CN106755260A - High-throughput screening method for anti-hydatid drugs - Google Patents

Abstract

The invention discloses a high-throughput screening method for anti-hydatid drugs by using mouse secondary echinococcus granulosus germinal layer cells. The technical scheme adopted is: Infect the experimental mice with the protoscole of Echinococcus granulosus, establish the secondary infection model of echinococcosis 8-10 months later, dissect the sick mice to take out the hydatid cysts in their bodies, separate the germinal layer cells and establish cell lines. The cell concentration was adjusted and plated in a 96-well culture plate, and the effect of the drug was evaluated by the changes in cell activity and the release of endogenous substances from the cells after the drug was applied. The invention greatly reduces the drug screening period, not only reduces the dosage of the drug to be tested, but also improves the screening accuracy and reduces the screening cost consumption.

Description

High-throughput screening method for anti-hydatid drugs

field of invention

The invention relates to a high-throughput screening method for anti-hydatid drugs, in particular to a method for screening anti-hydatid drugs by using germinal layer cells, and in particular to separating the germinal layer by using secondary echinococcus granulosus cysts in mice A method for high-throughput screening of anti-hydatid drugs by cells.

Background technique

Echinococcosis, commonly known as echinococcosis, is one of the most important zoonotic diseases in the world, which seriously endangers people's health and brings huge economic losses to animal husbandry. According to incomplete statistics, there are as many as 600,000 to 700,000 echinococcosis patients in my country, and more than 66 million people are at risk. Echinococcosis develops slowly in the patient's body, mainly producing space-occupying lesions, making the patient lose the ability to work, and causing serious damage to the patient's physical and mental health. Especially alveolar coccidiosis has a high mortality rate and is known as "parasitic tumor" and "second cancer". However, there are only two drugs, mebendazole and albendazole, for the treatment of echinococcosis at present, and the cure rate of these drugs is only about 30%. It is urgent to find new and effective alternative drugs, and finally achieve the goal of improving the efficacy of drug treatment and alleviating the suffering of patients.

At present, in vitro culture and animal models are mainly used for drug efficacy research at home and abroad. The establishment period of infection animal models is long, the cost is high, and it is difficult to apply to drug screening. The in vitro culture model is to investigate the killing effect of drugs on protoscolex, metacestode and germinal cells of echinococcosis. Among them, drug screening with protoscole and hydatid cysts has different degrees of shortcomings, and it is difficult to achieve high-throughput screening of drugs. Recent studies have shown that the culture and application of germinal layer cells can largely replace animal models, and it is economical, simple and safe. However, the establishment of echinococcus germinal layer cell lines reported so far is based on the materials of patients or sick animals in the epidemic area, and the acquisition of materials is limited; at present, no anti-hydatid drug screening system has been established on the basis of the germinal layer cell culture; Moreover, there is a lack of sensitive, high-throughput drug efficacy evaluation methods. Due to the lack of the above-mentioned technologies, there is no research and report on drug screening based on mouse secondary germinal layer cells.

In order to solve the above problems, the present inventors harvested echinococcus cysts by establishing an experimental animal model of protoscoleum secondary infection, and isolated germinal layer cells from them for drug screening, in order to achieve high-throughput screening of anti-hydatid drugs in a wide range.

Contents of the invention

The invention provides a high-throughput screening method for anti-hydatid drugs, comprising the following steps:

1) Establish a mouse model of secondary Echinococcus granulosus infection, isolate Echinococcus granulosus germinal layer cells and establish a germinal layer cell line;

2) Prepare echinococcus germinal layer cell suspension, and adjust the cell concentration to 5×10 ⁴ or OD ₅₀₈ =0.2;

3) Co-culture with the drug to be tested for 72 hours after preparing the adherent cells;

4) Determining the content of alkaline phosphatase in the supernatant and the activity of adherent cells to determine the anti-hydatid activity of the drug to be tested.

In the method of the present invention, in step 1) establishment of a secondary echinococcus granulosus infection model in mice, the sheep-derived protoscole is used to infect the mice, and then the mice are fed for 8-10 months, so as to obtain the echinococcus infection model.

Echinococcus granulosus was dissected from mice infected with ovine protoscole for 8-10 months, germinal layer cells were separated, cultured in RPMI 1640 medium at 37°C, 5% CO ₂ environment, and carried out after 21 days Passaging, after the passage is stable, passaging once every 2 weeks to establish the germinal layer cell line.

In the method of the present invention, the preparation method of echinococcus germinal layer cell suspension in step 2) comprises: taking adherent cells, adding 0.25% trypsin to digest for 5-30 minutes, adding culture medium and blowing to obtain cell suspension.

In the method of the present invention, step 3) inoculate the hydatid germinal layer cell suspension in a 96-well culture plate and culture for 24 hours, so that the cells are completely adhered to the wall, and prepared as adherent cells.

The adherent cells were co-cultured with the drug to be tested for 72 hours, and the drug to be tested was a candidate anti-hydatid drug.

In the method of the present invention, step 4) is: absorb the culture supernatant 72 hours after adding the drug to be tested, and measure the alkaline phosphatase content of the supernatant by conventional methods.

In step 4), the activity of the adherent cells was also determined by the MTT (tetramethylazolate) method.

The technical scheme of the present invention is based on cell culture technology, by comparing the growth conditions of cells in commonly used cell culture medium PRMI1640 and MEM medium, and finally selecting PRMI1640 medium as a suitable culture medium for germinal layer cells. In addition, by observing the proliferation cycle of the cells, the germinal layer cells were cultured in the above-mentioned medium for 21 days before passage, and after the passage was stable, they were passaged once every 2 weeks, so as to realize the secondary hydatid germinal layer of mice. Cell line establishment. Moreover, the accuracy and sensitivity of cell viability determination are ensured by adopting the traditional cell viability assay method and measuring the spillover of intracellular active substances after cell damage.

Specifically, the technical solution of the present invention is: a high-throughput screening method for anti-hydatid drugs using mouse secondary Echinococcus granulosus germinal layer cells, the steps are as follows:

1) Isolation of mouse Echinococcus granulosus germinal layer cells and establishment of cell lines:

Infect Kunming rats with sheep-derived protothecosis to establish a secondary Echinococcus granulosus infection model. After 8-10 months of inoculation, the Echinococcus granulosus is dissected to separate the germinal layer cells and placed in RPMI1640 medium at 37°C. Plate culture in an environment of 5% CO ₂ . The germinal layer cells were cultured in the above-mentioned medium for 21 days and then subcultured, and after the passage was stable, they were subcultured every 2 weeks to establish cell lines.

2) Screening of anti-hydatid drugs:

Preparation of echinococcus germinal layer cell suspension: take adherent cells and add 0.25% trypsin to digest for 5-30 minutes, then add culture medium and pipette to form cell suspension;

Adjustment of echinococcus germinal layer cell concentration: adjust to 5×10 ⁴ or OD ₅₀₈ =0.2 with a hemocytometer;

Screen the drug to be tested: inoculate the above suspension in a 96-well culture plate, and add the drug to be tested after 24 hours after the cells are completely adhered to the wall;

Detection: 72 hours after the addition of the drug, the supernatant of the culture solution was drawn to measure the content of alkaline phosphatase, and at the same time, the activity of the adherent cells was measured by the MTT (tetramethylazolium salt) method. The effect of the drug is evaluated by measuring the change of the absorbance value, and the inhibitory rate of the drug to be tested on the activity of the germinal layer cells is calculated.

MTT method to measure the activity of adherent cells:

Cell activity inhibition rate=[(absorbance value of control illuminance-absorbance value of zeroing well)-(absorbance value of drug group-absorbance value of zeroing well)]/(absorbance value of control illuminance-absorbance value of zeroing well)×100%

Alkaline phosphatase assay:

Cell activity inhibition rate=[(absorbance value of drug group-absorbance value of zero-adjusted well)-(absorbance value of control illuminance-absorbance value of zero-adjusted well)]/(absorbance value of control illuminance-absorbance value of zero-adjusted well)×100%

After 24 hours of germinal layer cells cultured according to the above method, the cells can be seen to adhere to the wall and protrude fusiform pseudopodia to the surroundings.

The beneficial effects of the present invention are: the establishment of a high-throughput screening system based on the determination of germinal layer cell activity lays the foundation for finding anti-hydatid drugs in a wider range. At the same time, it not only greatly reduces the dosage of the drug to be tested, but also improves the screening efficiency and reduces the screening cost consumption.

Brief description of the drawings

Figure 1 shows the morphology of germinal layer cells (A. stained cells; B. unstained cells).

detailed description

Example 1

1) Preparation of germinal layer cells:

(1) Establishment of secondary echinococcosis mouse model: Collect echinococcus cysts in endemic areas, absorb the cyst fluid containing the protoscole, pour the cyst fluid into a 50ml round-bottomed centrifuge tube, and let it settle naturally After 10 minutes, the supernatant was removed, and then the precipitate containing the protoscole was washed 5-8 times with physiological saline, 10 minutes each time. Add a certain amount of normal saline containing 500,000 u/L of penicillin sodium salt to the washed protoscole, suspend evenly, draw 10-20 μl, count under an inverted microscope, and observe the survival of the protoscole. When the number of survival accounts for more than 95%, it can be used for inoculation. When inoculating, dilute the original head section to 4000/ml for later use. Add a stirring bar into the beaker containing the Echinococcus granulosus suspension to be inoculated, place the beaker on the platform of a magnetic stirrer, and adjust the rotation speed to keep the protoscole and cyst tissue in suspension. Use a 1ml syringe to draw 0.5ml of the protoscoleus suspension (2000 protoscolees), wipe the skin of the mouse with 75% alcohol, and inject the protoscolene into the abdominal cavity of the mouse. The model can be used for follow-up experiments after 8-10 months of establishment.

(2) Separation of germinal layer cells: autopsy the above-mentioned mice, take out the hydatid cysts in the body, wash the cysts with normal saline for 3-5 times, cut them into pieces, add 0.25% trypsin, digest at 37°C for 10- After 30 minutes, the supernatant was centrifuged at 500 rpm for 5 minutes, then the supernatant was discarded, and the cell suspension was prepared with 1640 medium.

(3) Subculture of germinal layer cells: The germinal layer cells were cultured in the above medium for 21 days and then subcultured, and after the passage was stable, they were subcultured every 2 weeks to establish cell lines.

(4) Preparation of monolayer cell suspension: take adherent cells and add 0.25% trypsin to digest for 5-30 minutes, then add culture medium and pipette to form cell suspension, and adjust to 5× ¹⁰⁴ or Use a UV spectrophotometer to measure and adjust the OD ₅₀₈ value to 0.2.

(5) Identification of germinal layer cells: the cells were plated in a 96-well plate, and detected by means of enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA).

(6) Cryopreservation and recovery of germinal layer cells: The germinal layer cells were preserved according to the routine freezing and recovery methods of cells.

2) Simulation screening of anti-hydatid drug mebendazole:

Screen the drug to be tested: plate 100 μl of the adjusted germinal layer cell suspension, add mebendazole stock solution after 24 hours after the cells adhere to the wall, so that the final concentrations are 10 μg/ml, 5 μg/ml and 1 μg/m ( n=6).

Detection: Take 30μl of cell culture medium at 24h, 72h and 144h after administration, and add 170μl of alkaline phosphatase assay system (which contains 0.67M p-nitrophenyl phosphate, 1.0M diethanolamine and 0.50mM magnesium chloride, pH 9.8) , and then measure the absorbance at a wavelength of 405 nm with a UV spectrophotometer after placing it at room temperature in the dark for 1 h. Calculate the inhibitory rate of the test drug to the germinal layer cell activity according to the formula, and the results are shown in Table 1.

Cell activity inhibition rate=[(absorbance value of drug group-absorbance value of zero-adjusted well)-(absorbance value of control illuminance-absorbance value of zero-adjusted well)]/(absorbance value of control illuminance-absorbance value of zero-adjusted well)×100%.

Table 1 The activity inhibition rate of different concentrations of mebendazole on germinal layer cells

It can be seen from Table 1 that with the increase of administration concentration and drug action time, the inhibition rate of mebendazole to germinal layer cells also increases correspondingly, and this phenomenon also corresponds to the mechanism and characteristics of mebendazole.

Example 2

1) Take the preparation of the germinal layer cells in Example 1.

2) Simulation screening of anti-hydatid drug mebendazole:

Screening drug to be tested: same as Example 1.

Detection: Discard the medium after 24h, 72h and 144h after administration respectively, add 5mg/ml MTT solution (MTT 0.5g is dissolved in 100ml phosphate buffered solution (PBS) or medium without phenol red, use 0.22μm Filter through a membrane to remove bacteria in the solution, store 50 μl at 4 °C in the dark, place at 37 °C, add 100 μl DMSO after 2 hours, and measure the absorbance at 570 nm wavelength with a UV spectrophotometer after the formazan is completely dissolved. Calculate the inhibitory rate of the test drug to the germinal layer cell activity according to the formula, and the results are shown in Table 2.

Cell activity inhibition rate=[(absorbance value of control illuminance-absorbance value of zeroing well)-(absorbance value of drug group-absorbance value of zeroing well)]/(absorbance value of control illuminance-absorbance value of zeroing well)×100%

Table 2 Changes in activity of germinal layer cells treated with different concentrations of mebendazole

It can be seen from Table 2 that the results of MTT assay and alkaline phosphatase assay activity are basically consistent, that is, with the increase of administration concentration and drug action time, the inhibition rate of mebendazole to germinal layer cells also increases correspondingly.

Example 3

1) Take the preparation of the germinal layer cells in Example 1.

2) Simulation screening to kill the protoscolic drug nitazoxanide:

Screen the drug to be tested: plate in 96-well culture plate, add a certain concentration of nitazoxanide to the culture well according to the mode of Example 1, and act in an environment of 37° C. and 5% CO ₂ for 72 hours.

Detection: take 30 μl of cell culture medium, add 170 μl of alkaline phosphatase assay system (same as Example 1), and measure the absorbance at 405 nm wavelength with a UV spectrophotometer after 1 hour at room temperature in the dark. Calculate the inhibitory rate of the test drug to the germinal layer cell activity according to the formula, and the results are shown in Table 3.

Inhibition rate of cell activity=[(absorbance value of drug group-absorbance value of zero-adjusted well)-(absorbance value of control illuminance-absorbance value of zero-adjusted well)-]/(absorbance value of control illuminance-absorbance value of zero-adjusted well)×100%

Table 3 Changes in the activity of germinal layer cells treated with different concentrations of nitazoxanide

It can be seen from this table that nitazoxanide has almost no inhibitory effect on the germinal layer cells of Echinococcus, thus confirming its effect as an anti-hydatid drug that kills the protoscoleum rather than the activity of the germinal layer cells.

Example 4

1) Take the preparation of the germinal layer cells in Example 1.

2) Simulation screening to kill the protoscolic drug nitazoxanide:

Screening of the drug to be tested: plated in a 96-well culture plate, according to the pattern of Example 1, a certain concentration of the drug to be screened was added to the culture well and reacted in an environment of 37° C. and 5% _CO for 72 hours.

Detection: Discard the medium after 24h, 72h and 144h after administration respectively, add 5mg/ml MTT solution (MTT 0.5g is dissolved in 100ml phosphate buffered solution (PBS) or medium without phenol red, use 0.22μm Filter through a membrane to remove bacteria in the solution, store 50 μl at 4 °C in the dark, place at 37 °C, add 100 μl DMSO after 2 hours, and measure the absorbance at 570 nm wavelength with a UV spectrophotometer after the formazan is completely dissolved. Calculate the inhibitory rate of the test drug to the germinal layer cell activity according to the formula, and the results are shown in Table 2.

Cell activity inhibition rate=[(absorbance value of control illuminance-absorbance value of zeroing well)-(absorbance value of drug group-absorbance value of zeroing well)]/(absorbance value of control illuminance-absorbance value of zeroing well)×100%

Table 4 The activity inhibition rate of different concentrations of nitazoxanide on germinal layer cells

It can be seen from Table 4 that the results of MTT assay and alkaline phosphatase assay are basically consistent, and nitazoxanide has no obvious effect on germinal layer cells.

Claims (8)

1. a kind of anti-hydatid drugs high-throughput screening method, it is characterised in that including as follows Step：

1) mouse Secondary cases Echinococcus granulosus model, separation of fine particle echinococcus are set up Germinal layer cell and set up Germinal Cell Line；

2) echinococcus germinal layer cell suspension is prepared, adjustment cell concentration is 5 × 10⁴Or OD₅₀₈=0.2；

3) 72h is co-cultured after preparing attached cell with medicine to be measured；

4) content of alkaline phosphatase of supernatant and the activity of attached cell are determined to determine The anti-Echinococcus hydatid cyst activity of medicine to be measured.

2. anti-hydatid drugs high-throughput screening method as claimed in claim 1, wherein Step 1) described in the foundation of mouse Secondary cases Echinococcus granulosus model include：With Continue to raise mouse 8-10 month after the protoscolex infecting mouse of sheep source.

3. anti-hydatid drugs high-throughput screening method as claimed in claim 1, wherein Step 1) described in Germinal Cells From Hidatid Cyst of Echinococcus Granulosus separation and the germinal layer cell The foundation of system includes：The sheep source protoscolex infection mouse of 8-10 month is taken into particulate spine The ball larva of a tapeworm or the cercaria of a schistosome, isolates germinal layer cell, in 37 DEG C, 5%CO in RPMI1640 culture mediums₂ Bed board culture in environment, is passed on after 21 days, is passed within every 2 weeks after stabilization is passed on Once setting up Germinal Cell Line.

4. anti-hydatid drugs high-throughput screening method as claimed in claim 1, wherein Step 2) described in the preparation of echinococcus germinal layer cell suspension include：Take attached cell Add 0.25% pancreatin to add culture medium to be blown and beaten after digesting 5~30 minutes, obtain thin Born of the same parents' suspension.

5. anti-hydatid drugs high-throughput screening method as claimed in claim 1, wherein Step 3) described in prepare attached cell be to connect the echinococcus germinal layer cell suspension Plant in 96 well culture plate culture 24h, make cell completely adherent.

6. anti-hydatid drugs high-throughput screening method as claimed in claim 1, wherein Step 3) described in medicine to be measured be candidate's anti-hydatid drugs.

7. anti-hydatid drugs high-throughput screening method as claimed in claim 1, wherein Step 4) described in determine supernatant content of alkaline phosphatase include：Add medicine to be measured 72h draws nutrient solution supernatant after thing, determines the content of alkaline phosphatase of supernatant.

8. anti-hydatid drugs high-throughput screening method as claimed in claim 1, wherein Step 4) described in the determination of activity of attached cell use mtt assay.