Application of plant polyphenol picrol H in regulation and control of intestinal organ differentiation

Abstract

The invention discloses an application of plant polyphenol picrol H in regulation and control of differentiation of intestinal organs. The picrol H (Kuwanon H) is a flavonoid compound from mulberry. According to the method, Kuwanon H is added into a culture medium in an intestinal organ culture system, so that the growth condition of intestinal organs is remarkably enhanced, specifically, the budding number (namely, the number of goblet cells) is increased, and proliferation and differentiation related gene expression is up-regulated. The changes finally promote proliferation and differentiation of the intestinal organs, so that the growth of the intestinal organs is more vigorous, and the development process of the intestinal organs is effectively accelerated.

Description

Technical Field

[0001] The present invention belongs to the field of intestinal organoid culture, and specifically relates to the application of plant polyphenol picrol H in regulating the differentiation of intestinal organoids. Background Art

[0002] As the "first line of defense for physical health", the intestine is not only an important organ for nutrient digestion and absorption, but also the largest immune detoxification organ of the human body. It is crucial to maintaining the body's homeostasis. Imbalance in intestinal health can lead to intestinal diseases. Currently, most of the models used for intestinal development and disease are two-dimensional cell models, animal models, etc. In recent years, intestinal organoids, as a new in vitro intestinal model, can replace traditional colon adenocarcinoma cell lines to reproduce the key characteristics of complex intestinal physiology in vivo for development and disease research, as well as to evaluate drug metabolism and absorption in the small intestine. Intestinal organoids can be derived from the self-renewal of adult stem cells or pluripotent stem cells.

[0003] However, when the existing culture medium for intestinal organoids is used, the proliferation of various cells (intestinal stem cells, goblet cells) is relatively slow. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0005] Another object of the present invention is to provide the application of plant polyphenol pyrifos H in regulating the differentiation of intestinal organoids.

[0006] To this end, the technical solution provided by the present invention is:

[0007] Application of plant polyphenol picromelin H in regulating intestinal organoid differentiation. Picromelin H is a flavonoid compound derived from mulberry.

[0008] Preferably, in the application of the plant polyphenol pyrimidine H in regulating the differentiation of intestinal organoids, the molecular formula of the pyrimidine H is C 45 H 44 O 11 , its chemical formula is as follows:

[0009]

[0010] Preferably, the plant polyphenol picrol H is used in regulating the differentiation of intestinal organoids, wherein the regulating the differentiation of intestinal organoids includes regulating the proliferation of intestinal organoids.

[0011] Preferably, in the use of the plant polyphenol pyrifos H in regulating the differentiation of intestinal organoids, the concentration of pyrifos H is 4 nM.

[0012] The present invention has at least the following beneficial effects:

[0013] The present invention significantly enhances the growth of intestinal organoids by adding Kuwanon H to the intestinal organoid culture system, which is specifically manifested by an increase in the number of buds (i.e., the number of goblet cells) and an upregulation of the expression of genes related to proliferation and differentiation. These changes ultimately promote the proliferation and differentiation of organoids, making the growth of organoids more vigorous and effectively accelerating the development process of intestinal organoids.

[0014] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A schematic diagram showing microscope observation of the experimental results in the embodiments of the present invention.

[0016] Figure 2 A schematic diagram of a kuwanon H reference comparison experiment in an embodiment of the present invention is shown.

[0017] Figure 3 A schematic diagram of a reference experiment of different compounds in the examples of the present invention is shown. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0019] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0020] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0021] In order to enable those skilled in the art to better understand the technical solution of the present invention, the following embodiments are provided for further explanation:

[0022] See also Figure 1 - Figure 3 As shown, the application of plant polyphenol Kuwanon H in regulating the differentiation of intestinal organoids, wherein Kuwanon H is a flavonoid compound in mulberry, and is a non-peptide bombesin receptor antagonist. Kuwanon H can specifically inhibit the binding of intracellular gastrin-releasing peptide CRP to (GRP-preferring receptor) receptors. i The value is 290nM.

[0023] In one embodiment of the present invention, preferably, the molecular formula of kuwanon H is C 45 H 44 O 11 ; The chemical formula is as follows:

[0024]

[0025] In one embodiment of the present invention, preferably, the regulating the differentiation of intestinal organoids includes regulating the proliferation of intestinal organoids.

[0026] In one embodiment of the present invention, preferably, the kuwanon H is used for proliferation of intestinal organoid culture, and the concentration of the kuwanon H is 4 nM.

[0027] Raw material selection: First, determine the natural raw materials containing the target compound (such as "picryl alcohol H"), such as plants, microorganisms or marine organisms. This is usually based on previous research or literature reports.

[0028] The Kuwanon H is a flavonoid compound in mulberry, and is a non-peptide bombesin receptor antagonist. Kuwanon H can specifically inhibit the binding of intracellular gastrin-releasing peptide CRP to (GRP-preferring receptor) receptors. i The value is 290 nM, and kuwanon H is a commercially available product, and no further introduction is given. The product used in the present invention is the product HY-N2600 from MCE.

[0029] Example 1

[0030] In the process of culturing intestinal organoids, the present invention adds different key elements to the culture medium to observe the process of cell differentiation and proliferation. The detection process is as follows:

[0031] (1) The frozen intestinal organoids were quickly thawed in warm water at 37°C, and centrifuged with 4 times the volume of 90% Advanced DMEM / F12 plus 10% FBS (4°C, 200 rcf, 5 min). The Advanced DMEM / F12 was purchased from Gibco with the catalog number 12634-010. After that, the intestinal organoid culture medium was embedded with Matrigel at a ratio of 1:1, and cultured in a 24-well plate to form 20 μL droplets. The culture was placed in an incubator for 20 min to solidify, and 500 μL of intestinal organoid culture medium was added. After culturing for 3 days, the culture medium was a customized product of Hunan Ogred Biotechnology Co., Ltd., Crypt Boosting Medium. The frozen intestinal organoids were extracted from the jejunal tissue of the small intestine of newborn healthy pigs.

[0032] (2) After the pig's intestinal organoids were revived, they were passaged to P1. After the culture medium was aspirated, TE digestion solution was added for digestion. The TE digestion solution is Trypsin / EDTA Solution, which belongs to life technologies company, and the product number is R001100. After 3 minutes, 3-5 times the volume of 90% Advanced DMEM / F12+10% FBS was added for centrifugation (4°C, 200rcf, 5 minutes), and intestinal organoid culture medium and Matrigel were added in a 1:1 ratio for embedding. The culture was incubated in an incubator for 20 minutes to wait for solidification, and 500μL of culture medium was added. The intestinal organoids were able to grow stably, the organoid budding was stable, and the quantity met the experimental requirements;

[0033] (3) After absorbing the culture medium, add TE digestion solution for digestion. After 3 minutes, add 3-5 times the volume of 90% Advanced DMEM / F12+10% FBS for centrifugation (4°C, 200rcf, 5 minutes). Add intestinal organoid culture medium and Matrigel at a ratio of 1:1 for embedding. Incubate in the incubator for 20 minutes to wait for coagulation. Add 500 μL culture medium containing 4 nM Kuwanon H and culture until maturity. Observe the growth status of the 4 nM Kuwanon H treatment group and the control group without Kuwanon H treatment.

[0034] Figure 1 This is a bright field image of the growth status of the 4 nM Kuwanon H-treated group and the control group without Kuwanon H treatment. The results showed that the intestinal organoids treated with 4 nM Kuwanon H grew well compared with the control group without Kuwanon H treatment.

[0035] Example 2

[0036] (1) The frozen intestinal organoids were quickly thawed in warm water at 37°C, and centrifuged with 4 times the volume of 90% Advanced DMEM / F12 plus 10% FBS (4°C, 200 rcf, 5 min). The Advanced DMEM / F12 was purchased from Gibco with the catalog number 12634-010. After that, the intestinal organoid culture medium was embedded with Matrigel at a ratio of 1:1, and cultured in a 24-well plate to form 20 μL droplets. The culture was placed in an incubator for 20 min to solidify, and 500 μL of intestinal organoid culture medium was added. After culturing for 3 days, the culture medium was a customized product of Hunan Ogred Biotechnology Co., Ltd., Crypt Boosting Medium. The frozen intestinal organoids were extracted from the jejunal tissue of the small intestine of newborn healthy pigs.

[0037] (2) After the pig's intestinal organoids were revived, they were passaged to P1. After the culture medium was aspirated, TE digestion solution was added for digestion. The TE digestion solution is Trypsin / EDTA Solution, which belongs to life technologies company, and the product number is R001100. After 3 minutes, 3-5 times the volume of 90% Advanced DMEM / F12+10% FBS was added for centrifugation (4°C, 200rcf, 5 minutes), and intestinal organoid culture medium and Matrigel were added in a 1:1 ratio for embedding. The culture was incubated in an incubator for 20 minutes to wait for solidification, and 500μL of culture medium was added. The intestinal organoids were able to grow stably, the organoid budding was stable, and the quantity met the experimental requirements;

[0038] (3) After absorbing the culture medium, add TE digestion solution for digestion. After 3 minutes, add 3-5 times the volume of 90% Advanced DMEM / F12+10% FBS for centrifugation (4°C, 200rcf, 5 minutes). Add intestinal organoid culture medium and Matrigel at a ratio of 1:1 for embedding. Wait for solidification in the incubator for 20 minutes, and add 500μL culture medium containing 4nM Kuwanon H to culture until maturity. The number of buds in the 4nM Kuwanon H treatment group and the control group without Kuwanon H treatment was counted. Each group has 3 parallels.

[0039] Figure 2 Figure 1 is a statistical graph of the number of sprouts in the 4nM Kuwanon H treated group and the control group not treated with Kuwanon H. The results showed that by statistically analyzing the sprouting rate, Kuwanon H treatment promoted the sprouting of intestinal organoids, compared with the organoids not treated with Kuwanon H (e.g. Figure 2 ), the proportions of intestinal organoids with 1 to 3 buds, 4 to 6 buds, and 7 to 9 buds in the Kuwanon H-treated group were greater than those in the control group not treated with Kuwanon H. This indicates that Kuwanon H at a low concentration can promote the maturation of intestinal organoids.

[0040] Example 3

[0041] (1) The frozen intestinal organoids were quickly thawed in warm water at 37°C, and centrifuged with 4 times the volume of 90% Advanced DMEM / F12 plus 10% FBS (4°C, 200 rcf, 5 min). The Advanced DMEM / F12 was purchased from Gibco with the catalog number 12634-010. After that, the intestinal organoid culture medium was embedded with Matrigel at a ratio of 1:1, and cultured in a 24-well plate to form 20 μL droplets. The culture was placed in an incubator for 20 min to solidify, and 500 μL of intestinal organoid culture medium was added. After culturing for 3 days, the culture medium was a customized product of Hunan Ogred Biotechnology Co., Ltd., Crypt Boosting Medium. The frozen intestinal organoids were extracted from the jejunal tissue of the small intestine of newborn healthy pigs.

[0042] (2) After the pig's intestinal organoids were revived, they were passaged to P1. After the culture medium was aspirated, TE digestion solution was added for digestion. The TE digestion solution is Trypsin / EDTA Solution, which belongs to life technologies company, and the product number is R001100. After 3 minutes, 3-5 times the volume of 90% Advanced DMEM / F12+10% FBS was added for centrifugation (4°C, 200rcf, 5 minutes), and intestinal organoid culture medium and Matrigel were added in a 1:1 ratio for embedding. The culture was incubated in an incubator for 20 minutes to wait for solidification, and 500μL of culture medium was added. The intestinal organoids were able to grow stably, the organoid budding was stable, and the quantity met the experimental requirements;

[0043] (3) After absorbing the culture medium, add TE digestion solution for digestion. After 3 minutes, add 3-5 times the volume of 90% Advanced DMEM / F12+10% FBS for centrifugation (4°C, 200rcf, 5 minutes). Add intestinal organoid culture medium and Matrigel at a ratio of 1:1 for embedding. Incubate in the incubator for 20 minutes to wait for solidification. Add 500 μL culture medium containing 4 nM Kuwanon H and culture until maturity. Each group has 3 parallels.

[0044] (4) Collect mature intestinal organoids, wash them three times with PBS buffer, and centrifuge. Use the Trizol method to extract total RNA, as follows: resuspend the cells with 1ml Trizol, blow up and down until there are no cell clumps and the entire solution is clear; add 200μl chloroform, shake evenly on a vortexer, place at room temperature for 3min, wait for stratification, centrifuge at 12000g at 4℃ for 15min, and the solution will be stratified. Take the upper layer of liquid into a new EP tube. Be careful during the whole process to avoid contact with the white precipitate in the middle layer; add an equal amount of isopropanol to the aspirated liquid, mix it upside down, and let it stand at -20℃ overnight to facilitate RNA precipitation; centrifuge at 12000rpm for 10min; retain the white RNA precipitate at the bottom of the tube and carefully remove the supernatant; wash the white precipitate with 75% ethanol and centrifuge at 12000rpm for 5min; remove 75% ethanol as much as possible and let it evaporate at room temperature for 5min to prevent ethanol from contaminating the sample and affecting subsequent experiments; dissolve the RNA precipitate with DEPC water; measure the RNA concentration and purity, and dilute it accordingly, with a final concentration of 69ng / ul.

[0045] In the second step, mRNA was reverse transcribed into cDNA in a 50 μl system.

[0046] The third step was real-time PCR, with the ligand solution prepared according to the kit requirements, the final system was 5 μl, and the annealing temperature Tm was uniformly set at 58 °C;

[0047] Real-time fluorescence quantitative PCR was used to detect the expression of genes related to proliferation and differentiation marker proteins associated with intestinal organoids: chromophore protein A (ChgA), proliferating cell nuclear antigen (PCNA), human mucin (MUC2), tight junction protein (Occludin / ZO-1), intestinal stem cell (LGR5) and B-lymphoma Moloney murine leukemia virus insertion region-1 (BMI1) in the normal group without treatment with 4 nM Kuwanon H and the control group treated with 4 nM Kuwanon H (as shown in Figure 2). Figure 3 ). The results showed that compared with the control group, the levels of proliferation and differentiation markers in the 4nM Kuwanon H treatment group were significantly increased, proving that Kuwanon H can produce beneficial effects on the growth of intestinal organoids.

[0048] All of the above indicate that picrol H can promote the rapid proliferation and differentiation of intestinal organoids.

[0049] Although the embodiments of the present invention are disclosed above, they are not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the embodiments of the present invention. For those familiar with the art, other modifications can be easily realized. Therefore, without departing from the general concept defined by the claims and equivalent scopes, the embodiments of the present invention are not limited to the specific details and the legends shown and described here.

Claims

1. Application of plant polyphenol pyranol H in regulating intestinal organoid differentiation, characterized in that: Picrohol H is a flavonoid compound derived from mulberry.

2. The use of the plant polyphenol bitter alcohol H in regulating intestinal organoid differentiation according to claim 1, characterized in that: The molecular formula of the picryl alcohol H is C 45 H 44 O 11 , its chemical formula is as follows:

3. The use of the plant polyphenol bitter alcohol H in regulating intestinal organoid differentiation according to claim 1, characterized in that: The regulating the differentiation of intestinal organoids includes regulating the proliferation of intestinal organoids.

4. The use of the plant polyphenol pyranol H in regulating intestinal organoid differentiation according to claim 1, characterized in that: The concentration of picromelin H is 4 nM.

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