Multi-factor composition for regulating WNT-NOTCH signal pathway to improve dental ability of odontogenic stem cells

Abstract

The invention provides a multi-factor composition for regulating a WNT-NOTCH signal channel to improve the tooth forming ability of odontogenic stem cells, which is characterized in that an expression vector replaced by a promoter is firstly constructed, then the promoter-positive odontogenic stem cells are screened by the vector, and then the odontogenic stem cells are continuously stimulated by the multi-factor composition, so that the odontogenic ability of the odontogenic stem cells is improved, and the odontogenic ability of the odontogenic stem cells is improved. Therefore, the tooth forming capability of the odontogenic stem cells is improved. The dental implant has the excellent effects that the dental restoration capability is enhanced, the biocompatibility and the integration are improved, the regeneration of a natural tooth structure is promoted, the dental pulp infection risk is reduced, the long-term stability of treatment is improved, the discomfort of a patient is reduced, the recovery time of the patient is shortened, and the dental implant has wide clinical application potential.

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 improve 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: 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.

[0004] 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.

[0005] 3. Pulp infection and restoration issues: When a tooth is damaged, especially when there is a large area of ​​defect or pulp exposure, the pulp is susceptible to infection. Traditional treatments often rely on filling materials, which may not only cause further infection, but may also cause the tooth to lose its biological structure and function.

[0006] 4. Material aging and biocompatibility issues: The materials currently used for dental restorations may age, wear or experience biocompatibility issues after long-term use, which limits the durability of the restoration effect and the patient's quality of life.

[0007] 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 dentin defects. Summary of the invention

[0008] In view of 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 technology.

[0009] To achieve the above objectives, this application adopts the following technical solutions: The inventive point of the present 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.

[0010] The nucleotide sequence of the DLX6-AS1 promoter is shown as SEQ ID No. 1 in the sequence listing.

[0011] Optionally, in the above method, the nucleotide sequence of the DLX6-AS1 promoter is located at the 4836 bp-6835 bp position from the 5' end in the full-length sequence of the expression vector.

[0012] Optionally, in the above method, the starting vector CMV-EGFP-MCS-SV40-Puromycin is prepared according to the following steps: S1. Obtain the gene fragments of EGFP, MCS, SV40 promoter, and Puromycin resistance gene; S2. Select a basic vector, obtain a linearized vector backbone fragment after enzyme digestion, product separation and recovery; 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 connection product; S4. Transform the ligation product obtained in step S3 into competent cells, culture the colonies and screen out positive colonies; S5. Expand the positive colonies obtained in step S4, extract the plasmid, perform restriction digestion, and sequence identification to obtain the starting vector CMV-EGFP-MCS-SV40-Puromycin.

[0013] The detailed preparation method of the starting vector CMV-EGFP-MCS-SV40-Puromycin is as follows: 1) Obtaining EGFP gene fragment: By PCR amplification, the plasmid containing the EGFP gene was used as a template to design specific primers, and appropriate restriction endonuclease sites (such as EcoRI and BamHI) were introduced into the 5' and 3' ends of the primers, respectively. PCR amplification was performed using a high-fidelity DNA polymerase, and the reaction system included template DNA, primers, dNTPs, buffer, and polymerase. The reaction conditions are generally as follows: pre-denaturation at 95°C for 3-5 minutes; denaturation at 95°C for 30 seconds, annealing at 55-65°C for 30 seconds, extension at 72°C for 1-2 minutes (adjusted according to the fragment length), for a total of 30-35 cycles; and finally extension at 72°C for 5-10 minutes. The amplified products were separated by agarose gel electrophoresis, and the target fragments were recovered using a gel recovery kit.

[0014] 2) MCS (multiple cloning site) fragment acquisition: According to the required enzyme cutting site combination, a double-stranded DNA fragment containing multiple commonly used restriction endonuclease sites is artificially synthesized, such as HindIII, XbaI, SalI, etc., and restriction endonuclease sites matching the vector connection are also designed at both ends. The synthetic fragment is annealed to form a double strand and then purified.

[0015] 3) Obtaining SV40 promoter fragment: It is obtained from the genome of organisms or related plasmids containing the SV40 promoter by PCR amplification. Primers with appropriate restriction endonuclease sites (such as BamHI and XhoI) are also designed, and amplified and recovered under PCR reaction conditions similar to those of the EGFP gene fragment.

[0016] 4) Obtaining Puromycin resistance gene fragments: The plasmid containing the Puromycin resistance gene is used as a template and 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.

[0017] 5) Vector backbone selection and processing: Select a suitable basic vector, such as pUC series vectors. Use the corresponding restriction endonuclease (based on the subsequent fragment connection design, such as EcoRI and PstI) to double-digest the vector. The enzyme digestion system includes vector DNA, restriction endonuclease, buffer, etc., and react at an appropriate temperature (generally 37°C) for 1-3 hours. The digestion products are separated by agarose gel electrophoresis, and the linearized vector backbone fragments are recovered using a gel recovery kit to eliminate the possibility of vector self-circularization.

[0018] 6) Fragment connection: The recovered EGFP gene fragment, MCS fragment, SV40 promoter fragment, Puromycin resistance gene fragment and linearized vector backbone were mixed in a certain molar ratio, and ligated using T4 DNA ligase. The ligation system includes the above fragments, T4 DNA ligase, buffer and ATP, etc. The ligation is carried out at 16°C overnight or the reaction temperature and time are adjusted according to the ligase instructions.

[0019] 7) Transformation and screening: Transform the ligation product into competent E. coli cells (such as DH5α). Take the competent cells out of the -80℃ refrigerator, melt them in an ice bath, add the ligation product, mix gently, ice bath for 30 minutes, heat shock at 42℃ for 90 seconds, quickly ice bath for 2 minutes, then add LB medium without antibiotics, shake and culture at 37℃ for 1 hour. Spread the culture on an LB plate containing the corresponding antibiotics (such as Ampicillin, because the basic vector may carry the AmpR gene) and X-Gal, IPTG, and culture overnight at 37℃. Select white colonies for preliminary screening of colony PCR, use specific primers to amplify the target fragment, and culture the positive colonies later.

[0020] 8) Plasmid extraction and identification: The positive colonies screened were expanded and cultured, and plasmids were extracted using a plasmid extraction kit. The extracted plasmids were identified by restriction endonuclease digestion. The plasmids were digested with the restriction endonucleases introduced previously, and the digestion products were analyzed by agarose gel electrophoresis to observe whether the band size was in line with expectations. At the same time, sequencing identification can be performed, and the plasmids are sent to a sequencing company for sequencing and compared with the expected sequence to ensure that the vector construction is correct. If the sequence is correct, the starting vector CMV-EGFP-MCS-SV40-Puromycin is obtained.

[0021] The second invention of the present application is to provide an expression vector prepared according to the above method.

[0022] Optionally, the above-mentioned expression vector is NR_015448-promoter-EGFP-MCS-SV40-Puromycin.

[0023] NR_015448-promoter is the DLX6-AS1 promoter sequence.

[0024] The third invention of the present application is to provide the above-mentioned expression vector for screening DLX6-AS1 + Application in cells.

[0025] 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.

[0026] 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.

[0027] 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 selected by the aforementioned expression vector. + Human tooth germ mesenchymal stem cells and DLX6-AS1 + One or more of human dental pulp stem cells.

[0028] Optionally, the above-mentioned 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 ratio concentration.

[0029] 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.

[0030] 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 + The 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 + The odontogenic capacity of cells promotes the biological repair of teeth.

[0031] The technical research process of this application is: 1. First, through the analysis of human tooth germ single-cell transcriptome and spatial transcriptome data, the relay regulation rules of the WNT-NOTCH signaling pathway were discovered; 2. Human tooth germ single-cell transcriptome combined with spatial transcriptome data analysis revealed human odontogenic DLX6-AS1 +The cells have odontogenic potential; 3. Screening of human tooth germ mesenchyme DLX6-AS1 by lentiviral transfection marker promoter + Stem cells and human dental pulp DLX6-AS1 + Stem cells, this step is crucial for subsequent cell experiments and tissue engineering applications; 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; 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; 6. Construction of recombinant tooth germ (epithelial combination + tooth germ mesenchyme DLX6-AS1 + cells), transplanted under the renal membrane of nude mice, and the above-mentioned combination of factors was used to rhythmically stimulate the WNT-NOTCH signaling pathway, eventually forming a dentin-like structure, which fully demonstrated that DLX6-AS1 + The potential of cells for use in dental tissue engineering; 7. Construct a mouse open-pulp dentin injury model and inject human dental pulp with DLX6-AS1 + Cells, combined with WNT-NOTCH rhythmic stimulation, successfully induced the formation of reparative dentin in mouse teeth, providing a broader prospect for the clinical repair of tooth defects.

[0032] The multi-factor composition provided in the present application for regulating the WNT-NOTCH signaling pathway to improve the odontogenic ability of dental stem cells can produce the following excellent effects after being applied to dental stem cells: 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 showed stronger tooth formation and mineralization capabilities than traditional methods; 2. Improving biocompatibility and integration: using DLX6-AS1 + Cells are used for tooth repair, which improves the biocompatibility of the repair material and the native tooth tissue, reduces immune rejection, and enhances the integration of the repair tissue; 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 on artificial filling materials. 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 repair; 5. Improve long-term stability of treatment: Compared with traditional tooth 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.

[0033] 6. Reduce patient discomfort and recovery time: Since this technology improves the biocompatibility and natural regeneration ability of tooth restoration, patients' postoperative discomfort is reduced, recovery time is shortened, and the quality of life of patients is improved; 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 dysplasia, etc., and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] 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 A integrates single-cell transcriptome data from various stages of tooth development, and conducts an overall analysis of the epithelium and dental papilla. It is found that the overall trend of the WNT and NOTCH signaling pathways is that the epithelium leads the secretion, and the dental papilla is responsible for the follow-up; Figure 1B analyzes from the time dimension and finds that the WNT-NOTCH signaling pathway presents a relay phenomenon in the communication process between the epithelium and the dental papilla; Figure 1 C shows the expression levels of each ligand receptor, and the WNT ligand and NOTCH ligand with the highest expression levels are selected for separate presentation.

[0035] Figure 2 shows the analysis of human tooth germ mesenchymal DLX6-AS1 by single-cell transcriptome data of human tooth germ + Figure 2A: When analyzing the RNA dynamics of the single-cell transcriptome data of the dental papilla, it was found that there were two different differentiation fates in its differentiation trajectory; Figure 2B: Based on the differentiation fate, the cells were divided into DLX6-AS1 + 、DLX6-AS1 - Figure 2C shows the changes in the proportion of each subpopulation over the developmental 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 is a comparison of DLX6-AS1 +and DLX6-AS1 - The differentially expressed genes of the two groups of cells showed that the transcription factors of Wnt and Notch were highly expressed in the positive cells; Figure 2F shows the expression of Wnt and Notch receptors and transcription factors, and found that the expression levels in the positive cells were higher. The expression of Notch in odontoblasts is also worthy of attention.

[0036] 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 purification of DLX6-AS1 from human tooth germ mesenchyme + cells and verified the expression of DLX6-AS1; Figure 3B shows the purification of DLX6-AS1 from human dental pulp + cells and verified the expression level of DLX6-AS1.

[0037] 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, found DLX6-AS1 + 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 proved 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.

[0038] Figure 5 It 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 (upper) and Alizarin red staining (lower) results in Figure 5B showed that after WNT-NOTCH relay stimulation, the cells showed stronger mineralization ability.

[0039] Figure 6 It is shown that by constructing recombinant tooth germ (epithelial combined + 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.

[0040] Figure 7 The results show that a mouse open-pulp dentin injury model was constructed and injected with human dental pulp DLX6-AS1 + The cells, combined with WNT-NOTCH rhythmic stimulation, successfully induced the results of reparative dentin formation in mouse teeth.

[0041] Figure 8 Shown is the plasmid map of the starting vector GV814 (CMV-EGFP-MCS-SV40-Puromycin).

[0042] Fig. 9 Shown is the plasmid map of the expression vector NR_015448-promoter-EGFP-MCS-SV40-Puromycin. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below. However, it should be understood that the description here is only used to explain the present application and is not used to limit the scope of the present application.

[0044] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present application, and the terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art, which will not be repeated herein.

[0045] In order to further understand the present application, the present application is further described in detail below in conjunction with the best embodiment.

[0046] Example 1 The method for constructing an expression vector comprises the following steps: replacing the CMV promoter driving 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. The nucleotide sequence of the DLX6-AS1 promoter is shown in SEQ ID No. 1 in the sequence table.

[0047] The nucleotide sequence of the DLX6-AS1 promoter is located at 4836 bp to 6835 bp from the 5' end in the full-length sequence of the expression vector.

[0048] The starting vector CMV-EGFP-MCS-SV40-Puromycin was prepared according to the following steps: S1. Obtain the gene fragments of EGFP, MCS, SV40 promoter, and Puromycin resistance gene; 1) Obtaining EGFP gene fragment: By PCR amplification, the plasmid containing the EGFP gene was used as a template to design specific primers, and appropriate restriction endonuclease sites (such as EcoRI and BamHI) were introduced into the 5' and 3' ends of the primers, respectively. PCR amplification was performed using a high-fidelity DNA polymerase, and the reaction system included template DNA, primers, dNTPs, buffer, and polymerase. The reaction conditions are generally as follows: pre-denaturation at 95°C for 3-5 minutes; denaturation at 95°C for 30 seconds, annealing at 55-65°C for 30 seconds, extension at 72°C for 1-2 minutes (adjusted according to the fragment length), for a total of 30-35 cycles; and finally extension at 72°C for 5-10 minutes. The amplified products were separated by agarose gel electrophoresis, and the target fragments were recovered using a gel recovery kit.

[0049] 2) MCS (multiple cloning site) fragment acquisition: According to the required enzyme cutting site combination, a double-stranded DNA fragment containing multiple commonly used restriction endonuclease sites is artificially synthesized, such as HindIII, XbaI, SalI, etc., and restriction endonuclease sites matching the vector connection are also designed at both ends. The synthetic fragment is annealed to form a double strand and then purified.

[0050] 3) Obtaining SV40 promoter fragment: It is obtained from the genome of organisms or related plasmids containing the SV40 promoter by PCR amplification. Primers with appropriate restriction endonuclease sites (such as BamHI and XhoI) are also designed, and amplified and recovered under PCR reaction conditions similar to those of the EGFP gene fragment.

[0051] 4) Obtaining Puromycin resistance gene fragments: The plasmid containing the Puromycin resistance gene is used as a template and 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.

[0052] S2. Select the basic vector, obtain the linearized vector backbone fragment after enzyme digestion, product separation and recovery; specifically: Select a suitable basic vector, such as pUC series vectors; use the corresponding restriction endonuclease (designed according to the subsequent fragment connection, such as EcoRI and PstI) to double-digest the vector. The enzyme digestion system includes vector DNA, restriction endonuclease, buffer, etc., and react at an appropriate temperature (usually 37°C) for 1-3 hours; the enzyme digestion products are separated by agarose gel electrophoresis, and the linearized vector backbone fragments are recovered using a gel recovery kit to eliminate the possibility of vector self-circularization; S3. After mixing the gene fragments of step S1 and the vector backbone fragments of step S2, the fragments are connected with a ligase to obtain a connection product; specifically: The recovered EGFP gene fragment, MCS fragment, SV40 promoter fragment, Puromycin resistance gene fragment and linearized vector backbone were mixed in a certain molar ratio, and ligated using T4 DNA ligase; the ligation system included the above fragments, T4 DNA ligase, buffer and ATP, etc., and the ligation was carried out at 16°C overnight or the reaction temperature and time were adjusted according to the ligase instructions; S4. The ligation product obtained in step S3 is transformed into competent cells, and positive colonies are screened after culturing the colonies; specifically: Transform the ligation product into competent E. coli cells (such as DH5α), take the competent cells out of the -80℃ refrigerator, melt them in an ice bath, add the ligation product, mix gently, ice bath for 30 minutes, heat shock at 42℃ for 90 seconds, quickly ice bath for 2 minutes, then add LB medium without antibiotics, shake and culture at 37℃ for 1 hour; spread the culture on LB plates containing corresponding antibiotics (such as Ampicillin, because the basic vector may carry the AmpR gene) and X-Gal, IPTG, and culture at 37℃ overnight; select white colonies, perform preliminary colony PCR screening, use specific primers to amplify the target fragment, and culture the positive colonies later; S5. The positive colonies obtained in step S4 are expanded and cultured, and the plasmid is extracted, digested, and sequenced to obtain the starting vector CMV-EGFP-MCS-SV40-Puromycin; specifically: The positive colonies screened were expanded and cultured, and plasmids were extracted using a plasmid extraction kit. The extracted plasmids were identified by restriction endonuclease digestion. The plasmids were digested with the previously introduced restriction endonucleases, and the digestion products were analyzed by agarose gel electrophoresis to observe whether the band size was in line with expectations. At the same time, sequencing identification can be performed, and the plasmids are sent to a sequencing company for sequencing and compared with the expected sequence to ensure that the vector construction is correct. If the sequence is correct, the starting vector CMV-EGFP-MCS-SV40-Puromycin is obtained.

[0053] 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 proved that the screened cells can highly express DLX6-AS1).

[0054] The main target cells for screening are one or more of human tooth germ mesenchymal stem cells and human dental pulp stem cells.

[0055] 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, wherein the dental stem cells are selected from DLX6-AS1 selected by using an expression vector containing a 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.

[0056] The multi-factor combination was selected to activate WNT6, WNT7B, and WNT10B for the WNT signaling pathway, and JAG1 for relaying activation of the NOTCH signaling pathway.

[0057] Example 2 This patented technology is based on the discovery that rhythmic regulation of the WNT-NOTCH signaling pathway can effectively promote DLX6-AS1 + Further research found that during tooth development, WNT-NOTCH signaling exhibits a relay phenomenon, guiding DLX6-AS1 + Differentiation of cells into odontoblasts.

[0058] The technical steps involved in this discovery include: 1) Initial activation of WNT signaling: WNT6, WNT7B, and WNT10B act as initial signals to activate DLX6-AS1 + WNT signaling pathway in cells; 2) Relay activation of NOTCH signaling: Subsequently, JAG1 acts as a ligand of NOTCH signaling, relaying WNT signaling and further promoting DLX6-AS1 + Cell differentiation.

[0059] Operation process: 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 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; 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: (1) Determine the promoter region of DLX6-AS1 and construct the target sequence: NR_015448-promoter; The NR_015448-promoter sequence (DLX6-AS1 promoter) is shown in SEQ ID No. 1:

[0060] (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; The final vector construction diagram based on GV814 replacing the promoter is shown in the figure Figure 8 As shown; (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: (a) Co-transfection: Cell preparation: HEK293T cells were selected and seeded into a suitable culture vessel so that the cells reached 70-80% confluence at the time of transfection; the cell culture conditions were 37°C, 5% CO 2 Incubator with medium suitable for cell growth (DMEM + 10% FBS); 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); Co-transfection operation: Mix the diluted transfection reagent with the diluted plasmid, mix gently and incubate at room temperature for a certain period of time (30 minutes), then add the mixture dropwise into the cell culture wells, and gently shake the culture plate to evenly distribute the mixture; (b) Virus collection: After a certain period of time (72 h) after cotransfection, the cell culture supernatant was collected and centrifuged at low speed (300 g , 10 min) to remove larger cell debris; (c) Virus Concentration: Use an ultrafiltration centrifuge tube (0.45μm), add the virus supernatant into 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 molecules of proteins, etc.) will pass through the ultrafiltration membrane, while the virus particles will be retained in the ultrafiltration tube.

[0061] (4) Infect the target cells with lentivirus and screen out the DLX6-AS1 promoter-positive cells to determine the cell population where the DLX6-AS1 promoter is active; when the promoter is active in the cell under endogenous conditions, it will drive the expression of the reporter gene. If the promoter activity is strong, 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; DLX6-AS1 + The cells have stronger odontogenic ability than unpurified odontogenic cells ( Figure 4 ); 3. The combination of WNT6+WNT7B+WNT10B+JAG1 can relay and activate the WNT-NOTCH signaling pathway and promote DLX6-AS1 + Cell odontogenicity: The isolated DLX6-AS1 + The cells were exposed to the combined stimulation of WNT6, WNT7B and WNT10B to activate the WNT signaling pathway; subsequently, JAG1 stimulation was added to activate the NOTCH signaling pathway, promoting the differentiation of cells into odontoblasts ( Figure 5 ); 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: 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 ); 5. Repair of tooth injury model: Construct a mouse open-pulp dentin injury 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 the regeneration of dentin ( Figure 7 );in, The method for establishing a mouse open-endodontic dentin injury model includes the following steps: (1) Animal preparation: Select adult male nude mice and fix them on the operating table after anesthesia; (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; (3) After pulp opening, appropriate wound treatment is performed (e.g., injection of DLX6-AS1 stimulated by WNT6, WNT7B, and WNT10B)+ dental pulp cells, combined with JAG1 stimulation), and then observed the recovery of the mice.

[0062] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present application should be included in the protection scope 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 table.

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. The method according to claim 2, characterized in that The starting vector CMV-EGFP-MCS-SV40-Puromycin was prepared according to the following steps: S1. Obtain the gene fragments of EGFP, MCS, SV40 promoter, and Puromycin resistance gene; S2. Select a basic vector, obtain a linearized vector backbone fragment after enzyme digestion, product separation and recovery; 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 connection product; S4. Transform the ligation product obtained in step S3 into competent cells, culture the colonies and screen out positive colonies; S5. Expand the positive colonies obtained in step S4, extract the plasmid, perform restriction digestion, and sequence identification to obtain the starting vector CMV-EGFP-MCS-SV40-Puromycin.

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

5. The expression vector according to claim 4, characterized in that The expression vector is NR_015448-promoter-EGFP-MCS-SV40-Puromycin.

6. The expression vector according to claim 4 or 5 is used in screening DLX6-AS1 + Application in cells.

7. The use according to claim 6, characterized in that: The target cells for screening are selected from human tooth germ mesenchymal stem cells or human dental pulp stem cells.

8. A multi-factor composition for regulating the WNT-NOTCH signaling pathway to improve the odontogenic ability of dental stem cells, characterized in that: The dental stem cells are selected as DLX6-AS1 selected by the expression vector of claim 4 or 5. + Human tooth germ mesenchymal stem cells or DLX6-AS1 + Human dental pulp stem cells.

9. The multi-factor composition according to claim 8, characterized in that: The composition is selected from WNT6 protein, WNT7B protein, WNT10B protein and / or JAG1 protein.

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