CN116083341A - Thyroid organoid culture medium and application thereof - Google Patents
Thyroid organoid culture medium and application thereof Download PDFInfo
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- CN116083341A CN116083341A CN202111308823.7A CN202111308823A CN116083341A CN 116083341 A CN116083341 A CN 116083341A CN 202111308823 A CN202111308823 A CN 202111308823A CN 116083341 A CN116083341 A CN 116083341A
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Abstract
The invention provides a thyroid organoid culture medium and application thereof, wherein the culture medium comprises a basal culture medium and retinoic acid. According to the culture medium provided by the embodiment of the invention, the number of times of passage of the thyroid organoids can be increased, the thyroid organoids which have good growth state and retain the pedigree and molecular characteristics of fetal thyroid tissues can be obtained, thyroid hormone-secreting human thyroid organoids can be secreted, the growth and development processes of the fetal thyroid organoids can be well simulated, and scientific researches can be carried out on the basis of the model.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a thyroid organoid culture medium and application thereof, and more particularly relates to a culture medium for culturing thyroid organoids, application of retinoic acid in preparation of a kit, application of the culture medium in preparation of the kit, a construction method of thyroid organoids, a thyroid organoid and a method for screening medicines.
Background
Thyroid gland function has a profound effect on human life-long health. Both the deficiency and excess of thyroid hormone can lead to potentially devastating health consequences. Currently, few studies have focused on the development of human thyroid; this is mainly because the developing embryo utilizes maternal thyroid hormone to ensure development of the central nervous system, whereas pregnant women often take thyroxine supplements to overcome hypothyroidism during gestation. However, limited diagnosis of juvenile thyroid disease before its severity increases and signs are urgent and there is a need to improve understanding of human thyroid development knowledge. Since critical developmental events have not yet been established at the cellular or molecular level, it is difficult to determine whether juvenile thyroid disease is due to embryo developmental defects. In addition, whether thyroxine is safe for the fetal thyroid for pregnant women also requires careful assessment.
To date, mouse genetics has established several factors that determine thyroid specialization and morphogenesis. In contrast, human studies have only some reports of the expression pattern of specific genes in a sample. The genome-wide transcriptome profile of human thyroid development, particularly at single cell resolution levels, is highly desirable for understanding the potential signs of thyroid development and early diagnosis in humans and for treating thyroid disorders.
For the comprehensive functional study of human thyroid development, there is a need for an organ model system that can generalize human thyroid development. Although thyroid organoids of ESC and IPSC origin or adult tissue origin show major thyroid characteristics and potential graft feasibility, they fail to mimic the specific time window of thyroid development, which limits the use of these models in studying thyroid development and pediatric disease progression. Thus, it is important to create an in vitro maturation system to produce human fetal thyroid organoids from fetal tissue.
Disclosure of Invention
The present application is made based on the discovery and recognition by the inventors of the following facts and problems:
in the invention, the inventor establishes a long-term culture system of human fetal thyroid organoids (hFTOs) by utilizing fetal thyroid tissues through screening various small molecular substances. Thyroid organoids are obtained that can be passaged for long periods of time, while preserving the lineage and molecular characteristics of human fetal thyroid tissue, maintaining the ability of their mice to produce human thyroid follicles following kidney transplantation. The invention can also induce hFTOs to mature by activating cAMP signals, and obtain hormone-secreting human thyroid organoids in vitro, which can well simulate key thyroid developmental events.
To this end, in a first aspect of the invention, the invention proposes a medium for culturing thyroid organoids. According to an embodiment of the invention, the culture medium comprises a basal medium and retinoic acid. The retinoic acid can accelerate the growth of fetal thyroid cells into fetal thyroid organoids (hFTOs), promote the proliferation of the thyroid organoids and the maintenance of morphology and function, so that the fetal thyroid organoids can be passaged for a long time, and finally the obtained hFTOs can simulate the real situation of embryo thyroid in morphology, transcription characteristics and secretion function, and scientific research based on the model has higher reliability.
According to an embodiment of the present invention, the above-mentioned medium may further include at least one of the following additional technical features:
according to an embodiment of the invention, the final concentration of retinoic acid in the culture medium is 40-60ng/mL. The concentration of retinoic acid in the culture medium according to the specific embodiment of the present invention can lengthen the number of passages of the thyroid organoids.
According to an embodiment of the invention, the final concentration of retinoic acid is 50ng/mL. The number of passages of the thyroid organoids was significantly increased at a concentration of 50ng/mL in the medium, thereby obtaining the thyroid organoids of different growth periods, and related studies were conducted using this as a model.
According to an embodiment of the invention, the medium comprises Noggin. According to the specific embodiment of the invention, the fetal thyroid cells can grow in a culture medium containing Noggin, the Noggin can accelerate proliferation of the fetal thyroid cells, and after Noggin and retinoic acid are added in the basic culture medium, the fetal thyroid cells can be passaged for a long time, so that thyroid organoids in different growth stages are obtained, and the fetal thyroid cells are studied by taking the culture medium as a model.
According to an embodiment of the invention, the final concentration of Noggin is 95-105ng/mL, preferably 100ng/mL. According to an embodiment of the invention, the fetal thyroid cells proliferate faster when the Noggin final concentration is 100ng/mL.
According to an embodiment of the invention, the medium comprises R-Spondin1, EGF, FGF10 and A8301. The culture medium according to embodiments of the present invention comprises a series of small molecule substances that can accelerate the growth of fetal thyroid cells into fetal thyroid organoids.
According to an embodiment of the invention, the final concentration of R-Spondin1 is 195-205ng/mL, preferably 200ng/mL.
According to an embodiment of the invention, the final concentration of EGF is 45-55ng/mL, preferably 50ng/mL.
According to an embodiment of the invention, the final concentration of FGF10 is 95-105ng/mL, preferably 100ng/mL.
According to an embodiment of the invention, the final concentration of A8301 is 490-510ng/mL, preferably 500nM.
According to an embodiment of the invention, the medium further comprises: glutamine, n-acetylcysteine, Y-27632, B27 and penicillin-streptomycin.
According to an embodiment of the invention, the final concentration of glutamine is 1mM-5mM, preferably 2mM.
According to an embodiment of the invention, the final concentration of n-acetylcysteine is 0.7-1.4mM, preferably 1mM.
According to an embodiment of the invention, the final concentration of Y-27632 is 5-15. Mu.M, preferably 10. Mu.M.
According to an embodiment of the invention, the final concentration of B27 is 1.0% -3.0% (v/v), preferably 2.0% (v/v).
According to an embodiment of the invention, the final concentration of penicillin-streptomycin is 0.1% -0.5% (v/v).
According to an embodiment of the invention, the basal medium is DMEM/F-12.
According to an embodiment of the invention, the medium further comprises Forskolin. According to the specific embodiment of the invention, the Forskolin can induce the fetal thyroid gland to mature, so that the fetal thyroid organoid is induced and grows into a mature thyroid organoid, the development condition of the thyroid gland is truly simulated, and the relevant research on the development of the thyroid gland in a specific time window can be performed by using the model.
According to an embodiment of the invention, the final concentration of Forskolin is 5-20 μm, preferably 10 μm. According to the specific embodiment of the invention, when the final concentration of Forskolin in the culture medium is 10 mu M, the fetal thyroid gland can be induced to mature, and the obtained mature thyroid organoids maintain good structural morphology, gene expression level and secretion function.
In a second aspect of the invention, the invention proposes the use of retinoic acid and/or Noggin in the preparation of a kit. According to an embodiment of the invention, the kit is for promoting thyroid cell proliferation and/or differentiation. According to the kit provided by the embodiment of the invention, proliferation and/or differentiation of thyroid cells can be promoted, thyroid organoids can be further obtained, the thyroid organoids can simulate the real situation of embryo thyroid development in morphology, transcription characteristics and secretion functions, and scientific research based on the model has higher reliability.
In a third aspect of the invention, the invention proposes the use of retinoic acid and/or Noggin in the preparation of a kit. According to an embodiment of the invention, the kit is used for inducing growth of thyroid single cells into thyroid organoids. According to the kit provided by the embodiment of the invention, thyroid cells are induced and promoted, thyroid organoids can be further obtained, the time for growing the thyroid cells into the thyroid organoids can be obviously shortened, the passage times of the thyroid organoids are prolonged, the thyroid organoids can simulate the real conditions of embryo thyroid development in morphology, transcription characteristics and secretion functions, and scientific research is carried out on the basis of the model, so that the kit has higher reliability.
In a fourth aspect of the invention, the invention provides the use of Forskolin in the preparation of a kit. According to an embodiment of the invention, the kit is used for inducing differentiation of a fetal thyroid organoid into a mature thyroid organoid. The kit prepared by Forskolin according to the embodiment of the invention can differentiate the fetal thyroid organoid into the mature thyroid organoid, and the mature thyroid organoid can simulate the real situation of the mature thyroid in morphology, transcription characteristics and secretion functions, and the scientific research based on the model has higher reliability.
In a fifth aspect of the invention, the invention provides the use of the medium of the first aspect for the preparation of a kit for culturing thyroid organoids. According to the kit provided by the embodiment of the invention, thyroid organoids can be obtained in a short period of time, the thyroid organoids obtained by culture can simulate the real situation of embryo thyroid development in morphology, transcription characteristics and secretion functions, and scientific research based on the model has higher reliability. In a sixth aspect of the invention, the invention provides a method of constructing a thyroid organoid. According to an embodiment of the invention, the method comprises the step of culturing thyroid cells using the medium according to the first aspect. The thyroid organoids obtained by the specific method of the embodiment of the invention can simulate the real situation of embryo thyroid development in morphology, transcription characteristics and secretion functions, and the scientific research based on the model has higher reliability.
In a seventh aspect of the invention, the invention provides a thyroid organoid. According to an embodiment of the invention, the thyroid organoids are constructed using the method according to the fifth aspect. The thyroid organoids according to the embodiments of the present invention can simulate the true situation of embryo thyroid development in morphology, transcription characteristics, and secretion functions, and scientific research based on the model has high reliability.
In an eighth aspect of the invention, the invention provides a method of screening for a drug. According to an embodiment of the invention, the drug to be screened is contacted with thyroid cells or thyroid organoids of the seventh aspect, which thyroid organoids have been subjected to a disease modeling treatment in advance; comparing the disease states of thyroid organoids before and after contact, and judging whether the drug to be screened is a target drug. Comparing the disease states of thyroid organoids before and after contact, judging whether the drug to be screened is a target drug, if the expression level of at least one of the PAX8, nkx2-1, TSHR, TG, TPO, SLC A5, PDE7A and PDE10 genes in thyroid cells or thyroid organoids after contact returns to the normal level without disease state, then judging the drug to be the target drug. According to the method for screening medicaments provided by the embodiment of the invention, medicaments for effectively treating or relieving thyroid-related diseases can be obtained.
According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:
according to an embodiment of the invention, improvement of the disease state of thyroid organoids after contact is an indication that the drug to be screened is the target drug. The target drug according to an embodiment of the present invention can restore the function of the thyroid gland, so that the disease state is improved, such as the expression of at least one of the thyroid organoid PAX8, nkx2-1, TSHR, TG, TPO, SLC A5, PDE7A and PDE10 genes is restored to normal levels after contact.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a bright field image of the results of a first generation fetal thyroid organoid culture grown in Medium 1 and Medium 1+noggin, scale bar 50 μm;
FIG. 2 is a bright field image of the results of second and fifth generation fetal thyroid organoids cultured using Medium 1+noggin and Medium 1+noggin+retinoic acid, scale bar 50 μm;
FIG. 3 is a bright field picture of the stages of day 0 culture, days 1, 5, 15, and day7 (P7 day7 (> 3 montahs)) of a fetal thyroid organoid according to an embodiment of the invention, wherein Thyroid organoids represents the thyroid organoid, and Enlargement represents an enlarged image of the culture up to day 15, to a scale of 500 μm;
FIG. 4 is a graph of Real-time PCR results of fetal thyroid marker genes according to an embodiment of the present invention, wherein the abscissa represents PAX8, NKX2.1, TG, TSHR, and TPO genes, the ordinate mRNA relative expression (Log 10) represents the relative expression level of mRNA (Log 10), negative represents Negative control (bile duct organoids), organoid1 represents fetal thyroid Organoid biological repeat group 1, organoid2 represents fetal thyroid Organoid biological repeat group 2;
FIG. 5 is a schematic representation of the bright field pattern of induced mature thyroid organoids on days 1, 3, 7, 11, 15 and cross-sectional structure stained with hematoxylin and eosin on day 15 of culture in growth factor medium and Foskolin treatment conditions, wherein GF group represents the fetal thyroid organoids not stimulated with Forskolin, +Fors group represents the fetal thyroid organoids stimulated with Forskolin,
5-A shows the bright field plot of organoids grown for days 1, 3, 7, 11, 15 under growth factor medium and Foskolin treatment conditions, scale bar 100 μm,
5-B shows a structure of a section of the culture day 15 stained with hematoxylin and eosin, with a scale of 50 μm;
FIG. 6 is a graph showing the results of statistical analysis of Real-time PCR of thyroid cell markers and cAMP-related genes of mature thyroid organoids according to an embodiment of the present invention, wherein the abscissa represents different treatment groups (GF group represents fetal thyroid organoids not stimulated with Forskolin group, +Forskolin group represents fetal thyroid organoids stimulated with Forskolin group) and the ordinate represents the relative expression amount of mRNA.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present invention, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise.
The term "optionally" is used for descriptive purposes only and is not to be construed as indicating or implying relative importance. Thus, a feature defined as "optional" may explicitly or implicitly include or exclude that feature.
Herein, the term "disease modeling process" refers to the creation of a disease model that is helpful in studying the mechanisms of occurrence and development of a disease, as in this application 3D thyroid organoids models that resemble in vivo environments may be used to model a disease in a more physiologically relevant environment. The disease modeling process is not particularly limited, and any process that causes a disease state in the thyroid organoid may be used, and the disease may be any disease associated with thyroid disease.
Herein, the drug screening is not particularly limited, and any drug that can be screened with the thyroid organoids described herein is included in the scope of the drug screening described herein, e.g., drug high throughput screening. The drug high-throughput screening refers to a screening model based on a molecular level or a cell level, takes a microplate as a reaction carrier, combines an automatic operation system and a sensitive and rapid detection method, and efficiently completes detection of tens of millions of samples to be detected so as to obtain a target drug.
Herein, the term "disease state improvement" refers to the condition that the disease state of the thyroid organoid gradually disappears after the thyroid organoid previously subjected to disease modeling treatment is contacted with the target drug, and the structure, function and metabolism of the thyroid organoid are repaired or completely restored to normal. It should be noted that the disease state improvement is not particularly limited, and any thyroid disease detection index is significantly improved or restored to normal levels without disease, both of which are disease states improvement.
The inventor screens various small molecular substances, develops a culture system and a method for humanized embryo peptide thyroid organoids, wherein the culture system contains retinoic acid, a humanized embryo peptide thyroid organoid model is established by adopting the culture system, the culture method and embryo peptide thyroid cell culture, the embryo peptide thyroid cells are obtained by purchasing or separating waste human embryo thyroid cell group tissues after 8-12 weeks post-fertilization development, and the liver peptide thyroid organoids can better simulate the growth process of embryo thyroid and can be used as a research model of embryo thyroid, such as: the fetal thyroid organoid model may be used to screen a variety of drugs.
In this application, the inventors performed structural identification of the obtained fetal thyroid organoids and detection of PAX8, NKX2.1, TG, TSHR and TPO gene expression levels to determine if the fetal thyroid organoids were successfully constructed. The fetal thyroid organoids that were successfully constructed reproduced a structure of a single layer of follicular epithelium similar to the original thyroid gland structure, with a complete follicular structure formed of several columnar epithelial cells with high electron density jelly in the lumen. These highly polarized cell lumen sides contain abundant mitochondria and lysosomes. In addition, the major carrier of iodinated-TG in glands, low electron density colloidal vesicular particles, can be occasionally observed by electron microscopic examination. On the aspect of transcription characteristics, the embryo thyroid organoid obtained by the culture system and the culture method highly expresses transcription factors and cell markers specifically expressed in thyroid follicular cells, and can normally secrete thyroid hormone. The inventors transplanted hFTOs organoids under the kidneys of NSG mice, immunostaining showed that the grafts formed a number of epithelial follicular structures in the kidney envelope, with deposition of cytoplasmic TG and polarized expression of basement membrane outer membrane NIS in the chamber, confirming the development of mature thyroid follicles at the site of transplantation. Detection of thyroid hormone T4 also demonstrated hormone secretion function of transplanted thyroid.
Subsequently, the inventor performs maturation induction on the successfully constructed fetal thyroid organoids by adding Forskolin or thyroid stimulating hormone TSH to a primary culture medium, and also performs structural identification on the induced thyroid organoids and detection of the expression levels of PAX8, NKX2.1, TG, TSHR, TPO, SLC A5, PDE7A and PDE10a genes to determine whether the thyroid organoids are thyroid that induces maturation. Wherein, PAX8 is a specific gene involved in the development of thyroid follicular cells; NKX2.1 is a thyroid-specific transcription factor, regulating thyroid-specific gene expression; TG encodes thyroglobulin, a substrate for synthesizing thyroxine and triiodothyronine, specifically expressed in thyroid follicular cells; TSHR encodes the receptor for thyroid hormone, a major controlling factor for thyroid cell metabolism; TPO encodes a membrane-bound glycoprotein which plays a central role as an enzyme in the synthesis of thyroxine and thyroid hormone, etc. The mature thyroid organoids (hMTOs) include the following features: hFTOs are able to form larger follicular structures and the mRNA expression levels of the organoid thyroid markers SLC5A5, TSHR, TG and TPO, as well as T4 secretion levels in organoid supernatants, are all significantly up-regulated. In addition, by transcriptome comparison with thyroid tissue samples of fetuses and adults, it was found that the differentially expressed genes between hFTOs and hMTOs significantly overlapped with the differentially expressed genes between fetuses and adult thyroid, and that genes related to mineral absorption, hormone synthesis and secretion were significantly upregulated in hMTOs, indicating that hMTOs are functionally closer to adult thyroid.
By utilizing the culture and differentiation system of the embryo thyroid organoid, the thyroid diseases of some pediatrics or adolescents can be simulated and studied while the development of the fetal thyroid is studied.
Sources of reagents used in the present application:
retinoic acid employed in the examples of the present invention was purchased from MCE (MedChemExpress) company.
R-Spondin1 used in the examples of the present invention was purchased from Organregen.
Noggin used in the examples of the present invention was purchased from organoregen corporation.
EGF used in the examples of the present invention was purchased from Invitrogen corporation.
FGF10 employed in the examples of the present invention was purchased from Organregen.
A8301 used in the examples of the present invention was purchased from Tocres corporation.
The glutamine employed in the examples of the present invention was purchased from Invitrogen corporation.
The n-acetylcysteine used in the examples of the present invention was purchased from Sigma-Aldrich.
Y-2763 used in the examples of the present invention was purchased from Sigma-Aldrich.
B27 employed in the examples of the present invention was purchased from Invitrogen.
The following examples are provided to illustrate the general commercial products of reagents, sequences, software and instrumentation not specifically addressed in the following examples.
EXAMPLE 1 fetal thyroid organoids construction and culture
1. Thyroid organoid culture medium component screening
(1) The thyroid cells are purchased, and subjected to 3 times of cleaning, re-suspension and hole laying by Matrigel (Corning);
(2) To investigate the medium composition suitable for fetal thyroid organoids, the inventors made various attempts to first supplement penicillin-streptomycin (Invitrogen), HEPES buffer (10 mM, gibco), B27 supplement (2%, invitrogen), glutamine (2 mM, invitrogen), n-acetylcysteine (1 mM Sigma-Aldrich), Y-27632 (10. Mu.M) in Advanced DMEM/F-12 basal medium. On the basis, the inventor screens the following growth factors/small molecule substances: EGF, R-Spondin1, FGF10, A83-01. The specific screening process is as follows: adding EGF (50 ng/mL) as a growth factor to be screened on the basis of the culture medium, finding that the fetal thyroid organoids cannot grow, continuing to add R-Spondin1 (200 ng/mL, organRegen), finding that the thyroid organoids cannot grow, continuing to add FGF10 (100 ng/mL, organRegen), finding that the thyroid organoids cannot grow, continuing to add small molecule A83-01 (500 nM, tocres), and still finding that the thyroid organoids cannot grow, so far, the inventor refers to the culture medium added with the substances as the culture medium 1, and after adding Noggin (100 ng/mL, organRegen) on the basis of the culture medium 1, the organoids can finish the first-generation culture (figure 1), but cannot be passaged for a long time. Subsequently, the inventors, again on the basis of Noggin addition, added retinoic acid, when retinoic acid concentration was added to 50ng/ML (MCE), achieved long-term passage of fetal thyroid organoids (fig. 2). The inventors performed a photograph recording of the different stages of fetal thyroid organoid culture based on this formulation (fig. 3). The above results indicate that Noggin is critical for the formation of fetal thyroid organoids, while retinoic acid is critical for long-term passage of fetal thyroid organoids.
2. Fetal thyroid organoid related gene detection
Fetal thyroid organoids cultured in 24 well plates for 30 days (third generation) were collected, as described in experiment 1 of this example, with the negative control being biliary organoids, organoid1 group being fetal thyroid Organoid biological repeat group 1, organoid2 group being fetal thyroid Organoid biological repeat group 2, and the fetal thyroid organoids used in the repeat of group 2 being obtained in the same batch. Thyroid organoid RNA was extracted and purified using a RNAprep Pure Micro Kit (day root biotechnology) kit, and specific experimental procedures were performed according to the instructions. Purified RNAs were reverse transcribed into cDNA using a reverse transcription system (Promega) according to the manufacturer's instructions, and real-time quantitative PCR was performed on a CFX 384-touch system (BioRad) using TaqMan Universal PCR Master Mix (Bimake B21202) using GAPDH as an internal reference gene to detect experimentally-related genes of interest.
The specific experimental results are shown in fig. 4, wherein PAX8 is a specific gene involved in development of thyroid follicular cells; NKX2.1 is a thyroid-specific transcription factor, regulating thyroid-specific gene expression; TG encodes thyroglobulin, a substrate for synthesizing thyroxine and triiodothyronine, specifically expressed in thyroid follicular cells; TSHR encodes the receptor for thyroid hormone, a major controlling factor for thyroid cell metabolism; TPO encodes a membrane-bound glycoprotein which serves as an enzyme that plays a central role in the synthesis of thyroxine and thyroid hormone, all of which are thyroid characteristic expression gene sets; as shown in fig. 4, the relative expression levels of these characteristic genes in the fetal thyroid organoids were significantly increased compared to the control group, indicating that the fetal thyroid organoids have the characteristics of the expression profile of thyroid, i.e., were able to mimic the function of thyroid.
Example 2 induction of fetal thyroid organoids into mature thyroid organoids
1. Structural detection of induced mature thyroid organoids
Normal cultured fetal thyroid organoids and fetal thyroid organoids treated with induction medium for 7 days were selected for detection, and morphology observation and staining of organoids were performed by paraffin sections and hematoxylin-eosin (H & E). The specific experimental operation is as follows: the tissues were fixed in 4% paraformaldehyde overnight and then paraffin embedded; thyroid organoids were gently collected, fixed in 4% pfa solution for 2 hours, and embedded with 3% agarose gel prior to paraffin embedding. Subsequently, 5 μm thick sections were attached to slides. After dewaxing and rehydration, slides were stained, dehydrated and blocked with Mei Yeshi hematoxylin and images were taken using the cellSens system of an Olympus microscope.
The resulting images are shown in FIG. 5, where GF group represents normal cultured fetal thyroid organoids, +Fors represents fetal thyroid organoids treated with induction medium (10 μm Forskolin added on normal medium) for 7 days, and hematoxylin-eosin (H & E) staining results show that +Fors induced group develops larger follicles similar to mature thyroid indicating that fetal thyroid organoids trend to mature after induction.
2. Thyroid cell markers inducing mature thyroid organoids and detection of cAMP-related gene expression levels
And (3) selecting normal cultured fetal thyroid organoids and fetal thyroid organoids treated by an induction medium for 7 days for detection, and detecting thyroid cell markers and cAMP related gene expression levels of mature thyroid organoids by adopting a Real-time PCR technology.
Relative expression amounts of mRNA of PAX8, NKX2.1, TSHR, TG, TPO, SLC5A5, PDE7A and PDE10a were examined by RT-PCR. Wherein SLC5A5 encodes a sodium ion-glucose cotransporter responsible for the absorption of iodine by the thyroid; PDE7A encodes a cyclic nucleotide phosphodiesterase, playing a key role in a number of important physiological functions by regulating intracellular cAMP concentrations; PDE10 a-encoded proteins also belong to the cyclic nucleotide phosphodiesterase family, and act by a similar mechanism to PDX 7A. As shown in FIG. 6, compared with the normal cultured fetal thyroid organoids, the thyroid maturation characteristic genes in the thyroid organoids subjected to the induction treatment by adding Forskolin are obviously increased, which indicates that the addition of small molecule Forskolin can promote the maturation of the fetal thyroid, and the development process of the thyroid is simulated in vitro.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (15)
1. A culture medium for culturing thyroid organoids, wherein the culture medium comprises a basal medium and retinoic acid.
2. The medium of claim 1, wherein the final concentration of retinoic acid in the medium is 40-60ng/mL;
optionally, the final concentration of retinoic acid is 50ng/mL.
3. The culture medium of claim 1, comprising Noggin;
optionally, the final concentration of Noggin is 95-105ng/mL, preferably 100ng/mL.
4. The medium of claim 1, comprising R-Spondin1, EGF, FGF10 and a8301;
optionally, the final concentration of R-Spondin1 is 195-205ng/mL, preferably 200ng/mL;
optionally, the final concentration of EGF is 45-55ng/mL, preferably 50ng/mL;
optionally, the final concentration of FGF10 is 95-105ng/mL, preferably 100ng/mL;
optionally, the final concentration of A8301 is 490-510ng/mL, preferably 500nM.
5. The medium of claim 1, further comprising: glutamine, n-acetylcysteine, Y-27632, B27, and penicillin-streptomycin;
optionally, the final concentration of glutamine is 1mM-5mM, preferably 2mM;
optionally, the final concentration of n-acetylcysteine is 0.7-1.4mM, preferably 1mM;
optionally, the final concentration of Y-27632 is 5-15. Mu.M, preferably 10. Mu.M;
optionally, the final concentration of B27 is 1.0% -3.0% (v/v), preferably 2.0% (v/v);
optionally, the final concentration of penicillin-streptomycin is 0.1% -0.5% (v/v).
6. The medium of claim 1, further comprising: advanced DMEM/F-12.
7. The medium of claim 1, further comprising: the composition of Forskolin,
optionally, the final concentration of Forskolin is 5-20 μm, preferably 10 μm.
8. Use of retinoic acid in the preparation of a kit for promoting thyroid cell proliferation.
9. Use of retinoic acid in the preparation of a kit for inducing growth of thyroid single cells into thyroid organoids.
Use of forskolin in the preparation of a kit for inducing differentiation of a fetal thyroid organoid into a mature thyroid organoid.
11. Use of the medium of any one of claims 1-7 for the preparation of a kit for culturing thyroid organoids.
12. A method for constructing thyroid organoids, comprising the step of culturing thyroid cells using the medium according to any one of claims 1 to 7.
13. A thyroid organoid constructed by the method of claim 12.
14. A method for screening a drug, characterized in that the drug to be screened is contacted with a thyroid cell or the thyroid organoid of claim 13, which has been previously subjected to a disease modeling treatment;
comparing the disease states of thyroid organoids before and after contact, and judging whether the drug to be screened is a target drug.
15. The method of claim 14, wherein improvement in disease state of the thyroid organoid after contact is indicative of the drug to be screened as the drug of interest.
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