CN109402246B - L1TD1 gene and new application of expression product thereof - Google Patents

Abstract

The invention discloses a novel application of an L1TD1 gene and an expression product thereof, namely the invention discloses the L1TD1 gene and the expression product thereof as diagnosis and treatment targets of osteoarthritis. Therefore, the L1TD1 can be used for developing products for diagnosing osteoarthritis and medicines for treating osteoarthritis. The research result of the invention provides a theoretical basis for clinicians to formulate personalized treatment schemes and can provide a new drug target for the development of osteoarthritis drugs.

Description

L1TD1 gene and new application of expression product thereof

Technical Field

The invention relates to the field of biomedicine, in particular to application of an L1TD1 gene in preparing a product for diagnosing and treating osteoarthritis.

Background

Osteoarthritis (OA) is a chronic disease in which joint cartilage, which covers the ends of bones and forms the articular surface of a joint, gradually degrades over time. A number of factors have been reported to predispose patients to osteoarthritis, including genetic predisposition, obesity, accidental or athletic trauma, surgery, medications, and heavy physical demands. Osteoarthritis is believed to begin with damage to the articular cartilage. The two most common types of damage to joints are motion-related damage and long-term "repetitive use" joint damage. The joints most often affected by osteoarthritis are the knee, hip, and hand. In most cases, osteoarthritis in these joints is more likely to cause disability than osteoarthritis in the hands due to the necessary weight bearing functions of the knee and hip. As cartilage degradation progresses, secondary changes occur in the joint and other tissues surrounding the joint, including bone, muscle, ligaments, meniscus, and synovium. The net effect of primary failure of cartilage tissue and secondary damage to other tissues is that the patient experiences pain, swelling, weakness and loss of function of the affected joint. These symptoms often develop to the point of having serious effects, such as loss of labor or quality of life for the patient.

Articular cartilage is composed mainly of chondrocytes, type II collagen, proteoglycans and water. Articular cartilage has no blood or nerve distribution, and chondrocytes are the only cell type in this tissue. Chondrocytes are responsible for the production of type II collagen and proteoglycans of the cartilage matrix. The matrix thereafter has physicochemical properties that allow saturation of the matrix with water. The net effect of this structure-function relationship is that the articular cartilage has special wear characteristics and that virtually frictionless movement occurs between the articular cartilage surfaces. In the absence of osteoarthritis, articular cartilage often provides a lifetime of painless weight bearing and unrestricted joint movement even under high demand physical conditions.

As with all living tissues, articular cartilage is constantly undergoing a renewal process in which the "old" cells and matrix components are removed (catabolically active) and "new" cells and molecules are produced (anabolically active). The turnover rate of anabolism or catabolism of articular cartilage is low relative to most tissues. Long-term maintenance of the structural integrity of mature cartilage depends on an appropriate balance between matrix synthesis and degradation. Chondrocytes maintain matrix balance in response to chemical and mechanical stimuli from their environment. The appropriate and effective response of chondrocytes to these stimuli is essential for cartilage homeostasis. Disruption of homeostasis by either insufficient anabolic or excessive catabolic activity can lead to cartilage degradation and osteoarthritis (Adams et al, 1995, Nature377 Suppl: 3-174). Most tissues that are damaged and have increased catabolic activity are able to initiate an enhanced anabolic response, allowing tissue rejuvenation. Unfortunately, articular cartilage has a very limited ability to up-regulate its anabolic activity and increase the synthesis of proteoglycans and type II collagen in response to cartilage matrix damage or consumption.

Current osteoarthritis treatment methods include exercise, medication, rest and joint care, surgery, pain relief techniques, replacement therapy and weight control. Common drugs used in the treatment of osteoarthritis include non-steroidal anti-inflammatory drugs such as aspirin, ibuprofen, and the like; can be directly applied to skin for relieving topical pain such as cream, rubber, and spray. Surgery may be performed to resurface bone, reposition bone, and replace joints. Although various drug therapies have been used to treat diseases, they are ineffective for long-term control and prevention. Furthermore, since osteoarthritis occurs insidiously and progresses slowly, osteoarthritis is often identified late in disease progression rather than early in disease progression where potential treatments may be more effective. Further progression in the prevention, alteration or treatment of osteoarthritis disease processes therefore relies heavily on the identification of early diagnostic markers of the disease, allowing early intervention.

Disclosure of Invention

One of the purposes of the invention is to provide a method for realizing osteoarthritis diagnosis by detecting expression difference of L1TD1 gene.

It is another object of the present invention to provide a method for treating osteoarthritis by inhibiting the expression of the L1TD1 gene.

The invention also aims to provide a method for screening osteoarthritis treatment medicines.

The fourth purpose of the invention is to provide a medicament for treating osteoarthritis.

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

the invention provides application of a reagent for detecting L1TD1 in preparing a tool for diagnosing osteoarthritis.

Further, the reagent for detecting the L1TD1 comprises a reagent for detecting the expression level of the L1TD1 gene.

Further, the reagent for detecting L1TD1 includes a reagent capable of quantifying mRNA of the L1TD1 gene, and/or a reagent capable of quantifying the L1TD1 protein.

The reagent for quantifying the mRNA of the L1TD1 gene of the present invention can exert its function based on a known method using a nucleic acid molecule: such as PCR, e.g., Southern hybridization, Northern hybridization, dot hybridization, Fluorescence In Situ Hybridization (FISH), DNA microarray, ASO methods, high throughput sequencing platforms, etc. The assay can be performed qualitatively, quantitatively, or semi-quantitatively using the reagent.

Further, the PCR method is a known method, for example, ARMS (Amplification Mutation System) method, RT-PCR (reverse transcriptase-PCR) method, nested PCR method, or the like. The amplified nucleic acid can be detected by using a dot blot hybridization method, a surface plasmon resonance method (SPR method), a PCR-RFLP method, an in situ RT-PCR method, a PCR-SSO (sequence specific oligonucleotide) method, a PCR-SSP method, an AMPFLP (amplifiable fragment length polymorphism) method, an MVR-PCR method, and a PCR-SSCP (single strand conformation polymorphism) method.

The reagent capable of quantifying mRNA of the L1TD1 gene can be a specific primer of the L1TD1 gene or transcript, can also be a specific recognition probe of the L1TD1 gene or transcript, or comprises the primer and the probe.

The primers specific to the L1TD1 gene or transcript described above include those used in real-time quantitative PCR for specifically amplifying the L1TD1 gene. In a specific embodiment of the invention, the primer sequences are shown as SEQ ID NO.1 and SEQ ID NO. 2.

The primer can be prepared by chemical synthesis, appropriately designed by referring to known information using a method known to those skilled in the art, and prepared by chemical synthesis.

The probe may be prepared by chemical synthesis, by appropriately designing with reference to known information using a method known to those skilled in the art, and by chemical synthesis, or may be prepared by preparing a gene containing a desired nucleic acid sequence from a biological material and amplifying it using a primer designed to amplify the desired nucleic acid sequence.

The reagent for quantifying L1TD1 protein of the present invention can exert its function based on a known method using an antibody: for example, ELISA, radioimmunoassay, immunohistochemistry, Western blotting, etc. may be included.

The reagent for quantifying the L1TD1 protein comprises an antibody or a fragment thereof which specifically binds to the L1TD1 protein. An antibody or fragment thereof of any structure, size, immunoglobulin class, origin, etc., may be used so long as it binds to the target protein. The antibodies or fragments thereof included in the assay products of the invention may be monoclonal or polyclonal. An antibody fragment refers to a portion of an antibody (partial fragment) or a peptide containing a portion of an antibody that retains the binding activity of the antibody to an antigen. Antibody fragments may include F (ab')₂Fab', Fab, single chain fv (scfv), disulfide-bonded fv (dsfv) or polymers thereof, dimerized V regions (diabodies), or CDR-containing peptides. The reagent for quantifying the L1TD1 protein of the present invention may include an isolated nucleic acid encoding an amino acid sequence of an antibody or encoding a fragment of an antibody, a vector comprising the nucleic acid, and a cell carrying the vector.

Antibodies can be obtained by methods well known to those skilled in the art. For example, mammalian cell expression vectors that retain all or part of the target protein or incorporate polynucleotides encoding them are prepared as antigens. After immunizing an animal with an antigen, immune cells are obtained from the immunized animal and myeloma cells are fused to obtain hybridomas. The antibody is then collected from the hybridoma culture. Finally, a monoclonal antibody against L1TD1 protein can be obtained by subjecting the obtained antibody to antigen-specific purification using L1TD1 protein or a portion thereof used as an antigen. Polyclonal antibodies can be prepared as follows: an animal is immunized with the same antigen as above, a blood sample is collected from the immunized animal, serum is separated from the blood, and then antigen-specific purification is performed on the serum using the above antigen. The antibody fragment can be obtained by treating the obtained antibody with an enzyme or by using sequence information of the obtained antibody.

Binding of the label to the antibody or fragment thereof can be carried out by methods generally known in the art. For example, proteins or peptides may be fluorescently labeled as follows: the protein or peptide is washed with phosphate buffer, a dye prepared with DMSO, a buffer, or the like is added, and the solution is mixed and left at room temperature for 10 minutes. In addition, labeling may be carried out using commercially available labeling kits, such as biotin labeling kit, e.g., biotin labeling kit-NH 2, biotin labeling kit-SH (Dojindo laboratories); alkaline phosphatase labeling kits such as alkaline phosphatase labeling kit-NH 2, alkaline phosphatase labeling kit-sh (dojindo laboratories); peroxidase labeling kits such as peroxidase labeling kit-NH 2, peroxidase labeling kit-NH 2(Dojindo Laboratories); phycobiliprotein labeling kits such as phycobiliprotein labeling kit-NH 2, phycobiliprotein labeling kit-SH, B-phycoerythrin labeling kit-NH 2, B-phycoerythrin labeling kit-SH, R-phycoerythrin labeling kit-NH 2, R-phycoerythrin labeling kit SH (dojindo laboratories); fluorescent labeling kits such as fluorescein labeling kit-NH 2, HiLyte Fluor (TM)555 labeling kit-NH 2, HiLyte Fluor (TM)647 labeling kit-NH 2(Dojindo Laboratories); and DyLight 547 and DyLight647(Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody labeling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation), and EZ-marker protein labeling kit (Funakoshi Corporation). For proper labeling, a suitable instrument can be used to detect the labeled antibody or fragment thereof.

The obtaining of the sample for detecting the expression level of the L1TD1 gene is a routine technique in the field, and preferably can be achieved by a method which is non-invasive or minimally invasive.

The sample may be (but is not limited to): tissue, peripheral blood, bone marrow, lymph nodes, peritoneal cavity wash, urine, sweat. In a specific embodiment of the invention, the sample is from a tissue of a subject.

The invention also provides a tool for diagnosing osteoarthritis, which can detect the expression level of the L1TD1 gene.

Further, the means comprise a reagent capable of quantifying L1TD1 gene mRNA, and/or a reagent capable of quantifying L1TD1 protein.

Typically, the reagents are present in suitable containers. Each of the primers or probes can be adjusted to at least one desired amount of concentration using a diluent such as deionized water and dispensed into a container.

Further, the reagent capable of quantifying mRNA of the L1TD1 gene comprises a primer for specifically amplifying the L1TD1 gene, wherein the primer sequence is shown as SEQ ID NO.1 and SEQ ID NO. 2.

Further, the means for diagnosing osteoarthritis includes, but is not limited to, a chip, a kit, a strip, or a high throughput sequencing platform; the high-throughput sequencing platform is a special tool, and with the development of high-throughput sequencing technology, the construction of a gene expression profile of a person becomes very convenient work. By comparing the gene expression profiles of patients with diseases and normal people, the abnormality of which gene is related to the disease can be easily analyzed. Therefore, the knowledge that the abnormality of the L1TD1 gene is related to the occurrence of osteoarthritis in high-throughput sequencing also belongs to the novel application of the L1TD1, and is also within the protection scope of the invention.

The kit of the present invention may further comprise a reagent for extracting nucleic acid, a reagent for PCR, a reagent for staining or developing color, and the like. For example, such agents include, but are not limited to: an extraction solution, an amplification solution, a hybridization solution, a color development solution, a washing solution, and the like.

In addition, the kit can also comprise instructions and the like for describing a method for detecting the expression of the L1TD1 gene.

The kit of the present invention may contain a plurality of different reagents suitable for practical use (e.g., for different detection methods), and is not limited to the reagents listed so far, and any reagents for diagnosing osteoarthritis based on the detection of the L1TD1 gene or transcript are included in the scope of the present invention.

The present invention also provides a method for diagnosing osteoarthritis, the method comprising the steps of:

(1) obtaining a sample from a subject;

(2) detecting the expression level of the L1TD1 gene in a sample of the subject;

(3) correlating the measured expression level of the L1TD1 gene with the disease association of the subject.

(4) A statistically elevated level of L1TD1 gene expression compared to a normal control indicates that the subject is judged to have osteoarthritis, or that the subject is judged to be at high risk of having osteoarthritis.

The present invention also provides a method of treatment of osteoarthritis, the method comprising inhibiting the expression of L1TD1 gene, or inhibiting the activity of the expression product of L1TD1 gene.

The inhibition of the expression of the L1TD1 gene comprises inhibition of the expression of an L1TD1 gene transcript and inhibition of the expression of an L1TD1 protein.

The activity for inhibiting the expression product of the L1TD1 gene comprises the activity for inhibiting the transcript of the L1TD1 gene and the activity for inhibiting the protein of the L1TD 1.

The present invention also provides a method for screening a candidate drug for treating osteoarthritis, which can determine the effect of the candidate drug on improving prognosis by measuring the expression level of the L1TD1 gene or L1TD1 protein at a certain period after the test drug is added to model cells. More specifically, when the expression level of the L1TD1 gene or L1TD1 protein is decreased or restored to a normal level after the addition or administration of the test drug, the drug may be selected as a therapeutic drug for improving osteoarthritis.

The invention also provides a medicament for the treatment of osteoarthritis, the medicament comprising an agent that inhibits L1TD 1.

The agent for inhibiting L1TD1 of the present invention is not limited as long as the agent is capable of inhibiting the expression or activity of L1TD1 or a substance involved in the upstream or downstream pathway of L1TD1, and is a drug effective for the treatment of osteoarthritis.

The invention also provides application of the L1TD1 gene or an expression product thereof in preparing a medicament for treating osteoarthritis.

Further, the medicine comprises an interfering RNA aiming at the expression of the L1TD1 gene, or a negative regulation miRNA, a negative regulation type transcription regulation factor or a suppression type targeting small molecule compound.

The medicaments of the present invention may be used by formulating pharmaceutical compositions in any manner known in the art. Such compositions comprise the active ingredient in admixture with one or more pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the mode of administration and the dosage form envisaged. Therapeutically inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols such as polyethylene glycol, water, sucrose, ethanol, glycerol and the like, various preservatives, lubricants, dispersants, flavoring agents. Moisturizers, antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like may also be added as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration, formulations which may be used in such compositions may be in the form of their original compounds as such, or optionally in the form of their pharmaceutically acceptable salts, and the agents of the present invention may be administered alone, in various combinations, and in combination with other therapeutic agents. The compositions so formulated may be administered in any suitable manner known to those skilled in the art, as desired. When using pharmaceutical compositions, a safe and effective amount of the drug of the present invention is administered to a human, and the specific dosage will depend on factors such as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The medicine of the present invention may be prepared into various preparation forms. Including, but not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories for transdermal, mucosal, nasal, buccal, sublingual or oral use.

The route of administration of the drug of the present invention is not limited as long as it exerts the desired therapeutic or prophylactic effect, and includes, but is not limited to, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intratracheal, subcutaneous, transdermal, transpleural, topical, inhalation, mucosal, cutaneous, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, intrahepatic. In some cases, the administration may be systemic. In some cases topical administration.

The dose of the drug of the present invention is not limited as long as the desired therapeutic effect or prophylactic effect is obtained. The dose of the therapeutic agent or prophylactic agent of the present invention can be determined using, for example, the therapeutic effect or prophylactic effect on a disease as an index.

The "L1 TD1 gene" (NC _000001.11(62194802..62212328)) sequence of the present invention can be queried in the NCBI database.

In the context of the present invention, "osteoarthritis diagnosis" includes determining whether a subject has suffered from osteoarthritis, determining whether a subject is at risk of suffering from osteoarthritis, or determining that an osteoarthritis patient has relapsed.

As used herein, "treatment" encompasses treatment-related diseases or disease states in a mammal, such as a human, having the associated disease or disorder, and includes:

(1) preventing the occurrence of a disease or condition in a mammal, particularly when the mammal is susceptible to said disease condition but has not been diagnosed as having such a disease condition;

(2) inhibiting a disease or disease state, i.e., preventing its occurrence; or

(3) Alleviating the disease or condition, i.e., causing regression of the disease or condition.

The term "treatment" generally refers to the treatment of a human or animal (e.g., as applied by a veterinarian) wherein some desired therapeutic effect is achieved, e.g., inhibiting the progression of a condition (including slowing the progression, stopping the progression), ameliorating the condition, and curing the condition. Treatment as a prophylactic measure (e.g., prophylaxis) is also included. The use of a patient who has not yet developed a condition but who is at risk of developing the condition is also encompassed by the term "treatment".

The invention has the advantages and beneficial effects that:

the invention discovers and confirms the close correlation between the L1TD1 gene expression and the osteoarthritis generation for the first time, and has the advantages of large sample amount and accurate result. The relevance provides a new approach for the diagnosis and treatment of osteoarthritis.

Drawings

FIG. 1 shows a statistical plot of the differential expression of the L1TD1 gene in osteoarthritic and normal synovial tissues using QPCR;

FIG. 2 shows a statistical plot of the differential expression of L1TD1 protein in osteoarthritic and normal synovial tissues as measured by Western blot assay;

FIG. 3 shows a statistical chart of the inhibition rate of L1TD1 gene expression measured by Western blot experiment.

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 screening for differentially expressed genes in OA synovial tissue and Normal synovial tissue

1. Tissue collection

Synovial tissue from 7 OA patients was obtained from OA patients in hospital orthopedic knee replacement or synovectomy. The cases used met the diagnostic criteria for OA set forth by Altam. 7 cases of normal synovial tissues are from hospital orthopedics and are articular synovial tissues of trauma surgery patients. Clinical samples used in this study were informed and passed through the ethical committee of the hospital.

2. Tissue RNA extraction

Taking about 50mg of synovial tissue samples frozen at-80 ℃, putting the synovial tissue samples into liquid nitrogen for grinding, transferring the synovial tissue samples into a 1.5mL EP tube when no large-particle tissue exists, adding 1m L Trizol for total RNA extraction and real-time quantitative PCR analysis, and specifically carrying out the following processes:

1) adding 200 mu L of chloroform into the tissue suspension containing 1mL of Trizol, manually fully shaking and uniformly mixing, and standing at room temperature for 10 min;

2) centrifuging at 12000rpm and 4 deg.C for 15 min;

3) after the centrifugation is finished, the upper aqueous phase of the EP tube is gently sucked to a new 1.5mL EP tube, 600 muL isopropanol is added, the mixture is fully mixed by turning upside down, and the mixture is placed at room temperature for 20min (or placed at minus 20 ℃ for 2h, RNA precipitation can be increased);

4) centrifuging at 12000rpm and 4 deg.C for 15 min;

5) discarding the supernatant, adding 75% ethanol diluted by DEPC water, and slightly blowing and sucking the suspension precipitate by a gun head;

6) centrifuging at 12000rpm and 4 deg.C for 15 min;

7) discarding the supernatant, sucking the residual liquid by using a gun head, and airing at room temperature for 5 min;

8) dissolving 20 μ L of RNAase-free water in the precipitate, and using or freezing at-80 deg.C for use.

3. Quantification of RNA

Diluting the extracted total RNA 2 μ L in 98 μ L DEPC water, measuring absorbance values at wavelengths of 260nm and 280nm by using an ultraviolet spectrophotometer, judging the purity of the RNA according to the A260/A280 ratio, and calculating the concentration of the sample RNA according to the A260 value.

4. High throughput transcriptome sequencing

(1) RNA-seq read mapping

The clean reads were first removed from the low quality reads and then matched to the UCSC h. sapiens reference genome (hg19) using TopHat v1.3.1, a pre-constructed index of h.sapiens UCSC hg19 version was downloaded from the TopHat homepage and used as the reference genome, allowing multiple matching sites per read (default to 20) with up to 2 mismatches when matched to the genome using TopHat. TopHat builds a pool of possible cleavage sites based on the exon regions and GT-AG cleavage signals, from which reads that are not mapped to the genome are mapped to the genome. We use the system default parameters of the TopHat method.

(2) Transcript abundance assessment

The matched read files are processed by Cufflinks v1.0.3, and the Cufflinks v1.0.3 standardizes the number of RNA-seq segments to calculate the relative abundance of the transcript. The FPKM value refers to the number of fragments that match to a region of the exon 1kb long for a particular gene per million sequenced fragments. And calculating a confidence interval of the FPKM estimated value by a Bayesian inference method. The referenced GTF annotation file used by Cufflinks was downloaded from the Ensembl database (Homo _ sapiens. grch37.63. GTF).

(3) Detection of differentially expressed genes

The downloaded Ensembl GTF file and the original file matched by TopHat are transmitted to Cuffdiff, and the Cuffdiff uses the original matched file to re-estimate the expression abundance of the transcripts listed in the GTF file and detect differential expression. Only q values < 0.01 in the Cuffidff output, tests showed that successful comparisons were considered differential expression.

4. Results

The RNA-seq results showed that 859 genes with high expression and 473 genes with low expression were statistically significant in osteoarthritic synovial tissue compared to normal synovial tissue (P < 0.05).

Example 2 Large sample validation of expression of differentially expressed genes

Detection at the transcriptional level

1. Tissue collection

Osteoarthritis synovial tissue and normal synovial tissue were collected in 30 cases according to the criteria of example 1.

2. Tissue RNA extraction and identification

The procedure is as in example 1.

3. Design and preparation of primers

The real-time quantitative PCR primer sequences used in the present application were as follows (designed and synthesized by Competition Biotechnology (Shanghai) Ltd.):

l1TD1 gene primer:

an upstream primer: 5'-CACGAAGTTACGAAGTCA-3' (SEQ ID NO. 1);

downstream primer 5'-ATTCATCCTCTCCATCCT-3' (SEQ ID NO.2),

GAPDH gene primers:

an upstream primer: 5'-GACCTGACCTGCCGTCTA-3' (SEQ ID NO. 3);

a downstream primer: 5'-AGGAGTGGGTGTCGCTGT-3' (SEQ ID NO. 4).

4. Reverse transcription PCR

Reverse transcription was performed using a Takara reverse transcription kit (containing genomic DNA-removing enzyme) in the following reaction system:

reaction system for removing genomic DNA:

TABLE 1 reaction System for removing genomic DNA

Reagent	Volume of
		Total RNA	1.0μg
gDNA Eraser	1.0μL
		5*gDNA Eraser B buffer	2.0μL
RNase Free water	Make up to 10 mu L

The reaction was carried out at 42 ℃ for 5 min.

Reverse transcription PCR reaction System:

TABLE 2 reverse transcription PCR reaction System

The reaction was carried out at 37 ℃ for 15min and at 85 ℃ for 5 s.

5. Real-time quantitative PCR

Reaction system:

TABLE 3 reverse transcription PCR reaction System

Reagent	Volume of
		Primer(F+R)	1.0. mu.L + 1.0. mu.L (1.0. mu.L each)
SYBR Premix Ex Tap TM II	12.5μL
		cDNA	2μL
RNase Free water	8.5μL

Reaction conditions are as follows: pre-denaturation at 95 ℃ for 30 s;

15s at 95 ℃, 15s at 56 ℃ and 20s at 72 ℃, and the total cycle time is 40 times;

extension at 72 ℃ for 5min (GAPDH as internal control).

By using 2^-△△CtThe expression level of L1TD1 was analyzed by a relative quantitative method, and Ct is the intensity value of the fluorescence signal detected in the reaction system by the thermal cycler. The calculation method comprises the following steps: Δ Δ Ct ═ (Ct objective gene-Ct reference gene) osteoarthritis synovial tissue experimental group- (Ct objective gene-Ct reference gene) normal synovial tissue group, 2^-△△CtExpressed is the fold change of the expression of the target gene in the experimental group relative to the normal group, and the analysis of the experimental data was performed by the Bio-RAD analysis software.

6. Statistical analysis statistical software SPSS19.0 was used for data analysis, and a paired T test was used to determine whether there was a statistically significant difference in the expression of L1TD1 in samples of osteoarthritic and normal synovial tissues. The statistical tests are bilateral tests, and the difference is statistically significant when P is less than 0.05.

7. Results

Compared with the average level of normal synovial tissue, the expression of L1TD1 gene was significantly increased in 28 of 30 cases of osteoarthritic synovial tissue. Statistical results as shown in figure 1, the L1TD1 gene was significantly elevated in osteoarthritic synovial tissue compared to normal synovial tissue, with the difference being statistically significant (P < 0.05).

Secondly, detecting at protein level

1. Extracting the total synovial tissue protein

(1) Taking out the preserved osteoarthritis synovial tissue and normal synovial tissue from liquid nitrogen, cutting the tissue to about 100mg after thawing at room temperature, transferring to a tissue homogenizer and cutting the tissue into pieces by using clean scissors;

(2) about 300. mu.L of RIPA lysate containing 1% PMSF was added, followed by homogenization on ice;

(3) transferring the tissue homogenate to a 1.5mL EP tube, and standing on ice for 15 min;

(4) centrifuging at 12000rpm and 4 deg.C for 15 min;

(5) the supernatant was transferred to a new 1.5mL EP tube, 10. mu.L of the supernatant was quantitated for BCA protein, and the remainder was stored frozen at-20 ℃.

2. Electrophoresis

Beta-actin is used as an internal reference. After 50. mu.g of total protein was fractionated by SDS-PAGE, it was electrically transferred to a PVDF membrane and blocked by gentle shaking at room temperature for 1 hour using 1 XTSST containing 5% skimmed milk powder; respectively adding L1TD1 monoclonal antibody and beta-actin monoclonal antibody, and standing overnight at 4 ℃; washing the membrane with 1 × TBST for 4 times, adding a secondary antibody, and incubating at room temperature for 1 h; washing the film for 4 times with 1 × TBST, reacting in Super Signal chemiluminescence reagent for 2min, exposing with X-ray film in dark room, developing and fixing by conventional method, and selecting exposed picture for gray level analysis with Image Pro software.

3. Statistical treatment

The gray values of the protein bands were analyzed by using Image J software, and the gray values of the L1TD1 protein bands were normalized by using β -actin as an internal reference. The results were expressed as mean ± sd, statistically analyzed using SPSS13.0 statistical software, and the difference between the two was considered statistically significant when P <0.05 using the t-test.

4. Results

The levels of L1TD1 protein were significantly elevated in 28 of 30 osteoarthritic tissues compared to normal synovial tissue, with the differences being statistically significant. Statistical results as shown in figure 2, the levels of L1TD1 protein were significantly elevated in osteoarthritic synovial tissue compared to normal synovial tissue, with statistical significance for the differences (P < 0.05).

Example 2 interference with L1TD1 Gene expression

1. Interfering RNA design Synthesis

siRNA was designed and synthesized by Shanghai Jima pharmaceutical technology, Inc. based on the L1TD1 gene sequence. The Shanghai Jima pharmaceutical technology Co., Ltd simultaneously provided a negative control siRNA (siRNA-NC) having no sequence homology with L1TD1 gene.

siRNA-L1TD1：

The sense strand is 5'-UCAUAUAGGUGAUAUUUUCCU-3' (SEQ ID NO. 5);

the antisense strand is 5'-GAAAAUAUCACCUAUAUGAAA-3' (SEQ ID NO.6),

2. synovial tissue cell in vitro culture

Washing aseptically obtained synovial tissue with PBS, repeatedly shearing into tissue blocks of about 1mm x 1mm x 1mm by aseptic surgical scissors, adding collagenase II (0.5mg/ml) at 37 deg.C, digesting for 2 hr, filtering with 200 mesh gauze, centrifuging to remove supernatant, suspending cells in DMEM culture solution, standing at 37 deg.C and 5% CO₂Culturing in a cell culture box. When the cells grew into spindle-shaped and flaked, subculture was performed. After the cells are transmitted to 3 rd generation, FITC labeled mouse anti-human CD3, CD14, CD19 and PE labeled mouse anti-human CD11b are added for labeling, and flow cytometry detection and identification are carried out. The cells that were negative for all 4 markers described above were Fibroblast-like Synoviocytes (FLS) used in this study.

3. Transient transfection of cells

OA fibroblast-like synoviocytes were digested one day prior to transfection and seeded into 6-well plates at approximately 2X10 per well⁵The cells are cultured overnight, and the transient transfection of siRNA is carried out by observing the fusion degree of the cells (about 50-70%), which comprises the following steps:

1) the siRNA was diluted to 20. mu.M with RNase Free water as specified in the siRNA synthesis protocol;

2) marking two sterile EP tubes as A and B respectively, adding 125 mu L/hole serum-free culture medium and 5 mu L siRNA into the tube A, and adding 125 mu L/hole serum-free culture medium and 5 mu L Lipofectamine 3000 into the tube B;

3) mixing the solutions in tubes A and B, and standing at room temperature for 5 min;

4) replacing the culture medium in the 6-well plate with a serum-free culture medium, wherein each well is 2 mL;

5) adding the uniformly mixed solution A and B, and shaking in a cross manner to fully and uniformly mix the cells;

6) is placed in CO₂Performing conventional culture at 37 ℃ in an incubator;

7) after transfection for 48h, 1ml of Trizol or 200 μ L of RIPA was added to lyse cells, RNA and protein were extracted for real-time quantitative PCR and Western blot detection, and subsequent experiments were performed after analysis of interference effects.

4. Results

As shown in FIG. 3, compared with the negative control group (transfected siRNA-NC), the expression level of L1TD1 protein in the cells of the experimental group (transfected siRNA-L1TD1) is significantly reduced, and the difference is statistically significant (P < 0.05).

Example 4 Effect of L1TD1 Gene expression on proliferation of OA fibroblast-like synoviocytes

The cell proliferation is detected by CCK-8 method

(1) Transfection was performed according to the method of example 3;

(2) 24h after transfection, cells were digested with 0.25% trypsin, counted in crystal violet stain, resuspended in 1640 medium containing 0.5% FBS and adjusted to a cell concentration of 2X10⁵ml；

(3) Add 100. mu.l of cell suspension to each well of a new 96-well plate under sterile conditions, i.e., 2X10⁴Single cell, 37 ℃, 5% CO₂Standing for 48 hours;

(4) adding 10 mul of CCK solution into each hole respectively, and culturing for 2 h;

(5) the wavelength of 450nm, and the light absorption value of each experimental group is measured by an ultraviolet spectrophotometer.

As a result:

the results showed that the OD value of the negative control group (transfected siRNA-NC) cells was 0.2902 + -0.0021, and the OD value of the experimental group (transfected siRNA-L1TD1) cells was 0.1806 + -0.0013. The above results indicate that inhibition of L1TD1 expression, synovial cell proliferation was inhibited, and the difference was statistically significant (P < 0.05).

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.

Sequence listing

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<120> L1TD1 gene and new application of expression product thereof

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Claims (5)

1. Application of a reagent for detecting expression quantity of L1TD1 gene in preparation of a diagnostic tool for osteoarthritis.

2. The use according to claim 1, wherein the reagents comprise reagents capable of quantifying L1TD1 gene mRNA, and/or reagents capable of quantifying L1TD1 protein.

3. The use according to claim 2, wherein the reagents capable of quantifying mRNA from the L1TD1 gene comprise primers for the specific amplification of the L1TD1 gene used in real-time quantitative PCR; the reagent capable of quantifying the L1TD1 protein comprises an antibody specifically binding to the L1TD1 protein.

4. The use of claim 1, wherein the means comprises a kit, a chip, a strip, a high throughput sequencing platform.

5. Application of a reagent for inhibiting expression of L1TD1 gene in preparation of a medicament for treating osteoarthritis.

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