CN110564845B - Application of TARS (total amyloid receptor) as molecular marker for diagnosing male osteoporosis - Google Patents

Abstract

The invention provides application of TARS as a molecular marker for diagnosing male osteoporosis. The research of the invention shows that the expression of TARS in the blood of healthy control and male osteoporosis patients has obvious difference, and therefore the TARS can be used as a molecular marker for diagnosing the male osteoporosis. According to the research results, a product for early diagnosis of the male osteoporosis is developed, and the product has good detectable rate and is suitable for clinical popularization.

Description

Application of TARS (total amyloid receptor) as molecular marker for diagnosing male osteoporosis

Technical Field

The invention belongs to the field of biomedicine, relates to application of TARS, and particularly relates to application of TARS as a molecular marker for diagnosing male osteoporosis.

Background

Osteoporosis is a group of bone diseases caused by various reasons, bone tissues have normal calcification, calcium salts and matrixes are in a normal proportion, and metabolic bone lesions are characterized by reduction of the amount of the bone tissues in unit volume. In most osteoporosis, the reduction of bone tissue is mainly due to increased bone resorption. Is characterized by pain in the skeleton and susceptibility to fracture.

The world health organization has listed osteoporosis as the second most dangerous disease to human health next to cardiovascular disease. According to the blue book for preventing and treating osteoporotic fracture of 2013, which is newly released by the Chinese health promotion foundation, the osteoporotic fracture is a brittle fracture, namely, a fracture which can occur when the osteoporotic fracture suffers from slight trauma or low-energy trauma in daily activities. Common sites for osteoporotic fractures are the spine, hip, and distal forearm. About 6944 million people of people over 50 years old in China have osteoporosis, and about 2.1 hundred million people have low bone mass.

In China, the attention degree and the research depth of osteoporosis of old men are far from enough. However, osteoporosis in elderly men is not less harmful than in women, and the prevalence and fatality rate of hip fracture in men are significantly higher than in women.

Domestic and foreign research shows that the risk factors of male osteoporosis include:

(1) age is an independent risk factor for osteoporosis in men, age effects on bone metabolism may include relatively active bone resorption, with inhibition of bone formation resulting in greater bone resorption than bone formation, while kidney l α hydroxylase activity decreases with age, activity 1,25(OH)₂D₃Decrease, decrease of calcium absorption in intestinal tract, decrease of blood calcium, secondary hyperparathyroidism, and bone loss.

(2) Body Mass Index (BMI) BMI is an important indicator of the prevalence of osteoporosis in elderly men. An important indicator of the prevalence of osteoporosis in men. The study shows that the BMI is less than 20-25kg/m²The probability of osteoporosis is increased and the BMI is more than 25kg/m²The BMI is 15-20kg/m²The likelihood of a fracture of the patient's medulla increases. In addition, it was found that every 1kg/m increase in BMI²BMD was measured to increase 0.1SD, 0.11SD and 0.09SD in femoral neck, medulla and lumbar vertebrae, respectively. Increased BMI can cause increased mechanical load on the bone, stimulating bone formation and reducing bone resorption.

(3) Endocrine hormone effects a reduction in androgen levels is an important risk factor for osteoporosis in elderly men. Androgen receptors are present on bone cells and androgen acts directly on these receptors to promote osteoblast strengthening of the bone. Hypothalamus-pituitary-gonadal axis function is reduced, testicular function is reduced, testosterone levels are reduced, and sex hormone binding globulin levels are increased, causing a decrease in osteoblastic osteogenesis, resulting in a decrease in bone density in elderly men.

(4) Genetic factors play an important role in the onset of osteoporosis in men. At present, the genetic research of osteoporosis mainly comprises two major aspects of population genetics and molecular genetics, and the research is respectively carried out aiming at race, family and gene polymorphism. The most significant determinant of peak bone mass is the genetic factor, which may account for 60% -80% of the effects.

(5) The long-term eating habits of the patients with the nutritional level and the composition proportion of the food, particularly the content of calcium and vitamin D in the food, also play a certain role in the onset of osteoporosis. Meta analysis indicated that the population supplemented with calcium and vitamin D3 had an increased BMD and a reduced risk of fracture. Calcium agents can reduce bone mass loss, prevent vertebral body fractures, and may also be helpful in preventing non-vertebral body fractures.

In addition, lifestyle habits including exercise, smoking, and heavy drinking are also important risk factors for osteoporosis in men.

For the diagnosis of osteoporosis, first, a measurement of bone density must be carried out. Bone density is actually an indication of the health of the bone or, conversely, the degree of aging of the bone. Commonly used detection means include:

x-ray radiography: the history of use was the earliest, but the test results were not quantified and were gradually replaced due to radioactivity.

Single photon analyzer: low cost, convenience, small radiation amount and safety. But have been increasingly replaced due to the inability to measure the trunk bone and to distinguish between cortical, cancellous, soft tissue, etc.

A quantitative ultrasonic method: low cost, convenience and no radioactive damage. Is suitable for the bone state general investigation work of people of all ages. Not only can the bone density be detected, but also the quality of the bone can be known, and the microstructure and the elasticity of the bone can be reflected. But at present, the method is only used for calcaneus, shin bones and phalanges, and can not detect the skeleton of the whole body.

Dual energy X-ray absorption: the method is the most ideal determination method at present, is the gold standard for diagnosing osteoporosis, is universal at home and abroad, can measure bones of all parts of the whole body and can measure the thickness of soft tissues, but the equipment is less in quantity and expensive, so the economic burden is large for diagnosing osteoporosis patients. Therefore, the problem to be solved urgently is to find a method for diagnosing osteoporosis with high sensitivity, high accuracy and reasonable price.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a gene marker for diagnosing osteoporosis. The QPCR experiment proves that the expression level of TARS in the blood of a male osteoporosis patient is obviously higher than that of the blood of a healthy male, so that TARS can be used as a molecular marker for diagnosing male osteoporosis.

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

the invention provides an application of a reagent for detecting gene expression in preparing osteoporosis diagnosis products; the gene is TARS.

Further, the reagent comprises PCR amplification primers used for detecting the expression quantity of TARS by using SYBR Green, TaqMan probes, molecular beacons, double-hybrid probes or composite probes.

In a specific embodiment of the invention, the primer sequences are shown as SEQ ID NO.1 and SEQ ID NO. 2.

The invention provides a product for diagnosing osteoporosis, which comprises a reagent for detecting the expression level of TARS.

Further, the reagent comprises SYBR Green, a TaqMan probe, a molecular beacon, a double-hybridization probe or a PCR amplification primer used when a composite probe is used for detecting the TARS expression quantity.

In a specific embodiment of the invention, the primer sequences are shown as SEQ ID NO.1 and SEQ ID NO. 2.

Further, the aforementioned products include, but are not limited to, chips, kits, test strips, or high throughput sequencing platforms; the high-throughput sequencing platform is a special tool for diagnosing osteoporosis, and with the development of a high-throughput sequencing technology, the construction of an RNA expression profile of a person becomes very convenient work. By comparing the RNA expression profiles of patients with disease and normal populations, it is easy to identify which RNA abnormalities are associated with disease. Therefore, the application of TARS in high-throughput sequencing to the knowledge that the abnormal expression of TARS is related to osteoporosis also belongs to the application of TARS, and is also within the protection scope of the invention.

The kit comprises a reagent for detecting the expression quantity of the TARS, the reagent comprises nucleic acid combined with the TARS or a DNA sequence thereof, and the nucleic acid comprises SYBR Green, a TaqMan probe, a molecular beacon, a double-hybrid probe or a PCR amplification primer used when a composite probe is used for detecting the expression quantity of the TARS.

The chip comprises a reagent for detecting the expression level of the TARS, wherein the reagent comprises a nucleic acid combined with the TARS or a DNA sequence thereof, and the nucleic acid comprises a probe capable of detecting the expression level of the TARS.

The test strip comprises a reagent for detecting the expression level of the TARS, wherein the reagent comprises a nucleic acid combined with the TARS or a DNA sequence thereof, and the nucleic acid comprises a probe capable of detecting the expression level of the TARS.

The present invention provides a pharmaceutical composition for treating osteoporosis, comprising an agent that inhibits the expression of a TARS gene.

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

The reagent comprises siRNA, shRNA, inhibitory miRNA or inhibitory targeted small molecule compound.

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 application of the TARS gene in preparation of a medicament for treating osteoporosis. The medicament includes an agent that inhibits expression of a TARS gene. The agent is not limited as long as it can inhibit the expression level of TARS.

The invention also provides the application of the reagent for inhibiting TARS gene expression in preparing a medicament for treating osteoporosis.

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

(1) obtaining a sample from a subject;

(2) detecting the expression level of TARS in the subject sample;

(3) correlating the measured expression level of TARS with the presence or absence of disease in the subject.

(4) If the expression level of TARS is significantly increased compared to a normal control, the subject is judged to have osteoporosis or the risk of osteoporosis is judged to be high.

The present invention also provides a method for treating osteoporosis, which comprises inhibiting the expression level of TARS or inhibiting the regulatory activity of TARS.

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

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".

In the context of the present invention, a "TARS gene" includes a polynucleotide of the TARS gene as well as any functional equivalent of the TARS gene. The sequence of the TARS gene (Chromosome 5, NC-000005.10 (33440696..33468091)) can be queried in GeneBank, the International public nucleic acid sequence database.

The invention has the advantages and beneficial effects that:

the invention discovers the molecular marker for diagnosing the osteoporosis, and the molecular marker can be used for judging the osteoporosis at the early stage, thereby improving the cure rate of patients.

Drawings

FIG. 1 shows a statistical graph of the differential expression of TARS detected by QPCR;

FIG. 2 shows a statistical chart for detecting the effect of interference of TARS gene expression using immunoblotting;

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

Detailed Description

The following examples are intended to illustrate the invention in further detail with reference to the accompanying drawings and examples, and are not intended to limit the scope of the invention the experimental procedures, for which specific conditions are not indicated in the examples, are generally performed according to conventional conditions, such as those described in Sambrook et al, molecular cloning, A laboratory Manual (New York: Cold Spring Harbor L aboratoryPress,1989), or according to the manufacturer's recommendations.

Example 1 QPCR detection of differentially expressed genes

Firstly, collecting samples

30 male osteoporosis patients are selected, and the selection criteria are T < -2.5 of bone density measured by a double-energy X ray, T < -2.5 with the prior brittle fracture history, patients with normal liver and kidney functions, the exclusion criteria are ① patients who have used anti-osteoporosis drugs, ② patients with diabetes, thyroid or parathyroid disease, endocrine diseases such as adrenal gland and gonad, ③ secondary osteoporosis, the average age is 78 years old, the peripheral blood of the patients is extracted as an experimental sample, and the peripheral blood of 30 normal people is 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 in which 10 × erythrocyte lysate H is selected in a suitable volume according to the volume of the blood sample to be treated (for example, 140. mu.l of 10 × erythrocyte lysate H is selected when the volume of the blood sample to be treated is 200. mu.l), and RNase-Free ddH is used₂O diluted to 1 × erythrocyte lysate H.

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

Note that the volume of the mixture of blood and 1 × lysate H should not exceed 3/4 of the tube volume for optimal mixing, and that if the white blood cell content of the blood is high, the volume of the blood used can be scaled down proportionally, and the volume of 1 × lysate H used 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.400 400 × g) 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 × erythrocyte lysate H (2 times the volume of 1 × erythrocyte lysate H as compared to the 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.400 400 × g) 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 solution R L H (β -mercaptoethanol was added before use) was added to the leukocyte pellet, specifically in the amount shown in Table 1 below, and mixed by vortexing or using a pipette.

Note that if the blood is not whole blood of a healthy person, the required volume of lysate R L H is determined according to the number of leukocytes in the blood, and the cells should be completely lysed and the clumped cell pellet should disappear.

TABLE 1 lysate usage Scale

(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 13,400 × g) 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, 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 13,400 × g) 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 the adsorption column CR4, centrifuged at 12,000rpm (13,400 × g) for 30-60sec, and the waste liquid in the collection tube is discarded and the step 14 is directly performed.

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

(14) 500. mu.l of rinsing solution RW (previously checked for ethanol addition) was added to the adsorption column CR4, and the tube was left to stand at room temperature for 2min, centrifuged at 12,000rpm (13,400 × g) for 30-60sec, the waste liquid in the collection tube was discarded, and the adsorption column CR4 was returned to the collection tube.

(15) Step 14 is repeated.

(16) The column was centrifuged at 12,000rpm (13,400 × g) for 2min, the waste liquid was decanted, and the adsorption column CR4 was left at room temperature for several minutes to allow the rinsing liquid remaining in the adsorption material to dry thoroughly.

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) Transferring the adsorption column CR4 into a new RNase-Free centrifuge tube, adding 30-50 μ l RNase-freedH₂O was left at room temperature for 2min and centrifuged at 12,000rpm (. about.13,400 13,400 × g) 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, the ingredients are shown in Table 3. to ensure the accuracy of the reaction solution preparation, Master Mix was prepared in the amount of reaction +2, then 10. mu. L was dispensed into each reaction tube, and after gentle mixing, reverse transcription reaction was immediately performed.

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, duplicate wells were prepared in triplicate for each single gene of each sample, and the duplicate wells were sequentially added to a 96-well plate in ddH₂O as a negative control.

TABLE 4 QPCR reaction System

The primer sequences are as follows:

TARS

a forward primer: 5'-CAACCTGTGATGAATATG-3' (SEQ ID NO.1),

reverse primer: 5'-TTATACTGTGCTAACTGT-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 +54 ℃ for 34 seconds, for 45 cycles.

3) Ct values of each sample are obtained, and relative expression amounts of all target genes are calculated according to (2- △△ Ct), and reference genes GAPDH in the same group are respectively used as controls.

4. Results

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

EXAMPLE 2 preparation of Male osteoporosis diagnostic kit

The invention provides a kit for diagnosing male osteoporosis by detecting the expression condition of TARS gene according to the correlation between the TARS gene and the male osteoporosis, and the kit comprises a SYBR Green polymerase chain reaction system, a primer pair for amplifying the TARS gene and GAPDH gene, a forward primer sequence for amplifying the TARS gene is 5'-CAACCTGTGATGAATATG-3', a reverse primer sequence is 5'-TTATACTGTGCTAACTGT-3', a forward primer sequence for amplifying the GAPDH is 5'-GTGGACCTGACCTGCCGTCT-3', a reverse primer sequence is 5'-GGAGGAGTGGGTGTCGCTGT-3', a SYBR Green polymerase chain reaction system comprises PCR buffer solution, dNTPs and SYBR Green fluorescent dye, and the PCR buffer solution comprises 25 MKC L and 2.5mM MgC L₂、200mM(NH₄)₂SO₄。

Example 3 Effect of TARS 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, designed and synthesized by Shanghai Jima pharmaceutical technology Limited, 24h before transfection, hFOB1.19 cells are inoculated in a 6-well plate, 1.5m L DMEM/F12 complete medium is added, the adherence is overnight, transfection is carried out when the cell confluence reaches 80% -90%, before transfection is replaced by Opti-MEM medium without serum and G418, 250 mu L Opti-MEM medium is used for diluting siRNA (the final concentration is 33 nmol/L), the mixture is gently blown and sucked for 3-5 times, the transfection reagent is gently mixed and inverted, 250 mu L Opti-MEM medium is used for diluting 5.0 mu L L of fectamine TM 2000, the mixture is gently blown and sucked for 3-5 times, the mixture is stood for 5min at room temperature, the transfection reagent and the dilution solution are gently blown and sucked for 3-5 times, the mixture is stood for 20min at room temperature, the transfection complex is added into a 6-well cell plate, the cell plate is gently shaken before and after the cell plate is placed at 34 ℃, the volume fraction of the cell plate is evenly mixed, and₂culturing in an incubator. The transfection time is 6h, and DMEM/F12 complete medium is replaced.

The siRNA sequence against TARS (TARS-siRNA) is: forward sequence 5'-AAUCAUGUUCUGCUUUUAGUATT-3' (SEQ ID NO.5),

reverse sequence 5'-CUAAAAGCAGAACAUGAUUCCTT-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 mu l of PMSF (100 ×) and 10 mu l of lcooktail (100 ×) 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 using a Brandford method, mixing a proper amount of protein with a sample buffer solution, boiling for 5min, cooling for 5min, loading 30 mu g of protein to prepared 15% polyacrylamide gel for electrophoresis, transferring a membrane by using a Bio-Rad semi-dry transfer system, sealing overnight, adding a Western washing solution for washing, adding a monoclonal antibody shaking table for hybridization at room temperature for 2h, diluting the protein in a sealing buffer solution by using a Western goat anti-mouse secondary antibody diluent according to a proper proportion, incubating for 60min, washing the membrane for 3 times, developing by using an EC L reagent, fixing and detecting protein expression, calculating the gray level of a protein strip by using professional software, wherein the gray level is in direct proportion to the protein amount, setting the expression amount of a control group protein as a reference, and setting the gray level to be 1, and calculating the relative expression amount of TARS interfering group TARS.

4.3 results

As shown in FIG. 2, TARS-siRNA can effectively inhibit TARS protein expression, and the difference has statistical significance.

5. Osteoblast proliferation assay

The steps are that a 96-well plate is inoculated with hFOB1.19 cells, 2000 cells are inoculated in each well, 5 wells are added in each group, siRNA transfection is carried out according to the method described above, after 48h of transfection, 20 mu L/well of MTT liquid (5mg/m L) is added, incubation is carried out for 4h at 37 ℃, liquid in the wells is absorbed, 150 mu L/well of DMSO liquid 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 TARS 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 the function of osteoblasts, is one of the most common indexes for evaluating the secretory function of osteoblasts, and therefore, the influence of inhibiting TARS gene expression on osteoblast differentiation is evaluated by detecting the level of alkaline phosphatase in a culture solution.

Determination of alkaline phosphatase Activity in culture:

step 6 inoculation of 1 × 10 per well in the well plate⁵The siRNA transfection was performed according to the method described above, and the cell culture solution was aspirated for 24h of transfection, centrifuged at 2500r/min, centrifuged for 10min, and the supernatant was taken for determination using an alkaline phosphatase assay kit (determination of absorbance value-blank absorbance value)/(standard absorbance value-blank absorbance value)]× phenol standard substance concentration × 100m L× sample is diluted before measurement, absorbance value (A value) of each well is measured by selecting 520nm wavelength with a microplate reader, and relative A L P activity is calculated according to the measured A value.

As a result: the supernatant phosphatase activity of the TARS-siRNA group was significantly increased compared to the control group, indicating that inhibition of TARS expression promoted osteoblast differentiation, as shown in Table 5.

TABLE 5 statistics of supernatant alkaline phosphatase Activity

Item	NC-siRNA	TARS-siRNA
			Supernatant alkaline phosphatase	0.777±0.042	2.230±0.072

In summary, TARS-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 TARS gene, so that the inhibition of TARS gene expression 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

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Claims (3)

1. The application of the reagent for detecting gene expression in the preparation of diagnostic products for male osteoporosis; the gene is TARS.

2. The use of claim 1, wherein the reagent comprises PCR amplification primers used in detecting the gene expression amount by SYBR Green, PCR amplification primers used in detecting the gene expression amount by TaqMan probes, PCR amplification primers used in detecting the gene expression amount by molecular beacons, PCR amplification primers used in detecting the gene expression amount by double-hybrid probes, or PCR amplification primers used in detecting the gene expression amount by composite probes.

3. The application of claim 2, wherein PCR amplification primer sequences used for detecting the gene expression amount by SYBR Green are shown as SEQ ID No.1 and SEQ ID No. 2; the PCR amplification primer sequence used when the TaqMan probe detects the gene expression quantity is shown as SEQ ID NO.1 and SEQ ID NO. 2; the PCR amplification primer sequence used when the molecular beacon detects the gene expression quantity is shown as SEQ ID NO.1 and SEQ ID NO. 2; the sequences of PCR amplification primers used when the double-hybrid probe detects the gene expression quantity are shown as SEQ ID NO.1 and SEQ ID NO. 2; the PCR amplification primer sequence used when the composite probe detects the gene expression quantity is shown as SEQ ID NO.1 and SEQ ID NO. 2.

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