CN108578701B - Application of LYNX1 in promoting osseointegration of implant of diabetic patient - Google Patents

Abstract

The invention discloses an application of LYNX1 in promoting integration of an implant of a diabetic patient, which can promote integration of the implant of the diabetic patient based on insulin, and research a gene changed in the action process of the insulin, thereby further researching whether the targeted gene has the effect of promoting wound healing. The invention also discloses a method for screening candidate drugs for promoting the osteointegration of the implant of the diabetic patient and application thereof.

Description

Application of LYNX1 in promoting osseointegration of implant of diabetic patient

Technical Field

The invention belongs to the field of biological medicines, and relates to application of LYNX1 in promoting osseointegration of an implant of a diabetic patient.

Background

Insulin is considered to be the main regulator of energy metabolism in the body, and the main physiological action of insulin is to promote anabolism, so that insulin is the first choice drug for treating diabetes at present. The incidence and severity of periodontal diseases of diabetic patients are higher than those of non-diabetic patients, and the patients are more prone to tooth loss and other diseases. The artificial tooth implanting technology is effective means for repairing dentition defect and retaining oral cavity maxillofacial defect, the successful application of the artificial tooth implanting technology depends on the state of the implant forming osseointegration in the body, and the quality of the implant-osseointegration is influenced by the factors of the bone regeneration capacity of the body, the material, the shape and the stress environment of the implant, and the like. The bone tissue in the skeletal system can maintain balance of osteoblasts and their dominant bone formation and osteoclasts and their dominant bone resorption in bone reconstruction through self-regulation, thereby maintaining various physiological indexes of bone and their functional activities in a normal range, and maintaining the stability and order of the overall functions of the skeletal system, which is called bone homeostasis. Bone homeostasis is mainly achieved by periodic bone remodeling, which is a complex process affected by multiple factors and affects both the initial stage and long-term stability of dental implants. Therefore, the impaired bone regeneration in diabetic state is the root cause affecting the osseointegration of the implant, and thus becomes a bottleneck restricting the application of artificial dental implant technology to an increasing number of diabetic patients.

A large number of studies indicate that the bone structure and bone quality of type II diabetics are changed, and new bone around the implant is in a more immature and disordered state. Clinical studies and animal experiments show that insulin can be used as a bone metabolism factor to directly or indirectly participate in the regulation of bone formation. Previous studies (CN201010274095.8) of the applicant have demonstrated that the topical application of physiological concentration of insulin to the extraction sockets of diabetic rats can promote new bone formation and accelerate the healing of the extraction sockets, but there has been no clear research result on the mechanism of action. From the perspective that insulin has a promoting effect on bone healing, molecules playing an important role are researched, and a new way is provided for bone healing of an implant.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a gene marker integrated with the implant bone of a diabetic patient, and the gene marker can influence the healing of the implant bone by changing the expression level of a gene.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides application of LYNX1 in preparing a pharmaceutical composition for promoting osteointegration of an implant of a diabetic patient.

Further, the pharmaceutical composition includes an inhibitor of LYNX 1.

Further, the inhibitor is siRNA.

Preferably, the sequence of the siRNA is shown in SEQ ID NO. 3-4.

Further, the diabetes is type II diabetes.

The invention provides a pharmaceutical composition, which comprises an inhibitor of the functional expression of LYNX1, and/or other medicines compatible with the inhibitor, and a pharmaceutically acceptable carrier and/or auxiliary material. The inhibitor of functional expression of LYNX1 refers to any substance that can reduce the activity of LYNX1 protein, reduce the stability of LYNX1 gene or protein, down-regulate the expression of LYNX1 protein, reduce the effective duration of LYNX1 protein, or inhibit the transcription and translation of LYNX1 gene, and these substances can be used in the present invention as substances useful for down-regulating LYNX1, thereby promoting bone integration. For example, the inhibitor includes nucleic acid inhibitors, protein inhibitors, proteolytic enzymes, protein binding molecules. Wherein the nucleic acid inhibitor is selected from: an interfering molecule targeting LYNX1 or its transcript and capable of inhibiting LYNX1 gene expression or gene transcription, comprising: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid. The protein binding molecule is selected from: substances that specifically bind to LYNX1 protein, such as antibodies or ligands that inhibit the activity of LYNX1 protein.

Further, the inhibitor is siRNA.

Preferably, the sequence of the siRNA is shown in SEQ ID NO. 3-4.

The invention provides a method for screening a candidate drug for promoting osteointegration of an implant of a diabetic patient, which comprises the following steps:

treating a system expressing or containing the LYNX1 gene or protein encoded thereby with a substance to be screened; and detecting expression or activity of the LYNX1 gene or its encoded protein in said system.

Wherein, if the substance to be screened can reduce the expression or activity of LYNX1 gene or its encoded protein, preferably significantly (e.g. more than 20% lower, preferably more than 50% lower, more preferably more than 80% lower), it indicates that the substance to be screened is a candidate drug for promoting the implant osteointegration of diabetic patients.

In the present invention, the system includes (but is not limited to): a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system. Such drug candidates include (but are not limited to): interfering molecules, nucleic acid inhibitors, small molecule compounds designed against the LYNX1 gene or its upstream or downstream genes.

In the present invention, the steps further include: the obtained candidate drugs are subjected to further cell experiments and/or animal experiments to further select substances which promote osteointegration of the implant in diabetic patients from the candidate drugs.

The invention provides application of the method in screening candidate drugs for promoting osteointegration of implants of diabetic patients.

In the present invention, LYNX1 includes wild type, mutant or fragment thereof. In one embodiment of the invention, the nucleotide sequence of a representative LYNX1 gene is shown in LYNX1 (NM-001356370.1) in GeneBank, the current International public nucleic acid database. The full-length LYNX1 nucleotide sequence or its fragment can be obtained by PCR amplification, recombination or artificial synthesis.

In the present invention, as an alternative, the inhibitor of LYNX1 is an antibody that specifically binds LYNX 1. The specific antibody comprises a monoclonal antibody and a polyclonal antibody; the invention encompasses not only intact antibody molecules, but also any fragment or modification of an antibody, e.g., chimeric antibodies, scFv, Fab, F (ab') 2, Fv, etc. So long as the fragment retains the ability to bind to LYNX1 protein. The preparation of antibodies for use at the protein level is well known to those skilled in the art and any method may be used in the present invention to prepare such antibodies

In a preferred embodiment of the invention, the inhibitor of LYNX1 is a small interfering RNA molecule specific for LYNX 1. As used herein, the term "small interfering RNA" refers to a short segment of double-stranded RNA molecule that targets mRNA of homologous complementary sequence to degrade a specific mRNA, which is the RNA interference (RNA interference) process. Small interfering RNA can be prepared as a double-stranded nucleic acid form, which contains a sense and an antisense strand, the two strands only in hybridization conditions to form double-stranded. A double-stranded RNA complex can be prepared from the sense and antisense strands separated from each other. Thus, for example, complementary sense and antisense strands are chemically synthesized, which can then be hybridized by annealing to produce a synthetic double-stranded RNA complex.

When screening effective siRNA sequences, the inventor finds out the optimal effective fragment by a large amount of alignment analysis. The inventor designs and synthesizes a plurality of siRNA sequences, and verifies the siRNA sequences by transfecting related cell lines with transfection reagents respectively, selects siRNA with the best interference effect, and the siRNA sequences have sequences shown in SEQ ID NO.3 and SEQ ID NO.4 respectively, and further performs experiments at a cell level, and the result proves that the inhibition efficiency is very high for cell experiments.

The nucleic acid inhibitor of the present invention, such as siRNA, can be chemically synthesized or can be prepared by transcribing an expression cassette in a recombinant nucleic acid construct into single-stranded RNA. Nucleic acid inhibitors, such as siRNA, can be delivered into cells by using appropriate transfection reagents, or can also be delivered into cells using a variety of techniques known in the art.

In an alternative embodiment of the present invention, the inhibitor of LYNX1 may be a "Small hairpin RNA (shRNA)" which is a Small non-coding RNA molecule capable of forming a hairpin structure, and the Small hairpin RNA is capable of inhibiting gene expression via an RNA interference pathway. As described above, shRNA can be expressed from a double-stranded DNA template. The double-stranded DNA template is inserted into a vector, such as a plasmid or viral vector, and then expressed in vitro or in vivo by ligation to a promoter. The shRNA can be cut into small interfering RNA molecules under the action of DICER enzyme in eukaryotic cells, so that the shRNA enters an RNAi pathway. "shRNA expression vector" refers to some plasmids which are conventionally used for constructing shRNA structure in the field, usually, a "spacer sequence" and multiple cloning sites or alternative sequences which are positioned at two sides of the "spacer sequence" are present on the plasmids, so that people can insert DNA sequences corresponding to shRNA (or analogues) into the multiple cloning sites or replace the alternative sequences on the multiple cloning sites in a forward and reverse mode, and RNA after the transcription of the DNA sequences can form shRNA (short Hairpin) structure. The "shRNA expression vector" is completely available by the commercial purchase of, for example, some viral vectors.

The pharmaceutical composition of the present invention may contain any conventional non-toxic pharmaceutically acceptable carrier, adjuvant or excipient, and the pharmaceutically acceptable carriers include, but are not limited to, diluents, excipients, binders, wetting agents, absorption enhancers, surfactants, humectants, adsorptive carriers, lubricants, buffers, stabilizers, bacteriostats, isotonizing agents, chelating agents, pH controlling agents.

The pharmaceutical compositions of the invention may also be combined with other agents that promote osteointegration, and other therapeutic compounds may be administered simultaneously with the main active ingredient, even in the same composition. Other therapeutic compounds may also be administered alone in a composition or dosage form different from the main active ingredient.

Preferably, it can be carried out by means of gene therapy. For example, an inhibitor of LYNX1 may be administered directly to a subject by a method such as injection; alternatively, expression units carrying inhibitors of LYNX1 (e.g., expression vectors or viruses, etc., or siRNA or shRNA) may be delivered to a target in a manner that results in the expression of an active LYNX1 inhibitor, depending on the type of inhibitor, as will be appreciated by those skilled in the art.

In the present invention, the pharmaceutical composition may be in a form suitable for administration by injection, in a form suitable for oral ingestion (e.g., capsules, tablets, caplets, elixirs), in the form of an ointment, cream or lotion suitable for topical administration, in a delivery form suitable for use as eye drops, in an aerosol form suitable for administration by inhalation (e.g., by intranasal inhalation or oral inhalation), in a form suitable for parenteral administration, i.e., subcutaneous, intramuscular or intravenous injection.

For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1, 2 propylene glycol.

Some examples of suitable carriers, diluents, excipients and adjuvants for oral use include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatin and lecithin. In addition, these oral dosage forms may contain suitable flavoring and coloring agents. When used in the form of a capsule, the capsule may be coated with a compound that delays disintegration, such as glyceryl monostearate or glyceryl distearate.

Adjuvants typically include lubricants, emulsifiers, thickeners, preservatives, bactericides, and buffers.

Solid dosage forms for oral administration may comprise binders, sweeteners, disintegrants, diluents, flavouring agents, coating agents, preservatives, lubricants and/or time delay agents (time delay agents) acceptable in human and veterinary medical practice. Suitable binders include acacia, gelatin, corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharin. Suitable disintegrating agents include corn starch, methyl cellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavoring agents include peppermint oil, oil of wintergreen, cherry, citrus or raspberry flavors. Suitable coating agents include polymers or copolymers of acrylic and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delays include glyceryl monostearate or glyceryl distearate.

In a specific embodiment of the invention, the experiments were performed in at least 3 replicates, the results were expressed as mean ± sd, statistically analyzed using SPSS18.0 statistical software, the paired samples were analyzed using t-test, the multiple samples were analyzed using mean variance test (ANOVA), and it was considered statistically significant when P < 0.05.

Drawings

FIG. 1 is a graph showing the effect of siRNA interference on LYNX1 gene.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.

Example 1 insulin-stimulated wound healing test

1. Culture of THP-1 cells

The cells were cultured in complete medium (RPMI 1640: FBS: streptomycin diabody ═ 90: 10: 1, without addition of B mercaptoethanol) at 37 ℃ with 5% CO₂In the daily passage operation, when the cells grow well under the general condition, the culture solution is sucked to a new culture bottle, and a new culture medium is added; and (4) carrying out centrifugal passage every other day, centrifuging at 1000rpm for 3min, removing the old culture medium, washing with 1 multiplied by PBS (phosphate buffer solution) for 1 time, centrifuging at 1000rpm for 3min, and adding a new culture medium for continuous passage.

2. PMA induction of THP-1 cell differentiation

Culturing the THP-1 cell strain until 3 rd generation at 5 × 10⁶THP-1 cells are inoculated in a 12-hole plate and cultured in RPMI1640 culture medium containing 10% fetal bovine serum, 1% glutamine and 1% streptomycin, after the THP-1 cells are treated by 60 mu g/L Phorbol Myristate Acetate (PMA) for 48 hours, the cells are washed and the culture medium is replaced, the cell morphology is observed, the cells are changed from a suspension state to an adherent state, the cells are gradually spread from a circle in morphology and present a amoeba sample, which indicates that the cells are transformed from monocytes to macrophages.

3. Detection of phagocytic function of cells

Culturing macrophages: adding sterile glucose powder 0.969g per liter of RPMI1640 culture medium to prepare high sugar medium containing 16.5mmol/L glucose, placing cells in high sugar RPMI1640 culture medium containing 10% FBS, and culturing at 37 deg.C and 5% CO₂The incubator of (2) for cultivation.

The insulin treatment method comprises the following steps: in a cell super clean bench, insulin is mixed with complete culture medium, and the mixture is added into cell holes in sequence according to the concentration required by the experiment.

After the THP-1 monocytes induced to macrophages were washed 2 times with PBS, the cells were fully suspended and counted, 0.5mL of cells (5X 10)⁶Perml) and Porphyromonas gingivalis (P.g) 50. mu.L (5X 10)⁶CFU/mL), incubated at 37 ℃ for 60min at different concentrations of insulin (0,5,50,100,200,500,1000ng/mL), and the bactericidal rate was calculated by plating and recorded.

4. ROS production level detection

ROS production was measured using a redox sensitive fluorescent probe DCFH-DA. DCFH-DA was diluted in serum-free medium at a 1:1000 ratio to a final concentration of 10. mu.M. The cells were collected and suspended in diluted DCFH-DA at a cell concentration of 1X 10⁶mL, incubation in a 37 ℃ cell incubator for 20 min. The mixture was inverted every 3-5min to bring the probe into full contact with the cells. Cells were washed three times with serum-free cell culture medium to remove DCFH-DA well without entering the cells. THP-1 was incubated with bacteria in the presence of different concentrations of insulin (0,5,50,100,200,500,1000ng/mL) for 30min, and the cells were collected for detection of fluorescence intensity differences (using 488nm excitation wavelength, 525nm emission wavelength).

5. Results

1) The results of the effect of insulin on macrophage bactericidal rate are shown in table 1, where the bactericidal rate increased with increasing insulin concentration, the bactericidal rate reached the highest level at 200ng/ml insulin concentration, and then the bactericidal rate gradually decreased with increasing insulin concentration.

TABLE 1 Effect of insulin on macrophage Sterilization Rate

2) The effect of insulin on THP-1 cell ROS content under high glucose conditions the results are shown in Table 2, where ROS content increases with increasing insulin concentration, reaching a maximum at insulin concentrations of 200 ng/ml.

TABLE 2 Effect of insulin on ROS content in TPH-1 cells under high sugar conditions

The above results indicate that insulin can promote the phagocytic ability of neutrophils, thereby further promoting wound healing.

Example 2 screening of genes associated with osteointegration

1. Culture of cells

1) The procedure for culturing THP-1 cells was the same as in example 1;

2) PMA induced THP-1 cell differentiation procedure as in example 1;

3) insulin treatment of macrophages the procedure of example 1 was followed using insulin at a concentration of 200 ng/ml.

2. Extraction of RNA

1) Collection of cells

The cell culture solution is removed, the cells are quickly washed once by using PBS buffer solution, the PBS buffer solution is removed, and adherent cells can be collected by using a cell scraper. The collected cells were transferred to 1.5mL of EP tube with RNase-free, and 3 tubes of each group of cells were used as a parallel sample.

2) Adding TRIzol lysate into an EP tube to completely dissolve cells in TRIzol for full lysis,

3) transferring the lysate to a 1.5mL sterile centrifuge tube without RNase, adding 200. mu.l chloroform, violently shaking and mixing for 15s, incubating at room temperature for 5min, and centrifuging at 4 ℃ and 12000rpm for 15 min;

4) carefully taking out, carefully sucking the water layer, transferring the water layer into another centrifuge tube, adding 0.5ml of isoamyl alcohol, gently shaking and uniformly mixing, incubating at room temperature for 10min, and centrifuging at 12000rpm for 15min at 4 ℃;

5) gently removing the supernatant to prevent the precipitate from being sucked, adding l mL of 75% ethanol, suspending the precipitate, centrifuging at 4 ℃ and 7500rpm for 5min, and repeating the steps once;

6) gently removing the supernatant, cleaning as clean as possible to prevent the precipitate from being sucked, and air-drying at room temperature to volatilize ethanol;

7) adding 20-30 μ l DEPC water, and detecting RNA concentration and A260/A280 on a machine. The ratio should be maintained between 1.8-2.0 and the RNA concentration is recorded.

8) Mass analysis of RNA samples

The concentration and purity of the extracted RNA were determined using Nanodrop2000, RNA integrity was determined by agarose gel electrophoresis, and RIN was determined by Agilent 2100. The concentration is more than or equal to 200 ng/mul, and the OD260/280 is between 1.8 and 2.2.

3. Removal of rRNA

Ribosomal RNA was removed from total RNA using Ribo-Zero kit.

4. Construction of cDNA library

The construction of cDNA library was carried out using the Truseq RNA sample Prep Kit from Illumina, the detailed procedures were as described in the specification.

5. Sequencing on machine

The Illumina Hiseq x-ten second generation high throughput sequencing technology is used for sequencing mRNA, and the specific operation is carried out according to the instruction.

6. High throughput transcriptome sequencing data analysis

Performing bioinformatics analysis on the sequencing result, performing trim on 5 'and 3' segments of reads by using SeqPrep and Sickle, trim off bases with the mass of less than 20, deleting reads with the N of more than 10%, performing RNA-seq reading localization by using TopHat v1.3.1, quantifying the expression amount of mRNA by cuffquant and normalizing output, detecting differential expression by using cuffdiff, and considering that the mRNA is significantly differentially expressed when the p value is less than 0.05, | log2(Fold _ change) normalized | > 1.

7. Results

Sequencing results showed that LYNX1 was significantly down-regulated, log, in the insulin treated group₂(Fold _ change) was about 58, P was 0.0169, and the difference was statistically significant, suggesting that overexpression of LYNX1 is not conducive to efficient macrophage killing.

Example 3 silencing of LYNX1 Gene

1. Cell culture and differentiation procedures were as in example 1

2. Transfection

1) Treatment of cells prior to transfection

One day before transfection, 6-well culture plates are seeded with 3-5 multiplied by 10⁵Cells/well, cultured for one day in antibiotic-free medium, were changed to serum-free medium before transfection.

2) Design of siRNA

The sequence of the negative control siRNA sequence (siRNA-NC) is shown as SEQ ID NO. 1-2, and the sequence of siRNA1 is shown as SEQ ID NO. 3-4; the sequence of the siRNA2 is shown in SEQ ID NO. 5-6; the sequence of the siRNA3 is shown in SEQ ID NO. 7-8.

The experiment was divided into three groups: a control group (macrophage), a negative control group (siRNA-NC) and an experimental group (siRNA1, siRNA2 and siRNA3), wherein the siRNA of the negative control group has no homology with the sequence of LYNX1 gene.

3) Transfection

Transfection was performed using the Lipofectamine RNAiMAX kit from Invitrogen, the specific procedures were performed according to the instructions, and the silencing effect of interfering RNA was observed after transfection.

3. RNA extraction the same as in example 2

4. Reverse transcription

1) Mu.l dNTP mix 1. mu.l, Oligo dT primer 1. mu.l, total RNA 2. mu.g, RNase Free ddH₂O to make the total volume to 10 μ l, carrying out denaturation and annealing reaction on a PCR instrument at 65 ℃ for 5min, and placing at 4 ℃ after the reaction is finished.

2) A20. mu.l reaction system was constructed, and 5 XPrimerScript Buffer 4. mu.l, RNase Inhibitor 0.5. mu.l, Prime Script RTase 0.5. mu.l, RNase Free ddH were added₂O5.0. mu.l, and carrying out reverse transcription reaction on a PCR instrument according to the following conditions: and (3) 15-30 min at 42 ℃ and 5min at 95 ℃, and placing on ice after the reaction is finished.

3) Heating in water bath at 42 deg.C for 15min, heating at 95 deg.C for 3min, and storing at-20 deg.C for use.

5. QPCR detection of transcript level of LYNX1 gene

1) Design of primers

QPCR amplification primers were designed based on the coding sequences of LYNX1 gene and housekeeping GAPDH gene from Genebank and synthesized by Bomaide. Wherein, the primer sequence of the LYNX1 gene is shown in SEQ ID NO. 9-10; the primer sequence of the GAPDH gene is shown in SEQ ID NO. 11-12.

2) And (3) PCR reaction system: 1. mu.l each of forward and reverse primers, 10. mu.l of SYBR Green PCR master mix, 1. mu.l of cDNA, ddH₂O 7μl。

3) And (3) PCR reaction conditions: at 95 ℃ for 10min, (95 ℃ for 10s, 60 ℃ for 30s, 72 ℃ for 15s) multiplied by 40 cycles, at 65-95 ℃ and at a temperature rise rate of 0.5 ℃/5 s. PCR reactions were performed on a Bio-Rad iQ5 fluorescence quantitative PCR instrument, bands of interest were determined by melting curve analysis and electrophoresis, and relative quantification was performed by the Δ Δ CT method.

6. Results

As shown in FIG. 1, siRNA1 was selected for subsequent experiments because the experimental group showed the best interference effect of siRNA1 compared to the control group, and the difference was statistically significant (P < 0.05).

Example 4 Effect of silencing of LYNX1 Gene on macrophage Sterilization Rate

1. Cell culture and differentiation procedures were as in example 1

2. Grouping of cells

The experiment was divided into three groups: a control group (macrophage group/blank group), a transfection group (siRNA1) and a positive control group (insulin group), wherein the complete culture medium is added into the control group and the transfection cell group, the insulin is added into the positive control group in the same volume, and the final concentration of the insulin is 200 ng/ml.

3. Phagocytic function assay procedure of cells as in example 1

4. Results

The statistical result shows that the sterilization rate of siRNA1 group is 31.23% +/-0.37 and the sterilization rate of insulin group is 34.72 + -1.39, which indicates that LYNX1 plays an important role in the sterilization process of phagocytes and can be used for promoting the healing of dental implant wounds of diabetic patients.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

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Claims (5)

1. Use of an inhibitor of LYNX1 in the manufacture of a pharmaceutical composition for promoting healing of an implant wound in a diabetic patient, wherein the inhibitor promotes the bactericidal rate of macrophages; the inhibitor is siRNA.
2. 2. The use of claim 1, wherein the siRNA has a sequence as shown in SEQ ID No. 3-4.
3. 3. Use according to claim 1 or 2, wherein the diabetes is type II diabetes.
4. 4. A method for in vitro screening of candidate drugs for promoting wound healing in diabetic implants, comprising the steps of:

   treating a system expressing or containing the LYNX1 gene or protein encoded thereby with a substance to be screened; and

   detecting expression or activity of the LYNX1 gene or protein encoded thereby in said system;

   wherein, if the substance to be screened can reduce the expression or activity of the LYNX1 gene or the protein coded by the gene, the substance to be screened is a candidate drug for promoting the healing of the implant wound of the diabetic patient.
5. 5. Use of the method of claim 4 for in vitro screening of a candidate drug for promoting wound healing in an implant in a diabetic patient.

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