Method for detection of P-gp mediated Rh123 transport in 3D type organ and application thereof

A technology of rh123 and organoids, which is applied in the field of biomedicine, can solve the problems of time-consuming and cumbersome processes, and achieve the effect of easy operation, simple operation and realization of screening

Active Publication Date: 2016-02-17
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method can only monitor one 3D organoid at a time, and each monitoring requires real-time tracking ...

Method used

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  • Method for detection of P-gp mediated Rh123 transport in 3D type organ and application thereof
  • Method for detection of P-gp mediated Rh123 transport in 3D type organ and application thereof
  • Method for detection of P-gp mediated Rh123 transport in 3D type organ and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1C57

[0042] Example 1 Sorting of C57BL / 6 Mouse Crypts and Culture of 3D Organoids

[0043] (1) Take the small intestine of 6-8 week-old C57BL / 6 mice, divide it into several 5-8cm long segments, cut it from the middle, and wash it with pre-cooled phosphate buffered saline (PBS). Remove the small intestinal villi with a coverslip and transfer it to a 50 ml centrifuge tube containing an appropriate amount of PBS and place on ice.

[0044] (Note: All the following steps should be performed in a sterile environment, and the crypts should be placed on ice as much as possible.)

[0045] (2) In a biosafety cabinet, wash 3-4 times with PBS containing penicillin / streptomycin (P / S), then transfer the small intestine fragment to 25ml PBS containing 2mM EDTA, and digest it in a refrigerator at 4°C for 10min.

[0046] (3) Transfer the digested small intestine fragments to a 50ml centrifuge tube, add 25ml PBS containing P / S, shake vigorously 50 times, and collect the suspension; transfer the sma...

Embodiment 23D

[0052] Example 2 Verification of 3D organoid model

[0053] 2.1 Observation of 3D organoid morphology

[0054] When the crypts of C57BL / 6 mice were cultured to the fourth day, they were observed and photographed under an OlympusIX71 microscope equipped with an OlympusDP71 camera system, and the results were as follows figure 2 -A shown. From figure 2 In -A, it can be seen that the organoid structure formed by the differentiation of crypts, the center is surrounded by cells to form a spherical cavity, and the peripheral "buds" are new crypts formed by the differentiation of stem cells.

[0055] 2.2 mRNA level to prove the expression of P-gp in 3D organoids

[0056] (1) Extraction of mRNA from small intestine, crypts and 3D organoids of C57BL / 6 mice

[0057] Take an appropriate amount of homogenized small intestines, crypts, and four-day-cultured 3D organoids of mice, respectively, and place them in 1.5ml EP tubes, and add an appropriate amount of Trizol to it to extract t...

Embodiment 33D

[0091] Example 3 Detection of P-gp-mediated Rh123 in 3D organoids and the effect of Verapamil on Rh123 transport

[0092] (1) Establishment of Rh123 standard curve

[0093] Prepare a Rh123 stock solution with a concentration of 1 mM, and dilute it with PBS to 500, 200, 100, 50, 20, 10, and 5 nM standard solutions in sequence, and each concentration has three parallels. Utilize the multifunctional microplate reader (FLUOStarOPTIMA) to detect, the parameter is set as the excitation wavelength 485nm, emission wavelength Fluorescence intensity of Rh123 at different concentrations was measured at 535nm. With the concentration of Rh123 as the abscissa and the fluorescence intensity as the ordinate, perform linear regression, such as image 3 shown. In the range of 5-500nM, the concentration of Rh123 is linear to the fluorescence intensity, the regression equation is: y=55.27x+94.15, the correlation coefficient r 2 = 0.999, the weight factor is 1 / x 2 .

[0094] (2) Co-incuba...

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Abstract

The invention relates to a method for detection of P-glycoprotein (P-gp) mediated rhodamine 123 (Rh123) transport in a 3D type organ and an application thereof in screening of P-gp inhibitors. The method comprises the steps: firstly, sorting small intestinal crypts of C57BL/6 mice, inoculating a petri dish containing a matrix gel, culturing in an Advanced DMEM/F12 culture medium, and forming the 3D type organ; and then carrying out qualitative and quantitative detection of P-gp mediated Rh123 transport and influence of the P-gp inhibitor verapamil on Rh123 transport by using the 3D type organ model. The method particularly comprises the steps of respectively co-incubating the 3D type organ with (1) the P-gp substrate Rh123 and with (2) the Rh123 and the verapamil, releasing the Rh123 in the 3D type organ by using an ultrasonic crushing method, and finally detecting the concentration of the Rh123 on a multifunctional microplate reader. The method has the advantages of being simple, rapid, and high in sensitivity, can be combined with the 3D type organ model for carrying out research of the P-gp mediated drug transport, and also can perform research of in-vitro screening of the P-gp inhibitors.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a method for detecting Rh123 transport mediated by P-gp in 3D organoids and its application in screening P-gp inhibitors. Background technique [0002] At present, oral administration is the most common way of administration in clinical practice, and it has the advantages of relative safety and convenience compared with other ways of administration such as intravenous. When drugs enter the body, they generally go through the process of absorption, distribution, metabolism and excretion. For oral drugs, their absorption in the small intestine is the most important, because this is the prerequisite for the drug to exert its efficacy in the body. Studies have shown that in the process of new drug development, about 40% of lead compounds are rejected in the clinical stage. The most common reasons include unsatisfactory pharmacokinetic properties, especially poor absorp...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N33/68C12Q1/68
Inventor 王昕张远金曾之扬李大力刘明耀
Owner EAST CHINA NORMAL UNIV
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