CN108179181B

CN108179181B - Application of RDX gene in clinical medication

Info

Publication number: CN108179181B
Application number: CN201711469118.9A
Authority: CN
Inventors: 肖枫; 孙耀兰; 常鹏
Original assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Priority date: 2017-12-29
Filing date: 2017-12-29
Publication date: 2020-12-15
Anticipated expiration: 2037-12-29
Also published as: CN108179181A

Abstract

The invention relates to application of an RDX gene in clinical medication, in particular to new application of the RDX gene in clinical medication of postmenopausal osteoporosis and detection of postmenopausal osteoporosis. The inventor researches the molecular action mechanism of the pill of six ingredients with rehmannia on osteoporosis by adopting a high-throughput sequencing technology, explains the action mechanism of the pill of six ingredients with rehmannia from the perspective of genes, and selects and verifies a candidate gene RDX. The invention provides a molecular theoretical basis for postmenopausal osteoporosis and clinical medication, and has good clinical application value.

Description

Application of RDX gene in clinical medication

Technical Field

The invention relates to the field of biological medicines, in particular to application of an RDX gene in clinical medication, and more particularly relates to new application of the RDX gene in clinical medication of postmenopausal osteoporosis and detection of postmenopausal osteoporosis.

Background

Radixin (RDX) is a cell membrane-cytoskeleton connexin, is one of ERM (ezrin/radxin/moesin) family members connecting cell membranes and cytoskeletons, is expressed in liver cells, kidney cells and the like, and participates in various vital activities such as cell morphology regulation, signal transduction, cell movement and the like. RDX is composed mainly of 3 domains: the highly conserved globular amino-terminal end, which is connected to the cell membrane, has an alpha helical region in the middle, a positively charged carboxy-terminal end, which contains threonine residues that can be phosphorylated and dephosphorylated, and is the main regulatory site for RDX. Current studies indicate that RDX plays an important role in maintaining the polarity of hepatocytes (Re-defining ERM function in lymphocyte activation and migration, Immunol Rev, 2013, 256(1): 63-79).

Osteoporosis (osteoporotosis) occurs as a result of the combined action of genetic and environmental factors and is a complex multifactorial disease. In recent years, with intensive research on precise medicine, some related molecular markers have been found, and our previous research also found some osteoporosis-related genes, such as IFT52 gene (CN2015106280814), EPS8L3 gene (CN2015106280424), CERS2 gene (CN2015106293481), MTUS1 gene (CN 2015106280246), and the like. Postmenopausal osteoporosis (PMOP), which is one of osteoporosis, is related to aging, mainly occurs in postmenopausal women, and due to the fact that lack of estrogen causes reduction of bone mass and structural change of bone tissue, increased bone fragility, easy fracture, pain caused by fracture, bone deformation, complications and even death, the health and the quality of life of the old are seriously affected, and even the life is shortened, so that the financial and manpower burden of countries and families is increased.

In recent years, traditional Chinese medicine researches have proved that the onset of osteoporosis is closely related to kidney deficiency, and various kidney tonifying formulas, such as Liuwei Dihuang pills, have obvious curative effect on postmenopausal osteoporosis, have no obvious side effect and are gradually valued. The inventor researches the molecular action mechanism of the Liuwei Dihuang pill on osteoporosis by adopting a high-throughput sequencing technology, carries out sequencing analysis on peripheral blood samples before and after taking the Liuwei Dihuang pill and healthy people contrast peripheral blood samples for postmenopausal osteoporosis patients, explains the action mechanism from the gene angle, and selects a candidate gene RDX. The invention provides a molecular theoretical basis for postmenopausal osteoporosis and clinical medication, and has good clinical application value.

Disclosure of Invention

The invention aims to provide application of a reagent for detecting RDX gene and/or RDX protein in preparation of a clinical medicine gene detection preparation for postmenopausal osteoporosis.

In order to realize the purpose, the invention firstly screens candidate genes RDX by combining a high-throughput sequencing and bioinformatics method, then verifies that the RDX gene expression level of osteoporosis patients is obviously reduced after taking pills of six ingredients with rehmannia through a molecular biology method, shows that the genes are not only related to postmenopausal osteoporosis, but also are action targets of the pills of six ingredients with rehmannia, can be used for preparing a diagnosis preparation and a treatment target of postmenopausal osteoporosis, and has important clinical application value.

Furthermore, the sample group with high RDX gene and/or RDX protein expression is suitable for Liuwei Dihuang pills.

Further, the detection agent is used for detecting the expression of the RDX gene by one or more of the following methods: fluorescent quantitative PCR method, gene chip method, and high-throughput sequencing method.

The invention aims to provide application of a reagent for detecting RDX gene and/or RDX protein in preparing a postmenopausal osteoporosis diagnosis preparation.

Further, the RDX gene and/or RDX protein is highly expressed in postmenopausal osteoporosis samples.

Further, the postmenopausal osteoporosis diagnostic agent detects the expression of RDX gene and/or RDX protein in peripheral blood.

Further, the diagnostic preparation for postmenopausal osteoporosis is used for detecting the expression of the RDX gene by one or more of the following methods: fluorescent quantitative PCR method, gene chip method, and high-throughput sequencing method.

The fluorescence quantitative PCR method is characterized in that a PCR product is marked and tracked through a fluorescent dye or a fluorescence-marked specific probe, the reaction process is monitored on line in real time, the product can be analyzed by combining corresponding software, and the initial concentration of a sample template to be detected is calculated. The occurrence of fluorescence quantitative PCR greatly simplifies the quantitative detection process and truly realizes absolute quantification. The presence of multiple detection systems makes the assay more selective. The automatic operation improves the working efficiency, and the reaction is rapid, the repeatability is good, the sensitivity is high, the specificity is strong, and the result is clear.

Gene chips, also known as DNA microarrays (DNAmicroarray), can be divided into three main types: 1) nucleic acid probes or cDNA fragments immobilized on the surface of a polymer substrate (nylon membrane, nitrocellulose membrane, etc.) are usually hybridized with an isotope-labeled target gene and detected by a radioimaging technique. 2) The detection is carried out by hybridization with a target gene labeled with fluorescence using a DNA probe array immobilized on a glass plate by spotting. 3) An oligonucleotide probe array synthesized directly on a hard surface such as glass is hybridized with a target gene labeled with fluorescence for detection. As an advanced, large-scale and high-throughput detection technology, the gene chip is applied to the diagnosis of diseases, and has the advantages of the following aspects: firstly, high sensitivity and accuracy; secondly, the method is quick, simple and convenient; thirdly, can detect a plurality of diseases simultaneously.

High-throughput sequencing (also called next generation sequencing) is a revolutionary change to the conventional sequencing, and can perform sequence determination on hundreds of thousands to millions of DNA molecules at a time, thereby greatly improving the sequencing efficiency. The large-scale sequencing technology greatly improves the reading speed of genetic information of a plurality of species, and provides guarantee for acquiring sequence information of all mRNA and decrypting mRNA maps. High throughput sequencing at the same time makes it possible to perform a detailed global analysis of the transcriptome and genome of a species and is therefore also referred to as deep sequencing. Representative of high throughput sequencing platforms are the 454 sequencer (Roch GSFLX sequencer) by Roche (Roche), the Solexa Genome Analyzer (Illumina Genome Analyzer) by Illumina, and the SOLiD sequencer (ABI SOLiD sequencer) by ABI.

The product for detecting the RDX gene in postmenopausal osteoporosis by the fluorescent quantitative PCR method contains a pair of primers for specifically amplifying the RDX gene; the gene chip comprises a probe hybridized with a nucleic acid sequence of the RDX gene. Preferably, the upstream primer sequence of the pair of primers for specifically amplifying the RDX gene is SEQ ID NO.1, and the downstream primer sequence is SEQ ID NO. 2.

Further, the diagnostic formulation for postmenopausal osteoporosis further comprises an immunological method for detecting the expression of RDX protein. Preferably, the immunological method is used for detecting the expression of RDX protein in postmenopausal osteoporosis by a western blot and/or ELISA and/or colloidal gold method.

Enzyme-linked immunosorbent assay (ELISA) is a technique in which a known antigen or antibody is adsorbed on the surface of a solid phase carrier, and an enzyme-labeled antigen-antibody reaction is carried out on the surface of the solid phase. The technology can be used for detecting macromolecular antigens, specific antibodies and the like, and has the advantages of rapidness, sensitivity, simplicity, convenience, easy standardization of carriers and the like. The ELISA detection kit can be divided into indirect method, double antibody sandwich method, competition method, two-site one-step method, IgM antibody detection by capture method and ELISA using avidin and biotin according to the detection purpose and operation steps. The chromogenic substrate in the ELISA detection kit can be selected from horseradish peroxidase (HRP) or Alkaline Phosphatase (AP).

The commonly used detection technique of immune colloidal gold: (1) the immune colloidal gold optical lens staining method cell suspension smear or tissue section can stain with colloidal gold labeled antibody, or enhance labeling with silver developing solution based on the colloidal gold labeling, so that reduced silver atoms are deposited on the surface of the labeled gold particles, and the sensitivity of the colloidal gold labeling can be obviously enhanced. (2) The immune colloidal gold electron microscope staining method can combine the colloidal gold labeled antibody or anti-antibody with the negative staining virus sample or tissue ultrathin section, and then carry out negative staining. Can be used for observing virus morphology and detecting virus. (3) The dot immunogold filtration method uses microporous filter membrane as carrier, firstly, the antigen or antibody is spotted on the membrane, after closed, the sample to be detected is added, after washing, the corresponding antigen or antibody is detected by using colloidal gold-labeled antibody. (4) The colloidal gold immunochromatography fixes specific antigen or antibody on a membrane in a strip shape, a colloidal gold labeled reagent (antibody or monoclonal antibody) is adsorbed on a binding pad, when a sample to be detected is added on the sample pad at one end of a test strip, the sample moves forwards through capillary action, the colloidal gold labeled reagent on the binding pad is dissolved and then reacts with each other, when the sample moves to a region of the fixed antigen or antibody, a combination of the object to be detected and the gold labeled reagent is specifically combined with the sample to be detected and intercepted, and is gathered on a detection zone, and a color development result can be observed through naked eyes. The method is developed into a diagnostic test strip, and is very convenient to use.

Further, the ELISA method for detecting RDX protein is to use an ELISA detection kit. The antibody in the kit can adopt a commercially available RDX monoclonal antibody. Further, the kit comprises: the kit comprises a solid phase carrier coated with an RDX monoclonal antibody, an enzyme-labeled secondary antibody, an enzyme substrate, a protein standard substance, a negative control substance, a diluent, a washing solution, an enzyme reaction termination solution and the like.

Furthermore, the colloidal gold method for detecting the RDX protein uses a colloidal gold test strip, and the antibody adopts a commercially available RDX monoclonal antibody. Further, the colloidal gold test strip adopts a colloidal gold immunochromatography technique or a colloidal gold percolation method. Furthermore, the spraying point of the detection area (T) on the nitrocellulose membrane of the colloidal gold test strip is provided with an anti-RDX monoclonal antibody, and the spraying point of the quality control area (C) is provided with immunoglobulin IgG.

The invention aims to provide a fluorescent quantitative PCR kit for detecting postmenopausal osteoporosis, which is characterized in that the kit detects gene RDX, and adopts specific upstream primers and specific downstream primers, wherein the sequence of the upstream primers is SEQ ID NO.1, and the sequence of the downstream primers is SEQ ID NO. 2.

Furthermore, the PCR kit is suitable for all types of fluorescent quantitative gene amplification instruments on the market at present, has high sensitivity, quick and accurate quantification and good stability, and has good application prospect.

Further, the fluorescent quantitative PCR kit comprises the following components: specific primers, internal reference primers and fluorescent quantitative PCR reaction liquid. The specific primer comprises an upstream primer and a downstream primer, wherein the sequence of the upstream primer is SEQ ID NO.1, and the sequence of the downstream primer is SEQ ID NO. 2. The internal reference primer is a beta-actin internal reference primer, the sequence of an upstream primer is SEQ ID NO.3, and the sequence of a downstream primer is SEQ ID NO. 4.

The kit also comprises an RNA extraction reagent. Preference is given to

Reagent performs sample RNA extraction.

The invention aims to provide a postmenopausal osteoporosis detection kit, which is used for detecting RDX protein. Furthermore, the kit also comprises other detection reagents.

The invention aims to provide a gene chip for detecting postmenopausal osteoporosis, which comprises a probe hybridized with a nucleic acid sequence of an RDX gene.

The invention aims to provide a gene detection kit for clinical medicines for postmenopausal osteoporosis, which is used for detecting the expression of RDX genes and/or RDX proteins, and a sample group with high expression of the RDX genes and/or the RDX proteins is suitable for Liuwei Dihuang pills.

The invention aims to provide a gene chip for detecting clinical medication genes of postmenopausal osteoporosis, which comprises a probe hybridized with a nucleic acid sequence of an RDX gene, wherein a sample group with high expression of the RDX gene and/or RDX protein is suitable for Liuwei Dihuang pills.

The invention aims to provide application of RDX gene and/or protein inhibitor thereof in preparing a medicament for treating postmenopausal osteoporosis.

Further, the drug for treating postmenopausal osteoporosis refers to an agent which can inhibit the expression of RDX gene. It is well known to those skilled in the art that suppression of gene expression can generally be achieved by one or more of the following methods: through activating the suppressor gene of the RDX gene, activating the protein of the suppressor gene expression of the RDX gene, adopting an RNA interference technology to suppress the RDX gene expression, activating microRNA for promoting the mRNA degradation of the RDX gene, introducing molecules for promoting the protein degradation of the RDX gene coding, and inhibiting the expression of factors and proteins for promoting the RDX gene expression.

RNA interference (RNAi) refers to the phenomenon that exogenous and endogenous double-stranded RNA induces mRNA specific degradation of homologous target genes in an organism to cause post-transcriptional gene silencing, and is a technology which uses small double-stranded RNA to efficiently and specifically block the expression of a certain specific gene in the organism, promote the mRNA degradation and enable cells to show a specific gene deletion phenotype. After the siRNA design is finished, a direct synthesis method or a constructed siRNA expression vector can be adopted, and the prepared siRNA can transfect cells by a calcium phosphate coprecipitation method, an electroporation method, a DEAE-dextran and polybrene method, a mechanical method such as microinjection or a gene gun, a cationic liposome reagent method and the like.

Further, the siRNA target point for inhibiting the RDX gene expression is selected from one and/or several of the following sequences: SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO. 11. Preferably, the siRNA sequence is SEQ ID NO. 5.

Further, the siRNA sequence for inhibiting the RDX gene expression is selected from one and/or several of the following sequences: SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO. 13. Preferably, the siRNA sequence is SEQ ID NO.6, SEQ ID NO. 7.

The invention aims to provide a medicament for treating postmenopausal osteoporosis, which inhibits the expression of an RDX gene.

Furthermore, the medicine for treating postmenopausal osteoporosis also comprises a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier included in the present invention is a carrier generally used in the preparation, and includes, but is not limited to, lactose (lactose), dextrose (dextrose), sucrose (sucrose), sorbitol (sorbitol), mannitol (mannitol), starch, gum arabic, calcium phosphate, alginate (alginate), gelatin (gelatin), calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone (polyvinylpyrrolidone), cellulose (cellulose), water, syrup, methyl cellulose (methyl cellulose), methyl hydroxybenzoate (methyl hydroxybenzoate), propyl hydroxybenzoate (propyl hydroxybenzoate), talc, magnesium stearate (magnesium stearate), mineral oil (mineral oil), and the like.

The composition of the present invention may contain, in addition to the above components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. The carrier and the preparation which can be accepted in pharmacy are described in the complete Remington pharmaceutical book in detail.

The composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be administered by intravenous injection, intranasal injection, local injection, intracerebroventricular injection, spinal cavity injection, subcutaneous injection, intraperitoneal injection, transdermal administration, etc.

The appropriate dose of the composition of the present invention can be prescribed in various ways depending on factors such as the method of preparation, the mode of administration, the age, body weight, sex, disease state, food, administration time, administration route, excretion rate and reaction sensitivity of the patient, and the effective dose for the desired treatment or prevention can be easily determined and prescribed by a skilled physician.

The compositions of the present invention are formulated according to methods that can be readily practiced by those of ordinary skill in the art using pharmaceutically acceptable carriers and/or excipients, and can be prepared in unit dosage form or in multi-volume containers. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oily or aqueous medium, or may be in the form of a extract, powder, granule, tablet or capsule, and may further include a dispersant or stabilizer.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Drawings

FIG. 1 is a graph showing the relative expression amounts of the RDX gene in peripheral blood of postmenopausal osteoporosis patients and healthy persons, FIG. 2 is a graph showing the relative expression amounts of the RDX gene in peripheral blood of postmenopausal osteoporosis patients before and after administration of LIUWEIDIHUANG pill

FIG. 3 is a graph of relative expression levels of various groups of RDX mRNA following RNA interference: group C: blank control group; group C1: transfecting the liposome group; group C2: transfecting a nonspecific siRNA group; group S1, S2, S3: specific siRNA groups were transfected.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. Those of ordinary skill in the art will understand that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

Example 1 high throughput sequencing and analysis

Respectively collecting 3 cases of peripheral blood samples before and after taking Liuwei Dihuang pills for postmenopausal osteoporosis patients and 3 cases of healthy people contrast peripheral blood samples, carrying out RNA extraction, carrying out agarose gel electrophoresis after the RNA extraction, and preliminarily judging whether the quality of the extracted RNA sample is qualified or not from electrophoresis results, wherein the RNA sample can be used for further transcriptome analysis. And further detecting the extraction condition of the RNA sample by a NanoDrop1000 spectrophotometer, wherein the sample for RNA-seq sequencing requires: OD260/OD280 was 1.8-2.2.

The sequencing platform is a HiSeq 2500 high-throughput sequencing platform of Illumina company, high-throughput transcriptome deep sequencing is carried out, and after sequencing, the quality of sequencing data is integrally evaluated by using Fast-QC software, wherein the quality comprises the mass value distribution of bases, the position distribution of mass values, GC content, PCR amplification content, the frequency of kmer and the like. And during differential gene expression analysis, performing differential screening by adopting an internationally recognized algorithm EBSeq according to the obtained FPKM value. Wherein, during screening, LOG2FC is greater than 1 or < -1, and FDR is less than 0.05. To better understand the function of differentially expressed genes, we performed Gene ontology and signal pathway analysis on the differentially expressed genes, and performed functional annotation and protein interaction network analysis on the differentially expressed genes, and we screened the differentially expressed genes RDX in conjunction with literature in view of the results of the above data analysis. RDX is highly expressed in osteoporosis group and is low expressed after taking pill of six ingredients with rehmannia.

Example 2 postmenopausal osteoporosis patients before and after taking Liuwei Dihuang pill, peripheral blood RDX gene expression first, material and method

1. Material

22 postmenopausal osteoporosis patients were collected with consent, peripheral blood was collected at the initial stage of their examination, and half a year after taking liuwei di huang pills, and the peripheral blood was collected again, grouped and numbered.

2. Method of producing a composite material

2.1 extraction of Total RNA from postmenopausal osteoporotic peripheral blood and healthy human peripheral blood

By using

Reagent carries out sample RNA extraction, and the experimental operation is carried out according to the product instruction.

RNA quality determination criteria: the OD260/OD280 value of the RNA sample is between 1.8 and 2.2; the total RNA electrophoresis pattern has clear 28S and 18S bands; the electrophoresis pattern after the water bath heat preservation for 1 hour at 70 ℃ has no obvious difference with the pattern before the water bath heat preservation.

2.2 Synthesis of cDNA by reverse transcription

By using

III Reverse transcription of cDNA with Reverse Transcriptase (Invitrogen, cat # 18080-044)The experimental operation is carried out according to the product specification, and the specific operation is as follows:

using a reverse transcription kit, cDNA was synthesized by reverse transcription of l. mu.g of total RNA with reverse transcription buffer. A25-mu-l reaction system is adopted, 1 mu g of total RNA is taken from each sample as template RNA, and the following components are respectively added into a PCR tube:

5 ul of 5 XT buffer, 1.25 ul of 10mmol/l dNTP, 2.5 ul of 0.1mmol/l DTT, 2 ul of 30 ul mmol/l OligodT, 1.25 ul of 200U/ul MMLV, 1 ul of template RNA, and sterile water was added to the total volume of 25 ul. Incubate at 42 ℃ for 1 hour, 72 ℃ for 10 minutes, and briefly centrifuge. The cDNA was stored in a freezer at-20 ℃ for future use.

2.3 Real-Time PCR

2.3.1 Instrument and analytical method

The relative quantitative analysis of the data was performed by means of 2-delta CT using ABI 7500 type fluorescent quantitative PCR instrument.

2.3.2 primer design

The template sequence was NM-001260492.1 using online primer design software, synthesized by Invitrogen corporation after primer design. The specific primer sequences are as follows:

RDX primer:

5’-agtcattcctccaacaga-3’(SEQ ID NO.1)

5’-gttcttcctcgcttctatg-3’(SEQ ID NO.2)

the amplification length is 123 bp.

BETA-ACTIN primers:

5’-taatcttcgccttaatact-3’(SEQ ID NO.3)

5’-ccttcatacatctcaagt-3’(SEQ ID NO.4)

the amplification length is 103 bp.

The operation process is as follows:

reaction system: 2 × mix 10 μ l; 0.5. mu.l of each of the upstream primer (10uM) and the downstream primer (10 uM); 2 mul of template; sterile distilled water was added to make up to 25. mu.l.

By Power

Green PCR Master Mix (Invitrogen, cat # 4367659) was amplified and testedThe test operation is carried out according to the product specification.

The amplification procedure was: 95 ℃ 10min, (95 ℃ 15sec, 58 ℃ 60 sec). times.35 cycles.

Sample RealTimePCR assay: after 10-fold dilution of each sample cDNA, 2. mu.l of each sample cDNA was used as a template, and the target gene primer and the reference gene primer were used for amplification. At the same time, the dissolution curve analysis is carried out at 60-95 ℃. Second, experimental results

The real-time quantitative PCR results show (see fig. 1 in particular): comparing the RDX expression conditions of the RDX gene in peripheral blood before and after taking Liuwei Dihuang Wan by patients with postmenopausal osteoporosis, the RDX gene expression level after taking Liuwei Dihuang Wan is reduced by about one third compared with the RDX gene expression level before taking, and the RDX gene expression level is consistent with the trend of high-throughput sequencing results.

Example 4 RDX Gene expression in peripheral blood of postmenopausal osteoporosis patients and healthy persons

1. Material

Peripheral blood was collected from 32 postmenopausal osteoporosis patients and 29 healthy people, and was grouped and numbered.

2. Method of producing a composite material

The specific procedure is as in example 3.

3. Results

The inflection point of the real-time quantitative PCR amplification curve is clear, the overall parallelism of the amplification curve is good, the amplification efficiency of each reaction tube is similar, the limit is flat without raising, the slope of the exponential phase of the curve is larger, and the amplification efficiency is higher; the dissolution curves of the sample amplification products are all unimodal, which indicates that only one amplification product is specifically amplified; according to the relative quantitative formula of qRT-PCR, the qRT-PCR amplification result is stable, wherein the expression level of RDX in postmenopausal osteoporosis peripheral blood is higher than that in healthy people peripheral blood, the former is about 1.26 times of that in healthy people (see figure 2), and the result verifies the result of high-throughput sequencing.

EXAMPLE 5 culture of cell line MG-63

First, experimental material

Cell line MG-63 was purchased from Shanghai cell institute of Chinese academy of sciences.

Second, main solution

Cell culture solution: DMEM medium + 10% standard fetal bovine serum.

0.25% trypsin digest: adding 0.25g of trypsin into 100m of 1 deionized water, filtering and sterilizing a filter, and subpackaging for later use.

Thirdly, cell passage

1, discarding original culture solution in a culture bottle full of cells, adding 0.25% trypsin solution 1m1, covering a cell layer, sterilizing a bottle mouth, and covering;

observing cell change under an inverted microscope, gradually making the original adherent cells tend to be round along with the lapse of time, retracting intercellular substance, enlarging cell gap, discarding pancreatin when the cells do not float, and adding 5ml of culture solution containing 10% fetal calf serum to terminate digestion;

3, cell counting: taking 0.5mI of the cell suspension, properly diluting and dripping the cell suspension into a blood cell counting plate, counting the total number of cells in four squares of four corners according to a white blood cell counting method, only counting the cells with complete cell nucleuses and cell plasms during counting, calculating the piled cells according to one cell, and converting the total number of the cells in 4 squares into the number of the cells in each milliliter of the cell suspension according to the following formula: total number of cells/ml 4 large lattice cell number/4 × 10⁴X dilution factor;

4 according to the cell count result, further diluted with DMEM complete medium to contain 3X 10 per ml⁵The cell concentration was measured and distributed into culture flasks (8m 1/flask) placed at 37 ℃ in 5% CO₂Culturing in an incubator.

Example 6 RNAi inhibition of RDX Gene expression

First, experimental material

siRNA construction and Synthesis

Corresponding siRNA was designed based on the Sequence of the RDX gene in GenBank (NCBI Reference Sequence: NM-001260492.1). The design is sent to a synthesis company for synthesis. Nonspecific siRNA was provided by the synthetic company.

Second, Experimental methods

RNA interference technology for specifically inhibiting expression of RDX gene in MG-63 cell

1. Culture of MG-63 cells

The procedure is as in example 3.

2. Design and Synthesis of siRNA

The siRNA expression vector pSIREN-DNR contains a neomycin resistance gene and a GFP green fluorescent marker, and can monitor the transfection efficiency of the vector in cells in real time. Based on the desired mRNA sequence, 3 RNA interference target sequences were designed (Table 1). For each selected siRNA target sequence, siRNA sense and antisense strands are designed and linked by loop (9nt), called shRNA (short hairpin RNA). Synthesizing two single chains of each DNA template for coding shRNA, and annealing the DNA single chains to obtain the DNA double-chain template of the shRNA. The template chain is connected with RNAPIyIII polymerase transcription termination site behind, and BamHI and HindIII enzyme cutting sites are designed at two ends of the template chain respectively and can be cloned between the BamHI and HindIII enzyme cutting sites of the multiple cloning site of the siRNA vector. After double digestion of siRNA cargo with BamHI and HindIII, the vector was recovered by electrophoresis on a 1% agarose gel. The annealed DNA template is double-stranded into a linear vector. The molar ratio of insert to vector was about 3:1 using T4 ligase. The ligation product was transformed into DH 5. alpha. E.coli, plated on LB Amp medium and cultured overnight at 37 ℃. PCR identification; and (5) sequencing and identifying. Positive cloning vectors were column extracted and quantified.

TABLE 1 siRNA transcription template sequences

3. Cell grouping and transfection

(1) Grouping of cells

Group C: blank control group; group C1: transfecting the liposome group; group C2: transfecting a nonspecific siRNA group; group S1, S2, S3: specific siRNA groups were transfected.

(2) Transfection

According to Lipofectamine^TMThe procedure provided by 2000 transformation Reagent was performed.

24 hours before transfection, cells in logarithmic growth phase were digested with pancreatin and counted, and the cell concentration was adjusted to 1X 10 with DMEM medium⁵/ml, 2m1 was inoculated into a six-well plate and placed at 37 ℃ in 5% CO₂Cultured in an incubator and used for transfection when the cells reached 80% confluence. Serum-free DMEM medium was used for 3-4h prior to transfection.

Preparing transfection liquid:

solution A: diluting 4.0ug of DNA with 250u1 serum-free culture medium, and mixing;

and B, liquid B: diluting 10u1Lipofectamine with 250u1 serum-free culture medium, mixing gently, and standing at room temperature for 5 min;

③ transfection: mixing solution A and solution B, keeping the temperature at room temperature for 20min, directly adding the compound into each well, shaking the culture plate, and gently mixing. In CO₂The temperature of the incubator is kept at 37 ℃ for 24-48h, the liquid is changed after 6h, and a culture medium containing serum is added.

4. Verification of transfection efficiency

(1) Observation of cell morphology and transfection conditions under fluorescence inverted microscope

After 24h of transfection, the culture plate was placed under a fluorescence inverted microscope to observe the cell morphology and growth state, and the transfection condition was observed under green fluorescence.

(2) Detection of changes in RDX gene expression before and after transfection by Real-time PCR method

Constructing a standard curve: selecting 1 bottle of MG-63 cells cultured normally in 50mI culture bottle, extracting RNA, determining RNA concentration and purity, performing reverse transcription reaction, and diluting the DNA template generated by the reaction ten times to obtain 10⁴A DNA template of 100copies/ul is added with RDX primer and internal reference actin primer respectively to prepare a 25u1 reaction system, and a Real-time PCR amplification instrument is used for PCR amplification reaction. Standard curves for RDX and actin were obtained.

Secondly, detecting the change of RDX gene expression before and after transfection by using a Real-time PCR method: extracting RNA of each group of cells, determining the concentration and purity of the RNA, carrying out reverse transcription reaction, simultaneously carrying out Real-time PCR reaction of RDX and actin on each group of DNA template, and repeating the experiment for three times.

And thirdly, carrying out agarose gel electrophoresis on the PCR product.

Third, experimental results

The transfection efficiency was determined by Real-time PCR. The standard curves of RDX and actin are constructed by applying the Real-time PCR method, the linear relation is good, and the requirements are met. The expression of each set of RDX genes was compared by the double calibration curve method. The gene expression of the blank control group, the liposome transfection group and the nonspecific transfection group is basically similar, and the difference has no statistical significance. RDX-siRNA1, RDX-siRNA2 and RDX-siRNA3 all have the effect of inhibiting RDX gene expression, the effect of RDX-siRNA1 is more obvious, the inhibition efficiency reaches 75%, while the inhibition effects of RDX-siRNA2 and RDX-siRNA3 are 21% and 32% respectively, compared with a blank control group, a liposome transfection group and a non-specific transfection group, the difference is statistically significant, and P is less than 0.05 (figure 2).

The invention screens out the postmenopausal osteoporosis pathogenic related gene RDX by adopting high-throughput sequencing, and further analysis shows that the gene is related to the administration effect of the Liuwei Dihuang pill and has important significance in the aspect of clinical medication guidance. The invention provides a new diagnosis target point and a new medicine target point for clinical diagnosis and treatment of postmenopausal osteoporosis, and has good clinical application prospect.

Sequence listing

<110> Beijing, the deep biometric information technology GmbH

Application of <120> RDX gene in clinical medication

<160> 13

<170> SIPOSequenceListing 1.0

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gttcttcctc gcttctatg 19

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<212> DNA

<213> Homo sapiens

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taatcttcgc cttaatact 19

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<213> Homo sapiens

<400> 6

ucauuaaaug aaauauuucu g 21

<210> 7

<211> 21

<212> RNA

<213> Homo sapiens

<400> 7

gaaauauuuc auuuaaugac a 21

<210> 8

<211> 23

<212> DNA

<213> Homo sapiens

<400> 8

gagaatcaat aagcggattt tgg 23

<210> 9

<211> 21

<212> RNA

<213> Homo sapiens

<400> 9

aaaauccgcu uauugauucu c 21

<210> 10

<211> 21

<212> RNA

<213> Homo sapiens

<400> 10

gaaucaauaa gcggauuuug g 21

<210> 11

<211> 23

<212> DNA

<213> Homo sapiens

<400> 11

gacaaaaagg cacctgattt tgt 23

<210> 12

<211> 21

<212> RNA

<213> Homo sapiens

<400> 12

aaaaucaggu gccuuuuugu c 21

<210> 13

<211> 21

<212> RNA

<213> Homo sapiens

<400> 13

caaaaaggca ccugauuuug u 21

Claims

1. Application of a reagent for detecting RDX gene and/or RDX protein in preparing a postmenopausal osteoporosis diagnosis preparation.

2. Use according to claim 1, wherein the diagnostic agent detects the expression of RDX gene and/or RDX protein in peripheral blood.

3. Use according to claim 1, wherein the diagnostic agent is tested for expression of the RDX gene using one or more of the following methods: fluorescent quantitative PCR method, gene chip method, and high-throughput sequencing method.

4. The use according to claim 3, wherein the fluorescent quantitative PCR method uses a pair of primers for specific amplification of the RDX gene; the gene chip includes probes that hybridize to the nucleic acid sequence of the RDX gene.