WO2019144968A1 - 一种细胞诱导的方法 - Google Patents

一种细胞诱导的方法 Download PDF

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WO2019144968A1
WO2019144968A1 PCT/CN2019/073624 CN2019073624W WO2019144968A1 WO 2019144968 A1 WO2019144968 A1 WO 2019144968A1 CN 2019073624 W CN2019073624 W CN 2019073624W WO 2019144968 A1 WO2019144968 A1 WO 2019144968A1
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medium
inducing
hepatocytes
blebbistatin
fibroblasts
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PCT/CN2019/073624
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English (en)
French (fr)
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周琪
李伟
何正泉
王柳
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中国科学院动物研究所
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Priority claimed from CN201810083174.7A external-priority patent/CN110093309B/zh
Priority claimed from CN201810083568.2A external-priority patent/CN110093305B/zh
Priority claimed from CN201810083591.1A external-priority patent/CN110093310B/zh
Application filed by 中国科学院动物研究所 filed Critical 中国科学院动物研究所
Priority to JP2020562821A priority Critical patent/JP7473209B2/ja
Priority to CN201980005643.6A priority patent/CN111344392B/zh
Priority to EP19744381.5A priority patent/EP3747991A4/en
Priority to US16/965,395 priority patent/US20210230550A1/en
Publication of WO2019144968A1 publication Critical patent/WO2019144968A1/zh
Priority to JP2022117552A priority patent/JP7407469B2/ja

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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
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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
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    • C12N2510/00—Genetically modified cells
    • C12N2510/04—Immortalised cells

Definitions

  • the present invention relates to the field of biotechnology, and in particular to the field of cell induction technology, and more particularly to a method for inducing transdifferentiation of fibroblasts into adipocytes and an application thereof, a method for converting fibroblasts into immortalized cells, and an application thereof, And a method of inducing expansion of liver cells in vitro.
  • Transdifferentiation also known as differentiation and metastasis refers to the phenomenon of one type of differentiated cells transforming into another type of differentiated cells.
  • Different types of cells have been transdifferentiated, for example, transgenic embryonic fibroblasts, chondrocytes and retinal epithelial cells into myocytes, transgenic B-lymphocytes into macrophages, and fibroblasted mice.
  • the cells are transdifferentiated into functional nerve cells and the like.
  • Adipocytes are abundant in adults. Their tissues are also called adipose tissue. They are often white. They proliferate in a large number in early childhood and reach a peak in puberty. After that, the number generally does not increase. In normal animals and humans, adipose tissue mainly exists in the subcutaneous cavity and the subcutaneous part of the abdomen, but fat tissue in various parts of the body of obese patients, such as the kidney, mesentery, subcutaneous, abdominal cavity, etc., indicating that Cells that differentiate into adipocytes not only have fat precursor cells, but under certain conditions, some non-fat precursor cells can also differentiate into fat cells, which subverts the traditional notion that fat cells are differentiated from fat precursor cells. By transducing non-fatty precursor cells into adipocytes, the transformation mechanism of adipocytes can be more clearly understood, which in turn guides the development of anti-obesity drugs.
  • Fibroblast is the main cellular component of loose connective tissue, which is differentiated from embryonic mesenchymal cells. Transdifferentiation of fibroblasts into adipocytes has important applications in many fields. For example, transdifferentiating the most common types of myofibroblasts found in scar wounds into adipocytes may achieve scarring in the future, and in wrinkled skin. In the regeneration of fat cells, it is possible to derive a new anti-aging treatment strategy. Thus, it is important to realize the transdifferentiation of non-fat precursor cells, especially fibroblasts, into adipocytes.
  • CN104342401B discloses a method for promoting the transdifferentiation of fibroblasts into adipocytes using a defined combination of cytokines, the onset factors of which are epidermal growth factor, hepatocyte growth factor, dexamethasone, insulin and PPAR gamma agonists.
  • CN104372024A discloses a method for inducing transdifferentiation of bovine fibroblast/myoblast into adipocytes, comprising cloning a bovine transcription factor CCAAT enhancer binding protein C/EBP ⁇ gene, constructing a C/EBP ⁇ gene overexpression vector and packaging to obtain a recombinant gland.
  • Virus, adenovirus infects bovine fibroblasts and myoblasts to achieve rapid transdifferentiation of these cells into adipocytes.
  • the above method uses a large number of macromolecular substances or reprogramming processes, and the efficiency is low, and the safety also has a certain influence.
  • CN105754935A discloses an induction medium for inducing transdifferentiation of fibroblasts into adipocytes, the medium comprising a basal medium and an inducing small molecule combination, the induction small molecule combination being SG or 6TF, wherein S is SB431542 G is GSK126, 6 is E61541, T is phenylcyclopropylamine, and F is forskolin.
  • S is SB431542 G is GSK126
  • 6 is E61541
  • T is phenylcyclopropylamine
  • F is forskolin.
  • Normal tissue-derived somatic cells can grow and divide under normal in vitro culture conditions, but after a limited number of cell passages, proliferation, senescence and death cease, which limits the application of cell culture techniques.
  • Cell immortalization means that cells in the process of in vitro culture escape from the crisis of proliferative aging due to their own genetic changes or various external stimuli, avoiding the aging death process of normal cells, and can be subcultured for a long time. Split and multiply.
  • Targeted development of immortalization technology for specific cells can achieve long-term passage, infinite division and proliferation of normal somatic cells with fewer passages and slower proliferation and division, and increase cell cycle life. This not only helps to understand the law of cell growth, explores the causes of cell aging, but also has important clinical significance for solving organ transplantation problems.
  • cell immortalization is the premise of transformation into tumor cells, and it is a necessary stage for the transformation of normal cells into tumor cells. Studying the immortalization of cells can lay a solid foundation for treating tumors and controlling the proliferation of tumor cells.
  • the liver is the largest internal organ in the human body and is the main site of metabolism. Hepatocytes account for 85% of the liver. Hepatocytes have a strong regenerative potential in the body, and the normal liver can be resected to two centuries and then restored to its original volume by cell proliferation in just one week.
  • human liver can be rapidly regenerated in vivo, primary hepatocytes can only proliferate in a short period of time and cannot proliferate in vitro. So far, efforts to expand human liver cells in the laboratory have led to metabolic functions. Low immortalized cancer cells. The scarcity of human hepatocytes and the loss of function during hepatocyte expansion are the main bottlenecks in the development of science, medicine and pharmacy. Solving this problem will help promote the in vitro drug metabolism of liver cells, drug toxicity, end-stage liver disease cell therapy, and construction of organisms. Artificial liver to support research and applications such as patients waiting for transplantation, disease model construction.
  • the inventors have continually explored and unexpectedly found that the above object can be achieved by the Myosin inhibitor (-)-Blebbistatin or (S)-(-)-Blebbistatin O-Benzoate.
  • the invention relates to the use of a Myosin inhibitor for inducing cell transdifferentiation.
  • the transdifferentiation is to induce fibroblast transdifferentiation.
  • the transdifferentiation is to induce transdifferentiation of fibroblasts into adipocytes.
  • the Myosin inhibitor is (-)-Blebbistatin, abbreviated Ble or Bleb or Blebb.
  • the Myosin inhibitor is (-)-Blebbistatin O-Benzoate, abbreviated as S-Bleb-OB.
  • (-)-Blebbistatin (also denoted as (S)-(-)-Blebbistatin or S-Bleb) used in the present invention is a cell permeable inhibitor acting on non-myosin II ATPase, which does not inhibit muscle Globulin light chain kinase inhibits the contraction of cleavage furrows and does not interfere with the assembly of mitosis or contractile rings. Its structural formula is as shown in formula (I), and its molecular weight is 292.33.
  • (-)-Blebbistatin O-Benzoate also denoted as (S)-(-)-Blebbistatin O-Benzoate or S-Bleb-OB
  • S-Bleb-OB S-Bleb-OB
  • the present invention also relates to a method for inducing transdifferentiation of fibroblasts into adipocytes, comprising the steps of: culturing fibroblasts in a culture medium, adding a Myosin inhibitor and BMP4 to the culture medium, and continuing the culture until an adipocyte is obtained.
  • the medium includes any one or more of a basal medium, fetal calf serum, and a fat-inducing broth.
  • the base medium comprises high glucose DMEM, fetal calf serum and a double antibody.
  • the fat-inducing medium comprises N2B27 medium (DMEM/F12, a 1:1 mixture of Neurobasal), N2 additive, B27 additive, 2% bovine serum albumin, ⁇ -mercaptoethanol, GlutaMAX, insulin and double antibody , as well as serum substitutes.
  • N2B27 medium DMEM/F12, a 1:1 mixture of Neurobasal
  • N2 additive N2 additive
  • B27 additive 2% bovine serum albumin
  • ⁇ -mercaptoethanol ⁇ -mercaptoethanol
  • GlutaMAX insulin and double antibody
  • insulin insulin and double antibody
  • the invention also relates to an adipocyte obtained by the method described, or a reagent or research tool or diagnostic tool comprising the adipocyte.
  • the method of the invention only needs a single small molecule or single factor treatment, has simple operation, good repeatability, and can be efficiently performed in vitro and in vivo, does not involve transgenic operation, and has good safety of the obtained fat cells, and is suitable for tissue regeneration, Repair and other fields and applications in the industry.
  • the invention also relates to the use of a Myosin inhibitor for inducing cell immortalization.
  • the immortalization is to induce the conversion of fibroblasts into immortalized cells.
  • the Myosin inhibitor is (-)-Blebbistatin or (S)-(-)-Blebbistatin O-Benzoate.
  • the present invention also relates to a method for inducing transformation of fibroblasts into immortalized cells, comprising the steps of: culturing fibroblasts in a medium, adding a Myosin inhibitor to the medium, and continuing the cultivation until an eternal life is obtained Cells.
  • the medium includes any one or more of a basal medium, fetal calf serum, and an immortalized induction medium.
  • the present invention also relates to a medium for inducing transformation of fibroblasts into immortalized cells, which comprises a Myosin inhibitor, a basal medium, fetal calf serum and an immortalized induction medium.
  • the basal medium comprises high glucose DMEM and a double antibody
  • the immortalized induction medium comprises N2B27 broth: (DMEM/F12, a 1:1 mixture of Neurobasal), N2 additive, B27 additive 2% bovine serum albumin, ⁇ -mercaptoethanol, GlutaMAX, insulin and double antibody.
  • the immortalized induction culture solution further comprises one or more of KOSR, CHIR99021 and A83-01.
  • the invention also relates to immortalized cells obtained by the methods described, or reagents or research tools or diagnostic tools comprising the immortalized cells.
  • the invention also relates to the use of a gene in the preparation of a preparation for inducing proliferation or aging, characterized in that the gene comprises Sox2, Srrt, Yap, ⁇ -catenin, Mki67, Pcna, P19, P16ink4a, P15ink4b, Morf4l1, Elf5 One or several.
  • the proliferation-related gene comprises one or more of Sox2, Srrt, Yap, ⁇ -catenin, Mki67, and Pcna.
  • the aging-related gene comprises one or more of P19, P16ink4a, P15ink4b, Morf41, Elf5.
  • the above method of the invention only needs a single small molecule or single factor treatment, has simple operation, good repeatability, and can be efficiently performed in vitro and in vivo, does not involve transgenic operation, and the obtained immortalized cells have good safety and is suitable for understanding cell growth.
  • Regularity, exploring the causes of cell aging, and solving the problem of organ transplantation can also lay a foundation for the treatment of tumors and control the proliferation of tumor cells, which is of great significance.
  • the present invention also finds proliferation-related genes and aging-related genes, and provides a novel alternative for inducing cell proliferation and senescence.
  • the inventors have continually explored and unexpectedly found that the Myosin inhibitor can significantly promote long-term expansion of hepatocytes, thereby achieving the above object.
  • the present invention generally relates to the use of a Myosin inhibitor for inducing in vitro expansion of hepatocytes.
  • the invention also relates to the use of a Myosin inhibitor in the construction of a bioartificial liver.
  • the invention also relates to the use of a Myosin inhibitor in the construction of a liver disease model.
  • the Myosin inhibitor is (-)-Blebbistatin or (S)-(-)-Blebbistatin O-Benzoate.
  • the invention also relates to a method for inducing in vitro expansion of hepatocytes, which comprises the steps of: culturing hepatocytes in a medium, adding a Myosin inhibitor to the medium, continuing the culture, thereby inducing expansion of the hepatocytes in vitro increase.
  • the Myosin inhibitor is (-)-Blebbistatin or (S)-(-)-Blebbistatin O-Benzoate.
  • the invention also relates to hepatocytes obtained by the method.
  • the invention also relates to a reagent, bioartificial liver, research tool or diagnostic tool comprising the hepatocyte.
  • the present invention also relates to a liver disease model using hepatocytes obtained by in vitro expansion of the induced hepatocytes or obtained by the method.
  • the invention also relates to the use of hepatocytes obtained by the method in the construction of a bioartificial liver.
  • the invention also relates to the use of hepatocytes obtained by the method in constructing a liver disease model.
  • the above applications may be therapeutic or non-therapeutic.
  • the method of the invention requires only a single small molecule or single factor treatment, is simple to operate, has good repeatability, and can be efficiently performed in vitro, does not involve transcription factor reprogramming technology and complex transgenic operation, can be amplified for a long time, and is small molecule expansion. Increased hepatocytes are still functional.
  • FIG. 1A is an experimental flow of the first embodiment.
  • Figure 1B shows the fat cell oil red O staining induced by human foreskin fibroblasts.
  • Figure 1C and Figure 1D show the morphology of fat cells obtained by mouse embryonic fibroblasts and the ratio of oil red O staining and lipid droplet area, respectively.
  • Figure 1E and Figure 1F show oil red O staining and lipid droplet area statistics, respectively, after addition of different substances in the original fat-inducing medium.
  • Figures 2A and 2B show in vivo results of small molecule treatments showing significant increase in ventral fat content. Ble in the figure is (-)-Blebbistatin.
  • FIG. 3A is an experimental flow of the second embodiment.
  • Fig. 3B shows the results of observation by fluorescence microscopy.
  • Figures 3C and 3D show the plotted cell growth curves and calculated cell cycles.
  • Figures 3E and 3F show the results of cell cycle composition analysis.
  • Figures 3G and 3H show the results of detection of proliferation and senescence genes.
  • Figure 4A shows that cells were resuspended in mouse hepatocyte culture medium (5 ⁇ M Myosin II inhibitor (-)-Blebbistatin), inoculated on a dish with fibronectin, and one week later, green hepatocyte clusters with Alb expression were observed. .
  • Figure 4B shows the results of long-term expansion of AlB-GFP positive hepatocytes.
  • Figures 5A and 5B show the results of amplification after 12 days of passage (3 generations in total).
  • Figure 5C shows the results of albumin and alpha-fetoprotein gene expression after 12 days of passage (3 generations in total).
  • Figure 6A shows that after two days of continued culture, the small molecule group of cells was further expanded, while the control group showed no significant change.
  • Fig. 6B shows the number of cells of the small molecule group and the control group after 6 days of culture.
  • Fig. 6C shows the expression results of the proliferating cell nuclear antigen gene (PCNA) in the small molecule group and the control group.
  • PCNA proliferating cell nuclear antigen gene
  • Figure 6D shows the results of small molecule amplified adult hepatocytes expressing human hepatocyte specific gene albumin (ALBUMIN), alpha fetoprotein (AFP), CYP1A2 and CYP3A4.
  • ABUMIN human hepatocyte specific gene albumin
  • AFP alpha fetoprotein
  • CYP1A2 CYP3A4.
  • Figure 6E shows the results of subculture of human hepatocytes with 10 ⁇ m small molecule medium and control medium.
  • Figure 6F shows the expression of the CYP1A2 gene after induction of omeprazole by subcultured adult hepatocytes.
  • substantially free with respect to a particular component is used herein to mean that a particular component has not been purposefully formulated into the composition and/or is present only as a contaminant or in trace amounts.
  • the total amount of the particular component resulting from any accidental contamination of the composition is less than 0.05%, preferably less than 0.01%.
  • Most preferred are compositions in which the amount of a particular component is not detectable by standard analytical methods.
  • the term "about” is used to indicate that the value includes an inherent change in the error of the device that is used to determine the value or change that exists between subjects.
  • differential is the process by which less specialized cells become more specialized cell types.
  • Dedifferentiation is a cellular process in which a partially or terminally differentiated cell returns to an earlier stage of development, such as pluripotency or pluripotency.
  • Transdifferentiation is the process of converting one differentiated cell type to another. Typically, transdifferentiation occurs by programming without the cells undergoing an intermediate pluripotency stage - i.e., the cells are programmed directly from one differentiated cell type to another differentiated cell type.
  • the term "subject” or “subject in need” refers to a mammal, preferably a human, of a male or female of any age that requires cell or tissue transplantation.
  • a subject requires cell or tissue transplantation (also referred to herein as a receptor) due to a condition or pathology or undesired condition, condition or syndrome or body, morphology suitable for treatment via cell or tissue transplantation. Or physiological abnormalities.
  • BMP4 bone morphogenetic protein 4 (bmp4).
  • High-sugar DMEM a high-glycemic DMEM medium (dMEM), a commercial medium containing various glucoses and amino acids, was developed on the basis of MEM medium.
  • dMEM high-glycemic DMEM medium
  • N2B27 A well-defined cell culture medium containing DMEM/F12 basal medium and neurobasal basal medium mixed in a ratio of 1:1, containing N2 additive and B27 additive, reported to facilitate the differentiation of mouse embryonic stem cells into the nerve direction.
  • DMEM/F12 A commercial base medium mixed with DMEM medium and F12 medium 1:1, suitable for cloning density culture.
  • Neurobasal A commercial basal medium that facilitates the culture of nerve cells.
  • GlutaMAX A cell culture additive that directly replaces L-glutamine in cell culture media.
  • Penicillin and streptomycin are two commonly used antibiotics in cell culture to prevent bacterial contamination during cell culture.
  • N2 Additive A commercial serum-free cell culture additive.
  • B27 Additive A commercial serum-free cell culture additive.
  • KOSR Knockout serum replacement
  • CHIR99021 A GSK-3 ⁇ / ⁇ inhibitor commonly used as an activator of the Wnt signaling pathway.
  • A83-01 A selective TGF- ⁇ inhibitor that significantly inhibits the activity of ALK4, ALK5 and ALK7.
  • each well was prepared with 20 ⁇ g/ml Matrigel solution (BD, 354277) 1 ⁇ DMEM, coated for 12 hours, and the coating solution was removed and washed with 1 ⁇ PBS.
  • Matrigel solution BD, 354277
  • Mouse embryonic fibroblasts (C57, prepared by E13.5) or adult foreskin fibroblasts (HFF20y, Beijing stem cell bank) were uniformly seeded in each well, 1 ⁇ 10 4 cells per well, using basal medium (high) Sugar DMEM (Gibco, C12430500BT), double antibody) was incubated with 10% fetal bovine serum (Gibco, 16000-044) for 24 hours. The culture solution was removed and washed with PBS.
  • the fibroblasts treated as described above were added to a fat-inducing culture medium: (N2B27 medium: DMEM/F12 (Gibco, 10565018) mixed with Neurobasal (Gibco, 21103-049) 1:1, and added with N2 additive (100 ⁇ , Gibco) , 17502084), B27 additive (50 ⁇ , Gibco, 17504044), 2% bovine serum albumin (1000 ⁇ , sigma, A8022), ⁇ -mercaptoethanol (1000 ⁇ , Gibco, 21985023), GlutaMAX (200 ⁇ , Gibco, 35050-061), 1 ⁇ g/ml insulin (Roche, 11376497001), double antibody), 10% serum replacement (Gibco, 10828-028) added Myosin inhibitor (dimethyl sulfoxide solution (sigma, D2650) 100 mM thick Storage, -20 degrees Celsius in the dark for 1 month) and 10 ng / mL BMP4 (Peprotech, 10828-028),
  • the small molecule treatment group can significantly increase the content of ventral fat, as shown in Figures 2A and 2B.
  • (S)-(-)-Blebbistatin O-Benzoate substitution (-)-Blebbistatin treatment also increased ventral fat content, and the experimental results showed that the ventral fat content increased by about 50% without optimization.
  • each well was prepared with 20 ⁇ g/mL Matrigel solution (BD, 354277) 1 ⁇ DMEM, coated for 12 hours, and the coating solution was removed and washed with 1 ⁇ PBS.
  • Matrigel solution BD, 354277
  • Mouse embryonic fibroblasts (C57, prepared by E13.5) or tail-tip fibroblasts (prepared one week after birth or adult mice) were uniformly seeded in each well, 2 ⁇ 10 4 cells per well, and cultured in the base.
  • the solution high glucose DMEM (Gibco, C12430500BT), double antibody
  • the culture solution was removed and washed with PBS.
  • the fibroblasts treated as described above were added to the immortalization-inducing medium: (N2B27 medium: DMEM/F12 (gibco, 10565018) and Neurobasal (Gibco, 21103-049) were mixed 1 to 1, and N2 additive (100 ⁇ , Gibco, 17502084), B27 additive (50 ⁇ , Gibco, 17504044), 2% bovine serum albumin (1000 ⁇ , sigma, A8022), ⁇ -mercaptoethanol (1000 ⁇ , Gibco, 21985023), GlutaMAX (100 ⁇ , Gibco) , 35050-061), 1 ⁇ g/ml insulin (Roche, 11376497001), double antibody).
  • N2B27 medium DMEM/F12 (gibco, 10565018)
  • Neurobasal Gibco, 21103-049
  • the identification of immortalized cell SMPC mainly includes the following aspects:
  • the experimental method was as follows: First, the Stem Cell CDy1 Dye was diluted with PBS 1:40 to prepare a CDy1 dilution buffer, and then the CDy1 dilution buffer was diluted with the culture solution 1:100 to prepare a CDy1 staining solution. After the culture solution was removed, the CDy1 staining solution was added, and the cells were stained for 1 hour in a CO 2 incubator at 37 ° C, washed three times with PBS, and then added to the culture solution for 3 hours in a 37 ° C CO 2 incubator. Observation with a fluorescence microscope, the results are shown in Fig. 3B, in which Merge indicates a coincidence pattern. Untreated mouse fibroblasts were not stained by CDy1; cells treated with immortalized induction broth, we call them SMPCs, were able to be stained by CDy1, indicating that they obtained "dryness".
  • the cell growth curve is drawn.
  • the experimental method was as follows: The immortalized cell SMPC (second and fourth generation mouse embryonic fibroblasts as a control) was uniformly inoculated into a 12-well plate at 2 x 10 4 cells per well. The cell count plate was counted every 24 hours, the cell growth curve was plotted, and the cell cycle was calculated. The results are shown in Figures 3C and 3D. SMPC was able to proliferate rapidly at the same initial cell volume, and at 24, 48, 72, and 96 hours, the amount of cells was significantly higher than that of the control (P ⁇ 0.001). The calculated cell cycle showed that the cell cycle length of SMPC was 16 hours, which was significantly shorter than that of the second and fourth generation mouse embryonic fibroblasts: 36 hours and 45 hours (P ⁇ 0.001).
  • the experimental method is as follows: immortalized cells (mouse embryonic fibroblasts and mouse embryonic stem cells as control) were digested with 0.25% trypsin, terminated with DMEM + 10% FBS, centrifuged, discarded, and resuspended in culture medium. After 400 mesh cell sieves, the cell cycle was analyzed by a MoFlo XDP high-speed multicolor flow cell sorter. The results are shown in Figures 3E and 3F.
  • the immortalized SMPC homogeneity is enhanced, similar to mouse embryonic stem cells.
  • the immortalized cell SMPC is shorter than the mouse embryonic fibroblast G0/G1 phase, and the S phase is increased, similar to the mouse embryonic stem cell.
  • the experimental method was as follows: The immortalized cell SMPC (mouse embryonic fibroblasts as a control) was collected. (a) RNA was extracted by a kit method. An appropriate amount of TRIzol lysed cells were added to the cell pellet, 1/5 volume of chloroform was added, vortexed, and then allowed to stand on ice for 3 minutes, 10000 g, and centrifuged at 4 ° C for 15 minutes. Transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of 75% ethanol, transfer it to the adsorption column, centrifuge at 10,000g for 15 seconds, discard the liquid in the collection tube, wash it once after Wash Buffer I, add it to the adsorption membrane.
  • the PCR reaction system was as follows: 7.5 ⁇ L of SYBR Green Real Time PCR Master Mix, 2 ⁇ L of Plus solution, 0.5 ⁇ L of primer (upstream and downstream mixing), 0.5 ⁇ L of cDNA, and 4.5 ⁇ L of double distilled water.
  • the PCR reaction procedure was as follows: amplification curve: 95 ° C, 2 min, denaturation at 95 ° C for 15 seconds, annealing at 62 ° C for 15 seconds, 72 ° C extension for 45 seconds, and detection of fluorescence signal after the end of extension. Melting curve: denaturation at 95 ° C for 1 minute, annealing at 57 ° C for 30 seconds, and slow renaturation to 95 ° C for 30 seconds.
  • the experiment was performed on an Agilent MX3005P fluorescence real-time quantitative PCR instrument using Actb as an internal reference gene, and the results were treated by the ⁇ Ct method.
  • the results are shown in Figure 1G and Figure 1H.
  • the immortalized SMPC proliferation-related genes: Sox2, Srrt, Yap, ⁇ -catenin, Mki67, Pcna are more abundant than mouse embryonic fibroblasts, while aging-related genes: P19, P16ink4a, P15ink4b, Morf4l1, Elf5 are compared with mice. The expression level of embryonic fibroblasts is reduced.
  • Example 4 Isolation and in vitro expansion of mouse adult primary hepatocytes
  • Alb-Cre mice were introduced from Bangyao, Cre recombinase was only expressed in mature hepatocytes expressing albumin; Rosa26/mTmG mice were introduced from Vitallihua, which is a kind of red in the Rosa26 locus.
  • the mouse of the green fluorescence reporter system normally expresses the tomato red fluorescent protein in the cell, and in the case of cre recombination, it becomes green GFP fluorescence.
  • Alb-Cre adult mice were crossed with Rosa26/mTmG mice. Mature liver cells of Alb-Cre ⁇ Rosa26/mTmG mice were labeled green, and other tissue cells expressed red.
  • Alb-Cre ⁇ Rosa26/mTmG adult mice were sacrificed by cervical dislocation.
  • the liver page was taken out, cut into small pieces, washed repeatedly with 100 volumes of pre-cooled PBS, and the blood cells were removed until the washing solution was red. Add 3-5 times volume.
  • Collagenase IV (ThermoFisher, 17104019, 37 degrees Celsius preheat) was digested in a 37 ° C incubator for 30-40 minutes, 2-3 times in the middle, and the supernatant was removed by centrifugation.
  • the mouse hepatocyte culture medium includes: DMEM/F12 (gibco, 10565018), adding N2 additive (100 ⁇ , Gibco, 17502084), B27 additive (50 ⁇ , Gibco, 17504044), 5% bovine serum albumin (1000 ⁇ , Sigma, A8022), ⁇ -mercaptoethanol (1000 ⁇ , Gibco, 21985023), GlutaMAX (200 ⁇ , Gibco, 35050-061), non-essential amino acids (100 ⁇ , Gibco, 11140-050), 1 ⁇ g/mL insulin (Roche , 11376497001), hepatocyte growth factor (10 ng/mL, R&D, 294-HG-025), transforming growth factor beta inhibitor A83-01 (5 ⁇ M, stemgent, 04-0014), glycogen synthase kinase 3beta inhibitor Chir99021 ( 6 ⁇ M, stemgent, 04-0004-10), macrophage stimulating protein 1 and 2 inhibitor XMU-MP-1 (2-5 ⁇ M, MCE, HY-
  • Example 5 In vitro expansion of human embryonic hepatocytes
  • rat tail collagen Thermo scientific, A1048301, 3mg/mL coated plate concentration is 5 ⁇ g/cm 2 , taking 24-well plate as an example, the bottom area of each hole is 1.9cm 2 , then each hole needs 9.5 ⁇ g (about 3.2 ⁇ l), 3.2 ⁇ l of rat tail collagen was dissolved in 500 ⁇ l of 20 mM glacial acetic acid, added to the wells, and incubated for 1 hour in a 37-degree cell culture incubator. After aspiration, PBS was washed 3 times.
  • the inoculation density was 1 ⁇ 10 5 /24 well plate, and 500 ⁇ l of hepatocyte culture medium (control group) was added respectively.
  • the hepatocyte culture medium included: DMEM/F12 (gibco, 10565018), and N2 was added.
  • Additives 100 ⁇ , Gibco, 17502084), B27 additives (50 ⁇ , Gibco, 17504044), 5% bovine serum albumin (1000 ⁇ , sigma, A8022), ⁇ -mercaptoethanol (1000 ⁇ , Gibco, 21985023), GlutaMAX (200 ⁇ , Gibco, 35050-061), non-essential amino acids (100 ⁇ , Gibco, 11140-050), 1 ⁇ g/mL insulin (Roche, 11376497001), hepatocyte growth factor (10 ng/mL, R&D, 294-HG- 025), transforming growth factor beta inhibitor A83-01 (5 ⁇ M, stemgent, 04-0014), glycogen synthase kinase 3beta inhibitor Chir99021 (6 ⁇ M, stemgent, 04-0004-10), macrophage stimulating proteins 1 and 2 Inhibitor XMU-MP-1 (2-5 ⁇ M, MCE, HY-100526), hepatocyte growth factor 4 (10 ng/mL, R&D, 58
  • the first passage of human embryonic hepatocytes The cells were inoculated into a 24-well plate coated with rat tail collagen (as described above), and the seeding density was 3/5 of the number of cells cultured for 72 hours in the primary culture. 3 duplicate wells per group. After 72 hours of cell culture, the digestion was counted. The number of cells is greater than the number of cells at the time of inoculation, and the multiple of the first passage amplification can be obtained. A portion of the cells per well (2/5 of the cell count) was used for RNA extraction to detect human hepatocyte-associated gene expression.
  • the second passage of human embryonic hepatocytes The cells were inoculated into a 24-well plate coated with rat tail collagen (as described above), and the seeding density was 3/5 of the number of cells in the first subculture for 72 hours. Since most of the cells died after 72 hours of the first subculture of the control group, only a small number of cells remained, so all of these cells were inoculated. After changing the liquid every 2 days and continuing to culture for 144 hours (6 days), the cells were digested and counted. At this time, the number of cells was the second passage amplification factor than the number of cells at the time of inoculation. Cell culture supernatants from each well were used to detect human albumin concentration, and some cells (2/5 of cell count) were used for RNA extraction to detect human hepatocyte-associated gene expression.
  • the recovery efficiency was about 5.8%.
  • Hepatocyte-specific genes, and cells obtained after passage of control medium for 12 days detected very low albumin expression and no alpha-fetoprotein gene expression was detected, indicating that the cells at this time were not hepatocytes, and FIG. 5A and FIG. 5C. The results are consistent.
  • Example 6 Small molecule amplification of adult hepatocytes
  • the number of cells was estimated from the photograph on the second day. After 4 days, the 20 ⁇ m small molecule medium group was changed to a 10 ⁇ m small molecule medium. Cell counts were co-cultured for 6 days. A portion of the cells were used for RNA extraction to detect the expression of human hepatocyte-specific genes.
  • the adherence efficiency was similar at 5 hours after inoculation of adult hepatocytes.
  • the small molecule group began to show amplification clones, while the control group did not significantly expand.
  • the 20 ⁇ m medium group was changed with a 10 ⁇ m small molecule medium (named protocol#, that is, cultured at 20 ⁇ m for 4 days, and cultured at 10 ⁇ m for 2 days). After two days of culture, the small molecule group cell clones were further expanded, while the control group showed no significant change (Fig.
  • Small molecule amplified adult hepatocytes still express human hepatocyte specific gene albumin (ALBUMIN), alpha fetoprotein (AFP), CYP1A2, CYP3A4 (Fig. 6D).
  • the 10 ⁇ m small molecule medium can subculture human liver cells, while the control medium does not pass through adult liver cells (Fig. 6E).
  • Subcultured adult hepatocytes can increase the expression of CYP1A2 gene after induction with omeprazole, suggesting that small molecule-amplified hepatocytes still function (Fig. 6F).

Abstract

一种细胞诱导的方法，具体来说涉及：诱导成纤维细胞转分化为脂肪细胞的方法，包括以下步骤：将成纤维细胞培养在培养基中，在培养基中加入Myosin抑制剂和BMP4，继续培养，直至得到脂肪细胞。还涉及一种诱导成纤维细胞转化为永生化细胞的方法及其应用。所述方法包括以下步骤：将成纤维细胞培养在培养基中，在培养基中加入Myosin抑制剂，继续培养，直至得到永生化细胞。还涉及一种诱导肝细胞体外扩增的方法。还涉及Myosin抑制剂尤其是(-)-Blebbistatin或(S)-(-)-Blebbistatin O-Benzoate和通过所述方法获得的肝细胞在诱导肝细胞体外扩增、构建生物人工肝和构建肝脏疾病模型中的应用。

Description

一种细胞诱导的方法 技术领域
本发明涉及生物技术领域，具体涉及细胞诱导技术领域，更具体涉及一种诱导成纤维细胞转分化为脂肪细胞的方法及其应用、一种将成纤维细胞转化为永生化细胞的方法及其应用、以及一种诱导肝细胞体外扩增的方法。
背景技术
转分化(transdifferentiation，也称为分化转移)是指一种类型的分化细胞转变成另一种类型的分化细胞的现象。目前已经可以实现多种类型的细胞的转分化，例如：将胚胎成纤维细胞、成软骨细胞和视网膜上皮细胞转分化为肌细胞，将B淋巴细胞转分化为巨噬细胞，将小鼠成纤维细胞转分化为功能性的神经细胞等等。
脂肪细胞(adipocyte)在成人体内大量存在，其组织又称脂肪组织，常呈白色，在幼儿期大量增殖，到青春期数量达到巅峰，此后数量一般不再增加。在正常动物和人类的体内，脂肪组织主要存在于腹腔内和腹部的皮下内，但肥胖患者的机体各个部位均会出现脂肪组织，如肾脏、肠系膜、皮下、腹腔等部位的周围，这表明可能分化为脂肪细胞的细胞不仅有脂肪前体细胞，在一些特定条件下，一些非脂肪前体细胞也能够分化为脂肪细胞，这颠覆了脂肪细胞是由脂肪前体细胞分化而来的传统观念。通过将非脂肪前体细胞转分化为脂肪细胞，也可以更清楚地了解脂肪细胞的转化机制，进而指导抑制肥胖药物的开发。
成纤维细胞(fibroblast)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。将成纤维细胞转分化为脂肪细胞在诸多领域具有重要的应用，例如，将疤痕伤口中发现的最常见类型的肌成纤维细胞转分化为脂肪细胞可能在未来实现伤口不留疤痕，并且在皱纹皮肤中再生脂肪细胞，有可能衍生出新型抗衰老治疗策略。由此可见，实现非脂肪前体细胞尤其是成纤维细胞转分化为脂肪细胞具有重要的意义。
目前，现有技术已经公开了一些诱导成纤维细胞转分化为脂肪细胞的方 法。CN104342401B公开了利用确定的细胞因子组合促进成纤维细胞转分化为脂肪细胞的方法，组合物的起效因子为表皮生长因子、肝细胞生长因子、地塞米松、胰岛素和PPARγ激动剂。CN104372024A公开了一种诱导牛成纤维细胞/成肌细胞转分化为脂肪细胞的方法，包括克隆牛转录因子CCAAT增强子结合蛋白C/EBPβ基因，构建C/EBPβ基因过表达载体并包装获得重组腺病毒，腺病毒侵染牛成纤维细胞、成肌细胞实现上述细胞快速转分化为脂肪细胞。上述方法采用大量大分子物质或重编程过程，效率较低，安全性也存在一定的影响。
现已证实，小分子化合物也可以诱导成纤维细胞转分化为脂肪细胞，这能够提高转分化的速度、存活率和能力。例如，CN105754935A公开了一种诱导成纤维细胞转分化为脂肪细胞的诱导培养基，所述培养基包含基础培养基和诱导小分子组合，所述诱导小分子组合为SG或6TF，其中S为SB431542、G为GSK126、6为E61541、T为苯环丙胺、F为毛喉素。但仍然需要探索更多的小分子化合物来诱导成纤维细胞转分化为脂肪细胞以用于疾病的研究、治疗和更广泛的应用。
正常组织来源的体细胞在通常的体外培养条件下可生长和分裂，但经过有限次的细胞传代后，就会停止增殖，发生衰老和死亡，这就限制了细胞培养技术的应用。细胞永生化(cell immortalization)是指细胞在体外培养过程中，由于自身基因变化或者各种外界刺激因素，从增殖衰老的危机中逃离，避免了正常细胞的衰老死亡过程，可以长期传代培养、无限分裂增殖。
对于细胞永生化的机制和方法已经进行了大量的研究，已经证实，放射性因素、端粒酶激活、病毒基因转染、原癌基因与抑癌基因等均可以导致细胞的无限增殖分裂。但经过多年的研究发现，虽然永生化的机制有相似之处，但同样的永生化方法并非适用于所有细胞。例如，肝细胞永生化的方法有抑癌基因敲除、质粒转导和病毒转染、可恢复性永生化等，上皮细胞永生化的方法有DNA致瘤病毒转染等，心肌细胞永生化的方法有P16慢病毒载体和可逆SV40病毒转导途径成功的案例。
目前，现有技术已经披露了一些诱导成纤维细胞转化为永生化细胞的方法。王新文等(“皮肤成纤维细胞永生化的研究进展”，中国生物工程杂质，第22卷第4期，2002年8月)披露了皮肤成纤维细胞永生化的方法，除了以上常用的方法外，总的概括起来还有以下几种方法：HPV、四硝基喹啉一氧 化物、黄曲霉素等。
针对性发展对特定细胞的永生化技术可以实现具有传代次数少、增殖分裂慢的正常体细胞的长期传代、无限分裂增殖，增加细胞周期寿命。这不仅有助于了解细胞生长规律、探索细胞衰老原因，还对解决器官移植问题有着重要的临床意义。
另外，资料显示，细胞永生化是转化为肿瘤细胞的前提，是由正常细胞转化为肿瘤细胞所必经的阶段，研究细胞的永生化可以为治疗肿瘤、控制肿瘤细胞的增殖奠定坚实的基础。
因此，针对成纤维细胞转化为永生化细胞的研究有着广阔的应用前景。
肝脏是人体内最大的内部器官，其是代谢的主要场所。肝细胞在肝脏中占85％。肝细胞在体内具有强大的再生潜能，将正常的肝脏切除三分之二后可以在仅一周内通过细胞增殖恢复到原来体积。然而，遗憾的是，尽管人类肝脏在体内可以迅速再生，但原代肝细胞在体外培养条件下仅能短暂增殖而不能长期增殖，到目前为止，努力在实验室扩展人类肝细胞会导致代谢功能低的永生化癌细胞。人体肝细胞的稀缺和肝细胞扩展时功能丧失是科学、医学和药学发展的主要瓶颈，解决这一问题将有助于促进肝细胞体外药物代谢、药物毒性、终末期肝病细胞治疗、构建出生物人工肝来支持等待移植的患者、疾病模型构建等研究和应用。
目前，该领域内最常用的方法为转录因子重编程技术，但是此类技术由于外源基因片段的插入使得进入临床应用的风险较大。前期有研究发现，采用小分子重编程技术，中日两个研究团队分别成功地实现了原代肝细胞在体外向肝前体细胞的转变和快速增殖，经定向诱导分化后，增殖的前体样肝细胞可重新获得成熟肝细胞的功能，移植入小鼠体内可达70％以上的整合。采用小分子化合物重编程技术，所得到的肝细胞未经过任何的基因改造，因而更为接近体内细胞的初始状态，对于将来的临床应用更为安全有效。该领域仍然存在使用小分子化合物来诱导肝细胞体外扩增的强烈需求。
发明内容
本发明人经过不断的探索，意外地发现可以通过Myosin抑制剂(-)-Blebbistatin或(S)-(-)-Blebbistatin O-Benzoate实现上述目的。
因此，本发明涉及Myosin抑制剂在诱导细胞转分化中的应用。
优选地，所述转分化为诱导成纤维细胞转分化。优选地，所述转分化为诱导成纤维细胞转分化为脂肪细胞。
在一个实施方案中，所述Myosin抑制剂为(-)-Blebbistatin，缩写Ble或Bleb或Blebb。
在一个实施方案中，所述Myosin抑制剂为(-)-Blebbistatin O-Benzoate，缩写为S-Bleb-OB。
在本发明中使用的(-)-Blebbistatin(也表示为(S)-(-)-Blebbistatin或S-Bleb)是一种细胞渗透性抑制剂，作用于非肌球蛋白IIATPase，其不抑制肌球蛋白轻链激酶，抑制卵裂沟的缢缩，不干扰有丝分裂或收缩环的组装，其结构式如式(I)所示，分子量为292.33。
在本发明中使用的(-)-Blebbistatin O-Benzoate(也表示为(S)-(-)-Blebbistatin O-Benzoate或S-Bleb-OB)是(-)-Blebbistatin的衍生物，其结构式如式(II)所示。
Figure PCTCN2019073624-appb-000001
本发明还涉及一种诱导成纤维细胞转分化为脂肪细胞的方法，包括以下步骤：将成纤维细胞培养在培养基中，在培养基中加入Myosin抑制剂和BMP4，继续培养，直至得到脂肪细胞。
优选地，所述培养基包括基础培养液、胎牛血清和脂肪诱导培养液中的任一种或几种。
优选地，所述基础培养液包括高糖DMEM、胎牛血清和双抗。
优选地，所述脂肪诱导培养液包括N2B27培养液(DMEM/F12、 Neurobasal的1∶1混合物)、N2添加剂、B27添加剂、2％牛血清白蛋白、β-巯基乙醇、GlutaMAX、胰岛素和双抗，以及血清替代物。
本发明还涉及通过所述的方法获得的脂肪细胞，或者包含所述脂肪细胞的试剂或研究工具或诊断工具。
上述本发明的方法只需单个小分子或单因素处理，操作简单，重复性好，并且在体内体外均可高效进行，不涉及转基因操作，获得的脂肪细胞安全性好，适用于在组织再生、修复等领域和产业中应用。
本发明还涉及Myosin抑制剂在诱导细胞永生化中的应用。
优选地，所述永生化为诱导成纤维细胞转化为永生化细胞。
优选地，所述Myosin抑制剂为(-)-Blebbistatin或(S)-(-)-Blebbistatin O-Benzoate。
本发明还涉及一种诱导成纤维细胞转化为永生化细胞的方法，其特征在于，包括以下步骤：将成纤维细胞培养在培养基中，在培养基中加入Myosin抑制剂，继续培养，直至得到永生化细胞。
优选地，所述培养基包括基础培养液、胎牛血清和永生化诱导培养液中的任一种或几种。
本发明还涉及一种诱导成纤维细胞转化为永生化细胞的培养基，其特征在于，其包括Myosin抑制剂、基础培养液、胎牛血清和永生化诱导培养液。
优选地，所述基础培养液包括高糖DMEM和双抗，和/或，所述永生化诱导培养液包括N2B27培养液：(DMEM/F12、Neurobasal的1∶1混合物)、N2添加剂、B27添加剂、2％牛血清白蛋白、β-巯基乙醇、GlutaMAX、胰岛素和双抗。
优选地，所述永生化诱导培养液进一步包括KOSR、CHIR99021和A83-01中的一种或几种。
本发明还涉及通过所述的方法获得的永生化细胞，或者包含所述永生化细胞的试剂或研究工具或诊断工具。
本发明还涉及基因在制备诱导增殖或衰老的制剂中的应用，其特征在于，所述基因包括Sox2、Srrt、Yap、β-catenin、Mki67、Pcna、P19、P16ink4a、P15ink4b、Morf4l1、Elf5中的一种或几种。
优选地，增殖相关基因包括Sox2、Srrt、Yap、β-catenin、Mki67、Pcna中的一种或几种。优选地，衰老相关基因包括P19、P16ink4a、P15ink4b、Morf4l1、Elf5中的一种或几种。
上述本发明的方法只需单个小分子或单因素处理，操作简单，重复性好，并且在体内体外均可高效进行，不涉及转基因操作，获得的永生化细胞安全性好，适用于了解细胞生长规律、探索细胞衰老原因、解决器官移植问题，还能够为治疗肿瘤、控制肿瘤细胞的增殖奠定一定的基础，具有重要的意义。本发明还发现了增殖相关基因和衰老相关基因，为诱导细胞的增殖和衰老提供了一种全新的选择。
此外，本发明人经过不断的探索，意外地发现Myosin抑制剂能显著地促进肝细胞长期扩增，进而实现上述目的。
因此，本发明总体上涉及Myosin抑制剂在诱导肝细胞体外扩增中的应用。
本发明还涉及Myosin抑制剂在构建生物人工肝中的应用。
本发明还涉及Myosin抑制剂在构建肝脏疾病模型中的应用。
优选地，所述Myosin抑制剂为(-)-Blebbistatin或(S)-(-)-Blebbistatin O-Benzoate。
本发明还涉及一种诱导肝细胞体外扩增的方法，其特征在于，包括以下步骤：将肝细胞培养在培养基中，在培养基中加入Myosin抑制剂，继续培养，从而诱导肝细胞体外扩增。优选地，所述Myosin抑制剂为(-)-Blebbistatin或(S)-(-)-Blebbistatin O-Benzoate。
本发明还涉及通过所述方法获得的肝细胞。
本发明还涉及包含所述肝细胞的试剂、生物人工肝、研究工具或诊断工具。
本发明还涉及使用所述诱导肝细胞体外扩增的方法或通过所述方法获得的肝细胞的肝脏疾病模型。
本发明还涉及通过所述方法获得的肝细胞在构建生物人工肝中的应用。
本发明还涉及通过所述方法获得的肝细胞在构建肝脏疾病模型中的应用。
上述应用可以是治疗性应用，也可以是非治疗性应用。
本发明的方法只需单个小分子或单因素处理，操作简单，重复性好，并 且在体外可高效进行，不涉及转录因子重编程技术和复杂的转基因操作，能够长期扩增，并且小分子扩增的肝细胞也仍然具有功能。
附图说明
图1A为实施例一的实验流程。
图1B显示了由人包皮成纤维细胞诱导得到的脂肪细胞油红O染色。
图1C和图1D分别显示了由小鼠胚胎成纤维细胞得到的脂肪细胞形态及油红O染色以及脂滴面积比例。
图1E和图1F分别显示了在原脂肪诱导培养液中添加不同物质后的油红O染色和脂滴面积统计。
图2A和2B为小分子处理的体内实验结果，显示能显著提高腹侧脂肪的含量。图中的Ble为(-)-Blebbistatin。
图3A为实施例二的实验流程。
图3B显示了荧光显微镜观察的结果。
图3C和图3D显示了绘制的细胞生长曲线和计算出的细胞周期。
图3E和图3F显示了细胞周期组成分析的结果。
图3G和图3H显示了增殖和衰老基因的检测结果。
图4A显示将细胞用小鼠肝细胞培养液(加入5微摩Myosin II抑制剂(-)-Blebbistatin)重悬，接种于铺有fibronectin的培养皿，一周后观察有Alb表达的绿色肝细胞团。
图4B显示AlB-GFP阳性肝细胞长期扩增的结果。
图5A和图5B显示传代(共3代)培养12天后的扩增结果。
图5C显示传代(共3代)培养12天后白蛋白和甲胎蛋白基因表达的结果。
图6A显示继续培养两天后，小分子组细胞克隆进一步扩增，而对照组无明显变化。
图6B显示培养6天后，小分子组和对照组的细胞数。
图6C为小分子组和对照组中的增殖细胞核抗原基因(PCNA)的表达结果。
图6D为小分子扩增的成人肝细胞表达人肝细胞特异基因白蛋白(ALBUMIN)、甲胎蛋白(AFP)、CYP1A2和CYP3A4的结果。
图6E为10μm小分子培养基和对照培养基使人肝细胞传代培养的结果。
图6F为传代培养的成人肝细胞经奥美拉唑诱导后，CYP1A2基因的表达结果。
具体实施方式
下面将参照附图更详细地描述本发明的具体实施例。虽然附图中显示了本发明的具体实施例，然而应当理解，可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
需要说明的是，在说明书及权利要求当中使用了某些词汇来指称特定组件。本领域技术人员应可以理解，技术人员可能会用不同名词来称呼同一个组件。本说明书及权利要求并不以名词的差异来作为区分组件的方式，而是以组件在功能上的差异来作为区分的准则。如在通篇说明书及权利要求当中所提及的“包含”或“包括”为一开放式用语，故应解释成“包含但不限定于”。说明书后续描述为实施本发明的较佳实施方式，然所述描述乃以说明书的一般原则为目的，并非用以限定本发明的范围。本发明的保护范围当视所附权利要求所界定者为准。
如本文所用，就特定组分而言“基本上不含”在本文中用于表示特定组分未被有目的地配制到组合物中和/或仅作为污染物或以痕量存在。因此，由组合物的任何意外污染导致的特定组分的总量低于0.05％，优选低于0.01％。最优选的是其中特定组分的量用标准分析方法检测不到的组合物。
如在本说明书中所使用的，“一”或“一个”可以表示一个或多个。如权利要求中所使用的，当与单词“包含”一起使用时，单词“一”或“一个”可以表示一个或多于一个。
在权利要求中使用术语“或”用于表示“和/或”，除非明确指出仅指代替代方案或者替代方案是相互排斥的，尽管本公开内容支持仅指代替代方案和“和/或”的定义。如本文所用，“另一个”可以表示至少第二个或更多个。
贯穿本申请，术语“约”用于指示值包括装置的误差的固有变化，该方法用于测定该值或存在于研究对象之间的变化。
在本文中，“分化”是较不特化的细胞变成更特化的细胞类型的过程。“去分化”是这样的细胞过程，其中部分或终末分化的细胞回复到更早期发育阶段，如多能性或多潜能性。“转分化”是将一种分化细胞类型转化为另一种分 化细胞类型的过程。典型地，通过编程发生转分化而细胞不经过中间多能性阶段-即，细胞直接从一种分化细胞类型编程为另一种分化细胞类型。
如本文所用，术语“受试者”或“有需要的受试者”是指任何年龄的雄性或雌性的需要细胞或组织移植的哺乳动物，优选人。通常，受试者需要细胞或组织移植(在本文中也称为受体)，这是由于适合经由细胞或组织移植治疗的病症或病理或不期望的状况、状态或综合征或身体、形态学或生理学异常。
在本文中涉及的一些术语的定义如下：
BMP4：骨形态发生蛋白4(bone morphogenetic protein，bmp4)。
高糖DMEM：一种高糖型DMEM培养基(dulbecco′s modified eagle medium，DMEM)，即一种含各种葡萄糖和氨基酸的商品化的培养基，在MEM培养基的基础上研制的。
N2B27：一种以DMEM/F12基础培养基和neurobasal基础培养基以1∶1混合而成，包含N2添加剂和B27添加剂的成分明确的细胞培养液，报道有利于小鼠胚胎干细胞向神经方向分化。
DMEM/F12：一种以DMEM培养基和F12培养基1∶1混合而成的商品化的基础培养液，适于克隆密度的培养。
Neurobasal：有利于神经细胞培养的商品化基础培养基。
GlutaMAX：一种细胞培养添加剂，可直接替代细胞培养基中的L-谷氨酰胺。
双抗：青霉素和链霉素是细胞培养常用的两种抗生素，防止细胞培养过程中的细菌污染。
N2添加剂：一种商品化无血清的细胞培养添加剂。
B27添加剂：一种商品化无血清的细胞培养添加剂。
KOSR：商品化血清替代物(Knockout serum replacement，KOSR)。
CHIR99021：一种GSK-3α/β抑制剂，常用作Wnt信号通路激活剂。
A83-01：一种具有选择性的TGF-β抑制剂，能够显著抑制ALK4，ALK5和ALK7的活性。
实施例
以下通过具体实施例来详细阐述和说明本发明的实施方式，但以下内容不应理解为对本发明作任何限制，实施例中采用的物质等如果没有特殊说明 均为市售产品。
实施例一由成纤维细胞向脂肪细胞的转化
以12孔板为例(corning，3335)，每孔用20μg/ml基质胶溶液(BD，354277)1×DMEM配制，包被12小时，去掉包被液用1×PBS冲液洗一遍。
将小鼠胚胎成纤维细胞(C57，E13.5制备)或成人包皮成纤维细胞(HFF20y，北京干细胞库)均匀种于每一个孔，每孔1×10 4个细胞，用基础培养液(高糖DMEM(Gibco，C12430500BT)，双抗)加10％胎牛血清(Gibco，16000-044)培养24小时。去除培养液，PBS洗一遍。
将经上述处理后的成纤维细胞加入脂肪诱导培养液：(N2B27培养液：DMEM/F12(Gibco，10565018)与Neurobasal(Gibco，21103-049)1比1混合，加入N2添加剂(100×，Gibco，17502048)，B27添加剂(50×，Gibco，17504044)，2％牛血清白蛋白(1000×，sigma，A8022)，β-巯基乙醇(1000×，Gibco，21985023)，GlutaMAX(200×，Gibco，35050-061)，1μg/ml胰岛素(Roche，11376497001)，双抗)，10％血清替代物(Gibco，10828-028)加入Myosin抑制剂(二甲基亚砜溶解(sigma，D2650)的100mM浓储，-20摄氏度避光保存1月)以及10ng/mL BMP4(Peprotech，10828-028)，培养21天后鉴定，实验流程见图1A。
脂肪细胞的鉴定采用细胞油红染色，使用油红O染色液(细胞专用，Solarbio，G1262)，具体如下：去掉培养液，加入ORO Fixative固定10-15min后，去掉固定液，放在流动的空气中10-15分钟。此时按照ORO Stain A∶ORO Stain B＝3∶2的比例配置ORO Stain，混合后静置10min后加入孔中，浸染15min后去掉染色液，加入60％异丙醇漂洗20-30s，并蒸馏水清洗3次后于光学显微镜下拍照，中性脂肪呈橙红色或橘红色，磷脂呈粉红色。由人包皮成纤维细胞诱导得到的脂肪细胞油红O染色见图1B。
由小鼠胚胎成纤维细胞得到的脂肪细胞形态及油红O染色如图1C，可见橙红色脂滴的形成，标志中性脂肪的出现。脂滴面积比例近13％，显著高于对照组(P＜0.01)，统计结果见图1D。
在原脂肪诱导培养液中添加10μM A83-01(stemgent，04-0014)显著提高了脂肪诱导效率，高达20％(P＜0.001)；或添加10μM SB431542(stemgent，04-0010-10)也可显著提高脂肪诱导效率，达18％(P＜0.001)。若添加BMP 的抑制剂，脂肪诱导效率恢复至对照水平。油红O染色见图1E，脂滴面积统计见图1F。
体内实验：选取同批次6-8周ICR雌性小鼠，随机分成两组，每组5只，一组小分子(-)-Blebbistatin或(S)-(-)-Blebbistatin O-Benzoate(0.5mg/kg，溶于10％DMSO+2％Tween 80+生理盐水)注入小鼠腹侧，另一组注射与小分子组处理相同计量的DMSO，注射形式同实验组。每天注射一次，注射19天后，停药32天，颈椎脱臼处死小鼠观察腹侧脂肪含量。结果如图2所示，小分子处理组能显著提高腹侧脂肪的含量，具体见图2A、2B。此外，(S)-(-)-Blebbistatin O-Benzoate替代(-)-Blebbistatin处理也能提高腹侧脂肪含量，实验结果显示在未经优化的情况下，腹侧脂肪含量提高了约50％。
实施例二：由成纤维细胞向永生化细胞的转化
以12孔板为例(coming，3335)每孔用20μg/mL基质胶溶液(BD，354277)1×DMEM配制，包被12小时，去掉包被液用1×PBS冲液洗一遍。
将小鼠胚胎成纤维细胞(C57，E13.5制备)或尾尖成纤维细胞(出生后一周或成年小鼠制备)均匀种于每一个孔，每孔2×10 4个细胞，用基础培养液(高糖DMEM(Gibco，C12430500BT)，双抗)加10％胎牛血清(Gibco，16000-044)培养24小时。去除培养液，PBS洗一遍。
将经上述处理后的成纤维细胞加入永生化诱导培养液：(N2B27培养液：DMEM/F12(gibco，10565018)与Neurobasal(Gibco，21103-049)1比1混合，加入N2添加剂(100×，Gibco，17502048)，B27添加剂(50×，Gibco，17504044)，2％牛血清白蛋白(1000×，sigma，A8022)，β-巯基乙醇(1000×，Gibco，21985023)，GlutaMAX(100×，Gibco，35050-061)，1μg/ml胰岛素(Roche，11376497001)，双抗)。加入10％KOSR(gibco，12618013)、3μM的CHIR99021(stemgent，04-0004-10)、10μM的A83-01(stemgent，04-0014)和25μM的Myosin抑制剂(-)-Blebbistatin(MCE，HY-13441)或25μM的(S)-(-)-Blebbistatin O-Benzoate(TRC，B208070)。培养21～28天后鉴定，实验流程见图3A，利用包含(-)-Blebbistatin(MCE，HY-13441)或(S)-(-)-Blebbistatin O-Benzoate(TRC，B208070)永生化诱导培养液得到的细胞通过如下实验进行是否为永生化细胞SMPC的鉴定。
永生化细胞SMPC的鉴定主要包括以下几个方面：
干细胞标志物染色。实验方法如下：首先用PBS 1∶40稀释Stem Cell CDy1 Dye，制成CDy1稀释缓冲液，然后用培养液1∶100稀释CDy1稀释缓冲液制成CDy1染色液。去掉培养液后加入CDy1染色液，在37℃ CO 2培养箱中染色1小时，用PBS洗涤三次后，加入培养液在在37℃ CO 2培养箱中褪色3小时。用荧光显微镜观察，结果见图3B，其中Merge表示重合图。未处理的小鼠成纤维细胞不能被CDy1着色；经永生化诱导培养液处理的细胞，我们称之为SMPC，能够被CDy1着色，标志其获得了“干性”。
细胞生长曲线绘制。实验方法如下：将永生化细胞SMPC(第二代和第四代的小鼠胚胎成纤维细胞做对照)按照每孔2×10 4个细胞均匀接种于12孔板中。每隔24小时用血球计数板计数，绘制细胞生长曲线，并计算出细胞周期。结果见图3C和图3D。SMPC在起始细胞量相同的情况下，能够迅速增殖，在24、48、72、96小时，细胞量显著高于对照(P＜0.001)。经计算得到的细胞周期可见，SMPC的细胞周期长度为16小时，较第二代和第四代的小鼠胚胎成纤维细胞细胞周期长度：36小时和45小时显著缩短(P＜0.001)。
细胞周期组成分析。实验方法如下：将永生化细胞(小鼠胚胎成纤维细胞和小鼠胚胎干细胞做对照)用0.25％胰酶消化后，用DMEM+10％FBS终止后离心，弃上清，用培养液重悬后过400目细胞筛，经MoFlo XDP高速多色流式细胞分选仪分析细胞周期。结果见图3E和图3F。永生化细胞SMPC均一性增强，与小鼠胚胎干细胞相似。且永生化细胞SMPC较小鼠胚胎成纤维细胞G0/G1期缩短，S期增长，与小鼠胚胎干细胞相似。
实施例三：增殖和衰老基因的检测
实验方法如下：收集永生化细胞SMPC(小鼠胚胎成纤维细胞作对照)。(a)用试剂盒法提取RNA。向细胞沉淀中加入适量TRIzol裂解细胞，加入1/5体积的三氯甲烷，涡旋混匀后冰上静置3分钟，10000g，4℃离心15分钟。将上层水相转移到新的离心管中，加入等体积75％乙醇后一并转移到吸附柱中，10000g离心15秒，弃收集管中液体，Wash Buffer I洗一次后，在吸附膜上添加10μL DNase I+70μL Buffer RDD，室温孵育15分钟以消化DNA，随后用350μL Wash Buffer I洗一次，500μL Wash Buffer II洗一次，用RNase-Free Water洗脱RNA。用紫外/可见光分光光度计测定浓度。(b)将RNA 反转录为cDNA。在2μg的RNA样品中加入随机引物、dNTP后于65℃加热5分钟以去除RNA高级结构，随后于冰上淬火3分钟使RNA模板和随机引物结合。加入反转录酶、RNA酶抑制剂，于42℃反转录1小时得到cDNA。(c)进行实时荧光定量PCR检测增殖和衰老相关基因。
PCR反应体系如下：7.5μL SYBR Green Real Time PCR Master Mix，2μL Plus solution，0.5μL引物(上下游混合)，0.5μL cDNA，4.5μL双蒸水。PCR反应程序如下：扩增曲线：95℃，2min，95℃变性15秒，62℃退火15秒，72℃延伸45秒，并在在延伸结束后检测荧光信号。熔解曲线：95℃变性1分钟，57℃退火30秒，缓慢复性至95℃维持30秒。该实验在Agilent公司MX3005P荧光实时定量PCR仪上完成，以Actb作为内参基因，结果用ΔΔCt法处理。结果见图1G和图1H。永生化细胞SMPC增殖相关基因：Sox2，Srrt，Yap，β-catenin，Mki67，Pcna较小鼠胚胎成纤维细胞表达量升高，而衰老相关基因：P19，P16ink4a，P15ink4b，Morf4l1，Elf5较小鼠胚胎成纤维细胞表达量降低。
实施例四：小鼠成体原代肝细胞分离与体外扩增
Alb-Cre小鼠从邦耀生物引进，Cre重组酶只在表达白蛋白的成熟肝细胞中表达；Rosa26/mTmG的小鼠从维通利华引进，是一种在Rosa26位点整合一种红绿荧光报告系统的小鼠，正常情况下在细胞表达Tomato红色荧光蛋白，在有cre重组的情况下，会变成表达绿色GFP荧光。Alb-Cre成年小鼠与Rosa26/mTmG的小鼠杂交出生Alb-Cre×Rosa26/mTmG小鼠成熟的肝细胞被标记成绿色，其它组织细胞表达红色。
Alb-Cre×Rosa26/mTmG成年小鼠颈椎脱臼处死，取出肝页，剪碎成小块，用100倍体积的预冷PBS反复吹打洗涤，去除血细胞直至洗液无红色，加入3-5倍体积的胶原酶IV(ThermoFisher，17104019，37摄氏度预热)在37℃培养箱消化30-40分钟，中间吹打2-3次，离心去除上清。加入3-5倍体积的胰酶(Gibco，25300062)在37℃培养箱消化20分钟，加入2倍体积10％血清终止，离心取上清，将细胞用小鼠肝细胞培养液(加入5微摩MyosinII抑制剂(-)-Blebbistatin)重悬，接种于铺有fibronectin的培养皿，一周后观察有Alb表达的绿色肝细胞团，如图4A所示。小鼠肝细胞培养基包括：DMEM/F12(gibco，10565018)，加入N2添加剂(100×，Gibco，17502048)， B27添加剂(50×，Gibco，17504044)，5％牛血清白蛋白(1000×，sigma，A8022)，β-巯基乙醇(1000×，Gibco，21985023)，GlutaMAX(200×，Gibco，35050-061)，非必须氨基酸(100×，Gibco，11140-050)，1μg/mL胰岛素(Roche，11376497001)，肝细胞生长因子(10ng/mL，R&D，294-HG-025)，转化生长因子beta抑制剂A83-01(5μM，stemgent，04-0014)，糖原合成激酶3beta抑制剂Chir99021(6μM，stemgent，04-0004-10)，巨噬细胞刺激蛋白1和2抑制剂XMU-MP-1(2-5μM，MCE，HY-100526)肝细胞生长因子4(10ng/mL，R&D，5846-f4-025)双抗)。
不加Myosin II抑制剂(-)-Blebbistatin或(S)-(-)-Blebbistatin O-Benzoate，加入等体积DMSO做对照，分别培养传代传代3次(P3)、传代5次(P5)、传代7次(P7)，结果显示加入Myosin抑制剂(-)-Blebbistatin能显著促进AlB-GFP阳性肝细胞长期扩增(体外传代至少20代)，如图4B所示。此外，结果还显示(-)-Blebbistatin衍生物(S)-(-)-Blebbistatin O-Benzoate对肝细胞传代扩增也有类似的效果，处理之后肝细胞可以在体外传代20代以上。
实施例五：人胚胎肝细胞体外扩增
1、实验步骤
(1)包板 鼠尾胶原(Thermo scientific，A1048301，3mg/mL)包被培养板浓度为5μg/cm 2，以24孔板为例，每孔的底面积为1.9cm 2，则每孔需要9.5μg(约3.2μl)，取3.2μl鼠尾胶原溶于500μl 20mM冰醋酸，加入孔中，37度细胞培养箱中孵育1小时，吸弃后，PBS洗3遍。
(2)人胚胎肝细胞复苏 从液氮罐中取出人胚胎肝细胞(冻存日期为2014年1月15日，冻存液为cell banker 2，冻存细胞数为2×10 7/管)，迅速放入37度水浴锅中，待溶化后立即吸入含有5mL肝细胞培养基(对照组)的15mL离心管中，离心50g，4度，5分钟。弃上清，用500μl肝细胞培养基重悬计数为1.16×10 6，则细胞复苏率为5.8％。
(3)人胚胎肝细胞接种 接种密度为1×10 5/24孔板孔，分别加500μl肝细胞培养基(对照组)，肝细胞培养基包括：DMEM/F12(gibco，10565018)，加入N2添加剂(100×，Gibco，17502048)，B27添加剂(50×，Gibco，17504044)，5％牛血清白蛋白(1000×，sigma，A8022)，β-巯基乙醇(1000×，Gibco，21985023)，GlutaMAX(200×，Gibco，35050-061)，非必须氨基酸(100×，Gibco， 11140-050)，1μg/mL胰岛素(Roche，11376497001)，肝细胞生长因子(10ng/mL，R&D，294-HG-025)，转化生长因子beta抑制剂A83-01(5μM，stemgent，04-0014)，糖原合成激酶3beta抑制剂Chir99021(6μM，stemgent，04-0004-10)，巨噬细胞刺激蛋白1和2抑制剂XMU-MP-1(2-5μM，MCE，HY-100526)，肝细胞生长因子4(10ng/mL，R&D，5846-f4-025)双抗)。含10μM小分子(-)-Blebbistatin肝细胞培养基和含20μM小分子(-)-Blebbistatin肝细胞培养基，接种24小时后每组分别取3孔消化下来细胞计数，计算得到贴壁率。接种72小时后，每组分别消化3孔细胞并计数。72小时细胞数比24小时细胞数，则可得到细胞数变化倍数。分别取每孔一部分细胞(细胞计数的2/5)用于RNA提取检测人肝细胞相关基因表达。
(4)人胚胎肝细胞第一次传代 细胞分别接种到鼠尾胶原包被的24孔板中(如上所述)，接种密度为原代培养72小时细胞数的3/5。每组3个复孔。细胞培养72小时后，消化计数。该细胞数比接种时的细胞数，则可得到第一次传代扩增的倍数。取每孔一部分细胞(细胞计数的2/5)用于RNA提取检测人肝细胞相关基因表达。
(5)人胚胎肝细胞第二次传代 细胞分别接种到鼠尾胶原包被的24孔板中(如上所述)，接种密度为第一次传代培养72小时细胞数的3/5。由于control组第一次传代培养72小时后大部分细胞死亡，只剩一少部分细胞，所以这些细胞全部接种。每2天换液，继续培养144小时(6天)后，细胞消化计数，此时细胞数比接种时细胞数即为第二次传代扩增倍数。分别取每孔细胞培养上清液用于检测人白蛋白浓度，一部分细胞(细胞计数的2/5)用于RNA提取检测人肝细胞相关基因表达。
2、结果
人胚胎肝细胞液氮冻存3年半后，复苏效率约为5.8％。如图5A和图5B所示，经过小分子培养基传代(共3代)培养12天后，10μM小分子培养基可使其扩增约22.1倍(SD＝4.2)，20μM小分子培养基可使其扩增约13.0倍(SD＝3.39)。此时，细胞形态仍然为典型的肝细胞形态，呈现出不规则的多边形。然而，经对照组培养基传代培养后，由于在第一次传代培养后大部分细胞死亡，剩下的小部分细胞经过培养后在12天时扩增倍数达58.3倍(SD＝13.9)，但此时细胞呈典型的肝细胞形态，细长扁平状。如图5C，实时定量PCR检测人肝细胞特异的基因白蛋白(ALBUMIN)和甲胎蛋白(Alpha  fetoprotein)，结果显示，人肝细胞经10μM和20μM小分子培养基传代培养12天后，仍然表达人肝细胞特异的基因，而control培养基传代12天后得到的细胞检测到极低的白蛋白表达和检测不到甲胎蛋白基因表达，证明此时的细胞已经不是肝细胞，和图5A和图5C的结果一致。
实施例六：成人肝细胞小分子扩增
1、实验步骤
(1)包板方法如上所述。
(2)成人肝细胞复苏及培养 成人肝细胞(M00995-P Male human，BioreclamationIVT)从液氮罐中取出，迅速放入37度水浴锅中，待融化后加入到37度预热的5mL肝细胞接种培养基(InVitroGRO CP Medium)中，计数后，以9×10 4/孔接种于24孔板中，2-4小时肝细胞贴壁后，吸弃肝细胞接种培养基，分别加入肝脏细胞培养对照培养基、10μm小分子培养基和20μm小分子培养基。每2天换液。在第2天时通过照片估算细胞数。4天后20μm小分子培养基组改成10μm小分子培养基培养。共培养6天时细胞计数。取一部分细胞用于RNA提取检测人肝细胞特异基因的表达。
(3)成人肝细胞传代 将上述对照组培养的肝细胞4.7×10 4和10μm小分子培养基培养的肝细胞8×10 4分别重新接种到鼠尾胶原包被的24孔板中，每两天换液，培养6天，拍照记录细胞生长状况。
(4)10μm小分子培养基扩增的成人肝细胞CYP1A2诱导3×10 5肝细胞接种于鼠尾胶原包被的24孔板中，用10μm小分子培养基培养24小时后，换成含有50μm奥美拉唑(Omeporazole)的10μm小分子培养基，对照组为含有DMSO的10μm小分子培养基，48小时后，收集细胞检测CYP1A2基因的表达。
2、结果
成人肝细胞接种后5小时贴壁效率类似，2天后，对照组、10μm小分子组和20μm小分子组细胞均大量死亡。拍照并估算活细胞贴壁数(2.53×10 4，SD＝0.09)。而在4天时，小分子组细胞开始出现扩增克隆，而对照组无明显扩增。此时，20μm培养基组换液用10μm小分子培养基(命名为protocol#，即20μm培养4天，10μm培养两天)。继续培养两天后，小分子组细胞克隆进一步扩增，而对照组无明显变化(如图6A)。培养6天，细胞数分别为对照 组1.59×10 4(SD＝0.28)、10μm组为6.47×10 4(SD＝1.24)，#组为9.47×10 4(SD＝0.98)(图6B)。用第6天细胞数比第2天细胞数即为细胞扩增倍数，结果显示小分子对成人肝细胞具有明显的扩增作用(图6A和6B)。增殖细胞核抗原基因(PCNA)的表达小分子组显著高于对照组，进一步证明小分子对细胞增殖具有作用(图6C)。小分子扩增的成人肝细胞仍然表达人肝细胞特异基因白蛋白(ALBUMIN)、甲胎蛋白(AFP)、CYP1A2、CYP3A4(图6D)。10μm小分子培养基可以使人肝细胞传代培养，而对照培养基则不能使成人肝细胞传代(图6E)。传代培养的成人肝细胞经奥美拉唑诱导后，可以提高CYP1A2基因的表达，提示小分子扩增的肝细胞仍然具有功能(图6F)。
前面仅仅示出了本发明的原理，应理解，本发明的范围不预期限制在本文所述的示例性方面，而应包括所有当前已知的和未来开发的等同物。另外，应当指出，在不脱离本发明技术原理的前提下，还可以作出若干改进和修改，这些改进和修改也应被视为本发明的范围。

Claims (37)

  1. Myosin抑制剂在细胞诱导中的应用。
  2. 如权利要求1所述的应用，其中，所述在细胞诱导中的应用选自：Myosin抑制剂在诱导细胞转分化中的应用、Myosin抑制剂在诱导细胞永生化中的应用以及诱导肝细胞体外扩增中的应用中的任一种。
  3. 如权利要求2所述的应用，其中，所述转分化为诱导成纤维细胞转分化，优选地，所述转分化为诱导成纤维细胞转分化为脂肪细胞。
  4. 如权利要求2所述的应用，其中，所述永生化为诱导成纤维细胞转化为永生化细胞。
  5. Myosin抑制剂在构建生物人工肝中的应用。
  6. Myosin抑制剂在构建肝脏疾病模型中的应用。
  7. 如权利要求1-6中任一项所述的应用，其中，所述Myosin抑制剂为(-)-Blebbistatin或(-)-Blebbistatin O-Benzoate。
  8. 一种诱导成纤维细胞转分化为脂肪细胞的方法，其包括以下步骤：将成纤维细胞培养在培养基中，在培养基中加入Myosin抑制剂和BMP4，继续培养，直至得到脂肪细胞。
  9. 如权利要求8所述的方法，其中，所述培养基包括基础培养液、胎牛血清和脂肪诱导培养液中的任一种或几种。
  10. 如权利要求9所述的方法，其中，所述Myosin抑制剂为(-)-Blebbistatin或(-)-Blebbistatin O-Benzoate。
  11. 如权利要求9或10所述的方法，其中，所述基础培养液包括高糖DMEM、胎牛血清和双抗，和/或，所述脂肪诱导培养液包括N2B27培养液(DMEM/F12、Neurobasal的1∶1混合物)、N2添加剂、B27添加剂、2％牛血清白蛋白、β-巯基乙醇、Glutamax、胰岛素和双抗，以及血清替代物。
  12. 如权利要求9-11中任一项所述的方法，其中，所述脂肪诱导培养液进一步包括A83-01和/或SB431542。
  13. 如权利要求8-12中任一项所述的方法获得的脂肪细胞。
  14. 一种脂肪细胞的试剂或研究工具或诊断工具，其包含利用权利要求 8-12中任一项所述的方法获得的脂肪细胞或权利要求13所述的脂肪细胞。
  15. 一种诱导成纤维细胞转分化为脂肪细胞的培养基，其包括：
    Myosin抑制剂、BMP4、基础培养液、胎牛血清和脂肪诱导培养液。
  16. 如权利要求15所述的培养基，其中，所述Myosin抑制剂为(-)-Blebbistatin或(-)-Blebbistatin O-Benzoate。
  17. 如权利要求15-16中任一项所述的培养基，其中，所述基础培养液包括高糖DMEM、胎牛血清和双抗，和/或，所述脂肪诱导培养液包括N2B27培养液(DMEM/F12、Neurobasal的1∶1混合物)、N2添加剂、B27添加剂、2％牛血清白蛋白、β-巯基乙醇、Glutamax、胰岛素和双抗，以及血清替代物。
  18. 如权利要求15-17中任一项所述的培养基，其中，所述脂肪诱导培养液进一步包括A83-01和/或SB431542。
  19. 一种诱导成纤维细胞转化为永生化细胞的方法，其包括以下步骤：将成纤维细胞培养在培养基中，在培养基中加入Myosin抑制剂，继续培养，直至得到永生化细胞。
  20. 如权利要求19所述的方法，其中，所述培养基包括基础培养液、胎牛血清和永生化诱导培养液中的任一种或几种。
  21. 如权利要求19或20所述的方法，其中，所述Myosin抑制剂为(-)-Blebbistatin或(-)-Blebbistatin O-Benzoate。
  22. 如权利要求19-21中任一项所述的方法，其中，所述基础培养液包括高糖DMEM和双抗，和/或，所述永生化诱导培养液包括N2B27培养液(DMEM/F12、Neurobasal的1∶1混合物)、N2添加剂、B27添加剂、2％牛血清白蛋白、β-巯基乙醇、GlutaMAX、胰岛素和双抗。
  23. 如权利要求19-22中任一项所述的方法，其中，所述永生化诱导培养液进一步包括KOSR、CHIR99021和A83-01中的一种或几种。
  24. 如权利要求19-23中任一项所述的方法获得的永生化细胞。
  25. 一种永生化细胞的试剂或研究工具或诊断工具，其包含利用权利要求19-23中任一项所述的方法获得的永生化细胞或权利要求24所述的永生化细胞。
  26. 一种诱导成纤维细胞转化为永生化细胞的培养基，其包括：
    Myosin抑制剂、基础培养液、胎牛血清和永生化诱导培养液。
  27. 如权利要求26所述的培养基，其中，所述Myosin抑制剂为 (-)-Blebbistatin或(-)-Blebbistatin O-Benzoate。
  28. 如权利要求26-27中任一项所述的培养基，其中，所述基础培养液包括高糖DMEM和双抗，和/或，所述永生化诱导培养液包括N2B27培养液(DMEM/F12、Neurobasal的1∶1混合物)、N2添加剂、B27添加剂、2％牛血清白蛋白、β-巯基乙醇、GlutaMAX、胰岛素和双抗。
  29. 如权利要求26-28中任一项所述的培养基，其中，所述永生化诱导培养液进一步包括KOSR、CHIR99021和A83-01中的一种或几种。
  30. 基因在制备诱导增殖或衰老的制剂中的应用，其特征在于，所述基因包括Sox2、Srrt、Yap、β-catenin、Mki67、Pcna、P19、P16ink4a、P15ink4b、Morf4l1、Elf5中的一种或几种；优选地，增殖相关基因包括Sox2、Srrt、Yap、β-catenin、Mki67、Pcna中的一种或几种；优选地，衰老相关基因包括P19、P16ink4a、P15ink4b、Morf4l1、Elf5中的一种或几种。
  31. 一种诱导肝细胞体外扩增的方法，其包括以下步骤：将肝细胞培养在培养基中，在培养基中加入Myosin抑制剂，继续培养，从而诱导肝细胞体外扩增。
  32. 如权利要求31所述的方法，其特征在于，所述Myosin抑制剂为(-)-Blebbistatin或(-)-Blebbistatin O-Benzoate。
  33. 通过权利要求31或32所述的方法获得的肝细胞。
  34. 一种肝细胞试剂、生物人工肝、研究工具或诊断工具，其包含利用权利要求31或32所述的方法获得的肝细胞或权利要求33所述的肝细胞。
  35. 一种肝脏疾病模型，其是利用权利要求31或32所述的方法获得的或者利用权利要求33所述的肝细胞获得的。
  36. 通过权利要求31-32中任一项所述的方法获得的肝细胞或权利要求33所述的肝细胞在构建生物人工肝中的应用。
  37. 通过权利要求31-32中任一项所述的方法获得的肝细胞或权利要求33所述的肝细胞在构建肝脏疾病模型中的应用。
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