CN115948323A - Islet cell inducer, culture medium, method for obtaining islet beta cells and application of islet cell inducer and culture medium - Google Patents

Abstract

The invention discloses an islet cell inducer, a culture medium, a method for obtaining islet beta cells and application of the islet cell inducer and the culture medium, and belongs to the technical field of biomedicine. To provide a method for drug development using a small molecule compound. The invention provides a method for treating epithelial organs (EPOs) by using astragaloside materials, which can discover that the astragaloside monomer in the astragalus extract can remarkably promote the EPOs to generate islet cells, remarkably promote the expression of islet cell related genes, particularly insulin genes, the generated islet cells can respond to glucose stimulation to secrete insulin, and the hyperglycemia symptom of a diabetic mouse is relieved, and the generated insulin secreting cells have the physiological function of mature beta cells. The method can be used for preparing artificial islet system transplantation for treating diabetes.

Description

Islet cell inducer, culture medium, method for obtaining islet beta cells and application of islet cell inducer and culture medium

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to an islet cell inducer, a culture medium, a method for obtaining islet beta cells and application of the islet cell inducer and the culture medium.

Background

Diabetes Mellitus (DM) is a type of chronic metabolic disease characterized by hyperglycemia, and the resulting sustained high levels of blood glucose can cause widespread damage to the heart, kidneys, and neural tissues of a patient, leading to a variety of complications. Diabetes has become a worldwide important public health problem, and has a serious impact on economic development.

Islet transplantation is an effective method for treating diabetes, but is limited by the serious shortage of pancreas donors, and how to obtain functional islet cells is one of the problems facing the current treatment of diabetes by using cell replacement therapy. Wherein the successful construction of a pancreatic organoid culture system can be used to solve the problem of scarce sources of organ transplant donors. Organoids (organoids) refer to tissues that are similar in structure and function to the original organs by using the pluripotency of stem/progenitor cells, which are cultured in vitro to continually self-renew and self-organize. Hans Clever and the like successfully establish a pancreatic duct organoid based on adult stem cells and organoid technology, and can cause the pancreatic duct organoid to be endocrine and differentiate into insulin positive cells, but the differentiation efficiency is limited at present, so how to increase the pancreatic beta cell differentiation efficiency is an important problem to be solved urgently.

The traditional Chinese medicine Astragalus is a traditional common traditional Chinese medicine in China, the medicinal history of the traditional Chinese medicine Astragalus has been for more than 2000 years so far, and the traditional Chinese medicine Astragalus is a dried root of leguminous plant Astragalus astragaluses membrane araceus (Fisch.) bge. Modern pharmacological studies prove that the astragalus root mainly contains chemical components such as saponin, flavone and polysaccharide, and also contains various amino acids, folic acid, selenium, zinc, copper and other trace elements, and has the functions of enhancing the immune function of an organism, protecting the liver, promoting urination, resisting aging, resisting stress, reducing blood pressure and having wide antibacterial effect. Researches prove that the astragaloside contained in the astragalus has the effects of improving insulin resistance, regulating lipid metabolism and glycogen metabolism, relieving diabetic complication nephropathy and the like, and becomes an important direction for treating diabetes in recent years.

Disclosure of Invention

The invention aims to provide a method for obtaining islet cells in vitro by using astragaloside small molecules and epithelial cells. It is still another object of the present invention to provide a method for preparing artificial islets and for in vivo application in the treatment of diabetes, which comprises a physiologically functional plurality of islet cells, particularly beta cells.

The invention provides an inducer for islet cells, wherein the effective component of the inducer is astragaloside.

Further defined, the islet cells are islet alpha cells, islet beta cells, islet delta cells, or islet pp cells.

Further defined, the inducer is effective as any one or more of astragalosides I-VIII, isoastragalosides I, II and IV, acetylastragalosides E, F and G, agroaastragalosides I-IV and soyasaponin I, and derivatives thereof.

Further, the effective component of the inducer is isoastragaloside I.

The invention provides a culture medium for producing islet cells, which contains astragalosides.

Further defined, the culture medium further comprises an organoid proliferation medium.

Further defined, the islet cells are islet alpha cells, islet beta cells, islet delta cells, or islet pp cells.

Further defined, the medium contains isoastragaloside I.

Further limit, the concentration of the astragaloside is 0.1-8 μ M.

The invention provides a method for obtaining islet beta cells, which comprises the step of culturing epithelial cells in a culture medium containing astragaloside to differentiate so as to obtain the islet beta cells.

Further defined, the epithelial cells are human or mammalian liver, biliary, pancreatic, gastric, and intestinal epithelial cells.

Further limited, the effective components of the system of the astragalosides are any one or more of astragalosides I-VIII, isoastragalosides I, II and IV, acetyl astragalosides E, F and G, agroastragaloside I-IV and soyasaponin I and derivatives thereof.

Further, the effective component of the system of the astragalosides is isoastragaloside I.

Further limit, the concentration of the astragaloside is 0.1-8 μ M.

The present invention provides an islet β cell obtained by the method for obtaining an islet β cell described above.

The invention provides application of the islet beta cells in preparation of a cell medicament for treating diabetes.

Further defined, the dosage form of the cellular medicament comprises an injection.

Further defined, the cellular drug comprises an extracellular matrix compatible with the cells and a pharmaceutical carrier.

Further defined, the pharmaceutical carrier is a nanomaterial or a microfluidics. Further defined, the culture medium further comprises an organoid proliferation medium or a basal medium.

The basic culture medium is Advanced DMEM/F12, the cell culture basic additive components comprise glutamine and antibiotics, wherein the using concentration of 1 Xglutamine in the Advanced DMEM/F12 is 2mM, the antibiotics are penicillin-streptomycin, the using concentration of penicillin is 100U/mL, and the using concentration of streptomycin is 0.1 mg/mL.

Wherein the organoid proliferation medium comprises at least 1 selected from the group consisting of Wnt agonist, transforming growth factor-beta (TGF- β) inhibitor, epidermal Growth Factor (EGF), and Fibroblast Growth Factor (FGF), on a basal medium basis.

A Wnt agonist may be selected from one or more molecules consisting of the following: wnt1, wnt2b, wnt3a, wnt4, wnt5a, wnt5b, wnt6, wnt7a, wnt7b, wnt8a, wnt8b, wnt9a, wnt9b, wnt10a, wnt10b, wnt11, wnt16, R-spondin1.

BMP signaling pathway inhibitors include Noggin, dorsomorphin, LDN189, DMH-1, and combinations thereof, preferably Noggin.

The FGF comprises: FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, and combinations thereof, preferably FGF10.

Further defined, the medium comprises an extracellular matrix.

The Extracellular matrix (ECM) contains a wide variety of polysaccharides, water, elastin, and glycoproteins, such as collagen, nestin, fibronectin, laminin.

Further defined, the islet cells are islet alpha cells, islet beta cells, islet delta cells, or islet pp cells.

The invention provides a method for obtaining islet beta cells in vitro, which is characterized in that epithelial cells are differentiated in a system containing astragaloside to obtain the islet beta cells.

Further defined, the epithelial cells are human or mammalian liver, biliary, pancreatic, gastric, and intestinal epithelial cells.

Epithelial cell-initiating cells seeded in extracellular matrix, 5% CO at 37 ℃ in EM medium ₂ After 5 days of culture in an incubator, organoids are formed and can be continuously subcultured and cryopreserved.

Further, the astragalosides are any one or more of astragalosides I-I, isoastragalosides I, II and IV, acetylastragalosides E, F and G, agrogalosides I-IV and soyasaponin I, and derivatives thereof.

Further limit, the concentration of the astragaloside is 0.1-8 μ M.

The invention provides application of the islet beta cells obtained by the method in-vitro preparation of cell medicines for treating diabetes.

The invention provides application of the method for preparing the islet beta cells in preparing a medicament for inducing the islet beta cells in an animal body.

Further defined, the dosage form of the medicament comprises an injection.

In a further aspect, the injectable formulation includes sodium chloride, magnesium chloride, calcium sodium edetate and water for injection.

Further defined, the cellular drug further comprises a pharmaceutically acceptable carrier compatible with the cells.

Further limited, the medicine also contains medically acceptable auxiliary materials, and the medically acceptable auxiliary materials comprise adhesives, fillers, disintegrating agents, lubricants, antioxidants, flavoring agents, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators and colorants. A cell and a physiologically acceptable excipient or diluent including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, ethylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

Further defined, the medicament also contains pharmaceutically acceptable carriers, including extracellular matrices, which are a wide variety of polysaccharides, water, elastin, and glycoproteins, such as collagen, entactin, fibronectin, laminin, nanomaterials, and microfluidics. The nanometer material is selected from mesoporous silicon, liposome, polylactic-co-glycolic acid (PLGA), albumin, and exosome.

The invention provides an inducer for inducing epithelial cells, which contains astragaloside.

The invention provides a method for inducing epithelial cells to differentiate, which is to differentiate the epithelial cells in a system containing astragaloside.

Further defined, the epithelial cells are human or mammalian liver, biliary, pancreatic, gastric, and intestinal epithelial cells.

Further limited, the astragalosides are any one or more of astragalosides I-I, isoastragalosides I, II and IV, acetylastragalosides E, F and G, agrogalosides I-IV and soyasaponin I, and any one or more of derivatives thereof.

Further limit, the concentration of the astragaloside is 0.1-8 μ M.

A chemical small molecule composition comprising astragaloside, the chemical small molecule composition being a pharmaceutical composition, further comprising: a pharmaceutically acceptable carrier or excipient; preferably, the carrier or excipient comprises one or more selected from the group consisting of: water, saline, phosphate buffer, or other aqueous solvents; DMSO, glycerol, and ethanol or other organic solvents; microspheres, liposomes, microemulsions or polymeric surfactants; a colloidal drug delivery system or a polymer drug delivery system; preservatives, antioxidants, flavouring agents, fragrances, cosolvents, emulsifiers, pH buffering substances, binders, fillers, lubricants or other pharmaceutical excipients; or the chemical small molecule composition can be prepared into a pharmaceutical dosage form comprising: solid dosage forms, including powders, tablets, pills, capsules, sustained release agents, controlled release agents, or other solid dosage forms; liquid dosage forms, including injections, infusions, suspensions, or other liquid dosage forms; a gaseous formulation; or a semi-solid dosage form.

Comprises the application of the astragaloside in preparing preparations, reagents or culture media of islet cells; or used for preparing in vivo induced in situ epithelial cell differentiation into islet cells, and in situ differentiated pancreas duct cells promoting islet beta cell regeneration, reducing blood sugar, and treating diabetes related diseases (such as diabetic foot, diabetic nephropathy, poor skin healing caused by diabetes, diabetic cardiovascular disease, etc.); or for preparing islet cells for inducing epithelial cell differentiation in vitro.

The application of the chemical micromolecules comprising the astragaloside can be used for preparing a medicine box or a kit for inducing in-vivo and in-vitro islet cells, or a pharmaceutically acceptable carrier or an excipient is added based on the micromolecules, so that the micromolecules are used for inducing in-situ or gastrointestinal tract cells to be directly converted into islet cells in vivo, and tissue/organ pathological changes are reduced or reduced by the in-situ differentiation effect of the islet cells, so that the astragalus saponin can be developed or prepared into a medicine or a prodrug/medicine composition for treating clinical diabetes (such as diabetic foot, diabetic nephropathy, poor skin healing caused by diabetes, diabetic cardiovascular diseases and the like); or a differentiation medium or a kit for inducing the epithelial cells to directly differentiate into the islet cells, which is prepared by adding carriers or excipients such as an organic solvent/physiological saline/buffer solution/a cell basic medium and the like.

A kit or kit comprising astragalosides, said epithelial cells including but not limited to: human or mammalian gastrointestinal epithelial cells; including but not limited to human: extrahepatic and intrahepatic bile duct epithelial cells, pancreatic ductal epithelial cells, gastric epithelial cells, small and large intestine epithelial cells or epithelial cells of other tissues or organs of the human body; more preferably human pancreatic and bile duct epithelial cells.

Has the advantages that: the invention discloses astragalosides and a method for inducing intrahepatic bile duct epithelium, extrahepatic bile duct epithelium, pancreatic duct epithelium, gastric epithelium and intestinal epithelium to generate islet cells. By constructing epithelial organoids (EPOs), and treating the EPOs with astragalosides substances, the isoastragaloside I in the astragalus extract can obviously promote the differentiation of the EPOs and the expression of islet cell related genes, and the up-regulated expression of a pancreatic endocrine progenitor cell marker ngn3 is observed in the differentiation promotion process. The differentiated cells can respond to glucose stimulation, improve the blood sugar and glucose tolerance of diabetic mice, and have the physiological function of mature beta cells.

The isoastragaloside I can remarkably promote the expression of Mouse pancreatic ductal organoids (mPDOs) Insulin related genes Insulin 1 and Insulin 2, and the beta Cell differentiation ratio can reach 45 percent, which is improved by 30 times compared with the differentiation efficiency of 1.5 percent reported in the prior literature (Loomans CJM, et al, stem Cell Reports 2018. The secretion of the synthetic insulin marker C peptide can be detected by mPDOs after differentiation under the stimulation of glucose, and the physiological response of the synthetic insulin marker C peptide is proved to be the same as that of mature beta cells. The addition of the isoastragaloside I with the concentration lower than 8 mu mol/L has no influence on organoid growth and cell activity, and proves that the isoastragaloside I is an active astragalus molecule which is safe and effective and has good application potential. Meanwhile, the results of in vivo transplantation of human biliary tract organoids (hBDOs) pretreated by the isoastragaloside I prove that the human biliary tract organoids have the in vivo activity effects of relieving hyperglycemia symptoms of diabetic mice, improving glucose tolerance and the like. The isoastragaloside I has a remarkable effect of promoting beta cell differentiation when being singly added into an organoid culture medium, and the fact that the isoastragaloside I has more comprehensive effects of promoting pancreas regeneration and the like in vivo is suggested.

Drawings

FIG. 1 is the establishment of mouse pancreatic ductal organoids (mPDOs). FIG. 1A is a diagram of mPDOS setup mode; FIG. 1B is a diagram showing the state of cells at 5 th, 10 th, 14 th, 1 st and 10 th days in mPDOs culture;

FIG. 2 shows the use of RIP-cre; a PDOs model prepared from a Rosa26-mTmG hybrid mouse screens the astragalus saponin single molecules. FIG. 2A shows RIP-cre; a schematic diagram of the genetic background of the Rosa26-mTmG animal; FIG. 2B shows RIP-cre; cellular fluorescence images of Rosa26-mTmG PDOs added with radix astragali extract; FIG. 2E shows RIP-cre; adding Rosa26-mTmG PDOs into radix astragali extract, and quantifying; FIG. 2F shows RIP-cre; Q-PCR picture of Rosa26-mTmG PDOs added with radix astragali extract;

FIG. 3 is the structure of isoastragaloside I and its safety to mPDOs. FIG. 3A is the chemical structural formula of isoastragaloside I; FIG. 3B is a CCK8 chart for detecting mPDOS activity after different concentrations of isoastragaloside I are added;

FIG. 4 shows that isoastragaloside I can effectively promote mPDOs to differentiate into pancreatic cells in vitro. FIG. 4A is a Q-PCR image after adding isoastragaloside I and culturing mPDOs for 14 days; FIG. 4B is an immunostaining pattern of mPDOs cultured with isoastragaloside I for 14 days;

FIG. 5 shows that isoastragaloside I can effectively promote mPDOS to differentiate into functional beta cells in vitro. FIGS. 5A and B show the flow cytometry detection of C-pepti after adding isoastragaloside I and culturing mPDOs for 14 daysde ⁺ The proportion of cells; FIG. 5C shows the addition of isoastragaloside I, after 14 days of mPDOS incubation, glucose stimulation, ELISA for C-peptide secretion;

FIG. 6 is the establishment of human hepatobiliary pancreatic duct organoids. FIG. 6A is a schematic diagram of the establishment of human liver, gallbladder and pancreatic duct organs (hLDOs; hBDOs; hPDos); FIG. 6B shows the cell state diagrams of hBDOs at day 3, day 7, day 14, generation 5, and generation 20;

FIG. 7 shows the in vivo transplantation of hBDOs differentiated by isoastragaloside I induction to treat diabetes. FIG. 7A is a graph of the change in blood glucose after transplantation of hBDOs treated with isoastragaloside I to STZ induced renal cysts in diabetic mice; fig. 7B and C are graphs of mouse glucose tolerance changes following transplantation of hBDOs treated with isoastragaloside I; FIG. 7D is the change in the levels of Insulin in the sera of mice.

Detailed Description

"EM medium" means a basal cell culture medium supplemented with a Wnt signal transduction activator and a TGF β receptor inhibitor, and a growth factor for promoting organoid formation, for example, D/F12 medium, DMEM medium or MEM medium containing 2% of B27, 1% of Glutamax,1% of N2 supplement, A8301, noggin, nicotinamide and N-acetyl-L-cysteine, and with a cytokine such as EGF, R-spondin1, FGF10, PGE2, gastrin.

"EPOs" refers to organoids derived from the culture of epithelial cells from the gastrointestinal tract of hepatobiliary, pancreatic, hepatobiliary and pancreatic; "mPDOS" refers to organoids derived from the culture of mouse pancreatic ductal cells; "hBDOs" refers to organoids derived from the culture of human biliary epithelial cells; the organoids described above can be in primary culture (e.g., an undelivered culture), or can be in secondary or subsequent subcultures (e.g., a population of cells that have been subcultured or passaged one or more times).

"transformation" refers to the transformation of pancreatic ductal cells from a unipotent state to a pluripotent state.

"marker" refers to a Biomarker (Biomarker) that can mark a system, organ, tissue, cell, and subcellular structure, or a change in function or biochemical marker that may be altered. In the pancreatic endocrine cells, the beta cell marker is selected from insulin, the alpha cell marker is glucagon, the delta cell marker is somatostatin, and the pancreatic islet PP cell marker is pancreatic polypeptide.

"pancreatic somatic cells" refers to the types of pancreatic cells present in pancreatic tissue, including pancreatic ducts, islets, acinar cells. In a preferred embodiment, pancreatic ductal cells. The cells can be obtained from a mammal such as a human, monkey, pig, or the like, which can be transplanted from a donor.

Healthy Nu/Nu mice of 7-8 weeks of age were purchased from RIP-cre, a Beijing Wintolite laboratory animal technology, inc.; rosa26-mTmG hybrid mice were purchased from The Jackson Laboratory. Laboratory animal feeding and use was approved by the university of northeast forestry animal ethics committee, fed as required by the SPF-scale laboratory animal house, subjected to 12 hours of light/dark cycles at room temperature, and regularly fed with food and water by facility staff.

The astragalosides are purchased standard products, and can also be extracted and separated by water extraction, alcohol extraction, microwave-assisted purification, microwave-assisted water extraction, pre-column derivatization, resin adsorption, supercritical CO ₂ Extraction, ultrafiltration, and the like.

Pancreatic progenitor cells, embryonic stem cells, neural progenitor cells, bone marrow mesenchymal stem cells, liver stem cells, umbilical cord blood cells, blood-derived endometrial stem cells and dental pulp mesenchymal stem cells can be induced to differentiate into islet beta cells in vitro.

According to different methods for inducing islet-like cells in vitro, inducers are currently roughly divided into three categories, 1) cytokines such as EGF, bFGF, extendin-4, etc.; 2) Organic compounds such as nicotinamide, retinoic acid, DMSO, beta-mercaptoethanol, and the like; 3) Conditioned medium, such as rat regenerated pancreas extract.

In vitro induction of islet-like cells is a protocol that is differentiated by the presence and absence of serum, and by the induction step, and the like, and is typically represented by the "one-step" to "four-step" methods.

The one-step induction method mainly adopts an in-vitro induction culture medium with the same components to induce and differentiate the islet-like cells. For example, the mesenchymal stem cells are induced to be islet-like cells in vitro using serum-free medium, 0.6% glucose, 100. Mu.g/mL transferrin, 20nM progesterone, 25. Mu.g/mL insulin, 30nM selenium chloride, 60. Mu.M butanediamine, 2mM glutamine, 3mM sodium bicarbonate, 5mM HEPES buffer, 2. Mu.g/mL heparin, 20ng/mL EGF,20ng/mL bFGF,20ng/mL HGF,10mM nicotinamide and 100ng/mL activin A are added to D/F medium, and the cells are induced in vitro after 14 days.

"two-step induction method" means that the whole induction in vitro process is divided into two steps, such as inducing differentiation of bone marrow mesenchymal stem cells of rats directed to islet-like cells in vitro, wherein 10mmol/L nicotinamide and 1mmol/L β -mercaptoethanol are added in the first step, 20% FBS is induced for 24h in low sugar DMEM medium, 10mmol/L nicotinamide and 1mmol/L β -mercaptoethanol are added in the second step, and induction is continuously performed for 10h in serum-free high DMEM medium, and finally, in vitro induction is completed.

In the "three-step induction method", 1% of BSA was added to the D/F medium in the first step, 1% of PSA, activin A, ITS-X, sodium butyrate and β -mercaptoethanol; adding 1% BSA to the D/F medium in the second step, 1% PSA, ITS-X and taurine; in the third step 1% BSA was added to the D/F medium, 1% PSA, ITS-X, nicotinamide, taurine and GLP-1. After the three steps of in vitro induction, insulin-like secretory cells are finally formed.

In the "four-step induction method", the cells were treated for 24 hours by adding 0.2% FBS,2 ng/mL Wnt3A and 100ng/mL Activin-A, followed by adding 0.3% FBS and 100ng/mL Activin-A for 24 hours; in the second step, 2% FBS was added, 50ng/mL FGF was added, and induction was continued for 3 days; in the third step, 0.25. Mu. Mol/L SANT-1, 2. Mu. Mol retinoic acid, 100ng/mL Noggin and 1% of B27 are continuously induced for 4 days; in the fourth step, 100ng/mL Noggin,50nmol/L TPB and 1 mu mol/LALK5 inhibitor are added for continuous induction for 4-5 days, and finally in vitro induction can form islet-like cells. In conclusion, for the mesenchymal stem cell in vitro induction of the directional islet-like cells, the method has more usable inducers and more varieties, and the in vitro induction standard of the unified standard and the generally accepted inducers and unified induction steps are not existed internationally.

After continuously stimulating NOD mice, human glucagon-like peptide-1 (GLP-1) can promote differentiation and proliferation of pancreatic duct epithelial cells.

By adopting a low-sugar DMEM/F12 culture medium, D/F12 can keep the pH of the culture solution stable, and is more beneficial to the growth of epithelial cells compared with the culture media such as 1640. Glucose is the most important factor for stimulating gene expression in pancreatic islet beta cells, but its concentration too high may affect insulin expression and affect pancreatic islet beta cell differentiation.

The growth factor can not only accelerate cell division and enhance activity, but also promote the cell division into insulin secreting cells, for example, FGF7 is a mitogen, can promote the growth and rapid propagation of pancreatic duct epithelial cells, and also has the function of promoting the cell division into pancreatic stem cells; DMEM/F12 medium in combination with niacinamide promotes the growth of rat and pig ductal cells in vitro.

As commercially available ECMs, there are extracellular matrix proteins (Invitrogen), basement membrane preparations derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g., matrigel (BD biosciences)), and the like. ECM can be synthesized using ProNectin (SigmaZ 378666) or the like. In addition, mixtures of natural and synthetic ECMs may be used. Hydrogels, hydrogels for three-dimensional organoid culture, comprise hydrogels selected from hydrogels of polypeptides from Yeasen and the like commercially available for preparing organoids.

Example 1A Medium for culturing islet cells

Human expansion medium (hEM) formulation: 1% Penicillin-Streptomyces Solution (HyClone; SV 30010), 1% glutamine additive (gibco; 35050-061), 2% B27 with out vitamin A (Thermo; 12587010), 50ng/mL EGF (MCE; HY-P7067), 50ng/mL FGF10 (PeproTech; 100-26-25), 25ng/mL NOGGIN (PeproTech; 120-10C-20), 1mol/L N2 (Thermo; 17502001), 10nM Gastrin (SIGMA; G9145), 3 μ M PGE2 (SIGMA; P0409), 5 μ M A83-01 (MCE; HY-10432), 1mol/L Nicotinamide (Sigma; N0636), 1mol/L N-acetyl-L-cysteine (Sigma; A9165), 100ng/mL R-Spinmycin 1 (R & SpinD), 50% H/S7112 (Hy-711H) and 1 mL);

mouse expansion medium (moem) formulation: 1% Penicillin-Streptomyces Solution (HyClone; SV 30010), 1% glutamine additive (gibco; 35050-061), 2% B27 with out vitamin A (Thermo; 12587010), 50ng/mL EGF (MCE; HY-P7067), 50ng/mL FGF10 (PeproTech; 100-26-25), 25ng/mL NOGGIN (PeproTech; 120-10C-20), 1mol/L Nicotinamide (Sigma; N0636), 1mol/L N-acetyl-L-cysteine (Sigma; A9165), 100/mL R-Spondin-1 (R & D; 7150-RS), plus D/F12 to 40mL (HyClone; SH 30022.01); the EM culture medium contains one or more of astragalosides I-I, isoastragalosides I, II and IV, acetyl astragalosides E, cycloastragalosides F and G, agroastralosides I-IV and soyasaponin I and derivatives thereof with final concentration of 0.5, 1,2, 4 and 8 mu M.

Example 2A method for obtaining pancreatic islet beta cells

The method for obtaining the islet beta cells in vitro comprises the following steps:

1. culturing mouse pancreatic duct organoid

Mouse pancreatic ductal epithelial cells were initially seeded in extracellular matrix, using mEM medium at 37 deg.C, 5% ₂ Organoids were formed after 5 days of culture in incubator conditions.

(1) Primary culture of Mouse pancreatic ductal organoids (mPDOs): the extra-hepatic bile duct (PD) of the mouse was directly peeled off under a dissecting mirror by a mechanical method, and the PD obtained was placed in an EP tube containing collagenase IV and cut into pieces using a surgical scissors. Digesting for 20min at 37 ℃, taking out every 5min, repeatedly blowing and beating with a 1mL gun head, and stopping digestion on ice. The supernatant was discarded by centrifugation, washed 3 times with sterile PBS, and the digested cells were resuspended in Matrigel (Corning, 54234), and then dropped into a 24-well plate and allowed to stand at 37 ℃ for 5min to coagulate Matrigel. Each well was cultured by adding 800. Mu.L of mouse expansion medium (mEM). The specific operation steps are shown in FIG. 1A.

The cells obtained by the method are cultured in Matrigel, obvious organoid structure formation can be observed about 5 days, the cell density of mPDOs is obviously increased about 10 days along with the prolonging of the culture time, the cells are cultured for about 14 days until the cells are fully grown, and subculture and cryopreservation can be carried out.

(2) Subculturing of Mouse pancreatic ductal organoids (mPDOs): organoids were resuspended by blowing them into a pre-cooled high-glucose DMEM medium (HyClone; SH 30022.01), the supernatant was centrifuged off, and the pellet was subcultured using Matrigel and mEM. Passaging was performed every 7 to 8 days at a ratio of 1.

(3) Cryopreservation of Mouse pancreatic ductal organoids (mPDOs): the organoids were crushed and resuspended in precooled high-glucose DMEM medium (HyClone; SH 30022.01), after centrifugation to remove the supernatant, 1mL of precooled organoid cryopreservation solution was added, the resuspended organoids were transferred to a cell cryopreservation tube, the cryopreservation tube was placed in a cryopreservation box, then transferred to a-80 ℃ freezer overnight, and the cryopreservation tube was transferred to liquid nitrogen for storage the next day.

The mPDOs obtained in this study were passable for a long period of time, had stable proliferation and passability, and could maintain a good cell state even when the cells were passed to the 10 th passage, as shown in FIG. 1B. The structural formula of isoastragaloside I is shown in figure 3A.

Respectively utilizing EM culture medium containing one or more of astragalosides I-VIII, isoastragalosides I, II and IV, acetyl astragalosides E, cycloastragalosides F and G, agroastralosides I-IV and soyasaponin I and one or more of derivatives thereof with final concentration of 0.1, 0.5, 1,2, 4 and 8 mu M to culture the human single-tube epithelial cells for 14 days, and finally obtaining the islet cells.

The following experiments were used to verify the effect of the experiment:

(1) And (3) measuring fluorescence intensity: RIP-cre; mPDOS prepared from Rosa26-mTmG hybrid mouse at 1X10 ³ The density of the protein is paved in a 96-well plate, and RIP-cre is cultured by using mEM containing different astragalus extracts with concentration gradient; rosa26-mTmG PDOAfter s 14 days, the plates were placed in a TECAN microplate reader to measure the EGFP fluorescence intensity, ex/Em λ of EGFP: 488/507. RIP-cre without radix astragali extract; rosa26-mTmG PDOs were used as controls to calculate the percent fluorescence intensity.

As a result: according to the principle of cell lineage tracing, RIP-cre is utilized; rosa26-mTmG hybrid mice prepared mPDOs with an insulin promoter driving GFP expression, and established a mouse insulin expression fluorescence reporter system (FIG. 2A), wherein when the cells do not express an insulin gene, the cells express red fluorescent protein, and when added molecules promote the differentiation of mPDOs to beta cells (insulin-secreting cells), the differentiated cells can specifically express green fluorescence. According to the fluorescence change of RIP-cre-mTmG PDOs (shown in figure 2E-F), isoastragaloside I is screened from different astragaloside monomers.

(2) And (3) detecting the activity of the cells: the mPDOS was digested to single cells with trypsin and resuspended at 1X10 using precooled Matreigel ³ The density of each well is paved in a 96-well plate, mEM culture medium containing 0, 0.5, 1,2, 4, 8 and 10 mu M of isoastragaloside I is added, and CCK8 detection is carried out after 14 days of culture. On the day of detection, the medium in the 96-well plate was discarded, the Cell Counting Kit-8 reagent (bimake; B34302) and D/F12 were mixed well at a ratio of 1.

As a result: in order to determine the safe concentration for inducing mPDOS differentiation by using isoastragaloside I, the effect of isoastragaloside I on cell activity was detected by using CCK8, and it was found that the drug with concentration of 1-8 μmol/L did not have any effect on cell growth, and when the concentration reached 10 μmol/L, isoastragaloside I could significantly inhibit cell proliferation activity, indicating that the aforementioned 1 μmol/L concentration was very safe (FIG. 3B).

(3) Fluorescent quantitative PCR: after adding isoastragaloside I to mPDOs for 14 days, the mixture is collected by centrifugation and cleaved in TRIZOL to extract total RNA. Using Primerpcript ^RT The master kit (Vazyme, R323-01) inverts total RNA to cDNA. The reaction system and conditions were as described in the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) (see Table 1 for primers) and determined using Roche Light Cycle 480 fluorescenceAnd measuring the PCR by using a PCR instrument. Analysis Ct values were calculated by Abs Quant/2nd Derivative Max using the Light Cycler 480 self-contained software analysis module and using 2 ^-ΔΔCt The relative expression amount of mRNA was calculated.

TABLE 1 mouse primer sequences

(4) Immunostaining: after culturing mDOs with mEM containing 1. Mu.M of isoastragaloside I (MCE, HY-N0887) for 14 days, mDOs was washed with PBS, fixed with 4% PFA for 20min, repeated washing with cold PBS 3 times, the cell suspension was dropped onto the slide, and dried at 37 ℃ until the organoids were fixed on the slide. The membrane was punched with 0.3% Triton X-100 ℃ for 1h, antigen retrieval solution (Biyun, P0090) for 10min at room temperature, 10% horse serum for 1h at 37 ℃, primary antibody was added and incubated overnight at 4 ℃, secondary antibody was incubated for 2h at room temperature, hochest 33342 (1. Fluorescence photography was performed using the high resolution live cell imaging system DeltaVision. The antibody cargo numbers and the concentrations used are shown in Table 2.

TABLE 2 antibody sources and concentrations used

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As a result: after differentiation, the gene and protein expression of the hBDOs pancreas internal and external secretion markers are obviously up-regulated.

As a result: the isoastragaloside I can effectively promote mPDOS to differentiate towards pancreatic cells in vitro, and the gene and protein expression of the differentiated mPDOS pancreatic internal and external secretion markers are remarkably up-regulated (figure 4A-B).

(5) Flow cytometry: digestion of differentiated mPDOs into single cells using trypsin, 4% PFA fixation on ice for 15min, PBS wash, 0.1% TritonX-100 punch at room temperature for 10min, PBS wash, 5% BSA blocking at room temperature for 15min, after standing at room temperature for 15min, 3% BSA diluted primary antibody (C-peptide, 1.

(6) Glucose-stimulated C-peptide secretion: mPDOs were cultured for 14 days with mEM containing Isoastragaloside I (Isoastragaloside I) at a concentration of 1. Mu.M, then resuspended in organoids using a sugar-free Krebs solution, washed 2-3 times, placed in a low adsorption plate, and incubated overnight. Adding Krebs solution containing 2mM glucose, incubating for 10min, centrifuging, and collecting supernatant. After sugar-free Krebs is used for cleaning cells, krebs solution with 20mM glucose is added for heavy suspension, incubation is carried out for 10min, and supernatant is collected by centrifugation. The C peptide levels in the supernatant samples were analyzed using a mouse C peptide ELISA kit (mlbio, ml-1015542) according to standard protocols. As a result: the isoastragaloside I can effectively promote mPDOS to be differentiated into functional beta cells in vitro, and flow cytometry shows that the ratio of C-peptide + cells in the differentiated mPDOS is about 40% (shown in figures 5A-B), the differentiation efficiency is improved by about 30 times compared with the results reported in the literature, and the isoastragaloside I can respond to glucose stimulation to secrete C peptide (shown in figure 5C) and has the physiological response of the glucose stimulation of mature beta cells.

2. Culture of human bile duct organoid

Human extrahepatic bile duct cells were initially seeded in extracellular matrix, using hEM medium at 37 deg.C, 5% ₂ Organoids were formed after 5 days of culture under incubator conditions.

(1) Primary culture of human biliary organoids (hbds): the human Bile Duct (BD) was directly peeled off under a dissecting scope by a mechanical method, and the obtained BD was placed in an EP tube containing collagenase IV and cut into pieces using surgical scissors. Digesting at 37 deg.C for 20min, taking out every 5min, repeatedly beating with 1mL pipette tip, and stopping digestion on ice. The supernatant was discarded by centrifugation, washed 3 times with sterile PBS, and the digested cells were resuspended in Matrigel (Corning, 54234), and then dropped into a 24-well plate and allowed to stand at 37 ℃ for 5min to coagulate Matrigel. Each well was incubated with 800. Mu.L of proliferation medium (hEM). The detailed procedure is shown in FIG. 6A.

The cells obtained by the method are cultured in Matrigel, and proliferate for about 72h to form an obvious closed structure, and proliferate in a large amount within 2 weeks to form a saccular organoid with obvious morphology. When the cells grow to be filled with Matrigel, subculture and cryopreservation can be performed.

(2) Subculturing of human biliary organoids (human extrahepatic double product organoids, hbds): organoids were resuspended by blowing-up using pre-cooled high-glucose DMEM medium (HyClone; SH 30022.01), the supernatant was centrifuged off, and the pellet was subcultured using Matrigel and hEM. According to cell density, the cells were passaged every 7 to 8 days at a ratio of 1.

(3) Cryopreservation of human biliary organoids (human extrahepatic double product organoids, hbds): the organoids were crushed and resuspended in precooled high-glucose DMEM medium (HyClone; SH 30022.01), after centrifugation to remove the supernatant, 1mL of precooled organoid cryopreservation solution was added, the resuspended organoids were transferred to a cell cryopreservation tube, the cryopreservation tube was placed in a cryopreservation box, then transferred to a-80 ℃ freezer overnight, and the cryopreservation tube was transferred to liquid nitrogen for storage the next day.

The hBDOs obtained in this study could be passaged for a long period of time, had stable proliferation and passaging ability, and could maintain a good cell state even when it was passed to the 20 th passage, and the results are shown in fig. 6B. The structural formula of the isoastragaloside I is shown in figure 3A.

Respectively utilizing EM culture medium containing one or more of astragalosides I-VIII, isoastragalosides I, II and IV, acetyl astragalosides E, cycloastragalosides F and G, agroastralosides I-IV and soyasaponin I and one or more of derivatives thereof with final concentration of 0.1, 0.5, 1,2, 4 and 8 mu M to culture the human single-tube epithelial cells for 14 days, and finally obtaining the islet cells.

The experimental effect was verified using the following experiment:

(1) In vivo transplantation experiment: diabetes was induced by intraperitoneal injection of streptozotocin (160 mg/kg) 7 days before Nu/Nu mice transplantation. The glucometer measures non-fasting blood glucose in tail vein samples, and blood glucose levels elevated above 16.8mM were selected as diabetic model mice. Treating hBDOs with isoastragaloside I for 7 days, beating into single cells with pancreatin, and pressing at 10% ⁶ Transplantation of individual cells/cells to recipientIn the kidney cysts of mice, regular non-fasting blood glucose was measured every 7 days after transplantation. At the 8 th week of transplantation, a nephrectomy was performed to examine the effect of removing the transplanted organoids or islets on improvement of blood glucose.

(2) Glucose tolerance test: the glucose tolerance test is carried out according to a standard scheme, mice are starved overnight, 1g/kg of glucose is injected into the abdominal cavity, the blood glucose levels of 0min, 15min, 30min, 60 min, 90 min and 120min are detected, meanwhile, serum before and after the glucose injection is collected, and the change of the insulin content is measured by adopting an ELISA method.

As a result: when hBDOs induced and differentiated by 1 mu M of isoastragaloside I are transplanted into kidney cysts of STZ-induced diabetic mice, the reduction of the blood sugar of the mice can be obviously observed (figure 7A), and a glucose tolerance experiment shows that compared with a control group, the glucose tolerance of the transplanted hBDOs mice is relieved (figure 7B-C), and the level of Insulin in serum is improved (figure 7D). After nephrectomy of the transplantation, the blood glucose levels of mice were significantly elevated (fig. 7A), further demonstrating that the drop in blood glucose was indeed due to the transplanted hBDOs.

Example 3 use of islet beta cells

The cells can be used for preparing an artificial islet system and a method for treating diabetes by in vivo application:

mixing the islet beta cells obtained in example 2 with a medicinal carrier comprising extracellular matrix, nano material or microfluid, and implanting the islet beta cells into an animal body, or mixing the islet beta cells with sodium chloride, magnesium chloride, calcium sodium ethylene diamine tetraacetate and water for injection to support injection, and implanting the mixture into the animal body for treatment. The method comprises the following specific steps:

diabetes was induced by intraperitoneal injection of streptozotocin (160 mg/kg) 7 days before Nu/Nu mice transplantation. The glucometer measures non-fasting blood glucose in tail vein samples, and blood glucose levels elevated above 16.8mM were selected as diabetic model mice. Treating hBDOs with isoastragaloside I for 7 days, beating into single cells with pancreatin, and pressing at 10% ⁶ One cell/cell was transplanted into the kidney capsule of recipient mice, and regular non-fasting blood glucose was measured every 7 days after transplantation. The glucose tolerance test is carried out according to a standard scheme, the mice are hungry overnight, 1g/kg of glucose is injected into the abdominal cavity, and 0, 15, g,30. Blood glucose levels were measured by ELISA at 60, 90 and 120min, and serum was collected before and after glucose injection. At the 8 th week of transplantation, a nephrectomy was performed to examine the effect of removing the transplanted organoids or islets on improvement of blood glucose.

As a result: under the two-dimensional or three-dimensional culture condition, the isoastragaloside I can promote PDX1 positive cells in liver, gallbladder, pancreas and gastrointestinal tract epithelium, so that the formed islet-like cells are transplanted to diabetic animals, and can effectively reduce blood sugar, improve sugar tolerance and treat diabetes. The method can avoid hydrogel combination of polypeptide and its derivatives for immunological rejection, thereby reducing immunological rejection.

Example 4 an inducer for inducing differentiation of ductal cells

Human expansion medium (hum) formulation: 1% Penicilin-Streptomycin Solution (HyClone; SV 30010), 1% glutamine additive (gibco; 35050-061), 2% B27 with out vitamin A (Thermo; 12587010), 50ng/mL EGF (MCE; HY-P7067), 50ng/mL FGF10 (PeproTech; 100-26-25), 25ng/mL NOGGIN (PeproTech; 120-10C-20), 1 N2 (Thermo; 02017501), 10nM Gastrin (SIGMA; G9145), 3 μ M PGE2 (SIGMA; P0409), 5 μ M A83-01 (MCE; HY-10432), 1mol/L Nicotinamide (Sigma; N0636), 1mol/L N-acetyl-L-cysteine (Sigma; A9165), 100ng/mL R-Spondin-1 (R & D; 7150-RS), plus SH/F12 to 40.40. Mu.01; cloS 30022.01);

mouse expansion medium (moem) formulation: 1% Penicillin-Streptomyces Solution (HyClone; SV 30010), 1% glutamine additive (gibco; 35050-061), 2% B27 with out vitamin A (Thermo; 12587010), 50ng/mL EGF (MCE; HY-P7067), 50ng/mL FGF10 (PeproTech; 100-26-25), 25ng/mL NOGGIN (PeproTech; 120-10C-20), 1mol/L Nicotinamide (Sigma; N0636), 1mol/L N-acetyl-L-cysteine (Sigma; A9165), 100/mL R-Spondin-1 (R & D; 7150-RS), plus D/F12 to 40mL (HyClone; SH 30022.01); the prepared astragalosides with final concentration of 0.5, 1,2, 4, 8 μ M in the EM culture medium are astragaloside IV, astragaloside I, astragaloside II, astragaloside IV, isoastragaloside II or isoastragaloside I.

Example 5A method of inducing differentiation of catheter cells into islet cells

The ductal organoid culture stage (stage I) and the induced differentiation into islet cells stage (stage II).

(1) Stage I comprises the following steps: (a) Stripping under a dissecting mirror by using a mechanical method, obtaining intrahepatic bile duct epithelial cells, extrahepatic bile duct epithelial cells or pancreatic duct epithelial cells by using an enzyme digestion method, and embedding the intrahepatic bile duct epithelial cells, the extrahepatic bile duct epithelial cells or the pancreatic duct epithelial cells in matrigel; (b) Expanded in proliferation medium (EM), and passaged every 7 to 8 days after the cells were grown to be filled with matrigel (about 10 to 14 days).

Small molecules added to the propagation medium in stage I include B27 (no vitamin A type B-27 supplement), EGF (epidermal growth factor), FGF10 (fibroblast growth factor 10), NOGGIN (NOGGIN protein), N ₂ (Thermo; 17502001), gastrin (SIGMA; G9145), PGE2 (SIGMA; P0409), A83-01 (MCE; HY-10432), nicotinamide (Nicotinamide), N-acetyl-L-cysteine, and R-spondin1.

(2) Stage II comprises: adding 1 μ M concentration of astragaloside into EM medium of EPOs, and culturing for 14 days.

The astragalus extract added in the stage II comprises one or more of astragalosides I-VIII, isoastragalosides I, II and IV, acetylastragalosides E, F and G, astragalosides I-IV and soyasaponin I and one or more of derivatives thereof.

As a result: the pancreatic islet endocrine cells induced by the astragaloside function mainly comprise beta cells. In embodiments, the induced pancreatic islet endocrine cells express at least one cellular marker: insulin (INS) as a beta cell marker, glucagon (Glucagon; GCG) as an alpha cell marker, somatostatin (SST) as a delta cell marker, and Pancreatic Polypeptide (PP) as a PP cell marker. Meanwhile, the flow cytometry shown in FIG. 5 proves that the Differentiation-promoting efficiency of astragaloside of the present invention can reach 45%, which is improved by nearly 30 times compared with the Differentiation efficiency of 1.5% described in the previous literature [ Loomans C, giuliania N W, balak J, et al, expansion of Human functional Tissue YIELDS organic semiconductors Harboring promoter Cells with endothelial Differentiation position [ J ]. Stem Cell Reports,2018, 10 (3): 712 ].

SEQUENCE LISTING

<110> northeast university of forestry

<120> islet cell inducer, culture medium, method for obtaining islet beta cells and application thereof

<160> 14

<170> PatentIn version 3.5

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Claims (19)

1. An inducer for islet cells, which is characterized in that the effective component of the inducer is astragaloside.

2. The induction agent according to claim 1, wherein the islet cells are islet alpha cells, islet beta cells, islet delta cells, or islet pp cells.

3. The induction agent according to claim 1, wherein the induction agent is effective as any one or more of astragalosides i to i, isoastragalosides i, ii and iv, acetylastragalosides E, cycloastragalosides E, F and G, agroastralosides i to iv and daidzin i, and derivatives thereof.

4. The inducer according to claim 1, wherein the effective ingredient of the inducer is isoastragaloside i.

5. A culture medium for in vitro production of islet cells, comprising astragalosides.

6. The culture medium of claim 5, further comprising an organoid proliferation medium.

7. The culture medium of claim 5, wherein the islet cells are islet alpha cells, islet beta cells, islet delta cells, or islet pp cells.

8. The culture medium according to claim 5, wherein the culture medium contains isoastragaloside I.

9. The culture medium according to claim 5, wherein the concentration of astragaloside is 0.1-8 μ M.

10. A method for obtaining islet beta cells is characterized in that epithelial cells are cultured in a culture medium containing astragaloside to be differentiated to obtain the islet beta cells.

11. The method of claim 10, wherein the epithelial cells are human or mammalian liver, biliary, pancreatic, gastric and intestinal epithelial cells.

12. The method as claimed in claim 10, wherein the astragaloside system active ingredient is any one or more of astragalosides i to i, isoastragalosides i, ii and iv, acetylastragalosides E, cycloastragalosides E, F and G, agroastralosides i to iv and saponin i, and any one or more of their derivatives.

13. The method according to claim 10, wherein the effective component of the astragaloside system is isoastragaloside i.

14. The method of claim 10, wherein the concentration of astragaloside is 0.1-8 μ Μ.

15. Islet beta cells obtained by the method of any one of claims 10-14.

16. Use of the islet beta cell of claim 15 in the preparation of a cellular medicament for the treatment of diabetes.

17. The use of claim 16, wherein the cellular medicament is in a dosage form comprising an injectable formulation.

18. The use of claim 16, wherein the cellular drug comprises an extracellular matrix compatible with cells and a pharmaceutical carrier.

19. The use of claim 18, wherein the pharmaceutical carrier is a nanomaterial or a microfluidics.

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