Zebra fish model-based high-throughput evaluation method for hematopoietic toxicity of chemicals

Abstract

The invention discloses a high-throughput evaluation method for chemical hematopoietic toxicity based on a zebra fish model, and relates to the field of chemical toxicity evaluation technology and toxicology risk screening. According to the method, a transparent zebra fish strain marked by blood cells is constructed, an acute toxicity test is combined to determine a chemical concentration range, and an automatic imaging technology and a flow cytometry technology are utilized to perform high-throughput analysis on the number change of the blood cells, so that the hematopoietic toxicity of the chemicals is identified and evaluated. The method is low in cost, high in flux, high in reliability and suitable for large-scale toxicity screening and risk management and control of chemicals.

Description

Technical Field

[0001] The present invention relates to the technical field of high-throughput screening and evaluation of chemical toxicity, and specifically relates to a high-throughput evaluation method for the hematopoietic toxicity of chemicals based on a zebrafish model. Background Art

[0002] The normal function of the hematopoietic system is the basis for maintaining life activities, and its damage can cause serious diseases such as anemia, immune deficiency, and leukemia. The widespread use of chemicals such as industrial pollutants, drugs, and pesticides has made their potential toxicity to the hematopoietic system an important challenge in the fields of public health and environmental safety. Traditional hematotoxicity assessment methods mainly rely on mammalian models (such as mice and rats). Although they can reflect the toxic effects under the complex physiological environment in vivo, they have significant defects such as high breeding costs, long experimental cycles (usually several weeks to several months), low throughput (only a few compounds can be evaluated in a single experiment), and animal ethics restrictions, making it difficult to meet the rapid screening needs of tens of thousands of new chemicals added globally each year. In vitro cell experiments (such as the culture of hematopoietic stem cell lines) not only have high costs but also lack the in vivo metabolic environment and cell-cell interactions, and the correlation between experimental results and in vivo toxic effects is limited, unable to comprehensively simulate the overall impact of chemicals on the hematopoietic system.

[0003] As an emerging model organism, zebrafish exhibits unique advantages in hematotoxicity assessment: its genome has a 70% conservation rate with that of humans, and its hematopoietic development mechanisms (such as the differentiation of hematopoietic stem and progenitor cells and the regulation of blood cell formation) are highly similar to those of humans; the embryos are transparent and develop rapidly (hematopoietic stem cells are formed 32 hours after fertilization, and the hematopoietic system develops in 7 days), facilitating real-time observation of the blood cell formation process; transgenic labeling techniques at the single-cell level (such as fluorescent protein labeling of specific blood cells) combined with high-throughput imaging can achieve precise analysis of the number, distribution, and morphology of hematopoietic cells. In addition, zebrafish embryos / larvae can be cultured on a large scale in 96-well plates, combined with automated microscopic imaging and data analysis techniques, significantly improving the toxicity screening throughput (hundreds of samples can be processed in a single experiment) and reducing the unit cost (only 1 / 10 to 1 / 5 of mammalian experiments).

[0004] However, the existing methods for evaluating zebrafish hematotoxicity still have limitations: First, most studies focus on a single blood cell type (such as red blood cells or neutrophils), lacking a systematic analysis of multi-lineage cells such as hematopoietic stem cells and lymphocytes, and it is difficult to comprehensively reflect the damage of chemicals to the hematopoietic hierarchical network. Second, image acquisition and analysis rely on manual operations, with low efficiency and prone to introducing subjective errors, restricting the standardized implementation of high-throughput screening. Third, the toxicity evaluation endpoints are mostly based on morphological observations (such as malformation rate, mortality), lacking quantitative analysis at the single-cell level (such as cell cycle, apoptosis ratio), and it is difficult to analyze the molecular mechanism of toxic effects. For example, the existing methods for evaluating the developmental toxicity of lymphocytes focus on the morphological observation of the thymus at 120 hours after fertilization, lacking dynamic quantitative indicators; the detection of hematopoietic stem cells relies on tissue sections and cannot achieve in vivo high-throughput analysis.

[0005] In recent years, the development of automated microscopy techniques (such as high-throughput fluorescence microscopes) and flow cytometry has provided technical support for breaking through the above bottlenecks. The former can achieve automated imaging and regional recognition of zebrafish embryos / larvae (such as the caudal hematopoietic tissue, thymus), combined with image analysis algorithms (such as cell counting, fluorescence intensity quantification), significantly improving the efficiency and accuracy of blood cell quantitative analysis; the latter can prepare single-cell suspensions by enzymatic digestion and use fluorescently labeled antibodies or transgenic lines to accurately count rare cell populations such as hematopoietic stem cells and macrophages, making up for the deficiencies of imaging techniques. In addition, the construction of transparent zebrafish lines (such as Casper mutants) eliminates pigment interference, further optimizing the imaging quality and cell recognition accuracy.

[0006] In summary, the existing technologies have deficiencies in the throughput, comprehensiveness, and standardization of hematotoxicity evaluation, and there is an urgent need to develop an efficient evaluation method based on the zebrafish model. Through technology integration and process optimization, the present invention fills the gap in high-throughput analysis of multi-lineage blood cell toxicity and provides an innovative tool for chemical safety evaluation, environmental risk control, and anti-hematological disease drug research and development. Summary of the Invention

[0007] The embodiments of the present application provide a high-throughput evaluation method for the hematotoxicity of chemicals based on the zebrafish model. Based on the zebrafish model, the present application integrates technologies such as transgenic labeling, automated imaging, and flow cytometry to construct a multi-dimensional, high-throughput hematotoxicity evaluation system. By constructing a transparent and multi-blood cell-labeled zebrafish line, combined with concentration gradient exposure, automated image analysis, and single-cell flow cytometry detection, a full-lineage toxicity screening from early hematopoietic stem cells to mature blood cells is realized.

[0008] To solve the above technical problems, the technical solution proposed in the present application is:

[0009] The present invention provides a high-throughput evaluation method for the hematopoietic toxicity of chemicals based on a zebrafish model, comprising the following steps:

[0010] (1) Construct a transparent zebrafish genetic strain with blood cell labeling;

[0011] (2) Determine the exposure concentration range for evaluating the hematopoietic toxicity of the chemical to be tested;

[0012] (3) Expose zebrafish embryos;

[0013] (4) Use automated fluorescence microscopy imaging technology to conduct high-throughput identification of the hematopoietic toxicity of chemicals;

[0014] (5) Use flow cytometry technology to conduct further evaluation of the hematopoietic toxicity of chemicals with risks.

[0015] Furthermore, the types of blood cell labeling in step (1) include at least one of hematopoietic stem cells, neutrophils, lymphocytes, red blood cells, macrophages, and platelets.

[0016] Furthermore, the transparent zebrafish strain with blood cell labeling in step (1) is constructed in the following manner:

[0017] Hybridize transgenic zebrafish labeled with fluorescent protein with the transparent mutant zebrafish Casper (mitfa w2 / w2 ; mpv17 a9 / a9 ), and screen for a transparent and blood cell-labeled genetic strain after self-crossing the offspring for 2 generations.

[0018] Furthermore, the exposure concentration range is determined by acute toxicity testing in step (2), specifically:

[0019] The highest screening concentration (Cmax) is the concentration at which the sum of the mortality rate and malformation rate of zebrafish larvae exposed for 6 days does not exceed 20%, and 3-4 concentration gradients are set within the range of 1 / 100 Cmax to Cmax.

[0020] Furthermore, embryo exposure in step (3) starts from 1 day after fertilization and lasts for 4-6 days.

[0021] Furthermore, step (4) includes:

[0022] (i) Use automated microscopy imaging technology to collect fluorescence images of the caudal hematopoietic tissue (CHT) or the head thymus region of zebrafish;

[0023] (ii) Quantitatively analyze the number of neutrophils or the fluorescence area of lymphocytes, and statistically analyze significant changes to identify chemicals with hematopoietic toxicity risks.

[0024] Furthermore, the quantitative analysis of neutrophils is performed on the 4th day after exposure, i.e., 5 days after fertilization, and the quantitative analysis of lymphocytes is performed on the 6th day after exposure, i.e., 7 days after fertilization.

[0025] Furthermore, step (5) includes:

[0026] (i) Preparing an embryonic single-cell suspension containing fluorescently labeled blood cells;

[0027] (ii) Analyzing the changes in the numbers of hematopoietic stem cells, red blood cells, macrophages, and platelets by flow cytometry.

[0028] Furthermore, the method for preparing the single-cell suspension in step (5) includes:

[0029] Treating fluorescently labeled zebrafish larvae by enzymatic digestion and filtering through a 40-μm cell sieve to obtain.

[0030] Furthermore, in steps (4) and (5), the imaging data of at least 20 larvae and the flow cytometry data of at least 30 larvae are analyzed for each treatment concentration.

[0031] Furthermore, in step (4), the region for collecting zebrafish blood cell images using automated microscopy imaging technology is the caudal hematopoietic tissue (CHT) or the head thymus region, the region for collecting neutrophil images is the caudal hematopoietic tissue region, and the region for collecting lymphocyte images is the head thymus region.

[0032] Furthermore, in step (4), image quantitative analysis is performed using image processing software. The number of neutrophils is directly calculated, and the number of lymphocytes is represented by calculating the total area of fluorescent cells in the thymus region.

[0033] Furthermore, the time point for flow cytometry analysis in step (5) is the 4th day after exposure, i.e., 5 days after fertilization.

[0034] Furthermore, steps (4) and (5) finally evaluate the toxic effects of chemicals on hematopoietic stem cells, neutrophils, lymphocytes, red blood cells, macrophages, and platelets.

[0035] Compared with the prior art, the present invention has achieved the following beneficial technical effects:

[0036] (1) The present invention utilizes transparent and blood cell-labeled zebrafish and automated imaging to perform high-throughput image acquisition and analysis on the blood cells of zebrafish exposed to chemicals. It has the characteristics of high throughput, can quickly analyze a variety of chemicals, and meets the needs of large-scale chemical toxicity screening; (2) The present invention improves the specificity of hematotoxicity evaluation. Setting the exposure concentration range at a relatively low level for embryo death and teratogenic effects can avoid the systemic toxicity caused by high-dose exposure. Starting the embryo exposure after basic organ formation (24 hours after fertilization) can avoid the side effects of hematotoxicity caused by chemical damage to the early development of embryos. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0038] Figure 1 This is a zebrafish larva that is transparent and labeled with neutrophils according to the present invention. Among them, (A) a transparent and neutrophil-labeled zebrafish larva Tg(mpx:GFP); mitfa w2 / w2 ; roy a9 / a9 (5 dpf), the fluorescence signal is white; (B) a common pigmented neutrophil-labeled zebrafish larva Tg(mpx:GFP) (5 dpf), the fluorescence signal is white, and the arrow indicates the tail pigment cells.

[0039] Figure 2 This is the acute toxicity of HgCl2 to zebrafish embryos in the embodiment of the present invention.

[0040] Figure 3 This is the 3D-printed zebrafish larva imaging positioning comb in the embodiment of the present invention.

[0041] Figure 4 This is the imaging and quantitative statistical results of neutrophils in the caudal hematopoietic tissue region of zebrafish larvae exposed to HgCl2 in the embodiment of the present invention. Among them, (A) the imaging results of neutrophils (lyz + ) in the tails of larvae exposed to HgCl2 at concentrations of 100, 60, and 20 μg / L; (B) the quantitative and statistical results of neutrophils (lyz + ) in the tails of larvae exposed to HgCl2 at concentrations of 100, 60, and 20 μg / L, ***p < 0.001.

[0042] Figure 5Imaging and quantitative statistical results of lymphocytes in the head thymus region of zebrafish larvae exposed to HgCl2 in the embodiments of the present invention. Among them, (A) Imaging results of lymphocytes (rag2 + ) in the heads of larvae exposed to HgCl2 at concentrations of 100, 60, and 20 μg / L; (B) Quantitative and statistical results of lymphocytes (rag2 + ) in the heads of larvae exposed to HgCl2 at concentrations of 100, 60, and 20 μg / L, **p<0.01, ***p<0.001.

[0043] Figure 6 Flow cytometry quantitative and statistical results of hematopoietic stem cells and platelet cells in zebrafish larvae exposed to HgCl2 in the embodiments of the present invention. Among them, (A) Flow cytometry result graphs of hematopoietic stem cells (cd41 GFP low ) and platelet cells (cd41 GFP high ) in single-cell suspensions of larvae exposed to HgCl2 at concentrations of 100, 60, and 20 μg / L; (B) Flow cytometry quantitative statistical results of hematopoietic stem cells (cd41GFP low ) in larvae exposed to HgCl2 at concentrations of 100, 60, and 20 μg / L, *p<0.05, ****p<0.0001. (C) Flow cytometry quantitative statistical results of platelet cells (cd41 GFP high ) in larvae exposed to HgCl2 at concentrations of 100, 60, and 20 μg / L, *p<0.05, ****p<0.0001.

[0044] Figure 7 Flow cytometry quantitative statistical results of red blood cells in zebrafish larvae exposed to HgCl2 in the embodiments of the present invention. Among them, (A) Flow cytometry result graphs of red blood cells (gata1 + ) in single-cell suspensions of larvae exposed to HgCl2 at concentrations of 100, 60, and 20 μg / L; (B) Flow cytometry quantitative statistical results of red blood cells (gata1 + ) in larvae exposed to HgCl2 at concentrations of 100, 60, and 20 μg / L, *p<0.05, **p<0.01.

[0045] Figure 8 Flow cytometry quantitative statistical results of macrophages in zebrafish larvae exposed to HgCl2 in the embodiments of the present invention. Among them, (A) Flow cytometry result graphs of macrophages (mpeg + ) in single-cell suspensions of larvae exposed to HgCl2 at concentrations of 100, 60, and 20 μg / L; (B) Flow cytometry quantitative statistical results of macrophages (mpeg + ) in larvae exposed to HgCl2 at concentrations of 100, 60, and 20 μg / L, *p<0.05, **p<0.01. Detailed implementation manners

[0046] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0047] As Figure 1-8 shown, a high-throughput evaluation method for the hematopoietic toxicity of chemicals based on a zebrafish model includes the following steps:

[0048] (1) Construct a transparent zebrafish genetic strain with blood cell labeling;

[0049] (2) Determine the exposure concentration range for evaluating the hematopoietic toxicity of the chemical to be tested;

[0050] (3) Expose zebrafish embryos;

[0051] (4) Use automated fluorescence microscopy technology to conduct high-throughput hematopoietic toxicity identification of chemicals;

[0052] (5) Use flow cytometry technology to conduct further hematopoietic toxicity evaluation of chemicals with risks

[0053] Taking mercury chloride as an example, an embodiment of the high-throughput evaluation method for the hematopoietic toxicity of chemicals based on a zebrafish model is as follows:

[0054] (I) Evaluation of the hematopoietic toxicity of mercury chloride

[0055] 1. Construct a transparent zebrafish genetic strain with blood cell labeling

[0056] (1) Transparent zebrafish genetic strain with hematopoietic stem cell and platelet cell labeling:

[0057] Tg(cd41:GFP); mitfa w2 / w2 ; roy a9 / a9 ;

[0058] (2) Transparent zebrafish genetic strain with neutrophil labeling:

[0059] Tg(lyz:Dsred); mitfa w2 / w2 ; roy a9 / a9 ;

[0060] (3) Transparent zebrafish genetic strain with lymphocyte labeling:

[0061] Tg(rag2:Dsred); mitfa w2 / w2 ; roy a9 / a9 ;

[0062] (4) Transparent zebrafish genetic strains with red blood cell markers:

[0063] Tg(gata1:GFP); mitfa w2 / w2 ; roy a9 / a9 .

[0064] (5) Transparent zebrafish genetic strains with macrophage markers:

[0065] Tg(mpeg:DsRed); mitfa w2 / w2 ; roy a9 / a9 .

[0066] 2. Determine the range of exposure concentrations of the test compound

[0067] (1) Adult casper zebrafish are regularly cultured in a culture system. The culture water is treated to remove chlorine, with a pH value between 7 and 8. An appropriate amount of sodium chloride is added to maintain the conductivity between 500 - 800 μS / cm, the water temperature is 27 ± 2 °C, and the light cycle has a day-night ratio of 14h:10h;

[0068] (2) Female and male fish are placed in a breeding tank at a ratio of 1:1, separated by a partition in the middle. After the light is turned on the next day, the partition is removed, and the fertilized eggs are collected after the adult fish lay eggs;

[0069] (3) Unfertilized embryos are removed, and the fertilized eggs are cultured with 0.3x Danieau Medium (17 mM NaCl, 2 mM KCl, 0.12 mM MgSO4, 1.8 mM Ca(NO3)2, 1.5 mM HEPES, pH 7.6);

[0070] (4) Mercuric chloride solid powder is dissolved in DMSO to prepare a stock solution of 0.6 mg / mL. The stock solution is serially diluted with 0.3x Danieau Medium to obtain working solutions with concentrations of 37.5, 75, 150, 300, and 600 μg / L. The final concentration of DMSO in the working solutions is 0.1%, and the control group is 0.3x Danieau Medium containing 0.1% DMSO;

[0071] (5) When the fertilized casper embryos develop to 1 dpf, they are treated with mercuric chloride exposure solutions with concentrations of 37.5, 75, 150, 300, and 600 μg / L respectively. The embryos are placed in a six-well plate and 5 mL of exposure working solution is added;

[0072] (6) Record the number of dead and malformed embryos every day, remove the dead embryos, and replace the exposure working fluid.

[0073] (7) Wait until the 7th day of exposure, and count the mortality and malformation rates of the exposed embryos. As Figure 2 shown, the sum of the mortality and malformation rates of the embryos exposed to 150 μg / L of mercury chloride is 24.4%, and the sum of the mortality and malformation rates of the embryos exposed to 75 μg / L of mercury chloride is 11.1%. The highest concentration for the hematopoietic toxicity screening of mercury chloride is between 75 - 150 μg / L. Select 100 μg / L as the highest screening concentration, and conduct tests for the hematopoietic toxicity evaluation of mercury chloride in the range of 1 μg / L to 100 μg / L.

[0074] 3. Exposure of zebrafish embryos

[0075] (1) Set the screening concentrations of mercury chloride for hematopoietic toxicity to 20, 60, and 100 μg / L, and prepare the exposure working fluid.

[0076] (2) Collect transparent zebrafish embryos Tg(lyz:Dsred); mitfa w2 / w2 ; roy a9 / a9 and zebrafish embryos Tg(rag2:Dsred); mitfa w2 / w2 ; roy a9 / a9 , and perform exposure treatment on the embryos when they develop to 1 dpf.

[0077] (3) Record the number of dead and malformed embryos every day, remove the dead embryos, and replace the exposure working fluid.

[0078] 4. Identification of hematotoxic risk chemicals using high - throughput imaging analysis technology

[0079] (1) Wait until the embryos develop to 5 dpf, and perform automated imaging analysis on the centriole - labeled larvae. The specific method is (i) 3D print a zebrafish larva - orienting mold compatible with a 96 - well plate. As Figure 2 shown, it includes a bottom plate. There are 96 pins on the upper surface of the bottom plate. The top of each pin is a trapezoidal platform, and a protrusion in the shape of a zebrafish larva (5 dpf) is designed on the trapezoidal platform; (ii) Prepare 1% agarose gel, heat and melt it, and inject 70 μL into each well of the 96 - well plate. After cooling and solidifying, drop 3% methylcellulose containing 200 mg / L tricaine into each well, just enough to cover the zebrafish - shaped cavity; (iii) Anesthetize the larvae with 0.3x Danieau Medium containing 200 mg / L tricaine, and pick the fluorescent larvae under a fluorescence microscope; (iv)

[0080] Transfer approximately 70 - 100 μL of the solution containing fluorescent larvae into a 96 - well plate. Under a dissecting microscope, use a round - tipped glass needle that has been flame - fired to push the larvae into a cavity shaped like a zebrafish, and adjust the posture of the larvae so that the left - side view is facing up; (v) Place the 96 - well plate containing the embedded larvae on the stage of a fully automatic inverted fluorescence microscope Dmi8 (Leica, Germany). Open the imaging software, select the 96 - well plate scanning mode, set the white - light and fluorescence light paths, image the tail hematopoietic region, and adjust the exposure time to obtain the best signal - to - noise ratio of the fluorescence signal. The exposure time range is 150 - 300 ms; (vi) Browse the z - axis range of each larva, select the z - axis range that can cover the tail hematopoietic region of all larvae for scanning and imaging, and the z - axis step size is 20 μm; (vii) After setting the parameters, perform automated image acquisition. After the acquisition is completed, select the clearest image of neutrophils from the z - axis stack images, frame a region of a fixed size for analysis, process the images using ImageJ, and quantitatively analyze the number of neutrophils in the selected region.

[0081] (2) When the embryos develop to 7 dpf, perform automated imaging analysis on the lymphocyte - labeled larvae. The specific method is as follows: (i) 3D - print a zebrafish larva orientation mold compatible with a 96 - well plate, as Figure 2 shown, including a bottom plate. There are 96 pins on the upper surface of the bottom plate. The top of each pin is a trapezoidal platform, and a protrusion shaped like a zebrafish larva (7 dpf) is designed on the trapezoidal platform; (ii) Prepare 1% agarose gel, heat and melt it, and inject 70 μL into each well of the 96 - well plate. After cooling and solidifying, drop 3% methylcellulose containing 200 mg / L tricaine into each well, just enough to cover the zebrafish - shaped cavity; (iii) Anesthetize zebrafish larvae with 0.3x Danieau Medium containing 200 mg / L tricaine, and pick the fluorescent larvae under a fluorescence microscope;

[0082] (iv) Transfer approximately 70 - 100 μL of the solution containing fluorescent larvae into a 96 - well plate. Under a dissecting microscope, use a round - tipped glass needle that has been flame - fired to push the larvae into a cavity shaped like a zebrafish. Adjust the posture of the larvae so that the left - side view is facing up; (v) Place the 96 - well plate containing the embedded larvae on the stage of a fully automatic inverted fluorescence microscope Dmi8 (Leica, Germany). Turn on the imaging software, select the 96 - well plate scanning mode, set the white - light and fluorescence optical paths, image the head thymus region, and adjust the exposure time to obtain the best signal - to - noise ratio of the fluorescence signal. The exposure time range is 150 - 300 ms; (vi) Browse the range of each larva on the z - axis, select the z - axis range that can cover all the head thymus regions of the larvae for scanning and imaging, and the z - axis step size is 20 μm; (vii) After setting the parameters, perform automated image acquisition. After the acquisition is completed, select the clearest image of lymphocytes from the z - axis layer - scanned images, frame and select the region containing the thymus for analysis, process the images using ImageJ software, and quantitatively analyze the area of fluorescent cells in the thymus region.

[0083] (3) Statistical analysis to identify chemicals with high risk of hematotoxicity

[0084] (i) Statistically analyze the differences in the number of neutrophils between the larvae in different concentration exposure groups and the control group. As Figure 4 shown, exposure to 20, 60, and 100 μg / L of mercury chloride induced significant increases in the number of neutrophils in zebrafish larvae by 51%, 93%, and 141% respectively (p < 0.001), that is, there was a concentration - dependent increase in the number of neutrophils in mercury - chloride - exposed larvae, indicating that mercury chloride has a hematotoxic effect; (ii) Statistically analyze the differences in the total area of lymphocytes between the larvae in different concentration exposure groups and the control group. As Figure 5 shown, exposure to 20, 60, and 100 μg / L of mercury chloride induced significant decreases in the number of lymphocytes in zebrafish larvae by 9%, 10%, and 42% respectively (p < 0.001), that is, there was a concentration - dependent decrease in the number of lymphocytes in mercury - chloride - exposed larvae, indicating that mercury chloride has a hematotoxic effect.

[0085] 5. Evaluate the hematotoxicity of risk chemicals using flow cytometry

[0086] (1) Collect transparent zebrafish embryos Tg(cd41:GFP); mitfa w2 / w2 ; roy a9 / a9 、transparent zebrafish embryos Tg(gata1:EGFP); mitfa w2 / w2 ; roy a9 / a9 and transparent zebrafish embryos Tg(mpeg:Dsred); mitfaw2 / w2 ; roy a9 / a9 , When the embryos develop to 1 dpf, expose the embryos;

[0087] (2) Record the number of dead and malformed embryos every day, remove the dead embryos, replace the exposure working fluid, and wait for the exposed embryos to develop to 5 dpf;

[0088] (3) Prepare the exposed larvae into single-cell suspensions

[0089] (i) Wash 30 fluorescent larvae 3 times with Ringer's solution (116 mM NaCl, 2.9 mM KCl, 5 mM HEPES, 1 mM EDTA, pH 7.2, without Ca 2+ , without Mg 2+ ); (ii) Use a pipette to aspirate and blow the larvae to break the yolk. The pipette tip can be cut to make it wider for easier aspiration and blowing of the larvae. Centrifuge at 300 g for 5 minutes at room temperature and remove the supernatant; (iii) Wash once with D-PBS solution, centrifuge at 300 g for 5 minutes at room temperature, and remove the supernatant; (vi) Add 1 mL of D-PBS solution containing 0.26 UI / mL collagenase Liberase Blenzyme (Roche, 1119001), incubate at 32 °C for 0.75 - 1 h, and during this period, aspirate and blow the tissue mass with a 2 mL syringe every 15 minutes until there is no obvious blocky tissue; (v) Add 1 mL of D-PBS containing 1% FBS to terminate the reaction, centrifuge at 300 g for 5 minutes at 4 °C, remove the supernatant, collect the cells, and wash the cells 2 times with D-PBS; (vi) Add 1 mL of D-PBS and filter through a 40 μm cell strainer to obtain a single-cell suspension.

[0090] (4) Use flow cytometry to analyze the ratios of fluorescently labeled hematopoietic stem cells, platelet cells, red blood cells, and macrophages respectively, and perform statistical analysis with the control group to obtain the toxic effects of the risk compounds on hematopoietic stem cells and various mature blood cells. Among them, in Tg(cd41:GFP); mitfa w2 / w2 ; roy a9 / a9 , the cells with high GFP expression (GFP high ) are platelets, and the cells with weak GFP expression (GFP low ) are hematopoietic stem cells; in Tg(gata1:GFP); mitfa w2 / w2 ; roy a9 / a9 , the green fluorescently labeled gata1 + are red blood cells; in Tg(mpeg:dsRed); mitfa w2 / w2 ; roy a9 / a9 , the green fluorescently labeled mpeg + are macrophages.

[0091] (5) Statistical analysis of the hematotoxicity of risk chemicals

[0092] (i) Statistical analysis of the differences in the number of hematopoietic stem cells between the juvenile fish in different concentration exposure groups and the control group, as Figure 6 shown, exposure to 60 and 100 μg / L of mercury chloride induced a significant decrease in the number of hematopoietic stem cells in juvenile zebrafish by 15% (p < 0.05) and 27% (p < 0.0001) respectively, and exposure to 60 and 100 μg / L of mercury chloride induced a significant decrease in the number of platelets in juvenile zebrafish by 16% (p < 0.05) and 40% (p < 0.0001), that is, the numbers of hematopoietic stem cells and platelets in the mercury chloride-exposed juvenile fish both decreased; (ii) Statistical analysis of the differences in the proportion of red blood cells between the juvenile fish in different concentration exposure groups and the control group, as Figure 7 shown, exposure to 20, 60 and 100 μg / L of mercury chloride induced a significant decrease in red blood cells by 9% (p < 0.05), 17% (p < 0.01) and 17% (p < 0.01) respectively, that is, there was a concentration-dependent decrease in the number of red blood cells in the mercury chloride-exposed juvenile fish; (iii) Statistical analysis of the differences in the proportion of macrophages between the juvenile fish in different concentration exposure groups and the control group, as Figure 8 shown, exposure to 20, 60 and 100 μg / L of mercury chloride induced a significant increase in macrophages by 48% (p < 0.05), 63% (p < 0.05) and 75% (p < 0.01) respectively, that is, there was a concentration-dependent increase in the number of macrophages in the mercury chloride-exposed juvenile fish.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A high-throughput evaluation method for the hematopoietic toxicity of chemicals based on a zebrafish model, characterized in that, It includes the following steps: (1) Construct a transparent and blood cell-labeled zebrafish genetic strain; (2) Determine the exposure concentration range for the hematotoxicity evaluation of the chemical to be tested; (3) Expose zebrafish embryos; (4) Use automated fluorescence microscopy imaging technology to conduct high-throughput hematotoxicity identification of the chemical; (5) Use flow cytometry technology to conduct further hematotoxicity evaluation of the chemicals with risks.

2. The method according to claim 1, characterized in that, The types of blood cell labels in step (1) include at least one of hematopoietic stem cells, neutrophils, lymphocytes, red blood cells, macrophages, and platelets.

3. The method according to claim 1, wherein In step (1), the transparent and blood cell-labeled zebrafish strain is constructed in the following way: Cross the transgenic zebrafish labeled with fluorescent protein with the transparent mutant zebrafish Casper (mitfa w2 / w2 ; mpv17 a9 / a9 ), and after self-crossing the offspring for 2 generations, screen to obtain a transparent and blood cell-labeled genetic strain.

4. The method according to claim 1, wherein In step (2), the exposure concentration range is determined through acute toxicity tests, specifically: The highest screening concentration (Cmax) is the concentration at which the sum of the mortality rate and malformation rate of zebrafish larvae exposed for 6 days does not exceed 20%. Three to four concentration gradients are set within the range of 1 / 100 Cmax to Cmax.

5. The method according to claim 1, wherein In step (3), the embryo exposure starts from 1 day after fertilization and lasts for 4 - 6 days.

6. The method according to claim 1, wherein Step (4) includes: (i) Use automated microscopy imaging technology to collect fluorescence images of the caudal hematopoietic tissue (CHT) or the head thymus region of zebrafish; (ii) Quantitatively analyze the number of neutrophils or the fluorescence area of lymphocytes, and statistically significant changes are counted to identify chemicals with hematotoxicity risks.

7. The method according to claim 6, wherein The quantitative analysis of neutrophils is performed on the 4th day after exposure, that is, 5 days after fertilization, and the quantitative analysis of lymphocytes is performed on the 6th day after exposure, that is, 7 days after fertilization.

8. The method according to claim 1, wherein Step (5) includes: (i) Prepare a single-cell suspension of embryos containing fluorescently labeled blood cells; (ii) Analyze the changes in the number of hematopoietic stem cells, red blood cells, macrophages, and platelets through flow cytometry technology.

9. The method according to claim 8, wherein The method for preparing the single-cell suspension in step (5) includes: Treat fluorescently labeled zebrafish larvae by enzymatic digestion method and filter through a 40μm cell sieve to obtain.

10. The method according to claim 1, characterized in that, In steps (4) and (5), the imaging data of at least 20 larvae and the flow cytometry data of 30 larvae are analyzed for each treatment concentration.

11. The method according to claim 1, wherein In step (4), the area for collecting zebrafish blood cell images using automated microscopy imaging technology is the caudal hematopoietic tissue (CHT) or the head thymus region. The area for collecting neutrophil images is the caudal hematopoietic tissue region, and the area for collecting lymphocyte images is the head thymus region.

12. The method according to claim 1, characterized in that, In step (4), image processing software is used for image quantitative analysis. The number of neutrophils is directly calculated, and the number of lymphocytes is represented by calculating the total area of fluorescent cells in the thymus region.

13. The method according to claim 1, characterized in that, The time point for flow cytometry analysis in step (5) is the 4th day after exposure, that is, 5 days after fertilization.

14. The method according to claim 1, characterized in that, Steps (4) and (5) finally evaluate the toxic effects of the chemical on hematopoietic stem cells, neutrophils, lymphocytes, red blood cells, macrophages, and platelets.

Images