Application of methionine enkephalin in promoting liver maturation

Abstract

The invention relates to the technical field of biomedicine, in particular to application of methionine enkephalin in promoting liver maturation. The invention finds that the split product methionine enkephalin Met-ENK of the proenkephalin can promote the maturation of the liver, and can be directly injected in vivo or added into a culture medium for application, so that not only can the expression of embryo liver related genes be reduced, but also the expression of mature liver related genes can be increased, and the maturation of the liver can be promoted. The invention is helpful for research and application of liver organs in the aspects of disease pathology mechanism exploration, drug in-vitro screening and research and development and regenerative medicine.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the application of methionine enkephalin in promoting liver maturation. Background Art

[0002] The liver is one of the most important organs in the body, with multiple functions such as metabolism, biomacromolecule synthesis, detoxification, removal of aging red blood cells, immune surveillance and immune tolerance. Pathogenic microorganisms, drugs, overnutrition and other factors can lead to liver inflammation, fatty degeneration, cirrhosis and liver cancer and other diseases. These liver abnormalities can also lead to lesions in other tissues or even the whole body. The development of the liver has a postnatal maturation process. After mammals leave the mother's womb, diet, oxygen, hormones, intestinal microecology and other factors undergo drastic changes, causing multiple organs of the body to rapidly initiate the postnatal maturation process. The postnatal maturation of the liver is a key link, not only gradually transforming from a hematopoietic organ in the embryonic period to a metabolic and immune organ, but also has a profound impact on the postnatal maturation of other tissues, such as the postnatal maturation of the hypothalamus-pituitary-adrenal / thyroid axis and the establishment of the intestinal microecology. Taking mice as an example, nerve fibers begin to grow into liver tissue after birth. Seven days after birth, the number of hematopoietic cells in the liver gradually decreases. One to two weeks after birth, the transcriptome changes dramatically while the liver cells proliferate, gradually forming the structure and functional division of the liver lobule. Three weeks after birth and beyond, the proportion of immune cells such as T cells in the liver gradually increases, and the liver's own metabolic functions gradually improve. Humans also have a similar liver maturation process after birth, but it can last about two years.

[0003] Abnormal liver maturation is closely related to metabolic disorders in childhood and even in adulthood. For example, premature infants are deficient in vitamin A, and large intestinal doses cannot increase plasma vitamin A; premature infants or low birth weight infants have an increased risk of developing abnormal glucose metabolism and non-alcoholic fatty liver disease in adulthood. The immaturity of liver function is considered to be one of the important causes of these abnormalities.

[0004] Liver maturation disorders also exist in 3D culture of liver organoids based on human embryonic stem cells or induced pluripotent stem cells. These in vitro cultured liver organoids need to be transplanted into immunodeficient mice to significantly downregulate genes expressed in embryonic livers, such as AFP, but multiple function-related genes expressed in adult livers cannot be fully upregulated. This seriously restricts the application of liver organoids in the exploration of disease pathological mechanisms, in vitro drug screening and development, and regenerative medicine.

[0005] The maturation process of the liver is precisely regulated in time and space by a variety of factors inside and outside the liver, but people know very little about these regulatory mechanisms, which not only affects the development of intervention theories and strategies for premature and low birth weight infants, but also affects the optimization of treatment strategies for neonatal liver diseases such as hepatotropic virus infection. It also hinders the research and transformation in the field of liver repair and regeneration, and the in vitro simulation and mechanism research of liver diseases and drug liver metabolism. Therefore, in-depth and systematic research on the regulatory mechanism of postnatal maturation of the liver will be of great significance in the early prevention strategies of metabolic diseases, treatment strategies for premature and low birth weight infants, and the development and optimization of 3D culture and regeneration and repair programs of liver organoids.

[0006] Postnatal maturation of the liver, especially the maturation of hepatocytes, is subject to complex and subtle dynamic regulation at multiple levels, including transcriptional and posttranscriptional levels. For example, the liver transcription factor HNF4α plays a key regulatory role in liver development and maturation. At birth, Hnf4a The distal P2 promoter is converted to the proximal P1 promoter, upregulating Hnf4 Expression of the α1 / 2 spliceosome of Mettl3 is regulated within 2 weeks after birth Hnf4a、Smpd3 m of key molecules such as RNA 6 A modification and stability affect liver maturation, but Mettl3 down-regulates expression after 2 weeks and has no significant effect on adult liver homeostasis. Esrp2 is also up-regulated within 2 weeks after birth, and affects cell proliferation / differentiation, cell adhesion, liver zoning, and albumin production in liver maturation by regulating up to 20% of RNA alternative splicing.

[0007] The downregulation of fetal liver gene expression at 1-2 weeks after birth is also regulated by multiple factors. For example, the RNA binding protein IMP1 with RNA stabilization function is downregulated, the RNA binding protein with degradation-promoting function is upregulated, and the deadenylation induction mediated by Cnot3 is induced. Afp , Igf2、H19 The degradation of fetal liver gene mRNA and other genes; Cul4 and DCAF8 are upregulated, regulating the polyubiquitination of H3K79, promoting H3K79me2 / 3 and inhibiting fetal liver gene expression.

[0008] Abnormalities in these regulatory mechanisms can lead to liver maturation defects, such as hepatocyte-specific knockout Mettl3 , Cnot3, Srsf3, Esrp2 The mice still maintained high levels of Afp However, it is still unknown how the above regulatory mechanisms change at specific times and whether and how they are regulated by other factors.

[0009] Since the postnatal maturation of hepatocytes has spatiotemporal regulation characteristics, and the in vitro 3D culture of liver organoids based on induced pluripotent stem cells needs to be transplanted into the body to complete functional maturation, people generally believe that mechanisms other than hepatocytes, such as hormones, nutrition, and intestinal flora, are likely to play a crucial regulatory role in liver maturation, but there is a lack of in-depth and systematic research on which factors and how they regulate liver maturation. Only a few studies have found that insulin is crucial in the postnatal maturation of the liver, inducing polyploidization of hepatocytes and DNA demethylation of metabolism-related genes; testosterone also plays an important role in liver DNA demethylation 3 weeks after birth; lipid nutrients can promote DNA demethylation of genes related to fatty acid β-oxidation in the liver by activating PPARα; bile acids promote the expression of genes related to liver maturation by activating FXR, which is rapidly upregulated after birth, in collaboration with HNF4.

[0010] Immune cells play a variety of important roles in liver immunity, including tolerance, surveillance, protection, and damage repair. However, during the neonatal period, a variety of immune cells themselves are also undergoing a process of development and maturation. At the same time, some progenitor / precursor cells migrate from the liver to the bone marrow, and basically mature immune cells migrate to the liver. It is still unclear whether and how these dynamically changing immune cells are involved in regulating the postnatal maturation of the liver. Researchers have only made limited explorations of Kupffer cells (KC). KC is a tissue-resident macrophage of the liver, mainly located in the hepatic sinusoids. KC promotes erythroblast proliferation, differentiation, denucleation, and iron recycling in the fetal liver; it gradually acquires the function of clearing bacteria after birth. Csf1 and Csf1 The loss of macrophages due to knockout or mutation has no obvious effect on embryonic development, but can cause postnatal growth retardation, reduced hepatocyte proliferation at 3 weeks of age, delayed downregulation of fetal liver genes, and fatty degeneration of the liver. Although preliminary evidence shows that KC may regulate postnatal maturation of the liver by expressing insulin-like growth factor-1 (IGF-1) itself or by promoting hepatocyte proliferation and IGF-1 expression, it is unclear why KC specifically affects liver maturation in the neonatal period and by what molecular mechanism it regulates liver maturation. Summary of the invention

[0011] The present invention has found that the cleavage product of proenkephalin PENK, methionine enkephalin Met-ENK, can promote liver maturation. Based on this, the following invention content is proposed.

[0012] Firstly, the present invention provides the use of methionine enkephalin in promoting liver maturation for non-diagnostic therapeutic purposes.

[0013] Furthermore, the present invention provides the use of methionine enkephalin in preparing a product, wherein the product is used for promoting liver maturation or treating liver maturation disorders.

[0014] Preferably, the product is at least one of a medicine, a reagent or a kit, and a culture medium.

[0015] Furthermore, the present invention provides the use of methionine enkephalin in in vitro 3D culture of liver organoids.

[0016] Preferably, the present invention provides a method for culturing 3D liver organoids in vitro, comprising: culturing liver parenchymal cells in a culture medium containing methionine enkephalin.

[0017] Preferably, the culture medium containing methionine enkephalin comprises the following components: DMEM / F12 medium, 10% fetal bovine serum, insulin-transferrin-selenium-aminoethanol, penicillin and streptomycin, human epidermal growth factor, human hepatocyte growth factor, dexamethasone and methionine enkephalin.

[0018] Preferably, methionine enkephalin promotes liver maturation through at least one of the following pathways: (1) Inhibit the Notch signaling pathway; (2) Inhibit the growth of Hepa1-6 tumor cells; (3) Inhibit the differentiation and maturation of bile duct epithelial cells; (4) Promote the maturation of hepatocytes.

[0019] Preferably, methionine enkephalin reduces the expression levels of Notch signaling pathway related genes Sox9 and Hes1; methionine enkephalin reduces the expression levels of embryonic liver related genes Afp and Gpc3, and increases the expression levels of mature liver related genes Hnf4a, Esrp2, Alb, Ttr, Cyp1a2 and Ugt1a9.

[0020] Furthermore, the present invention provides the use of an opioid growth factor receptor antagonist in the preparation of a drug for inhibiting liver maturation.

[0021] Furthermore, the present invention provides an in vitro high-throughput drug screening and / or drug detection method, comprising: culturing liver parenchymal cells using a culture medium containing methionine enkephalin to obtain a liver organoid 3D culture; and then applying a drug to the liver organoid 3D culture for detection and analysis.

[0022] Furthermore, the present invention provides a method for constructing an animal model, comprising: injecting methionine enkephalin into the model animal to promote liver maturation of the model animal.

[0023] Preferably, the model animal is a rat or a mouse.

[0024] Furthermore, the present invention provides a 3D liver organoid culture obtained by the culture method in any of the above embodiments.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The present invention has found that the cleavage product of proenkephalin PENK, methionine enkephalin Met-ENK, can promote liver maturation, and can be directly injected in vivo or added to culture medium for application, which can not only reduce the expression of embryonic liver-related genes, reduce the expression of bile duct and Notch signal-related genes, but also increase the expression of mature liver-related genes, and promote liver maturation. The present invention is helpful for the research and application of 3D liver organoid culture in the exploration of disease pathological mechanisms, in vitro drug screening and development, and regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 CD4 in 1-3 week old livers + Foxp3 + Cell ratio and CD4 + Foxp3 - Changes in cell proportions.

[0027] Figure 2 The transcriptome sequencing results of Treg cells in the liver and spleen of 12-day-old wild-type mice were shown in Tbx21 and the expression of its regulated target genes.

[0028] Figure 3 Foxp3 in 12-day-old liver (nLiver), spleen (nSpleen), 7-week-old liver (aLiver), spleen (aSpleen) + UMAP plot of single-cell transcriptome sequencing analysis of T cells.

[0029] Figure 4 Foxp3 in 12-day-old liver (nLiver), spleen (nSpleen), 7-week-old liver (aLiver), spleen (aSpleen) + Projection of Treg cells on the UMAP map after TCR sequencing analysis.

[0030] Figure 5 The bubble chart shows the characteristic genes of each cell subpopulation.

[0031] Figure 6 The transcription factor regulatory network shows the transcription factor activity of each cell subset.

[0032] Figure 7 Display T-bet for RNA velocity+ Treg cells are in the terminal differentiation position, activated Treg (4th and 5th subgroups) or transitional Treg cells (10th subgroup) are T-bet + Precursors of Treg cells.

[0033] Figure 8 The transcription factor regulatory network shows Foxp3 in liver (nLiver), spleen (nSpleen), 7-week liver (aLiver), and spleen (aSpleen). + Comparative analysis of transcription factors of Treg cells.

[0034] Fig. 9 The bubble chart shows the ratio of each subgroup, nLiver / sSpleen and nLiver / aLiver.

[0035] Figure 10-11 Single-cell transcriptome was used to detect the transcription of Treg cells in the liver of 12-day-old T-cKO mice and jointly analyzed with Treg cells in the liver of 12-day-old WT mice, and the proportion of each subpopulation (T-cKO / WT ratio) was displayed through a bubble chart.

[0036] Figure 12-13 Four samples of liver and liver Treg cells from newborn WT and adult WT mice were jointly analyzed, and the expression of Penk in different subsets was shown.

[0037] Fig.14 The expression of PENK in various cell types was detected by qPCR.

[0038] Fig.15 The Nichenet package was used to analyze and compare the molecules upregulated in neonatal Tregs compared with adult Tregs.

[0039] Fig.16 Flow cytometry was used to compare the expression of PENK in liver Treg cells of T-cKO mice and liver Treg cells of WT mice.

[0040] Fig.17 The binding sites of T-bet and FOXP3 to the PENK promoter were predicted using the Jasper database.

[0041] Fig.18 Show 12 days old Penk fl / fl Foxp3 cre Mice and Penk w / w Liver weight and liver weight / body weight ratio of control mice.

[0042] Fig.19 Show 12 days old Penk fl / fl Foxp3 cre Mice and Penk w / w Hepatocytes from the liver of control mice (CD45 - CD31 - Ter119 - ) ratio and size, bile duct epithelial cells (EpCAM + CD45 - CD31 - Ter119 - ) and precursor cells (CD24 + CD45 - CD31 - Ter119 - ) ratio.

[0043] Fig. 20 The results of qPCR detection of liver maturation-related indicators in the livers of WT and P-cKO mice.

[0044] Fig.21 Liver weight and liver-to-body ratio of T-cKO mice after Met-ENK was supplemented by intraperitoneal injection.

[0045] Fig. 22 The proportion of hepatocytes in T-cKO mice after Met-ENK replenishment was detected by flow cytometry.

[0046] Fig.23 The liver maturation-related indicators were detected by qPCR after Met-ENK was supplemented in T-cKO mice.

[0047] Fig.24 is the effect of Met-ENK on liver 3D organoids.

[0048] Fig.25 The qPCR method was used to detect the upregulation of liver maturation-related indicators and the inhibition of bile duct cell differentiation and maturation-related indicators when Met-ENK was added during the 12-day culture of liver 3D organoids.

[0049] Fig.26 Flow cytometry was used to detect the expression of EPCAM at the protein level after adding Met-ENK during the 12-day 3D liver culture.

[0050] Fig. 27 qPCR was used to compare the expression of Ogfr and Oprd1, the two opioid receptors with the strongest affinity for Met-ENK, in the hepatocytes and bile duct cells of WT mice.

[0051] Fig.28 Flow cytometry was used to compare the proportion of hepatocytes and bile duct cells in WT mice after injection of DMSO (solvent) and opioid receptor inhibitors.

[0052] Fig.29 The expression of liver maturation markers and bile duct maturation markers were compared using qPCR.

[0053] Fig.30 The expression of bile duct markers and Notch signals involved in bile duct differentiation in T-cKO mice and WT mice was shown by population transcriptome.

[0054] Fig.31 The expression of Sox9, which characterizes the maturation of bile duct cells in P-cKO mice, was shown by qPCR.

[0055] Fig.32 The expression of Sox9, a marker of differentiation and maturation of cholangiocytes in 3D liver culture, was detected by qPCR after the addition of Met-ENK and the addition of receptor inhibitors at the same time as Met-ENK.

[0056] Fig.33 This is the expression of ADAM metalloproteinase 10 (Adam10) in T-cKO mice, P-cKO mice and in vitro 3D culture.

[0057] Fig.34 The heat map of differential gene analysis of the population transcriptome shows the liver mature gene expression and liver immature gene expression of T-cKO mice compared with WT mice.

[0058] Fig.35 qPCR was used to show the expression of Esrp2 in T-cKO mice and P-cKO mice, as well as in T-cKO mice injected with Met-ENK inhibitors and WT mice injected with receptor inhibitors.

[0059] Fig.36 The results of rMATS analysis of alternative splicing events in the transcriptome of P-cKO mice.

[0060] Fig.37 The results are from an attempt to add Met-ENK to Hepa1-6 mouse liver cancer cells using in vitro cell culture and to detect liver maturation-related indicators using qPCR. DETAILED DESCRIPTION

[0061] To make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. Those who do not specify specific techniques or conditions in the embodiments are all conventional methods or are carried out according to the techniques or conditions described in the literature in this field, or are carried out according to the product manual. Those whose manufacturers are not specified for the reagents and instruments used are all conventional products that can be purchased through regular channels.

[0062] 1. Reagent Preparation and Mice Acquisition The solution preparation method used in the following experiments is as follows: 1. 10× PBS buffer

[0063] Dissolve the above reagents in 500 mL ddH 2 O, adjust the pH to 7.2-7.4 and store. Dilute it to 1× PBS before use.

[0064] 2. Magnetic bead separation buffer

[0065] Dissolve it in 1× PBS to 50 mL and filter sterilize before use.

[0066] 3. BSS buffer

[0067] Dissolve the above reagents in 1769 mL ddH 2 O, adjust the pH to 7.2-7.4, then add fetal bovine serum at a concentration of 1%, mix well, and store in aliquots at 4°C.

[0068] 4. Red blood cell lysis buffer 1×ACK

[0069] Dissolve the above reagents in 500 mL ddH 2 O, adjust the pH to 7.2-7.4, and store at 4°C before use.

[0070] 5. Tbx21 fl / flMouse reference: Intlekofer AM, Banerjee A, Takemoto N, Gordon SM, Dejong CS, Shin H, Hunter CA, Wherry EJ, Lindsten T, Reiner SL. Anomalous type 17 response to viral infection by CD8+ T cells lacking T-bet and eomesodermin. Science. 2008 Jul 18;321(5887):408-11. doi: 10.1126 / science.1159806. PMID: 18635804; PMCID: PMC2807624. Construction. Penk fl / fl Mouse reference: Shan Zhang, Jianhui Chen, Qingqing Li, Wenwen Zeng, Opioid growth factor receptor promotes adipose tissue thermogenesis via enhancing lipid oxidation, Life Metabolism, Volume 2, Issue 3, June 2023, load018, https: / / doi.org / 10.1093 / lifemeta / load018 Construction. Foxp3 creMice refer to mice bred from Foxp3-GFP-Cre and Rosa26-loxP-Stop-loxP-YFP, and refer to Zhou X, Jeker LT, Fife BT, Zhu S, Anderson MS, McManus MT, Bluestone JA. Selective miRNA disruption in T reg cells leads to uncontrolled autoimmunity. J Exp Med. 2008 Sep 1;205(9):1983-91. doi: 10.1084 / jem.20080707. Epub 2008 Aug 25. PMID: 18725525; PMCID: PMC2526194. and Srinivas S, Watanabe T, Lin CS, William CM, Tanabe Y, Jessell TM, Costantini F. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol. 2001;1:4. doi: 10.1186 / 1471-213x-1-4. Epub 2001Mar 27. PMID: 11299042; PMCID: PMC31338. Prepared by Tbx21 w / w / Penk w / w Mice also refer to mice obtained by mating Foxp3-GFP-Cre and Rosa26-loxP-Stop-loxP-YFP. fl / fl With Foxp3 cre Tbx21 fl / fl Foxp3 cre (T-cKO) mice, Penk fl / fl With Foxp3 cre Mouse mating gets Penk fl / fl Foxp3 cre (P-cKO) mice.

[0071] Wild-type mice are C57BL / 6 mice that have not undergone any genetic modification or transformation.

[0072] 2. Experimental Methods 1. Identification of mouse genotype 1. Obtaining tissue Take a 0.1 cm mouse tail and cut it into pieces, add 100 μl 50 mM NaOH, shake and mix well, and then spin it instantly.

[0073] 2. DNA Extraction (1) Preheat the metal bath to 95°C in advance, and then place it in the 95°C metal bath for 10 minutes.

[0074] (2) Add 10 μl of 1 mol / L Tris-HCL (pH 8.0) and mix well to neutralize the NaOH.

[0075] (3) Centrifuge at 12000 rpm, 4°C for 5 min. Take 50 μl of the supernatant and add it to a new EP tube.

[0076] 3. DNA Amplification The primer sequences for mouse genotype identification are shown in Table 1.

[0077] Table 1

[0078] The PCR identification reaction system is shown in Table 2.

[0079] Table 2

[0080] Reaction conditions: (1) Foxp3 cre The PCR product band size was 235 bp, and the reaction cycle was 35 under the following conditions: 94°C for 30 s, 62°C for 30 s, 72°C for 30 s, and extension at 72°C for 7 min.

[0081] (2) Rosa26 YFP The PCR product band size was 575 bp, the reaction cycle was 30, and the reaction conditions were 94°C for 30 s, 60°C for 30 s, 72°C for 1 min, and extension at 72°C for 7 min.

[0082] (3) Penk flox / flox The product is 236 bp, Penk + / + The product was 174 bp, the reaction cycle was 30, and the reaction conditions were 94°C for 30 s, 60°C for 30 s, 72°C for 1 min, and extension at 72°C for 7 min.

[0083] (4) Tbx21 flox / flox The PCR product band size was 470 bp, Tbx21 + / + The band size was 650 bp, and the reaction cycles were 35: 94°C for 30 s, 60°C for 30 s, 72°C for 45 s, and extension at 72°C for 7 min.

[0084] 4. Agarose gel electrophoresis of amplified products (1) Weigh 0.8 g agarose, add 80 mL 1×TAE solution, heat in a microwave oven until completely melted and boiling, add Gelstain dye (final concentration 0.5 μg / mL), and prepare a 1% agarose gel.

[0085] (2) Pour the gel into the mold and let it solidify at room temperature for more than 30 minutes. Add sufficient 1×TAE electrophoresis buffer into the electrophoresis tank.

[0086] (3) Place the gel block in the electrophoresis tank, apply 10 μL of sample to each well, and perform electrophoresis at a constant voltage of 250 V for 15-25 min.

[0087] 2. Measurement of mouse liver-to-body ratio 12-day-old mice were weighed and then killed by cervical dislocation. After the gallbladder was removed, the whole liver was placed on tin foil, the blood was wiped off, the liver weight was weighed, and then the liver-to-body ratio was calculated.

[0088] 3. Acquisition of liver single cell suspension, liver parenchymal cells and liver lymphocytes (A) Obtaining liver single cell suspension 1. First, prepare liver digestion solution. Add 40 mg / ml collagenase IV (Col IV) at a concentration of 1:80 to RPMI1640, and add 0.5 mg / ml DNase I at a concentration of 1:20.

[0089] 2. Kill newborn mice of the corresponding days, dissect the abdomen and expose the portal vein. Use a 1ml syringe needle connected to a 10ml syringe to draw digestive fluid, insert it into the portal vein, and perform liver perfusion until the liver changes from red to white-brown.

[0090] 3. Remove the liver, chop it in a small volume of digestion solution, and then transfer it to a 15 ml tube, add digestion solution, mix it by inversion, and place it in a shaker at 37°C 200rpm for 15 minutes. Use a 100 mesh sieve to filter the digestion solution to remove undigested tissue and residues.

[0091] 4. After filtration, centrifuge at 4°C, 500g for 6 minutes and resuspend in BSS to obtain a liver single cell suspension.

[0092] (B) Acquisition of liver parenchymal cells 1. After obtaining the liver single cell suspension, count it, take a certain amount of cells, centrifuge at 4°C, 500g, 5min, resuspend the cells in BSS with anti-CD16 / CD32 added, incubate on ice for 10 minutes, and block the Fc receptors on the membrane surface.

[0093] 2. Centrifuge and remove the supernatant containing anti-CD16 / CD32.

[0094] 3. Resuspend the cells in BSS with FITC anti-CD45, FITC anti-Ter119, and FITC anti-CD31 added according to the ratio specified in the instruction manual and incubate on ice for half an hour.

[0095] 4. After centrifugation to remove the supernatant, resuspend the cells in magnetic bead sorting buffer with Anti-FITC magnetic beads added in a certain proportion and incubate on ice for 15 minutes.

[0096] 5. Obtain CD45 by magnetic bead sorting - CD31 - TER119 - Count the liver parenchymal cells.

[0097] (C) Acquisition of liver lymphocytes 1. After obtaining the liver single cell suspension, centrifuge at 500g for 5 minutes at 4°C and resuspend the liver cells in 40% Percoll (9.6 mL PBS + 0.64 mL 10×PBS + 5.76 mL Percoll stock solution).

[0098] 2. Centrifuge at 1000g for 11 minutes at room temperature, slowly rising and falling. The resulting solution is divided into three layers: the top layer is hepatocytes, the middle layer is Percoll, and the bottom layer is red blood cells and lymphocytes.

[0099] 3. Aspirate and discard the upper layer, pour out the middle layer, and resuspend the bottom layer of cells with BSS.

[0100] 4. After centrifugation, resuspend the cells with 1×ACK solution, lyse on ice for 3 minutes, stop with a large volume of BSS, and centrifuge at 4°C, 1500 rpm, for 5 minutes.

[0101] 5. Resuspend the cells in BSS to obtain lymphocytes and count them.

[0102] 4. 3D Culture and Identification of Liver Organoids 1. Prepare complete culture medium: DMEM / F12 + 10% fetal bovine serum (FBS) + 1× insulin-transferrin-selenium-aminoethanol (ITS-X) + 1× penicillin and streptomycin + 25 ng / mL human epidermal growth factor (hEGF) + 25 ng / mL human hepatocyte growth factor (hHGF) + 40 ng / mL dexamethasone.

[0103] 2. Mix the liver parenchymal cells obtained by magnetic bead sorting with matrix gel in a ratio of 1:1 and gently inoculate them in confocal microplates or 48-well plates, with 1x10 5of cells, making it arched.

[0104] 3. Change the medium every day for the first three days. On the fourth day, add different concentrations of Met-ENK to the above complete culture medium. Change the medium every two days. Culture for 12 days to obtain liver organoid 3D culture.

[0105] 4. Immunofluorescence staining (1) On the 12th day of culture, discard the original culture medium, add 1 ml of 1× PBS to wash the cells, discard the PBS, add 1 ml of 4% paraformaldehyde fixative to each dish, and let it stand for 20 minutes.

[0106] (2) Discard the 4% paraformaldehyde fixative and add 5% Triton-X100 to each dish for permeabilization and let stand for 30 minutes.

[0107] (3) Discard the permeabilization solution, add 1 ml of 1× PBS to each well to wash the cells, and then add 1 ml of 5% BSA to block the cells at room temperature for 2 h.

[0108] (4) Discard the blocking solution, dilute Mouse anti-SOX9 and Rabbit anti-ALB antibodies with 5% BSA, and incubate at 4°C overnight.

[0109] (5) Discard the anti-SOX9 / anti-ALB antibodies and wash three times with PBST.

[0110] (6) Use Goat anti-Rabbit secondary antibodies with different fluorescence, Goat anti-Mouse and Hoechst 33342 diluted in 5% BSA and incubate at room temperature in the dark for 1 hour.

[0111] (7) Discard the secondary antibody and wash three times with PBST.

[0112] (8) Add anti-fluorescence fading mounting medium to the confocal dish.

[0113] (9) Confocal microscopy is used to observe and photograph 3D structures.

[0114] 5. Extraction of RNA from Organoids (1) On the 12th day of culture, discard the organoid culture medium, wash three times with 1× PBS, add 1 mL TRIzol to resuspend the cells, save for RNA extraction, and store at -80°C.

[0115] (2) Add chloroform to the homogenate tube at a dose of 200 μL / 1 mL TRIzol, shake vigorously to mix thoroughly, leave at room temperature for 5 min, and then centrifuge at 4°C, 12,000 rpm for 15 min.

[0116] (3) After centrifugation, the sample is separated into three layers (upper colorless aqueous phase, middle protein layer and lower red organic phase). At this time, RNA is in the top layer. Slowly aspirate the upper aqueous phase several times (try to avoid aspirating the protein layer) into a new EP tube.

[0117] (4) Add an equal volume of isopropanol and 0.5 μl of glycogen, invert to mix, incubate at -20°C for 30 min, and then centrifuge at 4°C, 12,000 rpm for 10 min. A white RNA precipitate will be visible at the bottom of the tube.

[0118] (5) Discard the supernatant, add 1 mL of 75% ethanol, shake until the RNA precipitate is separated from the tube wall, and then centrifuge at 8500 rpm at 4°C for 10 min.

[0119] (6) Discard the supernatant, dry for 10 min, and then dissolve the RNA precipitate with an appropriate amount of DEPC water.

[0120] 6. RNA Reverse Transcription (1) Removal of genomic DNA: Prepare the reaction mix on ice using the reverse transcription kit according to Table 3.

[0121] Table 3

[0122] Place the above mix in a PCR instrument and set the reaction program to 42°C for 3 min. After completion, place on ice.

[0123] (2) Reverse transcription: Use a reverse transcription kit to prepare the reaction mix on ice according to Table 4. Add 10 μL of the prepared reverse transcription mix to the solution from which genomic DNA has been removed for each sample.

[0124] Table 4

[0125] Place the above mix in a PCR instrument and set the reaction program to 42°C for 15 min and 95°C for 3 min. The obtained cDNA can be diluted 3-5 times for subsequent experiments or stored at -20°C.

[0126] 7. qPCR identification of related mature gene expression (1) Prepare the reaction system (10 μL) according to Table 5 in a qPCR reaction tube and centrifuge.

[0127] Table 5

[0128] The primer sequences of fetal liver genes and adult liver function-related genes used are shown in Table 6.

[0129] Table 6

[0130] (2) Place the sample in the qPCR machine and select the relative quantification program. Set the reaction program as follows: 95℃ for 5 min; then 40 cycles of 95℃ for 15 s, 58℃ for 20 s, 72℃ for 20 s; 72℃ for 10 min. -ΔΔCt The relative content was calculated by the method.

[0131] (five) Tbx21 fl / fl Foxp3 cre Experiments on Met-ENK supplementation in (T-cKO) mice and intraperitoneal injection of Naltrexone in WT mice for Tbx21 fl / fl Foxp3 cre (T-cKO) mice were intraperitoneally injected with Met-ENK at 10 mg / kg daily starting from day 6 and were harvested on day 12 to measure the liver-to-body ratio, proportion and size of hepatocytes, proportion of bile duct cells and precursor cells, and liver maturity and immaturity-related markers to determine whether liver maturation was rescued.

[0132] For WT mice, Naltrexone was intraperitoneally injected at 15 mg / kg every day starting from the 6th day. The livers were harvested on the 12th day and the liver-to-body ratio, proportion and size of hepatocytes, proportion of bile duct cells and precursor cells, and liver maturity and immaturity-related markers were tested to determine whether liver maturation was affected.

[0133] (A) Immunofluorescence of liver tissue 1. Sampling and Fixation When the mice were 12 days old, one lobe of the liver was removed from the same position and fixed in 4% paraformaldehyde at room temperature for 24 h.

[0134] 2. Paraffin Embedding (1) Discard 4% paraformaldehyde, replace with 75% ethanol, and soak overnight; (2) Gradient dehydration: 85% ethanol for 30 min; 95% ethanol for 30 min; 100% ethanol I for 40 min, transfer the tissue into the embedding box; 100% ethanol II for 20 min; (3) Transparent (fume hood): Xylene I 20 min (preheat paraffin in embedding machine); Xylene II 20 min; Paraffin I 20 min; Paraffin II 30 min; (4) Prepare scissors, paraffin blocks, and plastic molds; use an embedding machine to embed and wait for the paraffin to solidify for about 1 hour; remove from the mold after the paraffin is completely solidified (about 3 hours) and store at 4°C; 3. Paraffin Sections (1) Pre-cool the tissue block in a -20℃ refrigerator for 2 hours, and preheat it in a slide machine (42℃) or a slide oven (60℃); (2) Fix the embedded block on the microtome head, adjust the blade, trim the slice by 10um first, and then slice it by 4um after trimming it to the appropriate position. Place the cut slices in the slide spreader, use the marked cationic slide to pick up the slices, shake off the water, and place them in the baking area for baking.

[0135] 4. Dewaxing and hydration of sections (1) Dewaxing: Dry the sheets at 70°C for 2 h, xylene I (20 min); xylene II (20 min).

[0136] (2) Hydration: 100% ethanol I (5 min); 100% ethanol II (5 min); 90% ethanol (5 min); 70% ethanol (5 min); distilled water (5 min, slow shaking on a shaker*3).

[0137] (B) Flow cytometry staining 1. Cell membrane surface molecule staining (1) Take a certain amount of hepatic parenchymal cells or hepatic lymphocytes and centrifuge them at 1500 rpm at 4°C for 5 min. Resuspend the cells in PBS with anti-CD16 / CD32 and LiveDead dye and incubate at 4°C for 10 min (this step can block the Fc receptors on the cell membrane surface and distinguish the live or dead cells).

[0138] (2) Centrifuge at 4°C, 1500 rpm for 5 min. Add the flow cytometry antibody to be tested to BSS in appropriate proportions to make a staining mix and mix thoroughly.

[0139] (3) Discard the supernatant, add an appropriate volume of staining mix to resuspend the cells, and incubate at 4°C in the dark for 30 min.

[0140] (4) Terminate staining with a large volume of BSS and centrifuge at 1500 rpm for 5 min at 4°C.

[0141] (5) Discard the supernatant, resuspend the cell pellet in BSS to the required concentration, filter through a 400-mesh sieve to remove adhesions, and place on ice away from light until loading.

[0142] 2. Cytokine staining in the cytoplasm (1) Add 100 μL IC Fixation Buffer to the stained cell pellet on the membrane surface, resuspend the cells, and fix them at 4°C in the dark for more than 30 min.

[0143] (2) Add 1 mL of 1× Permeabilization Buffer and permeabilize at 4°C in the dark for 10 min.

[0144] (3) Centrifuge at 1800 rpm and 4°C for 7 min. Add the flow cytometry antibody to be tested to 1× Permeabilization Buffer in an appropriate proportion to prepare a staining mix and mix thoroughly.

[0145] (4) Discard the supernatant, resuspend the cell pellet with the above staining mix, and stain for 20 min at room temperature in the dark.

[0146] (5) Use a large volume of BSS to terminate the staining and centrifuge at 1800 rpm for 7 min at 4°C. Discard the supernatant, resuspend the cells in BSS, filter through a 400-mesh sieve to remove adhesions, and keep away from light while waiting for loading.

[0147] 3. Transcription factor staining in the nucleus (1) Prepare nuclear fixative using Fixation / Permeabilization Concentrate and Fixation / Permeabilization Diluent in a volume ratio of 1:3.

[0148] (2) After the membrane surface molecules were stained, fix the cells with 200 μL of nuclear fixative at 4°C for more than 30 min.

[0149] (3) Add 1 mL of 1× Permeabilization Buffer and permeabilize at 4°C in the dark for 10 min.

[0150] (4) Centrifuge at 1800 rpm for 7 min at 4°C. Add the flow cytometry antibody to be tested to 1× Permeabilization Buffer in appropriate proportions to prepare a nuclear staining mix and mix thoroughly.

[0151] (5) Discard the supernatant, resuspend the cell pellet with the above staining mix, and stain for 20 min at room temperature in the dark.

[0152] (6) Terminate staining with a large volume of BSS and centrifuge at 1800 rpm for 7 min at 4°C.

[0153] (7) Discard the supernatant, resuspend the cells in BSS, filter through a 400-mesh sieve to remove adhesions, and keep away from light while waiting for loading.

[0154] (VI) Analysis of Treg single-cell transcriptome data 1. Sample acquisition (1) Take a certain number of newborn and adult mouse liver / spleen lymphocytes and centrifuge them at 4°C, 1500 rpm for 5 min.

[0155] The steps for obtaining liver lymphocytes are the same as above; the steps for obtaining spleen lymphocytes are as follows: a. Kill the mice on the corresponding days, remove the spleen, put it on a 400-mesh sieve, add BSS, cut it into pieces on the sieve, and grind it on ice to make a single-cell suspension.

[0156] b. Centrifuge at 4°C, 1500 rpm for 5 min and discard the supernatant.

[0157] c. Resuspend the cells in 1×ACK and lyse on ice for 3 min.

[0158] d. Then use a large volume of BSS to terminate the lysis and centrifuge again at 4°C, 1500 rpm for 5 min.

[0159] e. Discard the supernatant, resuspend the cell pellet in BSS, count and set aside.

[0160] (2) Discard the supernatant and use magnetic bead separation buffer at 1×10 7 Resuspend the cell pellet at a concentration of 10 cells / 90 μL.

[0161] (3) 10 μL / 1×10 7 Add CD4 positive selection magnetic beads to cells, mix thoroughly, and incubate at 2-8℃ in the dark for 10 min (at this time, 1 mL of magnetic bead sorting buffer can be used to rinse the LS separation column 3 times).

[0162] (4) Add the above suspension to the separation column, and then rinse the separation column with 5 × 1 mL magnetic bead separation buffer (the cell suspension flowing down is CD4 - cell).

[0163] (5) Remove the column, add 5 × 1 mL of magnetic bead separation buffer, and use the piston to push out the cells in the column, which are CD4 + T lymphocytes.

[0164] (6) Counting of CD4 + T lymphocytes, anti-CD4 flow cytometry staining, CD4 sorting + FOXP3-GFP + The cells are called Treg cells.

[0165] 2. Single-cell RNA-seq data preprocessing Treg cells from the liver and spleen of neonatal and adult mice were obtained by flow cytometry sorting, and then the libraries were constructed and sequenced according to the official 10×Genomics 5' end single-cell transcriptome and single-cell V(D)J sequencing library construction process. Cell Ranger ( https: / / support.10xgenomics.com / single-cell-gene-expression / software / The raw data were aligned and quantified using the standard process of R package Seurat (downloads / latest). After obtaining the cell-gene matrix, we used the R package Seurat (v4.3.0) for downstream data analysis. Throughout the analysis, all functions were run using default parameters unless otherwise stated. First, in order to filter out low-quality cells, strict filtering criteria (nFeature_RNA>200; nFeature_RNA<4200; mitochondrial gene proportion <10%) were used to filter Treg cells in the liver and spleen, and Cd68 hi or Cd19 + cells to remove non-T cell contamination.

[0166] 3. Single-cell RNA-seq integration and clustering The merge function of the Seurat package was used to integrate Treg data from the liver and spleen of newborn and adult mice. The data were then log-normalized with a scaling factor of 10,000, and then 2,000 highly variable genes were selected for downstream analysis using the vst method. After removing unnecessary sources of variation in single-cell data using the ScaleData function, principal component analysis was performed on the single-cell expression matrix containing highly variable genes. The first 20 principal components were used to correct for batch effects using harmony (v1.1.0). KNN (k-nearest neighbor) graphs were then used to embed cells into their structures. After attempting to divide the graph into highly interconnected "neighbors", the first 20 principal components were used for graph-based cell clustering (resolution = 0.5). These subpopulations were then visualized using UMAP (Uniform Manifold Approximation and Projection) dimensionality reduction.

[0167] 4. Determination of differentially expressed genes in subgroups and GSEA gene set enrichment analysis The FindAllMarkers function of the Seurat package was used to determine the differentially expressed genes between the subgroups, or the FindMarkers function was run to determine the differentially expressed genes between two organs or two subgroups. Then, the R package clusterProfiler (v4.6.2) was used to perform Gene Set Enrichment Analysis (GSEA) or GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis to determine the functions of each subgroup of cells.

[0168] 5. Developmental Trajectory Inference The developmental trajectories of each Treg cell subset were inferred by pseudo-time series analysis using the R package Monocle (v2.26.0). The newCellDataSet function (lowerDetectionLimit = 0.5; expressionFamily = negbinomial.size) was used to construct objects based on the differentially expressed genes of each subset identified by Seurat.

[0169] 6. SCENIC analysis The transcription factors (TFs) of single-cell transcriptome data were analyzed using the python package pySCENIC (v 0.9.11) with default parameters. Finally, TFs with significant regulatory intensity and core roles were screened out, and the related pathways and target genes regulated by them were analyzed, and a regulatory network with TFs as the driving center was established.

[0170] 7. Single-cell TCR-seq data processing Single-cell TCR data from liver and spleen of neonatal and adult mice were aligned and assembled using Cell Ranger to obtain the filtered_contig_annotations.csv file containing the CDR3-related information of TCR-α and TCR-β chains of single cells corresponding to the barcodes. The contig_annotation data of different samples were then combined into a list object using the R package scRepertoire (v1.8.0). The combined TCR contig list file was then integrated with the corresponding Seurat object of the scRNA-seq data using the combineExpression function. For cells containing more than two TCR chains, only the TCR-α and TCR-β chains with the highest expression were retained. The scRepertoire package was then used to analyze and compare the characteristics of TCR distribution between liver and spleen or between neonatal and adult mice, the frequency of different clonal types, and clonal diversity.

[0171] 8. Single-cell RNA velocity analysis The Velocyto program was used for cell RNA velocity analysis, which uses the relative ratio of unspliced ​​and spliced ​​mRNA abundance as an indicator of cell status. First, the standard process of velocyto.py was used to obtain the loom file of the sample containing the spliced ​​and unspliced ​​RNA conditions. Next, the loom files of different samples were merged, and downstream analysis was performed using the velocyto.R standard process. Finally, the predicted cell differentiation state was projected on the UMAP map.

[0172] 9. Analysis of Cell-cell Communication CellPhoneDB and NicheNet were used to analyze intercellular communication. First, the mouse genes were converted into the corresponding human gene names. Then, the metadata file and count file of the Seurat object were written, and the CellPhoneDB standard process and database were used to analyze intercellular communication and evaluate the potential communication ligand receptor pairs between Treg cells and other types of cells. Then, the NicheNet tool was used to compare the different ligand receptor activities in the newborn and adult states, with Treg cells as sender cells and other cells in the liver as receiver cells.

[0173] Statistical analysis was performed using GraphPad Prism 8.0.2 software to assess whether there was a significant difference between the two groups using paired or unpaired t-test, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.001, ns p>0.05. The data shown are mean ± SD.

[0174] 10. RNA-seq Mouse liver tissue was collected in EP tubes, frozen in liquid nitrogen and then transferred to -80℃. Tissue RNA was extracted and RNA integrity was quality controlled using Agilent 2100bioanalyze. The mRNA that passed the quality inspection was reverse transcribed to construct a cDNA library. The library was then preliminarily quantified using Qubit2.0 Fluorometer and diluted to 1.5 ng / ul. The insert size of the library was detected using Agilent2100 bioanalyzer. After meeting expectations, qPCR accurately quantified the effective concentration of the library (the effective concentration should be higher than 2 nM) to ensure the quality of the library. Illumina sequencing was performed according to the data volume requirements, followed by raw data filtering, sequencing error rate check, and GC content distribution check, and finally Clean Reads for subsequent analysis were obtained. Clean Reads were aligned with the reference genome using HISAT2 software to obtain the positioning information of Reads on the reference genome, and the proportion of Reads in the exons, introns, and intergenic regions of the genome were counted for alignment visualization and quantitative analysis of gene expression. (Completed by Beijing Novogene Company). Alternative splicing was performed using the rMATS program (version 3.2.5), and differential alternative splicing was determined using default parameters (FDR ≤ 0.10, junction read counts ≥ 10, PSI ≥ 10%, FPKM ≥ 1).

[0175] 11. Metabolomics testing Mouse liver tissue (greater than 50 mg) was collected and placed in an EP tube, quickly frozen in liquid nitrogen, and then stored at -80°C for later use.

[0176] 11.1 Precision Non-targeted Metabolomics Testing For non-targeted metabolome detection, the liver samples were first ground with a grinder and incubated at 4°C, 1500 rpm for 30 min. Then, the samples were centrifuged at 4°C, 12000 rpm for 10 min, and the supernatant was taken into a 1.5 mL EP tube and evaporated using a centrifugal concentrator. The samples were then reconstituted with 100 μL 1% acetonitrile for testing. All analyses were performed using an ultra-high pressure liquid chromatograph (Agilent 1290 II, Agilent Technologies, Germany) and a tandem high-resolution mass spectrometer (5600 Triple TOF Plus, AB Sciex, Singapore) in electrospray ionization (ESI±) mode. The primary mass spectrometry TOF full scan (Full sacn) and the secondary mass spectrometry information dependent acquisition (IDA) were used for data acquisition. MarkerView 1.3 (AB Sciex, Concord, ON, Canada) software was used to extract the peak area, mass-to-charge ratio, and retention time of the primary mass spectrometry raw data and generate a two-dimensional data array (filtering out isotope peaks). PeakView 2.2 (AB Sciex, Concord, ON, Canada) was used to extract the secondary mass spectrometry data and compare them with the Metabolites database, HMDB, METLIN, and standards to identify metabolite IDs, and the identified IDs were assigned to the corresponding ions in the primary mass spectrometry two-dimensional data array. The identified metabolome data were statistically analyzed and pathway analyzed using a self-written program based on the R language (completed by China Science and Technology Lipid Diagnostics Co., Ltd.).

[0177] 11.2 Lipidome analysis Lipids were extracted from liver samples using a modified Bligh / Dyer extraction method. The samples were then reconstituted in an isotope standard mixture and all analyses were performed using an Exion UPLC-QTRAP 6500 Plus (Sciex) LC / MS in electrospray ionization (ESI) mode. Phenomenex Luna silica 3 μm (150x2.0 mm ID) columns and Phenomenex Kinetex 2.6 μm C18 columns were used to separate various polar lipids and neutral lipids in specific mobile phases under certain conditions. Multiple reaction monitoring (MRM) mass spectrometry was established for polar lipids for identification and quantitative analysis of various lipids, and lipids were quantified by the addition of internal standards. Neutral lipids were quantified using neutral loss MS / MS technology using isotope internal standards. Free cholesterol and sterols and their corresponding esters were analyzed by HPLC-MS / MS in atmospheric pressure chemical ionization mode (APCI) and internal standard quantification (completed by Zhongke Zhidian Company).

[0178] 7. Construction of dual luciferase reporter gene system The pMSCV expression plasmid was used to construct the pMSCV-Tbx21 plasmid containing the full-length Tbx21 sequence. The pCMV7.1 expression plasmid was used to construct the pCMV7.1-Foxp3 plasmid containing the full-length Foxp3 sequence. Then, the pIRIGF dual-luciferase reporter plasmid was used to insert the PENK promoter sequence before the firefly luciferase sequence and after the Renilla luciferase to construct a dual-luciferase reporter gene plasmid. Then, pMSCV-Tbx21 / pMSCV-NC (empty) or pCMV7.1-Foxp3 / pCMV7.1-NC (empty) was co-transfected with the dual-luciferase reporter gene plasmid containing the PENK promoter sequence in 293 T cells, and the relative value of firefly luciferase compared with Renilla luciferase was detected.

[0179] 3. Experimental steps and results (I) Neonatal liver Tregs are rich in terminally differentiated T-bet + Treg cells Experimental steps: Figure 1 Flow cytometry was used to detect the proportion of Treg cells and Tconv cells in the liver of newborn WT mice aged 1-3 weeks. Figure 2 RNA-seq was used to detect the global transcriptome of Treg cells in the liver and spleen of 12-day-old WT mice. Figure 3-Figure 9 Single-cell transcriptome sequencing was used to detect the transcriptional characteristics of Treg cells in the liver and spleen of 12-day-old and 7-week-old adults. After dimensionality reduction clustering using Seurat, 11 subgroups were obtained. Comparative analysis between samples, comparative analysis between subgroups, and transcription factor regulation between samples and different subgroups were performed, and the potential differentiation relationship between different groups was predicted. In addition, the changes in the proportion of different subgroups between different samples were analyzed.

[0180] The results are as follows: Figure 1 CD4 in 1-3 week old livers + Foxp3 + Cell ratio and CD4 + Foxp3 - Changes in cell proportions; Figure 2 The transcriptome sequencing results of Treg cells in the liver and spleen of 12-day-old wild-type mice were shown in Tbx21 and the expression of its regulated target genes. Figure 3 Foxp3 in 12-day-old liver (nLiver), spleen (nSpleen), 7-week-old liver (aLiver), spleen (aSpleen) +UMAP plot of single-cell transcriptome sequencing analysis of T cells. Figure 4 Foxp3 in 12-day-old liver (nLiver), spleen (nSpleen), 7-week-old liver (aLiver), spleen (aSpleen) + Projection of Treg cells on the UMAP map after TCR sequencing analysis. Figure 5 The bubble chart shows the characteristic genes of each cell subpopulation. Figure 6 The transcription factor regulatory network shows the transcription factor activity of each cell subset. Figure 7 Display T-bet for RNA velocity + Treg cells are in the terminal differentiation position, activated Treg (4th and 5th subgroups) or transitional Treg cells (10th subgroup) are T-bet + Precursors of Treg cells. Figure 8 The transcription factor regulatory network shows Foxp3 in liver (nLiver), spleen (nSpleen), 7-week liver (aLiver), and spleen (aSpleen). + Comparative analysis of transcription factors of Treg cells. Fig. 9 The bubble chart shows the ratio of each subgroup, nLiver / sSpleen and nLiver / aLiver.

[0181] These results indicate that the terminally differentiated T-bet + Treg cells.

[0182] (ii) Endogenous opioid precursor proenkephalin (PENK) in neonatal liver T-bet + It is highly expressed in Treg cells and participates in the interaction between Treg cells and hepatocytes. T-bet and FOXP3 directly regulate the promoter region of PENK to regulate the expression of PENK.

[0183] Experimental steps: Figure 10-11 Single-cell transcriptome was used to detect the transcription of Treg cells in the liver of 12-day-old T-cKO mice and combined with Treg cells in the liver of 12-day-old WT mice for analysis. Figure 12-13 The combined analysis of liver Treg cells from newborn WT and adult WT mice found a molecule called PENK that was only upregulated in neonatal liver Treg cells, and showed the distribution of PENK in the subpopulations. Fig.14 The expression of PENK in various cell types was detected by qPCR. Fig.15The Nichenet package was used to analyze the interacting molecule pairs of mouse Treg cells as regulators and other liver cells as regulated parties, and the molecules upregulated in neonatal Tregs compared to adult Tregs were compared. PENK was also ranked high among them. Fig.16 Flow cytometry was used to compare the expression of PENK in liver Treg cells of T-cKO mice and liver Treg cells of WT mice. Fig.17 The binding sites of T-bet and FOXP3 with PENK promoter predicted by Jasper database and dual luciferase reporter gene system were used to co-transfect T-bet / FOXP3 plasmid and plasmid containing PENK promoter sequence, and the relative intensity of luciferase was compared with that of empty transfected plasmid and plasmid containing PENK promoter sequence to observe whether T-bet and FOXP3 have direct regulatory effects on PENK promoter region.

[0184] The results are as follows: Fig.10 Show 12 days old Tbx21 fl / fl Foxp3 cre Mice and Tbx21 w / w The UMAP diagram of the single-cell RNA sequencing results of control mouse liver Treg cells and the projection of the single-cell TCR sequencing results on the UMAP diagram showed that T-bet deficiency led to a decrease in the third subpopulation of Treg cells, and cell differentiation was blocked in the activated Treg cells and transitional cell subpopulations. Fig.11 Show 12 days old Tbx21 fl / fl Foxp3 cre Mice and Tbx21 w / w The bubble chart shows the ratio of each subset of liver Treg cells in the control mice, and the decrease of the third subset is also shown in the bubble chart. Fig.12 Comparison of the transcriptome of the third subgroup of liver and spleen Treg cells at 12 days and 7 weeks of age showed Penk It was only upregulated in 12-day-old liver Tregs. Fig.13 UMAP diagram showing Treg single-cell RNA sequencing results Penk Distribution of positive cells. Fig.14 Real-time quantitative PCR comparison at 12 days of age Tbx21 fl / fl Foxp3 cre Mice and Tbx21 w / w Treg cells in the liver and spleen of control mice, CD4 + CD25 -Non-Treg cells, 12 days old Tbx21 fl / fl Foxp3 cre Mice and Tbx21 w / w In the liver and spleen of control mice Penk expression level. Fig.15 Shows ligand pair analysis of subpopulation 3 liver Treg cells and single-cell transcriptome sequencing of 7-day-old mouse liver, showing the top 20 ligand pairs in neonatal Treg cells. Fig.16 The results showed that the expression of PENK in Treg cells in the liver of T-cKO mice was reduced compared with that in WT mice. Fig.17 It was shown that the protein level expression of PENK in liver Treg of T-cKO mice was significantly lower than that of WT mice. Fig.17 It shows that T-bet and FOXP3 can directly bind to the promoter region of PENK to regulate the expression of PENK.

[0185] The above results show that CD4 + Foxp3 + Treg cells promote liver maturation in 1-2 week old mice by expressing proenkephalin PENK.

[0186] 3. Newborn period Penk fl / fl Foxp3 cre Hepatic maturation disorder in (P-cKO) mice is improved by intraperitoneal injection of Met-ENK Tbx21 fl / fl Foxp3 cre Liver maturation in (T-cKO) mice Experimental steps: Fig.18 The liver weight and liver-to-body ratio of WT and P-cKO mice were measured. Fig.19 The characteristics of various liver cells in WT and P-cKO mice were detected by flow cytometry. Fig. 20 Liver maturation-related indicators in the livers of WT and P-cKO mice were detected by qPCR. Fig.21 Met-ENK (a decomposition product of PENK) was supplemented to T-cKO mice by intraperitoneal injection, and body weight, liver weight and liver-to-body ratio were tested. It was found that compared with T-cKO, the supplemented group achieved a certain recovery. Fig. 22 Flow cytometry revealed that the proportion of hepatocytes in T-cKO mice also recovered to a certain extent after Met-ENK was supplemented. Fig.23qPCR detected that liver maturation-related indicators in T-cKO mice recovered to a certain extent after Met-ENK was supplemented.

[0187] The results are as follows: Fig.18 Compared with wild-type controls in the same cage, 12-day-old Penk fl / fl Foxp3 cre The liver weight of mice was reduced, and the liver weight / body weight ratio was decreased. Fig.19 Compared with wild-type controls in the same cage, 12-day-old Penk fl / fl Foxp3 cre Mouse liver hepatocytes (CD45 - CD31 - Ter119 - ) decreased in proportion and number of cells, and the size of hepatocytes decreased. + CD45 - CD31 - Ter119 - ) increased, and the proportion of progenitor cells (CD24 + CD45 - CD31 - Ter119 - ) ratio increased. Fig. 20 Compared with wild-type controls in the same cage, 12-day-old Penk fl / fl Foxp3 cre Embryonic liver-related genes expressed in mouse hepatocytes Afp and Gpc3 Down-regulation of impaired, adult liver-related genes Hnf4a and Ugt1a9 Upward regulation disorder. Fig.21 A schematic diagram of the Met-ENK complementation experiment is shown. Specifically, 6-day-old Tbx21 fl / fl Foxp3 cre Mice were intraperitoneally injected with Met-ENK 10 mg / kg daily, and the mice were harvested and tested at 12 days of age. Compared with the solvent (DMSO) control group, the liver weight / body weight ratio of Met-ENK mice increased, but the liver weight was not significantly affected. Fig. 22 Compared with the solvent (DMSO) control group, the Met-ENK mouse hepatocytes (CD45 - CD31 - Ter119 - ) increased, and bile duct epithelial cells (EpCAM + CD45 - CD31- Ter119 - ) ratio is reduced. Fig.23 Compared with the solvent (DMSO) control group, the expression of Met-ENK in rat hepatocytes was Hnf4a and Ugt1a9 Expression increased, but Afp There was no significant difference in expression levels.

[0188] The above results show that the injection of methionine enkephalin Met-ENK improves Tbx21 fl / fl Foxp3 cre Liver maturation in newborn mice.

[0189] (IV) Met-ENK promotes hepatocyte maturation and inhibits cholangiocyte differentiation in in vitro 3D liver organoid culture.

[0190] Experimental steps: Fig.24 It is a method that uses in vitro cell 3D culture of primary hepatocytes to induce the formation of liver 3D organoids, adds Met-ENK, and uses immunofluorescence to determine the liver 3D formation effect after 12 days of culture. Fig.25 The qPCR method was used to detect the upregulation of liver maturation-related indicators and the inhibition of bile duct cell differentiation and maturation-related indicators when Met-ENK was added during the 12-day culture of liver 3D organoids. Fig.26 Flow cytometry was used to detect the protein level of EPCAM expression in 3D liver cultured for 12 days after the addition of Met-ENK.

[0191] The results are as follows: Fig.24 The effect of Met-ENK (2 μM) on liver 3D organoids is shown. Compared with the DMSO group, the group with Met-ENK showed an upregulation of ALB expression associated with hepatocyte maturation and a decrease in SOX9 expression associated with bile duct cell differentiation. Fig.24 It shows that 2 μM Met-ENK (labeled as Met-ENK in the figure) promotes the upregulation of mature liver function genes expressed by hepatocytes in 3D organoid culture. Fig.25 2uM Met-ENK was shown to inhibit the upregulation of differentiation genes of cholangiocytes in 3D organoid culture. Fig.26 It was shown that 2uM Met-ENK inhibited the expression ratio of EPCAM, a characteristic of bile duct cells, in 3D organoid culture.

[0192] The above results indicate that Met-ENK promotes the maturation of hepatocytes in in vitro liver 3D organoid culture and inhibits the differentiation of cholangiocytes in in vitro liver 3D culture.

[0193] (V) Met-ENK affects hepatocyte maturation and cholangiocyte differentiation through opioid receptors.

[0194] Fig. 27 qPCR was used to compare the expression of Ogfr and Oprd1, the two opioid receptors with the strongest affinity for Met-ENK, in hepatocytes and bile duct cells of WT mice. Fig.28 Flow cytometry was used to compare the proportion of hepatocytes and cholangiocytes in WT mice injected with DMSO (solvent) and opioid receptor inhibitors. Fig.29 The expression of liver maturation markers and bile duct maturation markers were compared by qPCR.

[0195] Fig. 27 The expression of Ogfr and Oprd1, two opioid receptors with the strongest affinity for Met-ENK, in hepatocytes and bile duct cells of WT mice is shown. Fig.28 It showed that after neonatal WT mice were injected with a broad-spectrum opioid receptor inhibitor, the proportion of hepatocytes decreased and the proportion of bile duct cells increased. Fig.29 The expression of hepatic maturation-related markers was decreased and markers of cholangiocyte differentiation and maturation were upregulated, similar to the characteristics of P-cKO mice.

[0196] (VI) Met-ENK inhibits Notch signaling through opioid receptors to affect bile duct cell differentiation Fig.30 The expression of bile duct markers and Notch signaling involved in bile duct differentiation in T-cKO mice and WT mice was shown by population transcriptome. Fig.31 The expression of Sox9, which characterizes the maturation of cholangiocytes, in P-cKO mice was shown by qPCR. Fig.32 The expression of Sox9, a marker of differentiation and maturation of cholangiocytes in 3D liver culture, was detected by qPCR after the addition of Met-ENK and when Met-ENK and receptor inhibitors were added simultaneously. Fig.33 This is the expression of ADAM metalloproteinase 10 (Adam10) in T-cKO mice, P-cKO mice and in vitro 3D culture.

[0197] Fig.30 It was shown that WT mice upregulated the expression of bile duct markers and corresponding Notch signaling-related genes involved in bile duct differentiation. Fig.31 It was shown that P-cKO mice upregulated the expression of the bile duct marker Sox9 compared to WT mice. Fig.32 It was shown that the addition of Met-ENK inhibited the expression of the bile duct marker Sox9, and the addition of a broad-spectrum opioid receptor inhibitor reduced this situation, indicating that Met-ENK affected the expression of Sox9 through opioid receptors. Fig.33The expression of Adam10, an enzyme involved in the cleavage of Notch nuclear import signals, is shown. The addition of Met-ENK reduces the expression of the cleavage enzyme and reduces the nuclear import of Notch protein fragments with transcriptional regulatory activity, thereby inhibiting the expression of Notch signals and Sox9 in bile duct cells. The use of broad-spectrum receptor inhibitors can reverse this situation.

[0198] The above results indicate that Met-ENK can affect the expression of Adam10, a cleavage enzyme involved in the nuclear import of Notch protein, through opioid receptors, and then affect the expression of bile duct cell markers and Notch signal downstream molecules.

[0199] (VII) Met-ENK promotes hepatocyte maturation by promoting the expression of the alternative splicing enzyme Esrp2 Fig.34 The heat map of differential gene analysis of the population transcriptome shows the liver mature gene expression and liver naive gene expression in T-cKO mice compared with WT mice. Fig.35 qPCR was used to show the expression of Esrp2 in T-cKO mice and P-cKO mice, as well as in T-cKO mice injected with Met-ENK inhibitors and WT mice injected with receptor inhibitors. Fig.36 Using rMATS to analyze the alternative splicing events of the transcriptome of the T-cKO and P-cKO mice, we found that the alternative splicing of the Esrp2-regulated gene Nav2 was significantly different from that of WT mice.

[0200] Fig.34 It was shown that the expression of liver mature genes was decreased and the expression of liver immature genes was upregulated in T-cKO mice compared with WT mice. Fig.35 It was shown that the presence of PENK led to upregulation of Esrp2 expression. Fig.36 The results show that T-cKO, P-cKO and WT mice have alternative splicing changes in the expression of the Esrp2-regulated gene Nav2, and exon skipping occurs.

[0201] 8. Met-ENK can promote the maturation of liver cancer cells Fig.37 The researchers used in vitro cell culture to try to add Met-ENK to Hepa1-6 mouse liver cancer cells, and used qPCR to detect liver maturation-related indicators.

[0202] Fig.37 It was shown that the addition of 500 nM Met-ENK to the hepatocellular carcinoma culture medium (DMEM + 10% FBS + 1x penicillin and streptomycin) promoted the upregulation of Hepa1-6 hepatocellular carcinoma cell line Hnf4a and CK18 , down Afp and Gpc3 transcription level.

[0203] The above results indicate that for hepatocytes, Met-ENK may affect the maturation of hepatocytes by affecting the expression of the alternative splicing enzyme ESRP2 through opioid receptors, thereby affecting the alternative splicing of liver maturation genes.

[0204] In summary, the present invention can promote liver maturation of 2-week-old mice by administering Met-ENK to mice within 1 week of birth. Compared with the solvent DMSO control, the liver weight / body weight ratio of mice that obtained Met-ENK increased, the proportion of hepatocytes increased, the proportion of bile duct cells decreased, the expression of embryonic liver-related genes decreased, and the expression of mature liver-related genes was upregulated. In addition, the present invention uses a 3D in vitro culture system prepared by 1-day-old hepatocytes. The addition of Met-ENK can reduce the expression of embryonic liver-related genes and increase the expression of mature liver-related genes, which may be achieved by regulating the expression of the alternative splicing enzyme Esrp2 of hepatocytes. At the same time, Met-ENK can also reduce the expression of bile duct differentiation genes and Notch signals, which may be caused by affecting the expression of Adam10, an enzyme that cuts Notch nuclear entry signals through opioid receptors, and affecting the nuclear entry of Notch proteins.

[0205] Therefore, the cleavage product of proenkephalin PENK, methionine enkephalin Met-ENK, can promote liver maturation by in vivo injection or addition to in vitro liver organoid 3D culture medium.

[0206] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of methionine enkephalin in promoting liver maturation for non-diagnostic therapeutic purposes.

2. Use of methionine enkephalin in preparing a product for promoting liver maturation or treating liver maturation disorders.

3. The use according to claim 2, characterized in that: The product is at least one of a medicine, a reagent or a kit, and a culture medium.

4. Application of methionine enkephalin in in vitro liver 3D culture.

5. A method for culturing an in vitro 3D liver, characterized in that: include: The liver parenchymal cells are cultured in a culture medium containing methionine enkephalin; preferably, the culture medium containing methionine enkephalin comprises the following components: DMEM / F12 medium, 10% fetal bovine serum, insulin-transferrin-selenium-aminoethanol, penicillin and streptomycin, human epidermal growth factor, human hepatocyte growth factor, dexamethasone and methionine enkephalin.

6. The use according to claim 1, characterized in that: Methionine enkephalin promotes liver maturation through at least one of the following pathways: (1) Inhibit the Notch signaling pathway; (2) Inhibit the growth of Hepa1-6 tumor cells; (3) Inhibit the differentiation and maturation of bile duct epithelial cells; (4) Promote hepatocyte maturation; Preferably, methionine enkephalin reduces the expression levels of Notch signaling pathway related genes Sox9 and Hes1; methionine enkephalin reduces the expression levels of embryonic liver related genes Afp and Gpc3, and increases the expression levels of mature liver related genes Hnf4a, Esrp2, Alb, Ttr, Cyp1a2 and Ugt1a9.

7. Use of opioid growth factor receptor antagonists in the preparation of drugs for inhibiting liver maturation.

8. A method for in vitro high-throughput drug screening and / or drug detection, characterized in that: include: Liver parenchymal cells are cultured in a culture medium containing methionine enkephalin to obtain a 3D liver culture; then a drug is applied to the 3D liver culture for detection and analysis.

9. A method for constructing an animal model, characterized in that: include: Injecting methionine enkephalin into model animals promotes liver maturation in model animals.

10. A 3D liver culture obtained by the culture method according to claim 5.

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