CN117987526A - Application of NF2 gene in repairing and regenerating defective skull - Google Patents

Abstract

The invention belongs to the field of regenerative medicine, and particularly relates to an application of NF2 genes in defective skull regeneration or repair. The inventor utilizes Cre/Floxp conditional knockout NF2 gene, osteoblast in-vitro culture, co-immunoprecipitation, micro-CT and other technologies to determine that Nf2 regulates and controls the mesenchymal stem cells of the cranium suture to participate in defective skull regeneration through interaction with FAK, and provides a new target point and a new idea for clinical treatment of defective skull regeneration.

Description

Application of NF2 gene in repairing and regenerating defective skull

Technical Field

The invention belongs to the field of regenerative medicine, and in particular relates to application of NF2 genes in defective skull regeneration.

Background

Skull defects are a relatively common clinical condition. Factors such as sports injuries, traffic accidents or diseases can cause various degrees of skull damage. In addition, birth defects can also cause defects in the skull, including craniosynostosis and hypoplasia. Birth defects of the skull are also one of the main causes of disability and death in children. For these skull defects, the traditional method is mainly used for bone regeneration reconstruction in clinic, including autograft, allograft, synthetic material use and the like.

In recent years, research has revealed that a population of mesenchymal stem cells exists in the skull suture, and the population of cells has self-proliferation capacity and strong differentiation potential and has the property of directional migration to defective tissues. The discovery provides a new thought and a new strategy for the regeneration of defective skull and the clinical treatment of birth defects of skull, and has important scientific value and clinical application prospect.

There are studies on gene profiles of different tissues of the skull, such as gene expression profiles of frontal bone from neural crest cells and parietal bone from mesoderm, which are found to have significant differences by high throughput sequencing techniques. Including Neurofibromin gene (NF 2), NF2 is mostly related to tumorigenesis, neurological diseases, organ development, and neurocellulose neurofibromin (NF 2) is differentially expressed in different tissues of the skull, but its specific function in skull development and defective skull regeneration has not been reported. Nf2 was first found in patients with clinical neurofibromatosis and contained two clinical types, type 1 (Nf 1) codes neurofibromin; type 2 (Nf 2) encodes Merlin protein, which belongs to a membrane cytoskeletal protein family member, with high homology to ERM proteins.

The research shows that Nf2 plays a regulating role in the connection of cell membranes and cytoskeleton, and relates to cell proliferation, signal transduction, tissue homeostasis and the like. Nf2 is the most interesting tumor suppressor, and can regulate the growth of tumor cells by inhibiting the Hippo signal pathway, and can also regulate the growth of tumor cells by interacting with Ras and RasGAP, mTOR and other signals. Intracellular Nf2-YAP interactions have a regulatory role in the tumor cell-induced iron apoptosis program. Thus, nf2 has a complex and diverse regulatory mechanism in tumorigenesis, and Nf2/Merlin is also considered as a potential target for development of antitumor drugs.

Early use of Nf2 as a promoter expression reporter gene lacZ, X-Gal staining showed that Nf2 had expression in different cells in early mouse embryo (E7.0-12.5), including neural tubes and migrating neural crest cells, suggesting that Nf2 has regulatory functions for neural crest cell-derived tissue and organ development. Recently, it was found that the expression of Nf2 in the neuroepithelium is essential for eye development, and that the Nf2-Hippo-YAP signaling pathway has a key regulatory role. Nf2 is involved in regulating the differentiation of inner cell mass in blasts. During development of ureteral buds Nf2 can inhibit YAP/TAZ activity by Hippo to promote bifurcation formation of the top cells. During bone system development, nf 1-deficient mice have increased bone formation and bone mass, but the specific function of Nf2 in defective skull regeneration has not been reported.

Experiments of the inventor find that Nf2 is an important factor for regulating and controlling FAK activity and defective skull regeneration; FAK is known to play a key role in cell migration as focal adhesion protein, and FAK and its mediated signaling pathway are also key signaling pathways that promote osteogenic differentiation. Thereby confirming that: nf2 regulates and controls the craniosynostosis mesenchymal stem cells to participate in the regeneration of defective skull through interaction with FAK to mediate signal transduction, thereby providing a new target point and a new idea for the clinical treatment of the regeneration of defective skull.

Disclosure of Invention

The invention illustrates the important role of NF2-FAK functional complex in defective skull regeneration.

The inventor uses Cre/Floxp-ER technology to specifically knock out NF2 gene in the skull of a mouse (C57 BL), and mainly comprises the steps of breeding a female mouse of F/F gene, mating a male mouse of F/+PRX1-CRE-ER+ gene, and obtaining an embryo (CKO) of F/F PRX1-CRE-ER+ gene knockout after the female mouse is pregnant for a certain period of time.

Extracting genome DNA, carrying out genotyping verification on animal phenotypes by a PCR technology, and detecting protein expression level by Western, so as to determine the animal phenotypes of NF2 knocked out (CKO) in mesenchymal stem cells.

Taking the skull cells of the mice as osteoblasts for in vitro culture. Taking the juveniles skull (frontal bone and/or parietal bone) to digest periosteum by pancreatin, then culturing the cells in vitro, observing the growth state of the cells, and collecting osteoblasts for detecting NF2 interaction proteins. And taking mesenchymal stem cells of the bone marrow cavity of the animal, obtaining the mesenchymal stem cells, knocking down the influence of NF2 analysis on the migration of the mesenchymal stem cells in the mesenchymal stem cells, and carrying out statistical analysis.

The method utilizes molecular cloning to construct NF2 and FAK over-expression plasmid in vitro, determines whether NF2-FAK forms an interaction relationship through an immune coprecipitation technology, and determines that the interaction relationship of NF2-FAK is direct through detection of a living cell protein interaction technology.

The regeneration condition of the skull is observed through a small animal CT technology, a skull defect model is constructed, the influence of the loss of NF2 in mesenchymal stem cells on the regeneration of the defect skull is analyzed, and further, how the interaction of NF2-FAK affects the regeneration of the defect skull is clarified.

The inventors have thus found that knocking down NF2 genes in mesenchymal stem cells results in a significant decrease in mesenchymal stem cell migration; meanwhile, through an inducible gene knockout mouse animal model technology, NF2 genes are knocked out in mesenchymal stem cells of skull suture, and found that the mesenchymal stem cells with Nf2 deficiency cause defective skull regeneration defects, and the data indicate that the NF2 genes are key factors for skull defect regeneration regulation. Further, according to a cell model and a conditional gene knockout mouse model, the defect of skull regeneration caused by knockout of Nf2 in craniosynostosis mesenchymal stem cells is found; abnormal migration and differentiation of NF 2-deficient mesenchymal stem cells; it was further found that Nf2 protein interacts with focal adhesion kinase (Focal adhesion kinase, FAK).

In a first aspect, the invention provides the use of an NF2 gene for the preparation of a reagent for diagnosing and/or treating a condition associated with defective skull regeneration defects.

Preferably, the defective skull regeneration defect related disorder is selected from a post-motor injury skull regeneration disorder or a post-surgical skull regeneration disorder.

Preferably, the application is selected from:

a) The NF2 gene is used as a target to be applied to the preparation of medicaments for treating the defect skull regeneration defect related diseases;

b) NF2 gene is used as a target to be applied to the screening of medicines for treating the defect skull regeneration defect related diseases; or (b)

C) NF2 gene is used as a target to prepare a reagent for diagnosing the defect skull regeneration defect related diseases.

In a second aspect, the invention provides a kit for aiding in the diagnosis of a condition associated with defective skull regeneration defects, characterized in that the kit comprises reagents for detecting a mutation or expression level of the NF2 gene.

Preferably, the kit comprises one or more of nucleic acid extraction reagents, PCR reagents, genome/transcriptome sequencing reagents, gene-specific primers or probes, antibodies specific for gene expression products, western Blot detection reagents, immunohistochemical staining detection reagents, protein chips, in situ hybridization or gene chip detection reagents.

Preferably, the PCR reagents, genome/transcriptome sequencing reagents, gene-specific primers or probes comprise an NF2 gene-specific primer pair or probes that hybridize to NF2 gene nucleic acid sequences.

Preferably, the specific antibody of the gene expression product comprises an antibody that specifically binds to a protein encoded by NF2 gene.

In a third aspect, the invention provides the use of an NF2 gene activator or modulator for the preparation of a medicament for the treatment of a condition associated with defective skull regeneration defects, wherein the NF2 gene activator or modulator is an agent that increases the level of NF2 gene expression.

Preferably, the defect skull regeneration defect related disorder is a post-motor injury defect skull regeneration disorder or a post-operation defect skull regeneration disorder.

In a fourth aspect, the invention provides the use of an NF2 gene activator or modulator in the manufacture of a medicament for promoting mesenchymal stem cell migration.

Drawings

Fig. 1: NF2 depletion results in mesenchymal stem cell migration defects: lentivirus mediated NF2 knockdown infected mesenchymal stem cells and cell scratch experiments (see fig. 1A) found that NF2 deficiency resulted in a significant slowing of mesenchymal stem cell migration, see fig. 1B (12 h, 24 h).

Fig. 2: co-immunoprecipitation experiments demonstrated that Nf2 interacted with FAK (fig. 2A); primary osteoblasts were cultured and an endogenous co-immunoprecipitation experiment was used to confirm that NF2 and FAK had an interactive relationship (fig. 2B); nanoBiT live cell protein interaction system it was further demonstrated that NF2-FAK had direct interactions in osteoblasts (fig. 2C).

Fig. 3: knocking out NF2 in craniosynostosis mesenchymal stem cells leads to defective skull regeneration defects; micro-CT scanning experiments of small animals show that NF2 knockout leads to obvious weakening of the regeneration capability of defective frontal bone and parietal bone tissues.

Detailed Description

In order to make the technical scheme and the beneficial effects of the application more obvious and understandable, the following detailed description is given by way of example. Wherein the drawings are not necessarily to scale, and wherein local features may be exaggerated or reduced to more clearly show details of the local features; unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

In a first aspect the invention relates to the use of the NF2 gene for the preparation of a reagent for diagnosing and/or treating a condition associated with defective skull regeneration defects.

In one embodiment, the defective skull regeneration defect related disorder is selected from a disorder of skull regeneration or repair following a sports injury, or a disorder of skull regeneration or repair following surgery.

In one embodiment, the application comprises:

a) The NF2 gene is used as a target to be applied to the preparation of medicaments for treating diseases or disorders related to defective skull regeneration defects;

b) NF2 gene is used as a target to be applied to the screening of drugs for treating the defect skull regeneration defect related diseases or disorders; or (b)

C) NF2 gene is used as a target to prepare a reagent for diagnosing the defect skull regeneration defect related diseases or disorders.

In another aspect, the invention also relates to a kit for aiding in the diagnosis of a condition or disorder associated with defective skull regeneration defects, wherein the kit comprises reagents for detecting a mutation or expression level of the NF2 gene.

In one embodiment, the agent that detects a mutation or expression level of an NF2 gene is any agent known in the art that can be used to detect a mutation or expression level of a gene; in particular embodiments, the reagents may be used in reagents for performing one or more of the following methods: real-time fluorescent quantitative PCR (RT-PCR), northern blotting, western blotting, genomic sequencing, transcriptome sequencing, biological mass spectrometry, or specific antibody detection, etc.

In some embodiments, the kit further comprises sample processing agents, such as sample lysing reagents, sample purification reagents, and nucleic acid extraction reagents, among others.

The transcriptome sequencing can rapidly and comprehensively obtain almost all transcripts and gene sequences of specific cells or tissues of a certain species under a certain state through a second generation sequencing platform, and can be used for researching gene expression quantity, gene functions, structure, alternative splicing, new transcript prediction and the like. Furthermore, by designing appropriate primers, the transcriptional expression level of a gene can be determined by PCR such as reverse transcription PCR. Protein expression levels of the individual genes can also be determined by immunoassays such as immunohistochemistry, ELISA, and the like, using antibodies specific for the gene proteins.

In one embodiment, the kit comprises one or more of nucleic acid extraction reagents, PCR reagents, genome/transcriptome sequencing reagents, gene specific primers or probes, specific antibodies to gene expression products, western Blotting detection reagents, immunohistochemical staining detection reagents, protein chips, in situ hybridization, or gene chip detection reagents.

In preferred embodiments, the PCR reagents, genome/transcriptome sequencing reagents, gene-specific primers or probes comprise an NF2 gene-specific primer pair or probes that hybridize to NF2 gene nucleic acid sequences.

In another preferred embodiment, the antibody specific for the gene expression product comprises an antibody that specifically binds to a protein encoded by the NF2 gene.

In another aspect, the invention also relates to the use of an NF2 gene activator or modulator in the manufacture of a medicament for treating a condition or disorder associated with defective skull regeneration defects, wherein the NF2 gene activator or modulator is an agent that increases the level of NF2 gene expression.

In one embodiment, wherein the condition or disorder associated with defective skull regeneration is a motor injury post-defective skull regeneration or repair disorder, or a post-operative defective skull regeneration or repair disorder.

In another aspect of the invention, the invention also relates to the use of an NF2 gene activator or modulator in the preparation of a medicament for promoting migration of mesenchymal stem cells, in particular embodiments, which are bone marrow derived mesenchymal stem cells.

The following definitions and descriptions are illustrative of terms used in the present invention; when describing the present invention, unless otherwise indicated, technical and scientific terms used herein should have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosures and documents referred to herein are incorporated by reference.

As used herein, the terms "comprises" or "comprising" mean that any illustrated steps/operations, components, elements, etc. of the recited method, structure, or composition include any recited volume, but do not exclude any other steps/operations, components, elements, etc.

In the context of the present invention, where a range of values is recited, it is understood that each intervening value, to the upper and lower limit of that range and between each and every other stated or intervening value in that range is encompassed within the invention. The upper and lower limits of these smaller ranges independently combinable with the range are also encompassed within the invention, subject to any specifically excluded limit in the stated range. When a specified range includes one or both of the limits, ranges excluding either of those included limits are also within the invention.

As used herein, the term "NF2 gene" is a gene located in the two bands (22 q 12) of the long arm 1 region of chromosome 22, about 110 000bp long, and the cDNA of the coding region is 1785bp long, containing 17 exons, producing two major proteins by alternative splicing, sub-Schwannomin or Merlin.

The term "FAK" refers to focal adhesion kinase, a non-receptor tyrosine kinase that transduces signals from a variety of stimuli (e.g., integrins, cytokines, chemokines, and growth factors) to control a variety of cellular pathways and processes, including cell proliferation, cell migration, cell morphology, and cell survival.

The term "expression level" of a gene is used interchangeably with "expression value" and refers to a parameter value that measures the degree of expression of a given gene. The expression value may be determined by detecting the mRNA level encoded by the gene of interest or by detecting the amount of protein encoded by the gene.

As used herein, the term "Mesenchymal Stem Cells (MSCs)" refers to a class of pluripotent stem cells having differentiation potential. It conforms to the following definition of the International Society of Cytotherapy (ISCT): 1) MSCs may grow in colony adhesion; 2) The MSC cell surface expresses markers CD105, CD73 and CD90 and does not express endothelial, hematopoietic or immune cell markers, such as CD45, CD34, CD14, CD11b, CD79 a, CD19 and HLA-DR. MSC cells can be obtained in different tissues, such as bone marrow tissue-derived BM-MSC, adipose tissue-derived AD-MSC, and umbilical cord tissue-derived UC-MSC, e.g., bone marrow tissue-derived mesenchymal stem cells of the present invention.

The invention will be better understood with reference to the following examples. These examples are representative of some specific embodiments of the invention and are not intended to limit the scope of the invention.

Example 1: CKO embryo acquisition

The NF2 gene is specifically knocked out in the skull of a mouse (C57 BL) by using Cre-floxp conditional knockdown technology, and the main steps comprise:

Female mice of the F/F gene are bred, and male mice of the F/+PRX1-CRE-ER+ gene are bred, so that F/F PRX1-CRE-ER+ gene knockout embryos (CKO) are obtained after the female mice are pregnant for a certain period of time.

Example 2: PCR genotyping

2.1 Extraction of rat tail DNA

Placing part of the rat tail tissue into a PCR tube, adding 50 μl of 50mM NaOH, treating at 98deg.C for 30min, and cooling in ice; to the solution was added 6. Mu.l of 1M Tris (pH 8.0) to neutralize the strong base, and the resulting solution, i.e., containing the tissue DNA, was used for PCR identification.

2.2 PCR genotyping (PCR genotyping)

Table 1: PCR reaction system

PCR reaction procedure:

1) Pre-denaturation at 94℃for 4min; 2) Denaturation at 94℃for 30 seconds, annealing at 55℃for 35 seconds, extension at 72℃for 60 seconds, and cycling for 35-40 times; 3) Extending at 72 ℃ for 10min; 4) Preserving at 4 ℃.

Table 2: genotyping primer sequences

1.5% Agaros gel was formulated. After cooling, adding nucleic acid dye liquor, mixing evenly and pouring glue. After cooling at room temperature, the sample was placed in an electrophoresis tank, and spotted at 10. Mu.l/well. And (3) turning on a power supply of the electrophoresis apparatus, connecting electrodes, setting the voltage to be 110V, and running the gel for about 30 min. UV was turned on in a gel analyzer for exposure photography and genotype was analyzed according to PCR fragment size.

Conditional Knockdown (CKO) mouse embryos or pups developing for different periods of time were obtained after genotyping by PCR, and the preservation phenotype was photographed.

Example 3: RNA, protein extraction from skull tissue

And (3) taking skull tissue extract RNA as RT-PCR to detect the transcriptional expression level of genes, and extracting protein as western to detect the translation expression level of related proteins.

3.1 RNA extraction

3.1.1 Homogenization treatment:

(1) Tissue: the tissue was ground in liquid nitrogen, 1ml TRIzol was added to each 50-100mg of the tissue, and homogenized with a homogenizer. The sample volume should not exceed 10% of the TRIzol volume.

(2) Monolayer culture of cells: TRIzol was directly added to the plate to lyse the cells, 1ml was added per 10cm2 area (i.e., 3.5cm diameter plate), and the cells were pipetted several times. The amount of TRIzol used will depend on the area of the plate and is not dependent on the number of cells. Insufficient TRIzol may result in DNA contamination of the extracted RNA.

(3) Cell suspension: cells were collected by centrifugation, 1ml TRIzol was added to each 5-10X 106 animal, plant, yeast cell or 1X 107 bacterial cell, and repeated pipetting was performed. Cells were not washed to avoid mRNA degradation prior to TRIzol addition. Some yeast and bacterial cells are treated with a homogenizer.

3.1.2 The homogenized sample was left at room temperature (15-30 ℃) for 5 minutes to allow complete separation of the nucleic acid protein complexes.

3.1.3 Optional steps: if the sample contains more protein, fat, polysaccharide or extracellular material (muscle, plant nodule, etc.), it can be centrifuged at 10000 Xg for 10 min at 2-8deg.C to obtain supernatant. The pellet obtained by centrifugation comprises cell outer membrane, polysaccharide, high molecular weight DNA, and RNA in supernatant. When treating adipose tissue, a large amount of oil should be removed from the upper layer. Taking the clarified homogenate for the next step.

3.1.4 0.2Ml chloroform was added to each 1ml TRIzol, and the mixture was vigorously shaken for 15 seconds and left at room temperature for 3 minutes.

3.1.5 Centrifugation at 10000 Xg for 15 minutes at 2-8 ℃. The samples were divided into three layers: the bottom layer is a yellow organic phase, and the upper layer is a colorless aqueous phase and an intermediate layer. RNA is predominantly in the aqueous phase, which is about 60% of the TRIzol reagent used.

3.1.6 Transfer of the aqueous phase to a fresh tube, where the organic phase can be preserved if DNA and protein are to be separated, after further manipulation. RNA in the aqueous phase was precipitated with isopropanol. 0.5ml of isopropyl alcohol was added to each 1ml of TRIzol, and the mixture was left at room temperature for 10 minutes.

3.1.7 Centrifugation at 10000 Xg for 10 min at 2-8℃no RNA precipitate was seen before centrifugation and after centrifugation colloidal precipitations appeared on the tube side and bottom. The supernatant was removed.

3.1.8 Washing RNA pellet with 75% ethanol. At least 1ml of 75% ethanol was added per 1ml of TRIzol used. Centrifuging at 2-8deg.C for 5 min at a temperature of no more than 7500 Xg, and discarding supernatant.

And (3) standing at 3.1.9 room temperature, drying or vacuum drying the RNA precipitate, and airing for about 5-10 minutes. Rather than vacuum centrifugal drying, too much drying results in a substantial decrease in RNA solubility. 25-200. Mu.l of RNase-free water or 0.5% SDS was added, and the mixture was sucked several times with a gun and left at 55-60℃for 10 minutes to solubilize the RNA. For example, RNA is used for the cleavage reaction without using SDS solution. RNA was also solubilized with 100% deionized formamide and stored at-70 ℃.

3.2 Protein extraction

3.2.1 Preparation of lysis night

(1) Dissolving PMSF (dissolving PMSF crystals in PMSF solvent, i.e., preparing 10m1100mM PMSF liquid)

(2) Preparation of lysate:

The composition is as follows: PIRA lysate + PMSF solvent + protease inhibitor, 10ml PIRA:100 μl PMSF: 1-sheet protease inhibitors

(3) The prepared lysate is placed at 4 ℃ for standby.

3.2.2 Grinding of tissue (parietal, frontal bone)

(1) The preparation method comprises the steps of preparing a mortar, a medicine spoon (2 handles), long tweezers, an insulation box (ice cubes are put in), a soup ladle, a centrifuge tube, a balance, gloves (2 pairs), liquid nitrogen, an ice tray and an oscillator.

(2) All tools that come into contact with the tissue are pre-cooled with liquid nitrogen.

(3) One person was responsible for giving tissue to the grinder and adding liquid nitrogen at any time (the person had to remember the number of tissue ground by each grinder), two persons ground tissue (multiple times fast), and one person pre-cooled and peeled the 1.5ml centrifuge tube number.

(4) The ground tissue was weighed in a corresponding centrifuge tube (the weight of each sample was recorded with white paper for ease of balancing during centrifugation) with the peel removed.

(5) Lysates (1 mg tissue plus 6. Mu.l lysate) were added.

(6) Shaking, mixing, placing on an ice tray, and shaking on a shaking table for 1h after all samples are added.

3.2.3 Centrifugal extraction of protein supernatant

(1) Pre-cooling the centrifuge (4 ℃) for 30min in advance;

(2) Balancing;

(3) Centrifuging, and setting out according to the set at the level (13000 r,5 min);

(4) The supernatant was taken and stored at-20℃for later use (1.5 ml centrifuge tube was numbered in advance).

Example 4: western immunoblotting

Preparing polyacrylamide gel: the glass plate is cleaned until water can flow freely on the plate, the thin plate and the thick plate are aligned and clamped into a glue making frame, and deionized water is used for detecting leakage. Preparing separating gel with a gel preparation kit, uniformly adding into a glass plate, adding deionized water for sealing, and standing until solidification. And (3) discarding deionized water on the upper layer after solidification, preparing concentrated glue by using a yase glue preparation kit, uniformly adding the concentrated glue to a glass plate, carefully inserting a comb, standing until solidification, placing the solidified glass plate into an electrophoresis tank, adding 1x SDS, extracting the comb, and waiting for sample loading.

Electrophoresis: according to a certain sequence, 5 mu L of protein Marker is added into one hole of the protein samples, and the samples are flattened in concentrated glue by using 80V constant pressure, and then 120V constant pressure is changed.

Transferring: the PVDF membrane was immersed in methanol for 10min and then washed with water until no bubbles were present. After electrophoresis, taking out protein gel and discarding concentrated gel, sequentially placing a foam-rubber cushion, filter paper, protein gel, PVDF membrane, filter paper and foam-rubber cushion on a membrane transferring clamping plate in order, wherein no air bubble exists between the PVDF membrane and the protein gel, clamping the membrane transferring clamping plate and buckling, paying attention that the positive electrode and the negative electrode are placed in a membrane transferring groove, immersing the membrane transferring groove with 1X membrane transferring liquid, and transferring the membrane at a low temperature of 120V for 110 min.

Closing: the PVDF film was taken out. Wash 2 times with TBST for 5min each, block with 5% nonfat dry milk formulated with TBST for 1.5h at room temperature.

Incubating primary antibody: the primary antibody was prepared by washing 3 times with TBST for 5min each time, and incubating overnight at a ratio of 1:1000 and 4 ℃.

The primary antibody was recovered and washed 5 times with TBST for 5min each time.

Secondary antibody was incubated with TBST at a ratio of 1:50000 for 1.5h at room temperature.

The secondary antibody was discarded and washed 3 times with TBST for 5min each.

Developing: equal amount of ECL luminous liquid A and B are evenly mixed and evenly dripped on a film, an ultrasensitive chemiluminescence imager is used for exposure, the exposure time is adjusted until clear stripes are swept out, a white light image with a Marker is reserved, and the stripes and the Marker are combined. And finally, analyzing the protein expression condition according to the result.

Example 5: cell culture

The inventor constructs NF2 gene knock-down mediated by slow virus, and cultures bone marrow mesenchymal stem cells in vitro by the following specific method:

Primary cell culture of mouse skull: the neck of the F1-generation female mice that had been pregnant for 18.5 days was sacrificed. The pregnant belly was carefully sheared off with scissors, E18.5 day embryo mice were removed under a stereoscope, washed in sterilized PBS, the scalp was peeled off with fine forceps, the required skull was carefully sheared off with surgical scissors, periosteum and connective tissue on both sides inside and outside the skull were gently peeled off, placed in a 1.5mL EP tube containing 1mL of alpha-1640 culture solution, labeled, and transferred to an ultra clean bench.

Taking 1.5mL of EP tube, adding 1mL of alpha-1640 culture solution, washing the skull tissue of the mice, repeatedly washing for 5 times, changing the EP tube and the culture solution each time, finally entering the EP tube with 1.5mL of 200 mu L of enzymolysis solution, and digesting for 20min in an incubator with 37 ℃. After 20min, the cells were removed, washed once with 1mL of a-1640 medium, transferred to another 1.5mL EP tube with 200. Mu.L of enzymatic hydrolysate, and digested in an incubator at 37℃for 30min. After 30min, the mice were removed, washed once with 1mL of a-1640 medium, transferred to another 1.5mL EP tube with 200. Mu.L of enzymatic hydrolysate, sheared to a non-massive tissue with blunt scissors, and digested for 90min in an incubator at 37 ℃. After 90min, the mixture was removed, 1mL of alpha-MEM Quan Pei was added to the EP tube, blown for 3s, and after 60s of standing, the upper suspension was transferred to a new 15mL EP tube. Then 1mL of alpha-MEM Quan Pei solution was added to the bone fragments, the procedure was repeated, the upper suspension was transferred to a 15mL EP tube, and finally only bone fragments remained in the 1.5mL EP tube. 1000rpm,4℃and 10 min. The supernatant was discarded, 2mL of a completely new solution of alpha-MEM Quan Pei was added, and the cells were completely suspended by blowing up and down, spread on a cell plate, and cultured in an incubator at 37 ℃. After overnight, all living cells adhere, and conventional cell culture procedures can be performed. Then taking the mouse skull cells as osteoblasts for in vitro culture.

Extracting hind limbs of animals for 2 months, taking out bone marrow cavity cell sap, performing cell culture, digesting for 2min by using pancreatin after 48h, obtaining mesenchymal stem cells, performing continuous culture, and collecting cells for cell migration experiments after the mesenchymal stem cells are full of growth.

The effect of NF2 gene knockdown on mesenchymal stem cell migration was statistically analyzed by using cell scratch experiments (fig. 1A), calculating scratch distance changes after different times. Therefore, the mesenchymal stem cells with the NF2 gene knockdown are found to have slower migration but no significance compared with the cell migration of the control group at different time points of 3-9 hours after the cell scratch; at 12h post cell streak, NF2 knockdown mesenchymal stem cell migration was significantly slowed down (P < 0.01); at 24h of cell scoring, NF2 knockdown mesenchymal stem cell migration was still significantly slowed down (P < 0.05) (fig. 1B).

Example 6: technique for interaction of living cell proteins

The Nf2-FAK protein interaction was confirmed to be direct by the living cell sensitive detection protein interaction technique (Promega, nanoBiT). The method comprises the steps of constructing over-expression plasmids, FAK-FLAG and FAK-HA fusion protein plasmids of mutants of different structural domains of Nf2 by molecular simulation of Nf2-FAK interaction structural domain information and by utilizing an overlap PCR technology, co-transfecting 293T cells, and determining the sites of the Nf2 and FAK interaction structural domains by an immune co-precipitation (coIP) experimental method.

6.1 Co-immunoprecipitated protein extraction

Taking the primary osteoblasts of D0, and washing 3 times with PBS. An appropriate amount of IP lysate was taken and PMSF was added to a final concentration of 1mM. PBS was removed from the cell plates, IP lysate was added, the cells scraped off and transferred to a 1.5mL EP tube and lysed on ice for 5min. Centrifuging at 12000rpm and 4 deg.C for 15min, collecting supernatant to obtain desired protein, and preserving at-80deg.C.

6.2 Preparation of co-immunoprecipitated proteins

Taking 2-tube protein samples, 500 mu L of each tube, and taking 25 mu L to 1.5mL of each EP tube to obtain input. 12.5. Mu.L of 5X Loading buffer was added to the input sample, and the mixture was boiled in a metal bath at 100℃for 10 minutes, and then stored at-20 ℃. mu.L of antibodies, such as FAK and rabbit IgG antibodies, were added to the 2-tube protein samples, respectively, and shaken overnight at 4 ℃. The next day 25. Mu.L of Protein A magnetic beads were added to the 2-tube Protein sample, mixed well and incubated with shaking at 4℃for 2h. After 2h, the EP tube was placed on a magnetic rack and after 1min the beads were completely adsorbed, the supernatant was aspirated and discarded. The EP tube was removed, the beads were washed by adding 200. Mu.L of IP lysate and allowed to stand on an ice box for 1min, taking care to wash slowly, avoiding damage to the beads. The EP tube was again placed on a magnet holder and the procedure repeated for a total of 3 washes. After the last removal of the IP lysate, 50. Mu.L of 1X Loading buffer prepared with the IP lysate was added, mixed with magnetic beads and boiled in a metal bath at 100℃for 10min. After boiling, the EP tube is placed on a magnetic rack for adsorption for 1min, the supernatant is transferred into a new EP tube with the volume of 1.5mL, and the EP tube is taken as the co-immunoprecipitated protein sample and is stored at the temperature of minus 20 ℃.

The inventor constructs NF2-HA and FAK-FLAG expression plasmids, and transfects 293T cells together, and through an immune coprecipitation experiment, the interaction between NF2 and FAK is shown (figure 2A). Likewise, by culturing primary osteoblasts, an endogenous co-immunoprecipitation experiment was used to confirm that NF2 and FAK are in an interactive relationship (fig. 2B). The presence of direct interactions of NF2-FAK in osteoblasts was further demonstrated by the NanoBiT live-cell protein interaction system (fig. 2C), thus functioning.

Example 7: imaging test of the percentage of defective skull healing

Using micro-CT scanning and reconstruction technology of small animals, analyzing the influence of NF2 deficiency on the regeneration of defective skull tissue, and statistically analyzing the healing condition of defective skull tissue:

A craniosynostosis Prx1+ mesenchymal stem cell gene knockout mouse model is established, a frontal bone and parietal bone defect (defect area 2mm & lt 2 & gt) model (2 m) is established, and the specific knockout of NF2 in Prx1+ mesenchymal stem cells is induced by continuously injecting tamoxifen. The skull tissue of the defect 3w is collected and fixed, and the healing condition of the skull tissue of the defect is detected by using a micro-CT scanning experiment of a small animal, so that the frontal bone and the parietal bone of the defect 3w of the control group are basically healed, the regeneration of the frontal bone and parietal bone of the defect is obviously delayed due to the loss of NF2 genes, and the result is shown in figure 3, namely, the defect of regeneration of the skull of the defect is caused by knocking out NF2 in mesenchymal stem cells of bone joints.

It should be understood that the above examples are illustrative and are not intended to encompass all possible implementations encompassed by the claims. Various modifications and changes may be made in the above embodiments without departing from the scope of the disclosure. Likewise, the individual features of the above embodiments can also be combined arbitrarily to form further embodiments of the invention which may not be explicitly described. Therefore, the above examples merely represent several embodiments of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims (10)

1. Use of the nf2 gene for the preparation of a reagent for diagnosing and/or treating a condition associated with defective skull regeneration defects.
2. 2. The use of claim 1, wherein the defective skull regeneration defect related disorder is selected from the group consisting of post-motor injury skull regeneration disorder or post-surgical skull regeneration disorder.
3. 3. The use of claim 1 or 2, wherein the use is selected from the group consisting of:

   a) The NF2 gene is used as a target to be applied to the preparation of medicaments for treating the defect skull regeneration defect related diseases;

   b) NF2 gene is used as a target to be applied to the screening of medicines for treating the defect skull regeneration defect related diseases; or (b)

   C) NF2 gene is used as a target to prepare a reagent for diagnosing the defect skull regeneration defect related diseases.
4. 4. A kit for aiding in the diagnosis of a condition associated with defective skull regeneration defects, the kit comprising reagents for detecting a mutation or expression level of the NF2 gene.
5. 5. The kit of claim 4, wherein the kit comprises one or more of nucleic acid extraction reagents, PCR reagents, genome/transcriptome sequencing reagents, gene-specific primers or probes, specific antibodies to gene expression products, western Blot detection reagents, immunohistochemical staining detection reagents, protein chips, in situ hybridization, or gene chip detection reagents.
6. 6. The kit of claim 5, wherein the PCR reagents, genome/transcriptome sequencing reagents, gene-specific primers or probes comprise an NF2 gene-specific primer pair or probes that hybridize to NF2 gene nucleic acid sequences.
7. 7. The kit of claim 5, wherein the antibody specific for the gene expression product comprises an antibody that specifically binds to a protein encoded by the NF2 gene.
8. Use of an nf2 gene activator or regulator for the preparation of a medicament for treating a condition associated with defective skull regeneration defects, wherein the NF2 gene activator or regulator is an agent that increases the level of NF2 gene expression.
9. 9. The use of claim 8, wherein the defective skull regeneration defect related disorder is post-motor injury defective skull regeneration disorder or post-surgical defective skull regeneration disorder.
10. Use of an nf2 gene activator or regulator in the preparation of a medicament for promoting mesenchymal stem cell migration.