CN115216437A - Application of retinoic acid in-vitro amplification of islet cell mass - Google Patents
Application of retinoic acid in-vitro amplification of islet cell mass Download PDFInfo
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- CN115216437A CN115216437A CN202110424916.XA CN202110424916A CN115216437A CN 115216437 A CN115216437 A CN 115216437A CN 202110424916 A CN202110424916 A CN 202110424916A CN 115216437 A CN115216437 A CN 115216437A
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- retinoic acid
- islet
- medium
- cell mass
- islet cell
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Abstract
The invention provides application of retinoic acid in-vitro amplification of islet cell mass, belonging to the technical field of biological medicine. The invention takes retinoic acid as the culture medium additive component to assist in realizing the in vitro amplification of the islet cell mass, compared with the islet cell mass cultured by the culture medium without retinoic acid, the islet cell mass is obviously increased, and related experiments prove that the islet cell mass can promote the proliferation of islet cells, improve the secretion of insulin, glucagon, somatostatin and the like, lay a solid foundation for preparing functional islet-like organs from islet cell sources, and therefore, the islet cell mass has good practical application value.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of retinoic acid in-vitro amplification of islet cell mass.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
In recent years, the prevalence rate of diabetes in China is increased sharply, the incidence of diabetes tends to be younger, and the range of harming people is larger and larger. Diabetes is classified into type 1 diabetes caused by insufficient insulin secretion and type 2 diabetes caused by insulin resistance, depending on the pathogenesis. Type 1 diabetes is caused by the destruction of islet beta cells due to an abnormality in the autoimmune system, and the body's blood sugar rises due to insufficient insulin secretion. At present, pancreas or islet transplantation is the main method for fundamentally treating type 1 diabetes, but due to the problems of the lack of donors, serious immunological rejection reaction after transplantation and the like, the large-scale application of the method in clinical treatment is limited. With the rise of organoid technology, it is hoped that tissues or organs can be cultured in vitro and finally diabetes can be treated by islet transplantation. Among the 5 major endocrine cells of the islets, the beta cell is of the greatest importance, since insulin released is the only hormone in the body that is capable of lowering blood glucose, and therefore the current research on functional islet cell regeneration is primarily based on the in vitro expansion of islet beta cells.
Currently, islet beta cells regenerated in vitro are mainly derived from pluripotent stem cells and non-beta adult cells, but the inventors found that the resulting beta-like cells often have problems of genomic or epigenetic instability (Kroon E, et al, 2008 nair gg, et al, 2019. This is largely avoided by the use of islet cell self-expansion. How to establish an in vitro functional islet model by means of an islet cell in vitro proliferation technology, and how to stably apply islet cells to drug screening and clinical treatment of diabetes on a large scale become problems to be solved in the field.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the application of retinoic acid in the in-vitro amplification of islet cell mass. According to research, the invention discovers that the culture medium component containing the all-trans retinoic acid can effectively realize the in-vitro amplification of rat islet cell mass when being used for culturing the islet cell mass, and retinoic acid plays an important role in the dedifferentiation and proliferation of the islet cell mass, so that a foundation is laid for preparing functional islet-like organs from islet cell sources.
Specifically, the invention relates to the following technical scheme:
in a first aspect of the invention, there is provided the use of retinoic acid for the in vitro expansion of islet cell mass.
Specifically, the application is as follows: retinoic acid promotes islet cell mass expansion in vitro.
In a second aspect of the present invention, there is provided a culture method for promoting the in vitro expansion of islet cell mass, comprising: retinoic acid is added to the medium.
In a third aspect of the invention there is provided the use and/or culture method as described above in any one or more of:
1) Constructing an islet organ model;
2) Constructing a diabetes animal model;
3) Screening and preparing diabetes-related medicaments;
4) Basic research on diabetes and related diseases;
5) Diabetes and related diseases.
The beneficial technical effects of one or more technical schemes are as follows:
the technical scheme reports that retinoic acid has the capacity of promoting the in-vitro amplification of islet cell mass for the first time, the retinoic acid is used as a culture medium adding component to assist in realizing the in-vitro amplification of the islet cell mass, compared with the islet cell mass cultured by a culture medium without retinoic acid, the islet cell mass is obviously increased due to the fact that the number of islet cells is obviously increased, the islet cell mass is obviously increased, and related experiments prove that the retinoic acid can promote the proliferation of islet cells, improve the secretion of insulin, glucagon, somatostatin and the like, and lay a solid foundation for preparing functional islet-like organs from islet cell sources, so that the retinoic acid has good practical application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a graph showing the phenotype of 0 day, 4 days and 8 days of DMEM/F12 (10% FBS), PIEM medium and PIEM (0.5. Mu.M retinoic acid added) medium in the examples of the present invention.
FIG. 2 shows the real-time quantitative PCR assay of Ki67 in the present example after 5 days of culture in the primary islet control, PIEM medium and PIEM (supplemented with 0.5. Mu.M retinoic acid) medium, respectively.
FIG. 3 shows the real-time quantitative PCR detection results of Marker related to dedifferentiated and progenitor cells such as SOX9 after 5 days of culture in PIEM medium and PIEM (with 0.5. Mu.M retinoic acid added) medium, respectively, in the examples of the present invention.
FIG. 4 shows the secretion of Insulin after 5 days of culture in rat islet and PIEM (supplemented with 0.5. Mu.M retinoic acid) media, respectively, in an example of the present invention.
FIG. 5 shows the secretion of Glucagon after 5 days of culture in rat islet and PIEM (supplemented with 0.5. Mu.M retinoic acid) media, respectively, in an example of the present invention.
FIG. 6 shows the secretion of somatotatin after 5 days of culture in rat islet and PIEM (supplemented with 0.5. Mu.M retinoic acid) media, respectively, according to the present invention.
FIG. 7 shows the real-time quantitative PCR detection results of Ki67 after 5 days of culture in the PIEM medium containing different concentrations of retinoic acid and the PIEM medium containing the original islet control in the present example.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. The experimental procedures, if specific conditions are not indicated in the following detailed description, are generally in accordance with conventional procedures and conditions of molecular biology within the skill of the art, which are fully explained in the literature. See, e.g., sambrook et al, "molecular cloning: the techniques and conditions described in the laboratory Manual, or according to the manufacturer's recommendations.
The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; materials, reagents and the like used in examples were commercially available unless otherwise specified.
As described above, how to establish an islet model with good in vitro functions by using an islet cell in vitro proliferation technology, and to stably apply islet cells in large scale to drug screening and clinical treatment of diabetes, is a problem to be solved in the art.
In view of the above, in one embodiment of the present invention, there is provided the use of retinoic acid in the in vitro expansion of islet cell mass.
In another embodiment of the present invention, the application is: retinoic acid can promote the in vitro amplification of islet cell mass;
more specifically, the research of the invention discovers that retinoic acid as a culture medium additive component can assist in realizing the in-vitro amplification of islet cell mass, the number of islet cells is obviously increased, the islet cell mass is obviously increased compared with the islet cell mass cultured by a culture medium without retinoic acid, and related experiments prove that the retinoic acid culture medium can promote the proliferation of islet cells and improve the secretion of insulin, glucagon, somatostatin and the like.
In yet another embodiment of the invention, the retinoic acid is all-trans retinoic acid; the concentration of retinoic acid added is controlled to be 0.1 to 4. Mu.M, more preferably 0.25 to 2. Mu.M, and most preferably 0.5. Mu.M. Experiments prove that the retinoic acid has the best effect of promoting the proliferation of islet cells when the addition concentration of the retinoic acid is 0.5 mu M.
In yet another embodiment of the present invention, the culture medium may be any medium that can be used for culturing islet cells, such as DMEM/F-12, and more preferably a medium having the function of promoting islet expansion, such as islet expansion medium PIEM; the PIEM takes DMEM/F-12 as a basic culture medium, and simultaneously adds RSPO1 conditioned medium, CHIR-99021, A8301, gastrin, N-acetylcysteine, Y-27632, forskolin, exendin-4, L-ascorbic acid, 5-iodotubercidin, recombinant human fibroblast growth factor-10, epidermal cell growth factor, nicotinamide and B27 cell culture additives.
In another embodiment of the present invention, there is provided a culture method for promoting the in vitro expansion of islet cell mass, comprising: retinoic acid is added to the medium.
Wherein the retinoic acid is all-trans retinoic acid; the concentration of retinoic acid added is controlled to be 0.1 to 4. Mu.M, more preferably 0.25 to 2. Mu.M, and most preferably 0.5. Mu.M. Experiments prove that the retinoic acid has the best effect of promoting the proliferation of islet cells when the addition concentration of the retinoic acid is 0.5 mu M.
In yet another embodiment of the present invention, the culture medium may be any medium that can be used for culturing islet cells, such as DMEM/F-12, etc., more preferably a medium with the ability to promote islet expansion, such as islet expansion medium PIEM; the PIEM takes DMEM/F-12 as a basic culture medium, and simultaneously adds RSPO1 conditioned medium, CHIR-99021, A8301, gastrin, N-acetylcysteine, Y-27632, forskolin, exendin-4, L-ascorbic acid, 5-iodotubercidin, recombinant human fibroblast growth factor-10, epidermal cell growth factor, nicotinamide and B27 cell culture additives.
In still another embodiment of the present invention, the culture method further comprises the step of subjecting the cultured cells to CO reduction at 30 to 40 ℃ (preferably 37 ℃), 1 to 8% 2 (preferably 5% CO) 2 ) Culturing under the culture conditions of (1).
In a further embodiment of the invention, there is provided the use and/or culture method described above in any one or more of:
1) Constructing an islet organ model;
2) Constructing a diabetes animal model;
3) Screening and preparing diabetes-related medicaments;
4) Basic research on diabetes and related diseases;
5) Diabetes and related diseases.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
ExamplesIn vitro islet cell mass expansion after exogenous retinoic acid addition
Experimental animals: all rats were raised and bred using Wistar rats as model animals, following strictly standard experimental conditions as specified by the regulations of the regulatory committee on laboratory animals.
The experimental method comprises the following steps:
isolation of islets from Wistar rats:
rats were sacrificed by cervical dislocation and the pancreas was isolated. After digestion of the pancreas with collagenase P (Roche, 11213873001) at 37 ℃ for 18-25min, digestion was stopped with ice Hank balanced salt solution (136.9mM NaCl 5.4mM KCl 1.3mM CaCl 0.8mM MgSO4 0.44mM KH2PO4 0.34mM Na2HPO4. Islets were collected manually using a stereomicroscope and cultured overnight in islet complete medium containing 5.6mM glucose (Biological Industries), 10% FBS (Gibco), 0.1% penicillin/streptomycin.
The islets are divided into cell clusters:
by controlling the enzyme concentration, digestion and sedimentation time, cell clumps of different diameters (20-150 μm) were obtained. Islets were naturally settled in KRBB buffer (135mM NaCl, 4.7mM KCl, 1.2mM KH2PO4, 10mM hepes, 3mM D-glucose; 5mM NaHCO3, 0.1% BSA, 1% penicillin/streptomycin, pH 7.4) for 1min, pre-heated EDTA-KRBB solution was added, gently blown for 2min, placed in a 33 ℃ water bath for 10min, and after removal of the supernatant, the tissues were digested with Dispase II (0.1-0.5U/mL, roche, 04942078001) in a 33 ℃ water bath for 3-8min to obtain islet cell masses of different diameters. Mechanical breakup was achieved by gentle pipetting (5-10 times) in a 15mL glass centrifuge tube using a glass pipette. Digestion was terminated using DMEM/F-12 (Biological Industries) medium containing 10% FBS (Gibco) and 1% penicillin/streptomycin. The pellet was collected by centrifugation (300 Xg, 5min, room temperature) to obtain the desired cell pellet.
Culturing of islet cell mass:
the separated islet cell mass was washed 2 times with DMEM/F-12 (Biological Industries), counted and gently mixed with Matrigel (Corning) in a glass centrifuge tube. After mixing, the mixture was dropped into a 24-well plate, and all cell clusters were randomly divided into 2 groups on average. After curing for 30min at Matrigel 37 ℃, 600 μ l of islet expansion medium (PIEM) was added to each well of one set and 0.5 μ M retinoic acid (MedChemExpress) in the other set. PIEM medium was DMEM/F-12 (1% GlutaMax, 1% penicillin-streptomycin) +15% RSPO1 conditioned medium, 3. Mu.M CHIR-99021 (BioGems), 1. Mu. M A8301 (Adooq Bioscience), 10nM gateway-1 (Medchem), 1.25mM N-acetyl cysteine (Sigma), 10. Mu. M Y-27632 (Adooq Bioscience), 10. Mu.M forskolin (targetMol), 50 ng/mLExedin-4 (ChinaPeptides), 50. Mu.g/mL L-ascobic (Sigma), 250nM 5-Iodotubulidin (Adooq Bioscience), 50ng/mL FGF10 (properm Tech), 50/mL, 10mM Tech (ThermoEGF), 27 mM Sigma (Sigma), or Nirmin A (Nirmstrong). The culture was incubated at 37 ℃ and 5% CO 2 Cultured in a cell culture box. During the culture period, the medium was renewed every three days.
And (3) pancreatic islet cell mass amplification detection:
the dedifferentiation and proliferation of the cell mass were detected by means of phenotypic pictures, real-time quantitative PCR, etc., and the results are shown in FIGS. 1-3.
And (3) detecting the physiological function of the islet cell mass:
the physiological function of the cell mass is detected by detecting the secretion of the cell mass insulin, glucagon and somatostatin, a glucose tolerance test after in vivo transplantation and the like, and the result is shown in figures 4-6. At the same time, it was further verified that 0.5. Mu.M retinoic acid was most effective in promoting islet cell proliferation (see FIG. 7).
The above results illustrate that: the retinoic acid can promote the in vitro amplification of islet cell mass.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. Application of retinoic acid in-vitro amplification of islet cell mass.
2. The application of claim 1, wherein the application comprises: retinoic acid promotes the in vitro expansion of islet cell mass.
3. The application of claim 1, wherein the application comprises: retinoic acid was used as an additive component of the medium.
4. The application according to claim 1, wherein the application specifically comprises:
1) Promoting islet cell proliferation;
2) Increase the secretion of insulin, glucagon and somatostatin.
5. The use of claim 1, wherein the retinoic acid is all-trans retinoic acid; the concentration of retinoic acid added is controlled to 0.1 to 4. Mu.M, more preferably 0.25 to 2. Mu.M, and most preferably 0.5. Mu.M.
6. The use according to claim 2, wherein the culture medium comprises DMEM/F-12 and islet expansion medium PIEM;
preferably, the PIEM takes DMEM/F-12 as a basic culture medium, and simultaneously adds RSPO1 conditioned medium, CHIR-99021, A8301, gastrin, N-acetylcysteine, Y-27632, forskolin, exendin-4, L-ascorbic acid, 5-iodotubercidin, recombinant human fibroblast growth factor-10, epidermal growth factor, nicotinamide and B27 cell culture additives.
7. A culture method for promoting the in vitro expansion of islet cell mass, comprising: retinoic acid is added to the medium.
8. The culture method of claim 7, wherein the retinoic acid is all-trans retinoic acid;
preferably, the concentration of retinoic acid added is controlled to be 0.1 to 4. Mu.M, more preferably 0.25 to 2. Mu.M, and most preferably 0.5. Mu.M.
9. The culture method according to claim 7, wherein the medium comprises DMEM/F-12 and islet expansion medium PIEM;
preferably, the PIEM takes DMEM/F-12 as a basic culture medium, and simultaneously adds RSPO1 conditioned medium, CHIR-99021, A8301, gastrin, N-acetylcysteine, Y-27632, forskolin, exendin-4, L-ascorbic acid, 5-iodotubercidin, recombinant human fibroblast growth factor-10, epidermal growth factor, nicotinamide and B27 cell culture additives;
preferably, the culture method further comprises the step of reducing CO by 1 to 8% at 30 to 40 ℃ (preferably 37 ℃), or 2 (preferably 5% CO) 2 ) Culturing under the culture conditions of (1).
10. Use according to any one of claims 1 to 6 and/or use according to any one of claims 7 to 9 of a culture method in any one or more of:
1) Constructing an islet organ model;
2) Constructing a diabetes animal model;
3) Screening and preparing diabetes-related medicaments;
4) Basic research on diabetes and related diseases;
5) Diabetes and related diseases.
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| CN1609198A (en) * | 2003-10-23 | 2005-04-27 | 陈立波 | Method of differentiating human bone marrow matrix stem cell to form insulin cell |
| CN1982440A (en) * | 2005-07-15 | 2007-06-20 | 北京市赛诺科生物工程技术有限公司 | Cultured pancreatic cell, its culturing method and use |
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