Epilepsy disease dynamic monitoring method and system based on brain organoid

Abstract

The invention discloses a brain organ-based epileptic disease dynamic monitoring method and system, and relates to the technical field of epileptic disease dynamic monitoring. The epilepsy disease dynamic monitoring method based on the brain organoid comprises the following steps: recombining transposons; culturing brain organoid; and verifying the to-be-detected medicine. A target gene MECP2 is respectively connected with a promoter A and a promoter B in series to respectively obtain an element A and an element B, then a first plasmid and a second plasmid are respectively inserted into chromosomes in a cell line A and a cell line B by utilizing transposase, and finally, after a first brain organ and a second brain organ are mature, the cell line A and the cell line B are constructed. The MECP2 protein content and change are respectively detected to verify the effect of the drug to be detected on the epilepsy disease, so that the effect of improving the screening accuracy of the neuronal active drug is achieved, and the problem of low screening accuracy of the neuronal active drug in the dynamic monitoring process of the epilepsy disease in the prior art is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of dynamic monitoring of epilepsy, and in particular to a method and system for dynamic monitoring of epilepsy based on brain organoids. Background Art

[0002] With the rapid development of neuroscience, biotechnology and information technology, the diagnosis and treatment of epilepsy are also constantly improving. When the activity of excitatory neurons to be tested is enhanced or the activity of inhibitory neurons to be tested is weakened, the electrical activity between the neurons to be tested loses balance, resulting in abnormal discharge activity, which will cause a series of neurological diseases, including epilepsy and paroxysmal sympathetic hyperactivity (PSH). Among them, epilepsy is one of the most common and serious brain diseases in the world, affecting more than 70 million people worldwide. Secondly, brain organoids are a biological model that simulates brain development and function through in vitro culture technology. They can reproduce the structural and functional characteristics of the brain to a certain extent, providing an ideal experimental platform for the study of neurological diseases such as epilepsy. Since brain organoids can better simulate the structure and some functions of the human brain, once they come out, brain organoids are widely used to study and simulate human brain development and evolution, neurodevelopmental disorders, mental illness, degenerative diseases, infectious diseases and other brain diseases, especially in combination with gene editing, whole genome screening, imaging and other technologies. Therefore, it is urgent to establish 3D cultured brain organoid technology for drug testing and screening as a powerful tool for drug efficacy and toxicity testing.

[0003] In the existing technology, the EEG activity in brain organoids is monitored in real time to capture the abnormal discharge pattern during epileptic seizures, and then the changes in epileptic activity in the corresponding ion channels are observed in real time to obtain the activity data of the neurons to be tested. Finally, the activity data of the neurons to be tested are identified and classified, thereby achieving real-time epilepsy diagnosis.

[0004] However, in the process of implementing the technical solution of the invention in the embodiments of the present application, the present application found that the above technology has at least the following technical problems:

[0005] In the prior art, the spatial resolution of electroencephalograms for epileptic waves is low, resulting in the inability to accurately capture changes in epileptic activity, and there is a problem of low accuracy in screening neuronal activity drugs during dynamic monitoring of epilepsy. Summary of the invention

[0006] The embodiments of the present application solve the problem of low accuracy in screening neuron-active drugs during dynamic monitoring of epilepsy in the prior art by providing a method and system for dynamic monitoring of epilepsy based on brain organoids, thereby improving the accuracy of screening neuron-active drugs.

[0007] The embodiment of the present application provides a method for dynamic monitoring of epilepsy based on brain organoids, comprising the following steps: S1, respectively concatenating the target gene MECP2 with the A promoter and the B promoter to obtain element A and element B respectively, using IL-PolyA-IR as a vector to perform transposon recombination on element A and element B to obtain a first plasmid and a second plasmid respectively; the A promoter and the B promoter are promoters of different strengths; S2, using a transposase to insert the first plasmid and the second plasmid into the chromosomes of cell line A and cell line B respectively, and differentiate and culture them to obtain a first brain organoid and a second brain organoid respectively; S3, after the first brain organoid and the second brain organoid are mature, adding a drug to be tested to the first brain organoid and the second brain organoid, and detecting the content and change of MECP2 protein respectively to verify the effect of the drug to be tested on epilepsy.

[0008] Furthermore, both cell line A and cell line B are cell lines that can verify the degree of toxicity of the drug to be tested on brain organoids.

[0009] Furthermore, the cell line A and / or cell line B are constructed by inserting a first expression element into the genome of the cell line cells using gene editing technology, and the first expression element is used to mark activated neurons; the first expression element is used to mark activated neurons specifically as follows: after expression, the first expression element indicates that the neuronal cells are mature and produce fluorescent substances.

[0010] Furthermore, the cell line A and / or cell line B are constructed by inserting a second expression element into the genome of the cell line cells using gene editing technology, and the second expression element is used to achieve quantifiable neuronal activity; the second expression element is used to achieve quantifiable neuronal activity specifically as follows: the second expression element is expressed to produce substance one only after the neurons mature, and the second expression element produces substance two throughout the entire cell growth stage, and the activity of the neurons can be quantified by detecting the amounts of substance one and substance two.

[0011] Furthermore, the cell line A and / or cell line B are constructed in the following manner: a stably transformed human embryonic stem cell line is constructed and divided into two groups, namely a first cell line and a second cell line; a first expression element is inserted into the genome of cells of the first cell line using gene editing technology, and the first expression element is used to mark activated neurons; the first expression element is used to mark activated neurons in the following manner: after expression, the first expression element indicates that the neuronal cells have matured and produce fluorescent substances; a second expression element is inserted into the genome of cells of the second cell line using gene editing technology, and the second expression element is used to achieve quantifiable neuronal activity; the second expression element is used to achieve quantifiable neuronal activity in the following manner: the second expression element is expressed to produce substance one only after the neurons mature, and the second expression element produces substance two throughout the entire cell growth stage, and the activity of the neurons can be quantified by detecting the amounts of substance one and substance two.

[0012] Furthermore, the first expression element is specifically: an E-SARE enhancer element in series with an ArcMini promoter in series with a fluorescent substance element; the fluorescent substance element is specifically: a fluorescent protein element, and the fluorescent protein element produces a specific color after expression.

[0013] Furthermore, the second expression element includes: a promoter related to neuronal activation, a sub-element one expressed after being activated by a promoter related to neuronal activation, a promoter that is continuously and stably expressed in cells, and a sub-element two expressed after being activated by a promoter that is continuously and stably expressed in cells; the sub-element one produces substance one after expression, and the sub-element two produces substance two after expression; the sub-element one is specifically: an E-SARE enhancer element in series with an ArcMini promoter in series with Gaussia luciferase; the sub-element two is specifically: a CAG promoter in series with a secretory luciferase Cypridina luciferase; the substance one is specifically: Gaussia luciferase produced after the expression of the E-SARE enhancer element in series with an ArcMini promoter in series with Gaussia luciferase; the substance two is specifically: Cypridina luciferase produced after the expression of the CAG promoter in series with the secretory luciferase Cypridina luciferase.

[0014] Furthermore, the A promoter is a CAG promoter, and the B promoter is a synapsin-Ⅰ promoter.

[0015] Furthermore, the transposase is Sleeping Beauty.

[0016] Furthermore, the first cerebral organoid and the second cerebral organoid were subjected to immunofluorescence staining respectively, and the content and changes of MECP2 protein were determined by the color after staining.

[0017] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0018] 1. Element A and element B were constructed by connecting the target gene MECP2 in series with promoters A and B of different strengths. This design enables researchers to precisely control the expression level of the MECP2 gene in different cell lines. Secondly, due to the different strengths of promoter A and promoter B, they will drive the MECP2 gene to transcribe at different rates and levels, thereby simulating the expression pattern of the MECP2 gene under different physiological or pathological conditions.

[0019] 2. By utilizing transposon recombination technology, element A and element B were integrated into the IL-PolyA-IR vector respectively to generate the first plasmid and the second plasmid. This vector not only has efficient transfection and integration capabilities, but also ensures the stable expression and inheritance of the target gene in the host cells. Through the action of transposase, these plasmids were accurately inserted into the chromosomes of cell lines A and B, thereby efficiently constructing a transgenic brain organoid model.

[0020] 3. After the brain organoids mature, the effects of these drugs on epilepsy can be evaluated by adding the drugs to be tested and detecting the content and changes of MECP2 protein. This method not only has the potential for high throughput and automation, but also can provide important information about the drug's mechanism of action, effectiveness and safety, which is of great significance for the development of new anti-epileptic drugs and the optimization of existing treatment options.

[0021] 4. By inserting the two expression elements of substance one and substance two into two cell lines respectively, double verification of neuronal activation status and activity is achieved. This design helps to reduce experimental errors. At the same time, by dynamically monitoring the activation status and activity level of neurons, researchers can reveal the abnormal activity patterns of neurons in epilepsy, provide clues for the development of new treatments, help evaluate the degree of toxicity of different drugs to brain organoids, and provide strong support for drug screening and safety assessment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A flowchart of a method for dynamic monitoring of epilepsy based on brain organoids provided in an embodiment of the present application;

[0023] Figure 2 The plasmid map of the vector plasmid 1 provided in the examples of the present application;

[0024] Figure 3 The plasmid map of the vector plasmid 2 provided in the examples of the present application;

[0025] Figure 4A diagram of the construction of a MeCP2-overexpressing organoid provided in an embodiment of the present application;

[0026] Figure 5 This is an analysis diagram of a neuronal activity drug test in a MeCP2 overexpression epilepsy disease model provided in an embodiment of the present application;

[0027] Figure 6 A screening flow chart of the drug to be screened provided in the embodiments of the present application;

[0028] Figure 7 A schematic diagram of the validation results of the brain organoid neurotoxic compound screening platform provided in the embodiments of the present application;

[0029] Figure 8 This is a compound test diagram for the brain organoid compound toxicity screening platform provided in the examples of the present application. DETAILED DESCRIPTION

[0030] The embodiment of the present application solves the problem of low accuracy of neuron-active drug screening in the process of dynamic monitoring of epilepsy in the prior art by providing a method and system for dynamic monitoring of epilepsy based on cerebral organoids. The target gene MECP2 is respectively connected in series with promoter A and promoter B to obtain element A and element B, and element A and element B are respectively subjected to transposon recombination using IL-PolyA-IR as a vector to obtain a first plasmid and a second plasmid, and then the first plasmid and the second plasmid are respectively inserted into the chromosomes of cell line A and cell line B using transposase, and differentiated and cultured to obtain a first cerebral organoid and a second cerebral organoid, and finally, after the first cerebral organoid and the second cerebral organoid are mature, the drug to be tested is added to the first cerebral organoid and the second cerebral organoid, and the content and change of MECP2 protein are respectively detected to verify the effect of the drug to be tested on epilepsy, thereby improving the accuracy of neuron-active drug screening.

[0031] The technical solution in the embodiment of the present application is to solve the problem of low accuracy in screening neuron active drugs in the above-mentioned dynamic monitoring process of epilepsy. The overall idea is as follows:

[0032] Element A and element B were obtained by concatenating the target gene MECP2 with promoter A and promoter B respectively, and then the first plasmid and the second plasmid were inserted into the chromosomes of cell line A and cell line B respectively using transposase. Finally, after the first and second cerebral organoids matured, the drugs to be tested were added to the first and second cerebral organoids, and the MECP2 protein content and changes were detected to verify the effect of the drugs to be tested on epilepsy, thereby improving the accuracy of screening for neuronal active drugs.

[0033] In order to better understand the above technical solution, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0034] like Figure 1 As shown, it is a flow chart of a method for dynamic monitoring of epilepsy based on brain organoids provided in an embodiment of the present application, the method comprising the following steps: S1, respectively concatenating the target gene MECP2 with the A promoter and the B promoter to obtain element A and element B respectively, using IL-PolyA-IR as a vector to perform transposon recombination on element A and element B to obtain a first plasmid and a second plasmid respectively; the A promoter and the B promoter are promoters of different strengths; S2, using a transposase to insert the first plasmid and the second plasmid into the chromosomes of cell line A and cell line B respectively, and differentiate and culture them to obtain a first brain organoid and a second brain organoid respectively; S3, after the first brain organoid and the second brain organoid are mature, adding the drug to be tested to the first brain organoid and the second brain organoid, and detecting the content and change of MECP2 protein respectively to verify the effect of the drug to be tested on epilepsy.

[0035] Among them, the drugs to be tested include high inhibitory drugs and low inhibitory drugs. The high inhibitory drugs are the drugs to be screened corresponding to the high activity inhibition in the activity inhibition level, and the low inhibitory drugs are the drugs to be screened corresponding to the medium activity inhibition in the activity inhibition level.

[0036] In this embodiment, the first cerebral organoid and the second cerebral organoid are stored in a cerebral organoid culture medium (i.e., a cerebral organoid model). The specific ratio process of the cerebral organoid culture medium is as follows: pretreatment of a 96-well plate: (1) adding 150 μL of 1% PVA polyvinyl alcohol to each well and placing it at room temperature for about 30 min; (2) preparing the culture medium: calculating the required volume of the culture medium, calculating it based on 150 μL per well, adding RC at a ratio of 1:100, and adding it to a 15 mL centrifuge tube for later use; (3) cell treatment: first aspirate the cell culture medium in the 6-well plate, add an appropriate amount of 0.5 μM EDTA, incubate at 37°C for 4 min, then aspirate the EDTA, add 500 μL Accutase, 37℃ for 4min, add an equal amount of culture medium to neutralize, gently blow down the cells, put them into a centrifuge tube, centrifuge at 200g for 2min, discard the supernatant, resuspend the cells with 1mL culture medium, count the cells, calculate the total number of cells required with 9000 cells per well, pipette the required cells into the prepared centrifuge tube (2), and mix by inversion; (4) Sample addition and centrifugation: adjust the range of the gun to 150μL, pour the prepared cell suspension into the sample addition slot, discard the Anti-adherence in the 96-well plate, add 150μL of cell suspension to each well, seal with sealing film, centrifuge at 100g for 5min. Remove the sealing film and place in a cell culture incubator for culture.

[0037] Specifically, a calcium ion indicator (such as Fluo-4) is added to the brain organoid culture medium, and a confocal microscope is used to record the calcium ion signal of the neurons to be tested in the brain organoid culture medium during activity to obtain the calcium ion signal intensity (directly obtained through the sensor). When the calcium ion signal intensity is greater than the reference calcium ion signal intensity, it indicates that the activity of the neuron to be tested is high; when the calcium ion signal intensity is not greater than the reference calcium ion signal intensity, it indicates that the activity of the neuron to be tested is low, wherein the reference calcium ion signal intensity is represented by the result of summing and averaging the historical calcium ion signal intensities in a preset database.

[0038] It should be noted that the culture medium of brain organoids needs to be replaced once a week to maintain its nutrient adequacy and sterility; the blank treatment is to not add the drug to be screened or only add the cell suspension in the culture medium of brain organoids; by comparing the differences in neuronal activity data with the corresponding reference neuronal activity data in the same time period, the inhibitory effect of the drug to be screened on neuronal activity can be evaluated. In order to ensure the accuracy and rigor of the data, the conditions of each experiment need to be kept consistent during the experiment (such as humidity, temperature, and light), so as to improve the accuracy of neuronal activity drug screening.

[0039] Furthermore, both cell line A and cell line B are cell lines that can verify the degree of toxicity of the drug to be tested on brain organoids.

[0040] It should be noted that the construction method of cell line A and / or cell line B is: (1) using gene editing technology to insert a first expression element into the genome of the cell line cells, and the first expression element is used to mark activated neurons; the first expression element is used to mark activated neurons specifically: after the first expression element is expressed, it indicates that the neuronal cells are mature and produce fluorescent substances.

[0041] (2) Using gene editing technology to insert a second expression element into the genome of a cell line cell, the second expression element is used to achieve quantifiable neuronal activity; the second expression element is used to achieve quantifiable neuronal activity in the following ways: the second expression element is expressed to produce substance one only after the neuron matures, and the second expression element produces substance two throughout the entire cell growth stage, and the activity of the neuron can be quantified by detecting the amounts of substance one and substance two.

[0042] (3) constructing a stably transformed human embryonic stem cell line and dividing it into two groups, namely a first cell line and a second cell line; using gene editing technology to insert a first expression element into the genome of cells of the first cell line, wherein the first expression element is used to mark activated neurons; the first expression element is used to mark activated neurons specifically: after expression, the first expression element indicates that the neuronal cells have matured and produce fluorescent substances; using gene editing technology to insert a second expression element into the genome of cells of the second cell line, wherein the second expression element is used to achieve measurable neuronal activity;

[0043] Specifically, the second expression element is used to achieve measurable measurement of neuron activity: the second expression element is expressed to produce substance one only after the neuron matures, and the second expression element produces substance two during the entire cell growth stage. The activity of the neuron can be measured by detecting the amount of substance one and substance two.

[0044] like Figure 2 As shown, it is a plasmid map of the vector plasmid 1 (i.e., the first plasmid) provided in the embodiment of the present application. At the same time, a promoter is connected in series before the first luciferase and the second luciferase, so that the measurable neuronal activity can be achieved, thereby obtaining the vector plasmid 2 (adeno-associated virus serotype 2 genomic integration site targeting neuronal activity dual luciferase measurement plasmid, AAVS1-Puro-E-SARE-Arcmini-Gaussia-CAG-Cypridina plasmid), as shown in FIG. Figure 3 As shown, it is a plasmid map of the vector plasmid 2 (i.e., the second plasmid) provided in an embodiment of the present application, wherein the first luciferase (Gaussia luciferase) is a monomeric enzyme with a length of 185 amino acids, which can be released from the endoplasmic reticulum of neurons to the outside of neuronal cells and emit 480nm fluorescence; the second luciferase (Cypridina luciferase) is composed of 555 amino acids, with a molecular weight of 62.171kDa, and the substrate is Vargulin, which can oxidize the substrate to produce blue light.

[0045] like Figure 4As shown, it is a schematic diagram of the structure of transposon recombination on the IL-PolyA-IR vector provided in an embodiment of the present application. The vector plasmid 1, vector plasmid 2 and gRNA vector plasmid on the plasmid map are electroporated into the H9 human embryonic stem cell line for subculture. When the neuronal cell fusion degree (i.e., the proportion of neuronal cells in the total cell population in the brain organoid culture medium) in the vector plasmid 1 and the vector plasmid 2 reaches 80% (preset fusion degree), it indicates that the cell line has been integrated into the corresponding neuronal genome to be tested, and genotype identification can be performed at this time. Otherwise, subculture needs to be continued until the measured neuronal cell fusion degree reaches 80% before genotype identification is performed, thereby achieving more accurate judgment of the genotype identification operation.

[0046] Furthermore, the first expression element is specifically: an E-SARE enhancer element in series with an ArcMini promoter in series with a fluorescent substance element; the fluorescent substance element is specifically: a fluorescent protein element, which produces a specific color after expression.

[0047] The second expression element includes: a promoter related to neuron activation, a sub-element one expressed after being activated by a promoter related to neuron activation, a promoter that is continuously and stably expressed in cells, and a sub-element two expressed after being activated by a promoter that is continuously and stably expressed in cells; sub-element one produces substance one after expression, and sub-element two produces substance two after expression.

[0048] The specific sub-element one is: E-SARE enhancer element tandem ArcMini promoter tandem Gaussia luciferase.

[0049] The second sub-element is specifically: CAG promoter tandem secretory luciferase Cypridina luciferase.

[0050] Substance 1 is specifically: Gaussia luciferase produced by the expression of E-SARE enhancer element in series with ArcMini promoter in series with Gaussia luciferase.

[0051] Substance 2 specifically refers to Cypridina luciferase produced by the expression of CAG promoter-tandem secretory luciferase Cypridina luciferase.

[0052] By performing immunofluorescence staining on the first and second cerebral organoids respectively, the content and changes of MECP2 protein were determined by the color after staining.

[0053] Specifically, the A promoter is the CAG promoter, the B promoter is the synapsin-Ⅰ promoter, and the transposase is SleepingBeauty. There are many transposon systems to choose from, such as piggyBac, SleepingBeauty, and Tol2. Different transposons correspond to different transposases. Among them, the strength of the CAG promoter is greater than that of the synapsin-Ⅰ promoter. The promoters described in this application, including the A promoter and the B promoter, are not limited to the above-mentioned specific types, and other promoters with similar functions can also be selected. For example, CMV, EF1a, Ubc, etc.

[0054] In this embodiment, the specific steps of the luciferase assay are as follows: (1) Preparation of substrates: Prepare the powders of Gaussia substrate Coelenterazine and Cypridina substrate Vargulin. Add 98 μL of acidified ethanol solution (20 μL / mL 3N HCl) to dissolve 500 μg of Coelenterazine to prepare a 12 mM coelenterazine stock solution. Add 200 μL of acidified n-butanol solution to 500 μg of Vargulin, and add 2-3 μL of 3N HCl solution until the yellow color disappears. Then dilute 500 times with PBS, place at room temperature and avoid light for 30 minutes for use.

[0055] (2) Diluting the culture medium: According to the preliminary experiment, the test culture medium was diluted with PBS by the gradient dilution method. The Gaussia sample was diluted 50 times and the Cypridina sample was diluted 2000 times. They were added to the ELISA plate, with 20 μL of the test sample in each well.

[0056] (3) ELISA detection: Add 50 μL of luciferase substrate working solution to each well, record the luminescence at 480 nm on an ELISA reader, and record the value.

[0057] (4) Data processing: The ratios of Gaussia fluorescence readings and Cypridina fluorescence readings in the experimental group and the control group were calculated, and then the fold changes in the experimental group and the control group were calculated. Two-way analysis of variance (ANOVA) was used for significance analysis. P < 0.05 was considered significant. After data analysis, expression histograms were prepared using GraphPad Prism 9 software. The standard deviation was used to reflect the repeatability of the data.

[0058] In the simulation scenario of actual epilepsy, after monitoring that the drug to be screened has an inhibitory effect, it is also necessary to simulate the MeCP2 duplication syndrome (Rett syndrome), especially to observe whether the MeCP2 overexpression organoids simulate the epilepsy clinical phenotype of this syndrome, in order to verify whether a brain organoid model of epilepsy can be constructed (the inhibitory drug to be screened NCH-51 is used in the verification process). Figure 5 As shown in the figure, it is a diagram of the construction of MeCP2 overexpression organoids provided in the embodiment of the present application, using the Sleeping Beauty (SB) transposon system to overexpress MeCP2 (CAG-MeCP2, SYN1-MeCP2) in the H9 cell line, wherein CAG and SYN1 are promoters of the MeCP2 gene, to construct an epilepsy disease model of neuronal overexcitation ( Figure 5 A: Schematic diagram of the construction of MeCP2 overexpressing cell lines); Western Blot (WB, Western Blot) was performed to verify the cell lines and cultured brain organoids. The results showed that in the constructed epilepsy organoid model, the expression of MECP2 protein was significantly increased ( Figure 5 B: Western blot results of MeCP2 overexpressing cells and organoids); immunofluorescence staining of brain organoids also revealed that the expression of MeCP2 in the overexpression group was significantly increased compared with the control group ( Figure 5 C: Immunofluorescence staining of MeCP2 on day 60 of culture, scale bar: 50 μm).

[0059] Specifically, the drug to be screened, NCH-51, is a histone deacetylase (HDAC) inhibitor and a potential clinical candidate drug. It has been reported that it can rescue the increase in neuronal action potentials caused by MeCP2 duplication and show a decrease in the activity level of MeCP2 duplication neurons that is almost the same as that of the control group; Figure 6 As shown, it is an analysis diagram of the neuronal activity drug test of the MeCP2 overexpression epilepsy disease model provided in the embodiment of the present application (wherein, n=8, two-way analysis of variance (Two-Way ANOVA), *P<0.5, **P<0.01, ***P<0.001, ****P<0.0001), where *P is a statistically significant marker value, **P is a higher statistically significant marker value, ***P is a highly statistically significant marker value, and ****P is an extremely highly statistically significant marker value; Figure 6D50 is the fluorescence intensity of MeCP2 in the overexpression group on the 50th day of the brain organoid culture medium, D64 is the fluorescence intensity of MeCP2 in the overexpression group on the 64th day of the brain organoid culture medium, and D78 is the fluorescence intensity of MeCP2 in the overexpression group on the 78th day of the brain organoid culture medium. According to the needs of the actual application scenarios of epilepsy diseases, it is also necessary to test the drug NCH-51 to be screened using the epilepsy brain organoid model. The specific process is: divide the organoids into groups of 8, and then treat the MeCP2-overexpressing brain organoids on day D50 with 2 μM NCH-51.

[0060] Fresh culture medium was replaced weekly and brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) were added. For fluorescent protein reporter gene brain organoids, the fluorescence intensity graph of d2GFP was taken; for luciferase reporter gene brain organoids, the culture medium of dual-luciferase organoids was collected to measure and count the luciferase content. Based on the changes in green fluorescence, we found that during one month of continuous drug treatment, organoids overexpressing MeCP2 showed a decrease in green fluorescence intensity and were close to that of the control group, which indicates that NCH-51 can rescue the neuronal overexcitation caused by MeCP2 to a certain extent ( Figure 6 A: Changes in green fluorescence after NCH-51 treatment of D50 brain organoids).

[0061] By measuring the changes in luciferase, it is helpful to more significantly observe that the phenotype of abnormal neuronal activation caused by MeCP2 overexpression after NCH-51 treatment has been corrected ( Figure 6 B: Changes in luciferase ratios at different days after adding BDNF and GDNF at D60).

[0062] Under the action of transposase, the transposon plasmid can be inserted into the host genome; the gene promoter is a key element in gene expression regulation, which determines the starting position and efficiency of gene transcription. Among them, the CAG promoter is a strong constitutive promoter that can drive efficient gene expression in a variety of cell types, and the SYN1 promoter is a specific promoter that mainly drives gene expression in neurons.

[0063] H9 cells are a commonly used human embryonic stem cell line with the potential to self-renew and differentiate into a variety of cell types. Transposase is an enzyme that can catalyze the insertion and excision of DNA fragments in the genome. After expressing transposase in H9 cells, the transposase will recognize the specific sequence on the transposon plasmid and insert it into the genome of H9 cells, thereby achieving stable integration and expression of the target gene.

[0064] If the neuronal activity of H9 cells after differentiation is greater than the reference expression value, it indicates that the H9 cells are successfully overexpressed (meaning that the target gene is effectively expressed and regulated in H9 cells). At this time, the H9 cells after successful overexpression are input into the brain organoid model. In the brain organoid model, the overexpressed H9 cells may cause abnormal neuronal activity and epileptic seizures. When the neuronal activity of H9 cells after neuronal differentiation is greater than the reference expression value of neuronal activity, it indicates that the neuronal activity to be tested has reached the conditions for epileptic seizures. At this time, the H9 cells after successful overexpression need to be input into the brain organoid model to obtain an epileptic brain organoid model. Otherwise, it indicates that the neuronal activity to be tested has not reached the conditions for epileptic seizures, and drug screening is required.

[0065] The steps for constructing a human embryonic stem cell line are as follows: (1) Plating: Thaw Matrigel overnight at 4°C. When it is completely thawed, it can be dispensed and stored at -20°C. Take 12mL of refrigerated DMEM / F12 in a 15mL centrifuge tube, prepare two 6-well plates, aspirate 1mL of DMEM / F12 in the centrifuge tube and gently blow until the Matrigel is thawed and mixed, aspirate the thawed and mixed Matrigel and add the remaining DMEM / F12 in the centrifuge tube, and use a 10mL pipette to repeatedly blow and mix again. Dispense 1mL / well into a 6-well plate, shake gently to mix, wrap with sealing film and place at 4°C overnight before use.

[0066] (2) Resuscitation of H9: After removing the cells from -80°C or liquid nitrogen, temporarily store them in dry ice. Then, gently shake them in a 37°C water bath until they are basically melted, transfer all the liquid to a 50mL centrifuge tube, gently shake and add 3mL of culture medium, centrifuge at 200g for 2 minutes, and discard the supernatant. Resuspend the cells in culture medium and transfer all of them to a culture dish treated with Matrigel. Add Y27632 (10mM) to the culture medium at a ratio of 1:1000, shake crosswise until the cells are evenly distributed, and culture in a 37°C constant temperature incubator.

[0067] (3) Cell passaging: Aspirate the culture medium, add an appropriate amount of 0.5 μM EDTA to cover the entire culture dish, incubate at 37°C for 4 min, and aspirate the EDTA. Use fresh culture medium to gently blow the cells off the dish wall, pass them as needed, and plate them into new culture dishes.

[0068] (4) Cell cryopreservation: After subculturing, the remaining cells can be cryopreserved as needed. 2 Gently pipette down the cells and freeze 200-300 μL per tube.

[0069] It should be understood that when evaluating the inhibitory effect on neuronal activity, when the luciferase content is positively correlated with the fluorescence signal intensity, it means that as the luciferase content increases, the fluorescence signal intensity also increases accordingly, indicating that the inhibitory effect on neuronal activity is weakened (the weakened inhibitory effect here is relative to the weakened inhibitory effect of the above-mentioned drug to be screened); when the luciferase content is negatively correlated with the fluorescence signal intensity, it means that as the luciferase content increases, the fluorescence signal intensity weakens instead, which usually means that the response ability of neurons to luciferase is reduced, that is, neuronal activity is inhibited. In this case, it can be clearly judged that the inhibitory effect on neuronal activity is enhanced, thereby achieving a more accurate analysis of the inhibitory effect on neuronal activity.

[0070] like Figure 7 , which is a schematic diagram of the verification results of the brain organoid neurotoxic compound screening platform provided in the examples of the present application. Through the verification of the experimental results, it was found that the cultured d2GFP organoids did not have green fluorescence in the early stages of culture (day 20, 40, 60, D20, D40, D60) without the addition of neurotrophic factors. However, after the addition of BDNF and GDNF neurotrophic factors at D60, strong green fluorescence expression could be observed in the organoids within 8 hours ( Figure 7 A). Immunofluorescence staining of d2GFP organoids at D70 revealed that GFP was significantly co-localized with the neuronal marker protein MAP2 and the synaptic marker protein PSD95 ( Figure 7 B). The dual-luciferase organoids were collected and cultured in D20, D40, and D60 culture media without adding neurotrophic factors, and in D70 culture media with BDNF and GDNF neurotrophic factors. The luciferase-catalyzed oxidation of their respective substrates to produce bioluminescence was detected using a microplate reader. The magnitude of the fluorescence value indicates the expression level of the two enzymes. The results showed that after adding BDNF and GDNF, the fluorescence value increased significantly, and the activity of neurons was significantly enhanced ( Figure 7 C). In addition, through WB analysis of organoids at different culture stages, it was observed that the expression trends of Gaussia protein and Cypridina protein were consistent with the results of the microplate reader test. In summary, genetically engineered brain organoids can quickly and sensitively display neuronal activity.

[0071] Among them, WB analysis can be performed through biological image analysis software (such as Image J software), that is, the grayscale value of the gene overexpression group in the biological image analysis software during the WB verification process. Through WB verification, the overexpression level of the target gene in the epilepsy brain organoid model in H9 cells can be accurately quantified. If the WB verification value is greater than the reference WB verification value, it indicates that the current experimental conditions (such as culture conditions) can meet the preset overexpression requirements, which helps to quickly screen out effective experimental conditions and save time and resources; if the WB verification results show that the gene overexpression does not meet the preset requirements, there is no need to further obtain the fluorescence intensity data of the neuronal area to be tested in the fluorescence intensity image. This strategy avoids unnecessary experimental steps and data collection, thereby reducing experimental costs and the complexity of data processing.

[0072] like Figure 8 As shown, it is a compound test diagram of the brain organoid compound toxicity screening platform provided in the embodiment of the present application. After the brain organoid neurons mature, BDNF and GDNF are added to activate the neurons, and then 10 μM of the test compound is added respectively. After the addition of the compound, the liquid is changed and the samples are collected at D0, D1, D3, and D5 respectively. According to the changes in green fluorescence and luciferase, it was found that 4-Aminopyridine and Gabapentin had no significant effect on neuronal activity, while Bupivacaine, Rufinamide, Rotigotine and Memantine Hydrochlotide caused a significant decrease in neuronal activity. In contrast, Aripiprazole Lauroxil increased neuronal activity within 72 hours, but decreased after 96 hours. By observing the green fluorescence graph, it was found that after the addition of the compound, the neurons showed obvious excitement, but the toxicity of the compound led to increased cell death, increased cell debris and decreased fluorescence intensity after 72 hours ( Figure 8 A, B). At the same time, due to individual differences between brain organoids, changes in luciferase allow us to observe changes in neuronal activity more accurately and with high throughput.

[0073] The above results show that the neuronal activity screening platform can be used to test the effects of different compounds on neurons for a long time without destroying brain organoids, by real-time fluorescence observation or testing the luciferase content secreted into the culture medium. In this experiment, no significant changes in neuronal excitability were observed after treatment with 10μM 4-Aminopyridine, so neurons may be insensitive to a dose of 10μM 4-Aminopyridine and show neuroprotective effects. This is consistent with past reports: 50μM and 2500μM 4-Aminopyridine have a neuroprotective effect on cerebellar granule neurons (CGNs) without obvious toxicity. It was observed that after treating brain organoids with 10μM Bupivacaine, the platform showed reduced fluorescence and reduced luciferase secretion.

[0074] In summary, the embodiment of the present application respectively concatenates the target gene MECP2 with the A promoter and the B promoter to obtain element A and element B, respectively, uses IL-PolyA-IR as a vector to perform transposon recombination on element A and element B to obtain the first plasmid and the second plasmid, respectively, then uses transposase to insert the first plasmid and the second plasmid into the chromosomes in cell line A and cell line B, respectively, and differentiates and cultures to obtain the first cerebral organoid and the second cerebral organoid, respectively, and finally, after the first cerebral organoid and the second cerebral organoid are mature, adds the drug to be tested to the first cerebral organoid and the second cerebral organoid, and detects the content and change of MECP2 protein to verify the effect of the drug to be tested on epilepsy, thereby achieving the improvement of the accuracy of neuronal active drug screening, and effectively solving the problem of low accuracy of neuronal active drug screening in the process of dynamic monitoring of epilepsy in the prior art.

[0075] Those skilled in the art will appreciate that embodiments of the present invention may be provided as methods, systems, or computer program products. Therefore, the present invention may take the form of a complete hardware embodiment, a complete software embodiment, or an embodiment combining software and hardware. Moreover, the present invention may take the form of a computer program product implemented on one or more computer-usable storage media (including but not limited to disk storage, CD-ROM, optical storage, etc.) containing computer-usable program code.

[0076] The present invention is described with reference to flowcharts and / or block diagrams of methods, devices (systems), and computer program products according to embodiments of the present invention. It should be understood that each process and / or block in the flowchart and / or block diagram, as well as the combination of processes and / or blocks in the flowchart and / or block diagram, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, a special-purpose computer, an embedded processor, or other programmable data processing device to produce a machine, so that the instructions executed by the processor of the computer or other programmable data processing device generate instructions for implementing the processes in the flowchart and / or block diagram. Figure 1 A process or multiple processes and / or boxes Figure 1 A device that provides the functions specified in a block or multiple blocks.

[0077] These computer program instructions may also be stored in a computer-readable memory capable of directing a computer or other programmable data processing device to operate in a specific manner, so that the instructions stored in the computer-readable memory produce an article of manufacture including an instruction device, which implements the process Figure 1 A process or multiple processes and / or boxes Figure 1 A function specified in one or more boxes.

[0078] These computer program instructions can also be loaded onto a computer or other programmable data processing device so that a series of operating steps are executed on the computer or other programmable device to produce a computer-implemented process, thereby providing instructions for implementing the process in the computer or other programmable device. Figure 1 A process or multiple processes and / or boxes Figure 1 The steps for the functions specified in one or more boxes.

[0079] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0080] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for dynamic monitoring of epilepsy based on brain organoids, characterized in that: The following steps are involved: S1, the target gene MECP2 is connected in series with the A promoter and the B promoter to obtain element A and element B respectively, and IL-PolyA-IR is used as a vector to perform transposon recombination on element A and element B to obtain the first plasmid and the second plasmid respectively; The A promoter and the B promoter are promoters of different strengths; S2, using transposase to insert the first plasmid and the second plasmid into the chromosomes of cell line A and cell line B, respectively, and differentiate and culture to obtain the first cerebral organoid and the second cerebral organoid, respectively; S3, after the first cerebral organoid and the second cerebral organoid are mature, adding the drug to be tested to the first cerebral organoid and the second cerebral organoid, and detecting the content and change of MECP2 protein respectively to verify the effect of the drug to be tested on epilepsy.

2. A method for dynamic monitoring of epilepsy based on brain organoids as claimed in claim 1, characterized in that: The cell line A and the cell line B are both cell lines that can verify the degree of toxicity of the drug to be tested on brain organoids.

3. A method for dynamic monitoring of epilepsy based on brain organoids as claimed in claim 2, characterized in that: The cell line A and / or cell line B are constructed as follows: Using gene editing technology to insert a first expression element into the genome of a cell line cell, wherein the first expression element is used to mark activated neurons; The first expression element is used to mark the activated neurons specifically: after the first expression element is expressed, it indicates that the neuron cells have matured and produced fluorescent substances.

4. A method for dynamic monitoring of epilepsy based on brain organoids as claimed in claim 2, characterized in that: The cell line A and / or cell line B are constructed as follows: Inserting a second expression element into the genome of a cell line cell using gene editing technology, wherein the second expression element is used to achieve measurable neuronal activity; The second expression element is used to achieve measurable neuronal activity in the following way: the second expression element is expressed to produce substance one only after the neuron matures, and the second expression element produces substance two during the entire cell growth stage. The activity of the neuron can be quantified by detecting the amount of substance one and substance two.

5. A method for dynamic monitoring of epilepsy based on brain organoids as claimed in claim 2, characterized in that: The cell line A and / or cell line B are constructed as follows: Stable human embryonic stem cell lines were constructed and divided into two groups, namely the first cell line and the second cell line; Using gene editing technology to insert a first expression element into the genome of cells of the first cell line, wherein the first expression element is used to mark activated neurons; The first expression element is used to mark the activated neurons specifically: after the first expression element is expressed, it indicates that the neuron cells have matured and produced fluorescent substances; Inserting a second expression element into the genome of a cell of a second cell line using gene editing technology, wherein the second expression element is used to achieve measurable neuronal activity; The second expression element is used to achieve measurable neuronal activity in the following way: the second expression element is expressed to produce substance one only after the neuron matures, and the second expression element produces substance two during the entire cell growth stage. The activity of the neuron can be quantified by detecting the amount of substance one and substance two.

6. A method for dynamic monitoring of epilepsy based on brain organoids as claimed in claim 3 or 5, characterized in that: The first expression element is specifically: an E-SARE enhancer element in series with an ArcMini promoter in series with a fluorescent substance element; The fluorescent substance element is specifically a fluorescent protein element, which produces a specific color after being expressed.

7. A method for dynamic monitoring of epilepsy based on brain organoids as claimed in claim 4 or 5, characterized in that: The second expression element comprises: a promoter associated with neuron activation, a sub-element 1 expressed after being activated by the promoter associated with neuron activation, a promoter stably and continuously expressed in cells, and a sub-element 2 expressed after being activated by the promoter stably and continuously expressed in cells; The sub-element 1 produces substance 1 after expression, and the sub-element 2 produces substance 2 after expression; The sub-element 1 is specifically: E-SARE enhancer element in series with ArcMini promoter in series with Gaussia luciferase; The second sub-element is specifically: CAG promoter tandem secretory luciferase Cypridina luciferase; The substance 1 is specifically: Gaussia luciferase produced by the expression of E-SARE enhancer element in series with ArcMini promoter in series with Gaussia luciferase; The second substance is specifically: Cypridina luciferase produced by expressing the CAG promoter in series with the secretory luciferase Cypridina luciferase.

8. A method for dynamic monitoring of epilepsy based on brain organoids as claimed in claim 1, characterized in that: The A promoter is a CAG promoter, and the B promoter is a synapsin-Ⅰ promoter.

9. A method for dynamic monitoring of epilepsy based on brain organoids as claimed in claim 1, characterized in that: The transposase is Sleeping Beauty.

10. A method for dynamic monitoring of epilepsy based on brain organoids as claimed in claim 7, characterized in that: By performing immunofluorescence staining on the first and second cerebral organoids respectively, the content and changes of MECP2 protein were determined by the color after staining.

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