CN111363715B - Mesenchymal stem cell induced islet secretion cell and insulin secretion detection method - Google Patents
Mesenchymal stem cell induced islet secretion cell and insulin secretion detection method Download PDFInfo
- Publication number
- CN111363715B CN111363715B CN202010193720.XA CN202010193720A CN111363715B CN 111363715 B CN111363715 B CN 111363715B CN 202010193720 A CN202010193720 A CN 202010193720A CN 111363715 B CN111363715 B CN 111363715B
- Authority
- CN
- China
- Prior art keywords
- induction
- culture solution
- incubator
- discarding
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/37—Digestive system
- A61K35/39—Pancreas; Islets of Langerhans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/12—Hepatocyte growth factor [HGF]
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
-
- C—CHEMISTRY; METALLURGY
- 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
- 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
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1369—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from blood-borne mesenchymal stem cells, e.g. MSC from umbilical blood
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/62—Insulins
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Urology & Nephrology (AREA)
- Zoology (AREA)
- Diabetes (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Genetics & Genomics (AREA)
- Food Science & Technology (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Veterinary Medicine (AREA)
- Endocrinology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Developmental Biology & Embryology (AREA)
- Emergency Medicine (AREA)
- Epidemiology (AREA)
- Nutrition Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Engineering & Computer Science (AREA)
- Physiology (AREA)
Abstract
The invention relates to a method for detecting islet secretion cells induced by mesenchymal stem cells and insulin secretion, wherein the induction method comprises the following steps: a first induction step: taking human umbilical cord mesenchymal stem cells at a ratio of 2×10 5 Spreading the cell number of each hole in a 6-hole cell culture plate for culture, discarding the original culture solution when the cell confluence reaches 70%, adding the induction culture solution I, and placing the culture solution I in an incubator for culture for 5d; a second induction step: discarding the induction culture solution I, adding the induction culture solution II, and continuously placing the mixture in an incubator for culturing for 3d; and a third induction step: discarding the induction culture solution II, adding the induction culture solution III, and continuously placing the mixture in an incubator for culturing for 5d; fourth induction step: discarding the induction culture solution III, adding the induction culture solution IV, culturing in an incubator for 14d to obtain target cell massAnd collecting and storing. The induction method provided by the invention has the advantages of less induction factors, simple steps and high induction rate.
Description
Technical Field
The invention relates to the technical field of cell engineering, in particular to a method for detecting islet secretion cells induced by mesenchymal stem cells and insulin secretion.
Background
Diabetes is a major illness that jeopardizes human health and is manifested by a relative or absolute deficiency of insulin, a chronic systemic metabolic disease characterized by persistent hyperglycemia as a major biochemical feature. At present, the main treatment method is oral hypoglycemic drugs or islet transplantation, the oral hypoglycemic drugs cannot fundamentally solve the problems, patients need to take the drugs for a long time or for a long time, and hidden dangers of drug side effects exist, so that islet transplantation is high in cost, lack of donors, high in risk and also has the problems of immune rejection.
Researches show that the type I diabetes and the type II diabetes are closely related to immune injury and inflammatory reaction, so that the effects of effectively inhibiting or blocking the immune injury can be achieved, and the effects of protecting beta cells from injury, maintaining islet function and correcting insulin resistance can be achieved. The mesenchymal stem cells have strong immunosuppressive effect, and reports and clinical researches show that the injection of the mesenchymal stem cells can relieve the illness state of diabetics. Meanwhile, research shows that the mesenchymal stem cells have the functions of tissue repair, angiogenesis promotion, antioxidation, diabetes metabolic disturbance correction, insulin resistance and the like, and can play an important role in diabetes.
However, islet transplantation is currently considered to be one of methods for curing diabetes, but because of the limitation of islet donor sources, the in vitro survival rate of islet cells and the clinical application of the significant limitation of immune rejection problems, the diabetes curing method still has a great room for improvement.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for detecting the mesenchymal stem cells induced into islet secretion cells and insulin secretion, which can provide low-cost and low-risk islet transplantation substitutes for clinical experiments.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a method of inducing mesenchymal stem cells into islet secreting cells, comprising:
a first induction step: taking human umbilical cord mesenchymal stem cells at a ratio of 2×10 5 Spreading the cell number of each hole in a 6-hole cell culture plate for culture, discarding the original culture solution when the cell confluence reaches 70%, adding the induction culture solution I, and placing the culture solution I in an incubator for culture for 5d;
a second induction step: discarding the induction culture solution I, adding the induction culture solution II, and continuously placing the mixture in an incubator for culturing for 3d;
and a third induction step: discarding the induction culture solution II, adding the induction culture solution III, and continuously placing the mixture in an incubator for culturing for 5d;
fourth induction step: and (3) discarding the induction culture solution III, adding the induction culture solution IV, continuously placing the mixture in an incubator for culturing for 14d, obtaining target cell clusters, and collecting and storing the target cell clusters.
As a preferred embodiment of the present invention, the induction culture solution i includes: 25mMol/L glucose, 10. Mu.g/L recombinant human activin-A, and 10. Mu.g/L epidermal growth factor.
As a preferred embodiment of the present invention, the induction culture solution ii includes: 25mMol/L glucose, and 15mMol/L niacinamide.
As a preferred embodiment of the present invention, the induction culture solution iii includes: 25mMol/L glucose, 15mMol/L niacinamide, and 10 μg/L hepatocyte growth factor.
As a preferred embodiment of the present invention, the induction culture solution IV comprises: 5.5 mmoles/L glucose, 10. Mu.g/L recombinant human activin-A, 15 mmoles/L niacinamide, 10. Mu.g/L hepatocyte growth factor, and 20nMOL/L Exendin-4.
As a preferred embodiment of the present invention, the fourth induction step is further followed by identification of islet secreting cells by dithizone staining.
As a preferred embodiment of the present invention, the fourth induction step is further followed by a genetic identification process of islet β cells.
In a second aspect, the present invention also provides an insulin secretion detection method of islet secretion cells, comprising:
step one: selecting 15 target cell clusters obtained in the fourth induction step, washing twice with a phosphate buffer solution, adding 500 mu L of glucose with the concentration of 5.5mMol/L, placing the mixture in an incubator, incubating for 24 hours, and collecting culture supernatant to be tested at the temperature of-80 ℃;
step two: selecting 15 target cell clusters obtained in the fourth induction step, washing twice with a phosphate buffer, adding 500 mu L of glucose and 25mMol of glucose, placing the mixture in an incubator for incubation for 24 hours, and collecting culture supernatant to be tested at the temperature of minus 80 ℃;
step three: insulin secretion after step one and step two stimulation was measured using the human insulin Elisa kit.
In a third aspect, the present invention also provides an islet secretory cell mass which is induced to form according to the induction method of any one of the above-described aspects.
In a fourth aspect, the invention also provides a medicament, reagent, composition or kit comprising the islet secretory cell mass.
In summary, compared with the prior art, the invention has the following beneficial effects:
the induction method provided by the embodiment of the invention adopts fewer induction factors, has simple steps and high induction rate, and has the final differentiation efficiency reaching 80% through detection, and only few undifferentiated mesenchymal stem cells and pancreatic stem cells exist; the insulin level secreted by glucose stimulation is also better, and islet secreting cells contained in the generated target cell mass can be used as a blood sugar reducing regulation function for treating diabetes model mice in animal experiments, so that low-cost and low-risk islet transplantation substitutes are provided for later clinical experiments.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing the morphology change of cells during induction in the first embodiment of the present invention.
FIG. 2 is a graph showing the results of the identification by the dithizone staining method in the first embodiment of the present invention.
FIG. 3 is a diagram showing the results of gene identification in the first embodiment of the present invention.
FIG. 4 is a graph showing insulin secretion results in the second embodiment of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A method of inducing mesenchymal stem cells into islet secreting cells, comprising:
a first induction step: taking human umbilical cord mesenchymal stem cells at a ratio of 2×10 5 The cell number of each hole is paved in a 6-hole cell culture plate for culture, when the cell confluence reaches 70%, the original culture solution is discarded, and an induction culture solution I is added, and the culture is carried out in an incubator for 5d, wherein the induction culture solution I comprises the following components: 25mMol/L glucose, 10. Mu.g/L recombinant human Activin-A (Activin A), and 10. Mu.g/L epidermal growth factor (Epidermal growth factor ).
A second induction step: discarding the induction culture solution I, adding the induction culture solution II, and continuously placing the mixture in an incubator for culturing for 3 days, wherein the induction culture solution II comprises: 25mMol/L glucose, and 15mMol/L Niacinamide (NIC).
And a third induction step: discarding the induction culture solution II, adding the induction culture solution III, and continuously placing the mixture in an incubator for culturing for 5 days, wherein the induction culture solution III comprises: 25mMol/L glucose, 15mMol/L niacinamide, and 10 μg/L Hepatocyte Growth Factor (HGF).
Fourth induction step: discarding the induction culture solution III, adding the induction culture solution IV, and continuously placing the mixture in an incubator for culturing for 14d to obtain target cell clusters, collecting and storing the target cell clusters, wherein the induction culture solution IV comprises: 5.5 mmoles/L glucose, 10. Mu.g/L recombinant human activin-A, 15 mmoles/L niacinamide, 10. Mu.g/L hepatocyte growth factor, and 20nMOL/L Exendin-4.
As shown in fig. 1, fig. 1 shows the process of cell morphology change in the induction process, umbilical cord mesenchymal stem cells are gradually rounded from the original long fusiform shape to form small cell clusters, the small cell clusters are aggregated to form large cell clusters, and the large cell clusters are further aggregated to finally form cell clusters capable of secreting insulin under the stimulation of glucose.
The identification of islet secreting cells is also performed by dithizone staining after the fourth induction step, the specific identification steps are: 50mg of Dithizone (DTZ) is dissolved in 5ml of dimethyl sulfoxide (DMSO), filtered and packaged in a dark place at the temperature of minus 20 ℃ to be stored as mother liquor, and Hanks liquid is used according to the following formula 1:100, mixing with target cell mass, culturing at 37deg.C for 30min, and performing microscopic examination; because dithizone is a chelating indicator, it can chelate lead, copper, zinc and the like, and the human and animal (except guinea pig) islet beta cells are rich in zinc, and after the dithizone reacts with it, it can be scarlet, and other cells are not colored, so that it can make specificity staining for islet cells by using dithizone. As shown in fig. 2, fig. 2 shows the microscopic examination result after the dithizone staining.
Meanwhile, the fourth induction step is followed by the genetic identification process of islet beta cells, which comprises the following specific steps:
(1) primer sequence:
H-INS-F：GGAACGAGGCTTCTTCTACA
H-INS-R：ACAATGCCACGCTTCTGC
H-MafA-F：TTCAGCAAGGAGGAGGTCAT
H-MafA-R：CGCCAGCTTCTCGTATTTCT
H-Nkx2.2-F：TCTACGACAGCAGCGACAAC
H-Nkx2.2-R：TTGTCATTGTCCGGTGACTC
H-β-Actin-F：AGAGCTACGAGCTGCCTGAC
H-β-Actin-R：AGCACTGTGTTGGCGTACAG
(2) RNA extraction: extracting total RNA of the uninduced mesenchymal stem cells and the target cell mass obtained in the fourth induction step by using an RNA extraction kit.
(3) Reverse transcription: the resulting cDNAs were reverse transcribed with 1. Mu.g total RNA per group using a reverse transcription kit and stored at-20℃for further use.
(4) The setting of qPCR reaction procedure was performed using a PCR reactor.
As shown in fig. 3, fig. 3 shows a schematic diagram of the result of gene identification.
Example two
An insulin secretion assay method for islet secreting cells comprising:
step one: selecting 15 target cell clusters obtained in the fourth induction step, washing twice with a phosphate buffer solution, adding 500 mu L of glucose with the concentration of 5.5mMol/L, placing the mixture in an incubator, incubating for 24 hours, and collecting culture supernatant to be tested at the temperature of-80 ℃;
step two: selecting 15 target cell clusters obtained in the fourth induction step, washing twice with a phosphate buffer, adding 500 mu L of glucose and 25mMol of glucose, placing the mixture in an incubator for incubation for 24 hours, and collecting culture supernatant to be tested at the temperature of minus 80 ℃;
step three: insulin secretion after step one and step two stimulation was measured using the human insulin Elisa kit.
As shown in fig. 4, fig. 4 shows a schematic diagram of insulin secretion results.
Example III
An islet secretory cell mass induced to form according to the induction method of example one.
Example IV
A medicament, agent, composition or kit comprising the islet secretory cell mass of embodiment three.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (3)
1. A method for inducing mesenchymal stem cells into islet secreting cells, comprising the following steps:
a first induction step: taking human umbilical cord mesenchymal stem cells at a ratio of 2×10 5 Spreading the cell number of each hole in a 6-hole cell culture plate for culture, discarding the original culture solution when the cell confluence reaches 70%, adding the induction culture solution I, and placing the culture solution I in an incubator for culture for 5d;
a second induction step: discarding the induction culture solution I, adding the induction culture solution II, and continuously placing the mixture in an incubator for culturing for 3d;
and a third induction step: discarding the induction culture solution II, adding the induction culture solution III, and continuously placing the mixture in an incubator for culturing for 5d;
fourth induction step: discarding the induction culture solution III, adding the induction culture solution IV, continuously placing the mixture in an incubator for culturing for 14d to obtain target cell clusters, and collecting and storing the target cell clusters;
the induction culture solution I is as follows: 25mMol/L glucose, 10. Mu.g/L recombinant human activin-A, and 10. Mu.g/L epidermal cell growth factor;
the induction culture solution II is as follows: 25mMol/L glucose, 15mMol/L niacinamide;
the induction culture solution III is as follows: 25mMol/L glucose, 15mMol/L niacinamide, and 10 μg/L hepatocyte growth factor;
the induction culture solution IV is as follows: 5.5 mmoles/L glucose, 10. Mu.g/L recombinant human activin-A, 15 mmoles/L niacinamide, 10. Mu.g/L hepatocyte growth factor, and 20nMOL/L Exendin-4.
2. The method of claim 1, wherein the fourth induction step is further followed by identification of islet secreting cells by dithizone staining.
3. The method of inducing mesenchymal stem cells into islet secreting cells according to claim 2, wherein the fourth inducing step is further followed by a genetic identification process of islet β cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010193720.XA CN111363715B (en) | 2020-03-19 | 2020-03-19 | Mesenchymal stem cell induced islet secretion cell and insulin secretion detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010193720.XA CN111363715B (en) | 2020-03-19 | 2020-03-19 | Mesenchymal stem cell induced islet secretion cell and insulin secretion detection method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111363715A CN111363715A (en) | 2020-07-03 |
| CN111363715B true CN111363715B (en) | 2023-07-18 |
Family
ID=71202717
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010193720.XA Active CN111363715B (en) | 2020-03-19 | 2020-03-19 | Mesenchymal stem cell induced islet secretion cell and insulin secretion detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111363715B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101570741A (en) * | 2009-06-15 | 2009-11-04 | 浙江大学 | Method and combined medium for preparing insulin-like secreting cells |
| CN102618491A (en) * | 2012-03-21 | 2012-08-01 | 协和干细胞基因工程有限公司 | Culture solution for inducing mesenchymal stem cells to differentiate into islet-like cells, and inducing method and application of culture solution |
| CN105670986A (en) * | 2015-11-23 | 2016-06-15 | 王意忠 | Culture medium for inducing human umbilical cord mesenchymal stem cells to differentiate into islet-like cells and induction method therefor |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11339372B2 (en) * | 2016-11-08 | 2022-05-24 | Cheng Li | Serum-free medium inducing differentiation of umbilical cord mesenchymal stem cell into insulin-secretion-like cell and preparation method and use thereof |
-
2020
- 2020-03-19 CN CN202010193720.XA patent/CN111363715B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101570741A (en) * | 2009-06-15 | 2009-11-04 | 浙江大学 | Method and combined medium for preparing insulin-like secreting cells |
| CN102618491A (en) * | 2012-03-21 | 2012-08-01 | 协和干细胞基因工程有限公司 | Culture solution for inducing mesenchymal stem cells to differentiate into islet-like cells, and inducing method and application of culture solution |
| CN105670986A (en) * | 2015-11-23 | 2016-06-15 | 王意忠 | Culture medium for inducing human umbilical cord mesenchymal stem cells to differentiate into islet-like cells and induction method therefor |
Non-Patent Citations (7)
| Title |
|---|
| 人脐带间充质干细胞体外分化为胰岛素分泌细胞;禹亚彬等;《江苏医药》;20130515(第09期);第1021-1024页 * |
| 人脐带间充质干细胞分化胰岛样细胞过程中胰岛素和巢蛋白的表达;申义等;《中国组织工程研究》;20161202(第50期);第7500-7506页 * |
| 体外定向诱导人脐带间充质干细胞分化为胰岛样细胞;禹亚彬等;《中国组织工程研究》;20130205(第06期);第1012-1017页 * |
| 体外定向诱导人脐带间充质干细胞分化为胰岛素分泌细胞的研究;李伟中等;《中国输血杂志》;20090331;第22卷(第3期);第193页第1.4.1-1.4.2，2.2.3-2.2.4节,图1 * |
| 干细胞诱导分化为产胰岛素细胞的研究进展;张辰等;《现代生物医学进展》;20160310(第07期);第3.1-3.5节 * |
| 李伟中等.体外定向诱导人脐带间充质干细胞分化为胰岛素分泌细胞的研究.《中国输血杂志》.2009,第22卷(第3期), * |
| 胎猪胰腺干细胞体外诱导分化为胰岛素分泌细胞及其移植治疗裸鼠糖尿病的研究;楚元奎等;《农业生物技术学报》;20110228(第01期);第9-17页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111363715A (en) | 2020-07-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109749986B (en) | Method for obtaining pancreatic precursor cells and pancreatic beta cells by differentiation of human pluripotent stem cells | |
| CN102899288B (en) | Method for constructing human islet-derived pancreatic stem cell line and method for differentiation of human islet-derived pancreatic stem cell line into insulin-producing cells | |
| US20190264179A1 (en) | Serum-free medium inducing differentiation of umbilical cord mesenchymal stem cell into insulin-secretion-like cell and preparation method and use thereof | |
| CN114874982B (en) | Culture method for enhancing umbilical cord mesenchymal stem cells to secrete vascular endothelial growth factors | |
| US20130084266A1 (en) | Methods for generating pancreatic tissue | |
| Liang et al. | Implantation underneath the abdominal anterior rectus sheath enables effective and functional engraftment of stem-cell-derived islets | |
| CN111363715B (en) | Mesenchymal stem cell induced islet secretion cell and insulin secretion detection method | |
| CN110462023A (en) | It include the Stem Cell Activity composition for promoting of histon deacetylase (HDAC) inhibitor and activity factor as effective component | |
| CN116574670B (en) | Method for inducing mesenchymal stem cells to differentiate into islet-like cells and application thereof | |
| CN113881624A (en) | Cell induction method | |
| CN112852946A (en) | Diagnostic biomarker and application thereof | |
| CN105062952A (en) | Method for transformation of mesenchymal stem cells into islet cells | |
| CN112251397B (en) | Culture method of newborn pig islet cells | |
| CN115595292A (en) | Method for in vitro induction of IPS (in-plane switching) cells to differentiate human islet beta cells | |
| CN106834213B (en) | Induction medium and induction method for inducing pluripotent stem cells to form hair papilla cells | |
| CN113736725B (en) | Cell differentiation medium composition, high secretion amount insulin producing cell and preparation thereof | |
| WO2023227068A1 (en) | A new site for transplantation | |
| KR20210093750A (en) | Inducing agent that induces differentiation of mesenchymal stem cells into islet cells | |
| KR101828696B1 (en) | Composition for insulin secreting cell transplantation and method for preparing the same | |
| CN113637630B (en) | Islet-like cell mass, and preparation method and application thereof | |
| KR101177273B1 (en) | Method for improving pancreatic islet cell survival or its activity | |
| CN106467918B (en) | Induction method and application of insulin secreting cells based on human skin cells | |
| CN104257690A (en) | Stem cell preparation for treating diabetes mellitus and preparation method of preparation | |
| CN114807013B (en) | Method for differentiating peripheral blood stem cells into endoderm cells and application thereof | |
| CN114015693B (en) | Annular RNA, detection reagent, kit, application and medicine thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20220309 Address after: 215000 room 201-202, building A5, bio nano Park, 218 Xinghu street, Suzhou Industrial Park, Jiangsu Province Applicant after: Suzhou Houpu Huikang Biomedical Technology Co.,Ltd. Address before: Room 201-206, building A5, bio nano Park, 218 Xinghu street, industrial park, Suzhou, Jiangsu 215000 Applicant before: HOPE BIOLOGICAL TECHNOLOGY (SUZHOU) Co.,Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |