中，所述人原代肝细胞来源于上海瑞德肝脏有限公司。具体的，所述人原代肝细胞在经TEM培养前经Percoll密度梯度离心联合流式分选排除掉CD24阳性和EpCAM阳性的前体细胞。
本实施例的所述步骤S1的TEM培养基中，以占HepX Basal培养基的含量计，含有1％的无血清添加剂N2(100×)，1％的无血清添加剂B27(50×)，20ng/mL的HGF，20ng/mL的EGF，20ng/mL的FGF，1.25μM的N-乙酰-L-半胱氨酸，10μg/mL的抗坏血酸，1μM的TGF-β信号抑制剂A8301，3μM的Wnt信号通路激动剂CHIR99021，10μM的ROCK激酶抑制剂Y27632，2％的青霉素-链霉素双抗(100×)和10％的胎牛血清FBS组成。其中：HepX Basal培养基、N2、B27、青霉素-链霉素双抗来源于上海源培生物科技股份有限公司；胎牛血清FBS来源于以色列Biological Industries公司；HGF来源于美国Sino Biological公司；EGF和FGF来源于美国Peprotech公司；A8301、CHIR99021和Y27632来源于上海陶速生物科技有限公司。
所述步骤S1中，对所述原代肝细胞进行培养的步骤包括：将所述人原代肝细胞使用TEM培养基重悬后以0.5-1×10 5/cm 2的密度接种于用Vitronectin XF TM(来源于加拿大干细胞技术有限公司)包板的6孔板中进行培养。培养进行的过程中，每2-3天更换一次TEM培养基。
所述步骤S2中，将经所述步骤S1后得到的细胞消化后按1：3的比例进行传代培养的步骤包括：使用0.25％的Trypsin-EDTA(来源于美国Gibco公司)进行消化，并按照1:3的比例接种入新的培养皿中扩增培养到第三代并直至细胞汇合率达80％。
所述步骤S4中，对所述细胞上清在300g离心力下离心10分钟以去除细胞碎片，得到所述第一肝巨噬细胞调节剂。
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本实施例通过流式细胞术对经所述步骤S2得到的人肝前体样细胞(HepLPCs)进行鉴别分析，得到的各肝前体相关标志物在HepLPCs细胞中的表达情况对比图请参见图34。参照图34可知，HepLPCs表达了肝前体相关标志物CK19和CD24以及肝系标志物ALB，表现出肝前体细胞特性。造血干细胞抗原CD34和白细胞共同抗原CD45的表达水平小于2％，表现出低免疫原性。
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本实施例对经TEM培养基培养10天后得到的细胞进行明场拍照，得到图66所示的HepLPCs的明场照片。参照图66可知，HepLPCs细胞呈梭形且贴壁生长，细胞长满视野且表现出高核/质比的肝前体细胞特征。
实施例3-2
本实施例提供了炎症细胞模型的建模方法，并对实施例1的第一种肝巨噬细胞调节剂(简记为HepLPCs-CM)与炎症细胞模型进行共培养，考察第一种肝巨噬细胞调节剂对M1型巨噬细胞的作用。
本实施例通过脂多糖LPS刺激巨噬细胞建立炎症细胞模型，具体过程包括： 获取小鼠原代骨髓来源巨噬细胞(BMDMs)，使用BMDM培养基重悬并接种后加入来源于近岸生物的小鼠粒细胞-巨噬细胞集落刺激因子GM-CSF，以对BMDMs进行为期7天的诱导分化直至细胞成熟，采用LPS对得到的成熟原代巨噬细胞进行6小时的体外定向极化诱导后消化巨噬细胞，得到炎症细胞模型。其中，LPS刺激的过程中控制LPS的浓度为100ng/mL。
具体的，将6～8周成年C57小鼠脊椎脱臼处死后取股骨髓腔中的骨髓团块；对上述骨髓团块经70μm细胞筛网过滤后在500g的离心力下离心5分钟后弃上清；用红细胞裂解液(来源于上海碧云天生物技术有限公司)重悬沉淀物后再次在500g的离心力下离心5分钟后弃上清得到二次沉淀物；用BMDM培养基对二次沉淀物进行反复的重悬和离心以充分洗涤沉淀中残余的红细胞裂解液以及细胞碎片。最后使用BMDM培养基对纯化后的细胞进行重悬后按(8～10)×10 5/孔接种入12孔板，接种时每孔加入小鼠GM-CSF并控制浓度为40ng/mL。接种完成后的6-8小时后，将细胞转移到新的培养皿中进行培养，每3天更换一次添加有40ng/mL小鼠GM-CSF的BMDM培养基直至培养至第7天，实现巨噬细胞的分化成熟。其中，BMDM培养基由500mL 1640培养液(来源于上海源培生物科技股份有限公司)、5％青霉素-链霉素双抗和10％FBS组成。
具体的，巨噬细胞分化成熟后，将培养基更换为含100ng/mL的LPS的BMDM培养基后进行6小时的培养，以完成体外定向极化诱导。
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本实施例采用流式细胞术对经小鼠GM-CSF诱导后得到的BMDMs以及经LPS体外定向极化诱导得到的BMDMs进行细胞鉴定，得到图67所示的总巨噬细胞占比分析结果以及图68所示的M1型巨噬细胞占比分析结果。参照图67和图68可知，经小鼠GM-CSF诱导后得到的BMDMs的巨噬细胞总表型标志物F4/80+的表达占比达到92.8％，即总巨噬细胞占比92.8％，符合BMDM实验细胞纯度；经LPS刺激6小时后得到的BMDMs中，M1表型标志物CD11c+以及F4/80+的表达占比为88.1％，即M1型巨噬细胞占比88.1％。可见BMDMs经本 实施例的小鼠GM-CSF诱导分化以及LPS刺激后得到的炎症细胞模型中，M1型巨噬细胞占比满足纯度要求。
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本实施例采用流式细胞术对未处理的BMDMs(Control组)以及经LPS体外定向极化诱导得到的BMDMs(DM+LPS组)进行细胞鉴定，得到图69所示的M1相关炎性基因表达情况对比图。参照图69，和对照组相比，经LPS刺激6小时后的BMDMs的M1相关基因表达水平均上调。其中，IL6表达水平上调为823.200±174.500，IL1β表达水平上调为8.389±0.029，iNOS表达水平上调为24.650±1.196。
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本实施例将HepLPCs-CM与前述经LPS体外定向极化诱导得到的BMDMs共培养6小时得到HepLPCs-CM+LPS组，对HepLPCs-CM+LPS组、Control组和DM+LPS组进行RNA提取和基因表达分析，得到图70所示的各组M1相关炎性基因表达情况对比图。参照图69和图70，相对于阳性对照处理组DM+LPS，经LPS体外定向极化诱导得到的BMDMs经HepLPCs-CM共培养后，M1相关基因表达显著下调。其中，IL6表达水平下调为346.300±20.810，IL1β表达水平下调为11.290±0.10，iNOS表达水平下调为169.800±9.711。
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本实施例对实验组、控制组和阳性对照处理组收集的细胞培养上清采用联科生物ELISA试剂盒进行细胞因子的浓度检测，得到图71所示的HepLPCs-CM+LPS组、Control组和DM+LPS组的细胞培养上清中M1相关炎性基因表达水平对比图。具体操作方法请参见试剂盒说明书。细胞培养上清的收集方法请参见实施例1，在此不做赘述。参照图71，相对于阳性对照处理组，经LPS体外定向极化诱导得到的BMDMs在经HepLPCs-CM处理后，上清中炎性因子分泌减少。其中，IL6分泌为138.700±32.130pg/(mL*105cell)，IL1β分泌为0.710±0.019pg/(mL*105cell)，iNOS分泌为0.095±0.001pg/(mL*105cell)。
目前，大量的实验和临床数据支持巨噬细胞在非酒精性脂肪性肝病(NAFLD)和非酒精性脂肪性肝炎(NASH)的发生和发展中的核心作用。巨噬细胞在不同因 素诱导下可出现两种经典的细胞亚群，即M1型巨噬细胞和M2型巨噬细胞。二者功能截然不同。业内通常认为，M1型巨噬细胞促进炎症反应，通过产生大量促炎性细胞因子如白细胞介素-1β(IL-1β)、TNF-α、白细胞介素-6(interleukin-6,IL-6)、以及一氧化氮(nitrogen monoxide,NO)和活性氧(reactive oxygen species,ROS)介导机体的炎症反应。
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从图70和图71以及各自的分析结果可知，HepLPCs-CM能够抑制LPS诱导的巨噬细胞炎性活化，显著减低炎症相关因子的基因及分泌蛋白表达。
实施例3
本实施例提供了修复型细胞模型的建模方法，并对实施例1的第一种肝巨噬细胞调节剂(简记为HepLPCs-CM)与修复型细胞模型进行共培养，考察第一种肝巨噬细胞调节剂对M2型巨噬细胞的作用。
本实施例通过来源于近岸生物的白介素4(IL-4)刺激巨噬细胞建立修复型细胞模型，具体过程包括：小鼠原代骨髓来源巨噬细胞(BMDMs)的获取方法以及使用小鼠GM-CSF对BMDMs进行为期7天的诱导分化的方法请参见实施例2。采用IL-4对得到的成熟原代巨噬细胞进行6小时的体外定向极化诱导后消化巨噬细胞，得到修复型细胞模型。其中，IL-4刺激的过程中控制IL-4的浓度为40ng/mL。其余具体实验步骤请参见实施例3-2。
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本实施例采用流式细胞术对经IL-4体外定向极化诱导得到的BMDMs进行细胞鉴定，得到图72所示的M2型巨噬细胞占比分析结果。参照图72可知，经IL-4刺激6小时后得到的BMDMs中，M2表型标志物CD206+以及巨噬细胞总表型标志物F4/80+的表达占比为97％，即M2型巨噬细胞占比97％。可见BMDMs经本实施例的小鼠GM-CSF诱导分化以及IL-4刺激后得到的修复型细胞模型中，M2型巨噬细胞占比满足纯度要求。
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本实施例采用流式细胞术对未处理的BMDMs(Control组)以及经IL-4体外 定向极化诱导得到的BMDMs(DM+IL-4组)进行细胞鉴定，得到图73所示的M2相关炎性基因表达情况对比图。参照图73，和Control组相比，经IL-4刺激6小时后的BMDMs的M2相关基因表达水平均上调。其中，CD206表达水平上调为114.000±3.579，IL10表达水平上调为2.634±0.028，ARG1表达水平上调为53.260±8.083。
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本实施例将HepLPCs-CM与前述经IL-4体外定向极化诱导得到的BMDMs在BMDM培养基中共培养6小时得到HepLPCs-CM+IL-4组，对HepLPCs-CM+IL-4、Control组和DM+IL-4组进行RNA提取和基因表达分析，得到图74所示的各组M2相关炎性因子IL10的分泌情况对比图。参照图73和图74，相对于DM+IL-4，经IL-4体外定向极化诱导得到的BMDMs经HepLPCs-CM共培养后，检测上清中炎性因子IL10分泌增加。IL10分泌为108.052±0.472pg/(mL*105cell)。
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业内通常认为M2型巨噬细胞主要产生免疫调节因子白细胞介素-10(interleukin-10,IL-10)等，参与Th2细胞型免疫应答，抑制炎症和纤维化，在组织的修复过程中具有重要作用。从图73和图74以及各自的分析结果可知，HepLPCs-CM能够促进IL-4诱导的修复型M2巨噬细胞基因的表达以及抗炎因子IL-10小幅度的增高。
综合实施例3-2和3-3可知，HepLPCs-CM通过影响巨噬细胞亚群的变化，起到抑制炎症反应并促进组织修复的作用。
实施例3-4
本实施例对实施例3-1的HepLPCs-CM中的外泌体成分(简记为HepLPCs-Ex)进行了提取，并考察其对实施例3-2的炎症细胞模型的作用。
本实施例使用ExoQuick-TC外泌体提取试剂盒对实施例3-1的HepLPCs-CM中的外泌体成分进行提取，得到HepLPCs-Ex。具体的提取方法请参见试剂盒说 明书。将提取得到的HepLPCs-Ex委托逍鹏生物进行NTA检测，结果表明提取的样本颗粒直径多集中于90-110nm之间，峰值在96nm。
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本实施例使用DIL标记HepLPCs-Ex后与巨噬细胞BMDMs混合后培养，考察了不同时间和不同外泌体浓度条件下考察5×10 5个巨噬细胞BMDMs对外泌体的吞噬效率，发现随时间延长，巨噬细胞吞噬外泌体越多，且所加入外泌体浓度越高，巨噬细胞吞噬越多。本实施例进一步对混合培养6小时后的样本用DAPI进行核染以及软件融合分析，得到图75所示的不同浓度外泌体DAPI核染后融合图。巨噬细胞BMDMs的制备方法请参见实施例2。参照图75，外泌体浓度从1.3ug/uL增加至5.2ug/uL并混合培养6小时后，巨噬细胞对外泌体的吞噬效果均显著，且外泌体浓度越高，巨噬细胞吞噬效率越好。
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本实施例在图75所示巨噬细胞对外泌体摄取情况指导下，控制BMDMs细胞数目为5×10 5个，外泌体浓度和HepLPCs-CM浓度均为1.3ug/uL。将HepLPCs-CM和HepLPCs-Ex分别加入到炎症细胞模型混合后培养6小时，形成CM+LPS组和EV+LPS组，通过qPCR考察Control组，DM+LPS组、CM+LPS组和EV+LPS组的M1相关炎性基因表达情况，得到图76所示的各组M1相关炎性基因水平对比图。
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参照图76，对照组DM+LPS组的M1相关炎性基因IL6、IL1β和iNOS的表达显著，通过HepLPCs-CM和HepLPCs-Ex干预均能够使得上述M1相关炎性基因表达水平显著下降，且HepLPCs-Ex干预使得上述M1相关炎性基因表达水平下降的程度与HepLPCs-CM的干预效果相当。
综上所述，HepLPCs-CM中的外泌体HepLPCs-Ex作为抑制成分，能够抑制M1型巨噬细胞的炎性活化。
实施例3-5
本实施例考察了实施例3-4的外泌体成分HepLPCs-Ex对实施例3-3的修复 型细胞模型的作用。
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本实施例在图75所示巨噬细胞对外泌体摄取情况指导下，控制BMDMs细胞数目为5×10 5个，外泌体浓度和HepLPCs-CM浓度均为1.3ug/uL。将HepLPCs-CM和HepLPCs-Ex分别加入到实施例3的修复型细胞模型混合后培养6小时，形成CM+IL4组和EV+IL4组，通过qPCR考察Control组，DM+IL4组、CM+IL4组和EV+IL4组的M2相关炎性基因表达情况，得到图77所示的各组M2相关炎性基因水平对比图。
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参照图77，相较于对照组DM+IL4组的M2相关炎性基因CD206、IL10和ARG1的表达水平，通过HepLPCs-CM和HepLPCs-Ex干预均能够使得上述M2相关炎性基因表达水平上调，且HepLPCs-Ex干预使得上述M2相关炎性基因表达水平上调的程度与HepLPCs-CM的干预效果相当。
综上所述，HepLPCs-CM中的外泌体HepLPCs-Ex作为促进成分，能够促进修复型巨噬细胞的产生。
实施例3-6
本实施例提供了NASH小鼠模型的建模方法。
本实施例使用购自上海灵畅生物科技有限公司的健康雄性5周龄野生型(WT)C57BL/6小鼠进行建模。小鼠的体重平均达到20g。每组7只小鼠，分组情况如下：
模型组：以2.5-3g/只的饲养量投喂CDAHFD高脂饲料，一周添加2-3次。
对照组：以2.5-3g/只的饲养量进行标准饮食投喂。
各组小鼠经检疫并预适应1周后，在无特定病原体级环境饲养(SPF级)饲养环境下饲养，控制室温20-26℃，湿度40-70％，光照12小时明暗交替，每笼动物不超过5只。
模型组各小鼠饲养3周后得到早期NASH小鼠模型；饲养6周后得到中晚期NASH小鼠模型。
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本实施例取早期NASH小鼠模型和对照组小鼠的肝脏组织进行石蜡包埋、切片制样和H&E染色，得到图78和图79分别所示的H&E染色照片，早期NASH小鼠模型相比对照组肝脏细胞出现气球样变，呈现明显的弥漫性脂肪样变。
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本实施例对早期NASH小鼠模型的肝脏组织进行石蜡包埋和切片制样后分别进行Masson染色和油红O染色，得到图80和图81分别所示的Masson染色照片和油红O染色照片可知，早期NASH小鼠模型的小鼠肝脏中存在大量脂质沉积。
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本实施例对早期NASH小鼠模型的肝脏组织进行石蜡包埋和切片制样后，分别进行M1型巨噬细胞特异性标志物CD68的免疫组化染色以及M2型巨噬细胞特异性标志物CD163的免疫组化染色，分别得到图82和图83所示的染色照片可知，早期NASH小鼠模型的小鼠肝组织伴随炎性细胞浸润。
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本实施例通过眼眶取血检测血液样本肝功相关指标AST、ALT、LDH以及检测离体肝脏组织中总胆固醇TC和肝脏甘油三脂TG含量，评估早期NASH小鼠模型和对照组小鼠的肝细胞脂质沉积情况，得到图84所示的早期NASH小鼠模型和对照组的血生化及TC、TG含量对比图。参照图84可知，早期NASH小鼠模型与正常饲料喂养的对照组相比，肝脏血生化指标AST/ALT较正常组上升6～7倍，LDH较正常组上升2倍，提示NASH小鼠出现较为明显的肝损伤。早期NASH小鼠模型与对照组TG含量无明显差别，△△P<0.05，无统计学意义；而TC含量则有明显降低。
可见，特殊高脂饮食喂养小鼠3周后NASH小鼠疾病模型构建成功。
实施例3-7
本实施例使用实施例3-1的HepLPCs对早期NASH小鼠模型进行干预，考察HepLPCs对NASH疾病的作用。其中早期NASH小鼠模型进行如下分组，每组7只小鼠：
模型组：口服生理盐水。
免疫抑制组：口服免疫抑制剂FK506，剂量为0.2mg/kg；
细胞低剂量治疗组：术前一天口服给予免疫抑制剂0.2mg/kg；术中每只腹腔注射200微升细胞注射液，使用生理盐水重悬0.5×10 6个HepLPCs得到细胞注射液；
细胞高剂量治疗组：和细胞低剂量治疗组的区别在于，每只注射的细胞注射液中含1×10 6个HepLPCs；
假手术组：每只脾脏注射200微升生理盐水；
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上述各组完成注射或口服后第10天处死小鼠，取肝脏组织进行石蜡包埋和切片后进行H&E染色和油红O染色，观察肝脏病理改变以脂质沉积情况，得到图85所示的假手术组、细胞低剂量治疗组和细胞高剂量治疗组小鼠的肝脏H&E染色照片和油红O染色照片对比图。参照图86，相较于假手术组，细胞低剂量治疗组和细胞高剂量治疗组显示肝脏的脂质沉积情况显著减轻。
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本实施例应用NAFLD活动度评分(NAS)对肝脏镜下病理改变进行分析，得到图86所示的假手术组、细胞低剂量治疗组和细胞高剂量治疗组的NAS评分对比图。参照图86，假手术组组NAS评分在4-6分之间，符合NASH的病理评定标准，而各细胞治疗组低于2分，显示可排除NASH。显示HepLPCs对早期NASH小鼠模型起到了积极的干预作用。
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本实施例对假手术组、细胞低剂量治疗组和细胞高剂量治疗组小鼠的肝脏切片进行Masson染色和肝纤维化区域统计，得到图87所示的假手术组、细胞低 剂量治疗组和细胞高剂量治疗组小鼠的肝脏切片的Masson染色照片以及图88所示的各组肝纤维化区域统计结果对比图可知，细胞低剂量治疗组和细胞高剂量治疗组小鼠肝脏中的纤维化区域相比较假手术对照组均显著减少，特别是细胞高剂量治疗组纤维减少情况最明显，显示HepLPCs治疗早期NASH小鼠模型能对肝脏纤维化起到较强的缓解能力。
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本实施例对假手术组、细胞低剂量治疗组和细胞高剂量治疗组小鼠的肝脏切片进行Ki67染色，得到图89所示的染色照片可知，相较于假手术组，HepLPCs促进了细胞低剂量治疗组和细胞高剂量治疗组小鼠的肝再生。
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本实施例对正常对照组NC、免疫抑制组CDA+FK506、口服生理盐水的模型组CDA+saline、假手术组CDA+sham、细胞低剂量治疗组CDA+HepLPCs(low dose)和细胞高剂量治疗组CDA+HepLPCs(high dose)小鼠进行眼眶取血，通过血生化水平分析考察了各组的ALT、AST、LDH指标，得到图90所示的各组ALT、AST、LDH指标水平对比图。与假手术组相比，两组细胞治疗组在ALT、AST、LDH三项指标均有一定量的下降。细胞低剂量治疗组肝功能指标ALT治疗窗口下降16％；细胞高剂量治疗组肝功能指标ALT治疗窗口下降31.5％；与假手术组相比，细胞低剂量治疗组肝功能指标AST治疗窗口下降15％；细胞高剂量治疗组肝功能指标AST治疗窗口下降22％；与假手术组相比，细胞低剂量治疗组肝功能指标LDH治疗窗口下降13.9％；细胞高剂量治疗组肝功能指标LDH治疗窗口下降17％。由此可见，HepLPCs可改善早期NASH小鼠模型的肝功能。
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本实施例对正常对照组、假手术组、细胞低剂量治疗组小鼠和细胞高剂量治疗组小鼠肝脏的TC和TG含量进行测定，得到图91所示的各组肝脏TC和TG含量对比图。与假手术组相比，细胞高剂量治疗组肝脏肝固醇TC含量下降23.4％；与假手术组相比，细胞低剂量治疗组肝脏甘油三酯TG含量下降17.6％；细胞高剂量治疗组肝脏甘油三酯TG无明显差异；从TC、TG检测来看，假手术组TC、 TG含量均较正常组高。尽管不同的细胞剂量缓解NASH肝脂代谢的能力不同，但总的来说，HepLPCs显示出可显著改善NASH肝脏脂代谢能力。
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本实施例对假手术组、细胞低剂量治疗组小鼠和细胞高剂量治疗组小鼠的肝脏组织进行抗CD163和抗CD68的免疫组化染色并对不同组肝脏中CD163 +巨噬细胞和CD68 +巨噬细胞数量进行了统计，得到图92所示的各组的抗CD163和抗CD68的免疫组化染色照片对比图，以及图93所示的各组肝脏中CD163 +巨噬细胞和CD68 +巨噬细胞数量对比图。两组细胞治疗组与假手术组小鼠相比较,肝脏内CD163 +巨噬细胞数量都明显增加，CD68 +巨噬细胞稍有减少。以上结果提示HepLPCs抑制了巨噬细胞相关的炎症反应，并促进修复型巨噬细胞的产生。
实施例3-8
本实施例使用实施例3-1的HepLPCs对中晚期NASH小鼠模型进行干预，考察HepLPCs对NASH疾病的作用。其中中晚期NASH小鼠模型的分组处理情况请参见实施例3，区别在于：细胞低剂量治疗组每只小鼠脾脏输注的细胞注射液含1×10 6个细胞，细胞高剂量治疗组每只小鼠脾脏输注的细胞注射液含2×10 6个细胞。
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本实施例对中晚期NASH小鼠模型的假手术组、细胞低剂量治疗组和细胞高剂量治疗组小鼠肝脏切片进行H&E染色，得到图94所示的染色照片可知，相较于假手术组，细胞低剂量治疗组和细胞高剂量治疗组小鼠肝脏显示脂质沉积得以改善。
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本实施例对中晚期NASH小鼠模型的假手术组、细胞低剂量治疗组和细胞高剂量治疗组小鼠肝脏切片进行Masson染色，并统计了纤维化区域,得到图95所示的各组Masson染色照片对比图，以及图96所示的各组肝纤维化区域统计结果对比图。细胞低剂量治疗组和细胞高剂量治疗组的小鼠肝脏中的纤维化区域相比较对照组均显著减少，特别是细胞低剂量治疗组的纤维减少情况最明显， 可见HepLPCs对中晚期NASH小鼠模型的肝脏纤维化起到较强的缓解能力。
实施例3-9
本实施例提供了炎症细胞模型的建模方法。
目前，大量的实验和临床数据支持巨噬细胞在非酒精性脂肪性肝病(NAFLD)和非酒精性脂肪性肝炎(NASH)的发生和发展中的核心作用。巨噬细胞在不同因素诱导下可出现两种经典的细胞亚群，即M1型巨噬细胞和M2型巨噬细胞。二者功能截然不同。业内通常认为，M1型巨噬细胞促进炎症反应，通过产生大量促炎性细胞因子如白细胞介素-1β(IL-1β)、TNF-α、白细胞介素-6(interleukin-6,IL-6)、以及一氧化氮(nitrogen monoxide,NO)和活性氧(reactive oxygen species,ROS)介导机体的炎症反应。
因此，有必要进行炎症细胞模型的建模，以通过炎症细胞模型深入考察HepLPCs对M1型巨噬细胞的作用。
具体的，将6～8周成年C57小鼠脊椎脱臼处死后取股骨髓腔中的骨髓团块；对上述骨髓团块经70μm细胞筛网过滤后在500g的离心力下离心5分钟后弃上清；用红细胞裂解液(来源于上海碧云天生物技术有限公司)重悬沉淀物后再次在500g的离心力下离心5分钟后弃上清得到二次沉淀物；用BMDM培养基对二次沉淀物进行反复的重悬和离心以充分洗涤沉淀中残余的红细胞裂解液以及细胞碎片。最后使用BMDM培养基对纯化后的细胞进行重悬后按(8～10)×10 5/孔接种入12孔板，接种时每孔加入小鼠GM-CSF并控制浓度为40ng/mL。接种完成后的6-8小时后，将细胞转移到新的培养皿中进行培养，每3天更换一次添加有40ng/mL小鼠GM-CSF的BMDM培养基直至培养至第7天，实现巨噬细胞的分化成熟。其中，BMDM培养基由500mL 1640培养液(来源于上海源培生物科技股份有限公司)、5％青霉素-链霉素双抗和10％FBS组成。
具体的，巨噬细胞分化成熟后，将培养基更换为含100ng/mL的LPS的BMDM 培养基后进行6小时的培养，以完成体外定向极化诱导。
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本实施例采用流式细胞术对经小鼠GM-CSF诱导后得到的BMDMs以及经LPS体外定向极化诱导得到的BMDMs进行细胞鉴定，得到图97所示的总巨噬细胞占比分析结果以及图98所示的M1型巨噬细胞占比分析结果。经小鼠GM-CSF诱导后得到的BMDMs的巨噬细胞总表型标志物F4/80+的表达占比达到92.8％，即总巨噬细胞占比92.8％，符合BMDM实验细胞纯度；经LPS刺激6小时后得到的BMDMs中，M1表型标志物CD11c+以及F4/80+的表达占比为88.1％，即M1型巨噬细胞占比88.1％。可见BMDMs经本实施例的小鼠GM-CSF诱导分化以及LPS刺激后得到的炎症细胞模型中，M1型巨噬细胞占比满足纯度要求。
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本实施例采用流式细胞术对未处理的BMDMs(Control组)以及经LPS体外定向极化诱导得到的BMDMs(DM+LPS组)进行细胞鉴定，得到图99所示的M1相关炎性基因表达情况对比图。参照图99，和对照组相比，经LPS刺激6小时后的BMDMs的M1相关基因表达水平均上调。其中，IL6表达水平上调为823.200±174.500，IL1β表达水平上调为8.389±0.029，iNOS表达水平上调为24.650±1.196。
实施例3-10
本实施例提供了修复型巨噬细胞模型的建模方法。
业内通常认为M2型巨噬细胞主要产生免疫调节因子白细胞介素-10(interleukin-10,IL-10)等，参与Th2细胞型免疫应答，抑制炎症和纤维化，在组织的修复过程中具有重要作用。
因此有必要建立修复型巨噬细胞模型，并通过修复型巨噬细胞模型深入考察HepLPCs对M2型巨噬细胞的作用。
本实施例通过IL-4刺激巨噬细胞建立修复型巨噬细胞模型，具体过程包括：小鼠原代骨髓来源巨噬细胞(BMDMs)的获取方法以及使用小鼠GM-CSF对BMDMs进行为期7天的诱导分化的方法请参见实施例3-2。采用IL-4对得到的成熟原代巨噬细胞进行6小时的体外定向极化诱导后消化巨噬细胞，得到修复型巨噬细胞模型。其中，IL-4刺激的过程中控制IL-4的浓度为40ng/mL。其余具体实验步骤请参见实施例5。
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本实施例采用流式细胞术对经IL-4体外定向极化诱导得到的BMDMs进行细胞鉴定，得到图100所示的M2型巨噬细胞占比分析结果可知，经IL-4刺激6小时后得到的BMDMs中，M2表型标志物CD206+以及巨噬细胞总表型标志物F4/80+的表达占比为97％，即M2型巨噬细胞占比97％。可见BMDMs经本实施例的小鼠GM-CSF诱导分化以及IL-4刺激后得到的修复型巨噬细胞模型中，M2型巨噬细胞占比满足纯度要求。
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本实施例采用流式细胞术对未处理的BMDMs(Control组)以及经IL-4体外定向极化诱导得到的BMDMs(DM+IL-4组)进行细胞鉴定，得到图101所示的M2相关炎性基因表达情况对比图，和Control组相比，经IL-4刺激6小时后的BMDMs的M2相关基因表达水平均上调。其中，CD206表达水平上调为114.000±3.579，IL10表达水平上调为2.634±0.028，ARG1表达水平上调为53.260±8.083。
实施例3-11
本实施例对实施例3-1的HepLPCs进行体外培养，从得到的体外培养上清中提取外泌体成分(简记为HepLPCs-Ex)，并考察其对实施例3-5的炎症细胞模型的作用。
实施例3-1的HepLPCs的体外培养以及获取体外培养上清(HepLPCs-CM)的过程包括：实施例1的传代培养结束后，将TEM培养基更换为使用无血清的 高糖DMEM培养基后，继续进行24小时的体外培养；所述体外培养结束后收集细胞上清，对所述细胞上清在300g离心力下离心10分钟以去除细胞碎片从而得到体外培养上清。
本实施例使用ExoQuick-TC外泌体提取试剂盒对HepLPCs-CM中的外泌体成分进行提取，得到HepLPCs-Ex。具体的提取方法请参见试剂盒说明书。将提取得到的HepLPCs-Ex委托逍鹏生物进行NTA检测，结果表明提取的样本颗粒直径多集中于90-110nm之间，峰值在96nm。
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本实施例控制BMDMs细胞数目为5×10 5个，外泌体浓度和HepLPCs-CM浓度均为1.3ug/uL。将HepLPCs-CM和HepLPCs-Ex分别加入到炎症细胞模型混合后培养6小时，形成CM+LPS组和EV+LPS组，通过qPCR考察Control组，DM+LPS组、CM+LPS组和EV+LPS组的M1相关炎性基因表达情况，得到图102所示的各组M1相关炎性基因水平对比图。
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参照图102，对照组DM+LPS组的M1相关炎性基因IL6、IL1β和iNOS的表达显著，通过HepLPCs-CM和HepLPCs-Ex干预均能够使得上述M1相关炎性基因表达水平显著下降，且HepLPCs-Ex干预使得上述M1相关炎性基因表达水平下降的程度与HepLPCs-CM的干预效果相当。
综上所述，HepLPCs在无血清基础培养基的诱导下释放的外泌体HepLPCs-Ex能够抑制M1型巨噬细胞的炎性活化。
实施例3-12
本实施例考察了实施例3-10的外泌体成分HepLPCs-Ex对前述修复型巨噬细胞模型的作用。
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本实施例控制BMDMs细胞数目为5×10 5个，外泌体浓度和HepLPCs-CM浓度均为1.3ug/uL。将HepLPCs-CM和HepLPCs-Ex分别加入到实施例6的修复 型巨噬细胞模型混合后培养6小时，形成CM+IL4组和EV+IL4组，通过qPCR考察Control组，DM+IL4组、CM+IL4组和EV+IL4组的M2相关炎性基因表达情况，得到图103所示的各组M2相关炎性基因水平对比图。
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参照图103，相较于对照组DM+IL4组的M2相关炎性基因CD206、IL10和ARG1的表达水平，通过HepLPCs-CM和HepLPCs-Ex干预均能够使得上述M2相关炎性基因表达水平上调，且HepLPCs-Ex干预使得上述M2相关炎性基因表达水平上调的程度与HepLPCs-CM的干预效果相当。
综上所述，HepLPCs在无血清基础培养基的诱导下释放的外泌体HepLPCs-Ex能够促进修复型巨噬细胞，即M2型巨噬细胞的产生。
以下实施例制备了免疫细胞的增殖抑制剂以及增殖抑制细胞制剂分别作为肝病调控制剂，并考察了这种肝病调控制剂的应用。具体如下：
实施例4-1
本实施例将来源于Invitrogen公司的供体1来源人原代肝细胞分别体外培养为肝前体样细胞HepLPC，并获取条件培养上清(HepLPC-CM)，然后将HepLPC-CM与ConA刺激的小鼠脾脏细胞共培养，考察HepLPC-CM对脾脏细胞的增殖抑制作用。
HepLPC的体外培养方法包括：将人原代肝细胞经Percoll密度梯度离心联合流式分选排除掉CD24阳性和EpCAM阳性的前体细胞后，先以2×10 4个细胞/cm 2的密度接种于Matrigel(Corning公司)包被的细胞培养6孔板中经含10％血清的WE培养基(Invitrogen公司)培养至贴壁，然后以2×10 4/cm 2的接种密度转移至TEM培养基中进行10天的培养，且每隔一天更换一次TEM培养基。
本实施例的TEM培养基组成和各组成成分的来源如下：DMEM/F12基础培养基(Invitrogen公司)，以及以占DMEM/F12基础培养基的含量计：体积含量1％ 的N2添加剂和体积含量1％的B27添加剂(Invitrogen公司)，1mmol/L丙酮酸钠(Invitrogen公司)，10μg/mL抗坏血酸(Sigma-Aldrich公司)，20ng/mL肝细胞生长因子HGF(Peprotech公司)，20ng/mL表皮细胞生长因子EGF(Peprotech公司)，10μmol/L ROCK激酶抑制剂Y27632(TargetMol公司)，3μmol/L Wnt信号通路激动剂CHIR99021(TargetMol公司)，1μmol/L TGF-β信号抑制剂A83-01(TargetMol公司)，1μmol/L一磷酸鞘氨酸S1P(Santa Cruz公司)以及5μmol/L吲哚乙酸LPA(Santa Cruz公司)。
HepLPC-CM的制备方法包括：消化使用0.25％的Trypsin-EDTA(来源于美国Gibco公司)对经TEM培养10天得到的前体样细胞进行消化，并按照1:3的比例接种入新的培养皿中扩增培养到第三代并直至细胞汇合率达80％，然后将TEM培养基更换为无血清的DMEM培养基进行24小时的体外培养；体外培养结束后收集细胞上清，对所述细胞上清在300g离心力下离心10分钟以去除细胞碎片，得到条件培养上清HepLPC-CM。
加入刺激剂的小鼠脾脏细胞悬液的制备方法为：断颈处死1周龄的C57BL/6小鼠后取脾脏在200目筛网研磨后使用淋巴分离液冲洗，收集包含淋巴细胞的分离液并使用含10％胎牛血清的RPMI-1640完全培养基重悬后在800g离心力下低温离心30分钟；离心结束后吸取淋巴细胞层后使用含10％胎牛血清的RPMI-1640完全培养基重悬后在250g离心力下低温离心10分钟，使用含10％胎牛血清的RPMI-1640完全培养基再次重悬得到的淋巴细胞层并加入含10％胎牛血清的RPMI-1640完全培养基重悬的刀豆蛋白A(ConA)悬液作为刺激剂。
共培养步骤包括：将前述加入刺激剂的小鼠脾脏细胞悬液以2×10 4个细胞/cm 2的密度接种于6孔板；使用HepLPC-CM以及使用含10％胎牛血清的RPMI-1640完全培养基重悬来自供体1的HepLPC-CM，得到HepLPC-CM浓度分别为100％、50％、25％、12.5％以及6.25％的HepLPC-CM悬液，将上述HepLPC-CM悬液分别加入到接种有小鼠脾脏细胞的6孔板中，控制ConA的终 浓度为2.5微克/毫升，形成以下共培养组：100％-CM组、50％-CM组、25％-CM组、12.5％-CM组以及6.25％-CM组。另外，以不添加HepLPC-CM的共培养组作为阳性对照组。
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本实施例分别使用qPCR和流式细胞术分析了供体1的人原代肝细胞和经TEM培养得到的HepLPC的基因表达情况，结果如图104和图105所示。人原代肝细胞在TEM培养基作用下，肝祖细胞基因Ck7、Ck19和Sox9的表达显著增加，而Alb、Cyp3a4和Hnf4α这类肝实质细胞标志物表达显著下降。Human-HepLPCs的肝细胞标志物HNF4α以及肝干细胞/肝祖细胞标志物CD24and CK19显著表达，造血干细胞抗原CD34、白细胞共同抗原CD45以及肝胎儿细胞标志物AFP的表达水平均小于2％。Human-HepLPCs不表达MHC II类抗原HLA-DP，HLA-DQ和HLA-DR，表现出低免疫原性。
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本实施例中，共培养72小时后，使用CFSE细胞增殖检测试剂盒对阳性对照组以及各共培养组进行处理后上流式仪检测，考察各共培养组中HepLPC-CM对脾脏细胞的增殖抑制情况，结果如图106所示。根据图106所示阳性对照组和各共培养组的增殖脾脏细胞占比情况可知，100％-CM组、50％-CM组、25％-CM组、12.5％-CM组以及6.25％-CM组中，HepLPC-CM对脾脏细胞增殖的抑制率分别为47％、50％、40％、37％和34％。可见，HepLPC-CM能够显著抑制小鼠脾脏细胞的增殖，且呈现剂量依赖性。
实施例4-2
本实施例将来源于Invitrogen公司的不同供体来源人原代肝细胞分别体外培养为肝前体样细胞HepLPC，并获取不同供体来源的条件培养上清，然后将不同供体来源的条件培养上清分别与ConA刺激的小鼠脾脏细胞共培养，考察HepLPC-CM对脾脏细胞的增殖抑制作用。
人原代肝细胞体外培养方法、条件培养上清制备方法、加入刺激剂的小鼠脾 脏细胞悬液制备方法请参见实施例4-1。共培养步骤与实施例1的区别在于：每种供体来源的HepLPC不使用含10％胎牛血清的RPMI-1640完全培养基重悬，而是直接与加入刺激剂的小鼠脾脏细胞共培养。
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本实施例中，共培养72小时后，使用CFSE细胞增殖检测试剂盒对阳性对照组以及含不同供体来源HepLPC-CM的共培养组进行处理后上流式仪检测，考察各供体来源HepLPC-CM的共培养组对脾脏细胞的增殖抑制情况与对应的的阳性对照组中脾脏细胞的增殖情况，结果如图107至图110所示。供体1、供体2、供体3和供体4来源的HepLPC-CM对脾脏细胞增殖的抑制率分别为42％、69％、59％和56％。可见，不同供体来源HepLPC-CM对脾脏细胞增殖的抑制效果体现出差异性，有利于后续开展免疫耐受药物的异质性研究。
实施例4-3
本实施例将来源于Invitrogen公司的供体1来源人原代肝细胞分别体外培养为肝前体样细胞HepLPC，然后将HepLPC与ConA刺激的小鼠脾脏细胞共培养，考察HepLPC-CM对脾脏细胞的增殖抑制作用。
HepLPC的体外培养方法、TEM培养基组成和各组成成分、加入刺激剂的小鼠脾脏细胞悬液的制备方法请参见前述实施例。
共培养步骤包括：前述TEM培养结束后细胞融合率达90％，再将得到的细胞按1×10 4个/平方厘米的接种密度在TEM培养基中以1：3的比例进行连续传代培养至第三代；使用0.05％胰酶/EDTA液消化前述传代培养至第三代且细胞汇合率达80％的HepLPC后，使用含10％胎牛血清的RPMI-1640完全培养基终止消化并重悬为单细胞悬液；使用PBS反复洗涤单细胞悬液以确保去除消化液；使用含10％胎牛血清的RPMI-1640完全培养基重悬并调整细胞浓度；将前述加入刺激剂的小鼠脾脏细胞悬液接种于6孔板，通过控制加入刺激剂的小鼠脾脏细胞悬液的加入量调整HepLPC和小鼠脾脏细胞的数量比分别为1:1，1:2， 1:5，1:10和1:20，从而得到若干共培养组。另外，以不添加HepLPC的共培养组作为阳性对照组，以添加终浓度10nM免疫抑制药物他克莫司FK506的共培养组作为FK506对照组。
本实施例分别使用qPCR和流式细胞术分析了供体1的人原代肝细胞和经TEM培养得到的HepLPC的基因表达情况请参见前述实施例。
[根据细则91更正 30.03.2022]　
本实施例中，共培养72小时后，使用CFSE细胞增殖检测试剂盒对阳性对照组、FK506对照组以及各共培养组进行处理后上流式仪检测，考察各共培养组中HepLPC和FK506对脾脏细胞的增殖抑制情况，结果如图111所示。根据图111所示阳性对照组、FK506对照组和各共培养组的增殖脾脏细胞占比情况可知，HepLPC和小鼠脾脏细胞的数量比分别为1:1，1:2，1:5，1:10和1:20的条件下，HepLPC对脾脏细胞增殖的抑制率分别为90％、87％、85％、80％和50％，可见HepLPC对脾脏细胞增殖的抑制作用具有剂量依赖性。尤其值得注意的是，FK506对照组对脾脏细胞增殖的抑制率为82％，当HepLPC和小鼠脾脏细胞的数量比在1:1-1:5的宽范围内，HepLPC所显示出的对小鼠脾脏细胞的增殖抑制效果与FK506相当甚至强于FK506的增殖抑制效果。
实施例4-4
本实施例将来源于Invitrogen公司的不同供体来源人原代肝细胞分别体外培养为肝前体样细胞HepLPC，将不同供体来源的HepLPC分别与ConA刺激的小鼠脾脏细胞共培养，考察HepLPC对脾脏细胞的增殖抑制作用。
人原代肝细胞体外培养方法、条件培养上清制备方法、加入刺激剂的小鼠脾脏细胞悬液制备方法以及共培养方法请参见实施例4-1。共培养步骤中控制每种供体来源的HepLPC与小鼠脾脏细胞的数量比分别为1:5。
[根据细则91更正 30.03.2022]　
本实施例中，共培养72小时后，使用CFSE细胞增殖检测试剂盒对阳性对照组以及含不同供体来源HepLPC的共培养组进行处理后上流式仪检测，考察各 供体来源HepLPC的共培养组对脾脏细胞的增殖抑制情况与对应的的阳性对照组中脾脏细胞的增殖情况，结果如图112至图115所示。供体1、供体2、供体3和供体4来源的HepLPC对脾脏细胞增殖的抑制率分别为91％、93％、85％和89％。可见，不同供体来源HepLPC对脾脏细胞增殖的抑制效果体现出差异性，有利于后续开展免疫耐受药物的异质性研究。
实施例4-5
本实施例使用实施例2得到的四种供体来源的HepLPC分别与人外周血单个核细胞(PBMC)共培养，并使用植物血凝素PHA刺激PBMC中T细胞的增殖，考察HepLPC对PBMC的增殖抑制作用。
首先，取健康男性外周血用适量D-PBS和Histopaque-1077稀释后在2000rpm下离心25分钟，吸取中间的白膜层加入D-PBS反复离心洗涤并弃上清，得到细胞沉淀；使用含10微克/毫升PHA、10％PBS、2mMGlutaMAX的RPMI-1640培养基重悬细胞沉淀，得到PBMC悬液。
本实施例的共培养过程如下：取实施例4-1的经传代培养和消化后得到的HepLPC，使用PBS重悬为单细胞悬液；使用PBS反复洗涤单细胞悬液以确保去除消化液；使用含10％胎牛血清的RPMI-1640完全培养基重悬并调整细胞浓度；将含10％胎牛血清的RPMI-1640完全培养基重悬的HepLPC接种于6孔板，加入PBMC悬液，通过控制PBMC悬液和HepLPC悬液用量以及各悬液浓度，使得HepLPC和PBMC的数量比为1:5，PHA的终浓度为5μg/ml。以不加入HepLPC悬液的PBMC培养组作为阳性对照组。
[根据细则91更正 30.03.2022]　
本实施例中，共培养72小时后，使用CFSE细胞增殖检测试剂盒对阳性对照组以及含不同供体来源HepLPC的PBMC共培养组进行处理后上流式仪检测，考察各供体来源HepLPC的PBMC共培养组对PBMC的增殖抑制情况与对应的的阳性对照组中PBMC的增殖情况，结果如图116所示。参照图116所示的阳 性对照组和各共培养组的增殖PBMC占比情况可知，供体1、供体2、供体3和供体4来源的HepLPC对PBMC增殖的抑制率分别为85％、83％、64％和44％。可见，不同供体来源HepLPC对PBMC增殖的抑制效果体现出差异性，有利于后续开展免疫耐受药物的异质性研究。
实施例4-6
本实施例使用实施例1的HepLPC进行细胞移植，考察HepLPC对ConA诱导的，T细胞和NKT细胞介导的小鼠自身免疫性肝炎模型的作用。
[根据细则91更正 30.03.2022]　
首先，按8毫克/公斤、20毫克/公斤和30毫克/公斤的注射剂量对不同组(每组8只)的6周龄、体重20-30g的C57BL/6雄性小鼠尾静脉注射PBS重悬的ConA，考察ConA浓度为小鼠生存的影响，得到图117所示的不同ConA注射剂量与小鼠生存率的关系曲线。从图117可以看到，ConA的致死剂量不低于20毫克/公斤，而8毫克/公斤为非致死剂量。
[根据细则91更正 30.03.2022]　
本实施例对8毫克/公斤注射剂量组的小鼠在不同时间眼眶取血，通过血生化水平分析考察了各组的ALT、AST和LDH指标，得到图118所示的ALT、AST、LDH指标水平对比图。参照图118，对于非致死剂量ConA处理的小鼠，血ALT、AST、LDH在注射后6h时血转氨酶最高。因此，按8毫克/公斤注射剂量尾静脉注射的小鼠在注射结束后的第6小时建模成功。本实施例的对照组NC的8只小鼠尾静脉注射同等剂量的PBS。
本实施例取实施例4-1的经传代培养和消化后得到的HepLPC，使用PBS进行反复重悬洗涤以确保纯化效果后，使用PBS重悬HepLPC，并分组如下：
实验组1：对8只C57BL/6雄性小鼠分别尾静脉注射PBS重悬的HepLPC悬液以及PBS重悬的ConA，控制HepLPC的注射剂量为10 6/只，ConA的注射剂量为8毫克/公斤的非致死剂量。
实验组2：与实验组1的区别在于，ConA的注射剂量为20毫克/公斤的致死剂量。
对照组：每只C57BL/6雄性小鼠尾静脉注射同等体积的PBS。
各组小鼠在注射完毕的6小时后仍存活。
[根据细则91更正 30.03.2022]　
本实施例在注射完毕的6小时后，对实验组1和对照组的各小鼠眼眶取血，通过血生化水平分析考察了各组的ALT、AST、LDH和ALP指标，得到图119所示的各指标水平对比图。HepLPC的移植显著改善了小鼠的ALT、AST和LDH水平。
[根据细则91更正 30.03.2022]　
本实施例在注射完毕的6小时后，对实验组2和对照组的各小鼠眼眶取血，通过血生化水平分析考察了各组的ALT、AST和LDH指标，得到图120所示的各指标水平对比图。参照图120，尽管ConA的注射剂量为致死剂量，HepLPC的移植也显著改善了小鼠的ALT、AST和LDH水平。

Claims (26)

  1. 一种肝病调控制剂，其特征在于，包含肝脏来源前体细胞或所述肝脏来源前体细胞的分泌上清。
  2. 根据权利要求1所述的肝病调控制剂，其特征在于，所述肝脏来源前体细胞为肝前体细胞或肝前体样细胞。
  3. 根据权利要求1所述的肝病调控制剂，其特征在于，所述肝脏来源前体细胞的分泌上清包括至少一种miRNA，所述至少一种miRNA为miRNA-182、miRNA-183和miRNA-574的至少一种，能够有效促进肝细胞增殖。
  4. 根据权利要求1所述的肝病调控制剂，其特征在于，所述肝脏来源前体细胞的分泌上清包含作用于JAK-STAT通路的起效成分，以抑制肝星状细胞活化或诱导所述肝星状细胞死亡。
  5. 根据权利要求4所述的细胞制剂，其特征在于，所述肝脏来源前体细胞的分泌上清包含白血病抑制因子、内皮素、集落刺激因子、双调蛋白和成纤维细胞生长因子的至少一种。
  6. 根据权利要求1所述的肝病调控制剂，其特征在于，所述肝脏来源前体细胞的分泌上清包含抑制成分和促进成分的至少一种，所述抑制成分用于抑制M1型巨噬细胞的炎性活化，所述促进成分用于促进M2型巨噬细胞的产生。
  7. 根据权利要求1所述的肝病调控制剂，其特征在于，所述肝脏来源前体细胞的分泌上清包含能够通过抑制所述免疫细胞的增殖来诱导受体建立有效免疫耐受的分泌成分。
  8. 根据权利要求7所述的肝病调控制剂，其特征在于，所述免疫细胞为巨噬细胞、B细胞、T细胞、NK细胞和NKT细胞的任意一种。
  9. 根据权利要求1所述的肝病调控制剂，其特征在于，所述肝脏来源前体细胞经体外培养基的培养得到所述肝脏来源前体细胞的分泌上清。
  10. 根据权利要求9所述的肝病调控制剂，其特征在于，所述体外培养基包括基础培养基，所述基础培养基为HepX Basal培养基、DMEM/F12细胞培养基、William’s E细胞培养基、Neurobasal Medium细胞培养基、MEM细胞培养基、DMEM细胞培养基、1640RPMI细胞培养基和F12细胞培养基的至少一种。
  11. 根据权利要求10所述的肝病调控制剂，其特征在于，所述体外培养基还包括血清类物质，以占所述基础培养基的体积含量计，所述血清类物质的含量不超过20％。
  12. 根据权利要求10所述的肝病调控制剂，其特征在于，所述体外培养基还包括无血清添加物、血清类物质、生长因子、TGF-β信号抑制剂、Wnt信号通路激动剂和ROCK激酶抑制剂的至少一种。
  13. 根据权利要求9所述的肝病调控制剂，其特征在于，所述肝脏来源前体细胞经肝细胞扩增转化培养基培养得到，所述肝细胞扩增转化培养基包括基础培养基、无血清添加物、血清类物质、生长因子、TGF-β信号抑制剂、Wnt信号通路激动剂和ROCK激酶抑制剂。
  14. 根据权利要求13所述的肝病调控制剂，其特征在于，以占所述基础培养基的含量计，所述生长因子的含量为0.1-100纳克/毫升，所述ROCK激酶抑制剂的含量为0.1-100微摩尔，所述Wnt信号通路激动剂的含量为0.1-50微摩尔，所述TGF-β信号抑制剂的含量为0.1-100微摩尔，所述血清类物质的含量不超过20％，所述无血清添加物的体积含量不超过2％。
  15. 根据权利要求9所述的肝病调控制剂，其特征在于，所述肝细胞扩增转化培养基还含有N-乙酰-L-半胱氨酸、抗坏血酸的至少一种。
  16. 根据权利要求1所述的肝病调控制剂的体外应用，其特征在于，包括将所述肝病调控制剂与目标细胞共培养。
  17. 根据权利要求16所述的肝病调控制剂的体外应用，其特征在于，所述目标细胞为原代肝细胞、肝星状细胞、巨噬细胞和免疫相关细胞的任意一种。
  18. 根据权利要求16所述的肝病调控制剂的体外应用，其特征在于，将所述肝病调控制剂与目标细胞共培养的步骤包括，使用共培养基对所述肝病调控制剂与所述肝星状细胞进行共培养，以占所述共培养基的体积百分比计，所述肝病调控制剂的含量不低于1％。
  19. 根据权利要求17所述的肝病调控制剂的体外应用，其特征在于，所述共培养基包括肝星状细胞活化剂。
  20. 根据权利要求16所述的肝病调控制剂的体外应用，其特征在于，将所述肝病调控制剂与目标细胞共培养的步骤包括，将所述肝病调控制剂与肝巨噬细胞模型进行共培养，所述肝巨噬细胞模型为炎症细胞模型或修复型细胞模型。
  21. 根据权利要求16所述的肝病调控制剂的体外应用，其特征在于，将所述肝病调控制剂与目标细胞共培养的步骤包括，将所述肝病调控制剂与所述免疫相关细胞共培养，并使用刺激剂诱导所述免疫相关细胞的增殖。
  22. 根据权利要求21所述的肝病调控制剂的体外应用，其特征在于，所述免疫相关细胞为外周血单个核细胞和脾脏细胞的任意一种。
  23. 根据权利要求21所述的肝病调控制剂的体外应用，其特征在于，将所述肝病调控制剂与所述免疫相关细胞共培养的步骤包括，使用共培养基对所述肝病调控制剂进行重悬，并控制所述肝病调控制剂占所述共培养基的体积浓度不低于5％，以使所述肝病调控制剂对所述免疫相关细胞的增殖抑制率不低于30％。
  24. 根据权利要求21所述的肝病调控制剂的体外应用，其特征在于，将所述肝病调控制剂与所述免疫相关细胞共培养的步骤包括，使用不同肝病调控制剂与所述免疫相关细胞共培养，所述不同肝病调控制剂中所含有的肝脏来源前体细胞的培养上清来源于不同供体。
  25. 根据权利要求1所述的肝病调控制剂在肝病治疗方面的应用，其特征在于，包括，将所述肝病调控制剂干预体内动物模型后考察对肝脏再生的影响。
  26. 根据权利要求25所述的肝病调控制剂在肝病治疗方面的应用，其特征在于，所述动物体内模型为四氯化碳诱导的小鼠急性肝衰竭模型、乙酰氨基酚诱导的小鼠急性肝衰竭模型、硫代乙酰胺诱导的哺乳动物肝硬化模型、四氯化碳诱导的哺乳动物肝硬化模型、哺乳动物非酒精性脂肪性肝炎模型、ConA诱导且T细胞和NKT细胞介导的小鼠自身免疫性肝炎模型、肝细胞或肝组织移植后的免疫排斥大鼠模型以及肝移植后急性宿主抗移植物反应模型中的任意一种。
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