WO2021047495A1 - 体内外化学诱导成纤维细胞直接重编程为肝细胞的化学小分子组合物及方法 - Google Patents

体内外化学诱导成纤维细胞直接重编程为肝细胞的化学小分子组合物及方法 Download PDF

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WO2021047495A1
WO2021047495A1 PCT/CN2020/113954 CN2020113954W WO2021047495A1 WO 2021047495 A1 WO2021047495 A1 WO 2021047495A1 CN 2020113954 W CN2020113954 W CN 2020113954W WO 2021047495 A1 WO2021047495 A1 WO 2021047495A1
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small molecule
inhibitor
fibroblasts
hepatocytes
inhibitors
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张培霖
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海门雨霖细胞科技有限责任公司
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C—CHEMISTRY; METALLURGY
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts

Definitions

  • the present invention belongs to the intersecting fields of cell biology, stem cell biology (cell reprogramming), medicine, and pharmacy; more specifically, the present invention relates to a gene that does not introduce or use any foreign gene/transcription factor/MicroRNA (miRNA), Or its genes combined with carriers, or its RNA, protein and polypeptide inducing factors, nor use any cytokines or growth factors. It only uses a combination of small chemical molecules (referred to as small molecules) to chemically induce fibroblasts in vivo and in vitro to regenerate directly. Chemical small molecule composition and method for programming (transdifferentiation) into hepatocytes.
  • miRNA foreign gene/transcription factor/MicroRNA
  • liver fibrosis liver fibrosis
  • liver fibrosis liver fibrosis
  • acute and chronic liver failure caused by various liver diseases, chemical drugs, trauma, etc.
  • He is critically ill and has a high case fatality rate.
  • Liver transplantation is an effective method for the treatment of end-stage liver disease or liver failure.
  • the lack of liver source makes many patients, especially patients with acute liver failure, lose treatment opportunities.
  • Hepatocyte transplantation, bioartificial liver and whole liver transplantation derived from bioengineering technology are important alternative methods of liver transplantation and have been concerned and studied. How to obtain sufficient sources of human liver cells for clinical treatment has become an international research hotspot.
  • the main ways to obtain functional hepatocytes are as follows: 1) Isolate primary hepatocytes from the donor liver; 2) Directly differentiate into hepatocytes from stem cells or induced pluripotent stem cells; 3) Transform from somatic cells such as fibroblasts Differentiate into hepatocytes and so on. Isolation of primary hepatocytes from donor livers is limited due to the severe lack of liver sources. Stem cells are directed to induce differentiation to obtain functional hepatocytes. Because of the immortal proliferation potential of stem cells, it has become the most effective means and way to obtain a sufficient number of hepatocytes.
  • the existing targeted differentiation methods have one or more of the following defects: such as low differentiation efficiency, low transformation purity, insufficient function of differentiated hepatocytes, possible immune rejection, incomplete differentiation of stem cells and potential carcinogenic risks And the high cost, etc., can not meet the clinical needs.
  • the direct reprogramming (transdifferentiation) of fibroblasts into hepatocytes has attracted attention because of avoiding the potential risks of reprogramming pluripotent stem cells into iPS cells (Induced pluripotent stem cells) and their applications.
  • the existing methods of transdifferentiating fibroblasts into hepatocytes still use the introduction of exogenous genes/transcription factors//MicroRNA (miRNA) genes, inducing factors such as small chemical molecules, various cytokines or growth factors, etc.
  • Substitution factors composed of multiple inducer combinations, induce fibroblasts to transdifferentiate hepatocytes.
  • the deficiencies in the method of directed differentiation of hepatocytes, iPS cell reprogramming, or transdifferentiation of hepatocytes are bottlenecks that urgently need to be resolved in the clinical application of hepatocytes.
  • Fibrotic diseases such as liver cirrhosis (liver fibrosis) caused by fibroblasts or fibroblast abnormalities are currently lacking effective treatment drugs and methods in clinic, and there is an urgent need to develop effective new methods and new drugs.
  • Cell reprogramming is the conversion of cells from one type to another. It is a process of inducing and regulating specific cell signaling pathways or epigenetic modifications to change the fate of cells.
  • epigenetic refers to the fact that the DNA sequence/structure does not change, but the gene expression has undergone heritable changes.
  • Induced cell reprogramming includes: (1) Reprogramming of induced pluripotent stem cells (iPS cells) that reverse the induced differentiation of cells to a pluripotent or pluripotent state; (2) Reprogramming of induced pluripotent stem cells (iPS cells) without going through the stage of pluripotent stem cells, from a differentiated cell type Cells that are directly transformed into another differentiated cell type are directly reprogrammed (also known as: transdifferentiation/lineage reprogramming).
  • iPS cells induced pluripotent stem cells
  • iPS cells induced pluripotent stem cells
  • Chemically induced direct cell reprogramming does not introduce or use any exogenous genes/transcription factors/MicroRNA (miRNA) genes, and induce factors such as RNA, proteins and peptides; only use chemical small molecule targeting The process of inducing and regulating cell signaling pathways and epigenetic modification to change the epigenetic modification to change the gene expression profile of the cell and directly transform one differentiated cell into another differentiated cell.
  • miRNA microRNA
  • small chemical molecules referred to as small molecules
  • small molecules can target and regulate specific signaling pathways and epigenetics, so that one type of cell can be transformed into another type of cell; it has good druggability, low cost, and stability With good performance and simple operation, it is the best candidate to replace induced transcription factors; therefore, chemically induced cell reprogramming has become one of the important goals in the field of stem cell scientific research.
  • the purpose of the present invention is to provide a method that does not introduce or use any exogenous genes/transcription factors/MicroRNA (miRNA) genes, and induce factors such as RNA, proteins or polypeptides; nor use any cytokines or growth factors, only Using an inducing composition composed of two (classes) chemical small molecule inhibitors: GSK3 ⁇ inhibitor and HMT inhibitor, the small molecule inducing composition that chemically induces and regulates fibroblasts directly reprogramming (transdifferentiation) into hepatocytes in vivo and in vitro And method.
  • miRNA microRNA
  • the present invention is an invention that has been disclosed before the present inventor: only 3 kinds of chemical small molecules (GSK3 ⁇ inhibitor, G9aHMT inhibitor, TGF ⁇ inhibitor) are used to induce direct reprogramming (transdifferentiation) of human fibroblasts into hepatocytes. Further innovations based on small molecule compositions and methods.
  • the use of the chemical small molecule composition includes: for preparing in vivo inducing in situ fibroblasts to transdifferentiate into hepatocytes, with the in situ transdifferentiation of hepatocytes to reduce or reduce the fibrosis of fibrotic tissues or organs, so it can be added
  • Drug carriers or excipients are developed and prepared into drugs/prodrugs/pharmaceutical compositions for clinical treatment of fibrotic diseases (such as liver cirrhosis/liver fibrosis); or, preparation of reagents or culture media for inducing fibroblasts to transdifferentiate hepatocytes ; Or used to transdifferentiate fibroblasts into hepatocytes to prepare transformed hepatocytes to provide a source of hepatocytes for scientific research applications, medical applications and clinical applications such as liver cell transplantation to treat liver failure.
  • composition of the present invention which is composed of only two small chemical molecules (GSK3 ⁇ inhibitor and HMT inhibitor), induces the transdifferentiation of fibroblasts into hepatocytes and the composition invented by the present inventor.
  • the former Compared with the composition and method for inducing fibroblast transdifferentiation into hepatocytes composed of two small molecules (GSK3 ⁇ inhibitor, G9aHMT inhibitor, TGF ⁇ inhibitor), the former not only has three small molecule compositions to induce fibroblast transformation In addition to the advanced and innovative nature of differentiated hepatocytes, it also has: (1) The former has the same or similar effect as the latter in inducing fibroblast transdifferentiation into hepatocytes in vitro; but the former induces fibroblast transdifferentiation in vivo in vivo.
  • fibroblast/fibroblast abnormal-related diseases such as liver fibrosis are the same as or better than the latter; (2) The small molecule combination structure of the former and the latter is different; 2 small molecules The composition of the composition has fewer components, simpler combined structure, better druggability, lower risk of side effects, lower development cost, and easier development as drugs for related diseases; (3) the former small molecule composition The number of effective small chemical molecules in the constituent HMT inhibitors has increased, and the range of candidate drug chemical small molecules has been wider.
  • a chemical small molecule composition for chemically inducing fibroblasts in vivo and in vitro to directly reprogram (transdifferentiate) into hepatocytes.
  • the chemical small molecule composition only includes chemical small molecule inhibitors: GSK3 ⁇ inhibitor and histone methyl transferase (HMT) inhibitor; or, the small chemical molecule composition only consists of chemical small molecules Inhibitor: GSK3 ⁇ inhibitor and HMT inhibitor composition;
  • no exogenous gene/transcription factor/MicroRNA (miRNA) gene, and inducing factors such as RNA, protein, or polypeptide are introduced or used in the fibroblast; or, in the chemical small molecule composition Does not contain or use any exogenous gene/transcription factor/MicroRNA (miRNA) gene, its transcribed RNA, or its translated protein and polypeptide inducing factors; nor does it contain or use any cytokine or growth factor;
  • the chemical small molecule HMT inhibitor includes (but is not limited to): G9a histone methyltransferase (G9aHMT) inhibitor, EZH2 histone methyltransferase (EZH2HMT) inhibitor; or Chemical small molecule compositions include:
  • G9aHMT inhibitor/EZH2HMT inhibitor Chemical small molecule GSK3 ⁇ inhibitor and HMT inhibitor (G9aHMT inhibitor/EZH2HMT inhibitor); or only composed of GSK3 ⁇ inhibitor and G9aHMT inhibitor/EZH2HMT inhibitor;
  • the chemical small molecule composition includes: chemical small molecule inhibitors: GSK3 ⁇ inhibitor and G9aHMT inhibitor/EZH2HMT inhibitor; or, the chemical small molecule composition: consists of only chemical small molecules Inhibitor: GSK3 ⁇ inhibitor and G9aHMT inhibitor or EZH2HMT inhibitor.
  • the chemical small molecule composition may also include chemical small molecule inhibitors: TGF ⁇ inhibitors; or, the composition may also be composed of chemical small molecule inhibitors: GSK3 ⁇ inhibitors, HMT inhibitors (G9aHMT inhibitor/EZH2HMT inhibitor) and TGF ⁇ inhibitor.
  • the chemical small molecule composition includes: a chemical small molecule inducing composition composed of chemical small molecule inhibitors: GSK3 ⁇ inhibitor, G9aHMT inhibitor/EZH2HMT inhibitor, and TGF ⁇ inhibitor.
  • the chemical small molecule composition includes: a chemical small molecule inducing composition composed of chemical small molecule inhibitors: GSK3 ⁇ inhibitor and G9aHMT inhibitor/EZH2HMT inhibitor; or,
  • the chemical small molecule composition includes: a chemical small molecule inducing composition composed of chemical small molecule inhibitors: GSK3 ⁇ inhibitor, G9aHMT inhibitor/EZH2HMT inhibitor, and TGF ⁇ inhibitor.
  • the chemical small molecule composition wherein the chemical small molecule inhibitors: GSK3 ⁇ inhibitor, HMT inhibitor, and TGF ⁇ inhibitor are:
  • GSK3 ⁇ inhibitor 5-80 parts by weight, preferably 10-70 parts by weight; or the final concentration in the solution state is 0.1-20 ⁇ M, preferably 0.5-10 ⁇ M;
  • HMT inhibitor 0.1-50 parts by weight, preferably 0.5-40 parts by weight; or the final concentration in the solution state is 0.01-20 ⁇ M, preferably 0.05-10 ⁇ M; or
  • TGF ⁇ inhibitor 0.1-50 parts by weight, preferably 0.5-40 parts by weight, or the final concentration in the solution state is 0.05-10 ⁇ M.
  • the chemical small molecule composition in terms of parts by weight, the small molecule inhibitor: GSK3 ⁇ inhibitor (such as CHIR99021, LiCl, CHIR-98014) and HMT inhibitor [G9aHMT inhibitor (such as BIX01294, UNC0638, UNC0642)/EZH2HKMT inhibitor (such as EPZ005687, GSK343, UNC1999)] is (5-80): (0.1-50); or the molar concentration ratio in the solution state is (0.1-20): (0.01- 20); or
  • the GSK3 ⁇ inhibitors such as CHIR99021, LiCl, CHIR-98014
  • HMT inhibitors such as BIX01294, UNC0638, UNC0642
  • EZH2HKMT inhibitors such as EPZ005687, GSK343, UNC1999
  • TGF ⁇ inhibitor such as SB431542, A83-01, RepSox
  • 0.1-50 0.1-50
  • molar concentration ratio in the solution state is (0.1-20): (0.01- 20): (0.01-20).
  • said small molecule composition may also include pharmaceutically acceptable carriers or excipients; or add organic solvents, physiological saline, buffers, cell-based culture Base and other carriers or excipients.
  • small molecule GSK3 ⁇ inhibitor, HMT inhibitor (G9aHMTase inhibitor/EZH2HMT inhibitor); or GSK3 ⁇ inhibitor, HMT inhibitor (G9aHMT inhibitor/EZH2HMT inhibitor) and TGF ⁇ inhibitor add The weight accounts for 0.01 to 99.9% of the total weight of the composition; more preferably 50 to 99.9%; 0.01 to 50% in the solution state, such as 0.01%, 1%, 5%, 10%, 20%, 30%, etc. .
  • the weight unit of the above weight part ratio can be any weight unit such as kilogram (kg), milligram (mg), microgram ( ⁇ g), etc.; the molar unit of the molar concentration ratio can be: mole (M), millimole (mM), Any molar concentration unit such as micromole ( ⁇ M).
  • the effective dose of large animals or humans (including the solid or solution dose conversion) is converted into the effective dose of large animals or humans (including the solid or solution dose conversion) according to the small animal dose through the corresponding professional conversion formula, which also belongs to The scope of protection of the present invention.
  • the chemical small molecule inhibitor refers to the general term for inhibitors that can target and inhibit the GSK3 ⁇ signaling pathway, including but not limited to: CHIR-99021, BIO, LiCl, IM-12, TWS119, 1-Azakenpaullone, CHIR-98014, Tideglusib, AR-A014418, LY2090314, SB216763, AZD1080, and other small molecule inhibitors of GSK3 ⁇ that induce inhibition of GSK3 ⁇ signaling pathway.
  • equivalent pharmaceutical products, analogs, isomers and/or salts, hydrates or precursors thereof, or combinations thereof preferably GSK3 ⁇ inhibitor CHIR-99021, LiCl, BIO, LY2090314.
  • HMT inhibitors including (but not limited to): G9aHMT inhibitors or EZH2HMT inhibitors, and other HMT small molecule inhibitors that inhibit HMT; or their equivalent pharmaceutical products, analogs, Isomers and/or salts, hydrates or precursors thereof, or combinations thereof; preferably G9aHMT inhibitors, EZH2HMT inhibitors.
  • the small chemical molecule G9aHMT inhibitor refers to the general term of inhibitors that can target and inhibit G9aHMT, including but not limited to: BIX01294, UNC0638, A-366, UNC0631, BRD4770, UNC0224, UNC0646, UNC0642, UNC0321, BRD4770, HKMTI- 1-247, HKMTI-1-248, CPUY074020, DCG066, and other G9aHMT small molecule inhibitors that inhibit G9aHMT.
  • EZH2HMT inhibitor refers to the general term that can target and inhibit EZH2HMT, including but not limited to: EPZ005687, GSK343, Tazemetostat (EPZ-6438), UNC1999, JQ-EZ-05 (JQEZ5), EBI- 2511, CPI-1205, EPZ011989, PF-06726304, EI1, GSK503, GSK126, CPI-360, CPI-169, and other EZH2HKMT small molecule inhibitors that inhibit EZH2HKMT. Or their equivalent pharmaceutical products, analogs, isomers and/or their salts, hydrates or precursors, or combinations thereof; preferably EPZ005687, GSK343, UNC1999.
  • TGF ⁇ inhibitors refer to the general term of inhibitors that can target and inhibit the TGF ⁇ signaling pathway, including but not limited to: SB431542, A83-01, SB525334, LY2109761, RepSox, SD-208, GW788388, SB505124, EW-7197, Galunisertib, and other small molecule inhibitors of TGF ⁇ that induce inhibition of TGF ⁇ signaling pathway. Or their equivalent pharmaceutical products, analogs, isomers and/or their salts, hydrates or precursors, or combinations thereof; preferably TGF ⁇ inhibitors SB431542, A83-01, RepSox or LY2109761.
  • the chemical small molecule composition is a pharmaceutical composition for chemically inducing fibroblasts in situ in vivo to directly reprogram (transdifferentiate) into hepatocytes, and to induce fibroblasts in situ in vivo Transdifferentiation into hepatocytes and its effects can reduce or reduce the abnormality of fibroblasts or fibroblasts in tissues or organs, and can be developed and prepared as drugs/prodrugs/drugs for the treatment of fibrotic diseases (such as liver cirrhosis/liver fibrosis) Composition; the same mechanism, the small molecule composition has been perfected through research and development, and should have similar effects on the treatment of pulmonary fibrosis, renal fibrosis, and other tissues, organs and tubes, and can be prepared as a corresponding drug or pro Body drug
  • the small molecule composition is a pharmaceutical composition, and further includes a pharmaceutically acceptable carrier or excipient.
  • the carrier or excipient includes (but is not limited to) one or more selected from the following group: water , Saline, phosphate buffer or other aqueous solvents; DMSO (dimethyl sulfoxide), glycerol and ethanol or other organic solvents; microspheres, liposomes, microemulsions or polymer surfactants; colloidal drug delivery systems or Polymer drug-carrying system; or preservatives, antioxidants, correctives, fragrances, co-solvents, emulsifiers, pH buffer substances, binders, fillers, lubricants or other pharmaceutical excipients.
  • the pharmaceutical dosage forms that can be prepared from the chemical small molecule composition include (but are not limited to): solid dosage forms, including (but not limited to): powders, powders, tablets, pills, capsules, suspensions Release agents, controlled immediate release agents; liquid dosage forms, including (but not limited to): injections, infusions, suspensions, or other liquid dosage forms; gas dosage forms; or semi-solid dosage forms.
  • the use of the chemical small molecule composition including: for preparing in vivo chemically induced in situ fibroblasts to directly reprogram (transdifferentiate) into hepatocytes, and to transform them in situ in vivo Differentiating hepatocyte effect, reducing or reducing the fibrosis of fibrotic cell tissues or organs, to treat fibroblasts or fibroblast-related diseases, namely fibrotic diseases (such as: liver cirrhosis/liver fibrosis) drugs/prodrugs /Pharmaceutical composition; or, for the preparation of a transdifferentiation medium or reagent for chemically inducing fibroblasts in vitro and in vivo to directly reprogram (transdifferentiation) into hepatocytes; or, for chemically inducing fibroblasts in vitro to directly reprogram (transdifferentiation) Differentiation) into hepatocytes to prepare transdifferentiated/transformed hepatocytes to provide a source of
  • a fibroblast that is not introduced or used, or does not contain or use any exogenous gene/transcription factor/MicroRNA (miRNA) gene in a small molecule composition, and Inducing factors such as RNA, protein, or polypeptide; also do not use cytokines or growth factors; only use chemical small molecule compositions, in vivo and in vitro chemically induce fibroblasts to directly reprogram (transdifferentiate) into hepatocytes, the method includes : A method for inducing fibroblasts to transdifferentiate into hepatocytes by using any of the chemical small molecule compositions described above; or, for preparing in vivo in situ induced fibroblasts to directly reprogram (transdifferentiate) into hepatocytes; A drug that reduces or reduces abnormalities of fibroblasts or fibroblasts in tissues and organs by transdifferentiating hepatocytes in situ in vivo to treat fibrotic diseases with abnormal fibroblasts or
  • miRNA gene/transcription factor/
  • the small molecule composition has been perfected through research and development, and should have a similar effect on the treatment of pulmonary fibrosis, renal fibrosis, and other tissues and organs’ fibrotic diseases.
  • Corresponding drugs or prodrugs can be prepared;
  • transdifferentiation medium or reagents for chemically inducing fibroblasts to directly reprogram (transdifferentiate) into hepatocytes Methods.
  • the provided method for preparing the transdifferentiation medium or reagent for inducing the direct reprogramming (transdifferentiation) of fibroblasts into hepatocytes and the experimental steps thereof include:
  • each component is dissolved in an organic solvent or an aqueous solvent to prepare a concentrated solution reagent;
  • the organic solvent includes dimethyl Sulfoxide;
  • the aqueous solvent includes: water, physiological saline, phosphate buffer;
  • fibroblast transdifferentiation hepatocyte culture medium Dilute the concentrated solution reagent in step (1) into cell basal medium containing 5-20% calf serum (make the concentration of each component meet the previous The final concentration defined in any of the small molecule compositions) to obtain a culture medium for inducing fibroblast transdifferentiation of hepatocytes; wherein, the percentage content of each component of the culture medium can also fluctuate by 50%; Float up and down 30% better; better up and down 20%, such as 10%, 5%;
  • step (3) Induce the transdifferentiation of fibroblasts into hepatocytes: suspend and plate fibroblasts in a cell basal medium containing 5-20% calf serum or a serum-free medium containing various cytokines or growth factors, After the cells adhere to the wall, change to the transdifferentiation medium of step (2), culture at 37°C, and change the medium every 2-4 days; passage once every 3-15 days.
  • Subculture of induced fibroblast transdifferentiated hepatocytes discard the original culture medium, wash once with PBS, add cell digestion solution to digest the cells, 37°C, 1-5 minutes, stop the cell digestion, centrifuge, discard the supernatant, and The cell pellet was resuspended and plated according to passage 1:1-1:3.
  • the digestive juice used includes pancreatin, EDTA, Acutase, TrypleE and so on. Passage once every 3-15 days.
  • kits or kit for chemically inducing fibroblasts in vivo and in vitro to directly reprogram (transdifferentiate) into hepatocytes includes: any one of the aforementioned chemical small molecule compositions; or a pharmaceutically acceptable carrier or excipient added based on the small molecule composition for in vivo chemical induction of in situ formation Fibroblasts are directly reprogrammed (transdifferentiated) into hepatocytes, and the effect of in situ transdifferentiation of hepatocytes reduces or reduces the fibrosis of fibrotic tissues/organs, and the developed or prepared treatments for human fibrotic diseases (such as liver cirrhosis/liver Fibrosis) drugs/prodrugs/pharmaceutical compositions; or by adding organic solvents/physiological saline/buffer/cell basal medium and other carriers or excipients to induce fibroblasts to directly reprogram (transdifferentiate) into liver Transdifferenti
  • the kit or kit does not include or does not contain: any exogenous gene/transcription factor/MicroRNA (miRNA) gene used to be introduced into fibroblasts or added to the small molecule composition/transdifferentiation medium, or Its RNA, protein, polypeptide and other components; or its combination with gene carrier.
  • miRNA gene/transcription factor/MicroRNA
  • the fibroblasts include but are not limited to: human fibroblasts or mammalian fibroblasts; preferably, including but not limited to humans: skin fibroblasts, liver fibroblasts (liver Hepatic stellate cells (HSC), lung fibroblasts, kidney fibroblasts, pancreatic fibroblasts, and fibroblasts of other tissues or organs in the human body.
  • they are human skin fibroblasts and liver fibroblasts (hepatic stellate cells).
  • FIG. 1 Morphological comparison of hepatic fibroblasts (hepatic stellate cells HSC) and their transdifferentiated hepatocytes (ciHep).
  • the right figure of Figure 1A shows the hepatocytes (ciHep) obtained from the transdifferentiation of mouse HSC induced by the transdifferentiation hepatocyte medium 1 (2F) of two small molecule compositions;
  • the right figure of Figure 1B shows the transdifferentiation of the three small molecule combinations Differentiation medium 11 (3F) induces HSC transdifferentiation of hepatocytes (ciHep); compared with the morphology of HSC in the left panels of Figures A and B, the morphology of transformed hepatocytes has changed significantly, which is consistent with the morphology of liver cells.
  • the transformed hepatocytes clearly showed double-cell nuclei. All HSCs have been transdifferentiated into hepatocytes (ciHep).
  • the experimental results show that the small molecule composition (2F) composed of only two chemical small molecules (GSK3 ⁇ inhibitor and HMT inhibitor) is compared with the previously invented small molecule (GSK3 ⁇ inhibitor, G9aHMT inhibitor) and only three small molecules (GSK3 ⁇ inhibitor and G9aHMT inhibitor).
  • the small molecule composition (3F) composed of TGF ⁇ inhibitors has the same or similar effect in inducing fibroblasts to transdifferentiate into hepatocytes in vitro.
  • FIG. 1 Glycogen staining (PAS) of hepatic stellate cells (HSC) and their transdifferentiation to obtain liver-like cells (ciHep).
  • Figure 2 Right panel: shows the glycogen staining of ciHep obtained from HSC transdifferentiation.
  • the left panel of Figure 2 shows that hepatic stellate cell (HSC) glycogen staining is negative; it indicates that the transdifferentiated liver-like cells have the same glycogen storage activity as human hepatocytes.
  • FIG. 3 Oil-red staining of hepatic stellate cells (HSC) and liver-like cells (ciHep) obtained by transdifferentiation.
  • HSC hepatic stellate cells
  • ciHep liver-like cells
  • the right panel of Figure 3 shows that the fatty oil red staining of ciHep obtained by transdifferentiation of hepatic stellate cells (HSC) is positive, and the positive staining shows the ability of transdifferentiated liver cells to metabolize fat.
  • the HSC fatty oil red staining was negative; the results showed that the transdifferentiated liver-like cells had the unique function of hepatocytes to metabolize fat.
  • Fibroblasts express hepatocyte-related genes after transdifferentiation into liver-like cells (ciHep). The results of this experiment showed that fibroblasts were transdifferentiated into liver-like cells and highly expressed hepatocyte-related genes; indicating that fibroblasts had been transdifferentiated into hepatocytes.
  • FIG. 6 Comparative experiment on the treatment of liver fibrosis mouse animal model by oral in vivo in situ fibroblast transdifferentiation reagent 7 and 10, and fix the liver tissues of two treatment groups (Treat) and control (Ctrl) mice respectively Section and stain with Sirius Red (according to the instructions of the kit).
  • Figure 6A Compared with the control group, mice in the treatment group treated with Fibroblast Transdifferentiation Reagent 7 (2F) had significantly reduced Sirius Red staining;
  • Figure 6B Compared with the control group, Fibroblast Transdifferentiation Reagent 10 (2F) was administered orally The Sirius red staining of mice in the 3F) treatment group was also significantly reduced.
  • the treatment conditions of Figure A have significantly better effects on reducing the degree of fibrosis than the treatment conditions of Figure B; it shows that the two small molecule compositions are better than the three small molecule compositions in vivo in situ Transdifferentiation of fibroblasts into hepatocytes and its effects can reduce or reverse liver fibrosis lesions better.
  • FIG. 7 Morphological comparison between human hepatic stellate cells (Lx2) and the transformed hepatocytes (Lx2-ciHep) obtained by transdifferentiation; the results show that after transdifferentiation, human hepatic stellate cells ((Lx2) have the characteristics of human hepatocytes Morphological characteristics.
  • the results showed that the hepatocytes obtained by transdifferentiation of human hepatic stellate cells stained positively with the primary hepatocytes, and possessed the unique functions of glycogen storage and fat metabolism.
  • FIG. 9 Flow cytometric analysis of human hepatocyte-specific markers of human hepatic stellate cells (Lx2) and transformed hepatocytes (Lx2-ciHep) obtained by transdifferentiation; the results show that the hepatocytes obtained by transdifferentiation (Lx2-ciHep) ) The specific markers of human hepatocytes were expressed, but the control group did not.
  • FIG 11 In vivo in situ fibroblast transdifferentiation hepatocyte oral reagent 12 in vivo in situ induction of mouse hepatic stellate cells (HSC) transdifferentiation into hepatocyte tracing comparative experiment.
  • Figure 11 Comparison of the control group in the upper image with the treatment group in the lower image.
  • the left image of the treatment group in the lower image shows that the hepatocyte tissue after transdifferentiation still shows red and the shape remains unchanged; the middle image shows the hepatic stellate cells after transdifferentiation into hepatocytes , The morphology has been transformed into hepatocyte morphology, but it still shows its green fluorescent label; the picture on the right shows that the transformed liver cell (green fluorescence) is integrated with the original liver cell tissue (red fluorescence), and the transformed liver cell still shows in situ HSC tracer labeled green fluorescence.
  • the experimental results fully show that the small molecule composition of the present invention can induce HSC transdifferentiation into hepatocytes in situ in vivo.
  • Fig. 12 Comparative experiment on the induction of fibroblast (HF) transformation by the individual small molecule components of the chemical small molecule composition.
  • Figure 12 shows that the fibroblast (HF) control group (Control) on the left side of the figure is compared with the small molecule treatment groups (Treat) on the right side of the figure.
  • Each small molecule component does not induce transdifferentiation of human fibroblasts into hepatocytes.
  • the effect of fibroblasts is basically unchanged before and after being induced and transdifferentiated, and there is no similarity with the morphology of hepatocytes.
  • Experimental results show that the individual components of the chemical small molecule composition: GSK3 ⁇ inhibitor, HMT inhibitor, and TGF ⁇ inhibitor alone do not have the function of inducing the transdifferentiation of fibroblasts into hepatocytes.
  • the inventors After in-depth research, the inventors have revealed a method that does not introduce or use any exogenous gene/transcription factor/MicroRNA (miRNA) gene, and its inducing factors such as RNA, protein or polypeptide; or, in chemical small molecule composition /The transdifferentiation medium does not contain or use any exogenous genes/transcription factors/MicroRNA (miRNA) genes, or its transcribed RNA, or its translated proteins and peptides, and other inducing factors; also do not use any cytokines Or growth factors.
  • miRNA gene/transcription factor/MicroRNA
  • fibrotic diseases such as liver cirrhosis/liver fibrosis
  • this small molecule composition is perfected through research and development, and is useful for the treatment of pulmonary fibrosis, kidney fibrosis, and other tissues and organs.
  • Fibrotic diseases also have similar effects, and can be prepared into corresponding drugs or prodrugs; or by adding aqueous or organic solvents, or carriers or excipients such as physiological saline/buffer/cell basal medium to prepare transdifferentiation reagents Or culture medium; it can also be used to chemically induce fibroblasts to transdifferentiate into hepatocytes in vitro, prepare transdifferentiated hepatocytes, and provide a source of hepatocytes for scientific research applications, medical applications and clinical applications such as liver cell transplantation.
  • aqueous or organic solvents, or carriers or excipients such as physiological saline/buffer/cell basal medium
  • transdifferentiation reagents Or culture medium it can also be used to chemically induce fibroblasts to transdifferentiate into hepatocytes in vitro, prepare transdifferentiated hepatocytes, and provide a source of hepatocytes for scientific research applications, medical applications and clinical applications such as liver cell transplantation.
  • the small molecule composition can directly reprogram (transdifferentiate) fibroblasts into hepatocytes by chemically in vivo and in vitro, which can be applied to human fibroblasts or mammalian fibroblasts; preferably, it includes but is not limited to humans: Skin fibroblasts, liver fibroblasts (hepatic stellate cells), lung fibroblasts, kidney fibroblasts, pancreatic fibroblasts or fibroblasts of other human tissues or organs; more preferably human skin fibroblasts or Liver fibroblasts (hepatic stellate cells).
  • the liver-like cells obtained by transdifferentiation in vitro have normal liver cell functions.
  • Cell reprogramming is the conversion of cells from one type to another. It is a process of inducing and regulating specific cell signaling pathways and epigenetic modifications to change the cell's fate through epigenetic changes. .
  • the present invention means that no exogenous genes/transcription factors/MicroRNA genes are introduced or used in fibroblasts or starting cells, or small molecule compositions/transdifferentiation media, and their transcribed RNA, or Translated proteins and peptides and other inducing factors; do not add any cytokines or growth factors.
  • the induction composition composed of small chemical molecules, GSK3 ⁇ inhibitor and HMT inhibitor (preferably G9aHMT inhibitor/EZH2HKMT inhibitor), two kinds of small molecule induction composition, and at the same time target to induce and regulate GSK3 ⁇ of fibroblasts
  • GSK3 ⁇ inhibitor and HMT inhibitor preferably G9aHMT inhibitor/EZH2HKMT inhibitor
  • the signaling pathway and its epigenetic enzyme HMT regulates new signaling pathways and epigenetic modifications, which changes the cell gene expression profile, thereby transdifferentiating fibroblasts into hepatocytes.
  • the small chemical molecule is a targeted small molecule compound, with stable properties, easy control of the time, dose and combination of action, stable and reliable action, and good druggability;
  • 2 The hepatocytes obtained by transdifferentiation have normal and mature human hepatocyte morphology and Function; 3
  • the fibroblasts of this method can be derived from the patient, so the hepatocytes obtained by transdifferentiation have personalized characteristics and two major advantages: First, it is easier to enter clinical applications, and minimize or avoid transforming liver cell transplantation The risk of immune rejection caused by the immune response; second, a representative liver cell bank can be constructed to be used in the safety evaluation and efficacy screening of new drugs for hepatotoxicity; 4 the method and induction combination for transdifferentiating fibroblasts into hepatocytes in vivo and in vitro It does not introduce or use any exogenous genes/transcription factors/MicroRNA genes, or its RNA, or its proteins and polypeptides, and other inducing factors; only small chemical molecules are used
  • the advanced and innovative nature of the present invention lies in that the present invention is at least composed of two chemical small molecules (GSK3 ⁇ inhibitor and HMT inhibitor) and the three small molecules (GSK3 ⁇ inhibitor) previously invented by the present invention.
  • the former Compared with the composition and method for inducing the transdifferentiation of fibroblasts into hepatocytes composed of G9aHMT inhibitors, G9aHMT inhibitors, and TGF ⁇ inhibitors, the former not only has 3 small molecule compositions to induce fibroblasts to transdifferentiate into hepatocytes.
  • the former and the latter have the same or similar effects in inducing fibroblast transdifferentiation into hepatocytes in vitro; but the former induces fibroblast transdifferentiation in vivo and its effects in in situ, reducing or The effect of improving fibroblast/fibroblast abnormality-related diseases such as liver fibrosis is the same as or better than the latter;
  • the combination structure of the small molecule composition of the former and the latter is different; the composition of 2 small molecules The composition is less, the combined structure is simpler, the druggability is better, the risk of side effects is lower, the development cost is lower, and it is easier to develop into related disease drugs;
  • the former small molecule composition component HMT inhibitor The number of effective small chemical molecules in the chemistry has increased, and the range of candidate drug chemical small molecules has been wider.
  • GSK3 ⁇ inhibitors preferably G9aHMT inhibitors/EZH2HKMT inhibitors
  • TGF ⁇ inhibitors in any of the aforementioned chemical small molecule compositions, wherein any small molecule group
  • the aforementioned small molecule inhibitors have the ability to specifically inhibit or block specific signaling pathways or epigenetic enzyme activities.
  • Other targeted small molecule inhibitors in each category are based on the specific induced regulation or inhibition.
  • Cell signaling pathways or enzyme activities can only be classified into the same category (the classification is completed by the inventors of small chemical molecules) when they perform effective regulatory functions, and are regulated by specific signaling pathways or epigenetic modification enzymes, and the performance
  • the function is the category name; for example, all small molecules in the GSK3 ⁇ inhibitor category have the common functional characteristics of targeting and inhibiting the GSK3 ⁇ signaling pathway.
  • small molecule inhibitors in the same category only the effective dose, activity size, and effect are different, but there is no essential difference in the ability to induce and regulate specific signal pathways or epigenetic enzyme activities. Therefore, due to the particularity that small molecule inhibitors of the same category can target and regulate the same specific cell signaling pathway or specific targets of epigenetic enzymes, it is basically the same that the same category of small molecule inhibitors function as separate components. ⁇ ; As a participating component of the composition, its effect in the organic whole of the composition is basically the same, and there will be no qualitative difference. The same applies to other small molecule inhibitors or small molecule compounds that target specific cell signaling pathways or specific epigenetic targets. This is common sense well-known to those in the field.
  • GSK3 ⁇ inhibitors include the induction and regulation of the same specific cell signaling pathway or its epigenetic modification enzymes, and exert Small molecule compounds in their respective categories with the same functional activity, effect and effect; the different combinations formed can induce fibroblasts to transdifferentiate into hepatocytes to varying degrees.
  • small molecule compounds in the same category that have the same function or induce the same target, or have the same effect on the same signaling pathway or its epigenetic enzymes, and constituted can induce and regulate the transdifferentiation of fibroblasts into hepatocytes
  • the small molecule combinations of are all within the protection scope of the present invention.
  • fibroblasts also known as fibroblasts, are the main cellular components of loose connective tissue and are differentiated from mesenchymal cells in the embryonic stage. Fibroblasts can be divided into fibroblasts and fibroblasts according to different functional activities; fibroblasts have vigorous functional activities, weakly basophilic cytoplasm, and obvious protein synthesis and secretion activities; mature or quiescent cells They are called fibroblasts; under certain conditions, the two can transform into each other.
  • Fibroblasts have different types and exist in various tissues or organs in the body; they have different names and characteristics in different tissues or organs, including: skin fibroblasts, liver fibroblasts (hepatic stellate cells), lungs Fibroblasts, pancreatic fibroblasts, and fibroblasts in other tissues or organs.
  • the fibroblasts induced by the method or the small molecule composition of the present invention include, but are not limited to: human fibroblasts or mammalian fibroblasts; preferably, include, but are not limited to, human fibroblasts : Skin fibroblasts, liver fibroblasts (hepatic stellate cells), lung fibroblasts, kidney fibroblasts, pancreatic fibroblasts, and fibroblasts of other human tissues or organs. More preferably, they are human skin fibroblasts or liver fibroblasts (hepatic stellate cells).
  • the inventor of the present invention proposes for the first time a kind of inducing factors that do not introduce or use any exogenous gene/transcription factor/MicroRNA (miRNA) gene, or its transcribed RNA, or its translated protein and polypeptide; nor Add any cytokines or growth factors.
  • miRNA gene/transcription factor/MicroRNA
  • a small molecule composition consisting of only chemical small molecule inhibitors: GSK3 ⁇ inhibitor and HMT inhibitor, chemically induces fibroblasts to directly reprogram (transdifferentiate) into hepatocytes in vivo or in vitro; that is, simultaneously inhibits GSK3 ⁇ signaling pathway to varying degrees , And inhibiting its HMT (G9aHMT or EZH2HMT) can induce and regulate new signal pathways and their epigenetic modification changes, thereby changing their gene expression profiles and making fibroblasts transdifferentiate into hepatocytes.
  • the above-mentioned small molecule composition is a pharmaceutical composition, and a TGF ⁇ inhibitor can also be added; the pharmaceutical composition can be used to chemically induce the transdifferentiation of fibroblasts in situ into hepatocytes by chemically inducing the effect of in situ transdifferentiation of hepatocytes in vivo to reduce or Reduce fibroblast abnormalities in tissues/organs, and achieve the effect of treating fibroblasts or fibroblast abnormal-related diseases (fibrotic diseases such as liver fibrosis/cirrhosis).
  • fibrotic diseases such as liver fibrosis/cirrhosis
  • drug carriers or excipients can be added to develop and prepare drugs/prodrugs/drug compositions for the treatment of liver fibrosis (liver cirrhosis); it can be deduced from this that the small molecule composition has been developed and perfected and is useful for the treatment of lung diseases. Fibrosis, renal fibrosis, and fibrotic diseases of other tissues and organs should also have similar effects; drug carriers or excipients can also be added to develop and prepare corresponding innovative drugs or prodrugs, so they should also be included in this Inventing.
  • small molecule inhibitors in the same category only differ in effective dose, activity size, and effect, but there is no essential difference in their ability to induce, inhibit or regulate specific signal pathways or epigenetic enzymes.
  • other small molecule inhibitors of GSK3 ⁇ that can target to inhibit the GSK3 ⁇ cell signaling pathway can also achieve the same technical effect and should also be included.
  • HMT inhibitors listed in the examples of the present invention, other chemical small molecule HMT inhibitors that target to induce and inhibit HMT can also achieve the same technical effect and should also be included in the present invention. ;
  • G9aHMT inhibitors/EZH2HKMT inhibitors listed in the examples of the present invention, other G9aHMT inhibitors or EZH2HKMT inhibitors that target, induce, regulate and inhibit G9aHMT/EZH2HKMT can also achieve the same technical effect. ; Should also be included in the present invention.
  • TGF ⁇ inhibitors listed in the examples of the present invention, other chemical small molecule TGF ⁇ inhibitors that target the inhibition of the TGF ⁇ signaling pathway can also achieve the same technical effects and should also be included in the present invention. in.
  • the term “consisting essentially of” means that in addition to the essential ingredients or essential components, the composition may also contain a small amount of minor ingredients and/or impurities that do not affect the active ingredients. For example, it may contain sweeteners to improve taste, antioxidants to prevent oxidation, and other pharmaceutical additives, carriers, and excipients commonly used in the art.
  • the “comprising GSK3 ⁇ inhibitor and HMT inhibitor” or “consisting of GSK3 ⁇ inhibitor and HMT inhibitor” includes “basically consisting of GSK3 ⁇ inhibitor and HMT inhibitor” and “mainly consisting of GSK3 ⁇ inhibitor And HMT inhibitors as the active ingredients”, “GSK3 ⁇ inhibitors and HMT inhibitors are the only active ingredients”, “basically GSK3 ⁇ inhibitors and HMT inhibitors are the active ingredients”.
  • pharmaceutically acceptable ingredients are substances that are suitable for humans and/or animals without excessive side effects (such as toxicity, irritation, and allergic reactions), that is, substances that have a reasonable benefit/risk ratio; Such as pharmaceutical carriers or excipients commonly used in the art.
  • the term "effective amount” refers to an amount that can produce function or activity on humans and/or animals and can be accepted by humans and/or animals.
  • a carrier refers to a system that can change the way and distribution of drugs into the body, control the release rate of drugs, and deliver drugs to targeted organs;
  • the drug carrier itself is not an essential active ingredient, and there is no excessive toxicity after administration.
  • Suitable carriers are well known to those of ordinary skill in the art, including but not limited to: water, saline, phosphate buffer and other aqueous solvents; DMSO (dimethyl sulfoxide), glycerol and ethanol and other organic solvents; microspheres, Liposomes, microemulsions, polymer surfactants; colloidal drug-carrying systems, new polymer drug-carrying systems, new drug carriers, and other pharmaceutical carriers; among which excipients refer to those in pharmaceutical preparations other than the main drug Add-ons can also be called auxiliary materials.
  • the pharmaceutical dosage form in the term "pharmaceutical dosage form prepared by the composition” refers to a pharmaceutical application form prepared to meet the needs of treatment or prevention, which is called a pharmaceutical dosage form;
  • parts by weight or “parts by weight” can be used interchangeably, and the parts by weight can be any fixed weight expressed in micrograms, milligrams, grams or kilograms (such as 1ug, 1mg, 1g, 2g, 5g, or kg, etc.).
  • a composition consisting of 1 part by weight of component a and 9 parts by weight of component b can be 1 gram of component a + 9 grams of component b, or 10 grams of component a + 90 grams of component b.
  • the percentage content of a certain component (parts by weight of this component/sum of parts by weight of all components) ⁇ 100%. Therefore, in a composition composed of 1 part by weight of component a and 9 parts by weight of component b, the content of component a is 10%, and the content of component b is 90%.
  • the above-mentioned "parts by weight” can also be converted into a "mole number”; and the “parts by weight ratio” can also be converted into a "molar concentration ratio”.
  • the weight unit of the weight ratio can be any weight unit such as kilogram (kg), milligram (mg), microgram (ug), etc.; the molar unit of the molar concentration ratio can be: mole (M), millimolar (mM) Any molar concentration unit such as, micromole (uM), etc.;
  • GSK3 ⁇ inhibitors such as CHIR99021, LiCl, CHIR-98014
  • HMT inhibitors such as BIX01294, UNC0638, UNC0642
  • EZH2HKMT inhibitors such as EPZ005687, GSK343, UNC1999)
  • TGF ⁇ inhibitors Such as SB431542, A83-01, RepSox
  • 5-80 (0.1-50): (0.1-50); preferably, (10-70): (0.5-40): ( 0.5-40) exists; or in the solution state, the molar concentration ratio (0.1-20): (0.01-20): (0.01-20); preferably, (0.5-10): (0.05-10): (0.05 -10) Exist.
  • the ingredients and parts by weight are as shown in Table 1 or the molar concentration is as shown in Table 2 (solution state).
  • the formula range in Table 1 and Table 2 can be used as a reference guide.
  • the effective dose of the composition used may vary with the mode of administration and the physical condition of patients with fibrosis (such as liver cirrhosis) to be treated, or the severity of the disease.
  • "weight/kg (body weight)” is usually used as the dosage unit; when the small molecule composition is applied to large animals and patients with liver disease, it is calculated by the corresponding professional conversion formula according to the small animal dosage
  • the effective dosage (including solid or solution dosage conversion) for large animals or humans also belongs to the protection scope of the present invention.
  • the examples are only for the small molecule induction and regulation composition, and representative small molecules of each category (such as for GSK3 ⁇ inhibitors, typically CHIR-99021 , LiCl, CHIR-98014 and other small molecule inhibitors to demonstrate) the concentration used in the experiment; therefore, the reasonable concentration range summarized in the claims of the patent application of the present invention naturally includes, but is not limited to, the categories in the examples Yes, the concentration range used in a specific representative small molecule experiment; this basic and simple principle is well known to those skilled in the art and should not be confused.
  • the GSK3 ⁇ inhibitor refers to the general term of small molecule inhibitors that can inhibit the GSK3 ⁇ signaling pathway in cells, including but not limited to: CHIR-99021, BIO, LiCl, IM-12, TWS119, 1-Azakenpaullone , CHIR-98014, Tideglusib, AR-A014418, SB216763, AZD1080, and other GSK3 ⁇ small molecule inhibitors or small molecule compounds that induce inhibition of GSK3 ⁇ signaling pathway;
  • the GSK3 ⁇ inhibitor is CHIR-99021, and its alias is CT99021; its molecular structure is shown in the following formula (I):
  • HMT inhibitors which is a general term for small chemical molecules that can target and inhibit HMT, including (but not limited to): G9aHMT inhibitors and EZH2HMT inhibitors;
  • the small chemical molecule G9aHMT inhibitor refers to the general term of small chemical molecule inhibitors that can target and inhibit G9aHMT, including but not limited to: BIX01294, UNC0638, A-366, UNC0631, BRD4770, UNC0224, UNC0646, UNC0642, UNC0321 , BRD4770, HKMTI-1-247, HKMTI-1-248, CPUY074020, DCG066, and other G9aHMT small molecule inhibitors or small molecule compounds that induce inhibition of G9aHMT;
  • the G9aHMT inhibitor is BIX01294 (or BIX-01294); its molecular structure is shown in the following formula (II):
  • the small chemical molecule EZH2HMT inhibitor refers to the general term of small chemical molecule inhibitors that can target and inhibit EZH2HMT, including but not limited to: EPZ005687, GSK343, Tazemetostat (EPZ-6438), UNC1999, JQ-EZ-05 (JQEZ5 ), EBI-2511, CPI-1205, EPZ011989, PF-06726304, EI1, GSK503, GSK126, CPI-360, CPI-169, and other chemical small molecule EZH2HMT inhibitors or small molecule compounds that inhibit EZH2HMT:
  • the chemical small molecule EZH2HMT inhibitor is a small molecule EPZ005687; its molecular structure is shown in the following formula (III):
  • the chemical small molecule TGF ⁇ inhibitors refer to the general term of chemical small molecule inhibitors that can inhibit the TGF ⁇ signaling pathway in cells, including but not limited to: SB431542, A83-01, SB525334, LY2109761, RepSox, SD-208, GW788388 , SB505124, EW-7197, Galunisertib, and other small molecule TGF ⁇ inhibitors or small molecule compounds that induce and inhibit the TGF ⁇ signaling pathway;
  • the small chemical molecule TGF ⁇ inhibitor is small molecule SB 431542 (or SB-431542); its molecular structure is shown in the following formula (IV):
  • the small chemical molecule TGF ⁇ inhibitor is the small molecule A83-01 (or A8301); its molecular structure is shown in the following formula (V):
  • the present invention also includes compounds, pharmaceutical products, analogs, and/or their salts, hydrates or precursors equivalent to the aforementioned small molecule compounds I, II or III, IV, and V; it also includes naturally-generated and artificially-synthesized compounds.
  • the analogs of the small molecule compound include, but are not limited to: isomers and racemates of the small molecule compound.
  • Compounds have one or more asymmetric centers. Therefore, these compounds can exist as racemic mixtures, individual enantiomers, individual diastereomers, mixtures of diastereomers, cis or trans isomers.
  • salts include but are not limited to: (1) salts formed with the following inorganic acids: such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, etc.; (2) salts formed with the following organic acids, such as acetic acid, oxalic acid, butadiene Acid, tartaric acid, methanesulfonic acid, maleic acid, or arginine, etc.
  • Other salts include salts with alkali metals or alkaline earth metals (such as sodium, potassium, calcium, or magnesium) and the like.
  • the "precursor of the compound” refers to a compound that can be converted into any of the above-mentioned compounds in a culture medium, animal, or human body after being applied or treated by an appropriate method, or the above-mentioned compound Any compound is a salt or solution composed of one compound.
  • the chemical small molecule composition of the present invention is a pharmaceutical composition used to induce the direct reprogramming (transdifferentiation) of fibroblasts into hepatocytes.
  • Drug carriers and excipients can be added to develop and prepare treatments involving fibroblasts or fibroblasts.
  • Drugs/prodrugs/pharmaceutical compositions for related diseases therefore, it also includes: a pharmaceutically acceptable carrier or excipient; preferably, the carrier or excipient includes one or more selected from the following group : Water, saline, phosphate buffer or other aqueous solvents; DMSO, glycerol and ethanol or other organic solvents; microspheres, liposomes, microemulsions or polymer surfactants; colloidal drug delivery systems or polymer drug delivery systems ; Preservatives, antioxidants, flavors, fragrances, cosolvents, emulsifiers, pH buffer substances, binders, fillers, lubricants or other pharmaceutical excipients; or
  • the dosage form of the chemical small molecule composition of the present invention is not particularly limited, and it can be any dosage form suitable for mammals; the dosage forms that can be prepared include: powders, powders, tablets, pills, capsules, sustained release agents, Controlled immediate release agents and other solid dosage forms; injections, infusions, suspensions and other liquid dosage forms; and other dosage forms such as gaseous dosage forms and semi-solid dosage forms.
  • the dosage form may be, but not limited to: solid dosage forms such as powders, granules, capsules, sustained-release preparations, and tablets, or liquid dosage forms such as injections, infusions, solutions, and suspensions.
  • the preparation method of the small molecule composition of the present invention is determined according to the dosage form and the route of administration to be prepared. After referring to the combination and the ratio provided by the present invention, those skilled in the art adopt the conventional preparation method of the pharmaceutical composition Then the chemical small molecule composition of the present invention can be prepared.
  • the present inventors proved for the first time that the small molecule composition of the present invention can induce the transdifferentiation of fibroblasts into hepatocytes in situ in vivo, and the effect of in situ transdifferentiation of hepatocytes in vivo reduces or reduces the fibroblasts or fibroblasts of tissues/organs.
  • drug carriers or excipients can be added to develop and prepare drugs or prodrugs or drug formulations for the prevention, improvement or treatment of fibrotic diseases such as liver fibrosis (cirrhosis);
  • the small molecule composition has been developed and perfected, and should have similar effects on the treatment of pulmonary fibrosis, renal fibrosis, and other tissues, organs and tubes; it can also add drug carriers or excipients to develop and prepare
  • the corresponding innovative drugs or prodrugs should therefore also be included in the present invention.
  • the effective dose of the composition used can vary with the mode of administration, the type of fibrotic disease to be treated, and the severity of the disease. .
  • the specific situation is determined according to the individual situation of the subject, which is within the scope of the judgment of a skilled physician or pharmacist.
  • the fibroblasts include, but are not limited to: human fibroblasts or mammalian fibroblasts; preferably, include but are not limited to humans: skin fibroblasts, liver fibroblasts (hepatic stellate Cells), lung fibroblasts, kidney fibroblasts, pancreatic fibroblasts, and fibroblasts of other tissues or organs in the human body. More preferably, they are human skin fibroblasts or liver fibroblasts (hepatic stellate cells).
  • the small molecule composition can be prepared by adding organic solvents, or physiological saline, or buffers, or cell basal media and other carriers/excipients/basic nutrient solutions to chemically induce fibroblasts to directly reprogram (transform) Differentiation) into a transdifferentiation medium or reagent for hepatocytes; a transdifferentiation medium for inducing fibroblasts to directly reprogram (transdifferentiate) into hepatocytes (hereinafter referred to as: fibroblast transdifferentiation hepatocyte medium).
  • a specific final concentration of the small molecule composition is selected for preparation.
  • the different components in the specific small molecule composition are dissolved in DMSO (dimethyl sulfoxide) or other organic solvents or aqueous solvents according to the different properties and different solubility of the solutes.
  • Concentrate reagents (ranging from 1:50-1:10,000); then according to the final concentration requirements of the specific small molecule composition, the small molecule organic solution concentrate reagents are diluted and added to the cell base containing 10% calf serum In the culture medium, the fibroblast transdifferentiation hepatocyte culture medium can be obtained.
  • the percentage content of each component of the medium can also fluctuate up and down 50%; preferably up and down 30%; more preferably up and down 20%, such as 10%, 5%. Unless otherwise stated, percentages are in v/v.
  • the cell basal medium includes but is not limited to: DMEM/F12, MEM, DMEM, F12, IMDM, RPMI1640, Neuronal basal or Fischers, etc., all of which are commercially available commodities.
  • a serum-free medium can also be used for preparation.
  • the "serum-free medium” refers to a cell culture medium that does not contain serum but contains various nutrients (such as growth factors, tissue extracts, etc.) that support cell proliferation and biological response. That is, additives such as various cytokines or growth factors other than serum are added to the cell culture medium composed of the cell basal culture medium.
  • the serum-free medium containing various cytokines or growth factors includes but not limited to: ITS, N2, B27, etc., all of which can be self-prepared or commercially available products.
  • the cell basal medium or serum-free medium is not limited to those exemplified in the present invention.
  • the "Fibroblast Transdifferentiation Hepatocyte Medium” is specifically prepared or formulated as follows:
  • 1 GSK3 ⁇ inhibitor such as CHIR99021: the final concentration is 0.1 ⁇ M-20mM; the preferred amount is: 0.5 ⁇ M-10mM; 2The final concentration of G9aHMT inhibitor (such as BIX01294) is 0.01-20 ⁇ M; the preferred amount is: 0.05-10 ⁇ M ; Mix to obtain the small molecule composition of the present invention for chemically inducing fibroblasts to directly reprogram (transdifferentiate) into hepatocytes.
  • the present invention also provides a reagent for injection or oral administration in experimental animals for chemically inducing in-situ fibroblast transdifferentiation of hepatocytes in vivo (hereinafter referred to as: in vivo in-situ fibroblast transdifferentiation hepatocyte reagent).
  • each small molecule composition calculates the corresponding dosage of each small molecule composition in any one of the aforementioned compositions by kilogram body weight, and dissolve it in Captisol (1-30%), Tween-80 (5%) In the solution, a reagent for in situ transdifferentiation of fibroblasts for experimental animal injection or oral administration is obtained. Preferably it is Captisol (1-30%).
  • the invention also discloses a chemical small molecule composition which chemically induces fibroblasts in vitro to directly reprogram (transdifferentiate) into hepatocytes to prepare a method for transforming hepatocytes.
  • the method steps include:
  • each component is dissolved in an organic solvent or an aqueous solvent to prepare a concentrated solution reagent;
  • the organic solvent includes dimethyl Sulfoxide;
  • the aqueous solvent includes: water, physiological saline, phosphate buffer;
  • the percentage content of each component of the medium can also fluctuate up and down 50%; preferably up and down 30%; more preferably up and down 20%, such as 10%, 5%.
  • Subculture to induce transdifferentiation of fibroblasts discard the original culture medium, wash once with PBS, add cell digestion solution to digest the cells, 37°C for 1-5 minutes, stop the cell digestion, centrifuge, discard the supernatant, and pellet the cells Resuspend and spread the plates according to 1:1-1:3.
  • the digestive juice used includes pancreatin, EDTA, Acutase, TrypleE and so on. Passage once every 3-15 days.
  • transdifferentiated hepatocytes After the above-mentioned experimental steps (3) and (4) transdifferentiation culture and subculture fibroblasts for 2-4 weeks, transdifferentiated hepatocytes can be obtained.
  • the hepatocytes can be used for other scientific research experiments; new drug toxicity and efficacy testing and evaluation; to provide a source of hepatocytes for the construction of bioartificial livers and clinical cell transplantation.
  • the transdifferentiated hepatocyte culture of fibroblasts follows the experimental steps of the above-mentioned culture method.
  • hepatocytes obtained by transdifferentiation the culture of fibroblasts of transdifferentiated hepatocytes is as described above, and the transformed hepatocytes obtained at different times of culture are used to test their related functions.
  • the present invention does not introduce or use any exogenous genes/transcription factors/MicroRNA (miRNA) genes, and inducing factors such as RNA, proteins or polypeptides; nor does it add any cells to the transdifferentiation medium or transdifferentiation reagents.
  • miRNA microRNA
  • Factors or growth factors only the combination of small chemical molecules inside and outside the body chemically induces fibroblasts to directly reprogram (transdifferentiate) into hepatocytes.
  • the small molecule composition can be prepared into a medicine or a prodrug or a pharmaceutical composition for treating abnormal fibroblast diseases (such as liver fibrosis/cirrhosis); or it can be prepared by chemically inducing fibroblasts to directly reprogram into hepatocytes transdifferentiation Culture medium or reagent; or used for chemically inducing fibroblasts to directly reprogram (transdifferentiate) into hepatocytes to prepare transformed hepatocytes, and provide hepatocyte source for scientific research application, medical application and clinical application.
  • abnormal fibroblast diseases such as liver fibrosis/cirrhosis
  • this method can also be widely used as a reference for the prevention and treatment of tissue or organ fibrosis and mechanism research; as well as a cell model for liver disease research, pharmacological and toxicological safety testing.
  • the obtained transdifferentiated hepatocytes can continue to undergo functional testing, pre-clinical research, etc.
  • This method not only opens up a new way for the research and development of new drugs for the prevention and treatment of liver fibrosis (cirrhosis), pulmonary fibrosis and other organ or tissue fibrosis diseases and their mechanism, but also for the medical application, clinical application and scientific research application of hepatocytes.
  • Providing a new source of hepatocytes has a wide range of application prospects; it also enriches the theory of stem cell reprogramming and expands its application range. It has important scientific significance and great application value.
  • Example 1 A small molecule composition for direct reprogramming (transdifferentiation) of fibroblasts induced by small molecules into hepatocytes, and preparation of transdifferentiation medium and reagents
  • composition or transdifferentiation medium which can be prepared by mole concentration or weight concentration:
  • GSK3 ⁇ inhibitor CHIR-99021 final concentration 3 ⁇ M
  • G9aHMT inhibitor BIX01294 final concentration 3 ⁇ M
  • GSK3 ⁇ inhibitor LY2090314 final concentration 0.3 ⁇ M
  • G9aHMT inhibitor UNC0642 final concentration 2 ⁇ M
  • GSK3 ⁇ inhibitor LiCl final concentration 10 ⁇ M
  • G9aHMT inhibitor BIX01294 final concentration 2.5 ⁇ M
  • GSK3 ⁇ inhibitor BIO final concentration 1uM
  • G9aHMT inhibitor UNC0638 final concentration 3uM
  • GSK3 ⁇ inhibitor Ly2090314 final concentration 1 ⁇ M
  • G9aHMT inhibitor UNC0638 final concentration 5 ⁇ M
  • G9aHMT inhibitor BIX01294 final concentration 2.5 ⁇ M
  • GSK3 ⁇ inhibitor Ly2090314 final concentration 0.4 ⁇ M
  • G9aHMT inhibitor BIX01294 final concentration 0.5 ⁇ M
  • GSK3 ⁇ inhibitor LiCl final concentration 20 ⁇ M
  • G9aHMT inhibitor BIX01294 final concentration 5 ⁇ M
  • GSK3 ⁇ inhibitor BIO final concentration 3 ⁇ M
  • G9aHMT inhibitor BIX01294 final concentration 2 ⁇ M
  • GSK3 ⁇ inhibitor Ly2090314 final concentration 4uM
  • G9aHMT inhibitor BIX01294 final concentration 2 ⁇ M
  • TGF ⁇ inhibitor SB431542 final concentration 10 ⁇ M
  • GSK3 ⁇ inhibitor BIO final concentration 3uM
  • G9aHMT inhibitor UNC0642 final concentration 2.5 ⁇ M
  • TGF ⁇ inhibitor A83-01 final concentration 2uM
  • GSK3 ⁇ inhibitor CHIR-99021 final concentration 6uM
  • G9aHMT inhibitor HKMTI-1-248 final concentration 5uM
  • TGF ⁇ inhibitor RepSox final concentration 0.5uM
  • GSK3 ⁇ inhibitor BIO final concentration 1uM
  • EZH2HMT inhibitor UNC1999 final concentration 2 ⁇ M
  • GSK3 ⁇ inhibitor CHIR-98014 final concentration 20 ⁇ M
  • Each specific small molecule composition is first dissolved in DMSO to prepare a concentrated solution reagent according to step (1) of the aforementioned "Cultivation Method".
  • the fibroblast transdifferentiated hepatocyte composition 1-15 of the above-mentioned experimental step 1 prepared by the DMSO concentrate reagent of each component of the "culture method" step (2), the selected cell basic medium is DMEM, supplemented with 10% Calf serum to obtain fibroblast transdifferentiation hepatocyte culture medium 1-15 (that is, the final concentration of the compound in medium 1 and composition 1 is the same, and the final concentration of the compound in medium 2 and composition 2 is the same,..., medium 15 Same as the final concentration of the compound of composition 15).
  • the DMSO concentrate of the fibroblast transdifferentiated hepatocyte composition 5, 7, 10, 12 was dissolved in 5% Captisol to prepare an oral reagent for in situ fibroblast transdifferentiated hepatocytes in vivo (with composition 5, 7, 10, The final concentration of compound 12 is the same).
  • HSC hepatic stellate cells
  • the original culture medium was discarded, and the treatment group was replaced with fibroblast transdifferentiation hepatocyte culture medium 1, 11, and cultured at 37°C.
  • the medium was changed every 3 days; passage was once every 3-7 days.
  • HSC hepatic stellate cells
  • Subculture steps discard the original culture medium, wash once with PBS, add cell digestion solution to digest the cells, stop the cell digestion for 3 minutes at 37°C, centrifuge, discard the supernatant, resuspend the cell pellet, and plate according to 1:2 passage.
  • the treatment group was cultured with fibroblast transdifferentiation hepatocyte medium 1, 11, and the medium was changed every 3 days.
  • the digestion solution used is pancreatin (EDTA, Acutase, TrypleE can also be used) and so on. Passage once every 3-7 days.
  • transdifferentiated hepatocytes After the fibroblast transdifferentiated hepatocyte culture and subculture for 2-4 weeks in the above experiment steps 1-2, transdifferentiated hepatocytes can be obtained, which can be used for further experiments.
  • liver fibroblasts hepatic stellate cells HSC
  • ciHep transdifferentiated hepatocytes
  • FIG 1 The right figure of A shows the hepatocytes (ciHep) obtained from HSC transdifferentiation induced by the transdifferentiated hepatocyte medium 1 (2F) of two small molecule compositions; the right figure of Figure 1B shows the transdifferentiation of the three small molecule combinations Hepatocyte culture medium 11 (3F) induced HSC transdifferentiation of hepatocytes (ciHep); comparing the morphology with the HSC in the left panels of Figures A and B respectively, the morphology of the transformed hepatocytes has changed significantly, which is consistent with the morphology of hepatocytes , Some transformed hepatocytes obviously have double nuclei. All HSCs have been transdifferentiated into hepatocytes (ciHep).
  • the experimental results show that the small molecule composition (2F) composed of only two chemical small molecules (GSK3 ⁇ inhibitor and HMT inhibitor) is compared with the previously invented small molecule (GSK3 ⁇ inhibitor, G9aHMT inhibitor) and only three small molecules (GSK3 ⁇ inhibitor and G9aHMT inhibitor).
  • the small molecule composition (3F) composed of TGF ⁇ inhibitors has the same or similar effect in inducing fibroblasts to transdifferentiate into hepatocytes in vitro.
  • Example 3 Glycogen staining of fibroblast transdifferentiation hepatocyte medium 2 inducing hepatic fibroblasts (hepatic stellate cells) to transdifferentiate into liver-like cells
  • Fibroblast transdifferentiation hepatocyte medium 2 chemically induces mouse liver fibroblasts (hepatic stellate cells) to transdifferentiate into hepatocytes.
  • the steps are the same as in Example 2.
  • the hepatic stellate cell (HSC) transdifferentiated liver-like cells were stained with glycogen.
  • the depth of staining shows the ability of liver cells to store glycogen.
  • the Schiff method was used for liver glycogen staining.
  • the specific methods are: (1) discard the cell culture medium and rinse with PBS once; (2) after fixing with 4% paraformaldehyde for 10 minutes, rinse with PBS for 5 minutes ⁇ 3 times; (3) add PAS-I solution for 10 minutes and rinse with running water ; (4) Add PAS-II solution for 1-2 min, rinse with running water; (5) Take pictures under microscope.
  • PAS Glycogen staining results of hepatocytes (ciHep) obtained by transdifferentiation of hepatic stellate cells (HSC) are shown in Figure 2; the left side of Figure 2 is the PAS results of hepatic stellate cells (HSC), and the right side is the transdifferentiation Glycogen staining results of transdifferentiated or transformed hepatocytes obtained in Medium 2.
  • the experimental results in Figure 2 show that the liver-like cells obtained from the transdifferentiation culture on the right side of the figure are stained positively for liver glycogen, while the liver stellate cells on the left side of the figure are stained negatively for liver glycogen. It is proved that the transdifferentiated hepatocytes obtained by the method of the present invention have the unique glycogen storage activity of hepatocytes.
  • Example 4 Oil-red staining experiment of fibroblast transdifferentiation hepatocyte medium 3 inducing hepatic fibroblasts (hepatic stellate cells) to transdifferentiate into liver-like cells
  • Fibroblast transdifferentiation hepatocyte culture medium 3 chemically induce fibroblast transdifferentiation into hepatocyte method and steps are the same as in Example 2.
  • Example 5 Chemically induced fibroblasts (HF) to express hepatocyte-related genes after transdifferentiation into hepatocyte-like cells
  • Fibroblast transdifferentiation hepatocyte medium 4 the method of chemically inducing fibroblast (HF) transdifferentiation into hepatocyte is the same as in Example 2. Collect RNA from the cells of the control group and the transdifferentiation group, and do RT-PCR to detect the expression of hepatocyte-related genes.
  • HF fibroblast
  • Example 6 Oral oral in vivo in situ fibroblast transdifferentiation reagent 5, in vivo chemical induction of in situ liver fibroblast transdifferentiation hepatocyte effect, treatment of liver fibrosis mouse animal model experiment
  • liver fibrosis Male C57/BL6 mice for 4 to 5 weeks were injected intraperitoneally with 5% CCI4 (olive oil solvent) at a dose of 5 ⁇ L/g body weight, 3 times a week for 84 days, about 12 Weekly make liver fibrosis model. At the 12th week of model building, a mouse was dissected, and liver tissues were fixed and sliced for HE staining (conventional staining) and Sirius red staining (according to the kit operating instructions) to confirm that the model of liver fibrosis was successfully made.
  • CCI4 live oil solvent
  • a total of 34 days of treatment about 5 weeks, during which CCI4 continued to be used. After the experiment, the blood and liver of the mice were collected for follow-up analysis.
  • Fig. 5 shows that the treatment group has significantly lower transaminase (alanine aminotransferase, aspartate aminotransferase) in the peripheral blood than the control group, indicating that the small molecule combination induces fibroblasts to transdifferentiate into hepatocytes in situ and has the effect of liver fibrosis The lesions were significantly reduced or reversed.
  • transaminase alanine aminotransferase, aspartate aminotransferase
  • Example 7 Oral in vivo in situ fibroblast transdifferentiation reagents 7, 10, in vivo chemical induction of in situ liver fibrosis cells transdifferentiation of hepatocytes and its effect, treatment of liver fibrosis mouse animal model comparative experiment
  • liver fibrosis male C57/BL6 mice for 4 to 5 weeks, intraperitoneally injected with 5% CCI4 (olive oil solvent), dose 5 ⁇ L/g body weight, 1 time/3 days for 84 days, approximately The liver fibrosis model was made in 12 weeks. At the 12th week of model building, a mouse was dissected, and liver tissues were fixed and sliced for HE staining (conventional staining) and Sirius red staining (according to the kit operating instructions) to confirm that the model of liver fibrosis was successfully made.
  • CCI4 live oil solvent
  • In situ fibroblast transdifferentiation hepatocyte oral reagent 7, 10 preparation and oral treatment experiment oral administration.
  • a total of 34 days of treatment about 5 weeks, during which CCI4 continued to be used. After the experiment, the blood and liver of the mice were collected for follow-up analysis.
  • mice in the treatment group of in situ fibroblast transdifferentiated hepatocyte oral reagent 7 have significantly reduced Sirius red staining
  • Fig. 6B in situ fibroblast transdifferentiated hepatocyte oral reagent 10 treatment
  • the Sirius red staining of mice in the group was also significantly reduced; however, the effect of the former in reducing the degree of fibrosis was significantly better than the latter.
  • the two small molecule compositions have the effect of transdifferentiation into hepatocytes in vivo in situ fibroblasts compared with the three small molecule compositions, and the effect of reducing or reversing liver fibrosis is the same or better.
  • Example 8 The morphological comparison experiment of human hepatic stellate cell (HSC) line Lx2 and the transdifferentiated hepatocyte (Lx2-ciHep) obtained by transdifferentiation
  • Transdifferentiation of fibroblasts in hepatocyte medium 6 chemically inducing liver fibroblast cell line Lx2 (hepatic stellate cell, HSC, Lx2 cell line) to transdifferentiate into hepatocytes is the same as in Example 2.
  • Lx2 hepatic stellate cell, HSC, Lx2 cell line
  • Example 9 Functional comparison experiment between human hepatic stellate cell (HSC) line Lx2 transformed hepatocytes (Lx2-ciHep) and human primary hepatocytes (PHH)
  • Transdifferentiation hepatocyte medium 8 chemically induces liver fibroblast cell line Lx2 (hepatic stellate cell, HSC, Lx2) to transdifferentiate into hepatocytes.
  • Lx2 hepatic stellate cell, HSC, Lx2
  • Transformed hepatocytes obtained by transdifferentiation of human hepatic stellate cells Lx2 (treatment group) were subjected to Oil-red staining (Oil-red) and glycogen staining (PAS), and the same was performed with human primary hepatocytes (PHH) Control staining comparison (the steps are the same as those in Examples 3 and 4), the experimental results are shown in Figure 8; the results show that the transformed hepatocytes (Lx2-ciHep) obtained by transdifferentiation of Lx2 and the primary hepatocytes (PHH) stained positively, indicating that the transformed liver Cells have the unique glycogen storage and fat metabolism functions of liver cells.
  • Example 10 Comparative experiment on the expression of specific markers of the human hepatic stellate cell (HSC) line Lx2 and the transdifferentiated hepatocytes (Lx2-ciHep) obtained by transdifferentiation
  • Transdifferentiation of fibroblasts in hepatocyte medium 9 chemically inducing liver fibroblast cell line Lx2 (hepatic stellate cell, HSC, Lx2) to transdifferentiate into hepatocytes is the same as in Example 2.
  • Lx2 hepatic stellate cell, HSC, Lx2
  • the human hepatic stellate cells Lx2 (control group) and the transdifferentiated hepatocytes (Lx2-ciHep) obtained by transdifferentiation (treatment group) were digested, centrifuged and fixed, immunostained with human hepatocyte specific antibody, and then the sample was flowed Cell analysis and comparison, the experimental results are shown in Figure 9. The results showed that the transdifferentiated hepatocytes (Lx2-ciHep) expressed specific markers of human hepatocytes, while the control group did not.
  • the experimental results show that the morphology and function (glycogen staining, oil red staining) of the transdifferentiated hepatocytes (Lx2-ciHep) obtained by transdifferentiation of human hepatic stellate cells Lx2 (treatment group) and the control group, as well as human hepatocytes Comparison of specific markers.
  • the results showed that after transdifferentiation of human hepatic stellate cells Lx2 into hepatocyte-like cells, they have the unique morphology, function and specific marker characteristics of human hepatocytes.
  • Example 11 Transdifferentiation hepatocyte culture medium 13, 14, 15 induce fibroblast transdifferentiation to obtain liver-like cells expressing hepatocyte-related genes
  • transdifferentiation hepatocyte culture medium 13, 14, 15 inducing skin fibroblasts to transdifferentiate hepatocyte-like cells are the same as in Example 2. Collect the RNA of the liver-like cells transdifferentiated from the control group (Control) and the transdifferentiated hepatocyte medium 13, 14, and 15 transdifferentiation treatment groups (marked as T1, T2, T3 in turn), and do RT-PCR to detect the liver. Expression of cell-related genes.
  • the results of the experiment are shown in Figure 10.
  • the results show that the hepatocyte-like cells obtained from the transdifferentiation hepatocyte medium 13, 14, and 15 induced fibroblast transdifferentiation, compared with the control group, the treatment group T1, T2, T3 high expression of hepatocytes is related Genes, including ALB, AAT, ASGPR1, 7A1.
  • Experimental results show that the two small molecule compositions of the present invention induce fibroblasts to transdifferentiate hepatocyte-like cells and highly express hepatocyte-related genes.
  • Example 12 In vivo in situ fibroblast transdifferentiation hepatocyte oral reagent 12 In vivo in situ induction of mouse hepatic stellate cell (HSC) transdifferentiation into hepatocyte tracing comparative experiment
  • the experimental procedure of the animal model of liver fibrosis disease is the same as in Example 6.
  • Transgenic mice mTmG/PDGFF1 carrying the tracer signal of labeled hepatic stellate cells were used to make animal models of liver fibrosis.
  • the only difference between the treatment conditions of the control group and the treatment group is that the treatment group oral in vivo fibroblast transdifferentiated hepatocyte oral reagent 12 contains the small molecule composition 12, while the oral reagent of the control group does not.
  • the experimental results are shown in Figure 11.
  • Figure 11 The upper panel of the control group, the left panel shows that the liver cells of the transgenic mice show red fluorescence, and the morphology is unchanged; the middle panel shows the orthotopic hepatic stellate cells (HSC) or liver fibroblasts of the transgenic mice, showing green Fluorescence, morphology has not changed; the figure on the right shows the fluorescence synthesis of the above two cell tissues;
  • HSC orthotopic hepatic stellate cells
  • FIG. 11 The treatment group in the lower figure.
  • the left figure shows that the liver cell tissue after transdifferentiation still shows red and the morphology is unchanged;
  • the middle figure shows that the hepatic stellate cell has transformed into a liver cell morphology after transdifferentiation, but still shows liver.
  • Stellate cells were originally labeled with green fluorescence;
  • the picture on the right shows that hepatic stellate cells are transdifferentiated into transformed hepatocytes and the original hepatocytes are integrated, and the transformed hepatocytes still show green fluorescence with HSC tracer in situ.
  • the experimental results fully show that the small molecule composition of the present invention can induce HSC transdifferentiation into hepatocytes in situ in vivo.
  • Example 13 Comparative Test of Inducing Cell Transformation by Each Individual Small Molecule Component of Chemical Small Molecule Composition [GSK3 ⁇ Inhibitor/HMT Inhibitor (G9aHMT Inhibitor/EZH2HMT Inhibitor)/TGF ⁇ Inhibitor]
  • the cell basal medium selected is DMEM, supplemented with 10% calf serum to obtain a small molecule component medium alone
  • Figure 12 shows that comparing the control group on the left side of the figure with the small molecule treatment groups on the right side of the figure, each individual small molecule component has no effect on human fibroblasts in inducing transdifferentiation of hepatocytes. Before and after the fibroblasts are induced to transdifferentiate, the morphology There is basically no change, and there is no similarity with the morphology of liver cells.
  • the experimental results show that the individual components of the chemical small molecule composition: GSK3 ⁇ inhibitor, HMT inhibitor (G9aHMT inhibitor/EZH2HMT inhibitor), and TGF ⁇ inhibitor alone do not induce fibroblasts to transdifferentiate into hepatocytes. Function.
  • the “chemical small molecule composition and method for direct reprogramming (transdifferentiation) of fibroblasts into hepatocytes in vivo and in vitro chemically induces the chemical small molecule composition and method of the present invention have the following beneficial effects:
  • the method of the present invention does not import or use any exogenous gene/exogenous transcription factor/MicroRNA (miRNA) gene, and its inducing factors such as RNA, protein or polypeptide; nor does it use any cytokine or growth factor; at least only Using an induction composition composed of two chemical small molecules, fibroblasts are induced to directly reprogram (transdifferentiate) into hepatocytes in vivo and in vitro.
  • the operation method is simple, the cost of transforming hepatocytes is low, the quality is good, and it is safer;
  • the chemical small molecule composition has many uses: (1) As a pharmaceutical composition for in situ in vivo induction of fibroblasts transdifferentiation into hepatocytes, it can be developed or prepared to treat human fibroblast abnormalities related diseases (such as liver (Fibrosis/liver cirrhosis) drugs/prodrugs/pharmaceutical compositions; (2) used to prepare transformation media or reagents for chemically inducing fibroblasts to transdifferentiate into hepatocytes; (3) used to chemically induce fibroblasts in vitro Transdifferentiation of cells into hepatocytes to prepare transformed hepatocytes to provide a source of hepatocytes for scientific research applications, medical applications and clinical applications.
  • human fibroblast abnormalities related diseases such as liver (Fibrosis/liver cirrhosis) drugs/prodrugs/pharmaceutical compositions
  • transformation media or reagents for chemically inducing fibroblasts to transdifferentiate into hepatocytes
  • the hepatocytes obtained by transdifferentiation in vitro have normal and mature human hepatocyte morphology and functions; they have albumin production, urea synthesis, glycogen storage, fat metabolism, P450 enzyme activity induction and other hepatocyte specific functions and human hepatocytes. Specific markers;
  • the present invention only uses a composition composed of two chemical small molecules at least, which has a simpler combined structure and better druggability; the risk of side effects is lower, the development cost is lower, and it is easier to develop drugs for related diseases.
  • the effect of in situ induction of fibroblasts to transdifferentiate hepatocytes in vivo is better for reducing or improving liver fibrosis/cirrhosis.
  • This method of transdifferentiated hepatocytes can be derived from the patients themselves.
  • Transformed hepatocytes have individual characteristics and have two major advantages: First, it is easy to enter clinical applications, and minimize or avoid the transformation/transdifferentiated hepatocyte transplantation. The risk of immune rejection; second, a representative liver cell bank can be constructed to be used in hepatotoxicity safety evaluation and drug efficacy screening of new drugs; therefore, it can provide a source of liver cells for clinical applications, medical applications and scientific research applications.
  • This method adopts small molecule chemical induction to directly reprogram cells, without the need for induced pluripotent stem cells to be reprogrammed to iPSC stage, avoiding the risk of carcinogenesis; it is convenient for clinical application.
  • the transdifferentiation method has good universality and repeatability; the method is simple, easy to operate, and low in cost; it is conducive to popularization and application.

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Abstract

提供了一种不导入任何外源基因/转录因子/MicroRNA基因，RNA、蛋白质或多肽等诱导因子，仅用化学小分子化学诱导体内外成纤维细胞直接重编程为肝细胞的方法及小分子组合物。该小分子组合物通过体内原位转分化肝细胞的效应达到治疗纤维化疾病如肝纤维化/肝硬化的效果。该小分子组合物可制备为治疗纤维化疾病的药物或前体药物或转分化试剂或培养基。

Description

体内外化学诱导成纤维细胞直接重编程为肝细胞的化学小分子组合物及方法 技术领域
本发明属于细胞生物学、干细胞生物学(细胞重编程)、医学、药学交叉领域；更具体地，本发明涉及一种不导入、不使用任何外源基因/转录因子/MicroRNA(miRNA)基因，或其结合有载体的基因，或其RNA、蛋白质及多肽等诱导因子，也不使用任何细胞因子或生长因子，仅用化学小分子(简称小分子)组合，化学诱导体内外成纤维细胞直接重编程(转分化)为肝细胞的化学小分子组合物及其方法。
背景技术
中国是肝病大国，甲肝、乙肝、脂肪肝、酒精肝、肝硬化(肝纤维化)等患病人数众多，而其中由各种肝病、化学药物、创伤等多种原因导致的急慢性肝衰竭，病情危重，病死率高。肝移植是治疗终末期肝病或肝功能衰竭的有效手段，然而肝源的缺乏使很多患者，尤其是急性肝衰竭患者失去治疗机会。肝细胞移植、生物人工肝和源于生物工程技术的全肝移植是肝移植的重要替代手段而被关注和研究，如何获得足够数量的可用于临床治疗的人肝细胞来源就成为国际研究热点。目前，获得功能肝细胞的途径主要有以下方式：1)从供肝分离原代肝细胞；2)由干细胞或诱导多能干细胞等定向分化为肝细胞；3)由成纤维细胞等体细胞转分化为肝细胞等。从供肝分离原代肝细胞因肝源的严重缺乏使其应用受限。干细胞定向诱导分化获得功能肝细胞，因为干细胞的无限增殖潜能而成为最可能获得足够数量肝细胞的有效手段和途径。然而现有的定向分化方法存在以下一个或者多个缺陷：如分化效率低、转化纯度低、分化获得的肝细胞功能欠缺、可能产生免疫排斥反应、存在干性细胞分化不彻底而有潜在致癌风险以及成本高昂等，不能满足临床的需求。
成纤维细胞直接重编程(转分化)为肝细胞，因为避免了多潜能干细胞重编程为 iPS 细胞(Induced pluripotent stem cell)及其应用的潜在风险而受到关注。但目前已有的将成纤维细胞转分化为肝细胞方法采用的仍然是导入外源基因/转录因子//MicroRNA(miRNA)基因，等诱导因子，结合化学小分子、各种细胞因子或生长因子等替代因子，组成的多种诱导因子组合物，诱导成纤维细胞转分化肝细胞。因导入外源基因或转录因子，存在破坏原有基因结构，故有致癌或致病风险，这使其仍然很难临床应用；且还存在操作复杂，实验环节增加，质量难以控制，成本增加，临床应用风险增加等等缺陷。因此，如何避免导入外源基因/转录因子/MicroRNA基因等诱导因子，仅用化学小分子，化学诱导成纤维细胞直接重编程为肝细胞，并克服上述导入外源基因/转录因子/MicroRNA基因诱导的定向分化、iPS细胞重编程，或转分化肝细胞方法的缺陷，是肝细胞临床应用亟待解决的瓶颈问题。而成纤维细胞或纤维细胞异常导致的纤维化疾病如肝硬化(肝纤维化)，目前临床更是缺乏有效治疗药物和手段，亟需研发有效的新方法及新药物。
细胞重编程(Cell reprogramming)是细胞从一种类型向另外一种类型的转换，是通过靶 向诱导调控特定细胞信号通路或表观遗传修饰改变，来改变细胞命运的过程。所谓表观遗传是指DNA序列/结构不发生变化，但基因表达却发生了可遗传的改变。诱导细胞重编程包括：(1)诱导分化的细胞逆转恢复到多能或全能性状态的诱导多潜能干细胞(iPS细胞)重编程；(2)不经过多潜能干细胞阶段，从一种分化细胞类型直接转化为另一种分化细胞类型的细胞直接重编程(又称：转分化/谱系重编程)。
化学诱导细胞直接重编程(转分化)：是在不导入、不使用任何外源基因/转录因子/MicroRNA(miRNA)基因，及其RNA、蛋白质及多肽等诱导因子；仅使用化学小分子靶向诱导调控细胞信号通路及表观遗传修饰，使其表观遗传修饰发生变化，以改变细胞的基因表达谱，将一种分化细胞直接转变为另一种分化细胞的过程。包括：(1)化学诱导多潜能干细胞重编程(Hongkui Deng等，Science.341,651-4，2013)；(2)化学诱导细胞直接重编程(转分化/谱系重编程)(Li X等，Cell Stem Cell；17(2):195-203，2015；Hu W等，Cell Stem Cell.17(2):204-212，2015)。
自从日本科学家山中伸弥(Shinya Yamanaka)于2006年利用病毒载体将四个转录因子(基因)Oct4(又称Oct，Oct3，或OCT-3多肽表达因子等)、Sox2、Klf4和c-Myc的诱导组合，导入分化的体细胞中，通过诱导多潜能干细胞重编程获得iPS细胞以来，由于外源导入转录因子/基因，存在破坏细胞原基因序列稳定，可导致突变致癌风险，以及细胞重编程转化率低、稳定性差、操作复杂等缺陷，因此诱导细胞重编程方法在不断改进，已逐步改为非整合以及仅导入1-2个外源转录因子诱导，结合使用MicroRNA(miRNA)基因及其RNA、蛋白多肽、化学小分子、细胞因子、生长因子等替代转录因子，组成多种因子诱导组合物诱导细胞重编程；而导入的外源转录因子/基因，是必须要在重编程过程中，首先使用基因载体导入外源转录因子/基因诱导起始细胞，同时将不需使用基因载体的其他组份如：化学小分子、各种生长因子、细胞因子等，同时、同培养条件诱导，才能重编程出目的细胞。因此，导入的外源诱导转录因子/基因，由于其不可替代性，仍是诱导重编程组合物的必需且最重要的诱导因子成分。
由于化学小分子(简称小分子)属于靶向性分子化合物，能够靶向诱导调控特定的信号通路和表观遗传，使一种细胞转化为另一种细胞；且成药性好、成本低、稳定性好、操作简单，因此成为替代诱导转录因子的最佳候选者；因此，化学诱导细胞重编程，就成为干细胞科研领域的重要目标之一。
发明内容
本发明的目的在于提供一种在不导入、不使用任何外源基因/转录因子/MicroRNA(miRNA)基因，及其RNA、蛋白质或多肽等诱导因子；也不使用任何细胞因子或生长因子，仅使用由2种(类)化学小分子抑制剂：GSK3β抑制剂和HMT抑制剂组成的诱导组合物，体内外化学诱导调控成纤维细胞直接重编程(转分化)为肝细胞的小分子诱导组合物及方法。
本发明是在本发明人之前已公开的发明：仅使用3种(GSK3β抑制剂、G9aHMT抑制剂、TGFβ抑制剂)化学小分子诱导人成纤维细胞直接重编程(转分化)为肝细胞的化学小分子组合物及其方法基础上的进一步创新发明。
本发明人发现，尽管组成该小分子诱导组合物的2种小分子(GSK3β抑制剂、HMT抑制剂)，包括可添加的TGFβ抑制剂，单独存在时并不具有诱导调控成纤维细胞转分化为肝细胞的功能，但它们作为组合物联合应用时却效果卓著。
该化学小分子组合物的用途包括：用于制备体内诱导原位成纤维细胞转分化为肝细胞、以其原位转分化肝细胞效应减少或降低纤维化组织或器官的纤维化，因此可添加药物载体或赋型剂研发制备成临床治疗纤维化疾病(如肝硬化/肝纤维化)的药物/前体药物/药物组合物；或，制备诱导成纤维细胞转分化肝细胞的试剂或培养基；或用于将成纤维细胞转分化为肝细胞，以制备转化肝细胞，为科研应用、医药应用及临床应用如肝细胞移植治疗肝衰竭提供肝细胞源。
该发明的先进性、创新性在于：本发明的仅由2种化学小分子(GSK3β抑制剂和HMT抑制剂)组成的诱导成纤维细胞转分化为肝细胞组合物与之前本发明人发明的3种小分子(GSK3β抑制剂、G9aHMT抑制剂、TGFβ抑制剂)组成的诱导成纤维细胞转分化为肝细胞的组合物及其方法相比较，前者不仅具有3种小分子组合物诱导成纤维细胞转分化肝细胞所具备的先进性和创新性外，还具有：(1)前者与后者体外诱导成纤维细胞转分化为肝细胞效果相同或相近；但前者体内原位诱导成纤维细胞转分化肝细胞及其效应，降低或改善成纤维细胞/纤维细胞异常相关疾病如肝纤维化疾病的效果与后者相同或更佳；(2)前者与后者的小分子组合结构不同；2个小分子组成的组合物的组成成分更少，组合结构更简单，成药性更好，产生副作用的风险更低，开发成本价格更低，更容易开发为相关疾病药物；(3)前者小分子组合物的组成成分HMT抑制剂中的有效化学小分子种类增加，数量更多，候选药物化学小分子的选择范围更广。
在本发明的第一方面，提供一种用于化学诱导体内外成纤维细胞直接重编程(转分化)为肝细胞的化学小分子组合物。所述的化学小分子组合物只包括化学小分子抑制剂：GSK3β抑制剂和组蛋白甲基转移酶(histone methyl transferase，HMT)抑制剂；或，所述的小分子组合物仅由化学小分子抑制剂：GSK3β抑制剂和HMT抑制剂组成；
其中，所述的成纤维细胞中不导入或不使用任何外源基因/转录因子/MicroRNA(miRNA)基因，及其RNA、蛋白质或多肽等诱导因子；或，所述的化学小分子组合物中不含有或不使用任何外源基因/转录因子/MicroRNA(miRNA)基因，及其转录的RNA，或其翻译的蛋白质及多肽等诱导因子；也不含有或不使用任何细胞因子或生长因子；
在一个优选例中，所述化学小分子HMT抑制剂包括(但不限于)：G9a组蛋白甲基转移酶(G9aHMT)抑制剂、EZH2组蛋白甲基转移酶(EZH2HMT)抑制剂；或所述化学小分子组合物包括：
化学小分子GSK3β抑制剂和HMT抑制剂(G9aHMT抑制剂/EZH2HMT抑制剂)； 或仅由GSK3β抑制剂和G9aHMT抑制剂/EZH2HMT抑制剂组成；
在一个优选例中，所述的化学小分子组合物包括：化学小分子抑制剂：GSK3β抑制剂和G9aHMT抑制剂/EZH2HMT抑制剂；或，所述的化学小分子组合物：仅由化学小分子抑制剂：GSK3β抑制剂和G9aHMT抑制剂或EZH2HMT抑制剂组成。
在一个优选例中，所述的化学小分子组合物还可添加包括化学小分子抑制剂：TGFβ抑制剂；或，所述的组合物还可由化学小分子抑制剂：GSK3β抑制剂、HMT抑制剂(G9aHMT抑制剂/EZH2HMT抑制剂)和TGFβ抑制剂组成。
在一个优选例中，所述的化学小分子组合物包括：由化学小分子抑制剂：GSK3β抑制剂、G9aHMT抑制剂/EZH2HMT抑制剂、TGFβ抑制剂组成的化学小分子诱导组合物。
在一个优选例中，所述的化学小分子组合物包括：由化学小分子抑制剂：GSK3β抑制剂和G9aHMT抑制剂/EZH2HMT抑制剂组成的化学小分子诱导组合物；或，
所述的化学小分子组合物包括：由化学小分子抑制剂：GSK3β抑制剂、G9aHMT抑制剂/EZH2HMT抑制剂、TGFβ抑制剂组成的化学小分子诱导组合物。
在一个优选例中，所述的化学小分子组合物，其中化学小分子抑制剂：GSK3β抑制剂、HMT抑制剂、TGFβ抑制剂为：
GSK3β抑制剂：5-80重量份，较佳地10-70重量份；或溶液状态下终浓度为0.1-20μM，较佳地0.5-10μM；
HMT抑制剂：0.1-50重量份，较佳地0.5-40重量份；或溶液状态下终浓度为0.01-20μM，较佳地0.05-10μM；或
TGFβ抑制剂：0.1-50重量份，较佳地0.5-40重量份，或溶液状态下终浓度为0.05-10μM。
在一个优选例中，所述的化学小分子组合物，按照重量份比，所述的小分子抑制剂：GSK3β抑制剂(如CHIR99021、LiCl、CHIR-98014)和HMT抑制剂【G9aHMT抑制剂(如BIX01294、UNC0638、UNC0642)/EZH2HKMT抑制剂(如EPZ005687、GSK343、UNC1999)】为(5-80)﹕(0.1-50)；或溶液状态下摩尔浓度比为(0.1-20)﹕(0.01-20)；或
按照重量份比，所述的GSK3β抑制剂(如CHIR99021、LiCl、CHIR-98014)、HMT抑制剂【G9aHMT抑制剂(如BIX01294、UNC0638、UNC0642)/EZH2HKMT抑制剂(如EPZ005687、GSK343、UNC1999)】和TGFβ抑制剂(如SB431542、A83-01、RepSox)为(5-80)﹕(0.1-50)﹕(0.1-50)；或溶液状态下摩尔浓度比为(0.1-20)﹕(0.01-20)﹕(0.01-20)。
在另一优选例中，上述的小分子组合物，所述的小分子组合物还可包含药学上可接受的载体或赋形剂；或添加包括有机溶剂、生理盐水、缓冲液、细胞基础培养基等载体或赋形剂。
在另一优选例中，小分子GSK3β抑制剂、HMT抑制剂(G9aHMTase抑制剂/EZH2HMT抑制剂)；或GSK3β抑制剂、HMT抑制剂(G9aHMT抑制剂/EZH2HMT抑制剂)和TGFβ抑制剂，相加的重量占组合物总重量的0.01～99.9％；更佳地为50～99.9％； 溶液状态下为0.01～50％，如0.01％，1％，5％，10％，20％，30％等。
上述重量份比的重量单位可以是：千克(kg)、毫克(mg)、微克(μg)等任一重量单位；摩尔浓度比的摩尔单位可以是：摩(M)、毫摩(mM)、微摩(μM)等任一摩尔浓度单位。
所述的小分子组合物应用于大动物和病人时，按小动物使用剂量通过相应的专业换算公式，换算出大动物或人的有效使用剂量(包括固态或溶液态的剂量换算)，也属于本发明的保护范围。
在另一优选例中，所述的化学小分子抑制剂：GSK3β抑制剂，指能够靶向抑制GSK3β信号通路的抑制剂总称，包括但不限于：CHIR-99021、BIO、LiCl、IM-12、TWS119、1-Azakenpaullone、CHIR-98014、Tideglusib、AR-A014418、LY2090314、SB216763、AZD1080，以及诱导抑制GSK3β信号通路的其他GSK3β小分子抑制剂。或与它们等效的药剂制品、类似物、异构体和/或其盐、水合物或前体，或其组合；较佳地为GSK3β抑制剂CHIR-99021、LiCl、BIO、LY2090314。
所述化学小分子抑制剂：HMT抑制剂，包括(但不限于)：G9aHMT抑制剂或EZH2HMT抑制剂，以及抑制HMT的其他HMT小分子抑制剂；或与它们等效的药剂制品、类似物、异构体和/或其盐、水合物或前体，或其组合；较佳地为G9aHMT抑制剂、EZH2HMT抑制剂。
所述的化学小分子G9aHMT抑制剂，指能够靶向抑制G9aHMT的抑制剂总称，包括但不限于：BIX01294、UNC0638、A-366、UNC0631、BRD4770、UNC0224、UNC0646、UNC0642、UNC0321、BRD4770、HKMTI-1-247、HKMTI-1-248、CPUY074020、DCG066，以及抑制G9aHMT的其他G9aHMT小分子抑制剂。或与它们等效的药剂制品、类似物、异构体和/或其盐、水合物或前体，或其组合；较佳地为G9aHMT抑制剂BIX01294(或BIX-01294)、UNC0638、UNC0642。
所述的小分子抑制剂：EZH2HMT抑制剂，指能够靶向抑制EZH2HMT的总称，包括但不限于：EPZ005687、GSK343、Tazemetostat(EPZ-6438)、UNC1999、JQ-EZ-05(JQEZ5)、EBI-2511、CPI-1205、EPZ011989、PF-06726304、EI1、GSK503、GSK126、CPI-360、CPI-169，以及抑制EZH2HKMT的其他EZH2HKMT小分子抑制剂。或与它们等效的药剂制品、类似物、异构体和/或其盐、水合物或前体，或其组合；较佳地为EPZ005687、GSK343、UNC1999。
所述的小分子抑制剂：TGFβ抑制剂，指能够靶向抑制TGFβ信号通路的抑制剂总称，包括但不限于：SB431542、A83-01、SB525334、LY2109761，RepSox、SD-208、GW788388、SB505124、EW-7197，Galunisertib，以及诱导抑制TGFβ信号通路的其他TGFβ小分子抑制剂。或与它们等效的药剂制品、类似物、异构体和/或其盐、水合物或前体，或其组合；较佳地为TGFβ抑制剂SB431542、A83-01、RepSox或LY2109761。
在另一优选例中，所述的化学小分子组合物是药物组合物，用于化学诱导体内原位成纤维细胞直接重编程(转分化)为肝细胞，以其体内原位诱导成纤维细胞转分化为肝细胞 及其效应，可减少或降低组织或器官的成纤维细胞或纤维细胞异常，可研发制备为治疗纤维化疾病(如肝硬化/肝纤维化)的药物/前体药物/药物组合物；同样机理，该小分子组合物通过研发完善，对治疗肺纤维化、肾纤维化，以及其他组织、器管的纤维化疾病也应有类似效果，并可制备成为相应的药物或前体药物；
因此，所述小分子组合物是药物组合物，还包含药学上可接受的载体或赋形剂，其载体或赋形剂包括(但不限于)选自下组的一种或多种：水、盐水、磷酸缓冲液或其它水性溶剂；DMSO(二甲基亚砜)、甘油和乙醇或其它有机溶剂；微球、脂质体、微乳液或高分子表面活性剂；胶体型载药系统或高分子载药系统；或防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、pH缓冲物质，黏合剂、填充剂、润滑剂或其它药物赋形剂。
在另一优选例中，所述的化学小分子组合物可制备的药物剂型包括(但不限于)：固体剂型，包括(但不限于)：粉剂、散剂、片剂、丸剂、胶囊剂、缓释剂、控速释剂；液体剂型，包括(但不限于)：注射剂、输液剂、混悬剂，或其它液体剂型；气体剂型；或半固体剂型。
在本发明的另一方面，提供所述的化学小分子组合物的用途，包括：用于制备体内化学诱导原位成纤维细胞直接重编程(转分化)为肝细胞，以其体内原位转分化肝细胞效应，减少或降低纤维化细胞组织或器官的纤维化，以治疗成纤维细胞或纤维细胞异常的相关疾病即纤维化疾病(如：肝硬化/肝纤维化)的药物/前体药物/药物组合物；或，用于制备化学诱导体内外成纤维细胞直接重编程(转分化)为肝细胞的转分化培养基或试剂；或，用于化学诱导体外成纤维细胞直接重编程(转分化)为肝细胞，以制备转分化/转化肝细胞，为科研应用、医药应用及临床应用如肝细胞移植治疗肝衰竭提供肝细胞源。
在本发明的另一方面，提供一种在成纤维细胞中不导入或不使用，或在小分子组合物中不含有或不使用任何外源基因/转录因子/MicroRNA(miRNA)基因，及其RNA、蛋白质或多肽等诱导因子；也不使用细胞因子或生长因子；仅用化学小分子组合物，体内外化学诱导成纤维细胞直接重编程(转分化)为肝细胞的方法，所述方法包括：应用上述任一所述的化学小分子组合物，体内外诱导成纤维细胞转分化为肝细胞的方法；或，为制备体内原位诱导成纤维细胞直接重编程(转分化)为肝细胞；以其体内原位转分化肝细胞效应减少或降低组织器官的成纤维细胞或纤维纤维细胞异常，以治疗成纤维细胞或纤维细胞异常的纤维化疾病(如：肝硬化/肝纤维化)的药物/前体药物/药物组合物的方法；
同样机理，该小分子组合物通过研发完善，对治疗肺纤维化、肾纤维化，以及其他组织、器管的纤维化疾病也应有类似效果，制备相应的药物或前体药物；
或以上述任何小分子组合物诱导成纤维细胞直接重编程为肝细胞，以制备转化肝细胞的方法；
或添加有机溶剂、或生理盐水、或缓冲液、或细胞基础培养基等载体或赋形剂，制备用于化学诱导成纤维细胞直接重编程(转分化)为肝细胞的转分化培养基或试剂的方法。
所提供制备诱导成纤维细胞直接重编程(转分化)为肝细胞的转分化培养基或试剂方法及其实验步骤，包括：
(1)浓缩液试剂配制：根据前面任一所述的小分子组合物，将各成分溶解于有机溶剂或水性溶剂中配制成浓缩液试剂；较佳地，所述的有机溶剂包括二甲基亚砜；较佳地，所述的水性溶剂包括：水，生理盐水，磷酸盐缓冲液；
(2)成纤维细胞转分化肝细胞培养基获得：将步骤(1)中的浓缩液试剂分别稀释入含5-20％小牛血清的细胞基础培养基中(使得各组分的浓度符合前面任一所述的小分子组合物中所限定的终浓度)，获得诱导成纤维细胞转分化肝细胞的培养基；其中，该培养基各组分的百分含量还可上下浮动50％；较佳地上下浮动30％；更佳地上下浮动20％，如10％，5％；
(3)诱导成纤维细胞转分化为肝细胞：将成纤维细胞在含5-20％小牛血清的细胞基础培养基或含有各种细胞因子或生长因子的无血清培养基中混悬、铺板，在细胞贴壁后换成步骤(2)的转分化培养基，37℃培养，每2-4天换液一次；3-15天传代一次。
(4)诱导成纤维细胞转分化肝细胞的传代培养：弃原培养液，PBS洗涤一次，加入细胞消化液消化细胞，37℃，1-5分钟，终止细胞消化，离心，弃上清，将细胞沉淀重悬，按1:1-1:3传代铺板。应用步骤(2)的转分化培养基，参照上述方法培养，每2-4天换液一次。所用消化液包括胰酶，EDTA，Acutase，TrypleE等。3-15天传代一次。
(5)诱导成纤维细胞转分化肝细胞细胞的收获：经上述实验步骤(3)、(4)转分化肝细胞培养和传代培养2-4周，即可将成纤维细胞转分化为肝细胞，并获得转分化后的肝细胞。
在本发明的另一方面，提供一种用于化学诱导体内外成纤维细胞直接重编程(转分化)为肝细胞的药盒或试剂盒。所述的药盒/试剂盒中包括：前面任一所述的化学小分子组合物；或基于该小分子组合物添加药学上可接受的载体或赋形剂，用于体内化学诱导原位成纤维细胞直接重编程(转分化)为肝细胞，以其原位转分化肝细胞效应减少或降低纤维化组织/器官的纤维化，而研发或制备的治疗人纤维化疾病(如肝硬化/肝纤维化)的药物/前体药物/药物组合物；或添加有机溶剂/生理盐水/缓冲液/细胞基础培养基等载体或赋形剂制备的诱导成纤维细胞直接重编程(转分化)为肝细胞的转分化培养基或试剂。
该药盒或试剂盒中不包括、不含有：用于导入成纤维细胞中，或添加到小分子组合物/转分化培养基中的任何外源基因/转录因子/MicroRNA(miRNA)基因，或其RNA、蛋白质及多肽等成分；或其与基因载体的结合物。
如前面任一方面，所述的成纤维细胞包括但不限于：人成纤维细胞或哺乳动物成纤维细胞；较佳地，包括但不限于人的：皮肤成纤维细胞、肝成纤维细胞(肝星状/形细胞hepatic stellate cell，HSC)、肺成纤维细胞、肾成纤维细胞、胰腺成纤维细胞，以及人体其他组织或器官的成纤维细胞。较佳地为人皮肤成纤维细胞及肝成纤维细胞(肝星状细胞)。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、肝成纤维细胞(肝星状细胞HSC)与其转分化所得肝细胞(ciHep)形态比较。图1A右侧图表示2种小分子组合物的转分化肝细胞培养基1(2F)诱导小鼠HSC转分化所得的肝细胞(ciHep)；图1B右侧图表示3种小分子组合的转分化培养基11(3F)诱导HSC转分化所得的肝细胞(ciHep)；分别与图A、B左侧图中HSC的形态比较，转化肝细胞形态均发生明显改变，与肝细胞形态吻合，部分转化肝细胞明显出现双细胞核。显示HSC都已转分化为肝细胞(ciHep)。实验结果显示，仅由2种化学小分子(GSK3β抑制剂和HMT抑制剂)组成的小分子组合物(2F)与之前本发明人发明的仅由3种小分子(GSK3β抑制剂、G9aHMT抑制剂、TGFβ抑制剂)组成的小分子组合物(3F)，在体外诱导成纤维细胞转分化为肝细胞的效果相同或相近。
图2、肝星状细胞(HSC)与其转分化获得肝样细胞(ciHep)的糖原染色(PAS)。图2右侧图：显示HSC转分化所得ciHep的糖原染色阳性。图2左侧图显示：肝星状细胞(HSC)糖原染色阴性；表明该转分化获得的肝样细胞具有人肝细胞相同的糖原储存活性特有功能。
图3、肝星状细胞(HSC)与其转分化获得肝样细胞(ciHep)的脂肪油红染色(Oil-red)比较。图3右侧图显示：肝星状细胞(HSC)转分化所得ciHep的脂肪油红染色阳性，阳性染色展现转分化肝细胞代谢脂肪的能力。而HSC脂肪油红染色阴性；结果表明该转分化所得肝样细胞具有肝细胞代谢脂肪特有功能。
图4、成纤维细胞(HF)转分化为肝样细胞(ciHep)后表达肝细胞相关基因。该实验结果显示，成纤维细胞转分化为肝样细胞后高表达肝细胞相关基因；表明成纤维细胞已经被转分化为肝细胞。
图5、通过口服体内原位成纤维细胞转分化肝细胞试剂5治疗肝纤维化小鼠动物模型实验，表明处理组(Treat)比对照组(Ctrl)的外周血中转氨酶(谷丙转氨酶(ALT)、谷草转氨酶(AST))显著降低，表明该小分子组合物诱导体内原位成纤维细胞转分化为肝细胞效应，使肝纤维化病变明显减轻或逆转。
图6、通过口服体内原位成纤维细胞转分化肝细胞试剂7、10治疗肝纤维化小鼠动物模型对比实验，分别取两处理组(Treat)和对照组(Ctrl)小鼠的肝脏组织固定切片，作天狼星红染色(按试剂盒操作说明)。图6A图：与对照组比较，口服成纤维细胞转分化试剂7(2F)的处理组小鼠其天狼星红染色显著减少；图6B图：与对照组比较，口服成纤维细胞转分化试剂10(3F)的处理组小鼠其天狼星红染色也显著减少。图6的A图和B图比较，A图的处理条件降低纤维化程度的效果显著比B图的处理条件更佳；表明该2种小分子组合物比3种小分子组合物的体内原位转分化成纤维细胞为肝细胞及其效应，使肝纤维化病变减轻或逆转效果更佳。
图7、人肝星状细胞(Lx2)与其转分化获得的转化肝细胞(Lx2-ciHep)的形态比较；结 果显示，人肝星状细胞((Lx2)转分化后，具人肝细胞的特有形态特征。
图8、人肝星状细胞系(Lx2)转分化获得的肝细胞(Lx2-ciHep)的油红染色(Oil-red)、糖原染色(PAS)；与人原代肝细胞(PHH)进行对照染色比较。结果显示，人肝星状细胞转分化获得的肝细胞与原代肝细胞染色均为阳性，具备糖原储存和脂肪代谢特有功能。
图9、人肝星状细胞(Lx2)与其转分化获得的转化肝细胞(Lx2-ciHep)的人肝细胞特异性标志物流式细胞分析；结果显示，转分化所获得的肝细胞(Lx2-ciHep)有人肝细胞的特异性标志物表达，而对照组则没有表达。
图10、实验结果显示，转分化肝细胞培养基13、14、15化学诱导成纤维细胞转分化后，高表达肝细胞相关基因(ALB、AAT、ASGPR1、7A1)。
图11、体内原位成纤维细胞转分化肝细胞口服试剂12体内原位诱导小鼠肝星状细胞(HSC)转分化为肝细胞示踪比较实验。图11上图对照组与下图处理组比较，下图处理组左侧图为转分化处理后的肝细胞组织仍然显示红色，形态不变；中间图显示肝星状细胞转分化为肝细胞后，形态已转变为肝细胞形态，但仍然显示其绿色荧光标记；右侧图显示转化肝细胞(绿色荧光)与原有肝细胞组织(红色荧光)整合为一体，且转化肝细胞仍然显示原位HSC示踪标记的绿色荧光。实验结果充分显示本发明的小分子组合物可体内原位诱导HSC转分化为肝细胞。
图12、化学小分子组合物的各单独小分子组成成分诱导成纤维细胞(HF)转化比较试验。图12显示，图左侧成纤维细胞(HF)对照组(Control)与图右侧各小分子处理组(Treat)相比较，各单独小分子成分对人成纤维细胞没有诱导转分化为肝细胞的作用，成纤维细胞被其诱导转分化前后，形态基本没有变化，更与肝细胞形态没有任何相似之处。实验结果表明，化学小分子组合物的各单独组分：GSK3β抑制剂、HMT抑制剂、TGFβ抑制剂单独存在时，不具有诱导成纤维细胞转分化为肝细胞的功能。
具体实施方式
本发明人经过深入的研究，揭示了一种不导入或不使用任何外源基因/转录因子/MicroRNA(miRNA)基因，及其RNA、蛋白质或多肽等诱导因子；或，在化学小分子组合物/转分化培养基中，不含有或不使用任何外源基因/转录因子/MicroRNA(miRNA)基因，或其转录的RNA，或其翻译的蛋白质及多肽等诱导因子；也不添加使用任何细胞因子或生长因子。仅使用化学小分子，体内外化学诱导成纤维细胞直接重编程(转分化)为肝细胞的小分子组合物及其方法；该小分子组合物可添加药学上可接受的载体或赋形剂，用于化学诱导体内原位成纤维细胞直接重编程(转分化)为肝细胞，以其体内原位转分化肝细胞效应，减少或降低组织或器官的成纤维细胞或纤维细胞异常，研发制备成临床治疗纤维化疾病(如肝硬化/肝纤维化)的药物或药物前体；同样机理，该小分子组合物通过研发完善，对治疗肺纤维化、肾纤维化，以及其他组织、器管的纤维化疾病也有类似效果，并可制备成为相应的药物或前体药物；或添加水性溶剂或有机溶剂，或生理盐水/缓冲剂/细胞基础培 养基等载体或赋型剂，制备成转分化试剂或培养基；还可用于体外化学诱导成纤维细胞转分化为肝细胞，制备转分化肝细胞，为科研应用、医药应用及临床应用如肝细胞移植提供肝细胞源。该小分子组合物，体内外化学诱导成纤维细胞直接重编程(转分化)为肝细胞的方法可应用于人成纤维细胞或哺乳动物成纤维细胞；较佳地，包括但不限于人的：皮肤成纤维细胞、肝成纤维细胞(肝星状细胞)、肺成纤维细胞、肾成纤维细胞、胰腺成纤维细胞或人体其他组织或器官的成纤维细胞；更佳地为人皮肤成纤维细胞或肝成纤维细胞(肝星状细胞)。而体外转分化获得的肝样细胞具有正常肝细胞功能。
基本机理
细胞重编程(Cell reprogramming)是细胞从一种类型向另外一种类型的转换，是通过靶向诱导调控特定细胞信号通路及表观遗传修饰，使其表观遗传发生变化来改变细胞命运的过程。
本发明即是在成纤维细胞或起始细胞中，或在小分子组合物/转分化培养基中不导入或不使用任何外源基因/转录因子/MicroRNA基因，及其转录的RNA，或其翻译的蛋白质及多肽等诱导因子；也不添加使用任何细胞因子或生长因子。仅使用化学小分子组成的诱导组合物GSK3β抑制剂和HMT抑制剂(较佳地为G9aHMT抑制剂/EZH2HKMT抑制剂)2种小分子组成的诱导组合物，同时靶向诱导调控成纤维细胞的GSK3β信号通路及其表观遗传酶HMT，调控出新信号通路及表观遗传修饰，使细胞基因表达谱发生改变，从而将成纤维细胞转分化为肝细胞。
本发明的小分子组合物及其方法具有以下先进性和创新性优点：
①化学小分子为靶向小分子化合物，性质稳定，作用的时间、剂量及组合方式易于控制，作用效果稳定可靠，成药性好；②转分化获得的肝细胞具有正常成熟的人肝细胞形态和功能；③该方法的成纤维细胞可取材于患者本人，因此转分化获得肝细胞具有个性化特征，并有两大优势：一是更易于进入临床应用，并最大限度降低或避免转化肝细胞移植引起的免疫排斥反应风险；二是可以构建具群体代表性的肝细胞库，应用于新药的肝毒安全评测和药效筛选；④该体内外转分化成纤维细胞为肝细胞的方法及诱导组合物，是在不导入、不使用任何外源基因/转录因子/MicroRNA基因，或其RNA，或其蛋白质及多肽，等诱导因子；仅使用化学小分子组成诱导组合物，避免了外源基因导入或结构基因改变引起新的致癌风险，更为安全可靠；而且也不使用任何细胞因子或生长因子，成本费用更低，操作也更简单。⑤该方法采取化学诱导直接重编程，不需要经过诱导多潜能干细胞(iPSC)重编程阶段，避免了诱导多潜能干细胞引起的致癌风险；⑥化学小分子为靶向小分子化合物，结构稳定，成药性好，易于应用于体内原位成纤维细胞转分化，以其体内原位转分化效应减少或降低纤维化组织/器官的纤维化，易于研发或制备成治疗临床纤维化疾病(如肝纤维化/肝硬化)的药物或前体药物；还可进一步研发制备治疗肾纤维化、肺纤维化、及 其他器官或组织纤维化疾病的药物或前体药物。
此外，本发明的先进性、创新性还在于：本发明最少仅由2种化学小分子(GSK3β抑制剂和HMT抑制剂)组成的组合物与之前本发明人发明的3种小分子(GSK3β抑制剂、G9aHMT抑制剂、TGFβ抑制剂)组成的诱导成纤维细胞转分化为肝细胞的组合物及其方法相比较，前者不仅具有3个小分子组合物诱导成纤维细胞转分化肝细胞的全部先进性和创新性外，还具有：(1)前者与后者体外诱导成纤维细胞转分化为肝细胞效果相同或相近；但前者体内原位诱导成纤维细胞转分化肝细胞及其效应，降低或改善成纤维细胞/纤维细胞异常相关疾病如肝纤维化疾病的效果与后者相同或更佳；(2)前者与后者的小分子组合物的组合结构不同；2个小分子组成的组合物的组成成分更少，组合结构更简单，成药性更好，产生副作用的风险更低，开发成本更低，更容易开发为相关疾病药物；(3)前者小分子组合物的组成成分HMT抑制剂中的有效化学小分子种类增加，数量更多，候选药物化学小分子的选择范围更广。
另一方面，前述任一化学小分子组合物中的GSK3β抑制剂、HMT抑制剂(较佳地为G9aHMT抑制剂/EZH2HKMT抑制剂)，以及可选择添加的TGFβ抑制剂，其中任一小分子组分单独存在时，并无任何诱导成纤维细胞转分化肝细胞的功能。
且至今未见任何文献报道上述化学小分子单独存在时，具有诱导成纤维细胞转分化为肝细胞的功能。
前述小分子抑制剂拥有特异性的抑制或阻断特定信号通路或表观遗传酶活性的能力，每个类别下的其他靶向性小分子抑制剂，都是根据其所诱导调控或抑制的特定细胞信号通路或酶活性，发挥有效调控功能，才划归为同一类别(该归类工作由化学小分子发明者完成)，并以所调控的特定信号通路或表观遗传修饰酶，以及所发挥功能为类别名称；例如，GSK3β抑制剂类别内的所有小分子都具有靶向抑制GSK3β信号通路的共同功能特点。而同类别内的小分子抑制剂之间，仅只是有效剂量、活性大小、作用效果存在差异，但诱导调控特定信号通路或表观遗传酶活性的能力并无本质区别。因此，由于同类别小分子抑制剂可靶向诱导调控同一特定细胞信号通路或其表观遗传酶特定靶点这一特殊性，使同一类别小分子抑制剂，其作为单独成分发挥功效是基本相同的；作为组合物的参与成分，其在组合物有机整体中发挥的功效也是基本相同的，而不会有质的差别。其他靶向诱导细胞特定信号通路或其表观遗传的特定靶点的小分子抑制剂或小分子化合物亦同此理。这为本领域科技人员所熟知的常识。
因此，GSK3β抑制剂、HMT抑制剂(G9aHMT抑制剂/EZH2HKMT抑制剂等)，以及可选择添加的TGFβ抑制剂，分别包含了诱导调控相同的特定细胞信号通路或其表观遗传修饰酶，以及发挥的功能活性、效应、效果一致的各自类别内的小分子化合物；所形成的不同组合，都能够不同程度地诱导成纤维细胞转分化为肝细胞。因此，其功能相同或诱导 靶点相同，或对同一条信号通路或其表观遗传酶起相同效应的同类别内的小分子化合物，以及所构成的能够诱导调控成纤维细胞转分化为肝细胞的小分子组合都属于本发明保护范围内。
另一方面，成纤维细胞(fibroblast)，也称为纤维母细胞，是疏松结缔组织的主要细胞成分，由胚胎时期的间充质细胞(mesenchymal cell)分化而来。成纤维细胞根据不同功能活动状态，可划分为成纤维细胞和纤维细胞；成纤维细胞功能活动旺盛，细胞质嗜弱碱性，具明显的蛋白质合成和分泌活动；处于成熟期或称静止状态的被称为纤维细胞；在一定条件下，二者可实现互相转化。成纤维细胞具有不同类型，存在与体内各种组织或器官中；在不同的组织或器官中有不同的名称和特性，包括：皮肤成纤维细胞、肝成纤维细胞(肝星状细胞)、肺成纤维细胞、胰腺成纤维细胞以及其它组织或器官中的成纤维细胞。
而本发明的方法或小分子组合物诱导的成纤维细胞，即前面所述的成纤维细胞包括但不限于：人成纤维细胞或哺乳动物成纤维细胞；较佳地，包括但不限于人的：皮肤成纤维细胞、肝成纤维细胞(肝星状细胞)、肺成纤维细胞、肾成纤维细胞、胰腺成纤维细胞，以及人体其他组织或器官的成纤维细胞。更佳地为人皮肤成纤维细胞或肝成纤维细胞(肝星状细胞)。
药物组合物及其应用
本发明人经过广泛的研究，首次提出一种不导入，不使用任何外源基因/转录因子/MicroRNA(miRNA)基因，或其转录的RNA，或其翻译的蛋白及多肽等诱导因子；也不添加使用任何细胞因子或生长因子。仅由化学小分子抑制剂：GSK3β抑制剂和HMT抑制剂组成的小分子组合物，体内或体外化学诱导成纤维细胞直接重编程(转分化)为肝细胞；即同时不同程度地抑制GSK3β信号通路，以及抑制其HMT(G9aHMT或EZH2HMT)，即可诱导调控出新的信号通路及其表观遗传修饰的改变，从而使其基因表达谱发生改变，使成纤维细胞转分化为肝细胞。
上述小分子组合物是药物组合物，还可添加TGFβ抑制剂；该药物组合物可用于化学诱导体内原位成纤维细胞转分化为肝细胞，以其体内原位转分化肝细胞效应，减少或降低组织/器官的成纤维细胞异常，达到治疗成纤维细胞或纤维细胞异常相关疾病(纤维化疾病如：肝纤维化/肝硬化)的效果。因此可添加药物载体或赋型剂，研发制备为治疗肝纤维化(肝硬化)疾病的药物/前体药物/药物组合物；由此推及，该小分子组合物经过研发完善，对治疗肺纤维化、肾纤维化，以及其他组织、器管的纤维化疾病也应有类似效果；亦可添加药物载体或赋型剂，研发制备相应的创新药物或前体药物，因此也应包含在本发明中。
应理解，同类别内的小分子抑制剂之间，仅只是有效剂量、活性大小、作用效果存在差异，但诱导抑制或调控特定信号通路或表观遗传酶的能力并无本质区别。除了本发明实 施例中所列举的具体、代表性的化学小分子GSK3β抑制剂以外的其它可靶向抑制GSK3β细胞信号通路的GSK3β小分子抑制剂，也可实现同样的技术效果，也应被包含在本发明中；
除了本发明实施例中所列举的具体的，代表性的HMT抑制剂以外的其它靶向诱导抑制HMT的化学小分子HMT抑制剂，也可实现同样的技术效果，也应被包含在本发明中；
除了本发明实施例中所列举的具体的，代表性的HMT抑制剂(G9aHMT抑制剂/EZH2HKMT抑制剂)以外的其它靶向诱导抑制HMT的化学小分子HMT抑制剂(G9aHMT抑制剂/EZH2HKMT抑制剂)，也可实现同样的技术效果，也应被包含在本发明中；
除了本发明实施例中所列举的具体的，代表性的G9aHMT抑制剂/EZH2HKMT抑制剂以外的其它靶向诱导调控抑制G9aHMT/EZH2HKMT的其他G9aHMT抑制剂或EZH2HKMT抑制剂，也可实现同样的技术效果；也应被包含在本发明中。
同样，除了本发明实施例中所列举的具体，代表性TGFβ抑制剂以外的其它靶向抑制TGFβ信号通路的化学小分子TGFβ抑制剂，也可实现同样的技术效果，也应被包含在本发明中。
如本文所用，术语“含有”或“包括”包括了“包含”、“基本上由……构成”、和“由……构成”。
如本文所用，术语“基本上由……构成”指在组合物中，除了含有必要成分或必要组份之外，还可含有少量的且不影响有效成分的次要成分和/或杂质。例如，可以含有甜味剂以改善口味、抗氧化剂以防止氧化，以及其他本领域常用的药物添加剂、载体、赋形剂。本发明中，所述“包括GSK3β抑制剂和HMT抑制剂”或“由GSK3β抑制剂和HMT抑制剂组成”包括了“基本上由GSK3β抑制剂和HMT抑制剂组成”、“主要以GSK3β抑制剂和HMT抑制剂为活性成分”、“以GSK3β抑制剂和HMT抑制剂为唯一活性成分”、“基本上以GSK3β抑制剂和HMT抑制剂为活性成分”的情形。
如本文所用，术语“药学上可接受的”的成分是适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)的，即有合理的效益/风险比的物质；如本领域常用的药物载体或赋形剂。
如本文所用，术语“有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
如本文所用，术语“药学上可接受的载体或赋形剂”，其中载体指能改变药物进入人体的方式和在体内的分布、控制药物的释放速度并将药物输送到靶向器官的体系；药物载体本身并不是必要的活性成分，且施用后没有过分的毒性。合适的载体是本领域普通技术人员所熟知的，包括但不限于：水、盐水、磷酸缓冲液以及其它水性溶剂；DMSO(二甲基亚砜)、甘油和乙醇以及其它有机溶剂；微球、脂质体、微乳液、高分子表面活性剂；胶体型载药系统、新型高分子载药系统、新型药物载体以及其他药学上的载体；其中赋形剂 指在药物制剂中除主药以外的附加物，也可称为辅料。如片剂中的黏合剂、填充剂、崩解剂、润滑剂；中药丸剂中的酒、醋、药汁等；半固体制剂软膏剂、霜剂中的基质部分；液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂等均可称为赋形剂。
对赋形剂的一般要求是性质稳定，与主药无配伍禁忌，不产生副作用，不影响疗效，在常温下不易变形、干裂、霉变、虫蛀、对人体无害、无生理作用，不与主药产生化学或物理作用，不影响主药的含量测定等。在Remington’s Pharmaceutical Sciences(Mack Pub.Co.，N.J.1991)中可找到关于药学上可接受的载体或赋形剂的充分讨论。
如本文所用，术语“组合物可制备的药物剂型”中的药物剂型指：为适应治疗或预防的需要而制备的药物应用形式，称为药物剂型；
如本文所用，“重量份”或“重量份数”可互换使用，所述的重量份可以是任何一个固定的以微克、毫克、克数或千克数表示重量(如1ug、1mg、1g、2g、5g、或kg等)。例如，一个由1重量份组分a和9重量份组分b构成的组合物，可以是1克组分a+9克组分b，也可以是10克组分a+90克组分b等构成的组合物。在所述组合物，某一组分的百分比含量＝(该组分的重量份数/所有组分的重量份数之和)×100％。因此，由1重量份组分a和9重量份组分b构成的组合物中，组分a的含量为10％，组分b为90％。
此外，在溶液状态时，上述“重量份”也可以换算成为“摩尔数”；“重量份比”也可以换算成为“摩尔浓度比”。所述重量份比的重量单位可以是：千克(kg)、毫克(mg)、微克(ug)等任一重量单位；摩尔浓度比的摩尔单位可以是：摩(M)、毫摩(mM)、微摩(uM)等任一摩尔浓度单位；
化学小分子：GSK3β抑制剂(如CHIR99021、LiCl、CHIR-98014)，HMT抑制剂【G9aHMT抑制剂(如BIX01294、UNC0638、UNC0642)/EZH2HKMT抑制剂(如EPZ005687、GSK343、UNC1999)】，TGFβ抑制剂(如SB431542、A83-01、RepSox)，以重量份比(5-80)﹕(0.1-50)﹕(0.1-50)；较佳地，(10-70)﹕(0.5-40)﹕(0.5-40)存在；或溶液状态下以摩尔浓度比(0.1-20)﹕(0.01-20)﹕(0.01-20)；较佳地，(0.5-10)﹕(0.05-10)﹕(0.05-10)存在。
例如，所述的组合物中，包括的成分及重量份比如表1或摩尔浓度比如表2(溶液状态)所示。
表1、重量份比(重量单位：kg、mg、μg……)
Figure PCTCN2020113954-appb-000001
表2、摩尔浓度比(摩尔单位：M、mM、μM……)
Figure PCTCN2020113954-appb-000002
表1和表2的配方范围可以作为参考性指导。但是应理解，当用于研发制备药物组合物时，所用的组合物的有效剂量可随施用的模式和待治疗纤维化(如肝硬化)疾病患者身体状况，或疾病严重程度而变化。且，体内使用时，通常使用“重量/公斤(体重)”作为剂量单位；所述的小分子组合物应用于大动物和肝病病人时，按小动物使用剂量通过相应的专业换算公式，换算出的大动物或人的有效使用剂量(包括固态或溶液态的剂量换算)，也属于本发明的保护范围。
还应理解，由于各种类别化学小分子各自的小分子成员较多，且各自小分子成员的有效剂量和活性大小差别较大，虽然本发明人长期研究化学小分子诱导调控干细胞定向分化，做了大量的实验，但说明书的实施例不可能全部例举，实施例仅是针对小分子诱导调控组合成分的，各自类别的代表性小分子(如针对GSK3β抑制剂，代表性地以CHIR-99021、LiCl、CHIR-98014等几个小分子抑制剂加以论证)在实验中的使用浓度；故本发明专利申请权利要求中所概括的合理浓度范围，自然是包括，但不限于实施例中各类别的，特定代表性小分子实验中的使用浓度范围；这一基本而浅显的道理，本领域专业技术人员都熟知，不可混淆。
如本发明所用，所述的GSK3β抑制剂是指能够抑制细胞中GSK3β信号通路的小分子抑制剂的总称，包括但不限于：CHIR-99021，BIO、LiCl、IM-12、TWS119，1-Azakenpaullone、CHIR-98014、Tideglusib、AR-A014418、SB216763、AZD1080，以及诱导抑制GSK3β信号通路的其他GSK3β小分子抑制剂或小分子化合物；
作为本发明的优选方式，所述的GSK3β抑制剂是CHIR-99021，其别名为CT99021；其分子结构式如以下式(I)所示：
Figure PCTCN2020113954-appb-000003
所述化学小分子抑制剂：HMT抑制剂，是能够靶向抑制HMT化学小分子的总称， 包括(但不限于)：G9aHMT抑制剂、EZH2HMT抑制剂；
所述的化学小分子G9aHMT抑制剂，是指能够靶向抑制G9aHMT的化学小分子抑制剂的总称，包括但不限于：BIX01294、UNC0638、A-366、UNC0631、BRD4770、UNC0224、UNC0646、UNC0642，UNC0321、BRD4770、HKMTI-1-247、HKMTI-1-248、CPUY074020、DCG066，以及诱导抑制G9aHMT的其他G9aHMT小分子抑制剂或小分子化合物；
作为本发明的优选方式，所述的G9aHMT抑制剂是BIX01294(或BIX-01294)；其分子结构式如以下式(II)所示：
Figure PCTCN2020113954-appb-000004
所述的化学小分子EZH2HMT抑制剂是指能够靶向抑制EZH2HMT的化学小分子抑制剂的总称，包括但不限于：EPZ005687、GSK343、Tazemetostat(EPZ-6438)、UNC1999、JQ-EZ-05(JQEZ5)、EBI-2511、CPI-1205、EPZ011989、PF-06726304、EI1、GSK503、GSK126、CPI-360、CPI-169，以及抑制EZH2HMT的其他化学小分子EZH2HMT抑制剂或小分子化合物：
作为本发明的优选方式，所述的化学小分子EZH2HMT抑制剂是小分子EPZ005687；其分子结构式如以下式(III)所示：
Figure PCTCN2020113954-appb-000005
所述的化学小分子TGFβ抑制剂，是指能够抑制细胞中TGFβ信号通路的化学小分子抑制剂的总称，包括但不限于：SB431542、A83-01、SB525334、LY2109761、RepSox，SD-208、GW788388、SB505124、EW-7197，Galunisertib，以及诱导抑制TGFβ信号通路的其他小分子TGFβ抑制剂或小分子化合物；
作为本发明的优选方式，所述的化学小分子TGFβ抑制剂是小分子SB 431542(或称为SB-431542)；其分子结构式如以下式(IV)所示：
Figure PCTCN2020113954-appb-000006
作为本发明的优选方式，所述的化学小分子TGFβ抑制剂是小分子A83-01(或称为A8301)；其分子结构式如以下式(V)所示：
Figure PCTCN2020113954-appb-000007
本发明还包括与上述小分子化合物Ⅰ、Ⅱ或Ⅲ、Ⅳ、V等效的化合物、药剂制品、类似物和/或其盐、水合物或前体；也包括其自然生成和人工合成化合物。
所述小分子化合物的类似物包括但不限于：所述小分子化合物的异构体、外消旋体。化合物具有一个或多个不对称中心。所以，这些化合物可以作为外消旋的混合物、单独的对映异构体、单独的非对映异构体、非对映异构体混合物、顺式或反式异构体存在。
所述的“盐”包括但不限于：(1)与如下无机酸形成的盐：如盐酸、硫酸、硝酸、磷酸等；(2)与如下有机酸形成的盐，如乙酸、草酸、丁二酸、酒石酸、甲磺酸、马来酸、或精氨酸等。其它的盐包括与碱金属或碱土金属(如钠、钾、钙或镁)形成的盐等。
所述的“化合物的前体”指当用适当的方法施用或处理后，该化合物的前体在培养基中，或动物，或人体内可转变成上述任一化合物的一种化合物，或上述任一化合物的一种化合物所组成的盐或溶液。
本发明的化学小分子组合物是用于诱导成纤维细胞直接重编程(转分化)为肝细胞的药物组合物，可添加药物载体、赋型剂，研发制备为治疗涉及成纤维细胞或纤维细胞相关疾病的药物/前体药物/药物组合物；因此还包括：药学上可接受的载体或赋形剂；较佳地，所述载体或赋形剂包括选自下组的一种或多种：水、盐水、磷酸缓冲液或其它水性溶剂；DMSO、甘油和乙醇或其它有机溶剂；微球、脂质体、微乳液或高分子表面活性剂；胶体型载药系统或高分子载药系统；防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、pH缓冲物质，黏合剂、填充剂、润滑剂或其它药物赋形剂；或
本发明所述的化学小分子组合物的剂型没有特别的限制，可以是任何适用于哺乳动物服用的剂型；可制备的剂型包括：粉剂、散剂、片剂、丸剂、胶囊剂、缓释剂、控速释剂及其它固体剂型；注射剂、输液剂、混悬剂及其它液体剂型；以及气体剂型、半固体剂型等其它剂型。优选的，所述的剂型可以是但不限于：粉末剂、颗粒剂、胶囊、缓释剂、片剂等固体剂型或注射剂、输液剂、溶液剂、混悬液等液体剂型。
本发明的小分子组合物的制备方法根据所需制备的剂型以及给药途径来决定，本领域技术人员在参考了本发明所提供的组合以及配比后，采用常规的药物组合物的制备方法即可制备出本发明的化学小分子组合物。
应理解，尽管在具体实施方式中，本发明人列举了几种组合物形式，但本领域人员 也可由此推导：本发明的其它任何一种小分子组合形式也是同样具有突出效果的。
本发明人首次证实了本发明的小分子组合物可体内原位诱导成纤维细胞转分化为肝细胞，以其体内原位转分化肝细胞效应减少或降低组织/器官的成纤维细胞或纤维细胞异常，因此可添加药物载体或赋型剂，研发制备预防、改善或治疗纤维化疾病如肝纤维化(肝硬化)药物或前体药物或药物配方；
应理解，该小分子组合物经过研发完善，对治疗肺纤维化、肾纤维化，以及其他组织、器管的纤维化疾病也应有类似效果；并可添加药物载体或赋型剂，研发制备相应的创新药物或前体药物，因此也应包含在本发明中。
当用于预防、改善或治疗肝纤维化(肝硬化)以及他器官或组织纤维化疾病时，所用的组合物的有效剂量可随施用的模式和待治疗纤维化疾病类型以及疾病严重程度而变化。具体情况根据受试者的个体情况来决定，这在熟练医师或药剂师可以判断的范围内。
本发明中，所述的成纤维细胞包括但不限于：人成纤维细胞或哺乳动物成纤维细胞；较佳地，包括但不限于人的：皮肤成纤维细胞、肝成纤维细胞(肝星状细胞)、肺成纤维细胞、肾成纤维细胞、胰腺成纤维细胞，以及人体其他组织或器官的成纤维细胞。更佳地为人皮肤成纤维细胞或肝成纤维细胞(肝星状细胞)。
制备转分化培养基和试剂
本发明还提供了小分子组合物可添加有机溶剂、或生理盐水、或缓冲液、或细胞基础培养基等载体/赋形剂/基础营养液，制备为化学诱导成纤维细胞直接重编程(转分化)为肝细胞的转分化培养基或试剂；诱导成纤维细胞直接重编程(转分化)为肝细胞的转分化培养基(以下简称：成纤维细胞转分化肝细胞培养基)。
按本发明所提供的化学小分子组合物终浓度配方，选择具体终浓度的小分子组合物进行配制。作为本发明的优选方式，将该具体小分子组合物中的不同成分，分别根据其溶质的不同性质和不同溶解度将其溶解于DMSO(二甲基亚砜)或其它有机溶剂或水性溶剂中配成浓缩液试剂(从1∶50-1∶10,000范围)；然后按该具体小分子组合物终浓度要求，将各小分子有机溶液浓缩液试剂稀释，添加入含10％小牛血清的细胞基础培养基中，即可获得所述的成纤维细胞转分化肝细胞培养基。其中，该培养基各组分的百分含量还可上下浮动50％；较佳地上下浮动30％；更佳地上下浮动20％，如10％，5％。除非另外说明，百分数以v/v计。
作为本发明的优选方式，所述的细胞基础培养基包括但不限于：DMEM/F12、MEM、DMEM、F12、IMDM、RPMI1640、Neuronal basal或Fischers等，均为市场上可购得的商品。
作为本发明的优选方式，前述转分化培养基特殊条件下，也可使用无血清培养基配制。所述“无血清培养基”指：不含血清而含有支持细胞增殖和生物反应的多种营养成分(如生长因子、组织提取物等)的细胞培养基。即将除血清以外的各种细胞因子或生长因子等 添加剂，添加到细胞基础培养基中组成的细胞培养基。
作为本发明的优选方式，所述的含有各种细胞因子或生长因子的无血清培养基包括但不限于：ITS、N2、B27等，均为可自行配制或商购产品。
应理解，本领域技术人员熟悉所述的细胞基础培养基或无血清培养基的配制或购买途径，因此，细胞基础培养基或无血清培养基并不限于本发明中所举例的这些。
作为本发明的优选方式，所述的“成纤维细胞转分化肝细胞培养基”具体制备或配制如下实施：
(1)将①GSK3β抑制剂(如CHIR99021)：终浓度为0.1μM-20mM；优选量为：0.5μM-10mM；②G9aHMT抑制剂(如BIX01294)终浓度为0.01-20μM；优选量为：0.05-10μM；混合，即可获得本发明用于化学诱导成纤维细胞直接重编程(转分化)为肝细胞的小分子组合物。
(2)将上述各小分子分别经过：溶解配制浓缩液→稀释到细胞基础培养基中→混合，即可配制获得“成纤维细胞转分化肝细胞培养基”。
本发明还提供了用于化学诱导体内原位成纤维细胞转分化肝细胞的实验动物注射或口服用试剂(以下简称：体内原位成纤维细胞转分化肝细胞试剂)。
作为本发明的优选方式，将前述任一组合物中的各小分子组合物，按公斤体重计算出相应的用药量，将其溶于Captisol(1-30％)，Tween-80(5％)溶液中获得实验动物注射或口服用的原位转分化成纤维细胞试剂。较佳地为Captisol(1-30％)。
制备转分化/转化肝细胞的培养方法
本发明还公开了一种化学小分子组合物，体外化学诱导成纤维细胞直接重编程(转分化)为肝细胞，以制备转化肝细胞的方法，所述方法步骤包括：
(1)浓缩液试剂配制：根据本发明中任一所述的组合物，将各成分溶解于有机溶剂或水性溶剂中配制成浓缩液试剂；较佳地，所述的有机溶剂包括二甲基亚砜；较佳地，所述的水性溶剂包括：水，生理盐水，磷酸盐缓冲液；
(2)培养基获得：将步骤(1)中的浓缩液试剂分别稀释入含5-20％小牛血清的细胞基础培养基中(使得各组分的浓度符合前面所述的组合物中所限定的浓度)，获得诱导成纤维细胞转分化肝细胞的培养基；
其中，该培养基各组分的百分含量还可上下浮动50％；较佳地上下浮动30％；更佳地上下浮动20％，如10％，5％。
(3)诱导成纤维细胞转分化为肝细胞：将成纤维细胞在含5-20％小牛血清的细胞基础培养基中；或无血清培养基中；混悬、铺板，在细胞贴壁后弃原培养基，换步骤(2)的成纤维细胞转分化肝细胞的培养基37℃培养，每2-4天换液一次；3-15天传代一次。
(4)诱导成纤维细胞转分化的传代培养：弃原培养液，PBS洗涤一次，加入细胞消化液消化细胞，37℃，1-5分钟，终止细胞消化，离心，弃上清，将细胞沉淀重悬，按1:1-1:3 传代铺板。按实验步骤第(2)和(3)步骤培养，每2-4天换液一次。所用消化液包括胰酶，EDTA，Acutase，TrypleE等。3-15天传代一次。
(5)诱导成纤维细胞转分化并获得转分化肝细胞：经上述实验步骤(3)、(4)转分化培养和传代培养成纤维细胞2-4周，即可获得转分化肝细胞。可用该肝细胞进行其他科研实验；新药药毒、药效检测评估；为构建生物人工肝，临床细胞移植等提供肝细胞源。成纤维细胞的转分化肝细胞培养如上述培养方法实验步骤。
转分化获得的肝细胞的功能检测：成纤维细胞的转分化肝细胞培养如上述，用培养不同时间获得的转化肝细胞检测其相关功能。
本发明的在不导入或不使用任何外源基因/转录因子/MicroRNA(miRNA)基因，及其RNA、蛋白质或多肽等诱导因子；也不在转分化培养基或转分化试剂中，添加使用任何细胞因子或生长因子；仅用化学小分子组合物体内外化学诱导成纤维细胞直接重编程(转分化)为肝细胞。该小分子组合物，可制备成治疗纤维细胞异常疾病(如肝纤维化/肝硬化)的药物或前体药物或药物组合物；或制备化学诱导成纤维细胞直接重编程为肝细胞的转分化培养基或试剂；或用于化学诱导成纤维细胞直接重编程(转分化)为肝细胞，以制备转化肝细胞，为科研应用、医药应用及临床应用提供肝细胞源。
此外，该方法还可广泛作为防治组织或器官纤维化的防治方法及机理研究的借鉴；以及肝病研究、药理及毒理安全性检测的细胞模型。所获得的转分化肝细胞可继续进行功能检测、临床前期研究等。该方法不仅为研发防治肝纤维化(肝硬化)、肺纤维化及其他器管或组织纤维化疾病新药及其机理研究开辟了一个新途径，还为肝细胞的医药应用、临床应用及科研应用提供新肝细胞源，具有广泛的应用前景；而且丰富了干细胞重编程理论，拓展其应用范围。具有重要的科学意义和极大的应用价值。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如[美]J.S.博尼费斯农等著；章静波，方瑾，王海杰等译，《精编细胞生物学实验指南[Short Protocols in Cell Biology]》，科学出版社2007；中所述的条件；或按照制造厂商所建议的条件。
实施例1、小分子诱导成纤维细胞直接重编程(转分化)为肝细胞的小分子组合物，及其转分化培养基和试剂的配制
设计如下组合物或转分化培养基，可按摩尔浓度或重量浓度进行配制：
1、成纤维细胞转分化肝细胞小分子组合物的配方
(1)成纤维细胞转分化肝细胞组合物1
GSK3β抑制剂CHIR-99021：终浓度3μM；
G9aHMT抑制剂BIX01294：终浓度3μM；
(2)成纤维细胞转分化肝细胞组合物2
GSK3β抑制剂LY2090314：终浓度0.3μM；
G9aHMT抑制剂UNC0642：终浓度2μM；
(3)成纤维细胞转分化肝细胞组合物3
GSK3β抑制剂LiCl：终浓度10μM；
G9aHMT抑制剂BIX01294：终浓度2.5μM；
(4)成纤维细胞转分化肝细胞组合物4
GSK3β抑制剂BIO：终浓度1uM；
G9aHMT抑制剂UNC0638：终浓度3uM；
(5)成纤维细胞转分化肝细胞组合物5
GSK3β抑制剂Ly2090314：终浓度1μM；
G9aHMT抑制剂UNC0638：终浓度5μM；
(6)成纤维细胞转分化肝细胞组合物6
GSK3β抑制剂CHIR-98014：终浓度4μM；
G9aHMT抑制剂BIX01294：终浓度2.5μM；
(7)成纤维细胞转分化肝细胞组合物7
GSK3β抑制剂Ly2090314：终浓度0.4μM；
G9aHMT抑制剂BIX01294：终浓度0.5μM；
(8)成纤维细胞转分化肝细胞组合物8
GSK3β抑制剂LiCl：终浓度20μM；；
G9aHMT抑制剂BIX01294：终浓度5μM；
(9)成纤维细胞转分化肝细胞组合物9
GSK3β抑制剂BIO：终浓度3μM；
G9aHMT抑制剂BIX01294：终浓度2μM；
(10)成纤维细胞转分化肝细胞组合物10
GSK3β抑制剂Ly2090314：终浓度4uM；
G9aHMT抑制剂BIX01294：终浓度2μM；
TGFβ抑制剂SB431542：终浓度10μM；
(11)成纤维细胞转分化肝细胞组合物11
GSK3β抑制剂BIO：终浓度3uM；
G9aHMT抑制剂UNC0642：终浓度2.5μM；
TGFβ抑制剂A83-01：终浓度2uM；
(12)成纤维细胞转分化肝细胞组合物12
GSK3β抑制剂CHIR-99021：终浓度6uM；
G9aHMT抑制剂HKMTI-1-248：终浓度5uM；
TGFβ抑制剂RepSox：终浓度0.5uM；
(13)成纤维细胞转分化肝细胞组合物13
GSK3β抑制剂BIO：终浓度1uM；
EZH2HMT抑制剂EPZ005687：终浓度3uM；
(14)成纤维细胞转分化肝细胞组合物14
GSK3β抑制剂CHIR-98014：终浓度4μM；
EZH2HMT抑制剂UNC1999：终浓度2μM；
(15)成纤维细胞转分化肝细胞组合物15
GSK3β抑制剂CHIR-98014：终浓度20μM；；
EZH2HMT抑制剂GSK343：终浓度6μM；
各具体小分子组合物按前述“培养方法”步骤(1)先溶解于DMSO中制成浓缩液试剂。
2、成纤维细胞转分化肝细胞培养基配制
将上述实验步骤1配制的成纤维细胞转分化肝细胞组合物1～15各成分DMSO浓缩液试剂按前述“培养方法”步骤(2)配制，选用的细胞基础培养基是DMEM，添加有10％小牛血清，获得成纤维细胞转分化肝细胞培养基1～15(即培养基1与组合物1的化合物终浓度相同，培养基2与组合物2的化合物终浓度相同，…，培养基15与组合物15的化合物终浓度相同)。
3、体内原位成纤维细胞转分化肝细胞口服试剂配制
将成纤维细胞转分化肝细胞组合物5、7、10、12的DMSO浓缩液溶于5％Captisol，配制成为体内原位成纤维细胞转分化肝细胞口服试剂(与组合物5、7、10、12的化合物终浓度相同)。
实施例2、成纤维细胞转分化肝细胞培养基1、11化学诱导肝成纤维细胞(肝星状细胞HSC)转分化为肝细胞比较实验
1、成纤维细胞转分化肝细胞培养
将小鼠肝星状细胞(HSC)即肝成纤维细胞在添加入含10％小牛血清的细胞基础培养基DMEM中混悬、铺板，37℃培养。设置对照组和处理组；
待肝星状细胞贴壁后，弃原培养基，处理组分别换成纤维细胞转分化肝细胞培养基1、11，37℃培养，每3天换液1次；3-7天传代一次。
2、肝星状细胞(HSC)转分化肝细胞的传代培养
传代培养步骤：弃原培养液，PBS洗涤一次，加入细胞消化液消化细胞，37℃，3分钟，终止细胞消化，离心，弃上清，将细胞沉淀重悬，按1:2传代铺板。处理组换成纤维细胞转分化肝细胞培养基1、11培养，每3天换液1次。所用消化液是胰酶(也可用EDTA， Acutase，TrypleE)等。3-7天传代一次。
3、转分化肝细胞的收获
经上述实验步骤1-2的成纤维细胞转分化肝细胞培养以及传代培养2-4周，可获得转分化肝细胞，可用于进行进一步的实验。
4、肝成纤维细胞(肝星状细胞HSC)与转分化所得肝细胞(ciHep)形态比较
将对照组肝星状细胞HSC与上述步骤3的处理组转分化所得肝细胞(ciHep)进行形态比较，结果如图1。
图1 A右侧图表示2种小分子组合物的转分化肝细胞培养基1(2F)诱导HSC转分化所得的肝细胞(ciHep)；图1B右侧图表示3种小分子组合的转分化肝细胞培养基11(3F)诱导HSC转分化所得的肝细胞(ciHep)；分别与其图A、B左侧图中的HSC进行形态比较，转化肝细胞形态均发生明显改变，与肝细胞形态吻合，部分转化肝细胞明显出现双细胞核。显示HSC都已转分化为肝细胞(ciHep)。
实验结果显示，仅由2种化学小分子(GSK3β抑制剂和HMT抑制剂)组成的小分子组合物(2F)与之前本发明人发明的仅由3种小分子(GSK3β抑制剂、G9aHMT抑制剂、TGFβ抑制剂)组成的小分子组合物(3F)，在体外诱导成纤维细胞转分化为肝细胞的效果相同或相近。
实施例3、成纤维细胞转分化肝细胞培养基2诱导肝成纤维细胞(肝星状细胞)转分化为肝样细胞的糖原染色
成纤维细胞转分化肝细胞培养基2，化学诱导小鼠肝成纤维细胞(肝星状细胞)转分化为肝细胞的方法步骤同实施例2。
将肝星状细胞(HSC)转分化所得肝样细胞进行糖原染色。染色的深浅展现肝细胞储存糖原的能力。用Schiff方法作肝糖原染色。具体方法为：(1)弃细胞培养液，PBS漂洗1次；(2)4％多聚甲醛固定10分钟后，PBS漂洗5分钟×3次；(3)加入PAS-I液10min，流水冲洗；(4)加入PAS-II液1-2min，流水冲洗；(5)显微镜取照片。
肝星状细胞(HSC)转分化所得肝细胞(ciHep)的糖原染色(PAS)结果如图2；图2左侧图为肝星状细胞(HSC)的PAS结果，右侧图为转分化培养基2所得转分化或转化肝细胞的糖原染色结果。
由图2实验结果显示，图右侧转分化培养所获肝样细胞的肝糖原染色阳性，而图左侧肝星状细胞肝糖原染色阴性。证明本发明的方法获得的转分化肝细胞具有肝细胞特有的糖原储存活性。
实施例4、成纤维细胞转分化肝细胞培养基3诱导肝成纤维细胞(肝星状细胞)转分化为肝样细胞的脂肪油红(Oil-red)染色实验
成纤维细胞转分化肝细胞培养基3，化学诱导成纤维细胞转分化为肝细胞的方法步 骤同实施例2。
使用脂肪油红(Oil-red)染色常规试剂盒，方法步骤见试剂盒。染色结果见图3。
由图3实验结果显示，右侧图显示肝星状细胞(HSC)转分化获得的肝细胞脂肪油红染色阳性，染色的深浅展现肝细胞代谢脂肪的能力；左侧图肝星状细胞(HSC)脂肪油红染色阴性；证明该2小分子组合物诱导肝星状细胞转分化所得肝样细胞具有肝细胞的代谢脂肪功能。
实施例5、化学诱导成纤维细胞(HF)转分化为肝样细胞后表达肝细胞相关基因
成纤维细胞转分化肝细胞培养基4，化学诱导成纤维细胞(HF)转分化为肝细胞的方法步骤同实施例2。分别收集对照组和转分化组的细胞的RNA，做RT-PCR，检测肝细胞相关基因的表达。
结果见图4，成纤维细胞转分化为肝样细胞后高表达肝细胞相关基因，包括ALB、HNF4a、AAT、CYP1A2、CYP3A4。表明成纤维细胞已经被转分化为肝细胞。
实施例6、口服体内原位成纤维细胞转分化肝细胞试剂5，体内化学诱导原位肝成纤维细胞转分化肝细胞效应，治疗肝纤维化小鼠动物模型实验
实验操作步骤：
1、肝纤维化小鼠动物模型制作：雄性4～5周C57/BL6小鼠，用5％CCI4(橄榄油溶剂)腹腔注射，剂量5μL/g体重，3次/周共84天，约12周制作肝纤维化模型。造模第12周取一只小鼠解剖，取肝脏组织固定切片，作HE染色(常规染色)和天狼星红染色(按试剂盒操作说明)，明确肝纤维化模型制作成功。
2、体内原位成纤维细胞转分化肝细胞口服试剂5配制及口服治疗实验：口服给药。将成纤维细胞转分化肝细胞组合物5的DMSO浓缩液溶于5％Captisol，配制成为原位成纤维细胞转分化肝细胞口服试剂(与组合物5的化合物终浓度相同)，1次/天作为处理组(n＝6)，将同等剂量DMSO溶于5％Captisol，口服给药作为对照(n＝6)。共处理34天，约5周，期间CCI4继续使用。实验结束后，收集小鼠血液、肝脏做后续分析。
3、取处理组和对照组小鼠的外周血，进行血常规生化检测其转氨酶。
结果见图5，表明处理组比对照组的外周血中转氨酶(谷丙转氨酶、谷草转氨酶)显著降低，表明该小分子组合物体内原位诱导成纤维细胞转分化为肝细胞效应使肝纤维化病变明显减轻或逆转。
实施例7、口服体内原位成纤维细胞转分化肝细胞试剂7、10，体内化学诱导原位肝纤维化细胞转分化肝细胞及其效应，治疗肝纤维化小鼠动物模型对比实验
实验操作步骤：
1、肝纤维化小鼠动物模型制作：雄性4～5周C57/BL6小鼠，用5％CCI4(橄榄油溶 剂)腹腔注射，剂量5μL/g体重，1次/3天共84天，约12周制作肝纤维化模型。造模第12周取一只小鼠解剖，取肝脏组织固定切片，作HE染色(常规染色)和天狼星红染色(按试剂盒操作说明)，明确肝纤维化模型制作成功。
2、原位成纤维细胞转分化肝细胞口服试剂7、10配制及口服治疗实验：口服给药。将成纤维细胞转分化肝细胞组合物7、10的DMSO浓缩液溶于5％Captisol，配制成为原位成纤维细胞转分化肝细胞口服试剂(与组合物7、10的化合物终浓度相同)，1次/天作为处理组(2n＝2*6)，将同等剂量DMSO溶于5％Captisol，口服给药作为对照(n＝6)。共处理34天，约5周，期间CCI4继续使用。实验结束后，收集小鼠血液、肝脏做后续分析。
3、分别取两处理组和对照组小鼠的肝脏组织固定切片，作天狼星红染色(按试剂盒操作说明)。
结果见图6，图6A原位成纤维细胞转分化肝细胞口服试剂7处理组小鼠与对照组比较，其天狼星红染色显著减少；图6B原位成纤维细胞转分化肝细胞口服试剂10处理组小鼠与对照组比较，其天狼星红染色也显著减少；但前者的降低纤维化程度的效果显著比后者更佳。表明该2个小分子组合物比3个小分子组合物的体内原位成纤维细胞转分化为肝细胞效应，使肝纤维化病变减轻或逆转效果相同或更佳。
实施例8、人肝星状细胞(hepatic stellate cell，HSC)系Lx2与其转分化获得的转分化肝细胞(Lx2-ciHep)的形态比较实验
成纤维细胞转分化肝细胞培养基6，化学诱导肝成纤维细胞系Lx2(肝星状细胞hepatic stellate cell，HSC，Lx2细胞系)转分化为肝细胞的方法步骤同实施例2。
将人肝星状细胞Lx2(对照组)与其转分化(处理组)获得的转化肝细胞(Lx2-ciHep)进行形态比较(实验结果见图7)；图7实验结果显示，该小分子组合物诱导人肝星状细胞转分化所得肝细胞具有肝细胞的特有形态特征。
实施例9、人肝星状细胞(hepatic stellate cell，HSC)系Lx2转分化获得的转化肝细胞(Lx2-ciHep)与人原代肝细胞(PHH)的功能比较实验
转分化肝细胞培养基8，化学诱导肝成纤维细胞系Lx2(肝星状细胞hepatic stellate cell，HSC，Lx2)转分化为肝细胞的方法步骤同实施例2。
将人肝星状细胞Lx2转分化(处理组)获得的转化肝细胞(Lx2-ciHep)进行油红染色(Oil-red)和糖原染色(PAS)，与人原代肝细胞(PHH)进行对照染色比较(步骤同实施例3、4)，实验结果见图8；结果显示Lx2转分化获得的转化肝细胞(Lx2-ciHep)与原代肝细胞(PHH)染色均为阳性，表明转化肝细胞具有肝细胞特有的糖原储存和脂肪代谢功能。
实施例10、人肝星状细胞(hepatic stellate cell，HSC)系Lx2与其转分化获得的转分化肝细胞(Lx2-ciHep)的特异标志物表达比较实验
成纤维细胞转分化肝细胞培养基9，化学诱导肝成纤维细胞系Lx2(肝星状细胞hepatic stellate cell，HSC，Lx2)转分化为肝细胞的方法步骤同实施例2。
将人肝星状细胞Lx2(对照组)与其转分化(处理组)获得的转分化肝细胞(Lx2-ciHep)消化，离心后固定，用人肝细胞特异性抗体作免疫染色，然后对样本作流式细胞分析比较，实验结果见图9。结果显示，转分化的肝细胞(Lx2-ciHep)表达人肝细胞的特异性标志物，而对照组则没有表达。
综上实验结果显示，人肝星状细胞Lx2转分化(处理组)获得的转分化肝细胞(Lx2-ciHep)与对照组的形态、功能(糖原染色、油红染色)，以及人肝细胞特异性标志物比较。结果显示，人肝星状细胞Lx2转分化为肝样细胞后，具有人肝细胞特有的形态、功能以及其特异性标志物特征。
实施例11、转分化肝细胞培养基13、14、15诱导成纤维细胞转分化获得肝样细胞表达肝细胞相关基因
转分化肝细胞培养基13、14、15诱导皮肤成纤维细胞转分化肝样细胞的方法步骤同实施例2。分别收集对照组(Control)和转分化肝细胞培养基13、14、15转分化处理组(依次标注为T1、T2、T3)转分化所得的肝样细胞的RNA，做RT-PCR，检测肝细胞相关基因的表达。
实验结果见图10，结果显示：转分化肝细胞培养基13、14、15诱导成纤维细胞转分化获得的肝样细胞，与对照组相比较，处理组T1、T2、T3高表达肝细胞相关基因，包括ALB、AAT、ASGPR1、7A1。实验结果表明，本发明的2小分子组合物诱导成纤维细胞转分化的肝样细胞，高表达肝细胞相关基因。
实施例12、体内原位成纤维细胞转分化肝细胞口服试剂12体内原位诱导小鼠肝星状细胞(hepatic stellate cell，HSC)转分化为肝细胞示踪比较实验
肝纤维化疾病动物模型实验操作步骤同实施例6。采用携带标记肝星状细胞示踪信号的转基因小鼠(mTmG/PDGFF1)制作肝纤维化小鼠动物模型。对照组与处理组的处理条件唯一区别是：处理组口服体内原位成纤维细胞转分化肝细胞口服试剂12含有小分子组合物12，而对照组的口服试剂没有。实验结果见图11。
图11上图对照组，左侧图显示转基因小鼠的肝细胞显示红色荧光，形态没有改变；中间图显示转基因小鼠的原位肝星状细胞(HSC)或称肝成纤维细胞，显示绿色荧光，形态没有改变；右侧图显示上述两种细胞组织的荧光合成图；
图11下图处理组，左侧图为转分化处理后的肝细胞组织仍然显示红色，形态不变；中间图显示肝星状细胞转分化后，形态已转变为肝细胞形态，但仍然显示肝星状细胞原有 绿色荧光标记；右侧图显示肝星状细胞转分化为转化肝细胞与原有肝细胞组织整合为一体，且转化肝细胞仍然显示原位HSC示踪标记的绿色荧光。实验结果充分显示本发明的小分子组合物可体内原位诱导HSC转分化为肝细胞。
实施例13化学小分子组合物的各单独小分子组成成分[GSK3β抑制剂/HMT抑制剂(G9aHMT抑制剂/EZH2HMT抑制剂)/TGFβ抑制剂]诱导细胞转化比较试验
(1)GSK3β抑制剂CHIR-99021，HMT抑制剂(G9aHMT抑制剂BIX01294、EZH2HMT抑制剂EPZ005687)、TGFβ抑制剂SB431542，各单独小分子成分培养基的配制：将GSK3β抑制剂CHIR-99021、HMT抑制剂(G9aHMT抑制剂BIX01294、EZH2HMT抑制剂EPZ005687)、TGFβ抑制剂SB431542，各单独小分子组成成分按照前述实验步骤1配制成为DMSO浓缩液试剂；按前述“培养方法”步骤(2)转分化肝细胞培养基的配制，选用的细胞基础培养基是DMEM，添加有10％小牛血清，获得单独小分子成分培养基
(2)诱导成纤维细胞转分化为肝细胞的培养方法步骤同实施例2；所使用细胞为人成纤维细胞(HF)。
(3)将各单独小分子成分培养基诱导人成纤维细胞转分化培养处理组(Treat)所获得的细胞形态与对照组(Control)的人成纤维细胞(HF)进行形态比较(对照组与处理组培养条件唯一差别是不添加化学小分子成分)。实验结果见图12。
图12显示，图左侧对照组与图右侧各小分子处理组相比较，各单独小分子成分对人成纤维细胞没有诱导转分化肝细胞作用，成纤维细胞被其诱导转分化前后，形态基本没有变化，更与肝细胞形态没有任何相似之处。
实验结果表明，化学小分子组合物的各单独组分：GSK3β抑制剂、HMT抑制剂(G9aHMT抑制剂/EZH2HMT抑制剂)、TGFβ抑制剂单独存在时，不具有诱导成纤维细胞转分化为肝细胞的功能。
综上，本发明的“体内外化学诱导成纤维细胞直接重编程(转分化)为肝细胞的化学小分子组合物及方法具有以下有益效果在于：
1、本发明方法是不导入、不使用任何外源基因/外源转录因子/MicroRNA(miRNA)基因，及其RNA、蛋白质或多肽等诱导因子；也不使用任何细胞因子或生长因子；最少仅使用由2种化学小分子组成的诱导组合物，体内外诱导成纤维细胞直接重编程(转分化)为肝细胞。操作方法简单，转化肝细胞成本低，质量好，更安全；
2、该化学小分子组合物具有多种用途：(1)作为体内原位诱导成纤维细胞转分化为肝细胞的药物组合物，可研发或制备成治疗人成纤维细胞异常相关疾病(如肝纤维化/肝硬化)的药物/前体药物/药物组合物；(2)用于制备化学诱导成纤维细胞转分化为肝细胞的转化培养基或试剂；(3)用于体外化学诱导成纤维细胞转分化为肝细胞，以制备转化肝细胞，为科研应用、医药应用及临床应用提供肝细胞源。
3、体外转分化获得的肝细胞具有正常成熟的人肝细胞形态和功能；具有白蛋白生成，尿素合成，糖原储存、脂肪代谢、P450酶活性诱导及其他人肝细胞特有功能和人肝细胞特异性标志物；
4、本发明最少仅使用2种化学小分子组成的组合物，具有组合结构更简单，成药性更好；产生副作用的风险更低，开发成本更低，更容易开发为相关疾病药物。且体内原位诱导成纤维细胞转分化肝细胞效应，减轻或改善肝纤维化/肝硬化效果更佳。
5、本转分化肝细胞方法可取材于患者自身，转化肝细胞具有个性化特征，并有两大优势：一是易于进入临床应用，并最大限度降低或避免转化/转分化肝细胞移植引起的免疫排斥反应风险；二是可构建具群体代表性的肝细胞库，应用于新药的肝毒安全评测和药效筛选；因此可为临床应用、医药应用及科研应用提供肝细胞源。
6、该方法采取小分子化学诱导细胞直接重编程，不需要经过诱导多潜能干细胞重编程为iPSC阶段，避免致癌风险；方便临床应用。
7、该转分化方法普适性和可重复性好；方法简单，易于操作，成本较低；有利于推广应用。
8、常规培养、周期短、适于批量生产和易于产业化等特点。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (12)

  1. 一种用于化学诱导体内外成纤维细胞直接重编程(转分化)为肝细胞的化学小分子组合物，其特征在于，所述的组合物包括化学小分子抑制剂：GSK3β抑制剂和HMT抑制剂；或，所述的组合物由化学小分子抑制剂：GSK3β抑制剂和HMT抑制剂组成；
    其中，所述成纤维细胞中不导入任何外源基因/转录因子/MicroRNA(miRNA)基因，及其RNA、蛋白质或多肽等诱导因子；或，所述的化学小分子组合物中不含有任何外源基因/转录因子/MicroRNA(miRNA)基因，或其转录的RNA，或其翻译的蛋白质及多肽等诱导因子。
  2. 如权利要求1所述的小分子组合物，其特征在于，所述化学小分子HMT抑制剂包括(但不限于)：G9aHMT抑制剂、EZH2HMT抑制剂；或所述化学小分子组合物包括：化学小分子GSK3β抑制剂和HMT抑制剂(G9aHMT抑制剂/EZH2HMT抑制剂)；或仅由GSK3β抑制剂和G9aHMT抑制剂/EZH2HMT抑制剂组成。
  3. 如权利要求1所述的化学小分子组合物，其特征在于，所述的小分子组合物还可添加包括化学小分子抑制剂：TGFβ抑制剂；或，所述的小分子组合物还可由化学小分子抑制剂：GSK3β抑制剂、HMT抑制剂(G9aHMT抑制剂/EZH2HMT抑制剂)和TGFβ抑制剂组成。
  4. 如权利要求1-3所述的化学小分子组合物，其特征在于，所述的化学小分子组合物包括：由化学小分子抑制剂：GSK3β抑制剂和G9aHMT抑制剂/EZH2HMT抑制剂组成的化学小分子诱导组合物；或，
    所述的化学小分子组合物包括：由化学小分子抑制剂：GSK3β抑制剂、G9aHMT抑制剂/EZH2HMT抑制剂、TGFβ抑制剂组成的化学小分子诱导组合物。
  5. 如权利要求1或3所述的化学小分子组合物，其特征在于，按照重量份比，所述的小分子抑制剂：GSK3β抑制剂和HMT抑制剂为(5-80)﹕(0.1-50)；或溶液状态下摩尔浓度比为(0.1-20)﹕(0.01-20)；或
    按照重量份比，所述的GSK3β抑制剂、HMT抑制剂和TGFβ抑制剂为(5-80)﹕(0.1-50)﹕(0.1-50)；或溶液状态下摩尔浓度比为(0.1-20)﹕(0.01-20)﹕(0.01-20)。
  6. 如权利要求1-3任一所述的化学小分子组合物，其特征在于，所述化学小分子抑制剂：GSK3β抑制剂是能够靶向抑制GSK3β信号通路的抑制剂，包括(但不限于)：CHIR-99021、LiCl、BIO、Ly2090314、IM-12、TWS119、1-Azakenpaullone、CHIR-98014、 Tideglusib、AR-A014418、SB216763、AZD1080，以及诱导抑制GSK3β信号通路的其他GSK3β小分子抑制剂，或与它们等效的药剂制品、类似物、异构体和/或其盐、水合物或前体，或其组合；较佳地为GSK3β抑制剂CHIR-99021、LiCl、BIO、LY2090314；
    所述化学小分子抑制剂：HMT抑制剂，包括(但不限于)：G9aHMT抑制剂、EZH2HMT抑制剂，以及抑制HMT的其他HMT小分子抑制剂，或与它们等效的药剂制品、类似物、异构体和/或其盐、水合物或前体，或其组合；较佳地为G9aHMT抑制剂；或
    所述的G9aHMT抑制剂，是能够靶向抑制G9aHMT的抑制剂，包括但不限于：BIX01294、UNC0638、A-366、UNC0631、BRD4770、UNC0224、UNC0646、UNC0642、UNC0321、BRD4770、HKMTI-1-247、HKMTI-1-248、CPUY074020、DCG066，以及抑制G9aHMT的其他G9aHMT抑制剂或小分子化合物，或与它们等效的药剂制品、类似物、异构体和/或其盐、水合物或前体，或其组合；较佳地为G9aHMT抑制剂BIX01294、UNC0638、UNC0642。或
    所述的EZH2HMT抑制剂是能够靶向抑制EZH2HMT，包括但不限于：EPZ005687、GSK343、Tazemetostat(EPZ-6438)、UNC1999、JQ-EZ-05(JQEZ5)、EBI-2511、CPI-1205、EPZ011989、PF-06726304、EI1、GSK503、GSK126、CPI-360、CPI-169，以及抑制EZH2HKMT的其他EZH2HKMT抑制剂或小分子化合物，或与它们等效的药剂制品、类似物、异构体和/或其盐、水合物或前体，或其组合；较佳地为EPZ005687、GSK343、UNC1999；
    所述化学小分子抑制剂：TGFβ抑制剂是能够靶向抑制TGFβ信号通路的抑制剂，包括(但不限于)：SB431542、A83-01、SB525334、LY2109761，RepSox、SD-208、GW788388、SB505124、EW-7197，Galunisertib，以及诱导抑制TGFβ信号通路的其他TGFβ小分子抑制剂，或与它们等效的药剂制品、类似物、异构体和/或其盐、水合物或前体，或其组合；较佳地为TGFβ抑制剂SB431542、A83-01、RepSox。
  7. 如权利要求1-3任一所述的化学小分子组合物，其特征在于，所述化学小分子组合物是药物组合物，还包括：药学上可接受的载体或赋形剂；较佳地，所述载体或赋形剂包括选自下组的一种或多种：水、盐水、磷酸缓冲液或其它水性溶剂；DMSO、甘油和乙醇或其它有机溶剂；微球、脂质体、微乳液或高分子表面活性剂；胶体型载药系统或高分子载药系统；防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、pH缓冲物质，黏合剂、填充剂、润滑剂或其它药物赋形剂；或
    所述的化学小分子组合物可制备的药物剂型包括(但不限于)：固体剂型，包括(但不限于)粉剂、散剂、片剂、丸剂、胶囊剂、缓释剂、控速释剂，或其他固体剂型；液体剂型，包括(但不限于)注射剂、输液剂、混悬剂，或其它液体剂型；气体剂型；或半固体剂型。
  8. 权利要求1-7任一所述的化学小分子组合物的用途，用于制备化学诱导体内外成 纤维细胞直接重编程或转分化为肝细胞的制剂、试剂或培养基；或用于制备体内化学诱导原位成纤维细胞转分化为肝细胞，以其原位转分化肝细胞效应减少或降低细胞组织/器官的纤维化，达到治疗组织或器官纤维化疾病(如肝硬化/肝纤维化)的药物/药物前体/药物组合物；或
    用于制备化学诱导体外成纤维细胞转分化的肝细胞。
  9. 一种化学诱导体内外成纤维细胞直接重编程(转分化)为肝细胞的方法，包括：应用权利要求1-7任一所述的化学小分子组合物体内外诱导成纤维细胞转分化为肝细胞。
  10. 如权利要求9所述的方法，其特征在于，所述方法是不导入或不使用外源基因/转录因子/MicroRNA及其RNA、蛋白质或多肽等诱导因子；也不使用细胞因子或生长因子；仅用化学小分子组合物的方法；或
    所述方法为制备体内原位诱导成纤维细胞直接重编程(转分化)为肝细胞的药物组合物/药物前体/药物的方法；或为制备体内外诱导成纤维细胞直接重编程为肝细胞的小分子组合物的方法；或为制备诱导成纤维细胞直接重编程为肝细胞的培养基或试剂的方法；或为以小分子诱导成纤维细胞直接重编程为肝细胞，以制备转化肝细胞的方法。
  11. 一种用于化学诱导体内外成纤维细胞直接重编程(转分化)为肝细胞的药盒或试剂盒，其特征在于，其中包括权利要求1-7任一所述的化学小分子组合物；或基于该小分子组合物添加药学上可接受的载体或赋形剂，用于体内化学诱导原位成纤维细胞直接重编程(转分化)为肝细胞，以其原位转分化效应减少或降低组织/器官的，而研发或制备成治疗临床纤维化疾病(如肝硬化/肝纤维化)的药物或前体药物/药物组合物；或添加有机溶剂/生理盐水/缓冲液/细胞基础培养基等载体或赋形剂制备的诱导成纤维细胞直接重编程(转分化)为肝细胞的转分化培养基或试剂。
  12. 如权利要求1-7所述的任一小分子组合物、权利要求8-10任一所述的用途或方法、权利要求11所述的药盒或试剂盒，其特征在于，所述的成纤维细胞包括但不限于：人成纤维细胞或哺乳动物成纤维细胞；较佳地，包括但不限于人的：皮肤成纤维细胞、肝成纤维细胞(肝星状细胞)、肺成纤维细胞、肾成纤维细胞、胰腺成纤维细胞或人体其他组织或器官的成纤维细胞；更佳地为人皮肤成纤维细胞或肝成纤维细胞(肝星状细胞)。
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