PFOS corticobrain organ injury model and construction method and application thereof

Abstract

The invention relates to a PFOS corticobrain organ injury model and a construction method and application thereof. The method comprises the following steps: (1) culturing corticobrain organs; (2) performing PFOS exposure on the cultured cortical brain organ to obtain a PFOS cortical brain organ injury model; (3) performing RNA-seq sequencing on the PFOS corticobrain organ injury model, and identifying whether the PFOS corticobrain organ injury model is successfully constructed or not through a molecular mechanism; the molecular mechanism comprises at least one of a P53signaling pathway and an axon guiding pathway, and the molecular mechanism comprises at least one of a P53signaling pathway and an axon guiding pathway; further, if the molecular mechanism appears, the PFOS cortical brain organ injury model is successfully constructed. The PFOS cortical brain organ injury model constructed by the method can promote deep research on PFOS injury brain pathological mechanisms, and a new perspective is provided for understanding the development process of diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a PFOS cortical brain organoid injury model and a construction method and application thereof. Background Art

[0002] Perfluorooctane sulfonic acid (PFOS) is a new type of environmental persistent organic pollutant. Due to its wide distribution in the environment, bioaccumulation potential and toxicological effects, it has become a research hotspot in recent years. PFOS not only causes reproductive toxicity and metabolic disorders, but also causes neurodevelopmental toxicity and induces neural damage. Previous studies have found that PFOS exposure has adverse effects on primary hippocampal neurons and cortical astrocytes in rats, leading to oxidative stress, apoptosis and damage to neuronal growth. PFOS has significant neurotoxicity, and further research is needed on the key mechanisms of neurotoxicity caused by PFOS and the search for treatments.

[0003] Brain organoids are a three-dimensional cell culture system derived from embryonic stem cells that simulates brain structure and function. In terms of the nervous system, overcoming the limitations of traditional two-dimensional culture, 3D brain organoids can simulate the complex characteristics of human brain neuronal tissue, including the diversity of cell types and the generation of characteristic regions (such as cortex, forebrain and hippocampus, etc.), while achieving long-term mature culture and providing an alternative to in vitro experiments that address ethical and technical barriers. At present, there are no reports on the use of cortical brain organoids to study the neurotoxicity of PFOS. Summary of the invention

[0004] The present invention provides a PFOS cortical brain organoid injury model and a construction method and application thereof. The method obtains a PFOS cortical brain organoid injury model by exposing cortical brain organoids cultured to day 30 to PFOS; RNA-seq sequencing is then performed on the constructed model to identify whether the model is successfully constructed through molecular mechanisms. The model and identification method provided by the present invention provide a better platform for studying the impact mechanism of PFOS on the nervous system.

[0005] In a first aspect, the present invention provides a method for constructing a PFOS cortical brain organoid injury model, comprising the following steps:

[0006] (1) Cultivation of cortical brain organoids;

[0007] (2) cultured cortical brain organoids were exposed to PFOS;

[0008] (3) performing RNA-seq sequencing on the PFOS cortical brain organoid injury model to identify whether the PFOS cortical brain organoid injury model is successfully constructed through molecular mechanisms; the molecular mechanism includes at least one of the P53 signaling pathway and the axon guidance pathway; further, if the molecular mechanism appears, the PFOS cortical brain organoid injury model is successfully constructed.

[0009] In the present invention, the concentration of PFOS is 10-200 μM.

[0010] In some specific embodiments of the present invention, the concentration of PFOS is 20-100 μM.

[0011] In some specific embodiments of the present invention, the concentration of PFOS is 20 μM.

[0012] In some specific embodiments of the present invention, the concentration of PFOS is 100 μM.

[0013] In some specific embodiments of the present invention, the exposure is performed on the 30th day of the cortical brain organoid culture, and the exposure time is 7 days.

[0014] In the present invention, the PFOS cortical brain organoid injury model can also be identified by nematodes after the molecular mechanism is identified.

[0015] In some specific embodiments of the present invention, the PFOS cortical brain organoid injury model identified by molecular mechanism is verified with nematodes. If the nematode dopaminergic neurons are abnormal and / or the nematode has movement disorders, the PFOS cortical brain organoid injury model is successfully constructed.

[0016] In a second aspect, the present invention provides a method for culturing the above-mentioned cortical brain organoids, comprising the following steps:

[0017] (1) When the confluence of cultured H9-GFP cells reached 60-80%, the cells were digested with Accutase to prepare a single-cell suspension. After cell counting, the cell clusters were added to a neural induction medium containing the ROCK inhibitor Y27632, and then 9000 cells / well were placed in a 96-well plate to form EBs;

[0018] (2) On days 8 to 12, EBs were transferred to 6-well plates and cultured in neural differentiation medium containing B27 supplement;

[0019] (3) On day 18, long-term culture was performed using neural maturation medium to obtain cortical brain organoids.

[0020] In the present invention, the neural induction medium includes at least one of DMEM / F-12, Knockout serum replacement, MEM-NEAA, Glutamax supplement, β-Mercaptoethanol, LDN-193189, SB431542 and XAV939;

[0021] And / or, the neural differentiation medium does not contain vitamin A; further, the neural differentiation medium comprises: at least one of DMEM / F-12, Neurobasal medium, N2 supplement, B27 supplement without vitamin A, MEM-NEAA, Glutamax supplement, Penicillin / Streptomycin, β-Mercaptoethanol, Insulin, CHIR and Matrigel;

[0022] And / or, the neural maturation medium includes at least one of DMEM / F-12, Neurobasal medium, N2supplement, B27 supplement, MEM-NEAA, Glutamax supplement, Penicillin-Streptomycin, Ascorbicacid, β-Mercaptoethanol, Ascorbic acid and BDNF.

[0023] In some specific embodiments of the present invention, the neural induction medium comprises 83% DMEM / F-12 medium, 15% Knockout serum replacement, 1% MEM-NEAA, 1% Glutamax supplement, 100 μM β-Mercaptoethanol, 100 nM LDN-193189, 10 μM SB431542, and 2 μM XAV939.

[0024] In some specific embodiments of the present invention, the neural differentiation medium comprises: 48% DMEM / F-12, 48% Neurobasal medium, 0.5% N2 supplement, 1% B27 supplement without vitamin A, 0.5% MEM-NEAA, 1% Glutamax supplement, 1% Penicillin / Streptomycin, 50μMβ-Mercaptoethanol, 0.025% Insulin, 3μM CHIR, and 1% Matrigel.

[0025] In some specific embodiments of the present invention, the neural maturation medium comprises 48% DMEM / F-12, 48% Neurobasal medium, 0.5% N2 supplement, 1% B27 supplement, 0.5% MEM-NEAA, 1% Glutamax supplement, 1% Penicillin-Streptomycin, 0.1% Ascorbic acid, 50 μM β-Mercaptoethanol, 200 μM Ascorbic acid, and 20 ng / ml BDNF.

[0026] In a third aspect, the present invention provides a PFOS cortical brain organoid injury model obtained by any of the above-mentioned methods.

[0027] In a fourth aspect, the present invention provides the application of the above-mentioned PFOS cortical brain organoid injury model in brain development research, nervous system disease research or drug screening.

[0028] Beneficial effects of the present invention:

[0029] (1) The present invention prepared a PFOS cortical organoid injury model by exposing the cortical organoids to 20 μM and 100 μM PFOS on the 30th day of culture, confirming that the cerebral cortical organoids are a powerful tool for evaluating the neurotoxicity of PFOS, and verifying whether the injury model was successfully constructed through the unique molecular mechanism caused by PFOS; in addition, it was further verified using nematodes.

[0030] (2) The model constructed by the present invention can promote in-depth research on the pathological mechanism of PFOS damage to the brain. It can observe in detail the pathological changes related to PFOS damage in a controlled environment, including the proliferation and apoptosis of brain organoids, the differentiation of neural progenitor cells and neural stem cells, etc., providing a new perspective for understanding the development process of the disease.

[0031] (3) The model can be used for drug screening and evaluation, accelerating the discovery and development of potential therapeutic drugs for PFOS neurological damage. By using human embryonic stem cells to construct homogeneous and reproducible cortical brain organoids, it will help promote the development of medical care. At the same time, the establishment and application of this model will reduce dependence on animal models and meet the requirements of modern scientific research ethics. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the culture process of brain organoids;

[0033] Figure 2 For identification of cortical brain organoids on day 30 by immunofluorescence staining;

[0034] Figure 3 This is a light microscopic comparison of brain organoids after PFOS exposure;

[0035] Figure 4 Comparison of immunostaining of proliferation marker (KI67) in brain organoids after PFOS exposure;

[0036] Figure 5 Comparison of immunostaining of apoptosis markers (TUNEL) in brain organoids after PFOS exposure;

[0037] Figure 6 Comparison of immunostaining of neuronal progenitor cells (SOX2) and neural stem cells (DCX) in brain organoids after PFOS exposure;

[0038] Figure 7 This is a comparison of immunostaining of the cortical marker interneuron (TBR2) in brain organoids after PFOS exposure;

[0039] Figure 8 This is a comparison of immunostaining of the deep layer neurons (CTIP2), a cortical marker, in brain organoids after PFOS exposure;

[0040] Fig. 9 Transcriptome analysis of brain organoids after PFOS exposure (cluster analysis);

[0041] Fig.10 Transcriptome analysis of brain organoids after PFOS exposure (Venn diagram);

[0042] Fig.11 KEGG enrichment bar graph of differentially expressed genes in the 20 μM PFOS treatment group;

[0043] Fig.12 KEGG enrichment bar graph of differentially expressed genes in the 100 μM PFOS treatment group;

[0044] Fig.13 Chord diagram for KEGG enrichment analysis;

[0045] Fig.14 The expression of GADD45A, CCNB1, CCNB2, CDK1, IGFBP3, and SERPINE1 was determined by qPCR;

[0046] Fig.15 GSEA enrichment analysis of Axon guidance pathway in 20 μM PFOS treatment;

[0047] Fig.16 GSEA enrichment analysis of Axon guidance pathway in 100 μM PFOS treatment;

[0048] Fig.17 To measure the expression of Netrin-1, DCC, and UNC5B by qPCR;

[0049] Fig.18 This is a comparison of immunostaining of the axon neuron marker TUJ-1;

[0050] Fig.19 This is the KEGG enrichment bar graph of differentially expressed genes in brain organoids treated with 10 μM AgNPs;

[0051] Fig. 20 KEGG enrichment bar graph of differentially expressed genes in brain organoids treated with 50 μM AgNPs;

[0052] Fig.21 KEGG enrichment histogram of differentially expressed genes in brain organoids treated with Nanoplastics;

[0053] Fig. 22 KEGG enrichment histogram of differentially expressed genes in brain organoids treated with Cadmium;

[0054] Fig.23 KEGG enrichment bar graph of differentially expressed genes in brain organoids treated with Di-(2-ethylhexyl)phthalate;

[0055] Fig.24 This is a hierarchical clustering heat map of Parkinson's disease signaling pathways;

[0056] Fig.25 This is a comparison of immunostaining of dopamine neuron markers (TH);

[0057] Fig.26 The effect of 20μM and 100μM PFOS treatment on the number of TH;

[0058] Fig. 27 The effect of PFOS treatment on the fluorescence of dopaminergic neurons in C. elegans;

[0059] Fig.28 Effects of PFOS treatment on nematode movement. DETAILED DESCRIPTION

[0060] The technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0061] Example 1 Construction of cortical brain organoids

[0062] A method for constructing a cortical brain organoid comprises the following steps:

[0063] (1) When the confluence of cultured H9-GFP cells reached 70%, the cells were digested with Accutase enzyme in a 37°C incubator for 10 min to prepare a single cell suspension. After cell counting, the cell clusters were added to neural induction medium (83% DMEM / F-12 medium, 15% Knockout serum replacement, 1% MEM-NEAA, 1% Glutamax supplement, 100 μM β-Mercaptoethanol, 100 nM LDN-193189, 10 μM SB431542, 2 μM XAV939) containing 50 μM ROCK inhibitor Y27632, and then 9000 cells / well were placed in an ultra-low adhesion 96-well plate to form embryoid bodies (EBs).

[0064] (2) Replace half of the culture medium every other day until day 10. On day 10, transfer EBs to a 6-well plate, add neural differentiation medium without vitamin A and containing B27 supplement (48% DMEM / F-12, 48% Neurobasal medium, 0.5% N2 supplement, 1% B27 supplement without vitamin A, 0.5% MEM-NEAA, 1% Glutamax supplement, 1% Penicillin / Streptomycin, 50μMβ-Mercaptoethanol, 0.025% Insulin, 3μM CHIR, 1% Matrigel), and place on a horizontal shaker for rotation culture to promote neuroepithelial proliferation.

[0065] (3) On day 18, long-term culture was performed using a neural maturation medium (48% DMEM / F-12, 48% Neurobasal medium, 0.5% N2 supplement, 1% B27 supplement, 0.5% MEM-NEAA, 1% Glutamax supplement, 1% Penicillin-Streptomycin, 0.1% Ascorbic acid, 50 μM β-Mercaptoethanol, 200 μM Ascorbic acid, 20 ng / ml BDNF). Through this directed differentiation, cortical brain organoids were generated.

[0066] (4) Draw a schematic diagram of the culture process of brain organoids (e.g. Figure 1 ), and on the 30th day, the cortical brain organoids were identified by immunofluorescence staining (e.g. Figure 2 ), neural progenitor cell markers (SOX2), microtubule-associated protein markers (MAP2), doublecortin (DCX), neuronal cells (TUJ1), cortical interneuron markers (TBR2), deep cortical neuron markers (CTIP2), and neural cadherin markers (N-cadherin) can be detected in organoids.

[0067] Example 2 Construction of PFOS cortical brain organoid injury model

[0068] 1 Methods

[0069] 1.1 Experimental plan

[0070] The experiment added 20μM and 100μM PFOs to 6-well ultra-low attachment plates containing 30-day-old cortical brain organoids and continued exposure for 7 days. The maturation medium was completely replaced every three days. During the culture period, light microscopy contrast images of brain organoids after PFOS exposure were recorded using an inverted microscope.

[0071] 1.2 Test indicators

[0072] The size changes of brain organoids after PFOS exposure were recorded under a light microscope; the neural progenitor cell proliferation marker Ki67 was detected by immunostaining and the cell apoptosis status was detected by TUNEL analysis; the differentiation of neural progenitor cells SOX2 and early neurons DCX was detected by immunostaining; the differentiation of cortical markers CTIP2 and TBR2 was detected by immunostaining.

[0073] 2 Results Analysis

[0074] PFOS exposure affects the proliferation and apoptosis of brain organoids

[0075] Light microscope comparison chart Figure 3As shown in Figure 3, PFOS exposure significantly reduced the size of brain organoids at d37; Figure 4 Co-staining of Ki67 and SOX2 confirmed that Ki67 in NPCs of 20μM PFOS and 100μM PFOS groups was significantly upregulated. + Proliferating cells decrease; e.g. Figure 5 As shown, TUNEL in the VZ-like region + The percentage of cells increased significantly, indicating that PFOS exposure can cause developmental toxicity in cortical organoids by inhibiting NPC proliferation and promoting NPC apoptosis.

[0076] PFOS exposure affects the differentiation of neural progenitor cells and neural stem cells in brain organoids

[0077] Co-staining of DCX and SOX2 can clearly show the neural rosette structure and neuronal structure in the VZ-like region of the organoid. Figure 6 , 100μM PFOS exposure significantly reduced the fluorescence intensity of DCX and the thickness of CP. In addition, the thickness of the VZ in the VZ-like area of ​​the cortical organoids in the 20μM PFOS and 100μM PFOS exposure groups was significantly reduced.

[0078] 2.3 PFOS exposure affects cortical differentiation of brain organoids

[0079] The 30-day-old cortical brain organoids are a critical period for cortical stratification. The intermediate area between the ventricular zone VZ and the cortical CP is the subcortical zone SVZ area, which mainly contains TBR2 intermediate neural precursor cells, and the cortical CP area mainly contains CTIP2 deep neurons. The results are as follows Figure 7 and Figure 8 , TBR2 in the SVZ, CTIP2 + The cell ratio and fluorescence intensity in CP were significantly lower than those in the control group. After exposure to 20 μM PFOS, only TBR2 was downregulated. + The proportion of cells in the SVZ affects the normal development and maturation of brain organoids and interferes with the normal stratification of the cortex.

[0080] The establishment of a PFOS cortical brain organoid injury model can promote in-depth research on the pathological mechanism of PFOS-induced brain damage. It can observe in detail the pathological changes related to PFOS damage in a controlled environment, including the proliferation and apoptosis of brain organoids, the differentiation of neural progenitor cells and neural stem cells, etc., providing a new perspective for understanding the development of the disease. In addition, the model can be used for drug screening and evaluation to accelerate the discovery and development of potential therapeutic drugs for PFOS neural damage.

[0081] Example 3 Using RNA-seq to identify whether the PFOS cortical brain organoid injury model is successfully constructed 1 Method

[0082] 1.1 Experimental Methods

[0083] On the 37th day, RNA-seq sequencing was performed on brain organoids treated with Control, PFOS 20 μM, and PFOS 100 μM. qPCR and immunostaining were used to verify the molecular mechanism. The neurotoxicity of PFOS was studied in combination with the Caenorhabditis elegans model organism.

[0084] On the 37th day, RNA-seq sequencing was performed on brain organoids treated with Control, 10 μM silver nanoparticles AgNPs, and 50 μM AgNPs (the treatment method was the same as the PFOS treatment method in Example 2).

[0085] 1.2 Test indicators

[0086] Analysis of the effects of PFOS exposure on the RNA-seq transcriptional profile of brain organoids, study of significantly enriched pathways; selection of significantly enriched genes in the pathways for qPCR validation; localization of the presence and distribution of specific genes and proteins in combination with immunostaining; RNA-seq sequencing analysis of brain organoids exposed to other pollutants such as AgNPs; and use of the same PFOS concentration to measure movement disorders and neuronal damage in the Caenorhabditis elegans model organism.

[0087] 2 Results Analysis

[0088] PFOS exposure alters the transcriptional profile of brain organoids

[0089] like Fig. 9 As shown in the figure, cluster analysis was used to determine the expression patterns of differentially expressed genes under three different experimental conditions: Control, PFOS 20 μM, and PFOS 100 μM. It was found that there was a close correlation between the 20 μM PFOS and 100 μM PFOS treatment groups. Fig.10 As shown, the distribution of DEGs and up-regulated and down-regulated genes were analyzed, and the Venn diagram showed that 719 genes overlapped between the two comparison combinations.

[0090] PFOS exposure induces cell cycle arrest in brain organoids

[0091] like Fig.11 and Fig.12 As shown, the differential gene enrichment in the organic system KEGG enrichment bar graph was drawn for the 20μM PFOS and 100μM PFOS treatment groups; the KEGG enrichment analysis chord diagram showed ( Fig.13 ): Cell Cycle and P53 signaling pathways are both related to cell proliferation and apoptosis, and significantly affected genes are combined with qPCR; Fig.14As shown, GADD45A expression was significantly upregulated in the 20μM and 100μM PFOS exposure groups, while CCNB1, CCNB2 and CDK1 expression was significantly reduced, which would enhance cell G2 / M phase arrest and inhibit cell proliferation and differentiation. qPCR results also showed that PFOS exposure significantly upregulated the expression of two paracrine aging inflammatory factors, IGFBP3 and SERPINE1.

[0092] PFOS exposure causes downregulation of axon guidance pathways in brain organoids

[0093] Both the 20μM PFOS and 100μM PFOS treatment groups were significantly enriched in the axon guidance pathway. Fig.15 and 16 GSEA enrichment analysis was performed on the KEGG data set of the 20μM PFOS treatment group and the 100μM PFOS treatment group. The analysis showed that Axonguidance was significantly negatively regulated by PFOS20μM and PFOS100μM. Fig.17 As shown, qPCR verified that PFOS exposure significantly reduced Netrin-1 and DCC and upregulated the expression of UNC5B; Fig.18 The immunostaining assay shown here shows that TUJ-1 expression, a marker for axonal growth, is involved in neural connectivity.

[0094] 2.4 RNA-seq analysis of brain organoids exposed to other pollutants (AgNPs, Nanoplastics, Cadmium, Di-(2-ethylhexyl)phthalate)

[0095] like Fig.19 and 20 As shown, KEGG enrichment histograms of differentially expressed genes were drawn for the 10μM AgNPs and 50μM AgNPs treatment groups, respectively. The effect of AgNPs treatment on brain organoids was enriched in signaling pathways such as P13K-Akt and Serotonergic synapse. In addition, in the literature (Chen, Shiqun et al. Toxic effects and mechanisms of nanoplastics on embryonic brain development using brain organoids model. The Science of the total environment vol. 904 (2023): 166913.), the effect of 0.025mg / mL and 0.05mg / mL Nanoplastics treatment on brain organoids was enriched in signaling pathways such as Hippo and Wnt ( Fig.21); In the literature (Huang, Yan et al. Exposure to cadmiuminduces neuroinflammation andimpairs ciliogenesis in hESC-derived 3D cerebral organoids. The Science of thetotal environment vol.797(2021):149043.), the effects of 40μM and 80μM Cadmium treatment on brain organoids were enriched in the mineral absorption signaling pathway ( Fig. 22 ); In the literature (Yang, Ling et al. Di-(2-ethylhexyl)phthalate exposure impairs cortical development in hESC-derived cerebral organoids. The Science of the total environment vol. 865(2023): 161251.), it was shown that the effects of Di-(2-ethylhexyl)phthalate treatment on brain organoids were enriched in ECM-receptor interaction and Focal adhesion signaling pathways ( Fig.23 ). As can be seen from the above, none of the above pollutants were significantly enriched in the 53signaling pathway and the Axon guidance pathway. Therefore, the signaling pathway and the Axon guidance pathway are unique characteristics of PFOS exposure, and this feature can be used to identify whether the PFOS cortical brain organoid injury model is successfully constructed.

[0096] 2.5 PFOS exposure causes dopaminergic neuron damage in brain organoids

[0097] like Figures 24-25 As shown in the figure, both PFOS treatment groups affected the Parkinson's disease signaling pathway, and dopaminergic neuron TH was significantly downregulated; further verified by qPCR, PFOS20μM and PFOS100μM treatment significantly downregulated the expression of TH gene ( Fig.26 ). The in vivo model was used to determine the changes of PFOS on dopamine neurons and motor behavior in Caenorhabditis elegans. The effect of PFOS on PD was further studied using the fluorescent-labeled Parkinson's disease model nematode BZ555. The results are as follows Fig. 27, 20μM PFOS and 100μM PFOS treatment significantly reduced dopaminergic neurons in nematodes. In addition, PFOS treatment caused movement disorders in C. elegans. Exposure to 20μM PFOS and 100μM PFOS significantly reduced the frequency of forward movement and omega movement of nematodes in a dose-dependent manner. Exposure to 100μM PFOS reduced the frequency of backward movement of nematodes ( Fig.28 ). In summary, it is confirmed that PFOS exposure downregulates dopaminergic neurons and increases the risk of PD. In addition, the nematode experiment can be used to further verify whether the PFOS cortical brain organoid injury model is successfully constructed.

[0098] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for constructing a PFOS cortical brain organoid injury model, characterized in that: The steps include: (1) Cultivation of cortical brain organoids; (2) The cultured cortical brain organoids were exposed to PFOS to obtain a PFOS cortical brain organoid injury model; (3) performing RNA-seq sequencing on the PFOS cortical brain organoid injury model to identify whether the PFOS cortical brain organoid injury model is successfully constructed through molecular mechanisms; the molecular mechanism includes at least one of the P53 signaling pathway and the axon guidance pathway; further, if the molecular mechanism appears, the PFOS cortical brain organoid injury model is successfully constructed.

2. The construction method according to claim 1, characterized in that: The concentration of the PFOS is 10 to 200 μM; further, the concentration of the PFOS is 20 to 100 μM.

3. The construction method according to claim 1, characterized in that: The concentration of PFOS was 20 μM.

4. The construction method according to claim 1, characterized in that: The concentration of PFOS was 100 μM.

5. The construction method according to claim 1, characterized in that: Exposure was performed on the 30th day of cortical brain organoid culture, and the exposure time was 7 days.

6. The construction method according to claim 1, characterized in that: The PFOS cortical brain organoid injury model identified by molecular mechanism is verified using nematodes. If the nematode dopaminergic neurons are abnormal and / or the nematodes have movement disorders, the PFOS cortical brain organoid injury model is successfully constructed.

7. The method for culturing cortical brain organoids according to claim 1, characterized in that: The steps include: (1) When the confluence of cultured H9-GFP cells reached 60-80%, the cells were digested with Accutase to prepare a single-cell suspension. After cell counting, the cell clusters were added to a neural induction medium containing the ROCK inhibitor Y27632, and then 9000 cells / well were placed in a 96-well plate to form EBs; (2) On days 8 to 12, EBs were transferred to 6-well plates and cultured in neural differentiation medium containing B27 supplement; (3) On day 18, long-term culture was performed using neural maturation medium to obtain cortical brain organoids.

8. The culture method according to claim 7, characterized in that: The neural induction medium comprises at least one of DMEM / F-12, Knockout serum replacement, MEM-NEAA, Glutamax supplement, β-Mercaptoethanol, LDN-193189, SB431542 and XAV939; And / or, the neural differentiation medium does not contain vitamin A; further, the neural differentiation medium comprises: at least one of DMEM / F-12, Neurobasal medium, N2 supplement, B27 supplement without vitamin A, MEM-NEAA, Glutamax supplement, Penicillin / Streptomycin, β-Mercaptoethanol, Insulin, CHIR and Matrigel; And / or, the neural maturation medium includes at least one of DMEM / F-12, Neurobasal medium, N2supplement, B27supplement, MEM-NEAA, Glutamax supplement, Penicillin-Streptomycin, Ascorbicacid, β-Mercaptoethanol, Ascorbic acid and BDNF.

9. A PFOS cortical brain organoid injury model, characterized in that: Obtained by the method according to any one of claims 1 to 6.

10. Use of the PFOS cortical brain organoid injury model according to claim 9 in brain development research, nervous system disease research or drug screening.

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