Construction method and application of animal model with high IgE syndromic bone malformation disease

Abstract

The invention relates to an application of an STAT3 agonist ColivelinTFA in the treatment of HIES (High Immunoglobulin E Syndrome) and osteoporosis. An osteoblast specific Stat3 knockout mouse is constructed through a Cre-loxp system, it is found that the mouse shows HIES-related bone deformity such as maxillofacial bone development deformity, spontaneous fracture and osteoporosis, and the mechanism that STAT3 inactivation causes HIES-related bone development deformity and osteoporosis by regulating osteogenic activity is proved. On the basis, it is proved that the STAT3 agonist ColivelinTFA can be used for treating osteoporosis caused by insufficient single dosage of STAT3 and stress loss. Compared with a diphosphate drug, the ColivelinTFA increases the bone mass by promoting bone metabolism instead of inhibiting bone metabolism, which may avoid a series of complications such as jaw bone necrosis caused by diphosphate to a certain extent.

Description

[0001] Divisional application This invention is a divisional application of the Chinese invention patent application with an application date of June 9, 2021, application number 202110644771.4, and name “Application of STAT3 agonist Colivelin TFA in the treatment of HIES and osteoporosis”. Technical Field

[0002] The present invention relates to the technical field of medical applications, and in particular to the application of a STAT3 agonist, Colivelin TFA, in the treatment of HIES and osteoporosis. Background Art

[0003] Inactivating mutations in the human STAT3 gene cause a complex primary immunodeficiency disorder characterized by elevated serum IgE concentrations, known as hyper-IgE syndrome (HIES). Spontaneous fractures are a common clinical manifestation of HIES. A cohort study showed that 57% of patients experienced at least three fractures, and this condition occurred across all age groups. Further analysis revealed that femoral and vertebral bone mineral density in patients with spontaneous fractures was significantly lower than normal. Clinically, drug treatments for osteoporosis fall into two categories: antiresorptive drugs, which slow bone resorption by osteoclasts, and anabolic drugs, which stimulate osteoblasts to form new bone. Bisphosphonates, as antiresorptive drugs, are the first-line treatment for osteoporosis and are also widely used in the treatment of HIES. Although there is evidence that bisphosphonates increase bone mineral density, they do not reduce the incidence of spontaneous fractures. Long-term use of bisphosphonates significantly increases the risk of poor bone healing and osteonecrosis of the jaw. Furthermore, long-term use of bisphosphonates is contraindicated in growing children. On the other hand, the only drugs that promote osteogenesis are full-length parathyroid hormone (PTH 1–84) and its N-terminal peptide, teriparatide (PTH 1–34). Currently, there are few reports on the efficacy of these two drugs in treating HIES-related osteoporosis and spontaneous fractures. In summary, HIES patients suffer from abnormal bone metabolism, limited treatment options, and poor treatment outcomes and prognosis, severely compromising their survival and quality of life, placing a heavy mental and financial burden on children and their families. Therefore, elucidating the underlying mechanisms is not only beneficial for the treatment of HIES-related bone diseases, but also holds significant implications for the treatment of osteoporosis.

[0004] Colivelin TFA is a brain-permeable neuroprotective peptide and a potent activator of STAT3. Colivelin inhibits neuronal death in vitro by activating STAT3. Colivelin TFA exhibits long-term beneficial effects on neurotoxicity, Aβ deposition, and neuronal apoptosis, particularly in neurodegenerative diseases. Previous studies have demonstrated the efficacy of Colivelin TFA in Alzheimer's disease and ischemic brain injury, but its effects on the skeletal system have not been elucidated. Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a use of a STAT3 agonist in the preparation of a medicament for treating HIES and osteoporosis, so as to solve the problems in the prior art.

[0006] To achieve the above objectives, the first aspect of the present invention provides the use of a STAT3 agonist in the preparation of a drug for treating HIES and osteoporosis.

[0007] Preferably, the STAT3 agonist is Colivelin.

[0008] The present invention also provides a composition for treating HIES and osteoporosis, characterized in that the composition comprises a STAT3 agonist as an active ingredient.

[0009] Preferably, the composition is in the form of tablets, powders, injections, capsules, suspensions, pastes, gels, coatings, films, sustained-release preparations or microspheres.

[0010] More preferably, the STAT3 agonist is Colivelin.

[0011] In the present invention, "pharmaceutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic or other untoward reactions when appropriately administered to animals or humans.

[0012] In the present invention, unless otherwise specified, the pharmaceutical dosage form is not particularly limited and can be prepared in the form of tablets, powders, injections, capsules, suspensions, pastes, gels, films, sustained-release formulations, or microspheres, and can be prepared by conventional methods. The choice of pharmaceutical dosage form should be compatible with the mode of administration.

[0013] The beneficial effects of the present invention are that the present invention constructs osteoblast-specific Stat3 Knockout mice, found that mice show HIES-related skeletal deformities such as maxillofacial bone development deformity, spontaneous fracture and osteoporosis, and confirmed the mechanism of STAT3 inactivation leading to HIES-related bone development deformity and osteoporosis by regulating osteogenic activity. On this basis, it is confirmed that the STAT3 agonist Colivelin TFA can be used to treat osteoporosis caused by STAT3 haploinsufficiency and stress loss. Compared with the existing bisphosphonate drugs for treating HIES, Colivelin TFA increases bone mass by promoting bone metabolism rather than inhibiting bone metabolism, which may to some extent avoid a series of complications such as jaw necrosis caused by bisphosphonates. BRIEF DESCRIPTION OF DRAWINGS

[0014] 图1 A. Stat3 Genome insertion of lox P sequences on both sides of exons 18-20, selection of osteoblast precursor cell-specific expression Osx Cre As a tool mouse; B. Western blotting detected STAT3 protein on Day 1 Osx Cre and Osx Cre ; Stat3 fl / fl Expression level of mouse calvarial cells; C. 8-week-old Osx Cre and Osx Cre ; Stat3 fl / fl Whole mouse observation; D-F. Day 1 Osx Cre and Osx Cre ; Stat3 fl / fl Whole mouse staining; G. 4-week-old Osx Cre and Osx Cre ; Stat3 fl / fl Mouse calvarial Micro-CT three-dimensional reconstruction image; H. 4-week-old mouse femur Micro-CT three-dimensional reconstruction image; I-M. Osx Cre and Osx Cre ; Stat3 fl / fl Statistical analysis results of mouse bone microstructure parameters, where the left side is Osx Cre Mouse data, right side is Osx Cre ; Stat3 fl / flMouse data, including the ratio of trabecular volume to total volume of the selected area (BV / TV), average trabecular thickness (Tb.Th), number of trabeculae (Tb.N), average width of the trabecular medullary cavity (Tb.Sp), and cortical bone thickness (Ct.Th).

[0015] 图2 A. Insert lox P sequences on both sides of exons 18-20 of the Stat3 genome, and select a gene specifically expressed in bone marrow stromal stem cells. Prx1 Cre As tool mice; B. Western detection of STAT3 protein at 4 weeks of age Stat3 fl / fl and Prx1 Cre ;Stat3 fl / fl Expression levels of mouse BMSCs; C. 8 weeks old Stat3 fl / fl and Prx1 Cre ;Stat3 fl / fl Overall view of mice; DE.2 weeks old Stat3 fl / fl and Prx1 Cre ;Stat3 fl / fl Whole-mount staining of mice; F. 1 week old Stat3 fl / fl and Prx1 Cre ； Stat3 fl / fl Safranin O staining of mice; G. Different age groups Stat3 fl / fl and Prx1 Cre ;Stat3 fl / fl The ratio of the length of the mouse femur, where the left side is Stat3 fl / fl Mouse data, right side Prx1 Cre ;Stat3 fl / fl Mouse data.

[0016] 图3 A. 4 weeks old Osx Cre and Osx Cre ; Stat3 fl / fl TRAP staining of mouse femur; B. 4 weeks old Osx Cre and Osx Cre; Stat3 fl / fl Results of the in vivo calcein-alizarin red labeling experiment on mouse femur; C. Day 1 Osx Cre and Osx Cre ; Stat3 fl / fl Immunohistochemical hybridization results of Col1a1 and Ocn in mouse femur; D. 1 week old Osx Cre and Osx Cre ; Stat3 fl / fl Immunofluorescence staining results of mouse femur; E. 4 weeks old Osx Cre and Osx Cre ; Stat3 fl / fl Mouse BMSCs were induced to differentiate into osteoblasts in vitro, and ALP staining was performed on day 7 and Alizarin red staining was performed on day 14; F. End point of osteoblast differentiation, RT-PCR was used to detect the expression of osteoblast-related marker genes, where the left side is Osx Cre Mouse data, right side Osx Cre ; Stat3 fl / fl Mouse data; G. 4 weeks old Stat3 fl / fl Mice were pre-stained with EGFP and Cre -EGFP adenovirus was used to induce differentiation, and ALP staining was performed on day 7 and Alizarin red staining was performed on day 14; H. At the end of osteogenic differentiation induction, the expression of osteogenic-related marker genes was detected by RT-PCR.

[0017] 图4 A. Day 1 Osx Cre and Osx Cre ; Stat3 fl / fl Results of RNA-seq of mouse calvaria tissue; B. Osx Cre ; Stat3 fl / fl Compared with mice Osx Cre Results of GO analysis of down-regulated genes in the mouse calvaria; C. Osx Cre ; Stat3 fl / fl Compared with mice Osx Cre Down-regulated osteogenic differentiation and ossification-related genes in mouse calvarial bone tissue; D.Day 1 Osx Cre and Osx Cre ; Stat3 fl / fl Mouse parietal bone Dlx5 Expression levels, where the left side is Osx Cre Mouse data, right side Osx Cre ; Stat3 fl / fl Mouse data; E. 1-week-old and 4-week-old Osx Cre and Osx Cre ; Stat3 fl / fl Immunohistochemical staining of DLX5 in mouse femur; F. 4 weeks old Osx Cre and Osx Cre ; Stat3 fl / fl Mouse BMSCs were treated with EGFP or DLX5 lentivirus in vitro and induced to differentiate into osteoblasts. ALP staining was performed on day 7 and Alizarin red staining was performed on day 14. G. End point of osteoblast differentiation, RT-PCR was used to detect the expression of osteoblast-related marker genes. From left to right are Osx Cre +EGFP, Osx Cre ;Stat3 fl / fl +EGFP, Osx Cre ;Stat3 fl / fl +DLX5 data.

[0018] 图5 A. Micro-CT 3D reconstruction of femur of 4-week-old mice; B. HE staining of femur of 4-week-old mice; C. Statistical analysis of bone microarchitecture parameters, including the ratio of trabecular volume to total volume of the selected area (BV / TV), average trabecular thickness (Tb.Th), number of trabeculae (Tb.N), average width between trabecular medullary cavities (Tb.Sp), and cortical thickness (Ct.Th). Osx Cre 、 Osx Cre ; Stat3 fl / fl +Vehicle, Osx Cre ; Stat3 fl / fl +Colivelin data; D. TRAP staining of femur of 4-week-old mice, from left to right: Osx Cre 、 Osx Cre ; Stat3 fl / fl +Vehicle, Osx Cre ; Stat3 fl / fl +Colivelin's data.

[0019] 图6 A. Micro-CT three-dimensional reconstruction image of the femur of a 5-week-old mouse; B. Statistical analysis results of bone microarchitectural parameters, including the ratio of trabecular volume to the total volume of the selected area (BV / TV), average trabecular thickness (Tb.Th), number of trabeculae (Tb.N), average width between trabecular medullary cavities (Tb.Sp), and cortical bone thickness (Ct.Th). From left to right, the data are Vehicle, TS+Vehicle, and TS+Colivelin; C. In vivo calcein-alizarin red labeling experiment results of the femur of a 5-week-old mouse; D. TRAP staining of the femur of a 5-week-old mouse. From left to right, the data are Vehicle, TS+Vehicle, and TS+Colivelin.

[0020] 图7 A. BMSCs from 4-week-old wild-type mice were cultured in vitro and induced into osteoblasts with the addition of PBS or 1 nM Colivelin. ALP staining was performed on day 7 and Alizarin red staining was performed on day 14. B. After 24 hours of osteoblastic differentiation with the addition of PBS or 1 nM Colivelin, p-STAT3 and STAT3 protein expression was detected by Western blotting. C. At the endpoint of osteoblastic differentiation, RT-PCR was used to detect the expression of osteoblast-related marker genes in the experimental and control groups. Data from the vehicle group are shown on the left, and data from the coliven group are shown on the right. DETAILED DESCRIPTION

[0021] The present invention will be further described below with reference to specific embodiments for a better understanding of the present invention.

[0022] Example Experimental materials: Colivelin TFA was purchased from MCE.

[0023] Experimental steps: (1) The model mouse construction method is as follows: using the Cre-Loxp system Stat3 Insert loxP sites on both sides of exons 18 to 20 of the gene to construct Stat3 fl / fl mice, and Osx Cre Mice (JAX, No. 006361) were mated and F1 intercrossed to obtain Osx Cre ； Stat3 fl / fl Mice.

[0024] (2) The model verification method is as follows: extraction Osx Cre Mice and Osx Cre ;Stat3 fl / fl Mice long bone marrow BMSCs, after 7 days of osteogenic induction, cell protein was extracted, Western blotting was used to detect the expression amount of STAT3 protein, and knockout was successfully verified Stat3 to make its protein expression amount decrease; the above evidence proves that the model mouse is successfully modeled.

[0025] For the obtained Osx cre ;Stat3 fl / fl Mice were used to carry out phenotype identification research, mainly including: (3) Newborn mouse whole bone staining: after removing the skin and internal organs of the newborn mouse and fixing it with 95% ethanol for 1 day, the newborn was stained in alcian blue solution for 42 hours, fixed and washed twice with 95% ethanol for 1 hour each time, then treated with 2% KOH for 4 hours; after staining with ARS solution for 4 hours, treat with 1% KOH in 20% glycerol, for 7-day-old mice, ARS staining needs 8 hours, then treat with 1% KOH in 20% glycerol.

[0026] (4) Micro-CT analysis of mouse craniofacial bones: harvest mouse skulls, remove soft tissue, store the remaining tissue in 70% ethanol, use 4-week-old Osx cre ;Stat3 fl / fl Male mice and littermates were subjected to Micro-CT analysis (SCANCO Medical AG, Bassersdorf, Switzerland, 10 pm).

[0027] (5) Micro-CT and parameter analysis of mouse long bones: after harvesting the hind limbs of the mouse, remove the soft tissue, store the remaining tissue in 70% ethanol, use 4-week-old Osx cre ;Stat3 fl / flFemora of male and littermate control mice were analyzed by micro-CT (SCANCO Medical AG, Bassersdorf, Switzerland, 10 μm). According to the manufacturer's guidelines, 100 slices were selected for measurement and analysis of cancellous bone microarchitectural parameters, including bone volume fraction (BV / TV), trabecular thickness (Tb.Th.), and trabecular number (Tb.N). Fifty slices from the mid-femur were selected for analysis of cortical bone thickness (Ct.Th.).

[0028] (6) Histological phenotype analysis: Bone tissues were fixed in 4% PFA and decalcified for 2 weeks before paraffin embedding. Tissue sections (6 μm) were used for H&E staining, tartrate-resistant acid phosphatase (TRAP) staining, Alizarin red staining, and immunohistochemical staining according to standard protocols (387A-1KT; Sigma-Aldrich, St-Louis, MO, USA). Images were captured using a microscope (BX51, Olympus, Tokyo, Japan).

[0029] (7) Analysis of osteogenesis-related phenotypes: Calcein-alizarin red double-labeling staining, Col1α1 / OCN in situ hybridization, OCN immunofluorescence staining, BMSCs-induced osteogenic differentiation Alizarin red staining, ALP staining, and osteogenesis-related gene RT-qPCR were used to analyze osteogenesis-related phenotypes.

[0030] (8) Analysis of osteoclast-related phenotypes: TRAP staining technique was used to analyze osteoclast-related phenotypes.

[0031] (9) Adenovirus transfection: We transfected 2×10 5 BMSCs were seeded in 12-well plates and cultured in α-MEM medium for 24 h. BMSCs were then infected with Cre-EGFP recombinase or EGFP adenovirus (Hanyuan Biotechnology Co., Ltd., Shanghai) at a multiplicity of infection of 30. After 4–6 h of infection, BMSCs were transferred to osteogenic medium (Cyagen, MUBMD-90021).

[0032] Osx Cre Stat3 fl / fl The results and analysis of the model mouse construction and phenotypic identification studies are as follows: a) Obtained by homologous recombination Stat3 flox / flox Mice. Use tool mice Osx Cre and Stat3 flox / flox Osteogenic precursor cell-specific knockout Stat3 ( Osx Cre ;Stat3 fl / fl ) of C57BL6 mice, and the process was as 图1 As shown in A.

[0033] b) Take Day 1 Osx Cre and Osx Cre ;Stat3 fl / fl Mouse calvarial bone cells, western blot detection of STAT3 protein expression level in calvarial bone, to verify whether the osteoblast precursor cell-specific Stat3 knockout mice were successfully constructed. The specific results are as follows 图1 As shown in middle B, the results show that mice with osteoblast precursor cell-specific Stat3 knockout were successfully constructed. Osx Cre and Osx Cre ; Stat3 fl / fl Overall view of mice 图1 As shown in C.

[0034] c) Take Day 1 Osx Cre and Osx Cre ; Stat3 fl / fl The whole body of mice was stained to study whether there were any differences in bones. The specific results are as follows 图1 As shown in DF, the whole-body staining revealed Osx Cre ;Stat3 fl / fl The parietal bones and clavicles of mice were compared with Osx Cre The mice showed obvious developmental abnormalities.

[0035] d) Take 6 pairs of 4-week-old male Osx Cre and Osx Cre ;Stat3 fl / fl Mouse skull and femur, Mico-CT 3D reconstruction of mouse skull and femur images, as shown in the following figure: 图1 As shown in G and H, the results of CT reconstruction show that: compared with Osx Cre mice, Osx Cre ;Stat3 fl / fl The shape of the mouse skull was obviously abnormal and porous. The femur 3D reconstruction and quantitative analysis showed that Osx Cre ;Stat3 fl / fl Mice showed obvious osteoporosis. Femur was quantitatively analyzed (in order to exclude the effect of estrogen on bone mass, male mice were selected for quantitative analysis of bone microstructure parameters), as shown in the following figure: 图1 IM, 3D CT quantitative analysis of 4-week-old Osx Cre Mice and Osx Cre ;Stat3 fl / fl Mice, found Osx Cre ;Stat3 fl / fl The mice showed obvious osteoporosis.

[0036] Use tool mouse Prx1 Cre and Stat3 flox / flox Bone marrow stromal stem cell-specific knockout Stat3 ( Prx1 Cre ;Stat3 fl / fl ) of C57BL6 mice ( 图2 Middle A), Western blot analysis at 4 weeks of age Stat3 fl / fl and Prx1 Cre ； Stat3 fl / fl STAT3 expression in mouse BMSCs. Prx1 Cre ;Stat3 fl / fl The expression of STAT3 in mice was significantly lower than Stat3 fl / fl Mouse ( 图2 Middle B) demonstrates the successful construction of bone marrow stromal stem cell-specific knockout Stat3 Observe 8-week-old mice Stat3 fl / fl and Prx1 Cre ;Stat3 fl / fl Mice, found Prx1 Cre ;Stat3 fl / fl Mice are significantly smaller than Stat3 fl / fl Mouse ( 图2 Middle C). Take 2-week-old Stat3 fl / fl and Prx1 Cre ;Stat3 fl / fl Whole body staining of mice revealed that, Prx1 Cre ;Stat3 fl / fl Significant abnormalities were found in both skull and femur of mice, and Prx1 Cre ;Stat3 fl / fl Multiple spontaneous fractures and poor healing were found in the limbs of mice 图2 Safranin O staining showed that 1-week-old Prx1 Cre ;Stat3 fl / fl Fracture of femur in mice 图2 F), further analysis of different age groups Stat3 fl / fl and Prx1 Cre ;Stat3 fl / fl The length of femur in mice was measured, and it was found that Prx1 Cre ;Stat3 fl / fl The degree of femur deformity in mice increased with age 图2 G).

[0037] e) Three pairs of 4-week-old Osx Cre and Osx Cre ;Stat3 fl / fl The difference between cortical bone and trabecular bone of femur in mice was observed after HE staining; the number of TRAP-positive cells was calculated after TRAP staining, and statistical analysis was performed to analyze the change of osteoclast activity, details 图3 A, by TRAP staining, it was found that Osx Cre ;Stat3 fl / fl The number of TRAP-positive cells in mice was much smaller than Osx Cre Mice, indicating that Osx Cre ;Stat3 fl / fl The osteoclast formation ability of mice is weaker than Osx Cre Mice.

[0038] f) 3-week-old male Osx Cre and Osx Cre ;Stat3 fl / flMice were intraperitoneally injected with 20 mg / kg calcein (Sigma, C0875-5G, 1 mg / ml in 2% NaHCO3 solution) and 40 mg / kg Alizarin Red S (Sigma, A5533-25G, 2 mg / ml in H2O) on Day 1 and Day 4, respectively. After 7 days, they were euthanized and their femurs were fixed, dehydrated, and embedded. The samples were cut into 5 μm thick pieces using a hard tissue cutter, and the images of the fluorescent markers were captured using a microscope. The distance between the two fluorescent lines at the same site in different samples was compared by analyzing the difference. Osx Cre and Osx Cre ;Stat3 fl / fl Mouse bone formation rate, specifically 图3 As shown in B, it can be seen from the figure Osx Cre ;Stat3 fl / fl The rate of new bone formation in mice is much lower than Osx Cre mouse.

[0039] g) Take Day 1 Osx Cre and Osx Cre ;Stat3 fl / fl Mouse femur, detection of osteoblast-related genes by immunoin situ hybridization Col1a1 and Ocn The expression, such as 图3 As shown in C, we can see Osx Cre ;Stat3 fl / fl Mouse osteogenesis-related genes Col1a1 , Ocn Expression below Osx Cre mouse.

[0040] h) Take 1 week old Osx Cre and Osx Cre ;Stat3 fl / fl Mouse femur was subjected to immunohistofluorescence staining of OPN, as follows 图3 As shown in D, we can see Osx Cre ;Stat3 fl / fl The expression of the osteoblast-related gene OPN in mice was lower than Osx Cre mouse.

[0041] i) Take 4 weeks old Osx Cre and Osx Cre ;Stat3 fl / fl Mouse BMSCs were cultured in vitro and induced to osteogenic differentiation. ALP and Alizarin red staining were performed at day 7 and day 14 of induction, respectively. At the end of induction, RNA was extracted from the cells for RT-PCR detection of osteogenesis-related indicators, as shown in Osx Cre and Osx Cre ； Stat3 fl / fl Group RNA, RT-PCR detection of osteogenesis-related indicators, as shown in 图3 From the results, it can be found that Osx Cre ； Stat3 fl / fl The osteogenic differentiation ability of mouse BMSCs is lower than that of Osx Cre mouse.

[0042] j) Take 4-week-old Stat3 fl / fl Mouse BMSCs were cultured in vitro and induced to osteogenic differentiation after pre-treatment of BMSCs with EGFP and Cre-EGFP. ALP and Alizarin red staining were performed at day 7 and day 14 of induction, respectively. At the end of induction, RNA was extracted from the cells for RT-PCR detection of osteogenesis-related indicators, as shown in 图3 From the results, it can be found that the osteogenic differentiation ability of BMSCs after Stat3 knockout is significantly weakened.

[0043] k) Collect 3 pairs of Day1 Osx Cre and Osx Cre ;Stat3 fl / fl Mouse calvarial tissue was extracted for RNA sequencing. The sequencing results are shown in 图4 As shown in A, sequencing found that compared with Osx Cre mouse, Osx Cre ;Stat3 fl / fl Mouse a large number of gene expression down-regulation. In order to find new possible Stat3 signal pathways for regulation, further GO analysis was performed on Osx Cre ;Stat3 fl / fl GO analysis was performed on the down-regulated genes in group, and the analysis results are shown in Figure 4As shown in B, most of the down-regulated genes were found to be related to ossification, osteoblast differentiation, and skeletal system development. Further analysis of the osteogenesis-related pathways in the GO results was performed to find Stat3 Possible downstream genes, such as Figure 4 As shown in C, Dlx5 Significant down-regulation was observed. Osx Cre and Osx Cre ; Stat3 fl / fl The expression of DLX5 in mice, such as Figure 4 As shown in D, Osx Cre ; Stat3 fl / fl Mouse skull bone tissue Dlx5 The expression of DLX5 was significantly downregulated, and the expression of DLX5 in mouse femur was further detected. Figure 4 As shown in E, Osx Cre ; Stat3 fl / fl The expression of DLX5 in mice was significantly lower than that in Osx Cre The above results indicate that Osx It is very likely that it further affects osteogenic differentiation by regulating the expression of Dlx5.

[0044] Take 4 weeks old ; Stat3 Cre and Osx Cre Osx fl / fl Mouse BMSCs were cultured in vitro and induced to differentiate into osteoblasts after being treated with EGFP or DLX5 lentivirus. Osx As shown in F and G, ; Stat3 Cre Figure 4 fl / fl After mouse BMSCs overexpressed DLX5, their osteogenic differentiation ability was restored to a certain extent, further proving that Stat3 may affect osteogenic differentiation by regulating the expression of Dlx5.

[0045] l) Take 1-week-old from the same litter Osx Cre and ; Stat3 Cre Osx fl / + Mice, divided into Osx Cre Group, ; Stat3 Cre+ PBS group, Osx Cre ； Osx fl / + + Colivelin group, in which Colivelin / PBS was administered by intraperitoneal injection at a dose of 1.5 mg / kg every other day, and the mice were sacrificed at the age of 4 weeks. The left leg of the mice was used for Micro-CT analysis of bone mass changes, and the right leg was fixed, dehydrated and embedded for further HE staining and Trap staining, as shown in Osx CT reconstruction ; Stat3 and quantitative analysis Figure 5 showed that Figure 5 Cre Figure 5 fl / fl After intraperitoneal injection of Colivelin in mice, the phenotype of reduced bone mass was partially recovered, and it was further found by HE staining Osx and TRAP staining ; Stat3 that Colivelin mainly increased bone mass by enhancing osteogenesis rather than weakening osteoclasts.

[0046] m) 4-week-old male C57BL / 6J mice were randomly divided into a blank control group, a TS+PBS group and a TS+colivelin group. The mice in the blank control group were injected intraperitoneally with PBS for 7 days, and the tails were not suspended. The mice in the TS+PBS group were suspended in a single plastic cage at an angle of about 30 degrees with the head downward for 7 days, while being injected intraperitoneally with PBS every day. The mice in the TS+colivelin group were suspended by the tail while being injected intraperitoneally with colivelin every day for 7 days at a dose of 1 mg / kg. After 7 days, the mice were analyzed by Micro-CT for bone mass changes, and the results are shown in Figure 5 CT reconstruction Figure 5 and quantitative analysis Figure 6 showed that the bone mass of the tail-suspended mice (TS+PBS group) was significantly reduced compared with that of the wild-type mice (blank control group); after intraperitoneal injection of Colivelin (TS+colivelin group), the phenotype of reduced bone mass was partially recovered. It was further found by calcein- alizarin red double-labeling Figure 6 and TRAP staining Figure 6 that Colivelin could increase bone mass by enhancing osteogenesis rather than affecting osteoclasts.

[0047] n) BMSCs from 4-week-old wild-type mice were cultured in vitro and treated with PBS / Colivelin to induce osteogenic differentiation. P-STAT3 and STAT3 protein expression was measured 24 hours after induction. ALP and Alizarin Red staining were performed on days 7 and 14 of differentiation induction. At the end of differentiation induction, RNA was extracted from the PBS and Colivelin groups, and osteogenic markers were detected by RT-PCR. The results are shown in the attached figure. Figure 6 After PBS / Colivelin treatment of mouse BMSCs for 4 hours, the expression of STAT3 and phosphorylated STAT3 (p-STAT3) was detected by western blot. The results showed that Colivelin could enhance the phosphorylation of STAT3 ( Figure 6 Middle B), further ALP and Alizarin red staining ( Figure 7 Middle A) found that after Colivelin treatment of BMSCs, the osteogenic ability was enhanced, and RT-PCR detection found that the expression of osteogenic-related genes was enhanced ( Figure 7 Middle C).

[0048] In summary, Figure 7 Cre Figure 7 Osx ; Stat3 fl / fl Mice exhibit HIES-related skeletal deformities, including maxillofacial malformations, spontaneous fractures, and osteoporosis. The study confirmed that STAT3 inactivation leads to HIES-related bone malformations and osteoporosis by regulating osteoblast activity. Furthermore, the STAT3 agonist Colivelin TFA has been shown to be effective in treating osteoporosis caused by STAT3 haploinsufficiency and stress loss.

[0049] While the specific embodiments of the present invention have been described in detail above, these are merely exemplary and the present invention is not limited thereto. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, any equivalent changes and modifications made without departing from the spirit and scope of the present invention are intended to be encompassed within the scope of the present invention.

Claims

1. A method for constructing an animal model of skeletal deformity caused by hyper-IgE syndrome, characterized in that: The construction method comprises: constructing osteoblast precursor cell-specific knockout by genetic engineering technology Stat3 mouse model.

2. The construction method according to claim 1, characterized in that The process of the genetic engineering technology includes: obtaining by homologous recombination Stat3 flox / flox Mice, using tool mice and the Stat3 flox / flox Osteogenic precursor cell-specific knockout Stat3 mouse model.

3. The construction method according to claim 2, characterized in that The homologous recombination process includes: using the Cre-Loxp system to Stat3 LoxP sequences were inserted on both sides of exons 18-20 of the genome.

4. The construction method according to claim 2, characterized in that The tool mouse is a protein specifically expressed in osteoblast precursor cells. Osx cre Mice; The mouse model is a specific knockout of osteoblast precursor cells Stat3 C57BL6 mice.

5. The construction method according to any one of claims 1 to 4, characterized in that The high IgE syndrome skeletal deformity disease is selected from at least one of maxillofacial bone developmental deformity, spontaneous fracture and osteoporosis.

6. The construction method according to any one of claims 1 to 4, characterized in that: The symptoms of the hyper-IgE syndrome-related skeletal deformity disease include abnormal development of the skull vault, abnormal development of the clavicle, and decreased expression of Dlx5 in skull vault bone tissue; and / or, The symptoms of the hyper-IgE syndrome-related skeletal deformity disease include decreased osteoclast formation capacity, decreased rate of new bone formation and decreased osteogenic differentiation capacity; and / or, The symptoms of the hyper-IgE syndrome-related skeletal deformity disease include decreased values ​​of the tissue volume to bone mass ratio BV / TV, trabecular thickness Tb.Th, trabecular number Tb.N, and cortical bone thickness Ct.Th, and increased value of trabecular spacing Tb.Sp.

7. The construction method according to any one of claims 1 to 4, characterized in that Symptoms of the hyper-IgE syndrome-related skeletal deformity disease include abnormal shape and porosity of the skull vault; and / or, Symptoms of the hyper-IgE syndrome-related skeletal deformity disease include decreased mRNA levels of run-binding protein 2 RUNX2, osteocalcin OCN, osteopontin OPN, alkaline phosphatase ALP, and bone sialoprotein Bsp.

8. Use of the animal model of hyper-IgE syndrome skeletal deformity disease constructed by the construction method according to any one of claims 1 to 7 in the study of hyper-IgE syndrome.

9. Use of the animal model of hyper-IgE syndrome skeletal deformity disease constructed by the construction method according to any one of claims 1 to 7 in screening drugs for the targeted treatment of hyper-IgE syndrome.

10. Use of the animal model of hyper-IgE syndrome skeletal deformity disease constructed by the construction method according to any one of claims 1 to 7 in the preparation of drugs for the targeted treatment of hyper-IgE syndrome.

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