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Construction method and application of p-glycoprotein model of human small intestine 3D organoid research

An organoid, small intestine technology, applied in artificial cell constructs, biochemical equipment and methods, gastrointestinal cells, etc., can solve the problems of cumbersome operations, large differences in real conditions, unfavorable high-throughput screening of drugs, etc., and achieve economic costs. Low, short training period, saving manpower and material resources

Active Publication Date: 2020-01-07
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the two methods reported above are cumbersome to operate, which is not conducive to the high-throughput screening of drugs.
More importantly, these two methods of studying drug transport both use small intestinal 3D organoids in mice, and there are species differences between mice and humans, and the data obtained from mouse 3D organoids are different from the real situation in humans larger

Method used

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  • Construction method and application of p-glycoprotein model of human small intestine 3D organoid research
  • Construction method and application of p-glycoprotein model of human small intestine 3D organoid research
  • Construction method and application of p-glycoprotein model of human small intestine 3D organoid research

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Human Small Intestine 3D Organoid Culture

[0051] (1) Suspend human small intestinal crypts containing small intestinal stem cells, Paneth cells, small intestinal epithelial cells and other cell types in Matrigel, with a density of 5-10 complete crypts per 1 μL.

[0052] (2) Add an appropriate volume of the above Matrigel to a preheated 96-well plate (5 μL) or 24-well plate (50 μL), and place in a 37° C. incubator for 15 minutes to allow the Matrigel to solidify.

[0053] (3) After Matrigel is solidified, add corresponding volume (100 μL for 96-well plate, 500 μL for 24-well plate) containing various growth factors (Recombinant Human R-Spondin-1, Recombinant Murine Noggin, Recombinant Mouse Epidermal Growth Factor (EGF), Recombinant Mouse Wnt-3a) ADMEM / F12 medium, and then place it in a 37°C incubator for cultivation. The medium was changed every two days.

Embodiment 2

[0054] Example 2 Morphological observation of human small intestine 3D organoids

[0055] The 3D organoids cultured for 0, 1, 2, 3, 4, 5, and 6 days were observed and photographed under an Olympus IX 71 microscope equipped with an Olympus DP 71 camera system. The results are as follows figure 1 shown. It can be seen from the figure that with the increase of culture days, the volume of crypts gradually increased and organoid structures were formed. When the volume of organoids increases to a certain extent, the stem cells in the crypts begin to differentiate to form new crypts, which is manifested as a "budding" phenomenon.

Embodiment 3

[0056] Example 3 Detection of P-gp protein expression at the mRNA level in human small intestine 3D organoids

[0057] (1) Acquisition of 3D organoids: After 2 days of culturing 3D organoids (in a 24-well plate) according to the culture method in Example 1, take them out from the cell culture incubator, discard the medium, and wash twice with PBS Add 1 mL of pre-cooled PBS to each well, gently blow off the matrigel, transfer to a 15 mL centrifuge tube, and centrifuge at 200×g for 5 min at room temperature. The precipitate is 3D organoids. Discard the supernatant and set aside.

[0058] (2) Acquisition of mRNA: 500 μL Trizol was added to each tube containing small intestine tissue, sorted crypts and 3D organoids, and total mRNA was extracted according to standard procedures. (Among them, the human small intestine tissue has been homogenized)

[0059] (3) Acquisition of cDNA: The extracted total mRNA was used as a template (1000 ng of template), reversed into cDNA under the act...

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Abstract

The invention discloses a method for building P-glycoprotein (P-gp) research models based on human small intestine 3D (three-dimensional) organoid. The method includes inoculating human small intestine crypts in matrigel at first, adding ADMEM / F12 culture media with specific growth factors into the matrigel and cultivating the human small intestine crypts to form 3D organoids; carrying out morphological observation and detecting expression of P-gp from mRNA [messenger RNA (ribonucleic acid)] and protein level; researching influence of Verapamil and Mitotane on Rh123 transportation by the aid of a co-incubation process by Rhodamine 123 (Rh123) which is used as a substrate. The method has the advantages that the P-glycoprotein research models based on human small intestine 3D organoid can be applied to P-gp-mediated medicine transport research and also can be widely applied to in-vitro high-throughput screening on P-gp inhibitors, and the method is high in efficiency and speed.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for constructing a P-glycoprotein model for human small intestine 3D organoid research and its application in the study of P-glycoprotein-mediated drug transport. Background technique [0002] Oral drugs enter the human body mainly through four processes of absorption, distribution, metabolism and excretion, and the three processes of absorption, distribution and excretion are usually completed with the participation of transporters. For oral drugs, the absorption rate and degree of drug absorption in the digestive tract will directly affect the efficacy of the drug. Among them, P-glycoprotein (P-glycoprotein, P-gp) plays a very important role in drug absorption. [0003] P-gp (ABCB1, MDR1) is an important member of the ATP-binding cassette (ATP-binding cassette, ABC) transporter superfamily, expressed on the membrane surface, with a molecular weight of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071G01N33/68C12Q1/686C12N15/11
CPCC12N5/0602C12N5/0625C12N5/0679C12Q1/68G01N33/68
Inventor 王昕赵军芳曾之扬张远金李大力刘明耀
Owner EAST CHINA NORMAL UNIV
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