CN110577995A

CN110577995A - Diagnostic marker for male osteoporosis

Info

Publication number: CN110577995A
Application number: CN201911029752.XA
Authority: CN
Inventors: 刘跃洪; 周宇; 陈曦; 王志聪; 张建军; 杨灵; 汪红; 江伟
Original assignee: Peoples Hospital of Deyang City
Current assignee: Peoples Hospital of Deyang City
Priority date: 2019-07-11
Filing date: 2019-10-28
Publication date: 2019-12-17
Anticipated expiration: 2039-10-28
Also published as: CN110184346A; CN110577995B

Abstract

The invention discloses a diagnosis marker for male osteoporosis, and also discloses a reagent, a kit and application for diagnosing male osteoporosis. The reagent for diagnosing male osteoporosis comprises a PCR primer pair specific to TES gene. The kit has the advantages of convenience in material taking, low cost, high sensitivity, good stability and the like, can timely and accurately detect the onset and risk of the male osteoporosis, has great clinical application value for rapid diagnosis of the early male osteoporosis, and provides technical support for further developing a rapid diagnosis kit used for patients with the early male osteoporosis.

Description

Diagnostic marker for male osteoporosis

Technical Field

The invention relates to the field of disease diagnosis, in particular to a diagnostic marker for male osteoporosis, and specifically relates to application of TES in male osteoporosis diagnosis.

Background

The incidence of osteoporosis (osteoporotis in men) in men is much lower than that in postmenopausal women, so that importance on osteoporosis in men has been far less than that in women. Recent research results show that men in the young age achieve higher peak bone mass than women, and the initial time of bone mass loss is obviously later than women, but both men and women have a bone loss process in phase with the age, namely senile osteoporosis (type II). Male osteoporosis shares many similarities with female osteoporosis, but there are still significant gender differences in etiology, pathology, and color pathology. Men over age 65 have a common occurrence of osteoporosis of varying degrees, with a severe complication of fracture.

In the United states, the incidence rate of hip fracture in men older than 65 years old is 4-5 per mill, and the incidence rate in women of corresponding ages is 8-10 per mill. Hip fractures occur at a rate of 1: 2 for men and women in northern europe, and relatively low in both sexes and other areas. Of all patients with hip fractures worldwide, about 30% are men, 51 million in 1990, and it is expected that the number of male hip fractures will increase to 116 million by 2025, and this rising trend will be more prominent in asia.

Cooper reported that men over age 50 had a lifetime risk of hip, vertebral and forearm fractures of 13.1% and women 39.7%. Neill reports 20% prevalence of vertebral fractures in 19 European countries 36 central 15570 cases of men 50-79 years old. According to the xushangzhong report about the prevalence rate of osteoporosis of male in China, the prevalence rate of male over 60 years old is 13.9%; wuqing in beijing area reported 13.4% (lumbar vertebrae); shenhuilan is reported to be 23.8% (lumbar).

osteoporotic fractures cause loss of living ability and life risk to patients, and Poor reports that mortality and morbidity of fractures are higher in men than in women.

like female osteoporosis, normal x-rays remain an essential means of diagnosing male osteoporosis and related fractures. The first manifestation is a decrease in bone density of the spine and pelvis. The cortical bone of the vertebral body becomes thin, trabecular bone in transverse motion is reduced or disappears, trabecular bone in longitudinal motion is relatively obvious, and the structure in the vertebral body disappears when the trabecular bone in longitudinal motion is serious; the vertebral body becomes flat, the upper and lower edges are concave, the intervertebral space is widened, the vertebral body is biconcave, and is compressed into wedge shape due to slight trauma. The thinning, reduction in the number of trabeculae, thinning of the cortex of the bone and the occurrence of delamination are observed on long bones. In severe cases, scattered and several mm-sized spot-like bright areas appear on the basis of diffuse bone density reduction, and the boundary is clear or fuzzy, so that bone destruction is easily misdiagnosed. However, the X-ray plain film has low sensitivity and accuracy for diagnosing osteoporosis, and is not helpful for early diagnosis of osteoporosis. Therefore, it is highly desirable to develop a method for diagnosing osteoporosis in an early stage of osteoporosis.

Disclosure of Invention

The invention relates to a diagnostic marker for male osteoporosis, and experiments prove that the content of TES gene mRNA in blood of male osteoporosis patients is obviously higher than that of normal people, so that the differential expression property of TES gene makes the TES gene become a molecular marker which can be used for diagnosing whether a subject suffers from male osteoporosis.

Specifically, the invention provides an application of a product for detecting TES in preparing a male osteoporosis diagnosis tool.

Further, the product for detecting TES comprises a product for detecting the expression level of TES gene in a sample.

Still further, the product for detecting TES includes a product capable of quantifying the mRNA of the TES gene in the sample, and/or a product capable of quantifying the TES protein in the sample.

The product for quantifying TES gene mRNA in a sample 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 product can be used to conduct the assay qualitatively, quantitatively, or semi-quantitatively.

The nucleic acid contained in the above-mentioned products can be obtained by chemical synthesis, or by preparing a gene containing a desired nucleic acid from a biological material and then amplifying it using a primer designed to amplify the desired nucleic acid.

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 product capable of quantifying the TES gene mRNA in the sample comprises primers for specifically amplifying the TES gene used in real-time quantitative PCR, and the sequences of the primers are shown as SEQ ID NO.1 and SEQ ID NO. 2.

The primers included in the product can be prepared by chemical synthesis, appropriately designed with reference to known information by using a method known to those skilled in the art, and prepared by chemical synthesis.

The above-mentioned nucleic acids may further include a probe which 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, or can be prepared by preparing a gene containing a desired nucleic acid sequence from a biological material and amplifying it using a primer designed for amplifying the desired nucleic acid sequence.

The product of the invention for quantifying the TES protein in a sample can perform its function based on known methods using antibodies: for example, ELISA, radioimmunoassay, immunohistochemistry, Western blotting, etc. may be included.

The products of the invention for quantifying the amount of TES protein in a sample comprise an antibody or fragment thereof that specifically binds TES 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. Products of the invention for quantifying the amount of TES protein in a sample may include isolated nucleic acids encoding the amino acid sequence of an antibody or encoding a fragment of an antibody, vectors comprising the nucleic acids, and cells carrying the vectors.

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 the TES protein can be obtained by subjecting the obtained antibody to antigen-specific purification using the TES 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.

As a sample of the detection product according to the present invention, a tissue sample or fluid obtained from a biopsy subject, for example, can be used. The sample is not particularly limited as long as it is suitable for the assay of the present invention; for example, it may comprise tissue, blood, plasma, serum, lymph, urine, serosal cavity fluid, spinal fluid, synovial fluid, aqueous humor, tears, saliva, or fractions or treated materials thereof.

In a particular embodiment of the invention, the sample is derived from blood.

Further, the product for quantifying the TES gene or TES protein may be a reagent for detecting the TES gene or TES protein, a kit, a chip, a test paper or the like containing the reagent, or a high-throughput sequencing platform using the reagent.

The present invention also provides a means of diagnosing male osteoporosis that is capable of detecting TES.

Further, the tool is capable of detecting the amount of TES gene expression in a sample.

Further, the means comprise an agent capable of quantifying the mRNA of the TES gene in the sample, and/or an agent capable of quantifying the TES protein in the sample.

Furthermore, the reagent capable of quantifying the TES gene mRNA in the sample is a primer for specifically amplifying the TES gene used in real-time quantitative PCR, and the sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO. 2. The reagent capable of quantifying the TES protein in the sample comprises an antibody that specifically binds to the TES protein.

Further, the means for diagnosing male osteoporosis includes, but is not limited to, a chip, a kit, a test strip, or a high throughput sequencing platform; the high-throughput sequencing platform is a special tool for diagnosing male osteoporosis, and with the development of a 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 use of TES in high-throughput sequencing to determine that the TES gene abnormality is related to male osteoporosis is also included in the protection scope of the present invention.

The number of amino acids recognized by the anti-TES antibody or fragment thereof used in the detection product, diagnostic tool of the present invention is not particularly limited as long as the antibody can bind TES.

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

(1) Obtaining a sample from a subject;

(2) Detecting the expression level of a TES gene or protein in a sample from the subject;

(3) The measured expression level of the TES gene or protein is correlated with the presence or absence of disease in the subject.

(4) When the expression level of the TES gene or protein is increased as compared with that of a normal control, the subject is judged to have a tendency to suffer from male osteoporosis, or has suffered from male osteoporosis, or a male osteoporosis patient is judged to have a relapse, or a male osteoporosis patient is judged to have a poor prognosis.

The invention also provides a pharmaceutical composition for treating osteoporosis, which comprises an agent that inhibits TES gene expression.

Further, the agent is not limited as long as it can inhibit the expression level of TES.

The agent may include siRNA, shRNA, inhibitory miRNA, or inhibitory targeted small molecule compounds.

The pharmaceutical composition of the present invention may be administered alone or together with other drugs as a medicine. The other drug that can be administered together with the pharmaceutical composition of the present invention is not limited as long as it does not impair the effect of the therapeutic or prophylactic pharmaceutical composition of the present invention.

The pharmaceutical composition of the invention can be prepared into various dosage forms according to requirements. 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 pharmaceutical composition of the present invention is not limited as long as it can exert 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, transmucosal, cutaneous, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, intrahepatic, intratumoral. In some cases, the administration may be systemic. In some cases topical administration.

the dosage of the pharmaceutical composition of the present invention is not limited as long as the desired therapeutic effect or prophylactic effect is obtained, and can be appropriately determined depending on the symptoms, sex, age, and the like. The dose of the therapeutic or prophylactic pharmaceutical composition of the present invention can be determined using, for example, the therapeutic effect or prophylactic effect on a disease as an index.

The invention also provides the application of the TES gene in preparing a medicament for treating osteoporosis.

further, the medicament includes an agent that inhibits the TES gene. The agent is not limited as long as it can inhibit the expression level of TES or inhibit the functional activity of TES.

The invention also provides the application of the TES gene expression inhibiting agent in preparing a medicine for treating osteoporosis.

In the context of the present invention, "diagnosing male osteoporosis" includes determining whether a subject has suffered from male osteoporosis, determining whether a subject is at risk of suffering from male osteoporosis, determining whether a male osteoporosis patient has relapsed, and determining the prognosis of a male osteoporosis patient.

The TES gene sequence of the invention (Chromosome 7, NC _000007.14(116210539..116258786)) can be queried on the NCBI website.

The invention has the advantages and beneficial effects that:

The invention discloses a molecular marker for diagnosing male osteoporosis, and the molecular marker can be used for judging the early occurrence of the male osteoporosis.

Drawings

FIG. 1 shows a statistical chart of TES gene differential expression at the mRNA level using QPCR;

FIG. 2 shows a statistical graph of the effect of interfering with TES gene expression;

FIG. 3 shows a statistical plot of the effect of interfering with the TES gene on osteoblast proliferation.

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 QPCR detection of differentially expressed genes

Firstly, collecting samples

Selecting 30 male osteoporosis patients, and adopting the selection standard: measuring the T < -2.5 of the bone density by dual-energy X rays; t < -2.5 with a previous history of brittle fracture; those with normal liver and kidney functions. Exclusion criteria: firstly, the previous use of anti-osteoporosis drugs; ② there are diabetes, thyroid or parathyroid gland disease, adrenal gland and gonad endocrine diseases; ③ secondary osteoporosis. The average age was 78 years. Peripheral blood of the above patients was extracted as a test sample, while peripheral blood of 30 normal persons was collected as a control sample.

Second, validation at mRNA level

1. total RNA extraction

total blood RNA was extracted using the RNAprep Pure high-efficiency blood total RNA extraction kit (cat # DP443) from Tiangen corporation, and the general procedure was as follows according to the instructions:

(1) dilution of erythrocyte lysate: selecting a suitable volume of 10 × erythrocyte lysate H according to the volume of the blood sample to be treated (for example, 140 μ l of 10 × erythrocyte lysate H when the volume of the blood sample to be treated is 200 μ l), and treating with RNase-Free ddH₂O diluted to 1 × erythrocyte lysate H.

(2) To 1 volume of human whole blood was added 5 volumes of 1 x erythrocyte lysate H (prepared from a suitable clean tube).

Note that: for optimal mixing, the volume of the mixture of blood and 1 x erythrocyte lysate H should not exceed 3/4 of the tube volume. If the white blood cell content of the blood is high, the volume of the used blood can be proportionally reduced, and the volume of the used 1 × erythrocyte lysate H in step 6 can be adjusted accordingly.

(3) Incubate on ice for 10-15min, vortex and mix well for 2 times during incubation.

Note that: the solution will become translucent during incubation, indicating red blood cell lysis. The incubation time can be extended to 20min if necessary.

(4) The supernatant was completely removed by centrifugation at 2,100rpm (. about.400 Xg) at 4 ℃ for 10 min.

Note that: the leukocytes may form globules after centrifugation, ensuring complete removal of the supernatant, and the presence of traces of erythrocytes makes the leukocyte globules red, which disappears during the subsequent rinsing step.

(5) To the leukocyte pellet, 1 Xerythrocyte lysate H (1 Xerythrocyte lysate H was added in a volume 2 times the amount of whole blood used in step 1) was added, and the cells were resuspended.

(6) The supernatant was completely removed by centrifugation at 2,100rpm (. about.400 Xg) at 4 ℃ for 10 min.

Note that: incomplete removal of the supernatant will affect cleavage and subsequent binding of the RNA to the membrane, resulting in a decrease in the final RNA yield.

(7) Lysis buffer RLH (beta-mercaptoethanol added prior to use) was added to the leukocyte pellet in the amounts specified in Table 1 below, either vortexed or mixed using a pipette.

Note: if the blood is not whole blood of a healthy person, the volume of lysis buffer RLH is determined according to the amount of leukocytes in the blood, and the cells are completely lysed and the clumped cell pellet disappears.

TABLE 1 lysate usage Scale

Lysate u.L	Healthy human whole blood (ml)	Number of leukocytes
			350	Up to 0.5	Up to 2 x 10⁶
600	0.5-1.5	2*10⁶To 1 x 10⁷

(8) The solution was transferred to the filtration column CS (the filtration column CS was placed in the collection tube), centrifuged at 12,000rpm (. about.13,400 Xg) for 2min, the filtration column CS was discarded, and the filtrate was collected.

Note that: to avoid aerosol formation, please adjust the pipettor to 750 μ l or more to ensure that all solutions are transferred to the filter column at one time, if there are too many cells, the lysate will be sticky, resulting in a situation of difficult aspiration.

(9) To the filtrate, 70% ethanol (usually 350. mu.l or 600. mu.l) was added in an amount of 1 volume, and mixed (precipitation may occur at this time), and the resulting solution was transferred together with the precipitate to an adsorption column CR4 (adsorption column CR4 was put into a collection tube), centrifuged at 12,000rpm (. about.13,400 Xg) for 30-60sec, the waste liquid in the collection tube was discarded, and the adsorption column CR4 was put back into the collection tube.

Note that: for 70% ethanol preparation, RNase-Free ddH2O was used, and if the volume of the filtrate was lost, 70% ethanol was used. The solution and precipitate were transferred to adsorption column CR4, where the volume was larger than the capacity of the adsorption column, and this was done in two steps.

(10) If DNase I digestion is not performed, 700. mu.l of deproteinizing solution RW1H (previously checked whether ethanol is added or not) may be directly added to adsorption column CR4, centrifuged at 12,000rpm (13,400 Xg) for 30-60sec, and the waste liquid in the collection tube is discarded to directly perform step 14.

DNase I digestion: 350 μ l of deproteinizing solution RW1H was added to the adsorption column CR4, centrifuged at 12,000rpm (. about.13,400 Xg) for 30-60sec, the waste liquid in the collection tube was discarded, and the adsorption column CR4 was returned to the collection tube.

(11) Preparing DNase I working solution: add 10. mu.l DNase I stock into a new RNase-Free centrifuge tube, add 70. mu.l RDD solution, mix gently.

(12) 80. mu.l of DNase I working solution was added to the center of the adsorption column CR4, and the mixture was left at room temperature for 15 min.

(13) 350 μ l of deproteinizing solution RW1H was added to the adsorption column CR4, centrifuged at 12,000rpm (. about.13,400 Xg) for 30-60sec, the waste liquid in the collection tube was discarded, and the adsorption column CR4 was returned to the collection tube.

(14) Add 500. mu.l of rinsing solution RW (previously checked for ethanol addition) to the adsorption column CR4, let stand at room temperature for 2min, centrifuge at 12,000rpm (13,400 Xg) for 30-60sec, discard the waste from the collection tube, and replace the adsorption column CR4 back into the collection tube.

(15) Step 14 is repeated.

(16) centrifuge at 12,000rpm (. about.13,400 Xg) for 2min and discard the waste. The adsorption column CR4 was left at room temperature for several minutes to thoroughly dry the residual rinse solution from the adsorption material.

note that: after centrifugation, the adsorption column CR4 was left at room temperature for a while to allow it to dry sufficiently. If the rinsing liquid remains, the subsequent experiments such as reverse transcription, fluorescence quantification and the like can be influenced.

(17) The adsorption column CR4 was transferred to a new RNase-Free centrifuge tube, 30-50. mu.l RNase-Free ddH2O was added and left at room temperature for 2min, and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min to obtain an RNA solution.

Note that: the volume of elution buffer should not be less than 30. mu.l, and too small a volume affects the recovery efficiency. The RNA solution was stored at-70 ℃.

2. Reverse transcription reaction

Total RNA 1000ng of the purified RNA was reverse-transcribed to synthesize cDNA using PrimeScript RT reagent Kit (TAKARA).

1) The genomic DNA was removed and the reaction mixture was prepared on ice as follows in Table 2.

TABLE 2 reaction System for removing genomic DNA

Reagent	Amount of the composition used
		5*gDNA Eraser Buffer	2.0μL
gDNA Eraser	1.0μL
		Total RNA	1μL
RNase-free water	To 10 μ L

2) reaction on PCR Instrument

42℃ 2min；

Storing at 4 ℃.

3) Reverse transcription reaction

The reaction solution was prepared on ice, and the composition is shown in Table 3. In order to ensure the accuracy of the preparation of the reaction solution, the Master Mix should be prepared in the amount of the reaction number +2, and then 10. mu.L of the Master Mix should be dispensed into each reaction tube. After gentle mixing, reverse transcription reaction is carried out immediately.

TABLE 3 reverse transcription reaction System

Reagent	Amount of the composition used
		Reaction solution of the previous step	10μL
PrimeScript RT Enzyme Mix I	1.0μL
		RT Primer Mix	1.0μL
5*PrimeScript Buffer 2(for Real Time)	4.0μL
		RNase Free dH₂O	4.0μL
General assembly	20μL

4) Reaction on a PCR instrument:

37℃ 15min；

85℃ 5sec；

Storing at 4 ℃.

3. real-time quantitative reverse transcription-polymerase chain reaction (QPCR)

1) A10. mu.L PCR reaction system (see Table 4, for example) was prepared using SYBR Premix Ex Taq Kit (TAKARA), reagents other than cDNA were premixed, triplicate wells were prepared for each single gene of each sample, and the wells were sequentially placed in a 96-well plate and ddH was added thereto₂O as a negative control.

TABLE 4 QPCR reaction System

The primer sequences are as follows:

TES

A forward primer: 5'-TTGAGCAATGAAGAGGAT-3' (SEQ ID NO.1),

Reverse primer: 5'-ATAGGTAACTGTATTGATGGA-3' (SEQ ID NO. 2);

GAPDH

A forward primer: 5'-GTGGACCTGACCTGCCGTCT-3' (SEQ ID NO.3),

Reverse primer: 5'-GGAGGAGTGGGTGTCGCTGT-3' (SEQ ID NO. 4).

2) program setting:

Pre-denatured Cycle:1

35 seconds at 95 ℃;

95 ℃ for 5 seconds +57 ℃ for 34 seconds for 40 cycles.

3) Ct values of each sample were obtained, and relative expression amounts of all target genes were calculated as (2-. DELTA.DELTA.Ct) with reference gene GAPDH of the same group as a control.

4. Results

as shown in FIG. 1, the mRNA level of TES gene was significantly up-regulated in the blood of all of the 25 male osteoporosis patients compared to the average level of the 30 normal control groups, and the difference was statistically significant (P < 0.05).

EXAMPLE 2 preparation of Male osteoporosis diagnostic kit

According to the relevance of TES gene and male osteoporosis, male osteoporosis can be diagnosed by detecting the expression condition of TES gene, so that the invention provides a kit for diagnosing male osteoporosis based on detecting the expression of TES gene, and the components in the diagnostic kit are as follows: SYBR Green polymerase chain reaction system; primer pairs for amplification of the TES gene and the GAPDH gene. The sequence of the forward primer for amplifying the TES gene is 5'-TTGAGCAATGAAGAGGAT-3', and the sequence of the reverse primer is 5'-ATAGGTAACTGTATTGATGGA-3'; the forward primer sequence for amplification of GAPDH is 5'-GTGGACCTGACCTGCCGTCT-3' and the reverse primer sequence is 5'-GGAGGAGTGGGTGTCGCTGT-3'. The SYBR Green polymerase chain reaction system comprises PCR buffer solution, dNTPs and SYBR Green fluorescent dye. The PCR buffer solution comprises the following components: 25mM KCL, 2.5mM MgCL₂、200mM(NH₄)₂SO₄。

example 3 Effect of TES Gene expression on osteoblast proliferation and differentiation

1. The reagents used

hfob1.19 (purchased from shanghai cell bank of chinese academy of sciences); DMEM (high glucose)/F12 (DMEM: F12 ═ 1:1, available from Gibco), newborn fetal calf serum (FCS, green season in hangzhou); MTT (bi yun sky); alkaline phosphatase kit (Nanjing construction company)

2. Human osteoblast culture

hFOB1.19 cells were plated on 5% CO₂The cells were cultured in an incubator at 34 ℃ in DMEM (high-sugar)/F12 complete medium (DMEM (high-sugar)/F12 supplemented with 10% final fetal bovine serum and 0.3% final G418).

3. Cell transfection

The siRNA gene sequence is introduced from GenBank and designed and synthesized by Shanghai Jima pharmaceutical technology GmbH. 24h before transfection, hFOB1.19 cells were inoculated in a 6-well plate, 1.5mL of DMEM/F12 complete medium was added, the cells were attached overnight, and transfection was performed when the cells reached 80% -90% confluence. Before transfection, the medium was changed to Opti-MEM medium without serum and G418. The siRNA (final concentration of 33nmol/L) was diluted in 250. mu.L LOpti-MEM medium and gently pipetted 3-5 times and mixed well. The transfection reagents were mixed by gentle inversion, diluted 5.0. mu.L Lipofectamine TM 2000 in 250. mu.LOpti-MEM medium, mixed by gentle pipetting 3-5 times, and allowed to stand at room temperature for 5 min. And mixing the transfection reagent and the siRNA diluent, gently blowing and sucking for 3-5 times, uniformly mixing, and standing for 20min at room temperature. The transfection complex was added to a 6-well cell plate and mixed well by gently shaking the cell plate back and forth. The cell plate was placed at 34 ℃ with a volume fraction of 5% CO₂Culturing in an incubator. The transfection time is 6h, and DMEM/F12 complete medium is replaced.

The siRNA sequence for TES (TES-siRNA) was: forward sequence

5’-AUAGGUAACUGUAUUGAUGGATT-3’(SEQ ID NO.5)，

Reverse sequence 5'-CAUCAAUACAGUUACCUAUGATT-3' (SEQ ID NO. 6). The universal negative control sequence (NC-siRNA) was provided by Shanghai Jima pharmaceutical technology, Inc.

4. Knock-out efficiency detection

4.1 extraction of Total cellular protein

1) After transfection for 48h, the old culture medium was aspirated away, 1ml of 4 ℃ PBS buffer solution was added to each well to wash the 6-well plate for 2-3 times, and the PBS was placed on ice for the last time;

2) Preparing fresh lysate in an EP tube, adding 10 ul of PMSF (100 x) and 10 ul of Cooktail (100 x) into 1ml of RIPA lysate, slightly blowing and uniformly mixing, and placing on ice for later use;

3) Discarding PBS in the 2 nd sample, adding 120 mul of the prepared lysis solution into each hole, gently shaking the 6-hole plate, and putting the 6 th-hole plate on ice for lysis for 40 min;

4) After the lysis is finished, quickly collecting cells, and transferring cell fragments and lysate into a 1.5ml sterile EP tube by using a sterilized gun head;

5) Centrifuging at 4 deg.C at 12000rpm for 20min, transferring the supernatant into another 1.5ml sterile EP tube, and labeling;

6) Diluting 3 mul of protein lysate by 10 times, and measuring the protein concentration by using a BCA protein quantitative kit;

7) Adding a certain amount of SDS loading buffer (6x) into the rest protein lysate according to volume, mixing well, and boiling in boiling water for 5-10 min. Storing at-80 deg.C for use.

4.2 immunoblotting

Quantifying by Brandford method, mixing appropriate amount with sample buffer solution, boiling for 5min, and cooling for 5 min; loading 30 mu g of protein to prepared 15% polyacrylamide gel for electrophoresis, transferring the membrane by using a Bio-Rad semi-dry transfer system, sealing the membrane overnight, adding a Western washing solution for washing, adding a monoclonal antibody shaker, and hybridizing the solution at room temperature for 2 hours; diluting the diluted solution with Western sheep anti-mouse secondary antibody in a sealing buffer solution according to a proper proportion, and incubating for 60 min; washing with the membrane washing solution for 3 times; and detecting protein expression by ECL reagent development and fixation. Calculating the gray level of the protein band by using professional software, wherein the gray level is in direct proportion to the protein amount, setting the gray level as 1 by taking the expression amount of the control group protein as a reference, and calculating the relative expression amount of TES protein of the TES interfering group.

4.3 results

As shown in FIG. 2, TES-siRNA can effectively inhibit TES protein expression, the TES protein amount of NC-siRNA group is about 5 times of that of TES-siRNA group, and the difference has statistical significance.

5. Osteoblast proliferation assay

The method comprises the following steps: the 96-well plate is inoculated with hFOB1.19 cells, 2000 cells per well, 5 wells per group, siRNA transfection is carried out according to the method described above, after 48h of transfection, 20 muL/well of MTT solution (5mg/mL) is added, incubation is carried out for 4h at 37 ℃, the liquid in the wells is aspirated, 150 muL/well of DMSO solution is added, oscillation is carried out for 10min, the absorbance value of each well is measured by selecting 570nm wavelength with a microplate reader, and the value is in direct proportion to the number of cells.

as a result: as shown in FIG. 3, inhibition of TES gene expression promoted hFOB1.19 cell proliferation, which was significantly different from the control group and statistically significant.

6. Osteoblast differentiation assay

Alkaline phosphatase is an enzyme protein secreted by osteoblasts, is a specific marker of osteoblast differentiation, has a content capable of indirectly reflecting functions of osteoblasts, is one of the most common indexes for evaluating secretion functions of osteoblasts, and therefore, the influence of TES gene expression inhibition on osteoblast differentiation is evaluated by detecting the level of alkaline phosphatase in a culture solution.

Determination of alkaline phosphatase Activity in culture:

The method comprises the following steps: 6 well plates were seeded at 1X 10 per well⁵/mL of hFOB1.19 cells. siRNA transfection was performed as described above, and cell culture fluid was aspirated 24h after transfection, centrifuged at 2500r/min for 10min, and the supernatant was assayed using an alkaline phosphatase assay kit. Alkaline phosphatase [ (measured absorbance value-blank absorbance value)/(standard absorbance value-blank absorbance value)]Xphenol standard concentration X100 mL X sample dilution before determination. The absorbance (A value) of each well was measured at a wavelength of 520nm by a microplate reader, and the relative ALP activity was calculated from the measured A value.

As a result: compared with a control group, the activity of the supernatant phosphatase of the TES-siRNA group is obviously improved, and the TES inhibition expression can promote osteoblast differentiation.

TABLE 5 statistics of supernatant phosphatase Activity

Item	NC-siRNA	TES-siRNA
			Supernatant alkaline phosphatase	0.777±0.042	1.469±0.064

In conclusion, TES-siRNA can promote the proliferation of hFOB1.19 osteoblasts and improve the activity of alkaline phosphatase to promote the differentiation of osteoblasts by reducing the expression of TES gene, so that the inhibition of the expression of TES gene can be used as a new method for treating osteoporosis.

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

<110> Deyang city people hospital

<120> diagnostic marker for osteoporosis in men

<150> 201910626067.9

<151> 2019-07-11

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Claims

1. The application of the product for detecting TES in preparing a tool for diagnosing male osteoporosis.

2. The use of claim 1, wherein the product for detecting TES comprises a product for detecting the amount of TES gene expression in the sample.

3. The use according to claim 2, wherein the product for detecting TES comprises a product capable of quantifying the mRNA of the TES gene in the sample and/or a product capable of quantifying the TES protein in the sample.

4. The use of claim 3, wherein the reagents capable of quantifying the mRNA of the TES gene in the sample comprise primers for the specific amplification of the TES gene used in real-time quantitative PCR.

5. The use according to claim 4, wherein the primer sequences are shown as SEQ ID No.1 and SEQ ID No. 2.

6. the use of claim 3, wherein the agent capable of quantifying the amount of TES protein in the sample comprises an antibody that specifically binds to TES protein.

7. The use according to any one of claims 2 to 6, wherein the sample is derived from blood.

8. A pharmaceutical composition for treating osteoporosis, comprising an agent that inhibits TES gene expression.

The application of TES in preparing medicine for treating osteoporosis.

10. The use of claim 9, wherein the medicament comprises an agent that inhibits TES gene expression.