CN110878354A - Biomarker for diagnosing and treating Parkinson's disease - Google Patents

Abstract

The invention discloses a biomarker for diagnosing and treating Parkinson's disease, wherein the biomarker is LXN. The invention proves that the LXN has differential expression in the Parkinson disease patient through QPCR experiments, constructs a PD cell model for further verifying the correlation of the LXN and the Parkinson disease, and proves that the interference of the LXN expression can reverse the damage effect of MPP + on cells, so the LXN can be used as a biomarker for diagnosing and treating the Parkinson disease. The research result of the invention provides a new method and strategy for clinical diagnosis or treatment of Parkinson's disease.

Description

Biomarker for diagnosing and treating Parkinson's disease

Technical Field

The invention belongs to the field of biomedicine, and relates to a biomarker for diagnosing and treating Parkinson's disease.

Background

The Parkinson's Disease (PD) is a nervous system degenerative disease which is well developed in middle-aged and old people and is also the most common extrapyramidal disease of the middle-aged and old people, the incidence rate of the PD is second in the middle-aged and old people, is only inferior to Alzheimer's Disease (AD), and has a trend of increasing year by year, the incidence rate of men is far higher than that of women.

However, the causes and exact pathogenesis of PD are still not completely understood, and in vitro and in vivo experiments of most scholars prove that the pathways such as mitochondrial dysfunction, oxidative stress and ubiquitination proteasome are related to the pathogenesis of PD, and the occurrence and development of PD may be the result of the interaction of mechanisms such as aging, heredity, environment, oxidative stress, infection, neurotrophic factor deficiency and immune abnormality. In recent years, researches show that the gene has important regulation and control effects on apoptosis of PD and dopaminergic neurons. Therefore, the intensive exploration of the pathogenesis and pathogenesis of PD and the search for effective neuroprotective agents are one of the important strategies for the future treatment of PD. This still requires further intensive research.

Disclosure of Invention

Compared with healthy people, the application proves that the expression of the LXN gene in the blood of a Parkinson disease patient is up-regulated, the LXN is suggested to be possibly used as a biomarker for diagnosing the Parkinson disease, in order to further research the correlation between the LXN gene and the Parkinson disease, an in vitro cell experiment is carried out, the influence of the inhibition of the expression of the LXN gene on the proliferation and apoptosis of the Parkinson cell model is observed by constructing the Parkinson cell model, and the result shows that the inhibition of the expression of the LXN gene reverses MPP + induced cell damage, promotes the cell proliferation and inhibits the cell apoptosis, which indicates that the LXN is a key diagnosis gene of the Parkinson disease and can be used as a diagnostic marker of the Parkinson disease. According to the research result, the protection content of the application is as follows:

according to one aspect of the present invention, there is provided a biomarker for diagnosing and treating parkinson's disease, the biomarker being an LXN gene or an expression product thereof.

According to another aspect of the invention, there is provided a reagent for detecting a biomarker as hereinbefore described.

Further, the reagent includes a reagent for detecting the level of mRNA of the LXN gene, or a reagent for detecting the level of the LXN protein.

Furthermore, the reagent for detecting the mRNA level of the LXN gene comprises a primer and/or a probe aiming at the mRNA of the LXN gene; reagents for detecting LXN protein levels include antibodies to LXN proteins.

According to a further aspect of the invention there is provided the use of a biomarker as hereinbefore described in the manufacture of a diagnostic tool for parkinson's disease.

According to a further aspect of the invention there is provided the use of an agent as hereinbefore described in the manufacture of a diagnostic tool for parkinson's disease.

Further, the tool comprises a chip, a kit, test paper, a high-throughput sequencing platform and a Parkinson disease diagnosis system.

Still further, the Parkinson's disease diagnosis system includes a diagnosis module that judges whether the subject has Parkinson's disease based on an expression level of the LXN gene or an expression product thereof of the subject.

According to a further aspect of the invention there is provided the use of a biomarker as hereinbefore described in the manufacture of a medicament for the treatment of Parkinson's disease.

Further, the medicament comprises an agent that inhibits expression of the biomarker.

Still further, the agents include, but are not limited to, siRNA, shRNA.

The medicament also comprises a pharmaceutically acceptable carrier. The effective components, the reagent for inhibiting the expression of the biomarkers and pharmaceutically acceptable carriers are prepared into the medicament by the conventional pharmaceutical technology.

According to a further aspect of the invention there is provided a method of treating parkinson's disease, said method comprising administering an agent which inhibits expression of a biomarker as hereinbefore described or administering a medicament as hereinbefore described comprising an agent which inhibits expression of a biomarker as hereinbefore described.

Drawings

FIG. 1 shows a statistical chart for detecting the mRNA expression level of LXN gene using QPCR;

FIG. 2 shows a statistical chart of LXN protein expression levels detected by immunoblotting;

FIG. 3 is a graph showing a statistic graph of the degree of inhibition of LXN gene expression using QPCR;

FIG. 4 shows a statistical graph of the activity of cells measured using MTT;

figure 5 shows a statistical plot of apoptosis detection using TUNEL.

Detailed Description

Nucleic acids and proteins

As used herein, the term "LXN gene" refers to a nucleic acid that encodes all or a portion of an LXN protein or is substantially identical to all or a portion of the nucleic acid sequence of NCBI gene ID:56925 or an analog thereof.

"LXN protein" refers to a protein that is homologous to all or part of NCBI accession numbers: the polypeptide sequence of NP-064554.3 or its analog is about the same, and has LXN biological activity.

By "about the same" is meant that the polypeptide or nucleic acid exhibits at least 75%, 85%, 90%, 95% or even 99% identity to the reference amino acid or nucleic acid sequence. For polypeptides, the aligned sequences are typically at least 35 amino acids, 45 amino acids, 55 amino acids, or even 70 amino acids in length. For nucleic acids, the aligned sequences are typically at least 60 nucleotides, 90 nucleotides, or even 120 nucleotides in length.

Sequence identity is typically determined using publicly available computer programs. Computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux et al, nucleic acids Research 12: 387, 1984), BLASTP, BLASTN, and FASTA (Altschul et al, J.mol.biol.215: 403, 1990). The well-known SmithWaterman algorithm can also be used to determine identity. BLAST programs are publicly available from NCBI and other sources (e.g., the BLAST handbook, Altschul et al, NCBI NLM NIH, Bethesda, MD 20894). These software programs match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions of amino acid alignments typically include substitutions in the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

As used herein, the term "portion" when referring to a protein (as in "a portion of a given protein") refers to a fragment of that protein. The fragments may vary in size from 4 amino acid residues to the entire amino acid sequence minus one amino acid (e.g., 4, 5, 6, n-1).

Detection of

The term "detecting the protein expression level" as used herein means detecting and identifying the presence and expression level of a diagnostic marker (protein) of parkinson's disease or a gene encoding the same in a biological sample. Examples of methods for detecting or comparatively analyzing proteins include, but are not limited to: protein chip assays, immunoassays, ligand binding assays, MALDI-T0F (matrix assisted laser desorption/ionization time-of-flight mass spectrometry), SELDI-T0F (surface enhanced laser desorption/ionization time-of-flight mass spectrometry), radioimmunoassays, radioimmunodiffusion, bidimensional immunodiffusion (Ouchterlony immunization), rocket immunoelectrophoresis, immunohistochemical staining, complement fixation assays, 2-D electrophoresis, liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), immunoblotting, and ELISA (enzyme-linked immunosorbent assay).

In the present disclosure, the reagent for detecting the expression level of a protein may comprise an antibody, an oligopeptide, a ligand, a PNA (peptide nucleic acid), or an aptamer that can specifically bind to the LXN protein.

The term "antibody" as used herein refers to a substance that specifically binds to an antigen to elicit an antigen-antibody reaction. For the purposes of this disclosure, the term "antibody" means an antibody that specifically binds to an LXN protein. Falling within the scope of the antibodies of the present disclosure are polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. These antibodies can be readily prepared using techniques well known in the art. Furthermore, antibodies useful in the present disclosure can be whole antibodies consisting of two full-length light chains and two full-length heavy chains, or functional fragments of a whole antibody molecule. The term "functional fragment" of an antibody molecule means a fragment that retains the binding function of the antibody, such as Fab, F (ab')2 and Fv.

The term "PNA (peptide nucleic acid)" as used herein refers to an artificially synthesized polymer similar to DNA or RNA, which was first introduced in 1991 by professor Nielsen, Egholm, Berg and Buchardt (university of Copenhagen, Denmark). DNA has a phosphoribosyl backbone, whereas the backbone of PNA is composed of repeating N- (2-aminoethyl) -glycine units linked by peptide bonds. Due to this structure, PNAs significantly enhance the affinity and stability of DNA or RNA, and thus are effectively used for molecular biological research, diagnosis, and antisense therapy. For a detailed description of PNA, reference is made to the literature [ Nielsen PE, EgholmM, BergRH, Buchardt0 (12 months 1991) ] "Sequence-selective catalysis of DNAbystranslational modification with catalysis-Substistent molecular Yamide". Science 254(5037): 1497-.

An "aptamer" as used herein is an oligonucleotide or peptide molecule that binds to a particular target molecule. For a detailed description of aptamers, reference may be made to BockLCetal, Nature355(6360):5646 (1992); Hoppe-Seyler F, ButzK "peptides: powerfulnewtol for molecular medicine". Jmol Med.78(8):42630 (2000); CohenBA, ColasP, BrentR. "Antifiticialcell-cycle inhibited random combinatorial approach. ProcNatlAcadSci USA.95(24):142727 (1998).

The term "detecting mRNA expression levels" as used herein means detecting and identifying the presence and expression levels of mRNA of a gene encoding a diagnostic marker (protein) in a biological sample. In the present disclosure, examples of analytical methods that can be used to detect mRNA expression levels include, but are not limited to, reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, Ribonuclease Protection Assay (RPA), northern blotting, and DNA chips.

In the present disclosure, the agent for detecting an mRNA expression level of a gene encoding LXN comprises a primer, a probe or an antisense nucleotide that specifically binds to mRNA of the gene encoding LXN. Information about LXN proteins is available through UniProt, and one skilled in the art can design primers, probes, or antisense nucleotides that specifically bind to the mRNA of the gene encoding the protein based on this information.

The term "primer" as used herein is a strand of short nucleic acid sequence that recognizes the target gene sequence, and includes a pair of forward and reverse primers. In particular, the "primers" include a pair of primers that provide an assay result of specificity and sensitivity. The primer is believed to provide a high degree of specificity when used to amplify a target gene sequence, but it does not cause amplification of non-target sequences that are not identical or complementary to the target gene sequence.

The term "probe" as used herein refers to a substance that specifically binds to a target to be detected in a sample. By this binding, the probe can determine the presence of the target in the sample. Any probe can be used in the present disclosure as long as it is generally used in the art. In particular, the probe may be a PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA, most preferably a PNA. In particular, the probe is a biological material, which may be derived from an organism or may be synthesized in vitro, or a mimetic thereof. For example, the probe may be an enzyme, protein, antibody, microorganism, animal or plant cell or organ, neuron, DNA or RNA. DNA may include cDNA, genomic DNA, and oligonucleotides. Likewise, genomic RNA, mRNA, and oligonucleotides may fall within the scope of RNA. Examples of proteins include antibodies, antigens, enzymes, and peptides.

The term "antisense" as used herein refers to an oligomer having a nucleotide base sequence and a subunit-subunit backbone that allows the antisense oligomer to hybridize to a target sequence in an RNA by Watson-Crick base pairing to form an RNA: oligomer heteroduplex nucleic acid molecule in the target sequence.

Reagent kit

Further, the present disclosure provides a kit for diagnosing parkinson's disease, comprising the composition for diagnosing parkinson's disease. For example, the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (multiple reaction monitoring) kit.

For example, the diagnostic kit may further comprise elements necessary for reverse transcription polymerase chain reaction. The RT-PCR kit contains a pair of primers specific for the gene encoding the marker protein. Each primer is a nucleotide having a sequence specific to the nucleic acid sequence of the gene, and may be about 7 to 50bp, more particularly about 10 to 39bp in length. In addition, the kit may further comprise a primer specific for the nucleic acid sequence of the control gene. In addition, the RT-PCR kit may comprise a test tube or suitable vessel, reaction buffers (different pH values and magnesium concentrations), deoxynucleotides (dntps), enzymes (e.g., Taq polymerase and reverse transcriptase), deoxyribonuclease inhibitors, ribonuclease inhibitors, DEPC-water, and sterile water.

In addition, the diagnostic kit of the present disclosure may contain elements necessary for the operation of the DNA chip. The DNA chip kit may comprise a substrate to which a gene or cDNA or an oligonucleotide corresponding to a fragment thereof is bound, and a reagent, a drug and an enzyme for constructing a fluorescently labeled probe. In addition, the substrate may comprise a control gene or cDNA or an oligonucleotide corresponding to a fragment thereof.

In some embodiments, the diagnostic kit of the present disclosure may comprise the elements necessary for performing an ELISA. The ELISA kit may comprise antibodies specific for the protein. The antibodies have high selectivity and affinity for marker proteins, are non-cross-reactive with other proteins, and may be monoclonal, polyclonal or recombinant. In addition, the ELISA kit may comprise an antibody specific for a control protein. In addition, the ELISA kit may further comprise reagents capable of detecting the bound antibody, e.g., a labeled secondary antibody, a chromophore, an enzyme (e.g., conjugated to an antibody), and substrates thereof or substances capable of binding the antibody.

The term "sample" used in connection with the detection refers to a biological sample that has a different level of protein or gene expression with the onset of Parkinson's disease, and examples of the sample include tissue, cells, serum, plasma, saliva, cerebrospinal fluid and urine, preferably blood, serum or plasma. In a particular embodiment of the invention, the sample is blood.

Medicine

The phrase "pharmaceutically acceptable carrier" is art-recognized and includes, for example, pharmaceutically acceptable materials, compositions or excipients, such as liquid or solid fillers, diluents, solvents or encapsulating materials, involved in carrying or transporting any subject composition from one organ or portion of the body to another organ or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the subject composition and not injurious to the patient. In certain embodiments, the pharmaceutically acceptable carrier is pyrogen-free. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered gum tragacanth; (5) malt; (6) gelatin; (7) talc powder; (8) cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) ringer's solution; (19) ethanol; (20) phosphate buffer; and (21) other non-toxic compatible materials used in pharmaceutical formulations.

"administration" refers to the method of administering a dose of a drug to a patient. The compositions used in the methods of the present invention may be administered by a route selected from, but not limited to: inhalation, ocular, parenteral, dermal, transdermal, buccal, rectal, sublingual, Perilingual (Perilingual), nasal, topical and oral administration. Parenteral administration includes intravenous, intraperitoneal, subcutaneous and intramuscular administration. The preferred method of administration may vary depending on factors such as the components of the composition being administered and the severity of the condition being treated.

The medicaments of the invention are prepared in a manner known to those skilled in the art, for example by means of conventional dissolving, lyophilizing, mixing, granulating or forming processes. Methods well known in the art for making formulations are shown, for example, in Remington: the Science and practice of Pharmacy, 20 th edition, eds A.R. Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology (Encyclopedia of Pharmaceutical Technology), J.Swarbrick and J.C. Boylan, 1988-1999, Marcel Dekker, New York.

The dosage of any agent used in the medicament of the present invention can be readily determined by one skilled in the art. Desirably, the dose of the agent in the medicament of the invention will be sufficient to alleviate the symptoms of Parkinson's disease in the patient. Alternatively, the dose will be sufficient to inhibit LXN in the cells of the patient.

Other terms

The term "diagnosing" as used herein is intended to encompass determining a subject's susceptibility to a disease or disorder, determining whether a subject has a disease or disorder, determining the prognosis or metric therapy (therametrics) of a subject having a disease or disorder (e.g., monitoring the status of a subject to provide information about the efficacy of a treatment). In particular, diagnosis as used herein means determining the onset or the likelihood (risk) of onset of parkinson's disease.

The term "treating" is well known in the art and includes preventing a disease, disorder or condition from occurring in an animal that may be predisposed to having the disease, disorder and/or condition but has not yet been diagnosed with the disease, disorder and/or condition; inhibiting a disease, disorder or condition, e.g., arresting its progression; and relieving the disease, disorder or condition, e.g., causing regression of the disease, disorder and/or condition. Treating a disease or condition includes ameliorating at least one symptom of a particular disease or condition, even if the underlying pathophysiology is not affected, such as treating a neurological condition such as multiple sclerosis and other diseases associated with oxidative stress such as kidney disease in a subject by administering an agent, although such agent does not treat the cause of the condition. The terms "treating", "treating" or "treatment" as used herein include curative therapy, prophylactic (e.g., disease-preventing) therapy, adjuvant therapy and palliative therapy.

The term "marker", "biomarker" or "diagnostic marker" as used herein means a label that allows for the differentiation of normal and disease states, or enables the outcome of a treatment to be predicted or objectively measured. In particular, in the context of association with parkinson's disease, a marker means an organic biomolecule, such as a polypeptide or nucleic acid (e.g., mRNA, etc.), a lipid, a glycolipid, a glycoprotein, a sugar (a monosaccharide, a disaccharide, an oligosaccharide, etc.), having a significantly increased or decreased level of protein or gene expression in a subject suffering from or at risk of developing parkinson's disease, as compared to a normal control (a subject not suffering from parkinson's disease).

As used herein, the terms "subject" and "patient" refer to any organism, including plants, microorganisms, and animals (e.g., mammals such as dogs, cats, livestock, and humans).

The term "siRNA" refers to short interfering RNA. In some embodiments, the siRNA comprises a duplex or double-stranded region, wherein each strand of the double-stranded region is about 18 to 25 nucleotides in length; the double-stranded region can be as short as 16 bases and as long as 29 base pairs in length, where the length is determined by the antisense strand. Typically, sirnas contain about 2 to 4 unpaired nucleotides at the 3' end of each strand. Sirnas appear to play a key intermediate in triggering RNA interference in invertebrates and vertebrates, and in triggering sequence-specific RNA degradation during post-transcriptional gene silencing in plants. At least one strand of the duplex or double-stranded region of the siRNA is substantially homologous or substantially complementary to the target RNA molecule. The strand complementary to the target RNA molecule is the "antisense" strand; the strand homologous to the target RNA molecule is the "sense" strand, and this strand is also complementary to the siRNA antisense strand. One strand in a double-stranded region need not have the exact length of the opposite strand, and thus, one strand may have at least one nucleotide less than the opposite complementary strand, resulting in the formation of a "bubble" or at least one unmatched base in the opposite strand. One strand of the double-stranded region need not be exactly complementary to the opposite strand; thus, the strand (preferably the sense strand) may have at least one mismatched base pair.

The siRNA may further comprise additional sequences; non-limiting examples of such sequences include a linker sequence, or loop, that connects the two strands of the duplex region. This form of siRNA may be referred to as "si-like RNA", "short hairpin siRNA" (where the duplex region of the siRNA is short) or "hairpin siRNA". Additional non-limiting examples of additional sequences present in the siRNA include stems and other folded structures. The additional sequences may or may not have a known function; non-limiting examples of such functions include increasing the stability of the siRNA molecule or providing a signal of cellular interest (cellular destination).

The present invention is further described with reference to the following embodiments, but the scope of the present invention is not limited thereto, and any person skilled in the art should be construed as the technical solutions and the inventive concepts equivalent to or modified within the scope of the present invention.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 correlation study of LXN Gene expression with Parkinson disease diagnosis

1. Study object

55 patients with primary PD, 30 men and 25 women, age 34-80 years, and course 6 months-20 years were collected. PD inclusion criteria: the diagnosis standards all accord with the PD clinical diagnosis standard (refer to the diagnosis standards of ' Jianyuping, Wangkang, Dingzheng, etc., primary Parkinson's disease, 2005, Chinese clinical neuroscience, 2006, 14:40 '). Exclusion criteria: (1) essential tremor; (2) secondary parkinsonism; (3) severe dementia, dysarthria; (4) patients suffering from other psychiatric disorders.

Normal group: 40 healthy volunteers of 34-80 years of age were selected, and 20 male and female were selected.

The difference between the two groups in age and gender has no statistical significance (P >0.10) and is comparable.

2. Extraction of total RNA from blood

(1) Homogenizing treatment (Homogenization)

Directly taking fresh blood (peripheral blood), adding erythrocyte lysate with 3 times volume, mixing uniformly, standing at room temperature for 10 minutes, and centrifuging at 10,000rpm for 1 minute. The supernatant was thoroughly aspirated and the leukocyte pellet was collected. 1ml of TRIzol was added to each 100-200. mu.l of the leukocyte pellet collected from the blood.

(2) Layering (Phase Separation)

a. After adding TRIzol, the sample was left at room temperature for 5min to allow the sample to be sufficiently lysed.

b. Adding 200 μ l chloroform into 1ml TRIzol, vigorously shaking, mixing, standing at room temperature for 3-5min, and naturally separating phases.

(3) RNA Precipitation (RNA Precipitation)

Centrifugation is carried out at 12,000rpm for 10-15min at 4 ℃. The sample will be divided into three layers: yellow organic phase, intermediate layer and colorless aqueous phase, RNA is mainly in the aqueous phase, and the aqueous phase (usually 550. mu.l can be pipetted) is transferred to a new tube.

b. The supernatant was added with an equal volume of ice-cold isopropanol and left at room temperature for 10-20 min. Centrifugation was carried out at 12,000rpm at 4 ℃ for 10min, the supernatant was discarded, and RNA was precipitated at the bottom of the tube.

(4) RNA rinsing (RNA Wash)

Add 1ml of 75% ethanol (prepared with RNase-free water) to the RNA pellet, gently shake the centrifuge tube and suspend the pellet. 1ml of 75% ethanol was added per 1ml of TRIzol.

b.4 ℃ 5,000 and 8,000rpm for 1-2min, and discarding the supernatant; and (4) performing short-time rapid centrifugation, carefully sucking and removing the supernatant by using a pipette, and placing the supernatant at room temperature for 1-2 minutes to dry and precipitate.

(5) Solubilizing RNA (redissolving the RNA)

Adding 50-100 μ l RNase-free water into the precipitate, flicking the tube wall to dissolve RNA sufficiently, and storing at-70 deg.C.

3. RNA quality and purity assays

RNA quality: as indicated by RNA integrity, integrity can be checked by normal agarose gel electrophoresis (electrophoresis conditions: 1.2% gel; 0.5 XTBE electrophoresis buffer; 150v, 15 min.).

Purity of RNA: the OD260/OD280 ratio is an index for the degree of protein contamination in the RNA sample. High quality RNA samples, OD260/OD280 (10mM Tris, pH7.5) around 2.0.

3. Reverse transcription

Mu.g of total RNA was reverse transcribed with reverse transcription buffer to synthesize cDNA. 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: DEPC water, 5 Xreverse transcription buffer, 10mmol/L dNTP, 0.1mmol/L DTT, 30. mu. mmol/L Oligo dT, 200U/. mu. L M-MLV, template RNA. Incubate at 42 ℃ for 1h, 72 ℃ for 10min, and centrifuge briefly.

4、QPCR

QPCR amplification primers were designed based on the coding sequences of LXN and GAPDH genes in Genbank and synthesized by Shanghai Bioengineering technology services, Inc. The specific primer sequences are as follows:

LXN gene:

the forward primer is 5'-GACTACACCATTCTACTTCAT-3' (SEQ ID NO. 1);

the reverse primer is 5'-GCAGACGGCTATTATGTT-3' (SEQ ID NO.2),

GAPDH gene:

the forward primer is 5'-TTTAACTCTGGTAAAGTGGATAT-3' (SEQ ID NO. 3);

the reverse primer was 5'-GGTGGAATCATATTGGAACA-3' (SEQ ID NO. 4).

The QPCR reaction was performed using 2.0. mu.l of cDNA. The amplification procedure was: 95 ℃ for 5min, (95 ℃ 15s, 54 ℃ 60s) 40 cycles. SYBR Green is used as a fluorescent marker, PCR reaction is carried out on a Light Cycler fluorescent real-time quantitative PCR instrument, and relative quantification is carried out by an delta CT method.

4. Western blot detection

Extracting the total cell protein. The total protein extracted was quantified using the BCA protein concentration kit. Mu.g of total protein was taken from each sample, and after electrophoresis for 1.5h on 12% SDS-PAGE, the membrane was transferred. 5% skimmed milk powder was blocked, primary antibody was incubated overnight at 4 ℃ and secondary antibody was incubated for 2h at 37 ℃. Performing electrochemiluminescence ECL luminescence liquid color development and exposing and developing by a gel imaging system; and (4) performing gray scale analysis, and measuring the gray scale value ratio of the relative expression of the target protein to GAPDH.

5. Results

(1) QPCR results

As shown in fig. 1, LXN gene mRNA levels were significantly increased in parkinson's disease patients compared to normal, with statistical significance for the difference (P < 0.05).

(2) Western blot results

As shown in fig. 2, LXN protein levels were significantly elevated in parkinson's disease patients compared to normal, with statistical significance for the difference (P < 0.05).

Example 2 inhibition of LXN Gene expression

1. Cell culture

SH-SY5Y cells were cultured in an incubator in DMEM high-glucose medium (GIBCO) containing 100U/ml penicillin, 100. mu.g/ml streptomycin and 10% fetal bovine serum under conditions of 37 ℃ and 5% CO 2. Cells were detached from the vial wall and passaged using 25% trypsin at a ratio of 1:2 to 1: 3. After the third generation, cells in good state are selected for freezing storage, and other passage cells enter a logarithmic phase to perform experiments.

2. Design and synthesis of siRNA for LXN

On the basis of the LXN mRNA sequence, on-line siRNA design software (http:// www.ambion.com) was used. According to the design principle provided by Ambion company, 1 pair of siRNA sequences of specific target LXN genes are screened out. The siRNA sequences were synthesized by Ribo Biotech, Inc., Guangzhou. The above sequences were verified by BLAST analysis to be non-homologous to other human coding sequences. A universal negative control sequence (siRNA-NC) was provided by Ruibo Biotech, Inc., Guangzhou.

siRNA-LXN：

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

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

3. cell transfection

Liposome Lipofectamine2000 was used as the transfection reagent. Experiment was divided into 2 groups: negative control group (siRNA-NC transfection), experimental group (siRNA-LXN transfection). SH-SY5Y cells in logarithmic growth phase are taken and inoculated on a 6-well cell culture plate. After 24h, the coverage rate of the cell culture plate is about 70-80%. The transfection protocol was performed according to Lipofectamine2000 instructions.

4. QPCR (quantitative polymerase chain reaction) experiment for detecting interference efficiency of siRNA-LXN (small interfering ribonucleic acid)

The procedure is as in example 1.

5. Results

The results are shown in fig. 3, the siRNA-LXN can significantly inhibit LXN expression, and the difference has statistical significance (P < 0.05).

Example 3 correlation study of LXN Gene expression with Parkinson's disease treatment

1. Preparation of MPP +

10mg of MPP + (sigma) was dissolved in 1ml of the cell culture solution to prepare a stock solution. 0.1ml of the stock solution was added to 0.9ml of a cell culture solution to prepare 3365.5. mu.M MPP + working solution.

2. Parkinson's disease cell model construction

Early experiments have proved that the SH-SY5Y cells are induced to be damaged for 24 hours by using the administration concentration of 4mM MPP +, the cell survival rate is about 60%, and therefore, a stable Parkinson disease cell model can be established by using the 4mM MPP +.

3. Interference on influence of LXN gene expression on MPP + -induced SH-SY5Y cell injury

The influence of interfering LXN gene expression on MPP + -induced SH-SY5Y cell damage was investigated by measuring cell survival rate and apoptosis rate.

3.1MTT assay

SH-SY5Y cells are adjusted to be at a proper concentration and inoculated into a 96-well plate, when the cell confluency reaches 70% -80%, siRNA is transfected according to the method of example 2, and MPP + is added for 24h after transfection for 48 h.

The experimental groups were as follows:

blank control group: not processing;

model group: adding MPP +;

negative control group: transfecting siRNA-NC, and adding MPP +;

treatment groups: transfecting siRNA-LXN, and adding MPP +;

then the MTT method is used for detecting the activity of the cells: dissolving MTT with PBS to a final concentration of 5mg/ml, discarding the solution in the wells, adding 100 μ l of culture medium, adding 10 μ l of MTT 5mg/ml per well, continuing culturing at 37 ℃ for 4h, discarding the solution in the wells, adding 100 μ l of DMSO per well, and incubating for 5-10min at room temperature by shaking. Reading the absorbance (OD) value of the background on a microplate reader at 570nm and 630nm double-wave removal, and determining the percentage of the OD value to the absorbance value of the control group as the percentage of cell activity. The percentage of cell activity was calculated as% OD value of experimental group/OD value of control group 100%.

3.2TUNEL experiment

SH-SY5Y cells are adjusted to be at a proper concentration and inoculated into a 12-well plate, when the cell confluency reaches 70% -80%, siRNA is transfected according to the method of example 2, and MPP + is added for 24h after transfection for 48 h.

The experimental groups were as follows:

negative control group: transfecting siRNA-NC, and adding MPP +;

treatment groups: transfecting siRNA-LXN, and adding MPP +;

then, apoptosis was measured using TUNEL apoptosis assay kit (color development method) (cat # C1091) from Byunnan, according to the protocol.

4. Results

4.1MTT assay

As shown in fig. 4, the LXN gene silencing in the treated group (survival rate of about 93%) resulted in decreased cell death rate compared to the model group (survival rate of about 64%), P <0.05, with statistical difference; the negative control group had a slightly decreased cell viability, P >0.05, and no statistical difference compared to the model group. The results show that the inhibition of the LXN gene can reverse the MPP + induced SH-SY5Y cell damage.

4.2TUNEL experiment

As shown in fig. 5, the LXN gene silencing in the treated group (about 6.1%) significantly reduced the percentage of TUNEL staining positive cells compared to the negative control group (about 24.4%), which was statistically significant. The inhibition of LXN gene expression is shown to inhibit the apoptosis of SH-SY5Y cells induced by MPP +.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

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Claims (10)

1. A biomarker for the diagnosis and treatment of parkinson's disease, wherein the biomarker is an LXN gene or an expression product thereof.

2. A reagent for detecting the biomarker of claim 1.

3. The agent according to claim 2, wherein the agent comprises an agent for detecting the level of mRNA of LXN gene or an agent for detecting the level of LXN protein.

4. The reagent according to claim 3, wherein the reagent for detecting the mRNA level of the LXN gene comprises a primer and/or a probe for the mRNA of the LXN gene; reagents for detecting LXN protein levels include antibodies to LXN proteins.

5. Use of the biomarker of claim 1 for the preparation of a diagnostic tool for parkinson's disease.

6. Use of the agent of any one of claims 2-4 in the manufacture of a diagnostic tool for parkinson's disease.

7. The use of claim 5 or 6, wherein the means comprises a chip, a kit, a dipstick, a high throughput sequencing platform, a Parkinson's disease diagnostic system.

8. The use of claim 7, wherein the Parkinson's disease diagnosis system comprises a diagnosis module which judges whether the subject suffers from Parkinson's disease or not according to the expression level of the LXN gene or the expression product thereof of the subject.

9. Detecting the application of the biomarker of claim 1 in preparing a medicament for treating Parkinson's disease.

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

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