CN113817680A

CN113817680A - Construction of rat nerve cell evaluation model and application of rat nerve cell evaluation model in evaluation of drug neurotoxicity

Info

Publication number: CN113817680A
Application number: CN202111145319.XA
Authority: CN
Inventors: 屈哲; 耿兴超; 霍桂桃; 田康; 林志; 杨艳伟; 张頔; 李波
Original assignee: National Institutes for Food and Drug Control
Current assignee: National Institutes for Food and Drug Control
Priority date: 2021-09-28
Filing date: 2021-09-28
Publication date: 2021-12-21

Abstract

The present invention discloses a method for evaluating neurotoxicity of a candidate compound, the method comprising: (1) culturing neural stem cell spheres in the presence of a candidate compound and determining the status of the neural stem cell sphere area; (2) culturing neural stem cells in the presence of the candidate compound and determining the DNA synthesis of the neural stem cells; (3) mixedly culturing neurons and astrocytes in the presence of the candidate compound, and determining the total length of neurites and the number of neurites; and (4) co-culturing neurons and astrocytes in the presence of the candidate compound, and determining the number of neurons and the change in the number of neurons and astrocytes as a percentage of the number of neurons. The method overcomes the defect that a single in vitro screening model and index cannot specifically evaluate multiple toxic targets of a candidate compound, comprehensively evaluates the neurotoxicity from four angles, and can be used for predicting drug development neurotoxicity and acute neurotoxicity.

Description

Construction of rat nerve cell evaluation model and application of rat nerve cell evaluation model in evaluation of drug neurotoxicity

Technical Field

The invention relates to the field of neurobiology, relates to a construction method of a rat nerve cell evaluation model, and also relates to application of the evaluation model in the aspect of evaluating drug neurotoxicity.

Background

Neurotoxicity (NT) is a common toxic and side effect of many new drugs and lead compounds thereof, environmental compounds, pesticides and the like, and early screening of neurotoxic drugs can avoid subsequent large investment of drug research and development enterprises and determine whether further drug Neurotoxicity evaluation work needs to be carried out. The nervous system is very sensitive to many endogenous and exogenous substances, and small changes in its structure and function can cause neurobiological and behavioral changes, especially in the neurodevelopmental stage, which can be permanent damage once damaged. The nervous system is relatively complex, many toxic targets and receptor pathways are involved, and the exact mechanism of neurotoxic effects of many drugs is not clear today. Nowadays, the guiding principles of the economic cooperation and development Organization (OECD) and the International harmonization society (ICH) for drug registration technical requirements on neurotoxicity evaluation are limited to in vivo experiments, mainly neurobehavioral observation and histopathological examination, and the results are highly subjective, the experimental operation is time-consuming and labor-consuming, and the neurotoxicity effect of the drug cannot be explained and evaluated from a toxicity mechanism. However, a single in vitro screening model or index cannot completely evaluate all toxic targets of the neurotoxins, so that an in vitro screening model aiming at different types of neurotoxins needs to be established, neurotoxicity effects of several types of neurotoxins are comprehensively evaluated or screened from multiple specific detection end points, and the in vitro screening model or index can be used for predicting acute neurotoxicity and developmental neurotoxicity of the drug in future.

Neural stem cells, because of their proliferative and multipotent differentiation, differentiate to produce three major cell types that make up the central nervous system: neurons, astrocytes and oligodendrocytes, are good tools for drug neurotoxicity screening. The invention constructs a primary rat neural stem cell and directional differentiation neural cell model, is used for in vitro evaluation research of nerve toxicants, and explores indexes of each detection end point of the neural cell model, such as neurite growth, total number of neural cells, percentage of the number of neurons and astrocytes in the number of the neural cells, and the like, in evaluating the specificity and sensitivity of different kinds of nerve toxicants.

The neural stem cells obtained from the embryonic brain tissue of the rat are cultured into a mature neuron and astrocyte mixed cell model with a certain differentiation proportion through a series of processes of digestion, passage, proliferation and differentiation in vitro. Nestin (Nestin), microtubule-associated Protein (MAP2, β -Tubulin III), Synaptophysin (Synaptophysin) and Glial Fibrillary Acidic Protein (GFAP) antibodies were used to identify neural stem cells, developmentally mature and early differentiated neurons, neurosynaptic and astrocytes, respectively. The neural stem cells are used for researching the toxicity of the drug influencing the growth of the neurospheres and the proliferation toxicity of EdU, the directionally differentiated neurons are used for evaluating the toxicity of the drug influencing the growth of the neurites, and the development neurotoxicity research of the drug in the process of differentiating the neural stem cells into the neurons and the astrocytes.

Compared with a cell line, the mixed cell model directionally differentiated from the rat primary cells is closer to the nerve growth mode of in-vivo in-situ cells, can detect a plurality of detection endpoints related to neurotoxicity, and can save the using number of animals greatly when being used for replacing or partially replacing animal experiments for evaluating the preclinical safety. The model can be used for effectively evaluating the neurotoxicity and developmental neurotoxicity of antitumor drugs, antiepileptic drugs, anesthetics and neurotoxic compounds.

The research result of the invention shows that the research on the application range of the neurotoxicity in-vitro screening model and the biological indexes can provide a quick and effective method for the early screening or evaluation of different neurotoxicity, is beneficial to the comparative research and verification among methodologies, and provides a new method and a new idea for the medicine supervision science by the comprehensive test and the combined application of various in-vitro detection indexes.

Disclosure of Invention

The invention mainly aims to provide a nerve poison in-vitro screening model, evaluate the sensitivity and specificity of each detection end point, screen various types of nerve poisons, provide a nerve poison in-vitro evaluation strategy for a supervision institution, solve the limitation of the existing in-vivo evaluation method, and comprehensively evaluate the multi-target toxicity of the nerve poisons from the morphological specificity end point, the developmental nerve toxicity end point and the like of a nerve cell model.

To achieve the above object, according to one aspect of the present invention, there is provided a method for evaluating neurotoxicity of a candidate compound, the method comprising:

(1) culturing neural stem cell spheres in the presence of a candidate compound and determining the status of the neural stem cell sphere area;

(2) culturing the neural stem cell in the presence of the candidate compound and determining the DNA synthesis of the neural stem cell;

(3) mixedly culturing neurons and astrocytes in the presence of the candidate compound, and determining the total length of neurites and the number of neurites; and

(4) culturing the neuron and the astrocyte in the presence of the candidate compound, and determining the number of nerve cells and the percentage change of the number of the neuron and the astrocyte in the number of the nerve cells.

Further, the candidate compound is selected from one or more of the following: vincristine, cisplatin, remifentanil, iron oxide nanoparticles, propofol, sodium valproate, phenytoin sodium, acrylamide, ethanol, and 9-cis retinoic acid.

Further, the method further comprises: culturing the neural stem cell sphere or neural stem cell, the neuron, and/or the astrocyte in the absence of the candidate compound, and comparing the condition with the condition in the presence of the candidate compound.

Furthermore, the neural stem cell ball or the neural stem cell is derived from a rat fetal brain tissue with the embryo age of 14-14.5 days.

Further, the method further comprises: adding rat neural stem cell-neuron cell-inducing differentiation medium and/or DMEM/F12 medium containing 10% fetal bovine serum to differentiate the neural stem cells into the neurons and/or the astrocytes.

Further, the area of the neural stem cell sphere was calculated using a CCD microscopy imaging system and analysis software.

Further, the DNA synthesis of the neural stem cells was determined using EDU cytotoxicity assay.

Further, the EDU cell proliferative toxicity assay was sampled at a time point of about 24 hours after administration of the candidate compound.

Further, the neural stem cell sphere is a neural stem cell of a first in vitro passage or a neural stem cell of a second in vitro passage.

Further, the sampling time points for determining the total neurite length and the number of neurites are about 48 hours and/or about 72 hours after administration of the candidate compound;

further, the sampling time points for determining the number of neural cells and the number of neurons and astrocytes as a percentage of the number of neural cells are about 48 hours and/or about 72 hours after administration of the candidate compound.

According to another aspect of the present invention there is provided a use according to the above method for assessing neurotoxicity of a drug.

The invention has the beneficial effects that:

the invention constructs an in vitro neural development model and a mature and stable neural cell mixed culture model for drug screening by obtaining the neural stem cells in the embryonic brain tissue of a rat and culturing the neural stem cells in vitro and proliferating and differentiating the neural stem cells into neurons and astrocytes. A high-content screening system is adopted, a rat neural stem cell and differentiated cell model is used for screening 11 medicines/compounds to influence the growth of neurospheres, the proliferation of neural stem cells, the growth of neurites, the differentiation of neural stem cells and the like, and the neurotoxicity and the development neurotoxicity of the medicines are screened in vitro at high flux and high content.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without exceeding the protection scope of the present application.

FIG. 1 is a diagram showing the results of nerve cell morphology and immune cell identification. FIG. A: neurospheres cultured in passage 1 for about 7 days (100 ×); and B: neurospheres generation 2 (100 ×); and (C) figure: neurosphere monolayer adherent growth (100 ×); FIG. D: anti-MAP-2 antibody labeled neuron positive (200 ×); FIG. E: anti-Nestin antibody labeled neural stem cell positive (200 ×); FIG. F: anti-GFAP antibody labeled astrocyte positive (100 ×); and (G) in the figure: neural stem cell immunohistochemical staining negative (200 ×); FIG. H: a synthetic plot (200X) of immunofluorescent staining for neural stem cells Nestin-IgG (Cy3) versus Hoechst nuclear staining; FIG. I: immunofluorescence staining synthetic map of differentiated neuron beta-Tubulin III-IgG-Alexa Fluor488 and differentiated astrocyte GFAP-IgG-Alexa Fluor594 (200X); FIG. J: synthetic map (200X) of immunofluorescence staining and Hoechst nuclear staining of differentiated neuron Synaptophysin-IgG-Alexa Fluor 594; FIG. K: MAP (200X) of differentiated neuron MAP-2-IgG-Alexa Fluor594 immunofluorescence staining and Hoechst nuclear staining; FIG. L: differential astrocyte GFAP-IgG-Alexa Fluor594 immunofluorescent staining was synthesized with Hoechst nuclear staining (200X).

Figure 2 shows a graph of the toxic effect of the drug on

neurite outgrowth

48h and 72h after administration. The total neurite length and number of neurites in each administration group were the percentage of the control group, and were expressed as the Mean ± standard deviation of four total wells analyzed in two independent experiments (Mean ± SD, n ═ 4). "+" indicates a significant difference in total neurite length (P <0.05), "#" indicates a significant difference in the number of neurites (P < 0.05).

FIG. 3 is a graph showing the experimental results of toxicity of drug-induced neural stem cell differentiation. The left Y-axis curve in the double Y-axis graph represents the total number of cells; the right Y-axis curve represents the percentage of total cells (astrocytes% and neurons%) occupied by astrocytes and neurons. (Mean ± SD, n ═ 4).

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are some, but not all, embodiments of the present application. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

As described in the background art, the existing in vivo evaluation method has limitations, and the in vitro screening model also has the problems of single evaluation index of toxic targets and lack of specificity. In order to solve the above problems, the present invention provides a method for evaluating neurotoxicity of a candidate compound, the method comprising:

In the present invention, the term "comprising" means "including" as a standard patent term, and does not exclude other components. Any of the various aspects of the invention described in connection with the term "comprising" also includes narrower embodiments in which the term "comprising" is replaced with the narrower term "consisting essentially of … … or" consisting of … …. As used in this specification, the terms "comprising" or "… … -comprising" should not be construed as limiting the invention, but rather as listing exemplary components.

In a preferred embodiment, candidate compounds selected for use in the present invention include, but are not limited to, the known neurotoxic antineoplastic drugs vincristine and cisplatin, the antiepileptic drugs sodium valproate and phenytoin sodium with developmental neurotoxic effects, the neurotoxic and developmental neurotoxic compounds ethanol and acrylamide, the anesthetic propofol and remifentanil, and the nano-formulation iron oxide nanoparticles capable of passing through the blood brain barrier, and the 9-cis retinoic acid control capable of promoting development and differentiation of the nervous system.

The candidate compounds selected for use in the present invention may also be other compounds with potential or known neurotoxicity and developmental neurotoxicity not mentioned in the present invention, as well as compounds not yet synthesized or discovered at the time of filing the present invention.

In a preferred embodiment, the method further comprises: culturing the neural stem cell sphere or neural stem cell, the neuron, and/or the astrocyte in the absence of the candidate compound, and comparing the condition with the condition in the presence of the candidate compound.

In a preferred embodiment, the neural stem cell sphere or the neural stem cell is derived from fetal brain tissue of a rat with an embryo age of 14-14.5 days.

In a preferred embodiment, the method further comprises: adding rat neural stem cell-neuron cell-inducing differentiation medium and/or DMEM/F12 medium containing 10% fetal bovine serum to differentiate the neural stem cells into the neurons and/or the astrocytes.

In a preferred embodiment, the area of the neural stem cell sphere is calculated using a CCD microscopy imaging system and analysis software.

In a preferred embodiment, the DNA synthesis of the neural stem cell is determined using the EDU cell proliferation toxicity assay.

Compared with the commonly used MTT colorimetric method, CCK8 kit method and Brdu method for detecting the cell proliferation capacity, the EdU (5-ethyl-2' -deoxyuridine) cell proliferation toxicity detection method adopted by the invention is faster and more sensitive.

In a preferred embodiment, the EDU cell proliferative toxicity assay is sampled at a time point of about 24 hours after administration of the candidate compound.

The term "about" or "approximately" with respect to a numerical value means ± 5% of the numerical value, but specifically includes the exact numerical value. For example, a time of "about" 24 hours refers to a time from 22.8 hours to 25.2 hours, but also specifically includes a time of exactly 24 hours.

In a preferred embodiment, the neural stem cell sphere is a first in vitro passaged neural stem cell or a second in vitro passaged neural stem cell.

In a preferred embodiment, the sampling time points for determining the total neurite length and the number of neurites are about 48 hours and/or about 72 hours after administration of the candidate compound.

The invention constructs primary rat neuron detection for the first time, and the result proves that the primary rat neuron detection also has better detectability and determines the optimal sampling time point. Different candidate compounds are selected at different sampling time points.

In a preferred embodiment, the sampling time points for determining the number of nerve cells and the number of neurons and astrocytes as a percentage of the number of nerve cells are about 48 hours and/or about 72 hours after administration of the candidate compound.

Among them, in vitro evaluation of developmental neurotoxicity of candidate compounds by determining the number of nerve cells and the number of neurons and astrocytes differentiated from neural stem cells as a percentage of the number of the nerve cells at a specific time point has not been reported in the prior art. Different candidate compounds are selected at different sampling time points.

Examples

1. Materials and methods

1.1 sources of cells

SD rats (E14-E14.5) pregnant for 14-14.5 days. Purchased from laboratory animal technology, Inc. of Wei Tong Li Hua, Beijing.

1.2 Main reagents and instruments

1.2.1 reagents

OriCell^TMSD rat neural stem cell complete medium (cat # RASNF-01001), manufacturer: science and technology of racing industry; OriCell^TMRat neural stem cell neuroblast induced differentiation medium (cat # RAXNX-90081), manufacturer: science and technology of racing industry; DMEM/F12 high-sugar medium (batch number: Lot. No. NAA1328), manufacturer: HycLone; fetal Bovine Serum (FBS) (lot No.: Lot. No.1565565), manufacturer: gibco; D-Hanks liquid (batch: Lot. No.8115074), manufacturer: invitrogen; pancreatin-EDTA (batch: Lot. No.20141211), manufacturer: beijing Beike Huiyu experiment equipment Co., Ltd; collagen I, rat tail (lot No.1834231), manufacturer: gibco; anti-Nestin antibody, clone mouse-401 (lot: lot. No.2780475), manufacturer: millipore; rabbits produced anti-MAP 2 polyclonal antibody (lot: Lot. No.2344988), manufacturer: millipore; rabbits produced anti-GFAP antibody (lot: Lot. No.2360000), manufacturer: abcam; mouse anti-beta-Tubulin III monoclonal antibody (lot: Lot. No.054M4819V), manufacturer: sigma; rabbits produced anti-Synaptophysin antibodies (batch: lot. No. 310333), manufacturer: sigma; goat anti-rabbit IgG-FITC (batch: Lot. No. G1314-M105), manufacturer: southern biotech; goat pAb to Ms IgG (Cy3) (batch No.: Lot. No. GR251063-6), manufacturer: abcam; mountainGoat anti-mouse IgG-Alexa Fluor488 (lot: Lot. No.1705900), manufacturer: thermo Fisher Scientific; goat anti-rabbit IgG-AlexaFluor 594 (lot: Lot. No.1704538), manufacturer: thermo Fisher Scientific;

EdU Alexa

488 flow cytometry test kit (50 tests), lot.1711804, manufacturer (brand): thermo Fisher (invitrogen).

1.2.2 instruments

Solid microscope: model MZ APO, manufacturer LEICA; the lamp type: LG-PS2, manufacturer OLYMPUS; and (3) inverting the microscope: the model is as follows: CKX31, manufacturer: japanese OLYMPUS; inverted fluorescence microscopy: the model is as follows: IX71, manufacturer: japanese OLYMPUS; an optical microscope: the model is as follows: BX-51, manufacturer: OLYMPUS; superclean bench: the model is as follows: BCN1360B, manufacturer: beijing Toyobo Harr Instrument manufacturing, Inc.; CO 2₂An incubator: the model is as follows: 10-0221, manufacturer: r.k.j; a high-speed refrigerated centrifuge: the model is as follows: h-500FRS, manufacturer: KOKUSAN, japan; CCD microscopic imaging system: the model is as follows: DP73-ST-SET, manufacturer: OLYMPUS; cell Sens standard analysis software: the manufacturer: OLYMPUS; flow cytometry: the model is as follows: FACS Calibur, manufacturer; in Cell data acquisition: the model is as follows: in CellAnalyzer 2000, manufacturer: GE Healthcare Life Sciences.

1.3 test substances

Vincristine sulfate for injection (abbreviated as vincristine, VCR) (lot No. lot: lot No.1405v1), manufacturer: shenzhen Wanle pharmaceutical Co., Ltd; cisplatin lyophilized form for injection (CDDP) (batch No.: Lot. No. H20023460), manufacturer: qilu pharmaceutical co ltd; remifentanil (remifenntanil) (171260-: a standard substance of the middle school; iron (II, III) Oxide, Iron Oxide Nanoparticles (ION-Oxide Nanoparticles, ION): specification: 10mL of a 5nm diameter magnetic nanoparticle solution, amine functionalized, 1mg/mL Fe in H₂Dispersion in O, batch number: MKBR2641V, manufacturer: sigma; propofol injection (Pr)opofol) (batch number: lot.no. h20040079), manufacturer: sichuan Muyu pharmaceutical industry, Inc.; sodium Valproate (Sodium Valproate) (lot No.100963-201302), manufacturer: a standard substance of the middle school; sodium Phenytoin (Phenyltoin Sodium) (Lot. No.100210-201303), manufacturer: a standard substance of the middle school; acrylamide (Acrylamide) (lot number: lot. No.20150427), manufacturer: chemical agents of the national drug group, ltd; ethanol (Ethanol) (batch No.: lot No.20150917), manufacturer: chemical agents of the national drug group, ltd; 9-cis retinoic acid (9c RA) (Lot. No. R4643), manufacturer: sigma; nerve Growth Factor (NGF) (lot: Lot. No. GF028), manufacturer: millipore; DMSO (batch: Lot. No. RNBD9351), manufacturer: sigma.

The nerve growth factor can promote the growth of neurospheres/nerve stem cells, and is used as a negative control drug in the invention. The 9-cis retinoic acid can promote the differentiation of neural stem cells.

The preparation of the test object: on the day of use of the test article, the test article was diluted with the cell culture medium to the set concentrations of the three dose groups, and the content of DMSO in the culture medium was 0.1%.

2. Construction of rat neural stem cell and differentiated cell model

2.1 rat Neural Stem Cells (NSCs) culture and differentiation

Anaesthetizing E14-14.5 SD rats with isoflurane, dissecting the abdominal cavity, completely discharging blood, picking the embryo tissues in the bilateral uterus together with the placenta and fetal membranes, and placing in 75% ethanol solution for 10 min. The soaking and sterilizing time is not suitable to be too long, otherwise, the embryo dehydration is caused. After soaking and disinfection, placing the embryo in an aseptic operation table, carefully peeling the fetal membranes and the placenta, transferring the complete embryo into D-Hanks liquid, repeatedly blowing and cleaning blood, placing the embryo cleaned by the D-Hanks liquid for 2-3 times under a stereomicroscope, peeling the skin, the skull and the meninges of the brain of the embryo by hairspring forceps or superfine precision forceps, and taking out the whole brain after the macroscopic great vessels are clear.

The brain tissues of all embryos are collected according to the method, and generally 1 pregnant mouse is pregnant with 15-20 fetal mice. After washing, the solution was pooled into D-Hanks solution on an ice plate. Cutting brain tissue into pieces with elbow eye scissors, addingAdding 0.25% pancreatin-EDTA 37 deg.C 5% CO₂The digestion was carried out in an incubator for 15min, and the digestion was stopped with DMEM/F12 medium containing 10% fetal bovine serum.

Blowing and beating the digested brain tissue into single cell suspension, filtering with 200 mesh screen, centrifuging at 1000rpm/min for 5min, removing supernatant, washing cells with 1mL SD rat neural stem cell complete culture medium for 2 times, adding appropriate amount of SD rat neural stem cell complete culture medium, blowing and beating into single cell suspension, adjusting cell count to 5 × 10 density⁵/cm²And inoculating the cells in 25mL or 75mL cell culture bottles.

Neural stem cells 5% CO at 37 ℃₂And (3) observing the growth state of the cells when the cells are cultured in the incubator for 3-4 days, gathering the neural stem cells into neurospheres, taking out the mixed solution of half of the cell culture medium and the neural stem cells by adopting a half liquid exchange method, placing the mixed solution into a new culture bottle, adding the same amount of new culture medium respectively, and continuing to culture for 3-4 days.

Passage of neural stem cells: and (3) centrifuging the neurosphere culture solution with a certain size for 5min at 1000rpm/min, repeatedly blowing and beating the neurosphere culture solution into a single cell suspension after heavy suspension, and continuously culturing the suspension in a new SD rat neural stem cell complete culture medium for 1 generation. At this time, the formed 2 nd generation neural stem cell ball growth stabilization can be used for the later experiment.

Directed differentiation of neural stem cells into neurons: and (3) inoculating single neural stem cells or neurospheres into a porous culture plate coated with polylysine to allow the cells to grow adherently. The culture medium is replaced by a rat neural stem cell adult neuron cell induced differentiation culture medium, and the cell morphology is observed and identified 6h, 24h and 48h after the culture medium is replaced respectively. Or changing the culture medium into a rat neural stem cell neuron cell induced differentiation medium during cell passage, then inoculating the cells into a porous culture plate coated with polylysine, and carrying out identification and detection experiments after the cells adhere to the wall.

Differentiation of neural stem cells into neurons and astrocytes: the rat neural stem cell and neuron cell induction differentiation medium was replaced with DMEM/F12 medium containing 10% fetal bovine serum. After the cells are attached to the wall, the astrocytes are identified by immunofluorescence using MAP2 antibody for neurons and GFAP antibody.

2.2 immunofluorescence technique for identifying nerve cells

The following steps are adopted to carry out immunofluorescence labeling on rat neural stem cells, directional differentiation neurons, differentiated neurons and astrocytes. The first antibody is used for labeling neural stem cells or neurospheres by adopting an anti-Nestin antibody, the anti-MAP 2 polyclonal antibody, an anti-beta-Tubulin III monoclonal antibody or an anti-Synaptophysin antibody is used for labeling neurons, and the anti-GFAP antibody is used for labeling astrocytes. The secondary antibody was developed with goat anti-rabbit IgG-FITC and goat pAb to Ms IgG (Cy3) or goat anti-mouse IgG-Alexa Fluor488 and goat anti-rabbit IgG-Alexa Fluor 594.

The method comprises the following specific steps: adherent cells were washed 3 times with pre-warmed PBS; fixing the cells with 4% paraformaldehyde at room temperature for 20 min; PBS washing for 3 times; permeabilizing 0.2% TritonX-100 for 15 min; PBS washing for 3 times; 5% sheep serum is sealed and incubated at 37 ℃ for 30 min; adding an anti-Nestin antibody diluted by 1:100 PBS respectively; 1:1000 dilution of anti-MAP 2 polyclonal antibody or 1:500 dilution of anti-beta-Tubulin III monoclonal antibody; 1:200 diluted anti-Synaptophysin antibody; 1:1000 dilution of anti-GFAP antibody. Overnight at 4 ℃; PBS washing for 3 times; adding a secondary antibody diluted by PBS 1:1000, and incubating for 30min at room temperature in a dark place; PBS washing for 3 times; and (3) staining nuclei by Hoechst, keeping out of the sun for 5min at room temperature, sealing by glycerol, observing under an inverted fluorescence microscope or directly entering an In CellAnalyzer 2000 high content analysis cell imaging system for image scanning after PBS is added.

3. Rat neural stem cell model evaluation study

3.1 Neurosphere growth toxicity

3.1.2 Experimental design and grouping

Culturing the first generation neurosphere for 3-4 days according to neurosphere density of 1 × 10⁴Transfer to 96-well plate. Each test substance was divided into a low-dose group, a medium-dose group and a high-dose group, and a blank control group and a positive control group (NGF group) were set at the same time. The concentrations of each test substance administered are grouped in table 1. The final medium + drug volume was made 100 μ L after each well dosing. 3 identical plates were inoculated simultaneously, 4 duplicate wells were set for each test group, and 50. mu.L of nerves were collected at 24h, 48h and 72h post-dose, respectivelyObserving the growth condition of neurosphere in the culture solution of neurosphere under microscope, measuring the size of neurosphere by using CCD microscopic imaging system and Cell Sens standard analysis software, and performing significance analysis (P) on average data among experimental groups by using SPSS statistical analysis software<0.05 marked difference, P<The 0.01 difference is very significant).

TABLE 1 Neurosphere growth toxicity test dosing group design

Note: "/" indicates that no group is set.

3.1.3 statistical methods

Data were statistically analyzed using One-way analysis of variance (One-way anova) according to the following method: (1) firstly, carrying out homogeneity test of variance by using tukey and REGWQ test methods, and if the data is uniform (test P is greater than 0.05), indicating that the variance is homogeneous; meanwhile, if the result of single-factor analysis of variance (F test) is obvious (P is less than or equal to 0.05), carrying out multiple comparison of tukey HSD by using a parameter method; if the one-way analysis of variance (F test) results were not significant (P >0.05), the test was terminated. (2) If the result of the analysis and the test of the homogeneity of the variance is remarkable (P is less than or equal to 0.05), the variance is represented as heterogeneity; meanwhile, if the result of the single-factor analysis of variance (F test) is obvious (P is less than or equal to 0.05), performing multiple comparison by a parameter method Dunnett 3; if the one-way analysis of variance (F test) results were not significant (P >0.05), the test was terminated.

3.2 neural Stem cell EdU proliferative toxicity

3.2.1 preparation before testing

The 2 nd generation neural stem cell is cultured for about 4-6 days to form a neurosphere again, and the distance from the neurosphere to the neurosphere is 25cm²/75cm²The mixed solution of neurospheres and the culture medium is sucked from the culture bottle, inoculated into a 12-hole plate according to the volume of 750 mu L per hole, and inoculated into 97 holes in total according to the experimental design (because each sample is more, one kit can only detect 50 samples, the experiment is carried out in two times), and the administration is prepared on the next day.

Reagent preparation and preservation:

all reagents in the kit require incubation at room temperature before use;

10mM EdU solution: 4mL of DMSO was added to the reagent A and mixed. After use, the residual solution is stored at the temperature of less than or equal to 20 ℃ and the stability is up to 1 year;

Alexa

488azide working solution: add 130. mu.L DMSO to reagent B and mix well. After use, the residual solution is stored at the temperature of less than or equal to 20 ℃ and the stability is up to 1 year;

500mL of 1 × Click-iT osmotic detergent: 50mL of reagent E was added to 450mL of 1% BSA solution. When prepared in small amounts, dilutions were made at a ratio of 1:10 for reagent E: 1% BSA solution. After use, the remaining solution was stored at 2-6 ℃ for 6 months at 1 × Click-iT osmotic detergent stability and 1 year at 10 × solution stability. Note that: the reagent E contains sodium azide, and generates high-toxicity azido acid under an acidic condition. Diluting azide compounds in tap water before liquid is discarded, and avoiding potential explosive sediments from being accumulated in a pipeline;

10 × Click-iT EdU buffer additive: 2mL of deionized water was added to the vial and stirred until the Click-iT EdU buffer was completely dissolved. After use, the remaining solution is stored at 20 ℃ or less with stability up to 1 year.

3.2.2 Experimental design and grouping

Drug delivery design and subject configuration: one blank well without EdU (for dye background analysis of flow assay data), one blank well with EdU added, and 11 test substance dose groups with EdU added for the remaining wells were designed for one experiment, with 3 duplicate wells for each sample. When each test object group is administrated, 250 mu L of test object and culture medium diluent is added into 750 mu L of neurosphere culture medium in each hole/12-hole plate, and after uniform mixing, the final volume of each hole is 1mL, so that the final concentration of each test object group is the same as the concentration of the test object in the low, medium and high dose groups in the neurosphere growth test. The preparation method of the test substance for the EdU cell proliferation toxicity test is shown in Table 2.

TABLE 2EdU cell proliferation toxicity test subject preparation method

Note: "/" indicates that the dose group was not set.

3.2.3 test procedure

EdU labeling of neural stem cells: neurospheres were suspended in media and administered at the optimal state for growth, and 12-well plates were removed 24h after administration, and 1 μ L of the prepared 10mM EdU solution was added to a super clean bench and mixed well so that the dilution ratio of the 10mM EdU solution to the media was 1:1000(10 μ M). The plates with the addition of EdU were again placed in the 37 ℃ incubator and incubated for 2 h. Note that the concentration of EdU added and the incubation time can be adjusted, and the incubation time is prolonged if the concentration of EdU is low, and shortened if the concentration is high. After incubation for 2h, the plates were removed and the neurospheres in each well were broken up into single cell suspensions by mechanical separation, which was gentle to avoid disrupting the cell structure. Transferring the single cell suspension into a flow tube, centrifuging for 3min at 1000r/m, and removing the supernatant.

Fixing and permeating: cells were washed once with 3mL PBS containing 1% BSA, centrifuged at 1000r/m for 3min, and the supernatant discarded. To the pellet was added 100. mu.L of Click-iT fixative (reagent D) and mixed well. Cells were incubated at room temperature for 15min in the dark. Cells were washed with 3mL of 1% BSA in PBS, centrifuged at 1000r/m for 3min, and the supernatant discarded. The pelleted cells were resuspended in 100. mu.L of 1 XClick-iT osmotic detergent, mixed well and incubated for 15 min.

Click-iT reaction: first, 1 XCLICK-iT EdU buffer additive was prepared and 10 XCLICK-iT EdU buffer additive was diluted 1:10 with deionized water. Next, the Click-iT reaction reagents were prepared as shown in Table 3. Note that the Click-iT reaction reagent is used within 15min after preparation. 0.5mL of Click-iT reagent was added to each sample and mixed well. The reaction mixture was incubated at room temperature for 30min in the absence of light. Washed once with 3mL of 1 XClick-iT osmotic detergent, centrifuged at 1000r/m for 3min, and the supernatant was discarded. 400-500. mu.L of 1 XClick-iT osmotic detergent was added to resuspend the cells, and the cells were prepared for analysis on a flow cytometer. Note that before the machine is started, the filter screen of 300 meshes is used for filtering out larger neural stem cell balls which are not separated sufficiently so as not to block the sample inlet pipe of the flow cytometer.

Approximately 5000 cells were collected from each sample using a flow cytometer Alexa

488azide detects EdU using a 405nm excitation with an ultraviolet emission filter (450/40 nm).

TABLE 3 Click-iT reaction reagent configuration

3.3 neurite outgrowth toxicity

3.3.1 Experimental design and grouping

When the 2 nd generation neural stem cells are gathered into neurospheres with proper sizes again, the neurospheres are scattered into single cell suspension by a mechanical separation method, and the density is 1-5 multiplied by 10⁵The cells were seeded in polylysine-coated 96-well plates, taking care that the cells were seeded with the 96-well plates in white at the four sides and either D-Hanks' solution or sterile PBS was added to prevent edge effects. After the single cells are attached to the wall, DMEM/F12 high-sugar medium is added to lead the neural stem cells to be directionally differentiated into neurons and astrocytes. The invention respectively detects the toxic effect of 48h and 72h cisplatin, remifentanil, iron oxide (II, III), propofol injection, sodium valproate, phenytoin sodium, acrylamide, ethanol and 9-cis retinoic acid on neurite growth. Cisplatin (lyophilized), remifentanil, iron oxide (II, III), propofol injection, sodium valproate, phenytoin sodium, acrylamide, ethanol, and 9-cis retinoic acid are grouped as shown in Table 4.

TABLE 4 grouping design of administration for neurite outgrowth toxicity experiment

Note: "/" indicates that no group is set.

3.3.2 immunofluorescent labeling of neurons and astrocytes

And labeling neurons and astrocytes in each cell well after administration by using an immunofluorescence labeling method. The primary antibody is labeled with an anti-beta-Tubulin III monoclonal antibody and an anti-GFAP antibody is labeled with astrocytes. The secondary antibody was developed using goat pAb to Ms IgG (Cy3) and goat anti-rabbit IgG-FITC.

The method comprises the following specific steps: adherent cells were washed 3 times with pre-warmed PBS; fixing the cells with 4% paraformaldehyde at room temperature for 20 min; PBS washing for 3 times; permeabilizing 0.2% TritonX-100 for 15 min; PBS washing for 3 times; 5% sheep serum is sealed and incubated at 37 ℃ for 30 min; adding an anti-beta-Tubulin III monoclonal antibody diluted by PBS 1:500 respectively; 1:1000 dilution of anti-GFAP antibody. Overnight at 4 ℃; PBS washing for 3 times; adding pAb to Ms IgG (Cy3) and anti-rabbit IgG-FITC secondary antibody diluted with PBS 1:1000, and incubating at room temperature for 30min in the dark; PBS washing for 3 times; hoechst staining nuclei was protected from light for 5min at room temperature. PBS is added into the cell hole and then directly enters an In CellAnalyzer 2000 high-flux high-content analysis cell imaging system for image scanning and acquisition.

3.3.3 high throughput screening of drug neurite outgrowth toxicity

3.3.3.1In Cell data acquisition

Open In Cell instrument, enter In Cell 1000 software system. Parameters are set under the Plate/Slide View window to preview the situation of each hole. And then, establishing and storing a Protocol for acquiring the image. And (4) operating the scheme on the target area in the Acquisition Mode, and taking a picture under an Acquisition Session window.

3.3.3.2 In Cell Analyzer 1000 data analysis

The image is analyzed for parameters such as Total Neurite Length (Total Neurite Length) and Neurite number (neuron Count) under a neuron outlet Analysis module In Cell Analyzer 1000 workflow software. See In Cell Analyzer 1000 workbench software specification for detailed procedures.

3.4 neural Stem cell differentiation toxicity

3.4.1 Experimental design and grouping

When the 2 nd generation neural stem cells are aggregated into neurospheres again, the neurospheres are scattered into single cell suspension by a mechanical separation method, and then the density is 1-510⁵Seeded in 96-well plates. After the single cells are attached to the wall, DMEM/F12 high-sugar medium is added to lead the neural stem cells to be directionally differentiated into neurons and astrocytes. In the present invention, 6 culture plates were inoculated in a total of 1 plate each of 24h and 48h 96-well plates to which cisplatin and remifentanil were administered, 1 plate each of 48h and 72h 96-well plates to which iron (II, III) oxide, propofol injection, sodium valproate and phenytoin were administered, and 1 plate each of 48h and 72h 96-well plates to which acrylamide, ethanol, 9-cis retinoic acid and nerve growth factor were administered. Cisplatin (lyophilized), remifentanil, iron (II, III) oxide, propofol injection, sodium valproate, phenytoin sodium, acrylamide, ethanol, 9-cis retinoic acid, and nerve growth factor were grouped as shown in Table 5.

TABLE 5 neural stem cell differentiation toxicity experiment administration grouping design

Note: "/" indicates that no group is set.

3.4.2 immunofluorescent labeling of neurons and astrocytes

The experimental work was referred to 3.3.2.

3.4.3 high throughput analysis of drug effects on neural Stem cell differentiation

3.4.3.1 In Cell data acquisition

The operation method is the same as the steps in 3.3.3.1, and comprises the steps of starting, lofting, previewing, establishing a scheme, saving a scheme file, imaging, photographing and closing.

3.4.3.2 In Cell Analyzer 1000 data analysis

Images were analyzed for changes In Cell number (Cell Count), percentage of differentiated neurons and astrocytes under the Multi Target Analysis Module In Cell Analyzer 1000 workshop software. See In Cell Analyzer 1000 workbench software specification for detailed procedures.

4 results and analysis

4.1 morphology observation and identification of rat neural stem cells and differentiated cells

The rat neural stem cells of generation 1 gather to form a neurosphere, and the sphere has better refractivity (shown in figure 1A). If the neurospheres are excessively gathered, the neural stem cells in the central part are lack of nutrition, the neurospheres are observed under a mirror to be dark in center (see figure 1B), the detection of various indexes later is not facilitated, and the neurospheres are difficult to separate and passage. Stem cells are stable by passage 2 or 3.

The culture plate is coated with polylysine and then attached to the neurospheres, the neural stem cells grow and extend outwards from the centers of the attached neurospheres, initially, the soma is polygonal and small, and synapses are short and not obvious (FIG. 1C and FIG. 1H). Directed differentiation into neuronal cells in spindle shape with constant elongation of the synapses and interlacing into networks (FIG. 1E, FIG. 1I, FIG. 1J, FIG. 1K). Differentiation into mixed cell growth included somatically fusiform, synaptically distinct neurons, and larger astrocytes (FIG. 1D, FIG. 1F, FIG. 1I, FIG. 1L). With time, the proportion of astrocytes increases. Cell immunochemical technology and cell immunofluorescence technology.

4.2 Neurosphere growth toxicity

Vincristine had the strongest toxic effect on neurosphere growth at 24h, 48h and 72h after administration, and neurosphere dissociation and fragmentation was seen in the 24h low dose group. Cisplatin, iron oxide nanoparticles, propofol injection and acrylamide are dissociated and broken from visible neurospheres in a high-dose group, and have obvious toxic effects on the neurospheres. Significant neurosphere reduction was seen in the high dose group of sodium valproate and phenytoin sodium, which had an inhibitory effect on neurosphere growth. Remifentanil, ethanol, 9-cis retinoic acid and nerve growth factor have no obvious nerve stem cell toxicity effect and nerve growth inhibition effect, and the nerve ball acted by the nerve growth factor is obviously enlarged along with the time extension of the ball body, so that the growth of the nerve ball is promoted.

Cell Sens standard analysis software measures the neurosphere size, SPSS statistical analysis software is used to analyze the significant difference between each test substance dose group and the blank control group and the positive control group, and the results are shown in Table 6.

The size (area) of the vincristine neurosphere of the low, medium and high dose groups is obviously reduced compared with the NGF group after 24h administration (the difference of P <0.05 is obvious). The administration is carried out for 48h and 72h, and the neurosphere of vincristine of each dose group is dissociated and broken.

After 24h of administration, the size (area) of cisplatin neurospheres in the high-dose group is remarkably reduced compared with that in the blank control group (P < 0.05); after 48 hours of administration, the size (area) of the cisplatin neurospheres in the low and high dose groups is greatly reduced compared with that in the blank control group (P <0.01), and the size (area) of the cisplatin neurospheres in the medium dose group is significantly reduced compared with that in the blank control group (P < 0.05); at 72h, the cisplatin neurosphere size (area) was significantly reduced in the high dose group compared to the blank control group (P < 0.05).

After 24h administration, the sizes (areas) of remifentanil neurospheres in the low, medium and high dose groups are remarkably reduced compared with those in the NGF group (P < 0.05); at 48h and 72h, the sizes (areas) of remifentanil neurospheres of the dose groups were not statistically different from those of the blank control group and the NGF group.

At the time of 48h administration, the size (area) of the nerve ball of the iron oxide nanoparticle in the medium dose group is greatly reduced compared with that in the blank control group and the NGF group (P < 0.01). After 24h of administration, the size (area) of the iron oxide nanoparticle neurosphere of each dose group is not statistically different from that of a blank control group and an NGF group. After 72 hours of administration, the sizes (areas) of the neurospheres of the iron oxide nanoparticles of the low and medium dose groups are not statistically different from those of the blank control group and the NGF group, and all the neurospheres of the iron oxide nanoparticles of the high dose group are dissociated and broken.

After 48h of administration, the size (area) of the propofol injection neurosphere in the low-dose group is remarkably and extremely remarkably reduced compared with that in the blank control group and the NGF group (P <0.05 and P <0.01), and the size (area) of the propofol injection neurosphere in the medium-dose group is remarkably reduced compared with that in the NGF group (P < 0.05). After 24h administration, the size (area) of the neurosphere of the propofol injection in each dose group is not statistically different from that of a blank control group and an NGF group. After 72 hours of administration, the sizes (areas) of the neurospheres of the propofol injection in the low and medium dose groups are not statistically different from those of the blank control group and the NGF group, and all the neurospheres of the propofol injection in the high dose group are dissociated and broken.

The high-dose sodium valproate neurospheres were significantly and significantly reduced in size (area) compared to the placebo group at 48h and 72h administration (P <0.01, P < 0.05); when the drug is administrated for 48 hours, the size (area) of the sodium valproate neurosphere in the high-dose group is remarkably reduced compared with that in the NGF group (P < 0.05). After 24h administration, the sizes (areas) of sodium valproate neurospheres of each dose group are not statistically different from those of a blank control group and an NGF group.

After 24h administration, the size (area) of the high-dose group phenytoin sodium neurospheres was significantly reduced compared to the NGF group (P < 0.05). After 48h of administration, the size (area) of the sodium phenytoin neurospheres in the high-dose group was significantly reduced compared with that in the blank control group (P <0.01), and the size (area) of the sodium phenytoin neurospheres in the medium-and high-dose groups was significantly and significantly reduced compared with that in the NGF group (P <0.05, P < 0.01). After 72h administration, the sizes (areas) of the phenytoin sodium neurospheres of each dose group are not statistically different from those of the blank control group and the NGF group.

At 24h, the size (area) of the acrylamide neurospheres in the high-dose group was significantly reduced compared with that in the NGF group (P < 0.05). After 48h administration, the size (area) of the acrylamide neurosphere of each dose group is not statistically different from that of the blank control group and the NGF group. After 72h of administration, the sizes (areas) of the acrylamide neurospheres of the low and medium dose groups are not statistically different from those of the blank control group and the NGF group, and all the acrylamide neurospheres of the high dose group are dissociated and broken.

The ethanol neurosphere size (area) of the medium-dose group was significantly reduced compared to the blank control group and the NGF group (P <0.05) at 48h of administration. No statistical difference was observed in the ethanol neurosphere size (area) of each dose group compared with the blank control group and the NGF group at 24h and 72h of administration.

When the drug is administered for 48h, the size (area) of the 9-cis retinoic acid neurospheres in the low-dose group is remarkably reduced compared with that in the NGF group (P < 0.05). No statistical difference was observed in the size (area) of 9-cis retinoic acid neurospheres in each dose group compared with the blank control group and NGF group after 24h and 72h of administration.

TABLE 6 Neurosphere growth/toxicity Effect observations

Note: "L" indicates the low dose group, "M" indicates the medium dose group, and "H" indicates the high dose group.

4.3 neural Stem cell EdU proliferative toxicity

24h after administration, EdU after administration of vincristine in the low, medium and high dose groups significantly reduced DNA synthesis (i.e., cell proliferation) of neural stem cells compared to the blank control group (P <0.05 difference is significant). Cisplatin was significantly reduced in the low, medium and high dose groups compared to the blank control group (P <0.05 difference was significant). Remifentanil was significantly reduced in the medium and high dose groups compared to the blank control group (P <0.05 difference was significant). Iron (II, III) oxide was significantly reduced in the high dose group compared to the blank control group (P <0.05 difference was significant). The propofol injection in the high-dose group is remarkably reduced compared with the blank control group (the difference is remarkable when P < 0.05). The high dose group had a significant decrease in sodium valproate (P <0.05 difference significant) compared to the blank control group. The phenytoin sodium in the high dose group was significantly reduced compared to the blank control group (P <0.05 difference was significant). Acrylamide in the high-dose group was significantly reduced compared to the blank control group (P <0.05 difference was significant). Ethanol in the medium and high dose groups is obviously reduced compared with that in the blank control group (P <0.05 difference is obvious). The low, medium and high dose groups showed no significant difference in 9-cis retinoic acid and nerve growth factor compared to the blank control group. Vincristine, cisplatin, remifentanil, iron oxide (II, III), propofol injection, sodium valproate, phenytoin sodium, acrylamide and ethanol all have EdU neural stem cell proliferation toxicity effects and are in dose-effect relationship.

4.4 neurite outgrowth toxicity

The total neurite length and neurite number were significantly increased (P <0.05) in the cisplatin 800ng/mL and 1600ng/mL dose groups compared to the blank control group at 48h administration (as shown in FIG. 2 a). At the administration time of 48h and 72h, the total neurite length and neurite number of the iron oxide nanoparticles in the 200 μ g/mL dose group were significantly increased (P <0.05) compared with the blank control group (as shown in FIG. 2c and FIG. 2 j). After administration of the drug for 48h, the total neurite length and neurite number of the propofol dose group at 200 μ g/mL were significantly increased (P <0.05) compared with the blank control group (as shown in FIG. 2 d). After being administrated for 48h, the number of neurites of the sodium valproate in the group with 200 mug/mL dose is remarkably increased (P <0.05) compared with that of the blank control group (as shown in figure 2 e). After 72h administration, the total neurite length of acrylamide in the group with 200 ug/mL dose was significantly increased (P <0.05) compared with that of the blank control group (as shown in FIG. 2 n). The remaining dosed groups were not significantly different or dose-related from the blank control group, as shown in the other graphs of fig. 2.

4.5 neural Stem cell differentiation toxicity

Cisplatin is administered for 24h and 48h, the total number of nerve cells is reduced along with the increase of the dose, the total number of nerve cells of 48h is obviously lower than 24h, and the dose effect relationship is more obvious. 24h, differentiating the neural stem cells into early neurons, wherein undifferentiated astrocytes are not found; at 48h, the toxic effect of the drug on neurons was more pronounced with increasing dose (as shown in figures 3a and 3 b).

Remifentanil is administrated for 24h, and the total number of nerve cells does not change significantly with the increase of the administration dose; 48h, a decrease in the total number of visible nerve cells given remifentanil at a dose of 1.6 μ g/mL; no difference was observed between the total number of nerve cells at 24h and 48h after administration. Similarly, the neural stem cells are differentiated into early neurons after 24h, and the undifferentiated astrocytes are not found; at 48h, the toxic effect of remifentanil given at a dose of 1.6 μ g/mL was more pronounced on astrocytes, and no significant neuronal and astrocytic toxic effect was seen in the remaining dose groups (as shown in figures 3c and 3 d).

The total number of nerve cells is reduced along with the increase of the administration dose after 48h and 72h of the administration of the iron (II, III), and the relationship between the total number of the nerve cells and the dose effect is not obviously different at two time points. At 48h and 72h, the toxic effects of the drug on neurons were more pronounced with increasing dose (as shown in figures 3e and 3 f).

The total number of nerve cells is reduced along with the increase of the administration dose (from the dose of 100 mu g/mL) by the administration of propofol for 48h and 72h, and the relationship between the total number of nerve cells and the dose effect is not significantly different at two time points. The toxic effect of the drug on neurons was more significant with increasing dose at 48h and 72h administration, and the trend of the toxic effect on neurons was more significant at 72h administration (as shown in fig. 3g and 3 h).

When sodium valproate is administrated for 48 hours, the total number of nerve cells has a tendency of decreasing with the increase of the administrated dose (from the dose of 100 mu g/mL), but when the sodium valproate is administrated for 72 hours, the total number of nerve cells is obviously decreased with the increase of the administrated dose. At 48h and 72h, the toxic effect of the drug on neurons was more pronounced with increasing doses (from 100 μ g/mL dose) (as shown in figures 3i and 3 j).

The total number of nerve cells of phenytoin sodium administered for 48h and 72h has a decreasing trend with increasing dose. At 48h and 72h, the administration of 200 μ g/mL dose of phenytoin sodium had a significant toxic effect on neurons (as shown in FIGS. 3k and 3 l).

After acrylamide is administrated for 48h and 72h, the total number of nerve cells is reduced along with the increase of the administrated dose, and the reduction trend is more obvious when the medicine is administrated for 72 h. At 48h and 72h, the toxic effect of the drug on neurons was more pronounced with increasing dose, and the trend of toxic effect on neurons was more pronounced at 72h administration (as shown in fig. 3m and 3 n).

After ethanol is administrated for 48 hours, the total number of nerve cells is not changed obviously along with the increase of the administrated dose; after 72h administration, the total number of nerve cells decreased with the increase of the dose. After the medicine is administrated for 48 hours, no obvious toxic effect is seen on neurons and astrocytes by using ethanol in each dose; after 72h administration, the drug had a toxic effect on both neurons and astrocytes as the dose was increased (as shown in FIGS. 3o and 3 p).

The total number of nerve cells did not change significantly with increasing dose for 48h and 72h of 9-cis retinoic acid administration (as shown in FIGS. 3q and 3 r).

Fig. 3s and 3t show that the negative control and the nerve growth factor set, both increasing the total number of nerve cells over time, and the negative control and the administration of nerve growth factor at 48h and 72h, differentiated higher proportion of astrocytes than neurons.

4.6 conclusion

And (3) a nerve growth toxicity test, wherein the size of the nerve ball is in direct proportion to the time when a blank control and a nerve growth factor are given, and the nerve growth factor promotes the growth and aggregation of the nerve ball obviously compared with the blank control. Sodium valproate and phenytoin sodium were shown to inhibit neurosphere growth and have developmentally neurotoxic effects. Vincristine, cisplatin, iron oxide nanoparticles, propofol injection and acrylamide directly cause nerve stem cell necrosis, and are not ideal evaluation indexes for the growth of neurospheres of the compounds with obvious neurotoxicity.

The invention adopts an EdU (5-ethyl-2' -deoxyuridine) cell proliferation toxicity detection method, can accurately replace thymine (T) to permeate into replicating DNA molecules in a cell proliferation period, and effectively detects the percentage of cells in an S phase. The specific reaction of EdU and Apollo fluorescent dye can quickly detect the DNA replication activity of cells, and can be used for high-throughput evaluation or screening of the drug-induced proliferation toxicity effect of neural stem cells. Compared with a BrdU detection method, the EdU dye is small in size, is 1/500 of a BrdU antibody, is easy to diffuse in cells, does not need DNA denaturation (acidolysis, pyrolysis, enzymolysis and the like), can effectively avoid sample damage, does not need antigen-antibody reaction, and can more accurately reflect the replication activity of DNA on the level of cells and tissues. The invention carries out the neural stem cell EdU proliferation toxicity research for 6h, 24h and 48h after the administration in the preliminary test, and the test result shows that no obvious dose effect relationship is observed between each dose group of the tested substances and the blank control group after the administration for 6 h. After the drug is administrated for 48 hours, the low-dose group of the test object can show obvious cell proliferation toxicity effect, and the medium-dose and high-dose groups can hardly detect the cell proliferation rate. And after 24h of administration, not only can obvious cell proliferation toxicity difference between the test object and the blank control group be seen, but also the dose groups of the test object have obvious dose effect relationship, so that the 24h of administration is a proper time point for screening the proliferation toxicity effect of the drug on the neural stem cells. In addition, the invention also carries out preliminary tests on the selection of the neural stem cells, and respectively carries out EdU proliferation toxicity research on the first generation neural stem cells and the neural stem cells of the first in vitro passage, and the result shows that the proliferation proportion of the neural stem cells in the S phase is about 80-90% when the tested substance acts on the EdU cell proliferation toxicity detection of the first generation neural stem cells, however, for some tested substances, because the neural stem cells are in a higher proliferation stage, the obvious dose effect relationship can not be detected, such as ethanol, and the obvious dose effect relationship can be detected in the neural stem cells of the passage, which is probably related to the stable state of multiple indexes such as the proliferation activity of the passage cells and the like, therefore, the neural stem cells with stable cell culture for the first passage or the second passage are used as carriers in the development neurotoxicity screening research test of the drug.

The invention firstly carries out the research on the neurite growth of neurons differentiated from primary rat neural stem cells through candidate compounds, and the result shows that the cisplatin, the ferric oxide nanoparticles, the propofol, the acrylamide and the 9-cis retinoic acid are expressed as promoting the neurite growth; remifentanil and phenytoin sodium have a tendency to inhibit neurite outgrowth. However, among them, cisplatin, iron oxide nanoparticles, propofol, and acrylamide also showed inhibition of neurosphere growth, having a neural stem cell proliferative toxicity effect, and a toxic effect on differentiation of neural stem cells into neurons and astrocytes at the same time point and dose level, indicating that these compounds may have a potential developmental neurotoxicity. This shows that the detection end point of neurosphere growth toxicity, cell proliferation toxicity and neural stem cell differentiation toxicity is more sensitive than the detection end point of neurite growth toxicity, but they lack the specificity of the detection end point of neurite growth, so that the four methods need to be combined according to the characteristics of neurotoxins with different action mechanisms, and the sensitivity and specificity of the combined application of the detection end points are improved.

In the neural stem cell differentiation toxicity research, when analyzing the influence of each administration dose group on the total number of nerve cells at two time points, the total number of the nerve cells is obviously reduced when cisplatin is administered for 48 hours compared with 24 hours in the low dose group, which indicates that the cisplatin low dose group has toxic effect on the nerve cells; compared with the administration time of 24h or48 h, the total number of nerve cells in the low-dose group of remifentanil, iron (II, III) oxide, propofol and phenytoin sodium has no significant difference, which indicates that the low-dose group of the medicines has a certain toxic effect on the number of nerve cells and influences the normal proliferation of the cells; the experimental group or low dose group of blank control, nerve growth factor, sodium valproate, acrylamide, ethanol and 9-cis retinoic acid increased the total number of nerve cells compared to 48h administration at 72h administration, indicating that the cells proliferated normally over time and were not affected by the action of the drug.

The effect of different dose levels of each drug on the number of nerve cells, if no dose-effect relationship was found for the total number of nerve cells, indicates that the drug has no toxic effect on both neurons and astrocytes, and that the ratio of neurons to astrocytes does not change significantly with dose changes, consistent with the results of this test, e.g., 48h and 72h dosing groups for 9-cis retinoic acid, 48h dosing groups for ethanol, and 24h dosing groups for remifentanil. If the total number of nerve cells is reduced along with the increase of the dose, whether the medicine has toxic effect on neurons or astrocytes needs to be analyzed, according to a curve chart of percentages of the neurons and the astrocytes, if the proportion of the neurons is reduced, the medicine mainly has a certain toxic effect on the neurons, otherwise, the medicine mainly has a toxic effect on the astrocytes, and if the proportion of the neurons and the astrocytes is not changed greatly, the medicine has a certain toxic effect on the neurons and the astrocytes. Therefore, the neurotoxic effect of the drug can be qualitatively and quantitatively evaluated by comparing the above results.

The in vitro high-flux high-content screening and evaluation of 11 test substances (drugs or compounds) on the effects of the growth of neurospheres, the proliferation of neural stem cells, the growth of neurites and the differentiation of neural stem cells are carried out by constructing a rat neural stem cell and differentiated cell model, and the evaluation results are mutually verified, which shows that: the neurotoxicity of the test substance (drug or compound) cannot be accurately evaluated by a single evaluation mode, and the neurotoxicity of the test substance (drug or compound) can be comprehensively reflected only by a multi-angle comprehensive evaluation system (as shown in table 7). For example, vincristine exhibits strong toxicity in neurosphere growth toxicity assays, and therefore neurite outgrowth is measured at this dose level without the need for proliferation and differentiation of neural stem cells. Cisplatin is also an anti-tumor drug, and the four indexes are used for detecting the toxicity of the cisplatin, which is more obvious, such as neural stem cells and neurons. The indexes of the anesthetic remifentanil and propofol show that the anesthetic has developmental neurotoxicity and shows inhibition effects in stem cell proliferation, differentiation and neurite growth, and the difference between the anesthetic and the propofol shows that remifentanil inhibits the differentiation of neural stem cells into astrocytes and propofol inhibits the differentiation of neural stem cells into neurons. The results of the other indexes of the antiepileptic drug sodium valproate except for neurite growth toxicity are consistent with the results of phenytoin sodium, and the results are expressed as neural stem cell proliferation and differentiation neuron toxicity. The neurotoxic compound acrylamide and ethanol have no obvious neurite growth toxicity, but show proliferation and differentiation toxicity of neural stem cells, and the ethanol shows bidirectional differentiation toxicity of neurons and astrocytes. Iron oxide nanoparticles exhibit neural stem cell proliferative and neurite outgrowth toxicity, as well as differentiated neuronal toxicity. Both retinoic acid and nerve growth factor did not exhibit neurotoxicity and developmental neurotoxicity as negative controls. The detection results of the nerve toxicants with different action mechanisms show that the nerve toxicants have different nerve toxicity action characteristics, and effective combination of nerve stem cell proliferation and differentiation and result correlation become main strategies for screening the nerve toxicants in vitro through nerve toxicity specificity detection indexes, namely neurite growth.

TABLE 7 summary of neurotoxicity in vitro evaluation index results

Note: "/" indicates no detection; "+" indicates positive; "-" indicates negative.

The foregoing detailed description of the embodiments of the present application has been presented to illustrate the principles and implementations of the present application, and the description of the embodiments is only intended to facilitate the understanding of the methods and their core concepts of the present application. Meanwhile, a person skilled in the art should, according to the idea of the present application, change or modify the embodiments and applications of the present application based on the scope of the present application. In view of the above, the description should not be taken as limiting the application.

Claims

1. A method for evaluating neurotoxicity of a candidate compound, the method comprising:

(2) culturing said neural stem cell in the presence of said candidate compound and determining the status of DNA synthesis of said neural stem cell;

(3) co-culturing neurons and astrocytes in the presence of the candidate compound and determining total neurite length and number of neurites; and

(4) culturing said neurons and said astrocytes in the presence of said candidate compound and determining the number of neurons and the percentage change in the number of neurons and astrocytes in said number of neurons.

2. The method of claim 1, wherein the candidate compound is selected from one or more of the following: vincristine, cisplatin, remifentanil, iron oxide nanoparticles, propofol, sodium valproate, phenytoin sodium, acrylamide, ethanol, and 9-cis retinoic acid.

3. The method according to claim 1 or 2, characterized in that the method further comprises: culturing said neural stem cell sphere or neural stem cell, said neuron, and said astrocyte in the absence of said candidate compound, and comparing said condition in the presence of said candidate compound.

4. The method according to claim 1, wherein the neural stem cell sphere or neural stem cell is derived from fetal brain tissue of a rat with an embryo age of 14-14.5 days.

5. The method of claim 4, further comprising: adding rat neural stem cell-neuron cell-inducing differentiation medium and/or DMEM/F12 medium containing 10% fetal bovine serum to differentiate the neural stem cells into the neurons and/or the astrocytes.

6. The method of claim 1, wherein the area of the neural stem cell sphere is calculated using a CCD microscopy imaging system and analysis software.

7. The method of claim 1, wherein EDU cell cytotoxicity assay is used to determine the status of DNA synthesis in said neural stem cells;

preferably, the EDU cell proliferation toxicity assay is sampled at a time point of about 24 hours after administration of the candidate compound.

8. The method of claim 7, wherein the neural stem cell sphere is a first in vitro passaged neural stem cell or a second in vitro passaged neural stem cell.

9. The method of claim 1, wherein the sampling time points for determining the total neurite length and the number of neurites are about 48 hours and/or about 72 hours after administration of the candidate compound;

preferably, the sampling time points for determining the number of neural cells and the number of neurons and astrocytes as a percentage of the number of neural cells are about 48 hours and/or about 72 hours after administration of the candidate compound.

10. Use of the method according to any one of claims 1 to 9 for evaluating drug neurotoxicity.