Biologicals containing c-kit and their use in the regulation of the development of tooth-derived organ tissues

Abstract

The application provides application of a c-kit gene and an overexpression vector thereof in preparation of a medicine for preventing and treating tooth-derived organs, including enamel development retardation / morphological deformity, and a nucleotide sequence of the c-kit gene is shown as SEQ ID No. 1. Compared with the prior art, the application has the following advantages: the application provides application of the c-kit gene and the overexpression vector thereof in preparation of the medicine for preventing and treating tooth-derived organ development retardation / morphological deformity, and a previously unidentified c-Kit-PI3K-Creb3l2 signal axis is recorded, which is crucial for normal tooth development; the path maintains the progenitor cell characteristics and proliferation capacity of dental epithelial cells, and ensures normal growth and pattern; the technology promotes the understanding of a tooth formation signal network, and is helpful for a wider field of regenerative dentistry, wherein manipulation of key signal pathways such as c-Kit and PI3K can provide information for tooth bioengineering or repair strategies.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a biological product containing c-Kit and application thereof in regulating development of odontogenic organ tissues. BACKGROUND

[0002] Tooth development or odontogenesis is a classic model for studying epithelial-mesenchymal interactions and the molecular mechanisms that control organogenesis. This highly coordinated process is orchestrated by multiple conserved signaling pathways, including BMPs, FGFs, SHH, and WNTs, which form a complex network to regulate cell proliferation, differentiation, and patterning. Disruption of these signaling cascades can lead to severe developmental defects, such as tooth agenesis, malformation, and other craniofacial abnormalities.

[0003] The receptor tyrosine kinase c-Kit (CD117) and its ligand stem cell factor (SCF) are recognized key regulators in many biological processes, including hematopoiesis, melanogenesis, and gametogenesis. Its role in stem cell maintenance and proliferation is well-documented. In the context of craniofacial and tooth development, c-Kit has been reported to be expressed in dental epithelium and mesenchyme, and mutations in the KIT gene are associated with gastrointestinal stromal tumors and piebaldism in humans. However, the precise functional role of c-Kit signaling in specific regions of the developing tooth bud and the detailed molecular pathways of its action have largely remained unexplored and poorly defined.

[0004] A key downstream signaling mediator commonly activated by c-Kit is the PI3K-Akt pathway. This pathway is a fundamental regulator of cell survival, growth, and metabolism. In developing organs, PI3K signaling is crucial for controlling progenitor cell dynamics. Moreover, the transcription factor CREB3L2 (cAMP response element-binding protein 3 Like 2), a member of the old astrocyte-specific inducible substance (OASIS) family, has recently been discovered as a potential effector of endoplasmic reticulum stress and PI3K signaling, involved in regulating collagen secretion and cell proliferation in certain tissues. However, its role in tooth development, particularly its potential link with c-Kit-PI3K signaling, has not been previously investigated. SUMMARY

[0005] In view of the above-mentioned technical limitations, the present application provides a biological product containing c-Kit and application thereof in regulating development of odontogenic organ tissues, and particularly relates to application of a c-kit gene and an overexpression vector thereof in preparing a medicine for preventing and treating odontogenic organ development retardation / morphological malformation. The present application overcomes the deficiencies and defects mentioned in the background art.

[0006] To achieve the above object, the present application adopts the following technical solutions:

[0007] The application point of the present application is the application of the c-kit gene and its overexpression vector in the preparation of a drug for preventing and treating the development retardation / morphological malformation of dental organs, wherein the nucleotide sequence of the c-kit gene is shown in SEQ ID No. 1.

[0008] SEQ ID No. 1 sequence:

[0009]

[0010] Optionally, the application, the c-kit gene overexpression vector is an adeno-associated virus vector AAV9-ckit overexpressing c-kit gene.

[0011] The receptor tyrosine kinase sequence encoded by the c-kit gene is shown in SEQ ID No. 2.

[0012] SEQ ID No. 2 sequence (NP_032443.2):

[0013] MTVATGDPVDEAAALPGHPQDTYDPEADHECCGRVVINISGLRFETQLKTLAQFPETLLGDPKKRMRYFDPLRNEYFFDRNRPSFDAILYYYQSGGRLRRPVNVPLDIFSEEIRFYELGEEAMEMFREDEGYIKEEERPLPENEFQRQVWLLFEYPESSGPARIIAIVSVMVILISIVSFCLETLPIFRDENEDMHGGGVTFHTYSNSTIGYQQSTSFTDPFFIVETLCIIWFSFEFLVRFFACPSKAGFFTNIMNIIDIVAIIPYFITLGTELAEKPEDAQQGQQAMSLAILRVIRLVRVFRIFKLSRHSKGLQILGQTLKASMRELGLLIFFLFIGVILFSSAVYFAEADERDSQFPSIPDAFWWAVVSMTTVGYGDMVPTTIGGKIVGSLCAIAGVLTIALPVPVIVSNFNYFYHRETEGEEQAQYLQVTSCPKIPSSPDLKKSRSASTISKSDYMEIQEGVNNSNEDFREENLKTANCTLANTNYVNITKMLTDV.

[0014] Optionally, the application, the nucleotide sequence of the AAV9-ckit is shown in SEQ ID No. 3.

[0015] SEQ ID No. 3:

[0016]

[0017] Optionally, the application described above, the drug prevents or treats the development retardation / morphological abnormalities of the dental organ by activating the PI3K-CREB3L2 transcriptional pathway in the dental organ through the c-kit gene, thereby enhancing the stemness and proliferation ability of the dental epithelial progenitor cells.

[0018] The second invention point of the present application is to provide the application of the receptor tyrosine kinase c-kit as a target for screening drugs for preventing and treating the development retardation / morphological abnormalities of the dental organ.

[0019] The third invention point of the present application is to provide the application of the reagent for detecting the level of tyrosine kinase c-kit in the preparation of early warning or clinical diagnosis reagent for the development retardation / morphological abnormalities of the dental organ.

[0020] The fourth invention point of the present application is to provide a drug for preventing and treating the development retardation / morphological abnormalities of the dental organ, wherein the drug contains a c-kit gene overexpression vector, and the nucleotide sequence of the c-kit gene is shown in SEQ ID No. 1.

[0021] Optionally, the drug described above, the c-kit gene overexpression vector is an adeno-associated virus vector AAV9-Ckit overexpressing the c-kit gene.

[0022] Optionally, the drug described above, the nucleotide sequence of the AAV9-Ckit is shown in SEQ ID No. 3.

[0023] Optionally, the drug described above, the drug contains an adeno-associated virus vector AAV9-Ckit-Gata2 overexpressing the c-kit gene and the GATA2 gene.

[0024] The overexpression vectors AAV9-Ckit and AAV9-Ckit-Gata2 are entrusted to Jikai Gene to construct, specifically AAV9-ckit (88352-6) (order number: GOSL0359423) and AAV9-Ckit-Gata2 (88353-1) (order number: GOSL0359424).

[0025] The sequence of AAV9-Ckit-Gata2 is shown in SEQ ID No. 4.

[0026] SEQ ID No. 4:

[0027]

[0028] Based on the reported expression of c-Kit in normal tooth germ epithelium and its role in the development of other organs, the applicant hypothesized that c-Kit signaling might also be involved in normal tooth formation and regulate progenitor cell populations through the PI3K pathway. To verify this hypothesis, the applicant first carefully mapped the expression pattern of c-Kit and its related signaling components in mouse tooth germs. Then, the applicant established a tooth epithelium-specific Kit conditional knockout mouse model to further determine its functional requirement in tooth formation. Further transcriptomic and functional analysis showed that c-Kit signaling is involved in tooth formation by regulating the PI3K-Creb3l2 axis, which in turn regulates the stemness and proliferative capacity of dental epithelial cells, thereby ensuring normal morphogenesis of dental epithelial cells and tooth growth.

[0029] Compared with the prior art, the present application has the following advantages:

[0030] The c-kit gene and its overexpression vector provided by the present application are used in the preparation of drugs for preventing and treating developmental retardation / morphological malformation of tooth-derived organs, which records a previously unrecognized c-Kit-PI3K-Creb3l2 signaling axis that is essential for normal tooth development; this pathway maintains the progenitor cell characteristics and proliferative capacity of dental epithelial cells, ensuring normal growth and pattern; this technology promotes the understanding of the signaling network of tooth formation, and may contribute to the wider field of regenerative dentistry, where manipulation of key signaling pathways such as c-Kit and PI3K can provide information for tooth bioengineering or repair strategies. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The expression pattern of c-kit in mouse molar and incisor bud stage, cap stage and bell stage is shown; wherein, Figure 1 A is the expression pattern of c-kit in the molar bud stage detected by RNAscope staining; Figure 1 B is the expression pattern of c-kit in the molar cap stage detected by RNAscope staining; Figure 1 C is the expression pattern of c-kit in the molar bell stage detected by RNAscope staining; Figure 1 D is the expression pattern of c-kit in the incisor bud stage detected by RNAscope staining; Figure 1 E is the expression pattern of c-kit in the incisor cap stage detected by RNAscope staining; Figure 1 F is the expression pattern of c-kit in the incisor bell stage detected by RNAscope staining (the image scale is 100 μm, and n=3 in each group).

[0032] Figure 2The construction experiment of epithelial specific kit gene knockout mice showed that the knockdown of c-kit gene in dental epithelium would cause tooth morphological malformation and tooth germ microsomia; Figure 2 A is the construction strategy of epithelial specific kit gene knockout mice; Figure 2 B and Figure 2 B' is Kit fl / fl and Krt 14Cre ; Kit fl / .fl Immunofluorescence staining and semi-quantitative analysis of c-kit in the middle of the tooth; Figure 2 C and Figure 2 C' is a visual image showing the development of PN4 mice and the effect of Kit fl / fl and Krt 14Cre ; Kit fl / .fl Semi-quantitative analysis of body length and body weight of mice; Figure 2 D is the incisor amputation test; Figure 2 E is an HE image showing Kit fl / fl and Krt 14Cre ; Kit fl / .fl Tooth development (the image scale is 100 μm, n=3 in each group; the data represent mean ± SEM, n=3 in each group; *P<0.05, **P<0.01, P<0.001, the statistical analysis of B', C' components was performed by unpaired t test).

[0033] Figure 3 Verification of gene knockout mice and gross phenotype are shown;

[0034] Among them, Figure 3 A is to detect the genotype of mice by gel electrophoresis; Figure 3 B is a micro-CT image showing Kit fl / fl and Krt 14Cre ; Kit fl / .fl Tooth development of 8-week-old mice; Figure 3 C is a negative control of secondary antibody; Figure 3 D' and Figure 3 D" is a morphological image showing the development of PN21 mice and the effect of Kit fl / fl and Krt 14Cre ; Kit fl / .flMouse body length and weight were semi-quantitatively analyzed (n=3 per group; data represent mean ± SEM, statistical analysis between D', D" groups was performed using unpaired t test, *P<0.05, **P<0.01, P<0.001).

[0035] Figure 4 PI3K signaling pathway was shown to be down-regulated in D' and D" groups, respectively. Kit fl / fl and Krt 14Cre ; Kit fl / .fl Down-regulated in D' and D" groups, respectively, wherein

[0036] Figure 4 A is based on Kit fl / fl and Krt 14Cre ; Kit fl / .fl E12.5 fetal mouse tooth dental epithelium (DE) bulk RNA sequencing sample schematic (n=3 per group) and kit gene knockout strategy was verified by genotyping snapshot;

[0037] Figure 4 B is a bar chart showing the differentially expressed transcripts obtained by bulk RNA sequencing analysis Kit fl / fl and Krt 14Cre ; Kit fl / .fl Differentially expressed transcripts between groups;

[0038] Figure 4 C is a volcano plot showing Kit fl / fl and Krt 14Cre ; Kit fl / .fl Up- and down-regulated genes between groups;

[0039] Figure 4 D is Kyoto Gene and Genome Encyclopedia pathway enrichment analysis shows that the PI3K pathway is at the top of the list of enriched pathways of differentially expressed genes in the two groups;

[0040] Figure 4 E and Figure 4 E' is gene set enrichment analysis shows that the expression of the PI3K signaling pathway is down-regulated in Kit fl / fl and Krt 14Cre ; Kit fl / .fl Down-regulated in D' and D" groups, respectively;

[0041] Figure 4E" is the quantitative reverse transcription polymerase chain reaction comparison Kit fl / fl and Krt 14Cre ; Kit fl / .fl The mRNA expression levels of PI3K pathway genes in the groups (n = 3 for each group; data represent mean ± SEM, statistical analysis was performed using unpaired t test, *P < 0.05, **P < 0.01, P < 0.001).

[0042] Figure 5 The results of the analysis of the epithelial sequencing after the knockout of kit are shown;

[0043] wherein, Figure 5 A is the principal component analysis of all expressed genes, each data point represents one mouse; Figure 5 B is a heatmap showing Kit fl / fl and Krt 14Cre ; Kit fl / .fl The top 20 up- and down-regulated genes in the groups; Figure 5 C is the GO enrichment analysis; Figure 5 D is the Reactome pathway enrichment analysis.

[0044] Figure 6 A and Figure 6 A' is the quantitative reverse transcription polymerase chain reaction comparison Kit fl / fl and Krt 14Cre ; Kit fl / .fl The mRNA expression levels of Ki67, Sox2, Heyl, Enpp6, Chitl and Gldc genes in the groups (n = 3 for each group; data represent mean ± SEM, statistical analysis was performed using unpaired t test, *P < 0.05, **P < 0.01, P < 0.001).

[0045] Figure 7 The expression levels of the genes associated with cell proliferation and stemness are significantly reduced in the experimental group compared to the control group, wherein Enpp6, Heyl and Creb3l2 expression is significantly down-regulated, and Gldc and Chitl expression is significantly up-regulated; wherein,

[0046] Figure 7 A, Figure 7 A', Figure 7 B, Figure 7 B, Figure 7 C, Figure 7 C' is Kitfl / fl and Krt 14Cre ; Kit fl / .fl immunofluorescence staining of Ki67 in the group;

[0047] Figure 7 D, Figure 7 D', Figure 7 E, Figure 7 E', Figure 7 F, Figure 7 F' is Kit fl / fl and Krt 14Cre ; Kit fl / .fl immunofluorescence staining of Sox2 and K14 in the group;

[0048] Figure 7 G, Figure 7 G', Figure 7 H, Figure 7 H', Figure 7 I, Figure 7 I' is Kit fl / fl and Krt 14Cre ; Kit fl / .fl immunofluorescence staining of Enpp6 and Heyl in the group;

[0049] Figure 7 J, Figure 7 J', Figure 7 K, Figure 7 K', Figure 7 L, Figure 7 L' is Kit fl / fl and Krt 14Cre ; Kit fl / .fl immunofluorescence staining of Gldc and Creb3l2 in the group;

[0050] Figure 7 M, Figure 7 M', Figure 7 N, Figure 7 N', Figure 7 O, Figure 7 O' is Kit fl / fl and Krt 14Cre ; Kit fl / .fl immunofluorescence staining of Chitl and K14 in the group;

[0051] Figure 7 P,Figure 7 Q, Figure 7 R, Figure 7 S, Figure 7 T, Figure 7 U is the semi-quantitative analysis of Ki67, Sox2, Heyl, Enpp6, Gldc and Creb3l2 (n=3 per group; data represent mean ± SEM, statistical analysis was performed using unpaired t test, *P<0.05, **P<0.01, P<0.001).

[0052] Kit showed the semi-quantitative analysis of Chitl protein (n=3 per group; data represent mean ± SEM, statistical analysis was performed using unpaired t test, *P<0.05, **P<0.01, P<0.001).

[0053] Krt AAV9-kit gene compensation can partially rescue the phenotype of kit gene deficiency; wherein, Kit A is a schematic diagram of rescuing epithelial cell kit gene deficiency by tail vein injection; Figure 7 B is HE staining showing the cervical loop of the incisor of PN4 mice in Figure 7 fl / .fl Group, Figure 7 14Cre ; Figure 7 fl / .fl developmental status of Group and AAV9-Kit group; Figure 7 C- Figure 7 C' is immunofluorescence staining showing the expression of Sox2 and Ki67 in the cervical loop of the incisor of PN4 mice in Kit fl / .fl Group, Krt 14Cre ; Kit fl / .fl expression and semi-quantitative analysis of Group and AAV9-Kit group; Figure 7 D is a schematic diagram of the mechanism of c-kit regulating tooth germ morphogenesis through the PI3K-Creb3l2 signaling pathway.

[0054] Figure 7 showed that kit combined with Gata2 combined to rescue gene deficiency; wherein,

[0055] Figure 7 A is a schematic diagram of rescuing epithelial cell kit gene deficiency by tail vein injection;

[0056] Figure 7 B is HE staining showing the cervical loop of the incisor of PN4 mice in Figure 7 fl / .fl Group, Figure 7 14Cre ; Kitfl / .fl Developmental status of the AAV9-Kit group and the AAV9-Kit-Gata2 group;

[0057] Krt C- Kit C' is the MicroCT image showing the cervical loop of incisor in PN4 mice of the AAV9-Kit group and the AAV9-Kit-Gata2 group. Figure 7 fl / .fl Group, Figure 7 14Cre ; Figure 7 fl / .fl Developmental status of the AAV9-Kit group and the AAV9-Kit-Gata2 group;

[0058] Figure 7 D is a schematic diagram showing the mechanism of c-kit regulating tooth germ morphogenesis through the PI3K-Creb3l2 signaling pathway. DETAILED DESCRIPTION

[0059] In order to make the purpose, technical solutions and advantages of the present application more clear and obvious, 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.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The reagents and instruments used herein are commercially available, and the characterization means involved can be referred to the related description in the prior art, which will not be described herein.

[0061] In order to further understand the present application, the present application is further described in detail below in combination with the best embodiments.

[0062] Example 1

[0063] 1. Materials and methods

[0064] 1.1 Animal study

[0065] The following animal experiments were approved by the Capital Medical University Animal Care and Use Committee (Beijing, China; license number: AEEI-2023-245). C57BL / 6J-Kitem1Cflox / Cya and C57BL / 6J-K14cre mice were obtained from Beijing Cyagen Company. The amplified region of F2 / R2 was the 5' arm end loxp site, which produced a 207 bp fragment; the amplified region of F1 / R1 was the 3' arm end loxp site, which produced a 200 bp fragment. When both positive bands were amplified, the targeting was fully indicated. The K14Cre tool mouse was crossed with the (flox / flox) homozygous mouse to obtain the [flox / +, Cre(+ / -)] mouse. The primers are shown in Table 1. The pregnant mice were taken at embryonic day 13.5 (E13.5), day 15.5, day 17.5, and postnatal day 4 (PN4).

[0066] Table 1

[0067]

[0068] 1.2 Morphological analysis of dental germ layer

[0069] The mandibles of E13.5, E15.5, E17.5, and PN4 groups of mice were collected. The visual image of the mouse was obtained with a stereomicroscope (Leica, Germany, MZ9.5) to obtain its approximate shape. Hematoxylin and eosin (HE) staining was used to analyze the cell morphology of the first molar (M1) and incisor of the decalcified mandible.

[0070] 1.3 Single molecule fluorescence in situ hybridization (smFISH)

[0071] Single molecule fluorescence in situ hybridization (smFISH) experiments were performed using the PinpoRNATM multi-channel fluorescence reagent kit (Pinpo PIF2000) to detect E13.5, E15.5, and E17.5 (Leica) 6 μm thick paraffin tooth sections, c-kit probes (Pinpo 165901-B1), negative control probes (Pinpo P0009), and positive control probes (Pinpo P0033) were detected according to the kit instructions. Images were obtained under an AX NIS-Elements 5.4 microscope at 20x and 40x. Image processing was performed using Fiji in ImageJ, and statistical data generation and processing were performed using GraphPad Prism 10.

[0072] 1.4 Immunofluorescence staining

[0073] Slices were treated with degumming and rehydration, followed by antigen retrieval. After blocking, the slices were incubated with primary antibodies overnight at 4°C, and then incubated with secondary antibodies for 1 hour at 37°C. Images were obtained using a confocal microscope (AX NIS-Elements 5.4; Nikon, Japan). The primary and secondary antibodies used are listed in Table 2.

[0074] Table 2

[0075]

[0076] 1.5 RNA sequencing and analysis

[0077] E12.5 tooth germs were digested with 30 mg / mL disase II, and tooth germs were isolated to obtain dental epithelial tissues. Total RNA was extracted from 6 tooth samples using RNeasy Micro Kit (Qiagen, Germany), and then purified using RNAClean XP Kit (Beckman Coulter, Inc, USA) and DNase I treatment (Qiagen). RNA-seq was constructed on the Illumina HiSeq2500 platform to construct libraries and perform sequencing.

[0078] 1.6 RT-qPCR

[0079] After three days of culture, total RNA was extracted from the cells using RNAprep Pure Cell / Bacteria Kit (DP430; TIANGEN, China). Reverse transcription was performed using FastKing gDNA diselling RT SuperMix (KR118; TIANGEN, China). Three qPCRs were performed using NovoStart SYBR qPCR SuperMix Plus (Novoprotein). GAPDH was used as an internal reference gene. Quantitative analysis was based on three measurements, and the relative expression level was determined using the 2-ΔΔCT method. The primer sequences (SEQ ID No. 11- SEQ ID No. 66) are shown in Table 3.

[0080] Table 3

[0081]

[0082] 1.7 Hydrodynamic tail vein injection

[0083] Krt 14Cre ; Kit fl / flTo facilitate the identification of the tail vein, liquid-powered tail vein injection was used. pAAV9-mKit-cherry and control virus (2 × 1013genome copies / kg, intravenous injection, pAAV9-mKit-cherry and control virus were purchased from Cyagen, China) were injected into the tail vein (5 mice in each group). The volume of sterile saline was 10% of the body weight, and the injection time was 8 ~ 10s. The mouse serum was collected at PN4 after injection, and the concentration of Kit in the serum was determined by ELISA.

[0084] 1.8 Statistical analysis

[0085] The data of three independent experiments were expressed as mean ± standard deviation. One-way ANOVA was performed by Prism 10 to evaluate statistical significance. Before one-way ANOVA, the data were evaluated for normality (one-sample Kolmogorov-Smirnov) and homogeneity of variance. p < 0.05 was considered statistically significant.

[0086] 2、Results

[0087] 2.1 C-Kit is highly expressed in the epithelium of the bud stage, cap stage and bell stage of tooth germ development.

[0088] The c-Kit pathway plays an important role in the development of many mammalian cells and organs. The research group previously analyzed the expression pattern of c-Kit in salivary gland epithelium from embryonic to mature stages. To further study its expression and mechanism in tooth development, the bud, cap and bell stages of tooth germ development were first selected, and the spatiotemporal expression profile of c-Kit was verified by RNAscope staining. C-kit was mainly expressed in the basement membrane of M1 bud and cap stage ( Figure 7 A, B), and in the inner enamel epithelium (IEE) of bell stage ( Figure 7 C). For incisors, c-kit was mainly expressed in the initial knot (IK) of incisor bud stage ( Kit D), in the enamel knot (EK) and IEE of tooth cap stage, especially in EK ( Krt E), and in IEE of bell stage ( Kit F).

[0089] 2.2 c-Kit-specific epithelial knockout can cause delayed development and morphological abnormalities of tooth-derived organs

[0090] To further study the role of c-Kit in tooth germ development, C57BL / 6J-Kitem1Cflox / Cya was crossed with C57BL / 6J-K14cre mice to produce epithelial-specific knockout mice. Exon 5 of the c-Kit gene was specifically knocked outFigure 7 A and Figure 7 A). In addition, we verified that the c-kit gene was successfully knocked out at the transcriptional and protein levels of the dental epithelium ( Figure 7 B-B'). At the same time, the naked eye observation and body weight measurement results showed that the gene knockout mice not only had a significantly smaller body size, but also had a significantly lower body weight than the wild type nest mice ( Figure 7 C-C' and Figure 7 C-D'). Through the incisor mechanical injury model, it was found that the incisor regeneration of the gene knockout mice was delayed ( Figure 7 D). Through HE staining observation, the M1 tooth germ of the knockout group was shorter and smaller from the bud stage to the bell stage, and the development was delayed.

[0091] 2.3 c-Kit regulates the development of dental epithelium through the PI3K signaling pathway.

[0092] In order to further study its mechanism, E12.5 Kit fl / fl and Krt 14Cre ; Kit fl / .fl RNA-seq sequencing was performed on mice. Further confirmed by snapshot, the c-Kit gene in the dental epithelium was successfully knocked out ( Figure 7 A and Figure 7 A). In Figure 7 fl / fl and Figure 7 14Cre ; Figure 7 fl / .fl A total of 1020 transcription factors were found to be significantly different between groups, of which 490 were significantly up-regulated and 530 were significantly down-regulated ( Figure 7 B and Kit A). The top 5 up-regulated genes were Chit1, Gldc, Nef1, Tfcp2l1 and Scg2, and the top 5 down-regulated genes were Gm36101, Hey1, Enpp6, Insc and Rspo4 ( Krt C and Kit B). Subsequent pathway enrichment analysis showed that the PI3K pathway was the most significantly down-regulated signaling pathway, and the transcription factor Creb3l2 in this pathway was also significantly down-regulated ( Figure 7 D-E' and Figure 7 C-D). At the same time, we further verified the sequencing results by RT-PCR and immunofluorescence experiments ( Figure 7 E", Figure 7 , Figure 7 and Figure 7 ). In addition, the proliferation ability and stemness characteristics of epithelial cells also showed significant down-regulation ( Kit A-F').

[0093] 2.4 Intravenous injection of c-Kit virus partially rescues tooth development in mice.

[0094] To further rule out non-specific interference, morphological changes were observed in mice by tail vein injection of AAV9-kit Krt A). By observing the incisor ring, AAV9-kit complement can partially rescue the development of the cervical loop (CL) of PN4 mice Kit B). Compared with Krt14Cre; the rescue group cells have significantly enhanced stemness and proliferation ability by expression of Sox2 and Ki67. In addition, its morphological development is more comparable to the WT group.

[0095] The experiments described in Example 1 show that epithelial c-Kit signaling is a key regulator of tooth morphogenesis, mainly by activating the downstream PI3K-Creb3l2 transcriptional pathway. The results of this experiment not only establish a functional link between c-Kit and key developmental processes in the tooth germ, but also reveal a previously unrecognized mechanism linking receptor tyrosine kinase activity to the maintenance of dental epithelial progenitor cells.

[0096] The severe developmental defects observed in this epithelium-specific Kit-cKO mouse, including significant developmental delay and abnormal tooth germ pattern, strongly support the indispensable role of c-Kit in tooth formation. This is consistent with previous reports of c-Kit expression in the tooth germ, but for the first time provides direct functional evidence of its specific requirement within the dental epithelium. Phenotypic abnormalities, particularly in morphogenesis, indicate that c-Kit is involved in the regulation of the enamel knot signaling center, which is responsible for directing morphogenesis. The absence of c-Kit can disrupt the precise spatiotemporal signaling network that affects morphogenesis.

[0097] Transcriptomic analysis shows that the PI3K signaling pathway is significantly down-regulated after Kit ablation. This finding is highly consistent with the established role of c-Kit as an upstream activator of the PI3K-AKT signal in various biological contexts. The innovation of the present application lies in identifying Creb3l2 as a key transcription factor downstream of this pathway during tooth development. The significant decrease in Creb3l2 expression in the Kit-cKO model indicates that it is the main effector that mediates the function of c-Kit / PI3K during tooth formation. Although Creb3l2 has been previously associated with stress response and collagen biosynthesis in secretory cells, its role in organ morphogenesis, particularly in dental progenitor cells, has not been reported. It is proposed that Creb3l2 is a new transcriptional effector that links mitogenic signals from c-Kit / PI3K to the maintenance of epithelial integrity and proliferation.

[0098] Furthermore, the simultaneous reduction in stem cell marker expression and the marked reduction in cell proliferation in Kit-cKO tooth germs highlights the c-Kit-PI3K-Creb3l2 axis as an important mechanism regulating tooth development. The dental epithelium contains progenitor cells that are critical for the continued growth and morphogenesis of the tooth. The experimental data suggest that disruption of c-Kit signaling impairs the maintenance of the progenitor cell pool, leading to developmental failure and morphogenetic failure. This is consistent with the known function of PI3K signaling in promoting cell survival and proliferation in developing tissues and extends it to the specific context of dental epithelial stem cells.

[0099] While the present application focuses on the epithelial component, it is important to note that tooth development relies on continuous epithelial-mesenchymal interactions. And while the model of the present application is specifically directed at epithelial Kit, the observed defects can indirectly affect mesenchymal signaling.

[0100] Example 2

[0101] The present technology can also be used in combination with a c-kit gene overexpression vector and a GATA2 gene overexpression vector, or a way to construct an adeno-associated virus vector AAV9-Ckit-Gata2 that can overexpress c-kit gene and GATA2 gene at the same time, to achieve the purpose of preventing and treating developmental retardation / morphological malformation of tooth-derived organs, as shown in the results of Figure 7 .

[0102] The specific operation process is as follows:

[0103] I. Pre-experiment preparation: check the experimental materials and reagents;

[0104] Vector preparation: confirm that the titer of AAV9-Ckit-Gata2 virus vector is 1x10 12 ~1x10 13 vg / mL, and store it on ice in the dark (AAV virus is sensitive to temperature, avoid repeated freezing and thawing).

[0105] Animal preparation: select 7-day pregnant mice for tail vein injection.

[0106] Instruments and reagents: prepare 1 mL insulin syringe, 75% ethanol cotton ball, sterile normal saline, sterile gauze, constant temperature pad, animal fixer.

[0107] Sterile and safety protection:

[0108] Wipe the experimental bench with 75% ethanol for disinfection, the syringe and needle are sterile disposable supplies to avoid cross contamination;

[0109] The operator wears sterile gloves and a mask, and if the vector is a virus, the dilution and extraction of the vector should be completed in a biological safety cabinet.

[0110] II. Mouse tail vein injection procedure:

[0111] 1. Mouse fixation and tail vein exposure:

[0112] Place the mouse into the fixator, adjust the tightness of the fixator so that the mouse's tail is fully extended out of the fixator.

[0113] Use a 75% ethanol cotton ball to repeatedly wipe the mouse's tail, and gently massage the tail to promote tail vein congestion.

[0114] 2. Use an insulin syringe to extract the target volume of carrier, and expel the air bubbles in the syringe after extraction.

[0115] 3. Tail vein puncture and injection:

[0116] Hold the syringe by hand, with the needle bevel facing upwards, and insert the needle from the tail tip 1 / 3~1 / 2; control the angle of insertion at 10°~15°, slowly push in a small amount of carrier about 20~30 μL, and observe whether there is a "white strip" in the tail blood vessels.

[0117] After confirming successful puncture, uniformly push the remaining carrier, with the injection time controlled at 10~20 seconds, to avoid rapid injection causing blood vessel rupture. If skin swelling is found in the tail during the injection process, indicating that the needle has pierced the blood vessel and the liquid has leaked subcutaneously, stop the injection immediately, replace the tail end or puncture the opposite blood vessel again (A). Figure 7 A).

[0118] 4. Post-injection treatment:

[0119] After injection, quickly pull out the needle, and gently press the puncture site with sterile gauze for 30~60 seconds to avoid bleeding or carrier leakage, without the need for bandaging.

[0120] Remove the mouse from the fixator and place it back in the feeding cage, observe for 10~15 minutes to confirm that the mouse has no abnormal reactions such as rapid breathing, convulsions, etc., and record the injection time, carrier volume, titer, etc. of each mouse.

[0121] III. Post-injection monitoring and sample collection:

[0122] Short-term monitoring (1~7 days):

[0123] Observe the general state of the mouse daily: body weight changes, food and water intake, activity ability, tail puncture site, and if abnormalities occur, record and evaluate whether to remove the mouse in time.

[0124] The mice were sacrificed at the experimental design time points, and tooth-derived organ tissues such as tooth germs, jaw bones, etc. were collected. The transduction efficiency of the vector was verified by detecting the expression of c-kit and GATA2 proteins by immunohistochemistry and detecting the mRNA expression of the target gene by real-time fluorescent quantitative PCR. At the same time, the development of the tooth-derived organs was observed by HE staining of tissue sections and Micro-CT scanning, and the prevention and treatment effect was evaluated.

[0125] The results of HE staining are shown in Figure 7 B, which are the cervical loops of incisors of PN4 mice in Figure 7 fl / .fl group, Figure 7 14Cre ; Figure 7 fl / .fl group; the development state of the AAV9-Kit group and the AAV9-Kit-Gata2 group;

[0126] The results of Micro-CT scanning are shown in Kit C- Krt C', which are the cervical loops of incisors of PN4 mice in Kit fl / .fl group, Figure 7 14Cre ; Figure 7 fl / .fl group; the development state of the AAV9-Kit group and the AAV9-Kit-Gata2 group;

[0127] From the result graphs of the above two, it can be seen that the development state of the AAV9-Kit-Gata2 group is better than that of the AAV9-Kit group, and has approached that of the Figure 7 fl / .fl group, which means that the adenovirus vector AAV9-Ckit-Gata2 which simultaneously overexpresses the c-kit gene and the GATA2 gene has indeed achieved the prevention and treatment of the delayed development / morphological abnormalities of the tooth-derived organs.

[0128] The mechanism of their joint action is shown in Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Kit Krt Kit Figure 7 Figure 7 Figure 7 D, which should be that the tooth germ morphogenesis is regulated through the PI3K-Creb3l2 signal pathway, and the development of the tooth-derived organs is promoted.

[0129] The above only describes the preferred embodiments of the present application and does not limit the present application. Any modification, equivalent replacement or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. Use of c-kit gene and its overexpression vector in the preparation of a medicament for preventing and treating the development retardation or morphological abnormality of dental organs, including enamel, characterized in that, The nucleotide sequence of the c-kit gene is shown as SEQ ID No.

1.

2. Use according to claim 1, characterized in that, The c-kit gene overexpression vector is an adeno-associated virus vector AAV9-ckit overexpressing the c-kit gene.

3. Use according to claim 2, characterized in that, The drug prevents or treats the developmental retardation or morphological malformation of the dental organ by activating the PI3K-CREB3L2 transcription pathway in the dental organ through the c-kit gene, and then enhancing the stemness and proliferation capacity of the dental epithelial progenitor cells.

4. A medicament for preventing the development retardation or morphological abnormality of tooth-derived organs, characterized by, The drug contains an adeno-associated virus vector AAV9-Ckit-Gata2 overexpressing the c-kit gene and the GATA2 gene simultaneously; and the sequence of the adeno-associated virus vector AAV9-Ckit-Gata2 is shown as SEQ ID No. 4.

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