CN115029299B - Application of JAK2 inhibitor in islet beta cell induced differentiation - Google Patents
Application of JAK2 inhibitor in islet beta cell induced differentiation Download PDFInfo
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- CN115029299B CN115029299B CN202210812902.XA CN202210812902A CN115029299B CN 115029299 B CN115029299 B CN 115029299B CN 202210812902 A CN202210812902 A CN 202210812902A CN 115029299 B CN115029299 B CN 115029299B
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Classifications
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0676—Pancreatic cells
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
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- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
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- C12N2500/00—Specific components of cell culture medium
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- C12N2500/38—Vitamins
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- C12N2501/70—Enzymes
- C12N2501/72—Transferases (EC 2.)
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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Abstract
The invention relates to the field of biotechnology, in particular to application of a JAK2 inhibitor in islet beta cell induction differentiation. The JAK2 inhibitor comprises at least one selected from CEP-33779, TG101209 and TG101348. The JAK2 inhibitor can greatly improve the differentiation efficiency of endocrine cells to islet beta cells, thereby regulating and controlling the differentiation of islet beta cells in vivo and being applied to the field of disease treatment.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to application of a JAK2 inhibitor in islet beta cell induction differentiation.
Background
Diabetes is one of the main diseases affecting human health, and insulin secretion deficiency and glucose metabolism disorder caused by pancreatic endocrine beta cell function decline are important causes of diabetes, and research of functional states of islet cells is an important part while researching pathogenesis. Among them, the use of islet beta cells is an effective method for treating diabetes.
However, differentiation to obtain islet β cells requires further investigation.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the related art to some extent. Therefore, an object of the invention is to propose an application of a JAK2 inhibitor in the field of islet beta cell induced differentiation and an application of the JAK2 inhibitor in preparing a medicament for preventing and/or treating diabetes.
The inventors of the present invention found during the course of the study that: JAK2 inhibitors can play an important role in the process of directional differentiation of pancreatic precursor cells (Pancreatic progenitors) into islet beta cells (pancreatic beta cells), and JAK2 inhibitors can greatly improve the efficiency of differentiation of endocrine cells into islet beta cells, so that acting cells express islet beta cell specific molecular markers Proinsulin (Proinsulin), insulin (Insulin), C Peptide (C-Peptide) and NKX6.1. Therefore, the JAK2 inhibitor can regulate the directional differentiation of islet beta cells in vivo or in vitro, and can be further used for preventing or treating diseases caused by islet beta cell deficiency or abnormality.
To this end, according to a first aspect of the present invention, there is provided the use of a JAK2 inhibitor in the field of islet β cell induced differentiation. The inventors found that JAK2 inhibitors can greatly improve the differentiation efficiency of endocrine cells into islet beta cells, thereby being capable of regulating and controlling the differentiation of islet beta cells in vivo; or can be used to induce differentiation into islet beta cells in vitro for use as a specific assay.
According to an embodiment of the present invention, the above JAK2 inhibitor may further be characterized by the following technical features in the field of islet β cell induction differentiation:
in some embodiments of the invention, the JAK2 inhibitor is a JAK2 protein kinase inhibitor.
In some embodiments of the invention, the JAK2 protein kinase inhibitor comprises at least one selected from CEP-33779, TG101209, TG101348. Representative substances of JAK2 inhibitors CEP-33779, TG101209, or TG101348 can inhibit JAK2 protein kinase, so that endocrine cells or pancreatic precursor cells can be induced to differentiate into islet beta cells, and thus can be used as an induced differentiation agent or regulatory factor to mediate differentiation of islet beta cells.
In some embodiments of the invention, the JAK2 inhibitors can be used to promote the directed differentiation of pancreatic endocrine precursor cells (pancreatic endocrine progenitor) into islet beta cells. The term "pancreatic endocrine precursor cells" as used herein refers to precursor cells having the potential to differentiate to obtain islet cells, which express a specific molecular marker of pancreatic endocrine.
In some embodiments of the invention, the JAK2 inhibitor is capable of promoting the directed differentiation of pancreatic precursor cells into islet beta cells.
According to a second aspect of the present invention there is provided the use of a JAK2 inhibitor in the manufacture of a medicament for the prevention and/or treatment of diabetes. JAK2 inhibitors can greatly improve the differentiation efficiency of pancreatic endocrine precursor cells into islet beta cells, so that the differentiation of islet beta cells in vivo can be regulated, and the JAK2 inhibitors can be used for treating diseases related to islet beta cell abnormality, for example, can be used for preventing or treating diabetes.
According to an embodiment of the present invention, the use of the JAK2 inhibitor in the preparation of a medicament for preventing and/or treating diabetes may further be characterized by the following technical features:
in some embodiments of the invention, the JAK2 inhibitor is a JAK2 protein kinase inhibitor.
In some embodiments of the invention, the JAK2 protein kinase inhibitor comprises at least one selected from CEP-33779, TG101209, TG101348.
In some embodiments of the invention, the JAK2 inhibitors can be used to promote the directed differentiation of pancreatic endocrine precursor cells into islet beta cells.
In some embodiments of the invention, the JAK2 inhibitor is capable of promoting the directed differentiation of pancreatic precursor cells into islet beta cells.
In a third aspect of the invention, the invention provides a pharmaceutical composition capable of inducing differentiation of cells into islet beta cells, the pharmaceutical composition comprising a JAK2 protein kinase inhibitor.
In some embodiments of the invention, the JAK2 protein kinase inhibitor comprises at least one of CEP-33779, TG101209, TG101348.
In some embodiments of the invention, the induced differentiation of islet beta cells described above further comprises: other pharmaceutically acceptable carriers and/or excipients.
In a fourth aspect of the invention, the invention provides a kit that can be used to promote differentiation of cells into insulin β cells, the kit comprising a JAK2 inhibitor.
According to an embodiment of the invention, the cells are selected from pancreatic endocrine precursor cells or pancreatic precursor cells.
According to an embodiment of the present invention, in the kit, the JAK2 inhibitor includes at least one selected from CEP-33779, TG101209, TG101348.
In a fifth aspect of the invention, the invention provides a method of promoting differentiation of cells into islet beta cells, comprising: the cells are cultured in contact with a JAK2 inhibitor to obtain islet beta cells.
In some embodiments of the invention, the above method of promoting differentiation of cells into pancreatic β cells may further comprise the following technical features:
in some embodiments of the invention, the cell is a pancreatic endocrine precursor cell or a pancreatic precursor cell.
In some embodiments of the invention, the cells and the JAK2 inhibitor are contacted and cultured for 5 to 7 days. The cells and the JAK2 inhibitor are continuously cultured for 5-7 days, so that pancreatic endocrine precursor cells or pancreatic precursor cells can be differentiated into islet beta cells, and the expression of the islet beta cells in vivo can be regulated. Can also be used to induce cell differentiation in vitro and thus act as an inducer.
Drawings
FIG. 1 is a flow cytometric diagram of the addition of JAK2 signaling pathway-related chemical small molecule inhibitors to boost the expression levels of islet beta cell specific molecular markers Proinsulin/instrument/C-Peptide and NKX6.1, provided in accordance with an embodiment of the present invention.
Fig. 2 is a graph of a significance analysis of the production of islet beta cells with respect to the addition of a small chemical molecule inhibitor associated with the JAK2 signaling pathway, provided in accordance with an embodiment of the present invention.
Fig. 3 is a graph showing the proportion of islet beta cells obtained by continuous addition of TG101348 for 6 days according to an embodiment of the present invention.
FIG. 4 is a graph showing the increase in the rate of islet beta cell production by addition of TG101348 according to an embodiment of the present invention.
Fig. 5 shows the content ratio of each molecular marker after TG101348 is added, wherein 5a shows the content ratio of NKX6.1 and proinsulin, insulin, C peptide after TG101348 is added, and 5b shows the content ratio of NKX6.1 and C peptide after TG101348 is added.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
The invention discovers that a JAK2 inhibitor plays an important role in the process of directional differentiation of pancreatic precursor cells (Pancreatic progenitors) into islet beta cells (pancreatic beta cells), thereby providing an application of the JAK2 inhibitor in the field of islet beta cell induced differentiation, greatly improving the differentiation efficiency of pancreatic endocrine precursor cells into islet beta cells, and expressing islet beta cell specific molecular markers Proinsulin (Proinsulin), insulin (Insulin), C Peptide (C-Peptide) and NKX6.1.
To this end, the present invention provides the use of JAK2 inhibitors in the manufacture of a medicament for the prevention and/or treatment of diabetes. According to one embodiment of the present invention, there is provided the use of a JAK2 protein kinase inhibitor in the preparation of a medicament for the prevention and/or treatment of diabetes. According to embodiments of the present invention, the JAK2 protein kinase inhibitor may be CEP-33779, TG101209 or TG101348.JAK2 inhibitors, particularly JAK2 protein kinase inhibitors, are capable of inducing differentiation of pancreatic precursor cells or pancreatic endocrine precursor cells into pancreatic islet beta cells, thereby allowing regulation of the balance of blood glucose for the prevention or treatment of diabetes.
Herein, the term "JAK2 inhibitor" means all inhibitor substances capable of acting on the JAK2 signaling pathway, which substances are capable of inhibiting the expression of signaling molecules in the JAK2 signaling pathway.
Herein, the term "JAK2 protein kinase inhibitor" means a protein kinase capable of inhibiting the activity of JAK2 protein kinase with JAK2 protein kinase as a target. According to an embodiment of the present invention, the JAK2 protein kinase inhibitor includes at least one selected from CEP-33779, TG101209, TG101348, as shown in table 1. According to an embodiment of the present invention, the JAK2 protein kinase inhibitor is at least two selected from CEP-33779, TG101209, TG101348.
TABLE 1 Structure of chemical small molecule inhibitors related to JAK2 Signal pathway and related information
According to one embodiment of the present invention, a pharmaceutical composition comprising a JAK2 protein kinase inhibitor is provided that is capable of inducing differentiation of cells into islet beta cells.
According to embodiments of the present invention, the pharmaceutical composition further comprises other pharmaceutically acceptable carriers and/or excipients. Different excipients are used for matching with the JAK2 protein kinase inhibitor to prepare different pharmaceutical compositions, and the pharmaceutical compositions can be prepared into different dosage forms according to the purpose of administration route. Examples of routes of administration include, but are not limited to: parenteral routes, such as intravenous, intradermal, subcutaneous, intramuscular, subcutaneous, oral, buccal, sublingual, inhalation, intranasal, transdermal, topical, transmucosal, intratumoral, intrasynovial and rectal administration. In particular embodiments, the compositions are formulated in accordance with conventional methods into pharmaceutical compositions suitable for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to humans. In a specific embodiment, the pharmaceutical composition is formulated for subcutaneous administration to a human in accordance with conventional methods. Typically, the compositions for intravenous administration are solutions formulated with sterile buffers. If desired, the pharmaceutical composition may also contain a solubilizing agent and a local anesthetic to reduce pain at the injection site.
The invention also provides a pharmaceutical dosage form. Examples of such pharmaceutical dosage forms include, but are not limited to: a tablet; a caplet; capsules, such as soft elastic gelatin capsules; a cachet; a lozenge; a dispersing agent; a suppository; an ointment; a paste; a powder; a dressing agent; a cream; a plaster; a solution; a patch; aerosols (e.g., nasal sprays or inhalants); gel; liquid dosage forms suitable for oral or mucosal administration to an individual, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions); a liquid dosage form suitable for parenteral administration to an individual; a solid dosage form.
Taking an oral dosage form as an example, the pharmaceutical compositions of the present invention suitable for oral administration are presented in a dispersion dosage form, including, but not limited to: tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). In certain embodiments, the oral dosage form is solid and is prepared with anhydrous ingredients under anhydrous conditions. Typical oral dosage forms of the invention are prepared by intimately mixing the active ingredient with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients may take a variety of forms depending on the form of formulation desired for administration. For example, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to: water, glycol, oil, alcohols, flavoring agents, preservatives, and coloring agents. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to: starch, sucrose, microcrystalline cellulose, diluents, granulating agents, lubricants, binders and disintegrants.
Examples of excipients that may be used in the oral dosage form of the present invention include, but are not limited to: binders, fillers, disintegrants and lubricants. Binders suitable for use in the pharmaceutical compositions and dosage forms include, but are not limited to: corn starch, potato starch or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose), polyvinylpyrrolidone, methyl cellulose, pregelatinized starch, hydroxypropyl methyl cellulose, microcrystalline cellulose, and mixtures thereof.
Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms described herein include, but are not limited to: talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextran, kaolin, mannitol, silicic acid, sorbitol, starch, pregelatinized starch, and mixtures thereof. Disintegrants are used in the compositions of the invention to disintegrate tablets when they are contacted with an aqueous environment. Tablets containing too much disintegrant disintegrate upon storage, while tablets containing too little disintegrant do not disintegrate at the desired rate or under the desired conditions. Accordingly, an appropriate amount of disintegrant that is neither too much nor too little to adversely affect the release of the active ingredients should be used to form the solid oral dosage forms of the present invention. Disintegrants that may be used in the pharmaceutical compositions and dosage forms of the invention include, but are not limited to: agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, potassium polcalide, sodium starch glycolate, potato or tapioca starch, pregelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof. Lubricants useful in the pharmaceutical compositions and dosage forms of the present invention include, but are not limited to: calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oils (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, and mixtures thereof.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
The Insulin precursors (Proinsulin, insulin (Insulin), C Peptide (C-Peptide) and NKX6.1, where NKX6.1 is a protein expressed by the NKX6.1 gene in islet beta cells, were examined by flow cytometry using CEP-3379, TG101209 and TG101348, respectively, in suspension with pancreatic precursor cells for 6 days, followed by further culture for 8 days:
1. method for differentiating pancreatic precursor cells into pancreatic endocrine precursor cells
Suspension culture methods were used to verify differentiation of pancreatic precursor cells into pancreatic endocrine precursor cells, and cultured for 6 days. CEP-3379, TG101209 and TG101348 were added to the medium containing pancreatic precursor cells, respectively, wherein the concentration of CEP-3379 was 0.05. Mu.M, the concentration of TG101209 was 0.5. Mu.M and the concentration of TG101348 was 0.2. Mu.M.
Wherein the suspension culture plate is a low adsorption six-well plate (beavirbio, cat#40406). The medium used was: DMEM (Gibco, cat#C11965500 CP) +50x vitamin A free B-27 (Gibco, cat#12587-010) +1% glutamate (Glutamax, invitrogen, cat# 35050079) +0.25mM 2 phosphorus-L-ascorbic acid trisodium salt (Phospho-L-ascorbic acid trisodium salt) (Sigma Aldrich; cat# 49752) +10μm ALK5 inhibitor II (Enzo Life Sciences; cat#ALX-270-445-M100), triiodo-L-thyronine (5-triodo-L-thronine) (Sigma, cat#T2877) +0.1μm gamma-Secretase inhibitor XX (EMD Millipore; cat#565789-1 MG).
Wherein, the culture conditions are as follows: 37℃、5%CO 2 And 88% humidity, at 90rpm.
And (3) obtaining pancreatic endocrine precursor cells through suspension culture, and then continuously adding CEP-3379, TG101209 or TG101348 into the cells to promote differentiation of the pancreatic endocrine precursor cells into islet beta cells through suspension culture.
2. Method for differentiating pancreatic endocrine precursor cells into islet beta cells
Suspension culture methods were used to verify differentiation of pancreatic endocrine precursor cells into islet beta cells. The above pancreatic endocrine precursor cells were continuously cultured in the following medium for 8 days, and whether the pancreatic endocrine precursor cells were differentiated into islet beta cells was examined.
Among them, the suspension culture plate was a low adsorption six-well plate (beavirbio, cat#40406). The medium used was: DMEM (Gibco, cat#C11965500 CP) +50Xcontains B-27 without vitamin A (Gibco, cat#12587-010) +1% Glutamax (Invitrogen, cat# 35050079) +10. Mu.M ALK5 Inhibitor II (Enzo Life Sciences; cat#ALX-270-445-M100). Through flow cytometry detection, the islet beta cells can be obtained after culturing for 4-8 days.
3. Flow cytometry detection method
The detection methods of Insulin precursors (Proinsulin), insulin (ins), C-Peptide (C-Peptide) and NKX6.1, which are specific molecular markers of islet beta cells, are as follows:
the differentiated cell pellet was digested with pre-warmed Ackutase cell digest cell isolate (Sigma, A6964-100 ML) for 5-10 min to single cells, washed once with PBS, and fixed with 200. Mu.L of fixed permeate (Fixation and Permeabilization Solution, BD; cat # 554722) for 20min.
Then 200. Mu.L BD permeate washing solution (BD Perm/Wash TM Buffer, cat # 554723) was washed twice and incubated with primary antibody for 2 hours at 4 ℃, wherein the primary antibody was formulated as follows: 200. Mu.L BD permeate washing solution (BD Perm/Wash) TM Buffer) +1 μl murine anti-NKX6.1 (mouse anti-NKX6.1, DSHB; cat#F55A12) +1μl rabbit C peptide antibody (Rat C-pep antibody, DSHB; cat#GN-ID 4).
After the incubation, 200. Mu.L BD permeate washing (BD Perm/Wash) TM Buffer) for two times by using fluorescenceThe light secondary antibody was incubated at 4 ℃ for 1 hour in the absence of light, wherein the fluorescent secondary antibody was formulated as follows: 200 mu L BD Perm/Wash TM Buffer+monkey anti-rabbit antibody with fluorescent dye Alexa Fluor 488 (Life Technologies; cat#A-21208) (Donkey anti-Rat IgG (H+L) Secondary Antibody) +Alexa with fluorescent dye647 conjoint (Life Technologies; cat#A-31571) monkey anti-Mouse antibody (Donkey anti-Mouse IgG (H+L) Secondary Antibody).
After incubation, 200. Mu.L BD Perm/Wash was used TM Buffer washing was performed twice, followed by 200. Mu.L BD Perm/Wash TM Buffer resuspension, transfer to flow tube, detection with flow cytometer (BD Calibur), flow analysis of results with FlowJo software.
The experimental results are shown in FIG. 1. As can be seen from fig. 1, the addition of a JAK2 signaling pathway-associated small chemical molecule inhibitor effectively increased the expression levels of islet beta cell specific molecular markers, proinsulin/instrument/C-Peptide and NKX6.1 (mainly as demonstrated by the increased levels of molecular markers located in Q2 and fluorescence intensity) compared to the control. As inhibitors of JAK2, CEP-33779, tg101209 and TG101348 were all able to effectively increase the differentiation efficiency of pancreatic endocrine precursor cells into islet beta cell directed induction figure 1.
Meanwhile, the proportion of JAK2 inhibitor to generate islet beta cells in the process of promoting directional differentiation of pancreatic endocrine precursor cells to islet beta cells is remarkably analyzed, as shown in figure 2. Wherein, both TG101209 and TG101348 can obviously improve the proportion of islet beta cells.
Furthermore, we studied the case where addition of TG101348 at different time nodes induced pancreatic endocrine precursor cells to produce islet β cells. Taking TG101348 as an example, as shown in fig. 3, the ordinate in fig. 3 represents different treatment times, where "1" represents one day of treatment with TG101348, "1-2" represents two days of treatment with TG101348 added on the first day, and so on "1-3" represents three days of treatment with TG101348 added on the first day. Likewise, "2-5" represents the addition of TG101348 treatment on the next day until the fifth day. As can be seen from the results presented in fig. 3, it was found that the proportion of islet β cells obtained was highest when the treatment was continued for 6 days with TG101348.
Example 2
At the same time we assessed the effect of JAK2 inhibitors on insulin cleavage, with no JAK2 inhibitors added as controls. The insulin shear test method used was as follows:
the differentiated cell pellet was digested with pre-warmed Ackutase cell digest cell isolate (Sigma, A6964-100 ML) for 5-10 min to single cells, washed once with PBS buffer, and fixed with 200. Mu.L of fixed permeate (Fixation and Permeabilization Solution) for 20min.
Then 200. Mu.L BD permeate washing solution (BD Perm/Wash TM Buffer) was washed twice and incubated with primary antibody for 2 hours at 4 ℃, wherein primary antibody was formulated as follows: 200. Mu.L BD permeate washing solution (BD Perm/Wash) TM Buffer) +1 μl murine anti-NKX6.1 (mouse anti-NKX6.1, DSHB; cat#F55A12) +1. Mu.L of a Pig anti-C peptide antibody (Pic anti-C Peptide antibody, abcam; cat#ab 30477).
After incubation, 200. Mu.L BD Perm/Wash was used TM Buffer wash twice, incubate with fluorescent secondary antibody at 4 ℃ in the dark for 1 hour, wherein the fluorescent secondary antibody is formulated as follows: 200 mu L BD Perm/Wash TM Buffer + with fluorescent dye AlexaDonkey anti-Mouse antibody (Donkey anti-Mouse IgG (H+L) Secondary Antibody) +Alexa depictinga fluorescent dye of 488conjugate (Life Technologies; cat#A-21202)>647conjugate (Life Technologies; cat#A-21450) Goat anti-Pig antibody (gold anti-Guinea Pig IgG (H+L) Secondary Antibody).
After incubation, 200. Mu.L BD Perm/Wash was used TM Buffer washing was performed twice, followed by 200. Mu.L BD Perm/Wash TM Buffer resuspension, transfer to flow tube, and perform with flow cytometer (BD Calibur)Detection, flow result analysis was performed with FlowJo software.
As shown in the flow result diagrams in fig. 4 and 5, compared with a control, the TG101348 is added to improve the generation ratio of islet beta cells and promote the shearing of proinsulin to insulin, so that the differentiated islet beta cells tend to be more functionally mature.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (2)
- Use of a JAK2 protein kinase inhibitor for the preparation of a medicament for preventing and/or treating diabetes, the JAK2 protein kinase inhibitor promoting differentiation of pancreatic endocrine precursor cells or pancreatic precursor cells into pancreatic islet beta cells, the JAK2 protein kinase inhibitor being selected from at least one of CEP-33779, TG101209, TG101348.
- 2. A method for promoting differentiation of cells into islet beta cells for non-therapeutic purposes, comprising:culturing the cells in contact with a JAK2 inhibitor for 5-7 days to obtain islet beta cells; the JAK2 inhibitor is a JAK2 protein kinase inhibitor;the cells are pancreatic endocrine precursor cells or pancreatic precursor cells; the JAK2 protein kinase inhibitor is at least one selected from CEP-33779, TG101209 and TG101348.
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