Application of STAT3 agonist colivelin TFA in preparation of medicine for treating AD-HIES related bone diseases

Abstract

The invention discloses application of an STAT3 agonist colivelin TFA in preparation of a medicine for treating AD-HIES related bone diseases, the bone diseases comprise AD-HIES related slight traumatic fracture, recurrent fracture and fracture healing disorder, the medicine takes the STAT3 agonist colivelin TFA as an active ingredient, and the invention further provides a medicine composition for treating the AD-HIES related bone diseases. It is found through experiments that in fracture healing disorders caused by STAT3 imbalance, CXCL12 is a key regulatory factor, an STAT3 agonist colivelin TFA can relieve AD-HIES related fracture healing delay, meanwhile, CXCL12 expression can be promoted, fracture healing is promoted, and a potential treatment thought is provided for treating AD-HIES related bone complications.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the use of a STAT3 agonist, colivelin TFA, in the preparation of a medicine for treating AD-HIES-related bone diseases. Background Art

[0002] Autosomal dominant hyperimmunoglobulin E syndrome (AD-HIES, also known as Job syndrome) is mainly caused by heterozygous mutations in the signal transducer and activator of transcription 3 (STAT3) gene, which leads to loss of function. AD-HIES patients often suffer from osteoporosis, recurrent fractures, and impaired fracture healing. Although previous studies have shown that STAT3 deficiency in osteoblasts can lead to AD-HIES-like phenotypes, the specific mechanism of how STAT3 causes recurrent fractures and fracture healing disorders in AD-HIES remains unclear. Clinical data show that about 50% of AD-HIES patients experience recurrent fractures, which are usually accompanied by significantly prolonged fracture healing time. Currently, bone marrow transplantation (BMT) has been used to treat AD-HIES-related infections. However, even if the infection is controlled, AD-HIES patients often have recurrent fractures after bone marrow transplantation, and there is no targeted treatment strategy for recurrent fractures or fracture healing disorders. Therefore, clarifying the role and potential mechanism of STAT3 in fracture healing is of great significance for the development of clinical intervention measures for AD-HIES-related skeletal complications.

[0003] STAT3 is a transcription factor widely expressed in the skeletal system and various tissues. Studies have shown that the loss of STAT3 in osteoblasts can lead to AD-HIES-like craniofacial deformities and osteoporosis. Since AD-HIES is caused by STAT3 heterozygous mutations, these mutations may partially retain activity and non-transcriptional functions. Therefore, osteoblast-specific STAT3 heterozygous deletion mice (Stat3 fl / + ;Osx Cre ) as a model for simulating human diseases. Studies have shown that Stat3 fl / + ;Osx Cre The mice showed an osteoporotic phenotype consistent with AD-HIES patients, proving that they can be used as a suitable model to study the mechanism of fracture healing. During fracture repair, stem cells / progenitor cells are mainly derived from the periosteum. Through genetic mouse models and lineage tracing studies, it was found that mouse lines driving Cre or Cre-ERT2 (such as Itm2a, Osx, Ctsk, PDGFRA, Mx1, Acta2, Gli1 and Prrx1) can mark periosteal cells during bone regeneration. In order to minimize interference during the developmental stage, Col1a2-Cre ERT2 (Col1Cre ERT2)A fracture model was established in inducible STAT3 knockout mice. Lineage tracing showed that Col1 CreERT2 was able to label perichondrial progenitor cells expressing CD200, Ctsk, Postn, and PDGFRα. Col1 CreERT2 positive cells contributed most of the chondrocytes and osteoblasts during fracture healing. After conditional knockout of STAT3 in Col1 + cells, impaired fracture healing was observed, including reduced Col1 + cell proliferation and impaired chondrogenic and osteogenic differentiation. These data suggest that STAT3 is crucial for the function of perichondrial stem / progenitor cells. Summary of the Invention

[0004] The main object of the present invention is to provide the use of the STAT3 agonist colivelin TFA in the preparation of a drug for treating AD-HIES-related skeletal diseases, to propose a new treatment method, and to solve the problem that there is no treatment for skeletal diseases such as minor traumatic fractures, recurrent fractures, and fracture healing disorders in the skeletal system of AD-HIES in the prior art.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect of the present invention, there is provided the use of the STAT3 agonist colivelin TFA in the preparation of a drug for treating AD-HIES-related skeletal diseases.

[0007] Preferably, the skeletal disease is AD-HIES-related minor traumatic fracture.

[0008] Preferably, the skeletal disease is AD-HIES-related recurrent fracture.

[0009] Preferably, the skeletal disease is AD-HIES-related fracture healing disorder.

[0010] Preferably, the drug uses the STAT3 agonist colivelin TFA as an active ingredient, and the effective dose is 0.1-5 mg / kg.

[0011] More preferably, the drug uses the STAT3 agonist colivelin TFA as an active ingredient, and the effective dose is 1 mg / kg.

[0012] Preferably, the drug further comprises a pharmaceutically acceptable carrier or diluent.

[0013] Preferably, the dosage form of the drug is selected from at least one of tablets, capsules, granules, dripping pills, suspensions, syrups, enteric preparations, emulsion suspensions, and injections.

[0014] More preferably, the drug is an injection.

[0015] In a second aspect of the present invention, there is provided a pharmaceutical composition for treating AD-HIES related skeletal diseases, which is characterized in that it contains an effective dose of the STAT3 agonist colivelin TFA.

[0016] Compared with the prior art, the present invention has the following beneficial effects: By combining a mouse bone injury model with single-cell RNA sequencing (scRNA-seq) analysis of callus tissues, it is found that in the fracture healing disorder caused by STAT3 dysregulation, CXCL12 is a key regulatory factor. The STAT3 agonist colivelin TFA can alleviate the delayed fracture healing related to AD-HIES, and at the same time it can promote the expression of CXCL12, thus promoting fracture healing, which is beneficial to shortening the fracture healing time and reducing the potential safety risks brought by the long fracture healing time for patients. It can also be extended to the treatment of conventional fracture healing, providing a potential treatment idea for dealing with AD-HIES related skeletal complications. Description of the Drawings

[0017] Figure 1 Experimental results showing delayed fracture healing in osteoblast precursor cell-specific STAT3 heterozygous knockout mice in the examples; (A) Schematic diagram of STAT3 deletion in Osx-expressing osteoblast precursor cells; (B) Schematic diagram of the experimental strategy; (C) Stat3 fl / + ; Osx cre Time-series X-ray imaging analysis of the fractured femurs of Stat3 fl / + ; Osx cre mice and control mice; (D) Callus index calculated according to X-ray data, showing Stat3 fl / + ; Osx cre Callus formation in Stat3 fl / + ; Osx cre mice and control mice at different time points, with n = 5 in each group, unpaired t-test was used; (E) μCT data reconstruction reflecting Stat3 cre ; Osx fl / + ; Osx cre(Mouse); (I) Schematic diagram of three-point bending test; (J) Stat3 fl / + ; Osx cre Representative load-displacement curves obtained from three-point bending tests on fractured femurs of Col1a2 and Stat3 mice and control mice at 28 dpf, n = 5; (K) Maximum load values measured in three-point bending tests, analyzed using two-tailed Student's t-test, data are presented as mean ± standard deviation (SD), n = 5; (L) Hematoxylin-eosin (H&E) staining showing Stat3 fl / + ; Osx cre Callus formation in fractured femurs of Col1a2 and Stat3 mice and control mice at 28 dpf, n = 5; (M) Immunofluorescence staining and quantitative analysis of OPN+ cells in fractured femurs at 28 dpf, analyzed using two-tailed Student's t-test, data are presented as mean ± standard deviation (SD), n = 5; (N) Safranin O staining showing Stat3 fl / + ; Osx cre Callus formation in fractured femurs of Col1a2 and Stat3 mice and control mice at 28 dpf, n = 5.

[0018] Figure 2 Results of the experiment showing that inducible STAT3-deficient mice in the examples exhibited blocked fracture healing and impaired endochondral ossification; (A) Schematic diagram of inducible STAT3 deletion in osteogenic lineage cells expressing Col1a2. When Stat3 Col1ERT2 mice were injected with tamoxifen (TAM), Cre protein translocated to the nucleus, resulting in deletion of the exon 18-20 region (TAM: tamoxifen); (B) Schematic diagram of the experimental strategy; 6-week-old Col1 CreERT2 and Stat3 Col1ERT2 mice were injected with TAM (100 mg·kg - -1·bw - -1) every other day, starting two weeks before fracture model construction; (C) Time-series X-ray imaging analysis of fractured femurs of Col1 CreERT2 and Stat3 Col1ERT2 mice; (D) Callus index calculated from X-ray data, showing callus formation in Col1 CreERT2 and Stat3 Col1ERT2 mice at different time points, n = 5 per group, analyzed using unpaired t-test, data are presented as mean ± standard deviation (SD); (E) μCT data reconstruction reflecting callus formation in Col1 CreERT2 and Stat3 Col1ERT2 mice at 14 and 35 days after fracture; (F) Showing Col1 CreERT2 and Stat3 Col1ERT2Bone volume and tissue volume of callus in mice at 14 dpf, n = 5, two-tailed Student's t-test, data are presented as mean ± SD; (G) non-union of fractures, Fisher's exact probability test was used, n = 15 (Col1 CreERT2 and Stat3 Col1ERT2 mice); (H) Hematoxylin-eosin (H&E) staining showed the formation of hard callus and histological quantitative analysis of bone area / callus area in the fractured femurs of Col1 CreERT2 and Stat3 Col1ERT2 mice at 14 dpf and 35 dpf, n = 5, two-tailed Student's t-test, data are presented as mean ± SD; (I) Safranin O staining showed the formation of cartilage callus in Col1 CreERT2 and Stat3 Col1ERT2 mice at 5, 7, 10, and 14 days after fracture, n = 5; (J) Quantitative analysis of cartilage area in the callus, n = 5 for each group, unpaired t-test was used, data are presented as mean ± SD; (K) Immunofluorescence staining and quantitative analysis of SOX9 CreERT2 cells in the callus of Col1 Col1ERT2 and Stat3 + mice 10 days after fracture; (L) Immunofluorescence staining and quantitative analysis of OPN CreERT2 cells in the callus of Col1 Col1ERT2 and Stat3 + mice 14 days after fracture; (M) Immunofluorescence staining and quantitative analysis of EMCN+ vascular endothelial cells in the callus of Col1 CreERT2 and Stat3 Col1ERT2 mice 10 days after fracture, n = 5 for each group (K to M), two-tailed Student's t-test, data are presented as mean ± SD.

[0019] Figure 3 are the experimental results that STAT3 deficiency in the examples affects the osteogenic differentiation of periosteal cells (PCs) and angiogenesis in vitro; (A) From 4-week-old male Stat3 fl / flAfter mouse-isolated periosteal cells were infected with adenovirus expressing EGFP or Cre-EGFP for 4 h, they were placed in chondrogenic induction medium for culture, and the results of Alcian blue staining after 21 days of chondrogenic induction culture; (B) the results of alkaline phosphatase (ALP) staining of periosteal cells after 7 days of osteogenic induction culture; (C) the relative mRNA expression levels of Runx2, Sp7, Alp, and Col1 genes of periosteal cells after 7 days of osteogenic induction culture, n = 9; (D) a schematic diagram of the current in vitro culture process of human umbilical vein endothelial cells (HUVEC); (E) representative images after 24 h of HUVEC scratch assay, the scratch edges of each group of cells were marked by dotted lines, and the histogram showed the average percentage of wound closure, n = 9 per group; (F) representative images of HUVEC Transwell migration assay, n = 9; (G) representative microscopic images of HUVEC tube formation assay on Matrigel and quantitative analysis of tube area, n = 9.

[0020] Figure 4 Experimental results showing that STAT3 deficiency impairs fracture healing by inhibiting stem cell mobilization in the examples; (A) schematic diagram of the loxP site-flanked STOP cassette and its under Cre recombination; (B) 6-week-old R26 tdTomato ; Col1-Cre ERT2 In unfractured femoral sections of mice, fluorescence labeling of Col1ERT2 lineage cells (tdTomato + cells), mice were injected with TAM (100 mg / kg) 24 h before euthanasia for analysis; (C) R26 tdTomato ; Col1-Cre ERT2 In unfractured femoral sections of mice, immunofluorescence staining of Postn + , CD200 + , Ctck + , PDGFA + cells; (D) Immunofluorescence staining and quantitative analysis of SOX9 tdTomato cells in fractured femoral sections of R26 ERT2 ; Col1-Cre + mice, n = 5; (E) Immunofluorescence staining and quantitative analysis of OPN tdTomato cells in fractured femoral sections of R26 ERT2 ; Col1-Cre + mice, n = 5; (F) R26 tdTomato ; Col1-Cre ERT2 mice and R26 tdTomato ; Stat3 Col1ERT2 mice, Col1ERT2 lineage cells (tdTomato+ Cell fluorescence labeling; CB: Cortical bone; BM: Bone marrow; (G) Col1 in the callus area + Quantitative analysis of the area, n = 5 mice at each time point, unpaired t-test, data are presented as mean ± standard deviation (SD); (H) Col1 CreERT2 and Stat3 Col1ERT2 Immunofluorescence staining and quantitative analysis of KI67 cells in the femur of mice 5 days after fracture + Immunofluorescence staining and quantitative analysis of Col1 CreERT2 and Stat3 Col1ERT2 EdU incorporation experiment in the periosteal region of mice 10 days after fracture. EdU administration started 1 day before fracture. The right figure is a high-magnification representative image of the callus area; (H) n = 5 mice in each group (I) and n = 3 mice, two-tailed Student's t-test, data are presented as mean ± standard deviation (SD).

[0021] Figure 5 Results of the experiment showing that STAT3 deficiency affects the proliferation and migration of periosteal cells (PCs) in the examples; (A) Periosteal cells isolated from 4-week-old male Stat3 fl / fl mice were infected with adenovirus expressing EGFP or Cre-EGFP for 4 h, and then the cell viability was evaluated using the CCK8 reagent at 24, 48, 72, and 96 h respectively; (B) After infection with adenovirus expressing EGFP or Cre-EGFP, the proliferation ability of periosteal cells was evaluated by EdU labeling experiment; (C) Representative images of the scratch assay of periosteal cells after 24 h. The dotted lines mark the scratch edges of each group of cells. The bar chart shows the average percentage of wound closure, n = 9 for each group; (D) Results of the Transwell migration assay of periosteal cells under different treatment conditions. The migration assay was performed after the membrane cells were infected with adenovirus expressing EGFP or Cre-EGFP.

[0022] Figure 6 Single-cell transcriptome sequencing (scRNA-Seq) analysis in the examples showed that CXCL12 + MSCs were significantly reduced in the fracture callus of STAT3-deficient mice; (A) Schematic diagram of the experimental procedure for single-cell transcriptome sequencing based on the droplet-based BD platform; (B) UMAP plot showing the unsupervised clustering results of 12,089 single cells. MSCs were further divided into different subpopulations; (C) Dot plot showing the expression of classical marker genes in major cell types; (D) Violin plot showing the expression level of CXCL12; (E) Stacked bar chart showing Col1 CreERT2 and Stat3 Col1ERT2 The relative proportions of each MSC subpopulation in the samples; (F) Pseudotime trajectory analysis of each MSC subpopulation; (G) CXCL12 +MSCs and Cxcl9 + Functional enrichment analysis of MSCs

[0023] Figure 7 Results of the experiment in which STAT3 regulates CXCL12 transcription by promoting promoter activity in the examples; (A) R26 tdTomato ; Col1-Cre ERT2 mice and R26 tdTomato ; Stat3 Col1ERT2 Immunofluorescence staining of CXCL12 cells in the femur of R26;Stat3 mice on day 10 after fracture + Quantitative analysis of CXCL12 cells at the fracture site, n = 5, two-tailed Student's t-test, data are expressed as mean ± standard deviation; (C) Changes in the relative mRNA levels of CXCL12 in periosteal cells (PCs) after infection with adenovirus expressing EGFP or Cre-EGFP, n = 9, independent sample t-test; (D) Stat3 + fl / fl ​After the periosteal cells were infected with EGFP or Cre-EGFP adenovirus for 4 h and cultured in osteogenic induction medium, the ELISA detection results of the concentration of CXCL12 in the culture supernatant, n = 5; (E) Alcian blue staining results of periosteal cells cultured in chondrogenic induction medium for 21 days after treatment with CXCL12 or control; (F) ALP staining results of periosteal cells cultured in osteogenic induction medium for 7 days after treatment with CXCL12 or control; (G) Relative mRNA levels of Runx2, Sp7, Alp, and Col1 genes in periosteal cells cultured in osteogenic induction medium for 7 days after treatment with CXCL12 or control, n = 9; (H) Cell viability was detected by CCK8 after adenovirus infection to express EGFP or Cre-EGFP and subsequent treatment with CXCL12 or control; (I) Cell proliferation was detected by EdU labeling experiment after adenovirus infection to express EGFP or Cre-EGFP and subsequent treatment with CXCL12 or control; (J) Representative diagram of Transwell migration experiment of periosteal cells treated with CXCL12 or control; (K) Representative diagram of scratch experiment of periosteal cells treated with CXCL12 or control, photographed after 24 h; The scratch edges of each group of cells are marked by a dashed line, and the right bar graph is the scratch closure ratio, n = 9; (L) Representative diagram of scratch experiment of HUVECs treated with CXCL12 or control, photographed after 24 h, the scratch edge is marked by a dashed line, and the right is the bar graph of the scratch closure ratio, n = 9; (M) Representative diagram of Transwell migration experiment of HUVECs treated with CXCL12 or control; (N) Representative diagram of tube formation experiment of HUVECs cultured with Matrigel matrix and treated with CXCL12 or control and quantitative analysis of the area of tubular structures; (O) Schematic diagram of the predicted STAT3 binding site on the CXCL12 promoter; (P) In C3H10 cells, ChIP experiment was used to detect the binding of STAT3 to the CXCL12 promoter; n = 3 in the IgG group, n = 3 in the STAT3 group; (Q) In 293T cells, STAT3, constitutively active STAT3 (STAT3-C), and dominant negative STAT3 mutant (STAT3-DN) were co-transfected with the luciferase reporter gene driven by the CXCL12 promoter respectively, and the luciferase activity was detected after 48 h, n = 3; (R) To detect the effect of mutation of the potential STAT3 binding site on the CXCL12 promoter on promoter activity, STAT3 was co-transfected with the wild-type CXCL12 promoter (CXCL12-Luc) or the mutant promoter (CXCL12-mu-Luc) with two predicted STAT3 binding sites deleted into 293T cells, and then the luciferase activity was detected, n = 3, two-tailed Student's t-test, data are expressed as mean ± standard deviation.

[0024] Figure 8In the example, CXCL12 alleviates impaired fracture healing in mice with inducible osteolineage cell-specific STAT3 deletion; (A) Schematic diagram of the experimental strategy; (B) μCT reconstruction images showing the hard callus formed on day 14 after fracture; (C) Region of interest (ROI) at the fracture site; (D) Chart analyzing bone volume, tissue volume, and bone volume fraction (BV / TV) of the callus on day 14, n = 5; (E) R26 tdTomato ; Col1-Cre ERT2 mice and R26 tdTomato ; Stat3 Col1ERT2 Fluorescence-labeled images of Col1ERT2 lineage cells (tdTomato + cells) at the injury site of the fractured femurs of mice on day 14 and quantitative analysis of the Col1 + region in the callus region, n = 5 mice per time point; (F) Immunofluorescence staining and quantitative analysis of SOX9 + cells in the femur on day 10 after fracture, n = 5, independent samples t-test; (G) Immunofluorescence staining and quantitative analysis of OPN + cells in the femur on day 14 after fracture, n = 5, independent samples t-test; (H) Immunofluorescence staining and quantitative analysis of EMCN + blood vessels in the femur on day 10 after fracture, n = 5, two-tailed Student’s t-test, data are presented as mean ± standard deviation (SD).

[0025] Figure 9 In the example, CXCL12 alleviates delayed fracture healing in mice with osteoprogenitor cell-specific STAT3 heterozygous deletion; (A) Schematic diagram of the experimental strategy; (B) μCT reconstruction images showing the hard callus formed on day 28 after fracture; (C) Region of interest (ROI) at the fracture site; (D) Chart analyzing bone volume, tissue volume, and bone volume fraction (BV / TV) of the callus on day 28, n = 5; (E) Safranin O staining results of the fractured femurs of Stat3 fl / + ; Osx cre mice and control mice on day 28, showing the formation of hard callus, n = 5; (F) Immunofluorescence staining and quantitative analysis of OPN + cells in the femur on day 28, n = 5; (G) Immunofluorescence staining and quantitative analysis of EMCN + blood vessels in the femur on day 28, n = 5, two-tailed Student’s t-test, data are presented as mean ± standard deviation (SD).

[0026] Figure 10In the example, the STAT3 agonist Colivelin alleviates the delayed fracture healing of heterozygous STAT3-deficient mice and promotes CXCL12 expression; (A) Schematic diagram of the experimental strategy; (B) μCT data reconstruction shows the formation of hard callus 28 days after fracture (dpf); (C) Region of interest (ROI) around the fracture site; (D) Chart showing the bone volume, tissue volume, and bone volume to tissue volume ratio (BV / TV) of the callus 28 days after fracture, n = 5; (E) 28 days after fracture, for Stat3 fl / + ; Osx cre Safranin O staining of the fractured femurs of mice and control mice 28 days after fracture shows the formation of hard callus, n = 5; (F) Immunofluorescence staining and quantitative analysis of OPN-positive cells on the fractured femurs 14 days after fracture, n = 5; (G) Immunofluorescence staining and quantitative analysis of EMCN-positive cells on the fractured femurs 10 days after fracture, n = 5; (H) Immunofluorescence staining and quantitative analysis of CXCL12-positive cells on the fractured femurs 10 days after fracture, n = 5. Two-tailed Student's t-test was used, and the data are expressed as mean ± standard deviation (SD). Detailed implementation mode

[0027] In order to more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below in combination with the accompanying drawings and specific examples. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. It should be noted that for those of ordinary skill in the art, other embodiments obtained without departing from the concept of the present invention all belong to the protection scope of the present invention.

[0028] In the following examples, it was verified that the STAT3 agonist colivelin TFA alleviates the delayed fracture healing related to AD-HIES and promotes CXCL12 expression. The CXCL12 protein used in the experiment was purchased from MCE Company. The specific experiments include:

[0029] (1) Using Stat3 fl / + ; Osx Cre Mice were used to simulate AD-HIES disease, and a femoral fracture model was constructed. The changes in the bone mass and mechanical properties of the callus in mice were analyzed by micro-CT and three-point bending experiments, and the changes in the endochondral ossification and angiogenesis abilities in mice were analyzed by immunofluorescence staining, etc.

[0030] (2) Using Stat3 Col1ERT2A femoral fracture model was constructed in mice. The changes in callus bone mass and mechanical properties of the mice were analyzed by micro-CT and three-point bending experiments. The changes in the ability of endochondral ossification and angiogenesis in the mice were analyzed by safranin-fast green and immunofluorescence staining, etc. An R26 tdTomato ; Stat3 Col1ERT2 fluorescent mouse fracture model was used to trace the changes in the number and distribution of osteogenic lineage cells after knocking out Stat3.

[0031] (3) Single-cell suspensions of Stat3 fl / fl and Stat3 Col1ERT2 mouse callus were prepared. A single-cell atlas of the callus was constructed by single-cell transcriptome sequencing. Differential cell subsets and their markers were screened and identified. The expression changes of CXCL12 were verified by qPCR, ELISA, and immunofluorescence staining; a rescue experiment was performed in vitro using CXCL12 protein.

[0032] (4) Stat3 Col1ERT2 fracture mice were selected and intervened by in-situ injection of CXCL12 protein. The changes in callus bone mass and bone volume fraction were analyzed by micro-CT, and the changes in the ability of endochondral ossification and angiogenesis in the mice were analyzed by histological experiments.

[0033] (5) Stat3 fl / + ; Osx Cre fracture mice were selected and intervened with CXCL12 protein. The changes in callus bone mass and bone volume fraction were analyzed by micro-CT, and the changes in the ability of endochondral ossification and angiogenesis in the mice were analyzed by histological experiments.

[0034] Example 1

[0035] Experimental steps:

[0036] 1. Mouse tail gene identification

[0037] (1) Tail sampling and numbering: Mice over 3 weeks old were taken out from the animal house. The mouse numbers were pre-written on the centrifuge tubes. The mice were taken out of the cage, and about 3 mm of the mouse tail was cut off with scissors from the end of the mouse tail. The cut samples were put into the corresponding numbered centrifuge tubes, and the gender of the mice was recorded at the same time.

[0038] (2) Preparation of lysis buffer: 4 mol of urea, EDTA-2Na-2H2O, 5 g of sodium dodecyl sulfate (SDS), 100 mL of Tris-HCL (1 M, pH 8.0), and 11.688 g of NaCl were added in sequence, and then double-distilled water was added to make up to 1000 mL.

[0039] (3) DNA extraction: Add 500 μL of lysis buffer (containing proteinase K, 20 μg of proteinase K is added to 50 mL of lysis buffer), place the centrifuge tube in an oven at 55 °C and digest overnight. Add 500 μL of isopropanol to the digested sample and invert it repeatedly. Generally, flocculent stratification can be seen. Then use a centrifuge to set at 13000 rpm and centrifuge for 10 min; discard the supernatant, add 750 μL of ethanol, use a centrifuge at 13000 rpm and centrifuge for 10 min, and repeat this operation once.

[0040] (4) Open the centrifuge tube, invert it and let it dry on the laboratory tissue paper. After drying, add 200 μL of double-distilled water to dissolve.

[0041] (5) Prepare the PCR mix. The single system is as follows: 10 μL of TAQ enzyme, 2 μL of DNA solution, 1 μL of primer R end, and 1 μL of primer F end. Make up to 20 μL with double-distilled water, and set up negative and positive controls.

[0042] (6) Set the PCR instrument program: Preheat at 94 °C for 4 min, denature at 94 °C for 1 min, anneal at 61 °C for 1 min, perform 30 cycles of the second to third steps, extend at 72 °C for 1 min, maintain at 72 °C for 7 min, and end at 4 °C.

[0043] (7) Agarose gel electrophoresis: Prepare the gel. Dissolve 2% agarose in TAE solution. Heat it in a microwave oven at high power for 3 min. After cooling, add 0.001% ethidium bromide (EB) and pour it onto the gel plate with a comb inserted.

[0044] (8) Load the sample. Add 10 μL of the sample to each well and add DNA marker as a reference to each column. Start electrophoresis at 140 V for 25 min.

[0045] (9) Gel imaging analysis.

[0046] 2. Construct a fracture model

[0047] (1) Preparation: Group the mice (5 mice in each group). After weighing, anesthetize them by intraperitoneal injection with 2.5 mL / kg (anesthetic: composed of 1.6 mL of Zoletil, 0.4 mL of dexmedetomidine, and 8 mL of normal saline), and shave the hair on the right leg of the mice.

[0048] (2) Build a fracture model: Cut the skin of the mice 1 cm near the knee joint, clamp the distal end of the femur with a curved hemostat, insert a 0.7×30 mm needle into the femoral medullary cavity, use a STRONG 204 electronic grinder with a 0.5 mm ball drill to grind and break half of the femur in the middle part of the mouse femur, cut the remaining part of the femur with an ophthalmic scissors, cut the part of the needle protruding from the knee, and suture the muscles and skin of the mice in sequence.

[0049] (3) Pre-experiment to evaluate the reliability and stability of mouse fracture modeling: Confirm the successful construction of the mouse fracture model through X-ray, micro-CT analysis, and histological sections, and analyze the within-group differences to confirm the modeling stability.

[0050] 3. Shooting and analysis of X-ray

[0051] (1) Turn on the X-ray scanner and the computer.

[0052] (2) Open the scanning software, fix the mouse on a 15-cm dish with tape, keep the leg with the fracture site as far away from the body as possible and close to the dish.

[0053] (3) After scanning, organize and analyze the pictures.

[0054] (4) Calculate the callus index (CI), which is the ratio of the maximum callus diameter to the bone diameter. This index reflects the development of callus tissue at the same level; when the callus index at the fracture site increases, it indicates that the healing is in progress because bone remodeling is also taking place at the fracture site. Regularly measure the callus index to observe the growth and remodeling process.

[0055] 4. Micro-CT scanning and analysis

[0056] Use Skyscan1172 to perform micro-CT scanning, set the voltage to 49 kV, the current to 179 μA, and the precision to high-precision and high-speed. After the image scanning is completed, outline the contour of the callus according to the scanning layer to generate a three-dimensional image, and calculate the corresponding volume and parameters.

[0057] 5. Safranin-fast green staining

[0058] (1) Prepare the staining solutions:

[0059] 0.05% fast green solution: Add 0.05 g of fast green to double-distilled water and make up to 100 mL;

[0060] 1% acetic acid differentiation solution: Add 1 mL of glacial acetic acid to double-distilled water and make up to 100 mL;

[0061] 0.5% safranin staining solution: Add 0.5 g of safranin to double-distilled water and make up to 100 mL.

[0062] (2) Select appropriate sections. After baking the paraffin sections at 65 °C for 45 min, dewax them with xylene, soak them twice, 15 min each time, and then sequentially soak them with 100% ethanol, 95% ethanol, 70% ethanol, and double-distilled water for 5 min each for gradient rehydration. After taking out the frozen sections from -20 °C, warm them to room temperature, soak them in PBS for 5 min, and repeat 3 times.

[0063] (3) Immerse in 0.05% fast green for 1.5 min for staining; immerse in acetic acid for 10 s for differentiation. Immerse in 0.5% safranin for 1 min for staining; immerse in 95% ethanol for 3 s for differentiation; then rinse with running water for 30 min; infiltrate with xylene twice, 15 min each time, to make the tissue transparent.

[0064] (4) Mount the slides with neutral balsam, place them in a fume hood to dry overnight, observe and take pictures under a microscope.

[0065] 6. Three-point bending test and analysis

[0066] Conduct a three-point bending test using an Instron 5569 universal tensile strength testing machine. Set the three-point bending span to 4 mm and the loading speed to 0.5 mm / s. Place the mouse femur in the sample area, start the machine, and stop the machine after the femur fractures. Measure the results through standardization, and based on the femur displacement data measured in real time under the loading load, plot the load-displacement curve and analyze the maximum load of the sample.

[0067] 7. Frozen immunofluorescence staining

[0068] (1) Preparation: Select appropriate sections. After taking the frozen sections out of -20°C, warm them to room temperature and immerse them in PBS for 5 min, repeating 3 times.

[0069] (2) Permeabilization: Wipe off the PBS on the surface of the sections, place them in a wet box to avoid excessive drying of the tissue surface. Drop about 200 μL of 0.5% Triton X-100 on each section for nuclear permeabilization treatment to ensure complete coverage of all tissues. Wash with PBS for 5 min, repeating 3 times.

[0070] (3) Blocking: Prepare the blocking solution by dissolving 3% BSA in 0.1% Triton X-100. After complete dissolution, drop the blocking solution on each section, about 200 μL per section, and place it in a wet box to block for 1 h.

[0071] (4) Primary antibody incubation: Pour off the blocking solution on the surface of the sections, wipe off the excess blocking solution, drop the prepared primary antibody on each section, about 100 μL per section. You can cover the sections with Parafilm to keep them moist and place them at 4°C overnight.

[0072] (5) Secondary antibody incubation: Take out the wet box, warm it for 30 min, immerse it in PBS for 5 min, repeating 3 times to wash off the primary antibody, and incubate the secondary antibody at room temperature for 1 h.

[0073] (6) Mounting: Wash with PBS for 5 min, repeating 3 times, and mount the slides with a DAPI-containing mounting medium.

[0074] (7) Observe, photograph and quantitatively analyze under a fluorescence microscope.

[0075] 8. Extraction of callus cells

[0076] (1) Preparation of digestive solution: Prepare the digestive solution and add each solvent in sequence: 49 mL of α-MEM medium, 1 mL of penicillin-streptomycin double antibody, 50 mg of type II collagenase, 100 mg of dispase II, 0.5 g of P188, and 50 μL of DNase I. Place it in a preheated water bath at 37 °C.

[0077] (2) Dislocate the cervical vertebrae of the mouse to death and soak it in 75% ethanol. Take out the leg bones of the mouse and place them in pre-cooled PBS. Cut open from the patellar ligament, separate the femur and tibia, and keep the joints intact, retaining both ends intact to avoid bone marrow exudation during subsequent separation.

[0078] (3) Enter the cell room, place the femur to be separated in a small amount of pre-cooled PBS to keep it moist. Use the flat end of the periosteal elevator to gently scratch a white mark on one section of the diaphysis, and soak the femur in PBS for 30 min. Then use the inclined plane of the periosteal elevator to peel off the periosteum along the direction of the mark. The periosteum is white and strip-shaped. Collect the periosteum with 1 mL of PBS and place it in a 1.5 mL centrifuge tube. Use a centrifuge to set at 3000 rpm and centrifuge for 3 min. Discard the supernatant, add 1 mL of digestive solution, place the centrifuge tube on a shaker at 37 °C, set at 280 rpm, and digest for 5 min.

[0079] (4) Take out the centrifuge tube, transfer the supernatant to 10 mL of pre-cooled complete medium to terminate digestion and place it on an ice bath. Add 1 mL of digestive solution to the centrifuge tube, place it on a shaker at 37 °C, set at 280 rpm, and digest for 5 min.

[0080] (5) Repeat the above step 3 times, for a total of 4 times of digestion in the digestive solution.

[0081] (7) Place the digested cells in a centrifuge tube, use a centrifuge to set at 800 rpm, 4 °C, and centrifuge for 4 min.

[0082] (8) Carefully discard the supernatant, and resuspend with 500 μL of PBS (containing 5% filtered FBS).

[0083] (9) Cell filtration and red blood cell lysis: After using a centrifuge to set at 800 rpm and centrifuge for 3 min, discard the supernatant; after resuspending with 2 mL of flow buffer, filter the cells through a 40 μm cell strainer, use a centrifuge to set at 800 rpm and centrifuge for 3 min, discard the supernatant; resuspend the cells with 2 mL of lysis solution, place the centrifuge tube on ice for 5 min, add 5 mL of PBS to terminate red blood cell lysis; use a centrifuge to set at 800 rpm and centrifuge for 3 min, and add 1 mL of flow buffer to resuspend.

[0084] (10) Cell counting: After resuspending with flow cytometry buffer, perform cell counting, calculate the total cell amount, evenly distribute the cells into 1.5 mL centrifuge tubes, adjust the volume of each tube to 100 μL, with 1×106 cells. The excess cells are placed on ice for use as negative controls.

[0085] (11) Staining:

[0086] a. Add antibodies according to the cell amount (single positive controls need to be set), record the cell number, antibody concentration and volume, and stain in the dark on ice for 30 min.

[0087] b. Add 1 mL of flow cytometry buffer to wash the cells, centrifuge at 800 rpm and 4 °C for 5 min using a centrifuge, and discard the supernatant.

[0088] c. Centrifuge at 800 rpm and 4 °C for 5 min using a centrifuge, discard the supernatant, add 100 μL of flow cytometry buffer to resuspend, select the corresponding antibody for staining, and stain in the dark on ice for 20 min.

[0089] d. Add 1 mL of flow cytometry buffer to wash the cells, centrifuge at 800 rpm and 4 °C for 2 min using a centrifuge, and discard the supernatant.

[0090] e. Add 100 μL of flow cytometry buffer to resuspend the cells, place them in the dark on ice, and detect using a flow cytometer.

[0091] 9. Flow cytometry sorting

[0092] (1) Loading:

[0093] a. Call the sorting template and uncheck restore laser delay and restore fluidics setup.

[0094] b. Set the storage path. Under Recording Settings on the left side of the software, create a folder for the experiment of the day in “Folder:” of FCs File.

[0095] c. Before loading, remove the original sample tube, press BACK FLUSH, wait for about 10 drops of liquid to drip out, then close BACKFLUSH, replace with the sample, open SAMPLE. Generally, set the pressure of SAMPLE at 18.0, and place the collection tube for receiving cells in the sorting chamber.

[0096] d. Click Acquire under Acquisition Dashboard in the software. After detection signals appear, adjust the voltage according to the current sample and draw gates. After setting the sorting population, sorting can be performed.

[0097] e. Storing data: Name the file in the FCS File. Click Record to store an original FCS file with 20,000 cells. Click the PDF icon under Worksheet to store real-time data.

[0098] (2) Sample change:

[0099] a. After the sorting of the current sample is completed, close the SAMPLE key to stop the sample injection. Click Stop in the Sort Layout interface of the computer software to stop sorting. Click Preview in the Sort Layout interface to store the sorted data as a PDF file. Take out the receiving tube in the sorting chamber, seal it with a sealing film, and temporarily place it on ice for preservation.

[0100] b. Remove the current sample tube, press BACK FLUSH, close BACK FLUSH after about 10 drops, replace it with the next sample, open SAMPLE, and place the receiving tube properly in the sorting chamber.

[0101] c. Click Reset in the software to refresh. After new detection signals appear, appropriately adjust the position and size of the gate according to the current sample. After setting the sorting population, sorting can be carried out.

[0102] d. Storing data: Select "File:" in the FCS File. After naming, click Record to store an original FCS file with 20,000 cells. Click the PDF icon under Worksheet to store real-time data.

[0103] (3) Sorting completion:

[0104] a. Press the SAMPLE key to stop the sample injection. Click Stop in the Sort Layout interface of the computer software to stop sorting. Click Preview in the Sort Layout interface to store the sorted data as a PDF file. Take out the receiving tube in the sorting chamber, seal it with a sealing film, collect it, and temporarily place it on ice for preservation.

[0105] b. Upload the data. Remove the current sample tube, press BACK FLUSH, close BACK FLUSH after about 10 drops, replace it with 3% penicillin-streptomycin double antibody, open SAMPLE, and open the lids of all sample tubes and throw them into the disinfection bucket in the buffer room.

[0106] 10. Single-cell transcriptome library construction and sequencing

[0107] The callus single-cell suspension was diluted to an appropriate concentration, and the BD Rhapsody system was used to isolate and capture single cells by means of micropore technology. After the micropores were captured, cell lysate was used to lyse the single cells to release mRNA. Magnetic beads were incubated. Each micropore could accommodate one magnetic bead, and reverse transcription primers were linked to the magnetic beads. The primers mainly consisted of the following three parts:

[0108] (1) Cell barcode (CB). The CB sequences of the primers on each magnetic bead were the same, while the CB sequences of the primers on different magnetic beads were different, so as to achieve the function of distinguishing single cells.

[0109] (2) Unique molecular identifier (UMI). The UMIs of the primers on each magnetic bead were different from each other and were used to distinguish mRNA molecules.

[0110] (3) Polydeoxythymidine (poly dT) was complementary to the 3'-polyadenylate tail (poly A) of mRNA. After the magnetic beads were incubated, the poly A of mRNA was complementary to the poly dT on the magnetic beads. After the magnetic beads were recovered, reverse transcription was carried out, and UMI and CL were introduced through complementary DNA (cDNA) synthesis, so that all mRNAs from the same cell source were labeled with the same CL, while different mRNA molecules from the same cell source were labeled with different UMIs. The target gene was amplified by multiplex PCR. First, PCR 1 primer (outer primer) was used to amplify the cDNA into larger fragments, and then PCR 2 primer (inner primer) was used to amplify the cDNA into smaller fragments, thereby increasing the specificity of the target gene amplification. Finally, index and sequencing adapters were added, and the library construction was completed. Nova-seq was used for second-generation sequencing, the sequencing mode was selected as PE150, and the data volume was 150G for each sample.

[0111] 11. Single-cell sequencing data analysis

[0112] (1) Quality control: The default parameter fast was used for scRNA-seq data to filter and delete low-quality sequencing data. The Seurat Package was used to remove dead cells, doublets, low-quality cells, and biases caused by batch effects for subsequent analysis.

[0113] (2) Cell clustering: For the cells grouped by the t-distributed stochastic neighbor embedding (tSNE) algorithm, based on the gene expression, unsupervised clustering was performed based on the Graph cluster or K Mean algorithm to further divide the single-cell population.

[0114] (3) RdsPlot Analysis: Using R language, specific genes and specific cell populations are selected to plot single-cell Uniform Manifold Approximation and Projection (UMAP) / tSNE plots, violin plots, bubble plots, etc.

[0115] (4) Cell Subclustering: After the initial division of single-cell populations, a differential screening algorithm is further used to obtain the Marker Gene population of single-cell Clusters. Through these Marker genes, we can speculate and identify the cell types to which each cell population belongs, and select the populations of interest for further subdivision.

[0116] (5) Pseudotime Analysis: Use machine learning strategies to learn the explicit principal graph constructed from single-cell genomics data to arrange the cell order, and accurately describe the trend and outcome of cell fate under mechanical action.

[0117] (6) Gene Function Analysis: Based on the database, genes falling into each cluster are analyzed by Gene Ontology (GO) from three aspects: biological process, cellular component, and molecular function, to obtain all GOs participated by the genes. The significance level of each GO is calculated using Fisher's test, so as to screen out the significance of gene enrichment.

[0118] (7) Signal Pathway Analysis: Based on the gene annotation database, signal pathways significantly enriched with differentially expressed genes are detected. Genes falling into each cluster are annotated by Pathway based on the KEGG database to obtain all Pathway Terms participated by the genes. The significance level of the Pathway is calculated using Fisher's test, so as to screen out the significant Pathway Terms of gene enrichment.

[0119] (8) Analysis through Gene Expression Quantification Set: The variance inflation factor algorithm is used to perform GSEA-like enrichment analysis on the gene set, and compare the enrichment differences of the same gene in different Clusters.

[0120] 12. Alkaline Phosphatase Staining

[0121] After cell induced differentiation, first remove the culture medium, fix with 4% PFA for 15 min, gently wash 3 times with PBS, and prepare the BCIP / NBT staining working solution in the dark according to the following ratio: 3 mL alkaline phosphatase chromogenic buffer, 10 μL BCIP solution (300×), and 20 μL NBT solution (150×). After adding the working solution to the well plate, place it in an oven at 37 °C and incubate in the dark for 20 - 30 min until the color development is complete. Aspirate the working solution, wash 3 times with PBS, dry, and observe and take pictures under the microscope.

[0122] 13. Real-time fluorescence quantitative polymerase chain reaction

[0123] (1) Remove the culture medium, rinse the cells twice with PBS, then add 1 mL of Trizol and transfer it to a centrifuge tube free of RNase.

[0124] (2) Pre-cool the centrifuge to 4 °C, add 200 μL of chloroform, shake the centrifuge tube vigorously, then let it stand for 5 min. Use the centrifuge to set at 12000 rpm and centrifuge for 15 min.

[0125] (3) Pipette the colorless and clear supernatant into a new centrifuge tube, add an equal volume of isopropanol, shake the centrifuge tube vigorously, and let it stand for 10 min. Use the centrifuge to set at 12000 rpm and centrifuge for 15 min.

[0126] (4) Discard the supernatant, add 1 mL of 75% ethanol (diluted with DEPC water), shake vigorously, use the centrifuge to set at 12000 rpm and centrifuge for 15 min. Repeat this step once.

[0127] (5) Aspirate the supernatant, invert the centrifuge tube on a clean tissue paper and dry it at room temperature.

[0128] (6) Add 20 μL of DEPC water to dissolve the RNA, and use a ultra-micro ultraviolet spectrophotometer to measure the RNA concentration.

[0129] (7) Reverse transcribe the RNA into cDNA: 500 ng of RNA, 2 μL of 5×ABScriptⅡRT Mix, make up to 10 μL with DEPC water. The reverse transcription reaction conditions are as follows: 25 °C for 5 min, 42 °C for 15 min, 85 °C for 5 s, and end at 4 °C.

[0130] (8) Real-time fluorescence quantitative polymerase chain reaction (qPCR): Each well contains 0.5 μL of cDNA, 3.8 μL of double-distilled water, 5 μL of 2×Universal SYBR Green Fast qPCR Mix, a 10 μL system. Set 3 replicates for each sample, use the centrifuge to set at 1000 rpm and centrifuge for 1 min, then put it into the PCR instrument to start the program. The program settings are as follows: Pre-denaturation at 95 °C for 1 min, 95 °C for 5 s, 60 °C for 30 s to collect fluorescence signals (a total of 50 cycles). The primer information is shown in Table 1.

[0131] Table 1

[0132]

[0133]

[0134] 14. Enzyme-linked immunosorbent assay

[0135] (1) Cell culture supernatant or other biological fluids: After collecting the liquid, centrifuge it at 1000×g for 20 min at 2 - 8°C to remove impurities and cell debris, and take the supernatant for detection.

[0136] (2) Set up standard wells, blank wells, and sample wells respectively. Add 100 μL of serially diluted standards to the standard wells, add 100 μL of standards and sample diluent to the blank wells, and add 100 μL of the sample to be tested to the remaining wells. Cover the ELISA plate with a film and incubate at 37°C for 90 min.

[0137] (3) Flick out the liquid in the wells without washing. Add 100 μL of biotinylated antibody working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 1 h.

[0138] (4) Flick out the liquid in the wells. Add 350 μL of washing solution to each well, soak for 1 - 2 min, aspirate or flick off the liquid in the ELISA plate, and pat dry on thick absorbent paper. Repeat this washing step 3 times.

[0139] (5) Add 100 μL of enzyme conjugate working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 30 min.

[0140] (6) Flick out the liquid in the wells and wash the plate 5 times.

[0141] (7) Add 90 μL of substrate solution to each well, cover the ELISA plate with a film, and incubate at 37°C in the dark for about 15 min.

[0142] (8) Add 50 μL of stop solution to each well to terminate the reaction.

[0143] (9) Measure the optical density of each well at a wavelength of 450 nm using an ELISA reader and analyze the experimental results.

[0144] 15. Scratch assay

[0145] (1) Seeding: After completely digesting the cells, add complete medium to terminate the digestion. Pipette the cells to make them evenly dispersed, then seed them in proportion and place the cell scratch insert into a 24-well plate.

[0146] (2) After 1 day, remove the cell scratch insert and change the medium. The insert forms a scratch of 500 μm.

[0147] (3) Harvest samples after an interval of 24 h. Wash the cells 2 times with PBS, fix the cells with 4% PFA for 15 min. Then wash the cells 2 times with PBS, 15 min each time. Add DAPI staining solution, observe and take pictures using a fluorescence microscope.

[0148] 16. Transwell migration assay

[0149] Performed using 24-well plate inserts to isolate Stat3 fl / fl The femurs of mice were used to extract PCs and BMSCs from the mice. Stat3 in the cells was knocked out by adenovirus. The supernatant of the cultured cells was taken and placed in the lower chamber, and 200 μL of HUVEC suspension with a density of 5×10 4 cells / well was inoculated in the upper chamber. After incubation for 24 h, non-migrating cells on the upper surface of the membrane were gently removed with a cotton swab, and the migrating cells on the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Three to five regions were randomly selected for photographing and counting.

[0150] 17. Tube formation assay

[0151] For the diluted Matrigel, repeated freeze-thaw cycles should be minimized as much as possible. Thaw the Matrigel on ice, place the 96-well plate on ice, and add 50 μL of Matrigel to each well to coat the 96-well plate.

[0152] HUVECs were digested with 0.25% EDTA trypsin. After the cells detached, they were neutralized with medium containing double FBS. At room temperature, the centrifuge was set at 1200 rpm and centrifuged for 3 min, and then resuspended in 3 mL of medium. The cell concentration of HUVECs was determined by counting the cells. HUVECs were thoroughly mixed at a cell density of 4×10 5 / mL and distributed into the coated 96-well plate. The plate was placed in an incubator at 37 °C and 5% CO2 for 4 - 6 h. The cells were visualized using an optical microscope, images of the capillary network were taken, and the tube length was calculated using Scion Image software.

[0153] 18. Chromatin immunoprecipitation

[0154] (1) Cell cross-linking: The cells were treated with 1% formaldehyde and shaken at room temperature for 10 - 15 min. Glycine was added to terminate the cross-linking, and the cells were incubated on ice for 5 min; washed twice with PBS for 5 min each time, the cells were collected, and the centrifuge was set at 2000 rpm and centrifuged for 5 min.

[0155] (2) Cell lysis: Lysis buffer (containing protease inhibitor) was added and the cells were lysed on ice for 10 min; the centrifuge was set at 5000 rpm, 4 °C, and centrifuged for 5 min, the supernatant was discarded, and the precipitated cell nuclei were collected.

[0156] (3) Chromatin fragmentation: The DNA was fragmented to 200 - 1000 bp using an ultrasonic disruptor, and a small amount of the sample was run on an agarose gel or detected for fragment size using a Bioanalyzer.

[0157] (4) Immunoprecipitation: Take 50 - 100 μg of chromatin, add 5 μg of the target antibody, and incubate with rotation at 4°C overnight. Add Protein A / G magnetic beads, place at 4°C, bind for 2 h, separate using a magnetic stand, and wash 3 times with low-salt / high-salt buffer to remove non-specific binding.

[0158] (5) Reverse cross-linking and DNA purification: Add 5 M NaCl and reverse cross-link overnight at 65°C. Digest proteins with proteinase K. Recover DNA using a DNA purification column.

[0159] (6) Data analysis: Use qPCR to detect the enrichment of the target DNA fragment (primers are designed at the predicted binding sites).

[0160] 19. Dual-luciferase reporter gene assay

[0161] (1) Cell lysis: After 24 - 48 h of plasmid transfection, discard the culture medium and gently rinse once with PBS; in a 24-well plate, add 80 μL of lysis buffer to each well, incubate with a shaker for 30 min to ensure complete cell lysis; centrifuge at 12,000 rpm for 5 min using a centrifuge, take the supernatant, and discard the pellet.

[0162] (2) Fluorescence detection: In a white opaque 96-well plate, add 15 μL of cell lysate to each well. Then add an equal volume of premixed Luciferase Assay Reagent II and immediately detect the fluorescence intensity (Firefly luciferase activity). After the detection is completed, add an equal volume of premixed Stop&Glo Reagent II to each well and measure the fluorescence intensity (Renilla luciferase activity).

[0163] (3) Data analysis: Record the two fluorescence intensity values, calculate the Firefly / Renilla fluorescence intensity ratio for normalization and statistical analysis.

[0164] 20. In vivo CXCL12 administration method and dose

[0165] (1) Dissolve CXCL12 in 10% FBS.

[0166] (2) Mice are given an in-situ injection at a dose of 100 ng dissolved in 50 μL every other day. The blank control group is injected with 10% FBS at the same dose, and samples are collected according to the experimental schedule to observe the phenotype.

[0167] 21. In vivo colivelin administration method and dose

[0168] (1) Dissolve the STAT3 agonist CLN in double-distilled water.

[0169] (2) Mice were intraperitoneally injected with a dose of 1 mg / kg daily, and the blank control group was injected with double-distilled water at the same dose. Samples were collected according to the experimental schedule to observe phenotypes.

[0170] Results

[0171] (1) Using Stat3 fl / + ; Osx Cre Mice were used to simulate AD-HIES disease and a femoral fracture model was constructed ( Figure 1 A-B). The changes in callus bone mass and mechanical properties of mice were analyzed by micro-CT and three-point bending experiments, and the changes in the ability of endochondral ossification and angiogenesis of mice were analyzed by immunofluorescence staining, etc. Compared with Osx Cre mice, Stat3 fl / + ; Osx Cre mice had a slow resorption rate of callus and a larger remaining callus volume 28 days after femoral fracture ( Figure 1 C-G), and the mechanical properties decreased ( Figure 1 I-K); the results of histological experiments showed that Stat3 fl / + ; Osx Cre mice had decreased abilities of endochondral ossification and angiogenesis ( Figure 1 L-N).

[0172] (2) Using Stat3 Col1ERT2 mice to construct a femoral fracture model ( Figure 2 A-B). The changes in callus bone mass and mechanical properties of mice were analyzed by micro-CT and three-point bending experiments, and the changes in the ability of endochondral ossification and angiogenesis of mice were analyzed by safranin-fast green and immunofluorescence staining, etc. A R26 tdTomato ; Stat3 Col1ERT2 fluorescent mouse fracture model was constructed to trace the changes in the number and distribution of osteogenic lineage cells after Stat3 knockout. The results showed that compared with Stat3 fl / fl mice, Stat3 Col1ERT2 mice did not form the maximum callus 14 days after femoral fracture, and the total volume, bone volume, and bone volume fraction of the callus were all reduced ( Figure 2 C-G); the number of osteogenic lineage cells in Stat3 Col1ERT2 mice was significantly reduced ( Figures 4 - 5 ); the results of immunofluorescence staining showed that Stat3 Col1ERT2 mice had decreased abilities of endochondral ossification and angiogenesis ( Figure 2 H-M and Figure 3 ).

[0173] (3) Preparation of single-cell suspensions of Stat3 fl / fl and Stat3 Col1ERT2 mouse calluses ( Figure 6(A), A single-cell atlas of callus was constructed by single-cell transcriptome sequencing, differential cell subsets and their markers were screened and identified, and the expression changes of CXCL12 were verified by qPCR, ELISA and immunofluorescence staining; a rescue experiment was performed in vitro using CXCL12 protein. The results showed that the single-cell atlas of mouse callus was divided into eight cell subsets. Further subdivision of the mesenchymal stem cell (MSC) subset revealed a differential subset CXCL12 + MSCs. The number of cells in this subset decreased after knocking out Stat3 ( Figure 6 (B-G), and its markers were identified by qPCR, ELISA and immunofluorescence staining ( Figure 7 (A-D); treatment with CXCL12 protein could effectively rescue the proliferation, migration, chondrogenic and osteogenic differentiation abilities of periosteal cells with Stat3 knocked out ( Figure 7 (E-J). Meanwhile, CXCL12 could significantly enhance the migration and tube formation abilities of vascular endothelial cells co-cultured with Stat3-knockout periosteal cells ( Figure 6 and Figure 7 (K-N), and STAT3 regulated its transcription by enhancing the activity of the Cxcl12 promoter ( Figure 7 (O-R).

[0174] (4) Stat3 Col1ERT2 fracture mice were selected and intervened by in-situ injection of CXCL12 protein ( Figure 8 (A-B). The changes in callus bone mass and bone volume fraction were analyzed by micro-CT, and the changes in the abilities of endochondral ossification and angiogenesis in mice were analyzed by histological experiments. The results showed that in Stat3 Col1ERT2 mice with in-situ injection of CXCL12, the total volume, bone volume and bone volume fraction of callus formed 14 days after fracture all increased ( Figure 8 (C-D), and its abilities of endochondral ossification and angiogenesis both increased ( Figure 8 (E-H).

[0175] (5) Stat3 fl / + ; Osx Cre fracture mice were selected and intervened with CXCL12 protein ( Figure 9 (A-B). The changes in callus bone mass and bone volume fraction were analyzed by micro-CT, and the changes in the abilities of endochondral ossification and angiogenesis in mice were analyzed by histological experiments. The results showed that in Stat3 fl / + ; Osx Cre mice with in-situ injection of CXCL12, the total volume and bone volume of callus formed 28 days after fracture both decreased ( Figure 9 (C-D), and its abilities of endochondral ossification and angiogenesis both increased ( Figure 9 (E-G).

[0176] (6) Select Stat3 fl / + ; Osx Cre For fracture mice, the STAT3 agonist colivelin (CLN) was intraperitoneally injected for intervention ( Figure 10 A - B). The changes in callus bone mass and bone volume fraction were analyzed by micro-CT, and the changes in the ability of endochondral ossification and angiogenesis in mice were analyzed by histological experiments. Stat3 fl / + ; Osx Cre After intraperitoneal injection of CLN in mice, the total volume and bone volume of the callus formed 28 days after fracture decreased ( Figure 10 C - D), while the ability of endochondral ossification and angiogenesis increased ( Figure 10 E - G). Meanwhile, the expression of CXCL12 in the callus increased ( Figure 10 H).

[0177] In summary, using Stat3 fl / + ; Osx Cre mice to simulate the fracture model of AD - HIES patients, the mice showed delayed fracture healing. On this basis, it was confirmed that colivelin can be used to treat AD - HIES fracture healing disorders ( Figure 1 and Figure 10 ), which can relieve the delayed fracture healing related to AD - HIES. At the same time, it can promote the expression of CXCL12, achieve the promotion of fracture healing, help shorten the fracture healing time, and reduce the potential safety risks brought by the long fracture healing time for patients. It can also be applied to the treatment of conventional fracture healing, providing potential treatment ideas for dealing with AD - HIES related skeletal complications.

[0178] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of STAT3 agonist colivelin TFA in the preparation of a medicament for treating AD-HIES related skeletal diseases.

2. The application according to claim 1, characterized in that, The skeletal disease is AD-HIES related minor traumatic fracture.

3. The application according to claim 1, wherein The skeletal disease is AD-HIES related recurrent fracture.

4. The application according to claim 1, characterized in that, The skeletal disease is AD-HIES related fracture healing disorder.

5. The application according to claim 1, characterized in that, The medicament uses STAT3 agonist colivelin TFA as the active ingredient, and the effective dose is 0.1 - 5 mg / kg.

6. The application according to claim 5, wherein The medicament uses STAT3 agonist colivelin TFA as the active ingredient, and the effective dose is 1 mg / kg.

7. The application according to claim 5, wherein The medicament further comprises a pharmaceutically acceptable carrier or diluent.

8. The application according to claim 7, characterized in that, The dosage form of the medicament is selected from at least one of tablets, capsules, granules, dripping pills, suspensions, syrups, enteric preparations, emulsion suspensions and injections.

9. The application according to claim 8, characterized in that The medicament is an injection.

10. A pharmaceutical composition for treating AD-HIES related skeletal diseases, characterized in that, It contains an effective dose of STAT3 agonist colivelin TFA.

Images