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Method for building BCRP (breast cancer resistance proteins) mediated medicine transport models for research on 3D (three-dimensional) organs of small intestines and application

A construction method and organoid technology, which can be applied in the fields of biochemical equipment and methods, artificial cell constructs, and microbial assay/inspection. and physiological functions, etc., to achieve the effect of efficient, fast, and simple operation.

Inactive Publication Date: 2017-08-04
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this model is considered to be the standard for studying BCRP drug transport in vitro, there are still shortcomings. First, caco-2 is a colon cancer cell line, which is quite different from normal intestinal epithelial cells in terms of morphology and physiological functions; second. Cell generation and culture conditions have a great influence on the stability of the monolayer cell transport model; finally, caco-2 needs a 21-day culture cycle to form a compact monolayer structure, and too long formation time is not conducive to BCRP substrates and inhibitors high-throughput screening of

Method used

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  • Method for building BCRP (breast cancer resistance proteins) mediated medicine transport models for research on 3D (three-dimensional) organs of small intestines and application
  • Method for building BCRP (breast cancer resistance proteins) mediated medicine transport models for research on 3D (three-dimensional) organs of small intestines and application
  • Method for building BCRP (breast cancer resistance proteins) mediated medicine transport models for research on 3D (three-dimensional) organs of small intestines and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Isolation of mouse small intestinal crypts and culture of small intestinal 3D organoids

[0075] (1) After 6-8 week old C57BL / 6 mice were killed by cervical dislocation, the small intestine was taken out and placed in pre-cooled PBS.

[0076] (2) After carefully removing the mesenteric adipose tissue with tweezers, cut it from the middle, wash the intestinal contents with pre-cooled PBS, transfer to a sterile 50mL centrifuge tube containing pre-cooled PBS, and place it on ice.

[0077] (3) In a biological safety cabinet, wash 3-4 times with pre-cooled PBS containing penicillin / streptomycin (P / S), transfer the small intestine to 25mL PBS containing 2mM EDTA, and digest in a refrigerator at 4°C for 25 minutes .

[0078] (4) Transfer the digested small intestine to a 50mL centrifuge tube containing 25mL pre-cooled PBS, shake for about 50 times, and collect the suspension.

[0079] (5) Repeat the previous step, filter the suspension into a 50 mL centrifuge tube ...

Embodiment 2

[0084] Example 2 Morphological observation of small intestine 3D organoids

[0085] Using Olympus DP 71 inverted microscope to observe and photograph the process of 3D organoid formation on days 1, 2, 3, 4, 5 and 6 of culture, the results are as follows figure 1 showed that isolated crypts gradually differentiated into 3D organoids with increasing culture time, and stem cells in the crypts began to differentiate and "bud out" to form new crypt structures.

Embodiment 3

[0086] Example 3 Detection of BCRP mRNA level

[0087] (1) Isolation of small intestinal villi: According to the method for isolating crypts in Example 1, after step 5, small intestinal villi in a 70 μm cell mesh were collected.

[0088] (2) Isolation of small intestinal crypts: the mouse small intestinal crypts were obtained according to the separation method in Example 1.

[0089] (3) Acquisition of 3D organoids: small intestinal crypts were isolated and cultured for 4 days to form 3D organoids, the culture medium was discarded, washed twice with pre-cooled PBS, and then Matrigel was broken with a 1mL pipette tip, and it was washed with PBS. Transfer to a 1.5mL centrifuge tube, centrifuge at 200g for 5 minutes at 4°C, discard the supernatant, and precipitate into 3D organoids.

[0090] (4) mRNA extraction and reverse transcription: Trizol was used to extract total RNA from small intestinal villi, crypts and cultured 3D organoids, and cDNA was synthesized using a reverse tra...

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Abstract

The invention discloses a method for building medicine transport models for research on BCRP (breast cancer resistance proteins) mediation for 3D (three-dimensional) organs of the small intestines of mice. The method includes separating crypts from the small intestines of the mice by means of digestion, suspending the crypts in matrigel and then joining the crypts with cell culture plates and promoting differentiation of the crypts by the aid of ADMEM / F12 media with Respondin-1, m-noggin and m-EGF cell differentiation growth factors to form the 3D organs; carrying out morphologic observation and detecting the expression level of BCRP genes and proteins to verify the feasibility of model theories; carrying out research on the trans-membrane transport activity of BCRP in the 3D organs by the aid of fluorescent substrates Hoechst 33342 of the BCRP and inhibitors Ko143 or YHO-13177 of the fluorescent substrates by co-incubation processes. The method has the advantages that the medicine trans-cell-membrane transport in-vitro models for the research on the BCRP mediation for the 3D organs of the small intestines of the mice are built for the first time, and the method for building the models is easy and convenient to implement and high in detection efficiency and speed and can be widely applied to screening BCRP substrates and inhibitors in an in-vitro manner.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to the construction of a small intestine 3D organoid model and its application in the study of BCRP-mediated drug transport. Background technique [0002] The ABC transporter superfamily, consisting of two transmembrane domains and two cytoplasmic ATP-binding domains, is involved in the transport of a variety of endogenous and exogenous substrates, such as cholesterol, peptides, drugs, toxins, bile salts, organic anions , nucleosides, iron, chloride ions, and sterols. So far, 49 human ABC transporter members are known to be discovered and classified into seven subfamilies (ABC-A to ABC-G). Among them, P-glycoprotein (P-gp, ABCB1), MDR-related protein-1 (MRP1, ABCC1) and breast cancer resistance protein (BCRP, ABCG2) are closely related to multidrug resistance, therefore, ABC transporters are considered It is an important target for overcoming tumor drug resistance....

Claims

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Application Information

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IPC IPC(8): C12N5/071G01N33/68G01N33/53C12Q1/68
CPCC12N5/0602C12N5/0625C12N5/0679C12N2500/30C12N2500/32C12N2501/10C12Q1/6886C12Q2600/106G01N33/53G01N33/68
Inventor 王昕张磊刘明耀
Owner EAST CHINA NORMAL UNIV
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