A pair of alpha-helical peptides that recognize and bind to each other and uses thereof

Abstract

The present application relates to the field of biotechnology, in particular to a pair of mutually recognizing and combining alpha helix peptides and application thereof. The alpha helix peptide 1 has an amino acid sequence as shown in SEQ ID No. 1, and the alpha helix peptide 2 has an amino acid sequence as shown in SEQ ID No. 2. The cells are combined with each other by a pair of mutually recognizing and combining alpha helix peptides, so that the cells are arranged in a desired manner. The suitable material concentration and action time are verified; the formed cell group morphology is observed by various microscopes; two kinds of models are successfully constructed; and the improvement of cell function by adding DPH material is verified.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a pair of mutually recognizing and binding α-helical peptides and their applications. Background Technology

[0002] Current methods for artificially regulating cell interactions include utilizing endogenous cell adhesion molecules (CAMs), using synthetic DNA molecules acting on cell membrane surfaces, protein-based antigen-antibody binding systems, and synthesizing matrix gels using nanomaterials. However, these methods all have limitations: native CAMs, such as cadherins, are limited by cross-reactivity and dual signaling; DNA materials are unstable outside the cell and require chemical modification, which is incompatible with genetic regulation; protein-based systems, including nanobody antigen pairs and synthetic receptors such as SynNotch, provide genetic encoding but face challenges in terms of scalability and orthogonality; and nanomaterial-synthesized matrix gels cannot function effectively for all cells. Helical peptides are stable, modular protein interaction domains with predictable pairing rules, and recent advances suggest they hold promise for mediating specific interactions between synthetic surfaces. However, their adaptability to dynamically control cell-cell and cell-surface interactions across different cell types, particularly in human systems, remains largely unexplored.

[0003] To overcome these limitations, this invention modified and synthesized a pair of alpha-helical peptides capable of recognizing and interacting with each other, with one end attached to a DMPE that can insert into the cell membrane. This ensures that the peptides are not limited by cell type and can stably position themselves on the cell membrane to form a stable coiled-coil structure. Using this pair of materials, this invention can obtain cell clusters, enabling cells that would otherwise not interact to interact, thus achieving the goal of artificially regulating cell interactions. The impact of these interacting cell clusters on the cells was also investigated. Furthermore, this invention constructed two cell interaction models: a "spherical model" and a "cell layer-by-layer assembly model," demonstrating the controllability of the materials in regulating cell interactions. Summary of the Invention

[0004] To address the aforementioned issues, this invention provides a pair of mutually recognizing and binding α-helical peptides and their applications. DPH1 / 2 material is a plug-and-play material; DMPE is inserted into the cell membrane, and the cells bind together through a pair of mutually recognizing and binding α-helical peptides, thereby arranging the cells in a desired manner. The appropriate material concentration and treatment time were verified; the morphology of the formed cell clusters was observed using various microscopes; two models were successfully constructed; the improvement of cell function by the addition of DPH material was verified; and transcriptome sequencing analysis was used to further analyze the mechanism of action of DPH material: the action of DPH material forms a coiled-coil structure, bringing cells closer together and enabling more cells to bind. This contact between cells leads to changes in the expression of proteins on the cell surface, and changes in the expression of genes related to these proteins, further enhancing cell-cell interactions.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] The present invention provides a pair of mutually recognizing and binding α-helical peptides, including α-helical peptide 1 and α-helical peptide 2, wherein the amino acid sequence of α-helical peptide 1 is shown in SEQ ID No. 1 and the amino acid sequence of α-helical peptide 2 is shown in SEQ ID No. 2.

[0007] This invention also provides the application of the α-helical peptide described in the above technical solution in the preparation of cell cluster products.

[0008] The present invention also provides a pair of polypeptides for preparing cell clusters, including DPH1 and DPH2, wherein DPH1 is α-helical peptide 1 in the above-mentioned technical solution with PEG and DMPE linked to it;

[0009] The DPH2 is the α-helical peptide 2 in the above-mentioned technical solution, to which PEG and DMPE are attached.

[0010] This invention also provides the application of the polypeptide described in the above technical solution in the preparation of cell cluster products.

[0011] The present invention also provides a method for constructing a spherical model cell cluster, comprising the following steps:

[0012] 1) Mix DPH1 in the polypeptide described in the above technical solution with cells to obtain DPH1-cells;

[0013] 2) Mix the DPH2 in the polypeptide described in the above technical solution with cells to obtain DPH2-cells;

[0014] 3) Mix the DPH1- cells obtained in step 1) with DPH2- cells and culture them to obtain a spherical model cell cluster.

[0015] Preferably, in step 1), DPH1 is mixed in the form of a DPH1 solution with a concentration of 300 μM, and the cells are mixed in the form of a cell suspension with a cell count of 5 × 10⁻⁶ cells. 5 The volume ratio of the DPH1 solution to the cell suspension is 42:458.

[0016] The mixing time is 10 minutes.

[0017] Preferably, in step 1), the DPH2 is mixed in the form of a DPH2 solution with a concentration of 300 μM, and the cells are mixed in the form of a cell suspension with a cell count of 5 × 10⁻⁶ cells. 5 The volume ratio of the DPH2 solution to the cell suspension is 42:458.

[0018] The mixing time is 10 minutes.

[0019] This invention also provides a method for constructing a cell cluster model of layer-by-layer cell assembly, comprising the following steps:

[0020] 1) After adhering to the culture vessel, the cells are mixed with DPH2 from the polypeptide described in the above technical solution and incubated.

[0021] 2) Then mix with DPH1 in the polypeptide described in the above technical solution and incubate for 10 min, then mix with cells to obtain cell clusters of the cell layer-by-layer assembly model.

[0022] Preferably, in step 1), the final concentration of DPH2 is 25 μM, and the number of cells is 5 × 10⁻⁶. 5 The incubation time is 10 minutes.

[0023] Preferably, in step 1), the final concentration of DPH1 is 25 μM, and the number of cells is 5 × 10⁻⁶. 5 The mixture was prepared in 1 hour and mixed with the cells.

[0024] The beneficial effects of this invention are:

[0025] In this invention, a method for rapidly preparing cell clusters is reported using a pair of mutually recognizing and binding α-helical peptides. Specifically, the following steps are taken: Polypeptide molecules SEQ ID No. 1: GEIAALEQENAALEQKIAALKWKNAALKQGGC and SEQ ID No. 2: CGGKIAALKQKNAAL-KYEIAALEQENAALEQG are designed and synthesized, and then linked to DMPE (which can insert into the cell membrane) via PEG to form DPH1 and DPH2. After incubating two groups of cells with 25 μM DPH1 and DPH2 for 10 minutes respectively, the two groups of cells are mixed. The α-helical peptides can recognize and bind to each other, thereby achieving the goal of binding the two groups of cells together to form cell clusters. Experimental results show that the addition of DPH1 and DPH2 can induce the formation of cell clusters from human acute lymphoblastic leukemia (CEM) cells and human pluripotent stem cell-derived pancreatic β cells. Further investigation revealed that the addition of DPH1 and DPH2 enables the assembled cell clusters to achieve similar functions to those induced cell clusters. Multiple cellular pathways and cell surface adhesion-related genes were significantly upregulated in cell clusters, such as the Hippo pathway, TGF-β pathway, and integrin genes.

[0026] This invention modifies existing helical peptide sequences and applies them to artificially regulate cell-cell interactions, constructing a cell model. The advantages of this invention are its plug-and-play nature, ease of use, lack of restrictions on cell types, and ability to artificially regulate cell-cell interactions.

[0027] The method disclosed in this invention has the following key points:

[0028] 1) A simple method for constructing cell clumps is provided, namely by using a pair of α-helical peptides that can insert into the cell membrane.

[0029] 2) Two cell assembly models were constructed: the cell sphere model and the cell layer-by-layer assembly model. These two models can be used as tools to study the interactions between cells. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0031] Figure 1 The structures of DPH1 and DPH2 are shown, where Ac stands for acetyl group.

[0032] Figure 2 LC-MS spectrum of DPH1 and 1 H-NMR spectrum;

[0033] Figure 3 LC-MS spectrum of DPH2 and 1 H-NMR spectrum;

[0034] Figure 4 Circular dichroism chromatogram results for DPH1, DPH2, and combinations of DPH1 and DPH2;

[0035] Figure 5 To illustrate the localization of DPH1 / 2 on the cell membrane, A: Localization of 25 μM DPH1 on the cell membrane at different time points; B: Confocal fluorescence statistics; C: Material distribution after long-term interaction of DPH1 with cells; D: Flow cytometry results of different concentrations of DPH1 interacting with cells for 10 minutes; E: Confocal fluorescence results of different concentrations of DPH1 interacting with cells for 10 minutes; F: Confocal fluorescence results showing the localization of different materials on the cell membrane; G: Flow cytometry results showing the localization of different materials on the cell membrane.

[0036] Figure 6 This study presents the morphology and statistical data of cell clusters formed under various microscopes in the presence and absence of DPH1 / 2 material. A: Cell aggregation of CEM cells and sp6 cells under a light microscope with and without DPH1 / 2 material; B: Cell aggregation of CEM cells and sp6 cells under a bioelectron microscope with and without DPH1 / 2 material; C: Cell aggregation of CEM cells and sp6 cells under a fluorescence confocal microscope with and without DPH1 / 2 material; D: Statistical analysis of CEM cell and sp6 cell cluster rates; E: Statistical analysis of CEM cell and sp6 cell cluster area.

[0037] Figure 7 To construct cell spheroid models and cell layer-by-layer assembly models using DPH1 / 2, A: Characterization of mCherry-IPS and GFP-IPS cells used in model construction; B: Cell spheroid model formation process; C: Cell spheroid model; D: Cell spheroid model control group; E: Cell layer-by-layer assembly model formation process; F: Cell layer-by-layer assembly model; G: Cell layer-by-layer assembly model control group;

[0038] Figure 8To investigate the effects of DPH1 / 2-mediated cell clusters on the differentiation of stem cells into pancreatic islet cells, the following data were analyzed: A: Effects of DPH1 / 2 on insulin secretion and glucagon expression in sp6 cells, as captured by fluorescence confocal microscopy, compared with the expression in stem cell-induced cell clusters; B: Flow cytometry results reflecting the effects of DPH1 / 2 on insulin secretion and glucagon expression in sp6 cells; C: Statistical analysis of flow cytometry results; D: Effects of DPH1 / 2 on insulin and glucagon gene expression in sp6 cells; E: Effects of DPH1 / 2 on key gene expression during sp6 cell induction into SC-β cells; F: Glucose-induced insulin secretion.

[0039] Figure 9 To illustrate the alterations in gene expression caused by DPH1 / 2-mediated cell-cell interactions, the following plots are presented: A: GO enrichment bar chart of CEM cell transcriptome sequencing; B: Volcano plot of differentially expressed genes from CEM cell transcriptome sequencing; C: KEGG enrichment scatter plot of differentially expressed genes from CEM cell transcriptome sequencing; D: RT-qPCR validation of differentially expressed genes in CEM cells by screening differentially expressed genes from transcriptome sequencing results; E: RT-qPCR results showing differences in cadherin gene expression on the surface of sp6 cells with and without DPH1 / 2; F: Immunofluorescence confocal microscopy results showing differences in cadherin gene expression on the surface of sp6 cells with and without DPH1 / 2; G: GO enrichment bar chart of sp6 cell transcriptome sequencing; H: KEGG enrichment scatter plot of differentially expressed genes from sp6 cell transcriptome sequencing; I: Volcano plot of differentially expressed genes from sp6 cell transcriptome sequencing; J: RT-qPCR validation of differentially expressed genes in sp6 cells by screening differentially expressed genes from transcriptome sequencing results. Detailed Implementation

[0040] The present invention provides a pair of mutually recognizing and binding α-helical peptides, including α-helical peptide 1 and α-helical peptide 2, wherein the amino acid sequence of α-helical peptide 1 is shown in SEQ ID No. 1 and the amino acid sequence of α-helical peptide 2 is shown in SEQ ID No. 2.

[0041] SEQ ID No.1: GEIAALEQENAALEQKIAALKWKNAALKQGGC;

[0042] SEQ ID No. 2: CGGKIAALKQKNAAL-KYEIAALEQENAALEQG.

[0043] This invention also provides the application of the α-helical peptide described in the above technical solution in the preparation of cell cluster products.

[0044] This invention also provides a pair of polypeptides for preparing cell clusters, including DPH1 and DPH2, wherein DPH1 is α-helical peptide 1 of the α-helical peptide described in the above-mentioned technical solution, with PEG and DMPE linked to it; and DPH2 is α-helical peptide 2 of the α-helical peptide described in the above-mentioned technical solution, with PEG and DMPE linked to it. This invention does not specifically limit the method for linking PEG and DMPE to α-helical peptide 1 or α-helical peptide 2; conventional methods can be used by those skilled in the art. PEG: polyethylene glycol; DMPE: 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine.

[0045] This invention also provides the application of the polypeptide described in the above technical solution in the preparation of cell cluster products.

[0046] The present invention also provides a method for constructing a spherical model cell cluster, comprising the following steps:

[0047] 1) Mix DPH1 in the polypeptide described in the above technical solution with cells to obtain DPH1-cells;

[0048] 2) Mix the DPH2 in the polypeptide described in the above technical solution with cells to obtain DPH2-cells;

[0049] 3) Mix the DPH1- cells obtained in step 1) with DPH2- cells and culture them to obtain a spherical model cell cluster.

[0050] This invention involves mixing DPH1 from the polypeptide described in the above-mentioned technical solution with cells to obtain DPH1-cells. In this invention, the DPH1 is preferably mixed in the form of a DPH1 solution (300 μM), and the cells are mixed in the form of a cell suspension, wherein the cell suspension contains 5 × 10-1 cells. 5 The volume ratio of the DPH1 solution to the cell suspension is 42:458; the mixing time is preferably 10 min. In this invention, the DPH2 is preferably mixed in the form of a DPH2 solution with a concentration of 300 μM, and the cells are mixed in the form of a cell suspension with a cell number of 5 × 10⁻⁶ cells. 5 The volume ratio of the DPH2 solution to the cell suspension is 42:458; the mixing time is preferably 10 min.

[0051] This invention provides a method for constructing a cell cluster model of layer-by-layer cell assembly, comprising the following steps:

[0052] 1) After adhering to the culture vessel, the cells are mixed with DPH2 from the polypeptide described in the above technical solution and incubated.

[0053] 2) Then mix with DPH1 in the polypeptide described in the above technical solution and incubate for 10 min, then mix with cells to obtain cell clusters of the cell layer-by-layer assembly model.

[0054] In this invention, adherent cells are cultured and then mixed with and incubated with DPH2 from the polypeptide described in the above-mentioned technical solution. In this invention, the final concentration of DPH2 is 25 μM, and the number of cells is 5 × 10⁻⁶. 5 The incubation time is preferably 10 minutes.

[0055] The present invention further involves mixing and incubating the DPH1 from the polypeptide described in the above-mentioned technical solution with cells to obtain a cell cluster of a layer-by-layer assembly model. In this invention, the final concentration of DPH1 is 25 μM, and the number of cells is 5 × 10⁻⁶. 5 The preferred mixing time with the cells is 1 hour.

[0056] The present invention does not specifically limit the type of cells.

[0057] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0058] Example 1

[0059] Taking DPH1 as an example, the designed molecular synthetic route is as follows:

[0060] The specific steps are as follows:

[0061] I. Basic Reaction Process

[0062] 1. Resin activation: Take 0.91g of 2-Cl resin into a clean and dry reaction tube, add 20ml of DMF, and activate for about 30min.

[0063] 2. Amino acid linking: Weigh the calculated amount of the first C-terminal amino acid Fmoc-Cys(Trt)-NH2 (protected) and 0.5 ml of DIEA into a reaction tube, and add excess DMF as a solvent. Add an insoluble catalyst according to the type of amino acid.

[0064] 3. Eluting Fmoc protection:

[0065] Step 1: Remove Fmoc

[0066] 20% piperidine

[0067] DMF 5min, once every 15min, once

[0068] Step 2: Wash with DMF x 2, MeOH x 2, DMF x 2 for 1 min / time.

[0069] Step 3: Couple AA / HBTU / NMM for 30 minutes

[0070] Step 4: Wash with DMF x 2, MeOH x 2, DMF x 2 for 1 min / time.

[0071] 4. Detection: In solid-phase peptide synthesis, the linkage efficiency is mainly determined by detecting free amino groups on the resin. The detection method is called the Kaiser method. The detection results show blue or reddish-brown (Pro, Ser, His) when there are free amino groups.

[0072] Kaiser's reagent includes: A, a 6% ninhydrin ethanol solution.

[0073] B, 80% phenol ethanol solution

[0074] C, 2% 0.001M KCN ​​pyridine solution

[0075] Take a small amount of the reacted resin, add 2-3 drops each of A, B, and C, and heat at 105-110℃ for 5 minutes. If the solution turns blue, or the resin turns blue or reddish-brown, it indicates that there are still free amino groups; otherwise, it indicates that the linkage is complete.

[0076] 5. After successful detection, continue with the ligation of the second amino acid at the C-terminus. The method is the same as above, starting from step 3.

[0077] 6. Cutting: Cut with trifluoroacetic acid cutting solution for 3 hours, filter the reaction solution to obtain a trifluoroacetic acid solution of the polypeptide.

[0078] 7. Precipitation: Precipitate with excess diethyl ether and centrifuge. Elute the centrifuged sample with diethyl ether multiple times and centrifuge again. Obtain the initial peptide sample.

[0079] 8. After initial dissolution, add a certain concentration of iodine reagent, stir and oxidize for 5 hours, detect the oxidation status by mass spectrometry, and add citric acid to terminate the oxidation.

[0080] 9. Purification: The crude peptide was purified by HPLC.

[0081] 10. Mass spectrometry analysis (detection)

[0082] 11. Freeze-drying: Rapid cooling with liquid nitrogen, followed by freeze-drying.

[0083] II. Quality Analysis (COA): High-performance liquid chromatography (HPLC) and mass spectrometry (MS) are used to perform quality analysis on the target compound for peptide sequence, molecular weight and chemical purity.

[0084] 3. Dissolve 80 mg of the peptide in water, and add acetonitrile until the peptide is completely dissolved. Dissolve 20 mg of DMPE and the peptide solution in DMF solution, and add acetonitrile until completely dissolved. Mix and react, adjusting the pH to 7.5-7.8, and react overnight at room temperature.

[0085] IV. After filtration through a filter membrane, the product is rapidly freeze-dried to obtain DPH1.

[0086] DPH2 is prepared using the same method described above.

[0087] Example 2

[0088] 1. DPH1 and DPH2 were successfully localized to the cell membrane, and it was confirmed that they bind cells together through the binding of α-helical peptides.

[0089] Select 1×10 5 Immunofluorescence and statistical results of CEM cells (CEM cells) treated with DPH1 (DPH1-FITC) containing green fluorescent molecules for 0 min, 2 min, 5 min, 10 min, and 15 min were collected. Figure 5 (A) shows that 10 minutes is the most appropriate reaction time; excessively long reaction times can cause material endocytosis. Additionally, a 1×10⁻⁶ ppm was selected. 5 The results of immunofluorescence and cell flow cytometry were obtained by co-processing individual cells (CEM cells) with 0 μM, 5 μM, 10 μM, 25 μM, and 50 μM DPH1 containing a green fluorescent molecule. Figure 5 Both C and D showed that 25 μM was the optimal concentration. DPH1 with green fluorescence: 1 mmol FITC was dissolved in 20 mL DMSO (dimethyl sulfoxide), 1 mL DIEA (N,N-diisopropylethylamine) was added, and the synthesized DPH1 was reacted with it at room temperature for 2 hours.

[0090] DPH2 is the same as above.

[0091] Furthermore, this embodiment also synthesized Rho B-helix peptide2, which cannot intercalate to the cell membrane without DMPE, to verify that its binding to DPH1-FITC relies on the direct interaction of this pair of α-helical peptides. Flow cytometry and confocal microscopy results ( Figure 5 Both E and F demonstrate that DPH1-FITC possesses DMPE, enabling it to insert into the phospholipid bilayer of the cell membrane. RhoB-H2 (peptide sequence 2 linked to rhodamine B, abbreviated as Rho B-H2), lacking DMPE, can only be visualized on the cell membrane by binding to DPH1-FITC; RhoB-H2 alone cannot be localized on the cell membrane. Figure 5 (F and G).

[0092] 2. DPH1 / 2 mediates cell clumping

[0093] Optical microscopy, scanning electron microscopy, and fluorescence confocal microscopy all showed that the addition of DPH1 / 2 caused dispersed cells to aggregate into cell clumps. Furthermore, the cell surfaces of these clumps were stretched, generating intercellular forces. The number and size of the cell clumps also demonstrated the significant effect of DPH1 / 2 on cell aggregation. Figure 6 ).

[0094] 3. By using DPH1 / 2 to form coiled-coil structures, artificial intervention in cell-cell interactions was used to construct spherical models and cell layer-by-layer assembly models.

[0095] This embodiment aims to obtain the desired cell aggregates by adding cells and DPH1 / 2 material in a predetermined order. To clearly distinguish the order of cell addition and to better observe the different components within the cell clusters under a fluorescence confocal microscope, two IPS cell lines labeled with GFP and mCherry, respectively, were used. 21 μl of 300 μL MDPH1 and 229 μl of [a specific ingredient] contained 1 × 10 [units of something]. 5 A mixture of mCherry-IPS cell suspensions; 21 μl of 300 μM DPH2 and 229 μl of a mixture containing 1 × 10⁻⁶ mCherry-IPS cells. 5 The cell suspensions of GFP-IPS were mixed (final concentration 25 μM), and then the mixture was further combined. The entire process was carried out on a shaker in a CO2 37℃ incubator. After 1 hour of incubation, images were taken under a fluorescence confocal microscope, revealing a cell spheroid model of randomly mixed red and green cells, consistent with expectations. Figure 7 ).

[0096] First, the mCherry-IPS cells were seeded in a confocal dish. The next day, after the cells had adhered to the dish, 42 μl of a mixture of 300 μM DPH2 and 458 μl of culture medium (final concentration 25 μM) was added. After incubation for 10 min, 42 μl of a mixture of 300 μM DPH1 and 458 μl of culture medium (final concentration 25 μM) was added.

[0097] Then, add the digested GFP-IPS cells (500 μl containing 1 × 10⁻⁶ cells). 5 A suspension of GFP-IPS cells was incubated for 1 hour before imaging. Layer-by-layer imaging under a fluorescence confocal microscope revealed distinct layers of red and green fluorescent cells, forming a layered, assembled cell structure. Figure 7 ).

[0098] 4. DPH1 / 2-mediated cell-cell interactions promote the differentiation of stem cells into pancreatic islet cells.

[0099] During the generation of pancreatic β cells derived from human pluripotent stem cells, some cells failed to aggregate into clusters. However, the addition of DPH1 / 2 material enabled them to aggregate well (refer to the literature "Generation of insulin-producing pancreatic β cells from multiple human stem cell lines"). Immunofluorescence, flow cytometry, and qPCR were used to detect insulin expression levels. The results showed that cell clusters formed with DPH1 / 2 and induced cell clusters had similar insulin and glucagon expression levels. High and low concentrations of glucose solutions were used to stimulate the experimental group (with DPH1 / 2 material forming coiled-coil structures) and the control sp6 cells (without DPH1 / 2) for 1 hour. The results showed that cells with coiled-coil structures responded better to both high and low concentrations of glucose solutions, suggesting that the formation of coiled-coil structures facilitates the differentiation of sp6 cells into SC-β cells. Furthermore, the expression levels of other important genes related to stem cell differentiation into SC-β cells were significantly increased. Figure 8 ).

[0100] 5. The effect of DPH1 / 2-mediated cell-cell interactions on gene expression

[0101] Several common genes related to cell adhesion and interaction were selected, and qPCR experiments were performed on CEM cell experimental and control groups for preliminary validation. qPCR results showed that, compared with the control group, many genes related to cell adhesion and interaction were significantly upregulated in the DPH1 / 2-added experimental group. Next, transcriptome sequencing was performed on CEM cells from both experimental and control groups. GO analysis of the CEM cell transcriptome revealed that the expression of genes related to cell surface and plasma membrane in coiled-coil-mediated CEM cell clusters was significantly affected. Simultaneously, cell-cell interactions also showed upregulation or downregulation of genes related to common cellular pathways such as the TGF-beta signaling pathway and the Hippo signaling pathway. RT-qPCR was then performed to validate the upregulated genes, and the upregulation trend was consistent with the transcriptome sequencing results. Further validation was performed on sp6 cells, selecting cadherin, a common cell adhesion-related protein. Immunofluorescence and qPCR results both showed that E-cadherin expression was upregulated in coiled-coil-mediated cells. Next, transcriptome sequencing was performed on sp6 cells. GO analysis showed significant changes in the expression of genes related to the cell surface and plasma membrane. Later, some upregulated genes were selected for RT-qPCR validation, and the upregulation trend was consistent with the transcriptome sequencing results. Figure 9 ).

[0102] Based on the above results, it is speculated that after DPH1 / 2 inserts into the cell membrane, it forms a coiled-coil structure, thereby bringing cells closer together and allowing cells that were originally unable to contact to aggregate. This, in turn, alters the gene expression of cells by affecting their behavior.

[0103] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A pair of mutually recognizing and binding α-helical peptides, characterized in that, It includes α-helical peptide 1 and α-helical peptide 2, the amino acid sequence of which is shown in SEQ ID No. 1 and the amino acid sequence of which is shown in SEQ ID No.

2.

2. The use of the α-helical peptide according to claim 1 in the preparation of cell cluster products.

3. A pair of polypeptides for preparing cell clusters, characterized in that, Includes DPH1 and DPH2, wherein DPH1 is α-helical peptide 1 of claim 1 with PEG and DMPE attached to it; The DPH2 is the α-helical peptide 2 in claim 1, to which PEG and DMPE are attached.

4. The use of the polypeptide according to claim 3 in the preparation of cell cluster products.

5. A method for constructing a spherical model cell cluster, characterized in that, Includes the following steps: 1) Mix DPH1 in the polypeptide of claim 3 with cells to obtain DPH1-cells; 2) Mix the DPH2 in the polypeptide of claim 3 with cells to obtain DPH2-cells; 3) Mix the DPH1- cells obtained in step 1) with DPH2- cells and culture them to obtain a spherical model cell cluster.

6. The method according to claim 5, characterized in that, In step 1), DPH1 is mixed in the form of a DPH1 solution with a concentration of 300 μM, and the cells are mixed in the form of a cell suspension with a cell count of 5 × 10⁻⁶ cells. 5 The volume ratio of the DPH1 solution to the cell suspension is 42:

458. The mixing time is 10 minutes.

7. The method according to claim 5, characterized in that, In step 1), DPH2 is mixed in the form of a DPH2 solution with a concentration of 300 μM, and the cells are mixed in the form of a cell suspension with a cell count of 5 × 10⁻⁶ cells. 5 The volume ratio of the DPH2 solution to the cell suspension is 42:

458. The mixing time is 10 minutes.

8. A method for constructing a cell cluster model of layer-by-layer cell assembly, characterized in that, Includes the following steps: 1) After adhering to the culture vessel, the cells are mixed with DPH2 from the polypeptide described in claim 3 and incubated. 2) Then mix with DPH1 in the polypeptide of claim 3 and incubate for 10 min, then mix with cells to obtain cell clusters of the cell layer-by-layer assembly model.

9. The method according to claim 8, characterized in that, In step 1), the final concentration of DPH2 is 25 μM, and the number of cells is 5 × 10⁻⁶. 5 The incubation time is 10 minutes.

10. The method according to claim 8, characterized in that, In step 2), the final concentration of DPH1 is 25 μM, and the number of cells is 5 × 10⁻⁶. 5 The mixture was prepared in 1 hour and mixed with the cells.

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