Preparation and application of ywhah active polypeptide

Abstract

The application discloses a YWHAH active polypeptide and a preparation method and application thereof, and belongs to the field of biological medicine and tissue engineering. The polypeptide sequence is LRDNLTLWTSDQQDE or TLWTSDQQDEEAGEG, which can target and block the GREM1 / YWHAH protein complex, and release the inhibition of GERM1 on the BMP signal pathway. Experiments prove that the polypeptide can significantly improve the osteogenic and dentinogenic differentiation ability of dental pulp mesenchymal stem cells, up-regulate the alkaline phosphatase activity and cell mineralization level, promote the expression of osteogenic and dentinogenic marker genes, and induce the formation of bone-like and dentin-like new tissues in vivo. The polypeptide has the advantages of small molecular weight, convenient chemical synthesis, good physicochemical stability, weak immunogenicity and the like, and can be used in stem cell culture preparations, composite tissue engineering scaffolds, local injection drugs and various dosage forms, and has a wide application prospect in various hard tissue defect repair scenes.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and tissue engineering, specifically to dental tissue regeneration technology, and more particularly to an active polypeptide for improving the performance of dental stem cells and promoting the regeneration of biological tooth roots, and its applications. Background Technology

[0002] Tooth loss is a common clinical condition in dentistry, severely impacting patients' physiological functions such as chewing and speech, and may even lead to facial deformities and psychological disorders, significantly reducing their quality of life. In the field of dental restoration, total tooth regeneration is considered the most ideal treatment goal. Its technical approaches are mainly divided into two categories: one is based on developmental biology principles, where dental mesenchymal cells and dental epithelial cells are recombined in vitro and then implanted into the body to induce regeneration according to the histological process of tooth development; the other is tissue engineering methods, which combine seed cells with a pre-designed biological scaffold with a complete tooth morphology, aiming to regenerate a substitute similar in appearance, structure, and function to a normal tooth.

[0003] However, both of the aforementioned total tooth regeneration strategies face technical bottlenecks. From an anatomical perspective, the tooth root is the core component supporting the tooth and performing chewing functions. Therefore, utilizing dental stem cells and biological scaffold materials to regenerate "biological tooth roots" with physiological anatomy and function similar to natural tooth roots through bioengineering principles, and then performing crown restorations on these roots, has become a more feasible alternative. Compared to total tooth regeneration, biological tooth root regeneration avoids technical challenges such as controlling tooth shape and eruption, while also avoiding the shortcomings of traditional implant dentures, such as lack of bioactivity and the presence of metallic foreign bodies. Furthermore, regenerated tooth roots possess periodontal ligament and dentin structures, enabling dual morphological and functional restoration, thus possessing unique advantages in dental tissue engineering research. However, currently, biological tooth root regeneration still faces problems such as low success rates, long regeneration cycles, and difficulties in reconstructing neurovascular function.

[0004] To address the aforementioned issues, improving the performance of seed cells (especially stem cells) is a crucial challenge that urgently needs to be overcome in this field. During the in vitro isolation, culture, and application of stem cells, the inability to fully replicate the in vivo microenvironment often leads to alterations in the self-renewal and differentiation capabilities of stem cells, thereby affecting regenerative efficacy. The stem cell microenvironment is not only a structural site but also plays a vital role in regulating the function and maintaining the stemness of mesenchymal stem cells. Different microenvironments, by regulating gene expression, ultimately determine the effectiveness of stem cell-mediated tissue regeneration. Therefore, in the in vivo regeneration process, if the relevant gene expression regulation in the microenvironment can be reproduced at the stem cell level, it will help optimize the functional state of stem cells and promote tissue regeneration.

[0005] Gene chip and bioinformatics analysis revealed significant changes in the expression of 2325 genes in cultured apical papilla stem cells (SCAPs) compared to apical papilla tissue. Among these, the expression of BMP2, BMP5, BMP6, and BMP7, members of the bone morphogenetic protein (BMP) family, was significantly downregulated, while the antagonistic gene Gremlin1 (GREM1) of the BMP signaling pathway was significantly upregulated in both cultured SCAPs and their cell sheets. Of these changes, BMP6 expression showed the most significant difference, and previous experiments have confirmed that BMP6 can significantly enhance the in vitro proliferation capacity and osteogenic / odontogenic differentiation function of SCAPs. Simultaneously, knocking down the GREM1 gene promoted the osteogenic / odontogenic differentiation function of SCAPs and upregulated the expression of BMP2, BMP6, and BMP7.

[0006] The GREM1 gene was initially cloned from the Xenopus laevis central nervous system spinal cord. The human GREM1 gene is located on chromosome 15q13-q15 and belongs to the BMP antagonist family. Its encoded protein has an 8-amino acid ring cysteine ​​loop at its C-terminus. Studies have shown that GREM1, as an endogenous BMP antagonist, can bind to BMP extracellularly and inhibit BMP secretion and function intracellularly. Its mechanism involves inhibiting Smad1 / 5 / 8 phosphorylation to block the classical BMP signaling pathway, and regulating the p38, ERK, and JNK pathways to inhibit the non-classical BMP signaling pathway. Furthermore, immunofluorescence experiments showed that the GREM1 protein is also localized in the nucleus of SCAPs, suggesting that it may affect the transcription of BMP2, BMP6, and BMP7 through a novel intranuclear regulatory mechanism, leading to changes in their mRNA levels. Further research revealed that GREM1 can form a complex with the YWHAH protein. YWHAH belongs to the 14-3-3 family and can bind to the transcription factor YAP in the cell nucleus. YAP has the function of regulating BMP transcription and osteogenic differentiation of mesenchymal stem cells. Experimental results show that knockdown of YWHAH in dental pulp stem cells (DPSCs) inhibits their osteogenic / odontogenic differentiation in vitro, while overexpression of YWHAH promotes this differentiation process.

[0007] However, the full-length YWHAH protein has a large molecular weight, making its preparation using recombinant protein technology difficult, resulting in strong immunogenicity. Furthermore, the protein structure is unstable and prone to degradation during transportation and storage. Therefore, the full-length YWHAH protein is not suitable as a drug candidate. In contrast, peptide drugs offer advantages such as convenient preparation, ease of modification, and well-defined effects. They can also overcome degradation and dilution issues through anchoring modifications, demonstrating unique application advantages.

[0008] However, there are currently no reports on using active peptides derived from YWHAH protein to regulate osteogenic / odontogenic differentiation of dental pulp mesenchymal stem cells and applying them to tooth regeneration and hard tissue repair. Summary of the Invention

[0009] To address the problems existing in the prior art, this invention designs and synthesizes a YWHAH active peptide. Experimental results show that the YWHAH active peptide can significantly promote osteogenic / odontogenic differentiation of dental pulp mesenchymal stem cells. The active peptide of this invention has the advantages of small molecular size, high stability, strong binding force, and ease of synthesis, providing a novel peptide drug for tooth regeneration. It can be applied to stem cell culture, tissue engineering scaffolds, and local injection formulations to improve regeneration efficiency, and has broad application prospects in the fields of tooth regeneration, osteoporosis, and other hard tissue regeneration.

[0010] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One objective of this invention is to provide an active polypeptide derived from YWHAH, wherein the amino acid sequence of the active polypeptide has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0011] Preferably, the amino acid sequence of the active polypeptide has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0012] More preferably, the amino acid sequence of the active polypeptide is as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0013] A second objective of this invention is to provide the application of the aforementioned active polypeptide in the preparation of products for treating bone-related diseases.

[0014] Preferably, the product includes a drug or a reagent kit.

[0015] Preferably, the bone-related diseases include osteoporosis.

[0016] Preferably, the bone-related diseases include dental tissue diseases.

[0017] More preferably, the dental tissue disease includes tooth loss, root defect, or dentin defect.

[0018] Preferably, the product can promote osteogenic differentiation and / or odontogenic differentiation of dental mesenchymal stem cells, enhance ALP activity, mineralization capacity and osteogenic / odontogenic gene expression, and promote the regeneration of osteoid and dentin-like tissues in vivo.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. Peptide drugs (usually referring to small molecule peptides formed by 2-50 amino acids linked by peptide bonds) and protein drugs (usually referring to large molecule proteins composed of more than 50 amino acids) both belong to the category of biological macromolecules. The former has several significant advantages over the latter. Peptide drugs have small molecular weight, simple structure, easily regulated spatial conformation, better stability, and are less prone to denaturation and inactivation; their preparation processes are mature and controllable, chemical synthesis allows for precise control of purity and modification sites, and large-scale production has low cost and short R&D cycle, which is superior to the complex gene engineering expression and purification processes of protein drugs.

[0020] 2. In terms of pharmacokinetics, peptide drugs are more likely to cross biological barriers, have high absorption efficiency and more flexible administration methods, strong distribution and targeting, reasonable metabolism and excretion, and are less likely to accumulate in the body; they have extremely low immunogenicity, good compatibility with the human body, better safety, strong modifiability, and greater application flexibility, can adapt to the treatment needs of a variety of diseases and can work synergistically with other drugs, making them more suitable for large-scale production and precision treatment, and with broader clinical application prospects.

[0021] 3. This invention provides, for the first time, an active polypeptide derived from YWHAH protein and its application in the field of tooth regeneration. Experiments show that YWHAH-peptide46 and YWHAH-peptide47 of this invention can target and block the binding of the GREM1 / YWHAH protein complex, relieve the inhibitory effect of GREM1 on the BMP signaling pathway, and significantly promote osteogenic / odontogenic differentiation of dental pulp stem cells. This invention fills the technological gap in utilizing YWHAH-derived active polypeptides to regulate the differentiation of dental pulp mesenchymal stem cells and applying them to the field of tooth regeneration. Attached Figure Description

[0022] Figure 1 The image shows the detection results of the binding sites of GREM1 and YWHAH protein in this invention. In the image, A is the hybridization signal image after the YWHAH peptide microarray chip and GREM1 protein are incubated, and the arrows indicate the positive binding sites. B is the quantitative analysis result of the spot density value of the corresponding positive binding site.

[0023] Figure 2 Here are the chemical structural formulas of YWHAH-peptide46 / 47 and YWHAH-peptide3 designed and synthesized in this invention, wherein A is YWHAH-peptide3; B is YWHAH-peptide46; and C is YWHAH-peptide47.

[0024] Figure 3 The image shows the CO-IP experimental results of YWHAH-peptide46 / 47 in this invention, where GAPDH is an internal reference.

[0025] Figure 4 The image shows the effect of YWHAH-peptide46 / 47 in promoting osteogenic / odontogenic differentiation of dental pulp stem cells in this invention. In the image, A is a bar chart of quantitative ALP activity and B is a photograph stained with Alizarin Red.

[0026] Figure 5 This is a Real-time RT-PCR quantitative bar chart showing the expression of osteogenic / odontogenic related genes in dental pulp stem cells promoted by YWHAH-peptide46 / 47 7 days after osteogenic induction in this invention. AE corresponds to the mRNA expression levels of BSP, DSPP, DMP1, OCN, and OPN, respectively.

[0027] Figure 6 This is a Real-time RT-PCR quantitative bar chart showing the expression of osteogenic / odontogenic related genes in dental pulp stem cells promoted by YWHAH-peptide46 / 47 14 days after osteogenic induction in this invention. AE corresponds to the mRNA expression levels of BSP, DSPP, DMP1, OCN, and OPN, respectively.

[0028] Figure 7 The image shows the scratch assay results of YWHAH-peptide46 / 47 promoting the migration ability of dental pulp stem cells in this invention. In the image, A represents the scratch migration assay and B represents the quantitative analysis.

[0029] Figure 8 The image shows the effect of YWHAH-peptide46 in promoting the formation of osteoid / dental tissue mineralization in dental pulp stem cells in vivo. In this invention, A is an HE-stained photograph, B is a quantitative bar chart of mineralized tissue, C, E, and G are immunohistochemical staining photographs of DSPP, DMP1, and BSP, respectively, and D, F, and H are quantitative bar charts of the proportion of positive cells of DSPP, DMP1, and BSP, respectively.

[0030] Figure 9 This is a diagram showing the sequence alignment results of human and rabbit YWHAH proteins in this invention.

[0031] Figure 10 This image illustrates the effect of YWHAH-peptide46 in promoting the mineralization of dentin-like tissue from dental pulp stem cells in this invention. A represents a micro-CT scan and 3D reconstruction; B is a quantitative histogram of regenerated tissue volume; C is a cross-sectional micrograph of the jawbone; D is a scanning electron microscope image (scale bar: 10 μm); E is an HE-stained image (top scale bar: 100 μm, bottom scale bar: 25 μm); and F is a Masson-stained image (top scale bar: 100 μm, bottom scale bar: 25 μm). In E and F, AB represents alveolar bone, rD represents regenerated dentin-like structures, and Od represents odontoblasts.

[0032] In the above figures, quantitative data are expressed as mean ± standard deviation; compared with the control group, P < 0.05 P < 0.01. Detailed Implementation

[0033] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0034] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0035] Example 1 1. The binding sites of the GREM1 / YWHAH protein complex were located using a peptide microarray. Three binding sites were found between GREM1 and YWHAH, two of which were positive binding sites. Figure 1 A). Sites with a spot density value exceeding 30% and a spot density value below 30% on the negative reaction membrane are considered positive peptide binding sites. Microarray hybridization grayscale analysis revealed that sites 46 and 47 exhibited the highest binding intensity among the three binding sites. Figure 1 B).

[0036] 2. Based on the binding site locations and sequences of the two positive reports above, and selecting a negative binding site as a negative control, the obtained peptide sequences were synthesized sequentially from the carboxyl terminus to the amino terminus under swelling conditions in resin and dichloromethane. The peptides were purified by liquid chromatography, lyophilized, and yielded three lyophilized peptide powders. The experimental group peptides were: YWHAH-46 peptide (SEQ ID NO.1: LRDNLTLWTSDQQDE), YWHAH-47 peptide (SEQ ID NO.2: TLWTSDQQDEEAGEG), and the control group peptide YWHAH-3 peptide (SEQ ID NO.3: ARLAEQAERYDDMAS). Figure 2 AC).

[0037] DPSCs were pretreated with peptides at a working concentration of 10 μg / mL for 24 hours, and then total protein samples from each group of DPSCs were extracted. A CO-IP experiment was constructed using YWHAH and IgG specific antibodies, and Western blot analysis was performed. The results showed that both active peptides, YWHAH-46 peptide and YWHAH-47 peptide at 10 μg / mL, could effectively open the binding of the GREM1 / YWHAH protein complex. Figure 3 ).

[0038] 3. The function of dental pulp stem cells (DPSCs) was regulated by controlling the binding of the GREM1 / YWHAH complex using the aforementioned small molecule bioactive peptides. Experimental group peptides: YWHAH-peptide46 (10 ug / ml), YWHAH-peptide47 (10 ug / ml); control group peptide: YWHAH-peptide3 (10 ug / ml). Compared with the blank group (no treatment) and the control group (with control group peptides), after 3 days of osteogenic induction, the ALP activity of DPSCs in the experimental group with added peptides YWHAH-peptide46 and YWHAH-peptide47 was significantly enhanced. Figure 4 A, P<0.01. Two weeks after osteogenic / dentition induction, alizarin red staining was performed. Compared with the blank group and the control group, the mineralization capacity of DPSCs in the experimental group was significantly increased (A, P<0.01). Figure 4 B). Real-time RT-PCR results showed that the expression levels of osteogenic / odontogenic genes BSP, DSPP, DMP1, OCN, and OPN in DPSCs of the experimental groups supplemented with peptides YWHAH-peptide46 and YWHAH-peptide47 were significantly increased at 7 and 14 days after osteogenic induction, and the promoting effect of peptide YWHAH-peptide46 was stronger than that of peptide YWHAH-peptide47. Figure 5 AE, Figure 6 AE, P<0.01).

[0039] 5. To investigate whether the small molecule peptides YWHAH-peptide46 / 47 GREM1 could regulate the migration and chemotaxis of DPSCs, a scratch assay was performed. The results of the 48-hour scratch assay showed that, compared with the blank group and the control group, the addition of peptides YWHAH-peptide46 and YWHAH-peptide47 significantly promoted the in vitro migration ability of DPSCs. Figure 7 AB, P<0.01).

[0040] 6. Aliquot HA / TCP bone powder into 1.5 mL cryovials (20 mg per tube). After autoclaving, immerse the bone powder in α-MEM medium containing 10% fetal bovine serum (FBS) and incubate at 37°C with 5% CO2 for 24 h. Centrifuge at 1100 r / min for 6 min and discard the supernatant. Then, centrifuge DPSCs at 8 × 10⁻⁶... 5 Cells were seeded per 10 cm culture dish and cultured at 37°C for 48 h in α-MEM complete medium containing 10% fetal bovine serum, 100 U / L penicillin, and 100 mg / L streptomycin. The cells were then divided into two groups: experimental group (DPSCs + YWHAH-46peptide) and control group (DPSCs + YWHAH-3peptide). The two groups of cells were seeded at a density of 1.5 × 10⁻⁶ cells / dish. 6 sh-Control and sh-SNHG8 cells mixed with bone meal were inoculated at a concentration of 1.5 × 10⁶ cells / mL into HA / TCP bone meal pre-soaked in α-MEM medium, and incubated at 37°C, 5% CO₂ for 2 h. The mixture was then centrifuged at 1100 r / min for 6 min, and the supernatant was discarded. Eight-week-old female nude mice were anesthetized with 4% chloral hydrate (1 mL / kg). The skin on the back of the mice was cut open, and sh-Control and sh-SNHG8 cells, respectively, mixed with bone meal, were implanted into the left and right sides of the mice's backs, at a density of 1.5 × 10⁶ cells / mL at each site. 6 Cells were sacrificed after 3 months, decalcified for 1 month, and then sectioned. Eight weeks later, subcutaneously re-implanted tissue was removed and stained with hematoxylin and eosin (HE). The results showed increased osteoid formation in the DPSCs+YWHAH-peptide46 group. Figure 8 AB, P<0.01). Immunohistochemical staining was then performed, and the results showed that the expression of DSPP, DMP1, and BSP in the DPSCs+YWHAH-peptide46 group was higher than that in the control group ( ). Figure 8 CD, EF, GH, P<0.01). This suggests that the small molecule peptide YWHAH-peptide46 can promote the in vivo mineralization of osteoid / dentinoid tissue by dental pulp stem cells.

[0041] 7. Comparative analysis of human and rabbit SFRP2 protein sequences showed that both human and rabbit SFRP2 proteins are 295 amino acids in length. A protein function search on the website (https: / / www.uniprot.org / ) revealed that their major functional domains are located in the same positions, indicating similar functions. Comparison of the full-length sequences of human and rabbit YWHAH proteins showed high homology, with a sequence identity of 88.21%. Figure 9 Furthermore, the human and rabbit sequences of the YWHAH-peptide46 peptide (SEQ ID NO.1: LRDNLTLWTSDQQDE) are completely identical.

[0042] 8. To facilitate further clinical application, this experiment used YWHAH-3peptide and YWHAH-46peptide to treat rabbit DPSCs. Fifth-generation dental pulp stem cells were resuscitated and divided into two groups: experimental group: DPSCs + YWHAH-46peptide; control group: DPSCs + YWHAH-3peptide. Each group was treated with 10 ug / ml of either the control peptide or the control peptide for 48 h. Subsequently, the cells were digested, and the cell concentration was adjusted to 2 × 10⁻⁶ cells / ml. 6 Cells were aliquoted per ml into sterile EP tubes, centrifuged at 1100 rpm for 6 minutes, the supernatant was discarded, and 20 μL of culture medium was added to resuspend the cells. The cell suspension was then transferred into EP tubes, labeled, and set aside. Hydrogel was thawed overnight at 4°C. 30 μL of hydrogel was added to each EP tube and mixed thoroughly with the cell suspension to obtain a cell-hydrogel mixture. This mixture was then implanted back into the extraction sockets of rabbits for further in vivo experiments. Four months post-surgery, intraoral observation showed good healing of the extraction sockets on the right upper and lower jaws of the rabbits. The rabbits were euthanized under general anesthesia, samples were taken, fixed, and MicroCT scans were performed. Statistical analysis of the total volume of regenerated high-density calcification in each group revealed that the DPSCs+YWHAH-peptide46 group had a significantly higher volume of high-density calcification than the control group (P<0.05). Figure 10 AB). When the samples were cut perpendicular to the long axis of the tooth, it was observed that the original extraction socket in the DPSCs+YWHAH-peptide46 group was filled with high-density hard tissue, with a hardness similar to dentin; however, only a small amount of high-density calcification was observed in the control group. Figure 10 C). Scanning electron microscopy of the regenerated tissue showed that both groups produced regenerated dentin matrix. The DPSCs+YWHAH-peptide46 group had a high degree of mineralization, with neatly arranged and regularly shaped dentinal tubules; while the control group had a low degree of mineralization, with sparse and disordered dentinal tubule structure. Figure 10 D). The samples were further decalcified, dehydrated, and embedded. Histological staining was used to evaluate the regenerated dentin structure within the jawbone. HE staining and Masson's staining results showed that the DPSCs+YWHAH-peptide46 group exhibited abundant regenerated dentin matrix within the extraction socket, with dentinal tubules observed under high magnification and arranged in a relatively regular pattern. The dentin sample was surrounded by an odontoblast layer, and periodontal ligament-like fibers were present. The control group also showed some regenerated dentin matrix, but under high magnification, the dentinal tubules were sparse and disordered. Figure 10 EF).

[0043] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An active polypeptide derived from YWHAH, characterized in that, The amino acid sequence of the active polypeptide has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The active polypeptide according to claim 1, characterized in that, The amino acid sequence of the active polypeptide has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.

2.

3. The active polypeptide according to claim 2, characterized in that, The amino acid sequence of the active polypeptide is shown in SEQ ID NO.

1.

4. The active polypeptide according to claim 2, characterized in that, The amino acid sequence of the active polypeptide is shown in SEQ ID NO.

2.

5. The use of the active polypeptide according to any one of claims 1-4 in the preparation of products for treating bone-related diseases.

6. The application according to claim 5, characterized in that, The products include drugs or reagent kits.

7. The application according to claim 5, characterized in that, The bone-related diseases mentioned include osteoporosis.

8. The application according to claim 5, characterized in that, The bone-related diseases include dental tissue diseases.

9. The application according to claim 8, characterized in that, The dental tissue diseases mentioned include tooth loss, root defects, or dentin defects.

10. The application according to claim 5, characterized in that, The product can promote osteogenic differentiation and / or odontogenic differentiation of dental mesenchymal stem cells, enhance ALP activity, mineralization capacity and osteogenic / odontogenic gene expression, and promote the regeneration of osteoid and dentin-like tissues in vivo.

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