Organic organelle for promoting closing of neural tubes and extraction method and application of organelle

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses an organelle for promoting closing of a neural tube and an extraction method and application of the organelle. The organelle for promoting the closing of the neural tube is named as a shaping body, is of a vesicular structure positioned in a polar region of a neural epithelial cell, has the diameter of about 500-2000nm, and contains RACK1, HNRNPA1 and other characteristic protein components. The invention comprises a method for identifying and extracting a plastic body, a method for promoting the generation of the plastic body by regulating and controlling the expression of RACK1 and HNRNPA1, and a therapeutic composition and a method which are developed on the basis of the method and are used for promoting the physiological closure of tissue structures such as neural tubes and the like. The invention provides application of the shaping body, the key components of the shaping body and the accelerant of the shaping body in preparation of drugs for treating the neural tube deformity, and a brand-new targeted therapy strategy is provided for related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an organelle that promotes neural tube closure, its extraction method, and its application. Background Technology

[0002] During embryonic development and organogenesis, stem cell populations construct complex three-dimensional functional structures through self-organization. This process is highly dynamic and diverse, relying on the precise spatiotemporal regulation of multiple developmental signaling pathways to coordinate biological behaviors such as cell proliferation, morphological changes, migration, and adhesion. However, whether a special subcellular structure exists in three-dimensional space capable of integrating multicellular collective movement to actively shape complex organ morphologies remains an unsolved scientific question in this field. Current research on tissue morphogenesis mechanisms is mostly limited to two-dimensional or simplified models; no cellular structures with clearly defined morphogenesis-promoting functions at the three-dimensional level have been reported.

[0003] The neural tube, a crucial structure formed early in the development of the central nervous system, has a tightly regulated closure process. Genetic factors or maternal exposure to adverse environments (such as infection, radiation, and malnutrition) can lead to incomplete neural tube closure, resulting in severe neural tube defects such as spina bifida and anencephaly. The incidence of this disease in newborns is approximately 0.1%, making it one of the deadliest congenital defects. About 70% of neural tube defects are related to genetic factors, and their closure requires the coordinated expression of numerous genes at specific times and spaces. Although hundreds of genes associated with neural tube defects have been identified, single or combined gene therapies remain limited in effectiveness for complex cases caused by multiple etiologies.

[0004] Currently, interventions for neural tube defects mainly fall into two categories: prevention and surgical repair, both of which have significant limitations. In terms of prevention, folic acid supplementation during pregnancy, as a primary prevention method, is effective in about 70% of cases, but about 30% are "folic acid resistant," and its effect is limited to the critical window of neural tube closure (within 28 days of conception), unable to reverse existing malformations. In terms of repair, while surgery (including intrauterine or postpartum surgery) can physically close defects and reduce risks such as hydrocephalus, it is essentially still a "physical repair," unable to restore damaged nerve function, and accompanied by high risks and significant trauma associated with maternal-fetal surgery. Furthermore, emerging regenerative medicine methods such as stem cell therapy, while theoretically possessing the potential to promote regeneration, face challenges in safety and efficacy, including tumorigenesis, immune rejection, low cell survival rates, and strict ethical regulations. While technologies such as tissue engineering and 3D bioprinting can construct three-dimensional structures, they still struggle to dynamically simulate and guide cells to autonomously complete precise morphogenesis processes.

[0005] In summary, how to directly and precisely regulate the three-dimensional morphogenesis ability of neuroepithelial cells to promote the autonomous and physiological closure of the neural tube remains a core challenge that current technologies have not yet overcome. Therefore, developing a targeted strategy that can directly empower neuroepithelial cells and reshape their three-dimensional morphogenesis ability to fill the fundamental gap in existing treatment modalities is of great significance. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides an organelle that promotes neural tube closure, its extraction method, and its application. This invention isolates and extracts plastids from tissues, organs, and cells, identifies the main molecules involved in plastid formation, and promotes plastid production by adenovirus transfection to enhance the expression of RACK1 and HNRNPA1 in tissue cells, thereby promoting neural tube morphogenesis. This is of great significance for the treatment of neural tube defects.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0008] This invention discloses an organelle that promotes neural tube closure, characterized in that it is a vesicle-like structure named "molding body" located in the polar region of the neuroepithelial cell, with a diameter of approximately 500-2000 nm, and containing RACK1 protein and HNRNPA1 protein.

[0009] Furthermore, the sculpting body facilitates the transformation of the neural plate from a planar to a three-dimensional shape, mediating the coordinated movement of neuroepithelial cells.

[0010] The present invention also discloses a method for preparing the above-mentioned shaped body, characterized by comprising the following steps: obtaining neural epithelial cells or embryonic neural tissue, lysing them, and then sequentially performing differential centrifugation and flow cytometry to collect the target components and obtain the shaped body.

[0011] Furthermore, the differential centrifugation conditions are as follows: centrifuge at 200g for 10 minutes, centrifuge at 3000g for 30 minutes, take the supernatant and continue to centrifuge at 17200g for 30 minutes, collect the precipitate and resuspend it in 1×PBS.

[0012] The present invention also discloses a method for promoting the formation of sculpted bodies, characterized by increasing the expression level or activity of RACK1 and HNRNPA1 proteins in target cells.

[0013] Furthermore, the method for increasing the expression level is to introduce nucleic acid molecules encoding RACK1 and HNRNPA1 proteins into the target cells.

[0014] The present invention discloses a pharmaceutical composition characterized in that it comprises effective amounts of RACK1 protein and HNRNPA1 protein, or their encoded nucleic acids, and a pharmaceutically acceptable carrier.

[0015] The present invention also discloses a pharmaceutical composition characterized by comprising the above-described molded body and a pharmaceutically acceptable carrier.

[0016] This invention discloses the use of the pharmaceutical composition described in any of the above claims in the preparation of a medicament for the prevention or treatment of neural tube defects.

[0017] Based on previous observations using multiple techniques including bulk electron microscopy, high-resolution light slide microscopy, long-term lattice light slide microscopy, live-cell panoramic super-resolution microscopy, and structured light illumination microscopy, we discovered and characterized a novel organelle in embryonic tissues and neuroepithelial cells that plays a crucial role in promoting neural plate bending and neural tube closure, which we named "molding body". This structure is a vesicle-like organelle formed by myosin transporting ribosomal translation components and glycolytic enzyme mRNA to the cell polar region, with a diameter of approximately 500-2000 nm. Its main components include ribosomal proteins, phase-separating proteins, glycolytic enzymes, chaperone proteins, and cytoskeletal proteins, and it possesses independent translation, metabolism, energy generation, and the ability to promote microfilament polymerization and contraction.

[0018] The organelle is distributed in the polar regions of cells and connected to the neuroepithelium via filamentous fibers. Its vesicles can fuse with each other and interweave with fibers to form an ordered network structure inside the neural tube lumen, continuously pulling on the cell tip and thus promoting the transformation of the planar cell layer into a three-dimensional tissue morphology. This organelle not only maintains the establishment of cell polarity in gene expression, energy metabolism, and cytoskeleton remodeling, but also achieves motor fusion through phase separation-mediated topological changes, thereby coordinating the aggregation and collective migration of multiple neuroepithelial cells and promoting the three-dimensional morphology of tissues and organs.

[0019] Further research revealed that the RACK1 and HNRNPA1 genes are core components of the morphogenetic body. Their high expression in cells significantly promotes the formation and function of the morphogenetic body, ultimately leading to tissue morphology repair. The formation process of the morphogenetic body integrates multiple events at the multicellular level, including translation, metabolism, and skeletal remodeling, forming a multi-stage "morphogenetic system" that can respond to changes in the microenvironment. This system possesses relatively independent energy supply and skeletal dynamic mechanisms, and its surface contains molecules that mediate spontaneous fusion, enabling it to dynamically sense and adapt to biochemical signals, regulate collective cell movement, and complete complex morphological construction.

[0020] More importantly, the malleable body is a common focal point for neural tube defects caused by a variety of genetic and environmental factors. Whether it is metabolic inhibition, translational arrest, or specific gene mutations (such as abnormalities in the retinoic acid pathway), the common pathological feature is the physical loss of the malleable body. Bioinformatics analysis shows that approximately 40% of known neural tube defect-related genes interact with malleable body proteins, further confirming that this organelle is a key bottleneck for normal neural tube closure. Therefore, restoring the integrity of the malleable body's structure and function is considered a broad and effective treatment approach for neural tube defects.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0022] In the complex processes of tissue and organ formation, morphosomes can precisely direct coordinated morphogenesis-related movements from cells in different regions through mutual fusion, thereby achieving the generation of complex morphologies. The formation of morphosomes integrates multicellular translation, metabolism, and the establishment of polarity in cytoskeleton remodeling, forming a multi-stage responsive morphogenesis system. Furthermore, morphosomes are structures generated by the cells themselves, possessing relatively independent energy supplies and skeletal dynamic systems. Simultaneously, the surface of morphosomes contains molecules capable of supporting spontaneous fusion. Therefore, morphosome-mediated multicellular collective movement can dynamically sense adaptive biochemical changes and regulate its own functions, representing a dynamic integration of tissue cells. Thus, in cases of structural birth defects and tissue / organ morphological damage, promoting the generation of morphosomes by morphogenesis-generating cells can achieve the effect of repairing tissue morphology.

[0023] This invention is the first to discover and target the "mold body," a core organelle that regulates the three-dimensional morphogenesis of the neural tube, achieving a fundamental shift from external support to internal program restart. The mechanism is innovative and precisely targeted. By supplementing key components of the mold body, this invention directly enhances the energy metabolism, protein translation, and collective movement capabilities of neuroepithelial cells, thereby driving the neural tube to achieve physiological and autonomous closure, rather than passive physical suturing. Crucially, this mechanism of action is independent of the folate metabolism pathway, providing a novel and effective treatment for "folate-resistant" neural tube defects, overcoming the bottleneck of existing prevention strategies. Regarding safety, the mold body, as a subcellular component, lacks the self-replication ability of intact cells, fundamentally avoiding the tumorigenic risks associated with stem cell therapy, and exhibiting low immunogenicity. Furthermore, the novel principle revealed in this invention, which guides tissue morphogenesis by regulating specific organelles, provides innovative therapeutic targets and approaches for the repair of neural tube defects and other tissue and organ morphological defects, demonstrating broad application prospects. Attached Figure Description

[0024] Figure 1 Scanning electron microscopy results of the molten body inside the neural tube of mouse embryos (left) and human embryos (right).

[0025] Figure 2 Extraction process of shaped objects.

[0026] Figure 3 The main components of a sculpted body.

[0027] Figure 4 In neural stem cells, overexpression of RACK1 and HNRNPA1 promotes the formation of body morphology.

[0028] Figure 5 Confocal imaging results of movement and fusion of the morphosome on the inner surface of the neural tube of E8.5 mouse embryos stained with F-actin.

[0029] Figure 6 Label-free tissue imaging results of the inner surface molding of the neural tube in E8.5 mouse embryos promoting neuroepithelial movement.

[0030] Figure 7 Intraamniotic injection of adenovirus RACK1 and HNRNPA1 into mouse embryos promotes the formation of morphosomes and the folding and bending of neural plates.

[0031] Figure 8 Overexpression of RACK1 and HNRNPA1 promotes plastid formation and neural tube closure. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0033] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0034] Example 1: Observation, extraction and identification of shaped objects.

[0035] I. Experimental Materials.

[0036] This invention selects neural tube tissues from mouse embryos (E9.5) and human embryos (CS12), as well as related chemical reagents for scanning electron microscopy sample preparation, single-cell suspension preparation, and flow cytometry sorting.

[0037] II. Experimental Methods.

[0038] 1. Observation of the plastinated embryos: Pregnant mice were euthanized by intraperitoneal injection of ketamine / hyoscyamine (200 mg / kg ketamine, 20 mg / kg hyoscyamine). Embryos were removed from the uterus of the pregnant mice and fixed in a mixture of paraformaldehyde (2%) and glutaraldehyde (2.5%). Human embryo samples were obtained from clinically discarded biological samples from voluntary abortions of healthy pregnant women. Immediately after separation from the abortion tissue, the samples were fixed in 2% paraformaldehyde (PFA) and 2% glutaraldehyde. Tissue samples were washed four times with phosphate buffer (0.1 M) and then incubated for 1 hour at room temperature with a solution of 1% osmium tetroxide and 1.5% tetrapotassium hexacyanoferrate(III). Tissue samples were washed four times with phosphate-buffered saline (0.1 M), then dehydrated for 15 minutes in a gradient of ethanol solutions (30%, 50%, 70%, 80%, 90%, 100%, 100%, 100%), and finally placed in a critical point desiccator. A 10 nm layer of platinum was sputtered onto the surface of the tissue samples. The inner surface of the neural tube of the tissue samples was scanned using a Zeiss Crossbeam 550 microscope.

[0039] 2. Extraction and Identification of Porphyrosomes: To isolate porphyrosomes, E8.5 embryonic tissue was minced and digested with collagenase (3 mg / mL) and DNase I (0.25 mg / mL) at 37°C for 30 minutes. The digested single-cell neural stem cells were incubated in 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG, 100 µM) for 30 minutes. The porphyrosome pellet was collected by differential centrifugation: 200 g, 10 min (cell removal), 3,000 g, 30 min (debris / large organelle removal), and 17,200 g, 30 min. The pellet was resuspended in 1×PBS. The pellet was analyzed using a flow cytometer (BDFACSymphony). TM The FITC channel of the S6SE was used for sorting, with gating based on an unlabeled control. The sorted components were the sculpted body components. The extracted sculpted body components were immobilized in a mixture of paraformaldehyde (2%) and glutaraldehyde (2.5%), and then incubated at room temperature for 1 hour with a solution of 1% osmium tetroxide and 1.5% tetrapotassium hexacyanoferrate(III). Tissue samples were washed four times with phosphate buffer (0.1M), followed by dehydration for 15 minutes in a gradient of ethanol solutions (30%, 50%, 70%, 80%, 90%, 100%, 100%, 100%), and then progressively impregnated in acetonitrile with EMbed-812 epoxy resin (or an equivalent low-viscosity resin) at concentrations of 25%, 50%, 75%, and 100%, each step lasting 1 hour. The samples were embedded in fresh resin and polymerized at 60°C for 48–72 hours. Ultrathin sections were used for transmission electron microscopy.

[0040] 3. Proteomic analysis of the morphosomes: E8.5 embryonic tissue was minced and digested with collagenase (3 mg / mL) and DNase I (0.25 mg / mL) at 37°C for 30 min. The digested single-cell neural stem cells were incubated in 2-(N-7-nitro-2,1,3-benzoxadiazole-4-amino)-2-deoxy-D-glucose (2-NBDG, 100 μM) for 30 min. Differential centrifugation was performed: 200 g, 10 min (cell removal), 3,000 g, 30 min (debris / large organelle removal), and 17,200 g, 30 min to collect the morphosome pellet. The pellet was resuspended in 1×PBS. Sorting was performed using the FITC channel of a BDFACSymphonyTMS6SE flow cytometer, with gating based on an unlabeled control. The sorted components were the body shaping components. The extracted body shaping component vesicles were lysed and proteins extracted using SDT (4% SDS, 100mM Tris-HCl, pH 7.6) buffer. Protein content was quantified using a BCA protein assay kit (Bio-Rad, USA). 20µg of protein from each sample was mixed with 5× loading buffer and boiled for 5 minutes. Proteins were separated on 4%–20% SDS-PAGE gels (180V, 45 minutes). Protein bands were visualized by Coomassie Brilliant Blue R-250 staining. Proteins were digested with trypsin according to the Filter-Assisted Sample Preparation (FASP) procedure described by Matthias Mann. Digested peptides from each sample were cultured on a C18 column (Empore). TM Desalting was performed on an SPE C18 column (standard density, bed inner diameter 7 mm, volume 3 mL, Sigma), followed by vacuum centrifugation and reconstitution in 40 µL of 0.1% (v / v) formic acid. Sample preparation for filtration aid (FASP digestion) procedure: DTT wash (final concentration 10 mM) was added to each sample, and the mixture was stirred at 600 rpm for 1.5 h (37 °C). After cooling to room temperature, IAA (final concentration 20 mM) was added to the mixture to block reduced cysteine ​​residues, and the mixture was incubated in the dark for 30 min. Next, the samples were transferred to filter membranes. The membranes were washed three times with 100 µL UA buffer, and then twice with 100 µL 25 mM NH4HCO3 buffer. Finally, trypsin (trypsin:protein (weight ratio) 1:50) was added to the samples, and the mixture was incubated at 37 °C for 15–18 h (overnight), collecting the resulting peptides as the filtrate. The peptides from each sample were then processed on a C18 column (Empore). TMDesalting was performed on an SPE C18 column (standard density, 7 mm inner diameter, 3 mL volume, Sigma), followed by concentration by vacuum centrifugation and dissolution in 40 µL of 0.1% (v / v) formic acid. The extinction coefficient of the 0.1% (g / L) solution was calculated to be 1.1 based on the frequencies of tryptophan and tyrosine in vertebrate proteins. The peptide content was estimated using the UV spectral density at 280 nm corresponding to this coefficient. LC-MS / MS analysis was performed using a QExactive mass spectrometer (ThermoScientific). Raw MS data from each sample were merged and searched using MaxQuant 1.6.14 software for protein identification and quantification.

[0041] III. Experimental Results.

[0042] The study observed malleable body-related structures in neural tube tissues of both mice and human embryos. Figure 1 ); and established a process for extracting shaped bodies ( ); Figure 2 The main components of the sculpted body were identified through proteomics analysis. Figure 3 ).

[0043] Example 2: Functional verification of the shaped body.

[0044] I. Experimental Materials.

[0045] This invention uses mouse neural stem cell C17.2 cells, adenovirus overexpressing mCherry-RACK1 and GFP-HNRNPA1 (Hanheng Biotechnology (Shanghai) Co., Ltd.), and related reagents and equipment for flow cytometry detection, amniocentesis, and in vitro embryo culture.

[0046] II. Experimental Methods.

[0047] 1. Plastosome Analysis: Mouse neural stem cells (C17.2) overexpressing mCherry-RACK1, GFP-HNRNPA1, and GFP-HNRNPA1-IDRDEL adenovirus were added, respectively. After passage for 48 hours, the mouse neural stem cells were cultured in 2-glycosyl-N-ethyl glucose for 30 minutes. Cells were treated with trypsin, resuspended, and centrifuged differentially: 200g, 10 min (cell removal), 3,000g, 30 min (debris / large organelle removal), and 17,200g, 30 min to separate the precipitate of blastosomes. The precipitate was resuspended in 1×PBS. Plastosome content was analyzed by flow cytometry using BD FACSAria III.

[0048] 2. Embryo In Vitro Culture and In Vivo Imaging: Early E8.5 embryos were retrieved after euthanasia in pregnant mice. Intact embryonic tissues were dissected and isolated in isolation medium (DMEM containing 10% FBS, 1 mg / mL glucose, pyruvate, and free of phenol red / glutamine). The embryonic culture medium consisted of 50% DMEM (containing 1 mg / mL D-glucose and pyruvate, free of phenol red and L-glutamine) with 1×Glutamax, 100 units / mL penicillin, 100 µg / mL streptomycin, 11 mM HEPES, and 50% rat serum. CellMask cytoskeleton dye (1:1000) was added to the embryonic culture medium and incubated for 30 minutes. Z-axis and time-series data of the tissues were acquired using a Zeiss confocal microscope and label-free live-cell imaging.

[0049] 3. Amniocentesis of Embryos: Early E8.5 embryos of the retinoic acid-induced mouse neural tube defect model were retrieved after euthanasia in pregnant mice. Embryos were dissected in isolation medium (DMEM containing 10% FBS, 1 mg / mL glucose, pyruvate, and phenol red / glutamine-free, pre-isolated at 37°C). The entire dissection process was performed on a heated platform for no more than 30 minutes to maintain viability. Embryos in the neural plate stage / early head fold stage with intact placental cones were selected, and adenovirus was injected into the amniocentesis cavity using a microinjection device. After 48 hours of in vitro culture, images were acquired using a stereomicroscope and light slide microscope.

[0050] 4. In vitro culture of embryos: Embryos were cultured under different gas phase conditions to match the metabolic needs of different developmental stages. The embryo culture medium consisted of 50% DMEM (containing 1 mg / mL D-glucose and pyruvate, phenol red-free and L-glutamine-free) plus 1×Glutamax, 100 units / mL penicillin, 100 µg / mL streptomycin, 11 mM HEPES, and 50% rat serum. The medium was aliquoted at 4°C and used within 2 months. The culture flasks were placed in a rotary culture system at 37°C, rotated at 30 rpm, and continuously purged with 20% O2 and 5% CO2 at 6.5 psi. 0–24 hours: 5–6 embryos are cultured in 2 mL of medium (50% DMEM, 50% rat serum, 1 mg / mL glucose) at 37°C with an air / CO2 ratio of 20% O2 / 5% CO2. 24–48 hours: Transfer to fresh medium with an additional 3 mg / mL D-glucose (total approximately 4 mg / mL), under an air / CO2 ratio of 30% O2 / 5% CO2. After 48 hours: Transfer to medium containing an additional 3.5 mg / mL glucose, under an air / CO2 ratio of 40% O2 / 5% CO2. Rotate the culture flask at 30 rpm. All media should be pre-equilibrated for at least 1 hour before use.

[0051] Immunofluorescence detection of virus-transfected tissues: After fixation in 4% PFA for 24 hours, tissues were embedded in paraffin. Dehydration and embedding were performed using a sequence of water, 70% ethanol, 80% ethanol, 95% ethanol, 95% ethanol, 100% ethanol, 100% ethanol, xylene, xylene-paraffin (1:1), paraffin (10 min each time). 4µm paraffin sections were prepared using a tissue sectioner, spread and mounted in a 50°C water bath, and dried in a 60°C oven for 2 hours. Dewaxing to water was performed using a sequence of xylene 15 min twice, 100% ethanol 5 min twice, 95% ethanol 1 min, 80% ethanol 1 min, 70% ethanol 1 min, and running water 3 min. Antigen retrieval was performed by heating in 0.01M sodium citrate buffer (pH 6) at 100°C for 10 minutes. The sections were removed from the heat source and allowed to stand in buffer at room temperature for 20 minutes. Samples were washed twice with ice-cold PBS. Tissue sections and cells were blocked with 5% serum or BSA at room temperature for 2 hours. Sections were incubated overnight at 4°C with diluted primary antibody, followed by incubation with secondary antibody at room temperature for 2 hours. Finally, they were stained with DAPI and rinsed with TBS. The slides were then mounted and images were acquired under a confocal microscope.

[0052] III. Experimental Results.

[0053] Overexpression of RACK1 and HNRNPA1 in neural stem cells significantly improved body regeneration. Figure 4 ); the movement of the apical neuroepithelium mediated by the plastic body ( Figure 5 and Figure 6 In a mouse embryonic model of neural tube defects, the formation of the plastic body and the normal establishment of neural tube curvature were reconstructed. Figure 7 ) and promotes the normal closure of the neural tube ( Figure 8 ).

[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An organelle that promotes neural tube closure, characterized in that, It is a vesicle-like structure named the plastic body, located in the polar region of neuroepithelial cells, with a diameter of approximately 500-2000 nm, and contains RACK1 and HNRNPA1 proteins.

2. The organelle according to claim 1, characterized in that, The sculpting body facilitates the transformation of the neural plate from a two-dimensional to a three-dimensional shape, mediating the coordinated movement of neuroepithelial cells.

3. A method for preparing the shaped body according to claim 1 or 2, characterized in that, Includes the following steps: Neural epithelial cells or embryonic neural tissue are obtained, lysed, and then subjected to differential centrifugation and flow cytometry to collect the target components, thereby obtaining the shaped body.

4. The method according to claim 3, characterized in that, The differential centrifugation conditions were as follows: centrifuge at 200 g for 10 minutes, centrifuge at 3000 g for 30 minutes, take the supernatant and continue centrifuging at 17200 g for 30 minutes, collect the precipitate and resuspend it in 1×PBS.

5. A method for promoting the formation of shaped bodies, characterized in that, This is achieved by increasing the expression level or activity of RACK1 and HNRNPA1 proteins in target cells.

6. The method according to claim 5, characterized in that, The target cells are neuroepithelial cells or neural stem cells.

7. A pharmaceutical composition, characterized in that, It contains effective amounts of RACK1 protein and HNRNPA1 protein, or their encoded nucleic acids, and a pharmaceutically acceptable carrier.

8. A pharmaceutical composition, characterized in that, It comprises the shaped body as described in claim 1 or 2, and a pharmaceutically acceptable carrier.

9. Use of the pharmaceutical composition of claim 7 or 8 in the preparation of a medicament for the prevention or treatment of neural tube defects.

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