Multifactor composition for regulating the WNT-NOTCH signaling pathway to enhance the odontogenic ability of dental stem cells

Abstract

This application provides a multifactor composition for regulating the WNT-NOTCH signaling pathway to enhance the odontogenic capacity of dental stem cells. The composition first constructs a promoter-replacement expression vector, then uses this vector to screen for promoter-positive dental stem cells. The multifactor composition then stimulates these stem cells to enhance their odontogenic capacity. This application has the potential for broad clinical application, with the following advantages: enhanced tooth repair capacity, improved biocompatibility and integration, promoted regeneration of natural tooth structure, reduced risk of dental pulp infection, improved long-term treatment stability, and reduced patient discomfort and recovery time.

Description

Technical Field

[0001] The present application relates to the technical field of signal pathway regulatory factors, and in particular to a multi-factor composition for regulating the WNT-NOTCH signal pathway to enhance the odontogenic ability of dental stem cells. Background Art

[0002] Existing technical studies have found that odontogenic cells play an important role in tooth development and regeneration, but our understanding of their fate-determining mechanisms remains limited.

[0003] Specifically, there are obvious research gaps and challenges in the following areas:

[0004] 1. The cell fate determination mechanism is unclear: How neural crest cells differentiate into specific odontogenic cell types and the molecular regulatory mechanisms in this process have not yet been fully elucidated.

[0005] 2. The mechanism of dentin formation is not fully understood: How odontogenic cells participate in the formation of dentin and their specific role in tooth development are key issues that still require further research.

[0006] 3. Pulp infection and restoration issues: When a tooth is damaged, especially when there is a large defect or exposed pulp, the pulp is susceptible to infection. Traditional treatments often rely on fillings, which can not only cause further infection but also cause the tooth to lose its biological structure and function.

[0007] 4. Material aging and biocompatibility issues: The materials currently used for dental restorations may age, wear, or experience biocompatibility issues after long-term use. These issues limit the durability of the restoration effect and the patient's quality of life.

[0008] 5. Lack of effective regeneration strategies: Although mild stimulation can promote the formation of reparative dentin, there is currently a lack of effective strategies to promote the regeneration of large-area dentin defects. Summary of the Invention

[0009] In response to the above-mentioned technical limitations, the present application proposes a multi-factor composition for regulating the WNT-NOTCH signaling pathway to improve the odontogenic ability of dental stem cells; it overcomes the deficiencies and defects mentioned in the background art.

[0010] To achieve the above objectives, this application adopts the following technical solutions:

[0011] The invention of this application is to provide a method for constructing an expression vector, comprising the following steps: replacing the CMV promoter that drives the expression of the target gene in the starting vector CMV-EGFP-MCS-SV40-Puromycin (purchased from Shanghai JiKai Gene Technology Co., Ltd., No. GV814) with the DLX6-AS1 promoter to obtain an expression vector.

[0012] The nucleotide sequence of the DLX6-AS1 promoter is shown as SEQ ID No. 1 in the sequence listing.

[0013] Optionally, in the above method, the nucleotide sequence of the DLX6-AS1 promoter is located at position 4836 bp to 6835 bp from the 5' end in the full-length sequence of the expression vector.

[0014] Optionally, in the above method, the starting vector CMV-EGFP-MCS-SV40-Puromycin is prepared according to the following steps:

[0015] S1. Obtain gene fragments of EGFP, MCS, SV40 promoter, and Puromycin resistance gene;

[0016] S2. Select a base vector, perform enzyme digestion, product separation, and recovery to obtain a linearized vector backbone fragment;

[0017] S3. After mixing the gene fragment of step S1 and the vector backbone fragment of step S2, the fragments are connected with a ligase to obtain a ligation product;

[0018] S4. Transform the ligation product obtained in step S3 into competent cells, culture the colonies and screen for positive colonies;

[0019] S5. Expand and culture the positive colonies obtained in step S4, extract the plasmid, perform enzyme digestion, and sequence identification to obtain the starting vector CMV-EGFP-MCS-SV40-Puromycin.

[0020] The detailed preparation method of the starting vector CMV-EGFP-MCS-SV40-Puromycin is as follows:

[0021] 1) Obtaining EGFP gene fragments:

[0022] PCR amplification is performed using a plasmid containing the EGFP gene as a template. Specific primers are designed with appropriate restriction endonuclease sites (e.g., EcoRI and BamHI) introduced at the 5' and 3' ends of the primers, respectively. PCR amplification is performed using a high-fidelity DNA polymerase. The reaction system includes template DNA, primers, dNTPs, buffer, and polymerase. Typical reaction conditions include: initial denaturation at 95°C for 3-5 minutes; denaturation at 95°C for 30 seconds, annealing at 55-65°C for 30 seconds, and extension at 72°C for 1-2 minutes (adjusted to fragment length) for 30-35 cycles; followed by a final extension at 72°C for 5-10 minutes. Amplified products are separated by agarose gel electrophoresis, and the desired fragments are recovered using a gel recovery kit.

[0023] 2) MCS (Multiple Cloning Site) Fragment Acquisition:

[0024] Based on the desired restriction endonuclease site combination, a double-stranded DNA fragment is synthesized containing multiple commonly used restriction endonuclease sites, such as HindIII, XbaI, and SalI. Restriction endonuclease sites that match the vector ligation site are also designed at both ends. The synthesized fragment is annealed to form a double strand and then purified.

[0025] 3) Obtaining the SV40 promoter fragment:

[0026] The EGFP gene fragment is obtained by PCR amplification from the genome of an organism or a related plasmid containing the SV40 promoter. Primers with appropriate restriction endonuclease sites (such as BamHI and XhoI) are also designed and amplified and recovered using PCR reaction conditions similar to those used for the EGFP gene fragment.

[0027] 4) Obtaining the Puromycin-resistant gene fragment:

[0028] Using the plasmid containing the Puromycin resistance gene as a template, the product is obtained by PCR amplification. Appropriate restriction endonuclease sites (such as XhoI and PstI) are added to both ends of the primers, followed by PCR amplification, electrophoresis separation and recovery.

[0029] 5) Vector backbone selection and processing:

[0030] Select an appropriate base vector, such as the pUC series. Double-digest the vector using the appropriate restriction endonucleases (designed for subsequent fragment ligation, such as EcoRI and PstI). The digestion system consists of vector DNA, restriction endonucleases, and buffer. Incubate the reaction at an appropriate temperature (generally 37°C) for 1-3 hours. Separate the digestion products by agarose gel electrophoresis, and use a gel recovery kit to recover the linearized vector backbone fragment to eliminate the possibility of self-circularization.

[0031] 6) Fragment connection:

[0032] Mix the recovered EGFP gene fragment, MCS fragment, SV40 promoter fragment, and Puromycin resistance gene fragment with the linearized vector backbone at a specific molar ratio and perform a ligation reaction using T4 DNA ligase. The ligation system contains the above fragments, T4 DNA ligase, buffer, and ATP. Ligation should be performed overnight at 16°C, or the reaction temperature and time should be adjusted according to the ligase instructions.

[0033] 7) Transformation and screening:

[0034] Transform the ligation product into competent E. coli cells (e.g., DH5α). Remove the competent cells from the -80°C freezer, thaw them on ice, then add the ligation product and gently mix. Incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, and quickly incubate on ice for 2 minutes. Then, add antibiotic-free LB medium and incubate at 37°C with shaking for 1 hour. Spread the culture onto LB plates containing the appropriate antibiotic (e.g., Ampicillin, as the base vector may carry the AmpR gene), X-Gal, and IPTG, and incubate at 37°C overnight. Select white colonies for preliminary screening by colony PCR. Use specific primers to amplify the target fragment, and positive colonies are cultured.

[0035] 8) Plasmid extraction and identification:

[0036] Positive colonies screened were expanded and plasmids were extracted using a plasmid extraction kit. The extracted plasmids were identified by restriction enzyme digestion using the previously introduced restriction enzymes. The digestion products were analyzed by agarose gel electrophoresis to verify that the band sizes met expectations. Sequencing can also be performed. The plasmids were sent to a sequencing company for sequencing and compared with the expected sequence to ensure that the vector was constructed correctly. If the sequence is correct, the starting vector CMV-EGFP-MCS-SV40-Puromycin was obtained.

[0037] The second invention of this application is to provide an expression vector prepared according to the above method.

[0038] Optionally, the above-mentioned expression vector is NR_015448-promoter-EGFP-MCS-SV40-Puromycin.

[0039] NR_015448-promoter is the DLX6-AS1 promoter sequence.

[0040] The third invention of this application is to provide the above expression vector for screening DLX6-AS1 + Application in cells.

[0041] DLX6-AS1 + The cells are DLX6-AS1 promoter-positive cells. Only promoter-positive cells can express DLX6-AS1, and qPCR experiments have proved that the screened cells can highly express DLX6-AS1.

[0042] Optionally, in the above application, the target cells for screening are selected from one or more of human tooth germ mesenchymal stem cells and human dental pulp stem cells.

[0043] The fourth invention of the present application is to provide a multi-factor composition for regulating the WNT-NOTCH signaling pathway to improve the odontogenic ability of dental stem cells, wherein the dental stem cells are selected as DLX6-AS1 screened by the aforementioned expression vector. + Human tooth germ mesenchymal stem cells and DLX6-AS1 + One or more of human dental pulp stem cells.

[0044] Optionally, the multi-factor composition is selected from one or more of WNT6 protein, WNT7B protein, WNT10B protein and JAG1 protein, and each protein is prepared in equal ratios.

[0045] The factors in the above-mentioned composition adopt a relay stimulation method, that is, the cells are first stimulated with WNT6 protein, WNT7B protein, and WNT10B protein to activate the WNT signaling pathway, and then relay stimulation with JAG1 to achieve the purpose of activating the NOTCH signaling pathway. After the relay stimulation, the dental stem cells show better tooth formation ability.

[0046] The research basis of this application is to focus on odontogenic DLX6-AS1 + Cells (DLX6-AS1 promoter positive cells) were selected to explore their odontogenic capacity in tooth development and adult dental pulp. The study found that under the rhythmic regulation of the WNT-NOTCH signaling pathway, these tooth germ mesenchymal DLX6-AS1 + Cells are able to form more dentin, and this process is still active in adult dental pulp, contributing to the formation of reparative dentin. This discovery provides a new strategy for the treatment of tooth defects. The applicant expects to activate and utilize DLX6-AS1 to + The odontogenic capacity of cells promotes the biological repair of teeth.

[0047] The technical research process of this application is:

[0048] 1. First, through the analysis of human tooth germ single-cell transcriptome and spatial transcriptome data, the relay regulation of the WNT-NOTCH signaling pathway was discovered;

[0049] 2. Through the analysis of human tooth germ single-cell transcriptome and spatial transcriptome data, we found that human odontogenic DLX6-AS1 + The cells have odontogenic potential;

[0050] 3. Screening of human tooth germ mesenchyme DLX6-AS1 by lentiviral transfection and promoter tagging + Stem cells and human dental pulp DLX6-AS1 + Stem cells, this step is crucial for subsequent cell experiments and tissue engineering applications;

[0051] 4. Compared with unpurified tooth germ mesenchyme or dental pulp cells, DLX6-AS1 + Cells showed stronger tooth-forming ability, and DLX6-AS1 was verified by cell experiments. + the potential of cells in tooth restoration;

[0052] 5. Relay stimulation was performed using WNT6+WNT7B+WNT10B+JAG1, which had the highest expression level in single-cell sequencing, and it was found that this combination could effectively increase DLX6-AS1 + odontogenic capacity of cells;

[0053] 6. Construction of recombinant tooth germ (epithelial combination + tooth germ mesenchyme DLX6-AS1 + cells) were transplanted under the renal membrane of nude mice, and the WNT-NOTCH signaling pathway was rhythmically stimulated by the above-mentioned combination factors, eventually forming a dentin-like structure, which fully demonstrated that DLX6-AS1 + The potential of cell applications in dental tissue engineering;

[0054] 7. By constructing a mouse open-pulp dentin injury model and injecting human dental pulp with DLX6-AS1 + cells, combined with WNT-NOTCH rhythmic stimulation, successfully induced the formation of reparative dentin in mouse teeth, providing broader prospects for the clinical repair of tooth defects.

[0055] The multi-factor composition provided in this application for regulating the WNT-NOTCH signaling pathway to enhance the odontogenic ability of dental stem cells can produce the following excellent effects after being administered to dental stem cells:

[0056] 1. Enhanced tooth restoration capabilities: This technology utilizes DLX6-AS1 +cells, significantly improving the ability of tooth repair; under the rhythmic regulation of the WNT-NOTCH signaling pathway, these cells exhibit stronger tooth formation and mineralization capabilities than traditional methods;

[0057] 2. Improve biocompatibility and integration: Utilize DLX6-AS1 + Cells are used for tooth repair, which improves the biocompatibility of the repair material and the native tooth tissue, reduces the immune rejection reaction, and enhances the integration of the repair tissue;

[0058] 3. Promote the regeneration of natural tooth structure: This technology activates and utilizes DLX6-AS1 + The natural odontogenic potential of cells promotes the natural regeneration of teeth, which is closer to physiological tooth restoration than relying solely on artificial filling materials.

[0059] 4. Reduce the risk of pulp infection: During the process of tooth defect repair, this technology effectively reduces the risk of pulp exposure and infection by promoting the formation of restorative dentin, thereby improving the success rate of tooth restoration;

[0060] 5. Improve long-term stability of treatment: Compared with traditional dental restoration materials, this technology provides longer-term stability and durability by promoting the natural regeneration of tooth tissue, reducing the need for further treatment or replacement in the future.

[0061] 6. Reduce patient discomfort and recovery time: Because this technology improves the biocompatibility and natural regeneration ability of tooth restorations, patients experience less postoperative discomfort, shorten recovery time, and improve their quality of life;

[0062] 7. Has broad clinical application potential: This technology is not only suitable for the repair of tooth defects, but can also be extended to the treatment of other dental diseases, such as periodontal disease, tooth agenesis, etc., and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 shows the rhythmic regulation of the WNT-NOTCH signaling pathway during human tooth development analyzed using single-cell and spatial transcriptome data; Figure 1 Figure A integrates single-cell transcriptome data from various stages of tooth development, performing a holistic analysis of the epithelium and dental papilla. The findings indicate that the overall trend of the WNT and NOTCH signaling pathways is that the epithelium leads the secretion, while the dental papilla takes over. Figure 1B analyzes the temporal dimension, revealing a relay-like pattern in the communication between the epithelium and the dental papilla. Figure 1 C shows the expression levels of various ligand receptors, and the WNT ligand and NOTCH ligand with the highest expression levels are selected for separate presentation.

[0064] Figure 2 shows the analysis of human tooth germ mesenchyme DLX6-AS1 by human tooth germ single cell transcriptome data. + Figure 2A shows that when analyzing the RNA dynamics of the single-cell transcriptome data of the dental papilla, two different differentiation fates were found in its differentiation trajectory; Figure 2B divides the cells into DLX6-AS1 according to their differentiation fate. + 、DLX6-AS1 - Figure 2C shows the changes in the proportion of each subpopulation over time; Figure 2D shows the highly expressed genes in each subpopulation, and it was found that many genes highly expressed in positive cells were also highly expressed in odontoblasts; Figure 2E shows the comparison of DLX6-AS1 + and DLX6-AS1 - The differentially expressed genes of the two cell groups showed that Wnt and Notch transcription factors were highly expressed in positive cells; Figure 2F shows the expression of Wnt and Notch receptors and transcription factors, and found that the expression levels were higher in positive cells. The expression of Notch in odontoblasts is also worthy of attention.

[0065] Figure 3 shows the purification of DLX6-AS1 from human tooth germ mesenchyme and dental pulp using lentiviral transfection and promoter tagging technology. + Figure 3A shows the isolation and purification of DLX6-AS1 from human tooth germ mesenchyme + cells and verified the expression of DLX6-AS1; Figure 3B shows the results of DLX6-AS1 isolation and purification from human dental pulp + cells and verified the expression level of DLX6-AS1.

[0066] Figure 4 DLX6-AS1 was verified by cell experiments + Compared with unpurified odontogenic cells, the cells have stronger odontogenic ability; Figure 4A compares tooth germ mesenchymal cells and DLX6-AS1 + Tooth germ mesenchymal cells, DLX6-AS1 was found + Tooth germ mesenchymal cells expressed higher levels of WNT and NOTCH downstream signaling proteins, as well as odontogenesis-related molecules such as DMP1 and DSPP. At the same time, Alizarin red staining results showed that they had stronger odontogenesis ability. Figure 4B compared with dental pulp cells, found that DLX6-AS1 + Dental pulp cells express higher levels of WNT and NOTCH downstream signals, as well as odontogenesis-related molecules such as DMP1 and DSPP.

[0067] Figure 5It shows that the combination of WNT6+WNT7B+WNT10B+JAG1 activates the WNT-NOTCH signaling pathway and promotes DLX6-AS1 + The WB results in Figure 5A showed that after WNT-NOTCH relay stimulation, the cells expressed higher levels of WNT and NOTCH downstream signals, as well as odontogenesis-related molecules such as DMP1 and DSPP; the ALP staining (top) and Alizarin red staining (bottom) results in Figure 5B showed that after WNT-NOTCH relay stimulation, the cells exhibited stronger mineralization ability.

[0068] Figure 6 It is shown that the recombinant tooth germ (epithelial junction + tooth germ mesenchyme DLX6-AS1 + The cells were transplanted under the renal membrane of nude mice and then stimulated with WNT-NOTCH rhythm, thereby forming a dentin-like structure.

[0069] Figure 7 The results show that a mouse open-pulp dentin injury model was constructed and human dental pulp DLX6-AS1 was injected + The cells, combined with WNT-NOTCH rhythmic stimulation, successfully induced the test results of reparative dentin formation in mouse teeth.

[0070] Figure 8 Shown is the plasmid map of the starting vector GV814 (CMV-EGFP-MCS-SV40-Puromycin).

[0071] Figure 9 Shown is the plasmid map of the expression vector NR_015448-promoter-EGFP-MCS-SV40-Puromycin. DETAILED DESCRIPTION

[0072] To make the purpose, technical solutions and advantages of this application more clear, this application is further described in detail below. However, it should be understood that the description herein is only used to explain this application and is not intended to limit the scope of this application.

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are intended solely for the purpose of describing specific embodiments and are not intended to limit this application. The reagents and instruments used herein are commercially available, and the characterization methods involved can be found in the relevant descriptions in the prior art and will not be further elaborated herein.

[0074] In order to further understand the present application, the present application is further described in detail below in conjunction with the best embodiment.

[0075] Example 1

[0076] The method for constructing an expression vector comprises the following steps: replacing the CMV promoter driving expression of the target gene in the starting vector CMV-EGFP-MCS-SV40-Puromycin (purchased from Shanghai GeneCare Gene Technology Co., Ltd., catalog number GV814) with the DLX6-AS1 promoter to obtain an expression vector. The nucleotide sequence of the DLX6-AS1 promoter is shown in SEQ ID No. 1 in the sequence listing.

[0077] The nucleotide sequence of the DLX6-AS1 promoter is located at 4836 bp to 6835 bp from the 5' end of the full-length sequence of the expression vector.

[0078] The starting vector CMV-EGFP-MCS-SV40-Puromycin was prepared according to the following steps:

[0079] S1. Obtain gene fragments of EGFP, MCS, SV40 promoter, and Puromycin resistance gene;

[0080] 1) Obtaining EGFP gene fragments:

[0081] PCR amplification is performed using a plasmid containing the EGFP gene as a template. Specific primers are designed with appropriate restriction endonuclease sites (e.g., EcoRI and BamHI) introduced at the 5' and 3' ends of the primers, respectively. PCR amplification is performed using a high-fidelity DNA polymerase. The reaction system includes template DNA, primers, dNTPs, buffer, and polymerase. Typical reaction conditions include: initial denaturation at 95°C for 3-5 minutes; denaturation at 95°C for 30 seconds, annealing at 55-65°C for 30 seconds, and extension at 72°C for 1-2 minutes (adjusted to fragment length) for 30-35 cycles; followed by a final extension at 72°C for 5-10 minutes. Amplified products are separated by agarose gel electrophoresis, and the desired fragments are recovered using a gel recovery kit.

[0082] 2) MCS (Multiple Cloning Site) Fragment Acquisition:

[0083] Based on the desired restriction endonuclease site combination, a double-stranded DNA fragment is synthesized containing multiple commonly used restriction endonuclease sites, such as HindIII, XbaI, and SalI. Restriction endonuclease sites that match the vector ligation site are also designed at both ends. The synthesized fragment is annealed to form a double strand and then purified.

[0084] 3) Obtaining the SV40 promoter fragment:

[0085] The EGFP gene fragment is obtained by PCR amplification from the genome of an organism or a related plasmid containing the SV40 promoter. Primers with appropriate restriction endonuclease sites (such as BamHI and XhoI) are also designed and amplified and recovered using PCR reaction conditions similar to those used for the EGFP gene fragment.

[0086] 4) Obtaining the Puromycin-resistant gene fragment:

[0087] Using the plasmid containing the Puromycin resistance gene as a template, the product is obtained by PCR amplification. Appropriate restriction endonuclease sites (such as XhoI and PstI) are added to both ends of the primers, followed by PCR amplification, electrophoresis separation and recovery.

[0088] S2. Select a base vector, and obtain a linearized vector backbone fragment after enzyme digestion, product separation, and recovery; specifically:

[0089] Select an appropriate base vector, such as the pUC series. Double-digest the vector using the appropriate restriction endonuclease (designed for subsequent fragment ligation, such as EcoRI and PstI). The digestion system includes vector DNA, restriction endonucleases, and buffer. Incubate the reaction at an appropriate temperature (generally 37°C) for 1-3 hours. Separate the digestion products by agarose gel electrophoresis, and use a gel recovery kit to recover the linearized vector backbone fragment to eliminate the possibility of vector self-circularization.

[0090] S3. After mixing the gene fragments from step S1 and the vector backbone fragments from step S2, the fragments are ligated with a ligase to obtain a ligation product; specifically:

[0091] Mix the recovered EGFP gene fragment, MCS fragment, SV40 promoter fragment, and Puromycin resistance gene fragment with the linearized vector backbone in a certain molar ratio and perform a ligation reaction using T4 DNA ligase. The ligation system contains the above fragments, T4 DNA ligase, buffer, and ATP. Ligation should be carried out at 16°C overnight or the reaction temperature and time should be adjusted according to the ligase instructions.

[0092] S4. The ligation product obtained in step S3 is transformed into competent cells, and positive colonies are screened after culturing the colonies; specifically:

[0093] Transform the ligation product into competent E. coli cells (e.g., DH5α). Remove the competent cells from the -80°C freezer, thaw them in an ice bath, add the ligation product, mix gently, and ice bath for 30 minutes. Heat shock the cells at 42°C for 90 seconds, quickly ice bath for 2 minutes, then add antibiotic-free LB medium and culture at 37°C with shaking for 1 hour. Spread the culture onto an LB plate containing the appropriate antibiotic (e.g., Ampicillin, as the basic vector may carry the AmpR gene), X-Gal, and IPTG, and culture at 37°C overnight. Select white colonies for preliminary colony PCR screening. Use specific primers to amplify the target fragment, and positive colonies are cultured subsequently.

[0094] S5. The positive colonies obtained in step S4 were expanded and cultured, and the plasmids were extracted and digested by enzymes and sequenced to obtain the starting vector CMV-EGFP-MCS-SV40-Puromycin; specifically:

[0095] Positive colonies screened were expanded and plasmids were extracted using a plasmid extraction kit. The extracted plasmids were identified by restriction enzyme digestion using the previously introduced restriction enzymes. The digestion products were analyzed by agarose gel electrophoresis to verify that the band sizes met expectations. Sequencing can also be performed. The plasmids were sent to a sequencing company for sequencing and compared with the expected sequence to ensure that the vector was constructed correctly. If the sequence is correct, the starting vector CMV-EGFP-MCS-SV40-Puromycin was obtained.

[0096] The expression vector (DLX6-AS1 promoter-EGFP-SV40-Puromycin) constructed by the above method can be used to screen DLX6-AS1 + cells (DLX6-AS1 + The cells are DLX6-AS1 promoter-positive cells. Only promoter-positive cells can express DLX6-AS1, and qPCR experiments have proven that the screened cells can highly express DLX6-AS1).

[0097] The main target cells for screening are one or more of human tooth germ mesenchymal stem cells and human dental pulp stem cells.

[0098] The present application also describes a multi-factor composition for regulating the WNT-NOTCH signaling pathway to improve the odontogenic ability of dental stem cells. The dental stem cells are selected as DLX6-AS1 selected by using an expression vector containing the DLX6-AS1 promoter. + Human tooth germ mesenchymal stem cells, and DLX6-AS1 screened using an expression vector containing the DLX6-AS1 promoter + Human dental pulp stem cells.

[0099] The multi-factor combination is selected to activate WNT6, WNT7B, and WNT10B, which activate the WNT signaling pathway, and JAG1, which relays to activate the NOTCH signaling pathway.

[0100] Example 2

[0101] This patented technology is based on the discovery of rhythmic regulation of the WNT-NOTCH signaling pathway, which can effectively promote the expression of DLX6-AS1 + Further research found that during tooth development, WNT-NOTCH signaling showed a relay phenomenon, guiding DLX6-AS1 + Differentiation of cells into odontoblasts.

[0102] The technical steps involved in this discovery include:

[0103] 1) Initial activation of WNT signaling: WNT6, WNT7B, and WNT10B act as initial signals to activate DLX6-AS1 + WNT signaling pathway in cells;

[0104] 2) Relay activation of NOTCH signaling: Subsequently, JAG1 acts as a ligand for NOTCH signaling, relaying WNT signaling and further promoting DLX6-AS1 + Cell differentiation.

[0105] Operation process:

[0106] 1. Data analysis: Using single-cell transcriptome data, we analyzed gene expression patterns during human tooth development to identify DLX6-AS1 + Key features of cells; results such as Figure 1 It was found that the overall trend of the WNT and NOTCH signaling pathways is that the epithelium dominates secretion, while the dental papilla is responsible for taking over. The WNT-NOTCH signaling pathway presents a relay phenomenon in the communication between the epithelium and the dental papilla; Figure 2 Discovery of DLX6-AS1 + Cells are the main cells that receive epithelial-derived WNT-NOTCH signals;

[0107] 2. Cell isolation and purification: DLX6-AS1 was isolated and purified from human tooth germ mesenchyme and dental pulp using lentiviral transfection and promoter tagging technology. + cells, such as Figure 3 As shown, the specific method is:

[0108] (1) Determine the promoter region of DLX6-AS1 and construct the target sequence: NR_015448-promoter;

[0109] The NR_015448-promoter sequence (DLX6-AS1 promoter) is shown in SEQ ID No. 1:

[0110]

[0111] (2) Replace the CMV promoter of the original GV814 vector element sequence CMV-EGFP-MCS-SV40-Puromycin with the target sequence NR_015448-promoter to construct the final vector element sequence of the target plasmid: NR_015448-promoter-EGFP-SV40-Puromycin;

[0112] The final vector construction diagram based on GV814 replacing the promoter is shown in the figure Figure 8 As shown;

[0113] (3) The target plasmid and packaging envelope plasmid are co-transfected into 293T cells, and the viral particles are collected and concentrated. The specific operations are as follows:

[0114] (a) Co-transfection:

[0115] Cell preparation: HEK293T cells were seeded into a suitable culture vessel to a confluence of 70-80% at the time of transfection. Cell culture conditions were 37°C, 5% CO2 in an incubator using a medium suitable for cell growth (DMEM + 10% FBS).

[0116] Transfection reagent and plasmid preparation: Dilute the constructed plasmid containing the target gene (NR_015448-promoter-EGFP-SV40-Puromycin) and auxiliary packaging plasmids (pHelper 1.0 and 2.0) in serum-free medium (DMEM).

[0117] Co-transfection procedure: Mix the diluted transfection reagent and the diluted plasmid, mix gently and incubate at room temperature for a certain time (30 minutes), then add the mixture dropwise to the cell culture wells and gently shake the culture plate to evenly distribute the mixture;

[0118] (b) Virus collection:

[0119] After a certain period of time (72 h) after co-transfection, the cell culture supernatant was collected and subjected to low-speed centrifugation (300g , 10 min) to remove larger cell debris;

[0120] (c) Virus concentration:

[0121] Use an ultrafiltration centrifuge tube (0.45μm), add the viral supernatant to the ultrafiltration tube, and centrifuge at an appropriate speed (5000g) in a centrifuge until the desired concentration multiple is reached; during the ultrafiltration process, small molecules (such as salts in the culture medium, small molecular proteins, etc.) will pass through the ultrafiltration membrane, while the virus particles are retained in the ultrafiltration tube.

[0122] (4) Infect target cells with lentivirus and screen out cells positive for the DLX6-AS1 promoter to determine the cell population in which the DLX6-AS1 promoter is active. When the promoter is active in cells under endogenous conditions, it drives the expression of the reporter gene. If the promoter is highly active, it usually indicates that the promoter is highly active in the cell and that the cell can strongly express the specific gene downstream of the promoter.

[0123] DLX6-AS1 + The cells have stronger odontogenic ability than unpurified odontogenic cells ( Figure 4 );

[0124] 3. The combination of WNT6+WNT7B+WNT10B+JAG1 can relay activate the WNT-NOTCH signaling pathway and promote DLX6-AS1 + Cell odontogenic capacity: isolated DLX6-AS1 + The cells were exposed to the combined stimulation of WNT6, WNT7B, and WNT10B to activate the WNT signaling pathway; subsequently, by adding JAG1 stimulation, the NOTCH signaling pathway was activated, promoting the differentiation of cells into odontoblasts ( Figure 5 );

[0125] 4. Construction and transplantation of recombinant tooth germs: DLX6-AS1 stimulated by WNT signaling + Tooth germ mesenchymal cells were combined with non-odontogenic epithelium to construct reconstructed tooth germs, which were transplanted under the renal membrane of nude mice. Beads incubated with JAG1 were added to the transplanted area. The finished beads were composed of: PLGA (75:25); Synonyms: lactic acid / glycolic acid copolymer (75:25); poly (lactic-co-glycolic acid) (75:25); Manufacturer's Product Number: MCE, HY-B2247A, to continuously provide NOTCH signal stimulation ( Figure 6 );

[0126] 5. Repair of tooth damage model: Construct a mouse open-pulp dentin damage model to simulate the clinical situation of tooth defect; inject DLX6-AS1 stimulated by WNT6, WNT7B and WNT10B + Dental pulp cells, combined with JAG1 stimulation, promote the repair of damaged areas and regeneration of dentin ( Figure 7 );in,

[0127] The method for establishing a mouse open-endodontic dentin injury model includes the following steps:

[0128] (1) Animal preparation: Select adult male nude mice and fix them on the operating table after anesthesia;

[0129] (2) Pulp-opening operation: Use a high-speed dental drill to open the surface of the molar crown of the mouse to expose the pulp tissue;

[0130] (3) After pulp opening, appropriate wound treatment should be performed (such as injection of DLX6-AS1 stimulated by WNT6, WNT7B and WNT10B) + dental pulp cells, combined with JAG1 stimulation), and then the recovery of the mice was observed.

[0131] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements or improvements made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A method for constructing an expression vector, comprising the following steps: replacing the CMV promoter driving the expression of the target gene in the starting vector CMV-EGFP-MCS-SV40-Puromycin with the DLX6-AS1 promoter to obtain an expression vector; the nucleotide sequence of the DLX6-AS1 promoter is shown in SEQ ID No. 1 in the sequence listing.

2. The method according to claim 1, characterized in that The nucleotide sequence of the DLX6-AS1 promoter is located at the 4836 bp to 6835 bp position from the 5' end in the full-length sequence of the expression vector.

3. An expression vector prepared according to the method according to any one of claims 1-2.

4. The expression vector according to claim 3, characterized in that The expression vector is DLX6-AS1 promoter-EGFP-MCS-SV40-Puromycin. 5 . Use of the expression vector according to claim 3 or 4 in screened DLX6-AS1 promoter-positive human dental germ mesenchymal stem cells or DLX6-AS1 promoter-positive human dental pulp stem cells.

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