Drug screening method

Abstract

The invention belongs to the technical field of medicine screening, and particularly relates to a medicine screening method which comprises the following steps: inversely placing molten agarose gel in a template printed with patterns, enabling the agarose gel to be completely attached to the template, forming and cutting into blocks; dropwise adding a mixed solution of type I collagen and sodium periodate on the pattern surface, and incubating the collagen; drying the agarose, and copying a pattern on the agarose; manufacturing a polyacrylamide film containing fluorescent particles, and covering the film with the copied pattern; after the film is polymerized and formed, dropwise adding PBS, and sterilizing to obtain a culture containing a polyacrylamide film; inoculating cells into the culture; adding a medicine capable of stimulating the cells to deform; microscopic examination: calculating the integral traction force of the cell cluster according to the displacement of the fluorescent particles before and after dosing; the cellular morphology and activity are evaluated according to the traction force, effective drugs and half effective concentration are screened out according to the cellular morphology and activity, and a new method is provided for screening candidate drugs for clinical diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of drug screening, and in particular to a drug screening method. Background Art

[0002] As an in vitro biological model, cells are widely used in biological toxicity and preclinical drug research. However, most of the previous cell analysis systems were based on cell screening, using label tracking to determine the functions of various cells, such as radioisotope labeling, enzyme labeling, fluorescent labeling technology, etc. A large number of experimental studies have shown that a class of traditional methods often have great limitations in the application process. These methods usually require labeling aptamers, enzyme substrates or tracer molecules, which may complicate experimental conditions. At the same time, the cost of washing and labeling cells is very high and takes a lot of time. Another type of traditional detection method, such as the tetramethylrhodamine microenzyme reaction colorimetric method for determining cell viability and the cell viability detection kit, is an endpoint method that will cause irreversible damage to cells and cannot detect the cell response process to drugs in real time.

[0003] In summary, the existing traditional drug screening methods have the defects of being unable to achieve high-throughput and unable to intuitively display the morphology of cells. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a drug screening method.

[0005] A drug screening method comprises the following steps:

[0006] Place the melted agarose gel upside down on the template printed with the pattern, make the agarose gel completely fit on the template, shape it, and cut it into pieces;

[0007] Add a mixture of type I collagen and sodium periodate to the pattern surface and incubate the collagen;

[0008] Blow dry the agarose that has been incubated with collagen, and then copy the pattern on the agarose;

[0009] Prepare a polyacrylamide film containing fluorescent particles, and then cover the copied pattern onto the film;

[0010] After the film is polymerized and formed, PBS is added dropwise for sterilization to obtain a culture containing a polyacrylamide film;

[0011] inoculating cells into the culture medium containing the polyacrylamide film;

[0012] adding a drug that can stimulate the cells to undergo deformation;

[0013] Microscopic examination, the overall traction force of the cell cluster was calculated based on the displacement of fluorescent particles before and after drug addition;

[0014] The cell morphology and activity are evaluated based on the traction force, and effective drugs are screened based on the cell morphology and activity.

[0015] Preferably, the agarose gel is prepared from agarose and deionized water, and the mass volume ratio of agarose to deionized water is 5-10 g:100 mL.

[0016] Preferably, agarose and deionized water are mixed and then heated at 80-100° C. for 1-3 minutes.

[0017] Preferably, the agarose gel is fitted to the template at 80° C. and cooled and solidified after there are no bubbles in the gel.

[0018] Preferably, the concentration of type I collagen is 100-300 μg / mL.

[0019] Preferably, the concentration of sodium periodate is 3-4 mg / mL.

[0020] Preferably, the particle size of the fluorescent particles is 0.1-0.2 μm.

[0021] Preferably, the concentration of inoculated cells is 0.1-1.0×10 5 / cm 2 .

[0022] Preferably, microscopic examination is performed using a fluorescence microscope.

[0023] Preferably, the model of the fluorescence microscope is Nikon ECLIPSE Ti2.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention has developed a non-labeling, non-destructive, quantitative, simple, and rapid high-throughput drug screening technology. By evaluating the contraction / relaxation, adhesion, and activity of the drug on the cell, it can monitor the living cells in real time for a long time and distinguish the cell morphology. It overcomes the problems of the existing drug screening methods, such as irreversible loss of the test sample, cost and time-consuming cleaning and labeling, low cell survival rate after detection, and inability to intuitively show cell changes.

[0026] According to the contraction / relaxation tension or adhesion force of the adherent cells, polyacrylamide films of different stiffness are selected to simulate a suitable detection environment. For cells attached to polyacrylamide films with uniform fluorescent particles, the traction force caused by deformation such as contraction / relaxation / shedding of the cells autonomously or after drug stimulation is calculated with the help of the displacement of the fluorescent particles. According to the traction force, the contraction / relaxation or activity changes of the cells are reflected, the degree of drug stimulation on the cells is evaluated, the effect of the drugs is measured and the half effective dose is calculated. The present invention combines a collagen pattern copied with a tiny pattern, attaches single cells or multiple cells of regular size, uses the fluorescent particles on the polyacrylamide film, and uses the traction force fluorescence microscope to detect the displacement of the fluorescent particles, and calculates the overall traction force of the cells on each pattern. The changes in cell mechanics are used to evaluate the effects of drugs of different concentration gradients on cells, screen effective drugs and calculate EC50.

[0027] This drug screening method greatly improves the efficiency of early screening in drug development, saves a lot of time and testing costs, and provides great help for new drug development and animal testing and even clinical trials of various candidate drugs. This method can greatly improve the development of pharmacy, drug analytical chemistry, drug screening, drug development and other fields.

[0028] The fluorescence microscope is used to calculate the force generated by the deformation of cells caused by drug stimulation, the change in cell deformation, and the effect of the drug on the cells. Some drugs can stimulate the cells to deform accordingly when they come into contact with cells, and cell deformation requires the participation of force. Through the fluorescence microscope, the mechanical changes caused by drug stimulation of cells are calculated to reflect the deformation size of the cells after drug stimulation, and then the effect of the drug on the cells is evaluated.

[0029] The drug screening process is non-invasive, label-free, and high-throughput. Non-invasive detection helps to culture, screen, amplify, and perform subsequent tests on the tested cells; label-free facilitates subsequent experiments without the need to wash markers, and at the same time, there is no irreversible loss to cells, allowing cells to maintain normal morphology and activity; microarray patterns meet the requirements of high-throughput drug screening, making it easy to screen thousands of drugs, saving a lot of time and money. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The process of making and analyzing the traction culture dish. Figure A shows the process of making the traction culture dish. Figure B shows the arrangement of the traction culture dish microarray. Figure C shows the calculation process of the MATLAB analysis program.

[0031] Figure 2The figure shows the change process of traction force caused by terbutaline inhibiting acetylcholine contraction. Figure A shows the change of cell morphology before and after drug addition; Figure B shows the force field cloud map obtained by MATLAB analysis program; Figure C shows the acetylcholine efficacy curve; Figure D shows the terbutaline efficacy curve;

[0032] Figure 3 To compare the experimental results, Figure A shows the result of collagen incubation with 500 μg / mL type I collagen, Figure B shows the result of collagen incubation with 300 μg / mL type I collagen, Figure C shows the result of collagen incubation with 100 μg / mL type I collagen, Figure D shows the result of collagen incubation with 50 μg / mL type I collagen, Figure E shows the result of collagen incubation when the mass volume ratio of agarose to deionized water is 2.5 / 100, Figure F shows the result of collagen incubation when the mass volume ratio of agarose to deionized water is 5 / 100, Figure G shows the result of collagen incubation when the mass volume ratio of agarose to deionized water is 10 / 100, and Figure H shows the result of collagen incubation when the mass volume ratio of agarose to deionized water is 20 / 100. DETAILED DESCRIPTION

[0033] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0034] It is known that acetylcholine (Ach) can cause contraction of airway smooth muscle cells and is used to construct an asthma model, while terbutaline (TB) can activate β2 receptors, causing airway relaxation and relieving asthma.

[0035] The present invention uses rat airway smooth muscle cells (ASMC) as the research object, produces φ300 μm micro-patterns, and realizes cell clusters as research samples.

[0036] In the "mass / volume ratio of agarose and deionized water" described in the following examples, the mass unit of agarose is g, and the volume unit of deionized water is mL.

[0037] The model of the fluorescence microscope used was Nikon ECLIPSE Ti2.

[0038] In the present invention, the culture dish containing polyacrylamide is also called a traction culture dish.

[0039] Example 1

[0040] The screening method of acetylcholine drugs comprises the following steps:

[0041] (1) Figure 1 As shown in A, a mixture of agarose and deionized water is prepared, and the mass / volume ratio of agarose to deionized water is 5 / 100;

[0042] (2) After mixing agarose and deionized water, heat in a microwave oven at 80°C for 1 minute to complete the gelling process;

[0043] (3) placing the melted agarose gel in (2) upside down into a template printed with a pattern;

[0044] (4) Place the template in (3) on an 80°C hot plate until the agarose is completely attached to the template, then transfer to 4°C for molding;

[0045] (5) Use a scalpel to cut the agarose gel formed in (4) into small pieces of 0.5×0.5 cm;

[0046] (6) adding a mixture of type I collagen and sodium periodate to the patterned surface of the small block in (5) and incubating for 60 minutes, wherein the concentration of type I collagen in the mixture is 100 μg / mL and the concentration of sodium periodate is 3.5 mg / mL;

[0047] (7) drying the agarose incubated with collagen in step (6), and then copying the pattern on the agarose onto a cover glass;

[0048] (8) preparing a polyacrylamide film containing 0.2 μm fluorescent particles and a stiffness of 10 kPa, and then covering the film with the cover glass printed with the pattern in (7);

[0049] (9) After the film covered with the cover glass with the pattern printed thereon in (8) is polymerized, deionized water is added dropwise and the cover glass is removed;

[0050] (10) Discard the deionized water, add PBS dropwise, and sterilize under ultraviolet light to obtain a culture dish containing polyacrylamide;

[0051] (11) Inoculate 0.5×10 airway smooth muscle cells into the culture dish containing the polyacrylamide film in (10). 5 Pieces / cm 2 ;

[0052] (12) Before drug administration, the cells were continuously tested under a fluorescence microscope. Different concentration gradients (10 -5 / 10 -4 / 10 -3 / 10 -2 / 10 -1 / 1 / 10 3nM) acetylcholine, with PBS added as the control group, and then continued to test for 15 minutes. Figure 1 As shown in B, high-throughput detection is achieved;

[0053] (13) Figure 1 As shown in C, the displacement of fluorescent particles before and after drug addition is calculated through the traction force calculation program to obtain the force field heat map, and then the overall traction force of the cell cluster is calculated;

[0054] (14) Cell morphology and activity were evaluated based on traction force. Effective drugs that can improve diseases caused by changes in cell morphology or activity were screened out, and the median effective concentration of the effective drugs was calculated using the pharmacodynamic curve. Figure 2 As shown in C, the half effective concentration of acetylcholine that causes airway smooth muscle cell contraction is 0.1475 nM. The dose of acetylcholine that causes airway smooth muscle cell contraction is screened based on the half effective concentration.

[0055] Example 2

[0056] The screening method of terbutaline drug comprises the following steps:

[0057] (1) Figure 1 As shown in A, a mixture of agarose and deionized water is prepared, and the mass / volume ratio of agarose to deionized water is 10 / 100;

[0058] (2) After mixing, place in a microwave oven and heat at 80°C for 1 minute to complete the sol process;

[0059] (3) placing the melted agarose gel in (2) upside down into a template printed with a pattern;

[0060] (4) Place the template in (3) on an 80°C hot plate until the agarose is completely attached to the template, then transfer to 4°C for molding;

[0061] (5) Use a scalpel to cut the agarose gel formed in (4) into small pieces of 0.5×0.5 cm;

[0062] (6) adding a mixture of type I collagen and sodium periodate to the patterned surface of the small block in (5) and incubating for 60 minutes, wherein the concentration of type I collagen in the mixture is 300 μg / mL and the concentration of sodium periodate is 3.5 mg / mL;

[0063] (7) drying the agarose incubated with collagen in step (6), and then copying the pattern on the agarose onto a cover glass;

[0064] (8) preparing a polyacrylamide film containing 0.2 μm fluorescent particles and a stiffness of 10 kPa, and then covering the film with the cover glass printed with the pattern in (7);

[0065] (9) After the film covered with the cover glass with the pattern printed thereon in (8) is polymerized, deionized water is added dropwise and the cover glass is removed;

[0066] (10) Discard the deionized water in (9), add PBS dropwise, and sterilize under ultraviolet light to obtain a culture dish containing polyacrylamide;

[0067] (11)) Inoculate 0.5×10 cells into the culture dish containing the polyacrylamide film in (10). 5 Pieces / cm 2 ;

[0068] (12) When adding drugs after inoculating cells, continuous monitoring under a fluorescence microscope was performed before adding drugs. Terbutaline was added to the asthma model within the first minute, and then continuous monitoring was performed for 15 minutes. Figure 1 As shown in B, high-throughput detection is achieved; the asthma model is constructed by acetylcholine;

[0069] (13) Figure 1 As shown in C, the displacement of fluorescent particles before and after drug addition is calculated through the traction force calculation program to obtain the force field heat map, and then the overall traction force of the cell cluster is calculated;

[0070] (14) Cell morphology and activity were evaluated based on traction force. Effective drugs that can improve diseases caused by changes in cell morphology or activity were screened out, and the median effective concentration of the effective drugs was calculated using the pharmacodynamic curve. Figure 2 As shown in D, the half effective concentration of terbutaline that causes relaxation of airway smooth muscle cells is 0.5483 nM. The dose of terbutaline that relaxes airway smooth muscle cells is screened out based on the half effective concentration.

[0071] In the traction culture dish inoculated with airway smooth muscle cells, the model group was induced to contract airway smooth muscle cells by dropping 1 μM acetylcholine; while the treatment group was dripped with 1 μM Ach, different concentration gradients (10 -4 / 10 -3 / 10 -2 / 10 -1 / 1 / 10 1 / 10 2 / 10 3 nM)TB, detect the changes in cell clusters and fluorescent particle displacement at the pattern before and after drug addition, and use the independently developed analysis program to analyze the changes in traction force of each group of cell clusters before and after drug addition, and calculate the EC / IC50 of effective drugs.

[0072] like Figure 2As shown in Figure A, the overall contraction traction force of the Ach group (marked as -∞) is about 30Pa. As the concentration of terbutaline increases, the traction force of the cell cluster gradually decreases. As shown in Figure B, after the addition of 0.1nM TB, the traction force changes from contraction force to relaxation force. By drawing the drug effect curve for the traction force of each TB concentration group, the TB half effective concentration is calculated to be 0.5483nM, and the drug dose is screened according to the half effective concentration.

[0073] Comparative experiment:

[0074] An experimental exploration was conducted on the effects of different type Ⅰ collagen concentrations and different agarose concentrations on the experimental results.

[0075] like Figure 3 As shown in AD, 50 / 100 / 300 / 500 μg / ml type I collagen was used for collagen incubation and A549 cells were inoculated. The results showed that most of the cells in the pattern incubated with 50 μg / ml type I collagen were defective, and this concentration was not enough for the cells to attach to the pattern ( Figure 3 A); Most of the cells in the pattern incubated with 100μg / ml type I collagen were attached ( Figure 3 B); cells perfectly adhered to the pattern incubated with 300μg / ml type I collagen ( Figure 3 C); In the pattern incubated with 500μg / ml type I collagen, cells attached too much and even to areas outside the pattern ( Figure 3 D).

[0076] like Figure 3 As shown in EH, agarose gels with a mass volume ratio of agarose to deionized water of 2.5:100, 5:100, 10:100, and 20:100 were selected, incubated with collagen, and then copied onto glass slides. Airway smooth muscle cells (ASMC) were subsequently inoculated for experiments. The results showed that 2.5% agarose was too soft, which was not conducive to printing patterns and easily led to pattern deformation (such as ellipse, irregular arc, etc.). At the same time, agarose was too soft and difficult to grasp ( Figure 3 E); 5% and 10% agarose have moderate hardness, the printing process is easy to operate, and the pattern is regular ( Figure 3 F. Figure 3 G); 20% agarose is too hard to absorb collagen type I. The concentration of collagen type I in the printed pattern is insufficient, which makes it difficult for cells to adhere and some wrinkles appear ( Figure 3 H).

[0077] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0078] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0079] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A drug screening method, characterized in that: The following steps are involved: Place the melted agarose gel upside down on the template printed with the pattern, make the agarose gel completely fit on the template, shape it, and cut it into pieces; Add a mixture of type I collagen and sodium periodate onto the pattern surface and incubate the collagen; Blow dry the agarose that has been incubated with collagen, and then copy the pattern on the agarose; Prepare a polyacrylamide film containing fluorescent particles, and then cover the copied pattern onto the film; After the film is polymerized and formed, PBS is added dropwise for sterilization to obtain a culture containing a polyacrylamide film; inoculating cells into the culture medium containing the polyacrylamide film; adding a drug that can stimulate the cells to undergo deformation; Microscopic examination, the overall traction force of the cell cluster was calculated based on the displacement of fluorescent particles before and after drug addition; The cell morphology and activity are evaluated based on the traction force, and effective drugs are screened based on the cell morphology and activity.

2. A drug screening method according to claim 1, characterized in that: Agarose gel is prepared from agarose and deionized water, and the mass volume ratio of agarose to deionized water is 5-10 g:100 mL.

3. A drug screening method according to claim 2, characterized in that: After agarose and deionized water are mixed, they are heated at 80-100°C for 1-3 minutes to complete the sol process.

4. A drug screening method according to claim 1, characterized in that: When the agarose gel is fitted to the template, it is done at 80°C and cooled and solidified after there are no bubbles in the gel.

5. A drug screening method according to claim 1, characterized in that: The concentration of type I collagen is 100-300 μg / mL.

6. A drug screening method according to claim 1, characterized in that: Sodium periodate concentration is 3-4 mg / ml.

7. A drug screening method according to claim 1, characterized in that: The particle size of the fluorescent particles is 0.1-0.2 μm.

8. A drug screening method according to claim 1, characterized in that: The inoculated cell concentration was 0.1-1.0×10 5 Pieces / cm 2 .

9. A drug screening method according to claim 1, characterized in that: Microscopic examination was performed using a fluorescence microscope.

10. A drug screening method according to claim 1, characterized in that: The model of the fluorescence microscope is Nikon ECLIPSE Ti2.

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