CN104862310A - Schizophrenia biomarker, screening method and kit - Google Patents
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Abstract
The invention belongs to the biological technical field and relates to a schizophrenia biomarker, a screening method for the schizophrenia biomarker and a kit for detecting the content of the schizophrenia biomarker. The schizophrenia biomarker comprises miRAN-1273d, miRAN-1303, miRAN-3064-5p, miRAN-3131, miRAN-3687, miRAN-4428, miRAN-4725-3p and miRAN-5096. Meanwhile, the invention further provides a biomarker capable of predicting schizophrenia drug therapeutic effects, a screening method for the biomarker and a kit for detecting the content of the biomarker. The biomarker comprises miRAN-21-3p and miRAN-3916. The invention further discloses a kit for identifying schizophrenia, generalized anxiety disorder, depression and mental retardation. The biomarker, the screening method and the kit have the advantages that the schizophrenia biomarker and the schizophrenia drug therapeutic effect predicting biomarker are provided for the first time, high specificity and sensitivity are provided, and high diagnosis and reference values are provided.
Description
Technical field
The invention belongs to biological technical field, relate to schizophrenia biomarker, screening method and test kit.
Background technology
Schizophrenia (schizophrenia, SZ) be one of the most serious mental disorder, with the positive symptoms such as illusion, vain hope, psychomotor excitement and (or) with affective dullness, apathy, the negative symptoms such as Social Withdrawal, hypobulia for main clinical characteristics, its cognition to patient, emotion, behavior and social function cause serious harm.
At present, though have certain understanding to the cause of disease of SZ, as investigator relate to inherited genetic factors according to the result of technology to SZ Study of Etiology such as neuroimaging, genomics, protein science, immunohistochemical methods, socio-psychological factor, neural biochemical pathology say and brain pathology and brain structural changes theory etc.But sufficient understanding is still lacked to its pathologic process, which prevent the progress to SZ diagnosis, treatment, rehabilitation technique.Large quantity research display, inherited genetic factors plays a major role in SZ generation, prognosis, curative effect, recurrence, from the familial aggregation of SZ, the difference that patient's risk level of suffering from again of compatriot and same ovum, dizygotic twins are suffered from altogether is inferred, inherited genetic factors accounts for 65% ~ 85% of SZ pathogenic factors.Therefore, to the understanding of SZ genetic mechanism and regulation process thereof, significant to its pathologic process of announcement, to the Diagnosis and Treat of SZ, also there is good prospect.
There are some researches show, if schizophrenia patients can clarify a diagnosis in early days and be effectively treated in time, significantly can improve the psychology of patient, physiology, social function and working efficiency, and greatly reduce medical expense.But, schizoid diagnosis is still relies on the subjective evaluation of its clinical symptom at present, lack effective objective diagnosis " gold standard ", thus mistaken diagnosis clinically, control and happen occasionally by mistake.On the other hand, because prior unknown patient is to which kind of medicaments insensitive, have to clinically with " trial and error " administration, this not only affects the result for the treatment of of patient, and reduces its drug compliance, and causes huge medical resource waste.Therefore, be necessary to research and develop a kind of biomarker with Sensitivity and Specificity, improve schizoid diagnosis science, and its hypotype is distinguished, so that carry out drug intervention targetedly and carry out clinical science research.
MiRNA is a kind of micromolecular non-coding RNA, and length is for being approximately 22 bases.The generation of miRNA comprises two steps and shears: first endonuclear miR-96 gene is transcribed into pri-miRNA, is cut into miRNA precursor pre-miRNA by endonuclear enzyme DroshaRNase.Translocator in core is by pre-miRNA transporte to cells matter, then the another kind of enzyme Dicer in tenuigenin cuts into double-strand miRNA miRNA precursor, by partially or completely with 3' non-translational region (3'UTR) complementary pairing of said target mrna, stop its translation or make it degrade, thus the effect regulated after playing genetic transcription.About have the protein coding gene of 50% to be subject to the accuracy controlling of miRNA, multiple miRNA molecule can regulate the expression of a gene jointly, the expression of miRNA molecule also hundreds of, thousands of genes of controllable.Increasing research proves, in the growth of brain, neural generation, neuronal maturation and Synaptic formation process, miRNA plays crucial regulating and controlling effect.Further research shows, in schizoid generation, development, miRNA also plays an important role, and has dependency with the curative effect of antipsychotics.Therefore, some MicroRNA can become the biomarker of schizophrenia diagnosis and prediction antipsychotic drug result for the treatment of.
Summary of the invention
The technical problem to be solved in the present invention is: provide schizophrenia biomarker, screening method and test kit.
In a first aspect of the present invention, provide schizophrenia diagnosis biomarker, described marker comprises miRNA-1273d, miRNA-1303, miRNA-3064-5p, miRNA-3131, miRNA-3687, miRNA-4428, miRNA-4725-3p and miRNA-5096.
In a second aspect of the present invention, provide the screening method of above-mentioned schizophrenia diagnosis biomarker, comprise the following steps: schizophreniac and the normal healthy controls group of choosing equivalent are respectively several, blood sampling, therefrom extract total serum IgE, extract the total serum IgE that obtains through Ploly A tailing, then use biotin labeling further, RNA and the RNA chip hybridization marked also goes out the miRNA of differential expression by the examination of RNA chip; Choose some routine schizophreniacs, be set to case group; Choose some routine normal healthy controls persons, be set to control group; Blood sample collection respectively, reverse transcription and the fluorescence quantitative PCR detection of miRNA is carried out after extracting the total serum IgE in peripheral blood lymphocytes, the data obtained Using statistics method is analyzed, the differential expression miRNA obtained by the examination of RNA chip is verified, obtains the miRNA kind in significant difference between case group and control group.
In a third aspect of the present invention, provide a kind of schizophrenia diagnosis test kit, it can measure the content of miRNA-1273d, miRNA-1303, miRNA-3064-5p, miRNA-3131, miRNA-3687, miRNA-4428, miRNA-4725-3p and miRNA-5096 in blood.
Preferably, described schizophrenia diagnosis test kit contains the primed probe of miRNA-1273d, miRNA-1303, miRNA-3064-5p, miRNA-3131, miRNA-3687, miRNA-4428, miRNA-4725-3p and miRNA-5096, and described probe sequence is as shown in SEQ ID NO.1, SEQ ID NO.2, SEQID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ IDNO.8.
Described test kit can also comprise PCR and react common agents, as Taq enzyme, and reversed transcriptive enzyme, damping fluid, dNTPs, MgCl
2with DEPC water etc.; Standard substance and/or reference substance can also be contained.
In a fourth aspect of the present invention, additionally provide the purposes of the probe of sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ IDNO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, it is for the preparation of schizophrenia diagnosis test kit.
In a fifth aspect of the present invention, provide a kind of biomarker predicting Stability of Schizophrenia, described marker comprises miRNA-21-3p and miRNA-3916.
In a sixth aspect of the present invention, provide the screening method of the biomarker of above-mentioned prediction antipsychotic drug curative effect, comprise the following steps: choose some routine schizophreniacs and carry out clinical medicine intervention follow-up observation, clinical conventional antipsychotic drug is imposed respectively to the patient chosen, difference blood sample collection before and after medication, reverse transcription and the fluorescence quantitative PCR detection of miRNA is carried out after extracting the total serum IgE in peripheral blood lymphocytes, the data obtained Using statistics method is analyzed, obtain the miRNA that expression level before and after medication has significant difference.
In a seventh aspect of the present invention, provide a kind of biomarker detection kit predicting antipsychotic drug curative effect, it can measure the content of miRNA-21-3p and miRNA-3916 in blood.
Preferably, the biomarker detection kit of described prediction antipsychotic drug curative effect contains the primed probe of miRNA-21-3p and miRNA-3916, and described probe sequence is as shown in SEQ ID NO.9 and SEQID NO.10.
Described test kit can also comprise PCR and react common agents, as Taq enzyme, and reversed transcriptive enzyme, damping fluid, dNTPs, MgCl
2with DEPC water etc.; Standard substance and/or reference substance can also be contained.
In a eighth aspect of the present invention, additionally provide the purposes of the probe of sequence as shown in SEQ ID NO.9 and SEQ ID NO.10, it is for the preparation of the test kit of prediction antipsychotic drug curative effect.
In a ninth aspect of the present invention, provide the test kit differentiating schizophrenia and generalized anxiety disorder, dysthymia disorders, mental retardation, described test kit contains the primed probe of miRNA-26b, and described probe sequence is as shown in SEQ ID NO.11.
In a tenth aspect of the present invention, additionally provide the purposes of the probe of sequence as shown in SEQ ID NO.11, it is for the preparation of the test kit differentiating schizophrenia and generalized anxiety disorder, dysthymia disorders, mental retardation.
Described test kit can also comprise PCR and react common agents, as Taq enzyme, and reversed transcriptive enzyme, damping fluid, dNTPs, MgCl
2with DEPC water etc.; Standard substance and/or reference substance can also be contained.
Beneficial effect of the present invention is:
1, schizoid diagnosis will not rely on subjective experience to judge, but can pass through this objective indicator inspection of microRNA expression level, make a definite diagnosis, and improve accuracy rate of diagnosis, Cultivation process;
2, be expected to change specific miRNA expression level by miRNA stand-in and antagonist (modified oligonucleotide) and abnormal gene regulatory network and signal path normalizing can be made, and then treatment schizophrenia;
3, be expected to predict by detecting the curative effect of specific miRNA expression level to antipsychotic drug; 4, be expected to by detecting specific miRNA expression level and to lapse to schizophreniac and prognosis situation being predicted;
5, be expected to carry out immunotherapy targeted autoantibody by miRNA stand-in and antagonist to certain specific symptoms of specific schizophrenia.
Accompanying drawing explanation
Fig. 1 is 9 miRNA expression of results contrasts between schizophreniac and collator.
Embodiment
One, research object
1. case group: the patient that year May in August, 2012 to 2014 accepts for medical treatment continuously in PLA the 102nd hospital outpatient and Psychiatric nursing.Enter group standard: 1. meet Americanism medical diagnosis on disease and statistic handbook the 4th edition (DSM-IV) GAD standard; 2. starting patient or enter to organize first 3 months and do not take antipsychotics; 3. 15 ~ 80 years old age.Exclusion standard: 1. suffer from other mental disorderes; 2. the bodies such as cerebral trauma or nervous system disorders is suffered from; 3. excessive drinking or drug abuse history is had; 4. enter to organize in first 1 month and have blood transfusion history; 5. enter to organize in first 3 months and used modified electro-convulsive therapy person (MECT).Enter the routine patient of group 82 altogether, wherein the male sex 39 example, women 43 example.
2. control group: from PLA the 102nd corpsman,hospital, health examination personnel, except a routine sex is not mated, all the other mate one by one with case group age, sex and nationality, eliminate the error that the factors such as sex, age and nationality are brought to greatest extent.Inclusive criteria: the disease that is 1. a cup too low family history; 2. in nearly 1 month without significant wound event; 3. nothing blood transfusion history in nearly January.Enter group 43 example altogether, wherein the male sex 20 example, women 23 example.
Before conducting a research, first the Psychiatric department doctor and postgraduate that participate in this problem are giveed training.Adopt schizophrenia Positive and Negative Symptom Scale (PANSS), substantially measuring scale (GAS), Clinical Global Impression (CGI) before case enters group, pharmacological agent 3 weeks afterwards, pharmacological agent assesses after 6 weeks respectively.According to DSM-IV-TR, patient is diagnosed by two attending psychiatrists, if there is inconsistent situation, then determine diagnosis after asking a psychiatric department chief physician jointly to discuss.
Generalized case (name, sex, age, nationality, schooling, occupation, income level, marital status, drug habit history and mental disorder family history etc.) is recorded when entering to organize.Equally, Normal group generalized case is recorded.
This research obtains the Medical Ethics Committee approval of hospital of the Chinese People's Liberation Army the 102nd, and all experimenters or tested family members (guardian) all sign Informed Consent Form.
Two, research method
1. scale is used
Schizophrenia Positive and Negative Symptom Scale (PANSS): in 19 end of the centurys, first proposed by Hughlings-Jackson, arranges through descendant, formally delivers, now widespread use in 1987.PANSS is severity for evaluating dissimilar the symptoms of schizophrenia and relates to and standardized measuring scale, merge reorganization by BPRS and spiritual pathology measuring scale to form, have 33 entries, wherein positive symptom entry 7, negative symptoms entry 7, general spirit pathology scale 16, compound scale 3.
Measuring scale (GAS) substantially: measuring scale is the scale evaluating all kinds of psychiatric disorders substantially, is most widely used one in similar scale.This research uses U.S.'s Mental Health Research Institute (NIMH) Spitzer version of 1976, has 10 grades of evaluations point (1 ~ 10 point), evaluates according to patient clinical symptom.Total score is higher, illustrates that the state of an illness is lighter.Use the Chinese version of GAS scale to assess in this research, this version, through the check analysis of letter validity, reaches psychometrics standard.
Clinical Global Impression (CGI): Clinical Global Impression is designed by WHO, for the research of international mental disorder pilot investigation (IPSS), existing in order to evaluate clinical efficacy, be applicable to any psychiatry treatment and research object.Scale is divided into coincident with severity degree of condition (severity of illness, SI), curative effect general comment (globalimprovement, GI) and therapeutic index (efficacy index, EI) three subscales.Use the Chinese version of CGI scale to assess in this research, this version, through the check analysis of letter validity, reaches psychometrics standard.Disease severity is used to divide (severity of illness, SI) and curative effect overall score (globalimprovement, GI) to carry out statistical study in this research.
2. Data acquisition,
Generalized case (name, sex, age, nationality, schooling, occupation, income level, marital status, drug habit history and mental disorder family history etc.) is recorded when entering to organize.Use schizophrenia Positive and Negative Symptom Scale (PANSS) and Clinical Global Impression (CGI) before medication by 3 attending psychiatrists or doctor and medication more than 6 weeks is evaluated case group is tested.Before evaluation, all participation researchists carry out unifying training, unified approach etc., stdn rotating detection process.Wherein HAMD uses total score and Factor minute statistics, and CGI uses curative effect overall score (global improvement, GI) statistics.
3. drug intervention
In 82 routine cases, random selecting 16 example is carried out clinical medicine and is intervened follow-up observation.Daily olanzapine (dosage range 5mg-20mg), 'naolijing ' (dosage range 800mg-1600mg), Ziprasidone (dosage range 40mg-80mg) oral administration are adopted to it.
4. main agents and consumptive material
RNA extracts test kit: miRNeasy serum/plasma extracts test kit (German Kai Jie company, article No. NO.217184); To participate in the experiment outward agent: miRNeasy serum/plasma is joined outward (German Kai Jie company, article No. .NO.219610); Fluorescence quantification PCR primer: TaqMan MicroRNA Assays (American AB I company); The general mix reagent box II (American AB I company, article No. NO.121207) of quantitative fluorescent PCR reagent: TaqMan; Reverse Transcription box: TaqMan MicroRNA Reverse Transcription box (American AB I company, article No. NO.1302146R).
5. key instrument
Scanner (instrument is originated: Affymetrix, model: Scanner 3000); Hybrid heater (instrument is originated: Affymetrix, model: Hybridization Oven 640); Washing workstation (instrument is originated: Affymetrix, model: Fluidics Station 450); 2100 (instrument is originated: Agilent, model: G2939A); 2100 vibrators (originate: Agilent, model: 9600) by instrument; NanoDrop (Thermo, 2000); PCR instrument (ABI, 9700); Whizzer (eppendorf, 5418); Concentrating instrument (eppendorf, 5301); Whizzer (its woods Bel, LX-200); Whizzer-1 (its woods Bel, LX-300); Vibrator-1 (its woods Bel, GL-88B); Magnetic stirring apparatus (its woods Bel, GL-3250B); Metal bath-2 (rich day, HB-100); Metal bath-3 (rich day, CHB-100); Electro-heating standing-temperature cultivator (smart grand experimental installation, XMTD-8222); Refrigerator (Rongshida, BCD-265F); ABI9700 type PCR instrument (American AB I company); It beautiful CT14RD type table-type high-speed refrigerated centrifuge (Shanghai Tianmei Biochemistry Instrument Engineering Co., Ltd.); NanoDrop1000 ultramicron ultraviolet spectrophotometer (attached computer 1 overlaps) (Thermo company of the U.S.); ABI 7900HT FastRead-Time PCR System (attached computer 1 overlaps) (American AB I company); The miniature whirlpool mixed instrument of WH-2 (Shanghai Hu Xi analytical instrument Co., Ltd., Factory); JS-400A constant-temperature metal bath (Shanghai Peiqing Science Co., Ltd); DK-8D digital display thermostat water bath (Medical Instruments factory of Jintan City); The single one side clean work station of SW-CJ-IFD type (Su Jing treating plant company limited);-81 DEG C of Ultralow Temperature Freezers (SANYO GS company);
6. gene chip examination
With AffymetrixmiRNA 3.0 chip, to GAD patient 3 people, normal control 3 people is totally 6 pattern detection and analysis.Sample total serum IgE utilizes NanoDrop ND-2100 (Thermo Scientific) quantitatively and detects RNA integrity through Agilent 2100 (Agilent Technologies).After RNA quality inspection is qualified, the mark of sample, the hybridization of chip and wash-out are with reference to chip standard flow process.First, total serum IgE through Ploly A tailing, then uses biotin labeling further.The RNA marked and chip hybridization, utilize AffymetrixScanner 3000 (Affymetrix) scanning to obtain original image after washing and dyeing.Data analysis: raw data is imported Expression Console software (version 1.3.1, Affymetix), the result obtained after utilizing the method for RMA to carry out stdn, comprises original signal value, normalized signal value, detects situation and detailed annotation information.Before screening difference miRNA, first carry out probe filtration, each probe has at least an identified as samples to be designated as " Detected " to be then preserved for follow-up screening.To biology replicate analysis, then the significance of difference P value utilizing T to check to obtain and the fold differences Fold change value of normalized signal value are screened, and standard is Fold change value >=2.0 and P value≤0.05.The analysis repeated for not having biology, only utilization variance multiple Fold change value is screened, and standard is Fold change value >=2.0.
7. total serum IgE extracting and quantitative fluorescent PCR: the miRNA expressed 7 species diversity in chip screening results carries out follow-up PCR and verifies.All tested use EDTA anticoagulant tubes adopt ulnar vein blood 5ml, shake anticoagulant tube gently antithrombotics and blood are mixed after blood sampling, and all blood samples carry out RNA extraction in blood sampling in latter 2 hours.First placed by Ficoll-Paque PLUS liquid chamber temperature (15-20 DEG C), all centrifugal processes also should complete in room temperature.Get 2mlEDTA anticoagulation and equal-volume balance liquid fully to mix (cumulative volume 4ml) with transfer pipet (or pipettor) in 15ml centrifuge tube.Extract 3mlFicoll Paque PLUS solution in new 15ml centrifuge tube with needle tubing, use transfer pipet (or pipettor) slowly to drip ready sample (4ml) on laminated fluid level along tube wall gently, note keeping clearly liquid level.Room temperature (18-20 DEG C) is with the centrifugal 30-40 minute of 400 × g.Blood plasma and the thrombocyte of the superiors is drawn with new transfer pipet (or pipettor).Monokaryon lymphocyte is placed in cryopreservation tube, and-80 DEG C save backup.By laboratory technician's time recording every day refrigerator temperature.TRIzol (
uSA) method extracts the total serum IgE (comprising miRNA) in Blood Mononuclear lymphocyte, and concrete operations are carried out according to test kit specification sheets.Reverse transcription reaction is carried out according to RNA Reverse Transcriptase kit (TaqMan RNA Reverse Transcription box, American AB I company) specification sheets.Reaction cumulative volume is 15 μ L (total serum IgE 5 μ L, TaqManMicroRNA Assay 3 μ L, nuclease free water 4.16ul, RNase inhibitor 0.19 μ L, damping fluid 1.5 μ L, Multiscribe reversed transcriptive enzyme 1 μ L and dNTP0.15 μ L), (16 DEG C at different temperatures, 42 DEG C, 85 DEG C, 4 DEG C) carry out different duration (30mins, 30mins, 5mins, 10mins) reaction.Real-time fluorescence quantitative PCR carries out according to TaqMan test kit (TaqMan general mix reagent box II, American AB I company) specification sheets.PCR amplification system cumulative volume is 10 μ L, reaction totally 40 circulations.PCR is reacted Ct value and is measured by 7900 real-time fluorescence quantitative PCR instrument (American AB I company), each reaction repetition twice.Use SDS 2.4 and DataAssist v3.0 software to carry out digital independent and analysis, after weeding out defective sample, select RNU48 to be that internal reference carries out data normalization, case group and control group are carried out miRNA Differential expression analysis.
Real-time quantitative PCR part is completed by base, U.S. GOPATH company Changzhou.
8. statistical procedures
All data uses DataAssist v3.0, SPSS v17.0 and Graphpad Prism 5.01 (GraphpadSoftware Inc., San Diego, CA, USA) statistical software to carry out statistical study.Δ CT value is calculated with the difference of threshold value ring between miRNA and internal reference RNU48 (threshold cycle, Ct).Use Wilcoxon rank test respectively comparative analysis 10 kinds of miRNA case groups and control group whether variant.Calculate Δ Ct value with the difference of threshold value ring between miRNA and outer ginseng miRNA-39 (threshold cycle, Ct), represent the relative expression levels of miRNA with 2-Δ Ct; With the miRNA relative expression levels of control group before using Mann-Whitney U rank test difference comparative analysis medication, after medication; Z test is used to analyze the comparison of case group miRNA expression level before medication, after medication.With P ﹤ 0.05 (two-tailed) for difference has statistical significance.
Three, result of study
1, by gene chip examination, find that 33 kinds of ripe miRNA there are differences expression between case group and control group, wherein 32 kinds of rises, a kind of downward.In conjunction with domestic and foreign literature, choose wherein 10 miRNA and carry out subsequent quantitation PCR checking.In schizophrenia group PMBC the CT value of 10 kinds of miRNA all comparatively control group be low, point out its actual expression level all comparatively control group increase, 10 kinds of miRNA expression levels are consistent with the trend of chip screening results.Except miRNA-3916, all the other miRNA are significant difference between study group and control group; Concrete data are as shown in table 1:
The miRNA of table 1 case group differential expression
2, wilcoxon rank test is adopted to case group and control group 10 kinds of miRNA expression levels, result is as shown in table 2: compared with normal people, and in schizophreniac, the expression amount of miRNA-1273d, miRNA-1303, miRNA-3064-5p, miRNA-3131, miRNA-3687, miRNA-4428, miRNA-4725-3p and miRNA-5096 significantly raises (P < 0.05) in schizophreniac.The expression level of miRNA-3916 and miRNA-21-3p does not find significant difference (P=0.093,0.572) between case group and control group.The results are shown in Table 2:
The expression of table 2 miRNA index in SZ patient and normal people (Δ CT median)
Often kind of miRNA is separately as schizophrenia diagnosis biomarker, susceptibility and specific degree data as follows: (susceptibility is 0.645 to miRNA-1273d, specific degree is 0.710), (susceptibility is 0.668 to miRNA-1303, specific degree is 0.719), (susceptibility is 0.6121 to miRNA-21-3p, specific degree is 0.6980), (susceptibility is 0.607 to miRNA-3064-5p, specific degree is 0.677), (susceptibility is 0.711 to miRNA-3131, specific degree is 0.788), (susceptibility is 0.704 to miRNA-3687, specific degree is 0.739), (susceptibility is 0.764 to miRNA-4428, specific degree is 0.825), (susceptibility is 0.819 to miRNA-4725-3p, specific degree is 0.907), (susceptibility is 0.697 to miRNA-5096, specific degree is 0.731), when miRNA-1273d, miRNA-1303, miRNA-3064-5p, miRNA-3131, miRNA-3687, miRNA-4428, miRNA-4725-3p, miRNA-5096 are as schizophrenia diagnosis associating biomarker, its susceptibility is 0.893, specific degree is 0.946, there is susceptibility and the specific degree of height, be expected to the associating biomarker becoming schizophrenia diagnosis.
For diagnosing schizoid test kit to comprise primed probe shown as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ IDNO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, can be buied by Life Technologies company; The common agents that can have needed for corresponding round pcr can also be had, as: Taq enzyme, reversed transcriptive enzyme, damping fluid, dNTPs, MgCl
2with DEPC water etc., these are all well known to those skilled in the art; Standard substance and/or reference substance can also be contained in addition.
3, the ROC curve display of miRNA-26b in schizophrenia and mental retardation, the ROC area under curve of miRNA-26b is 0.765 (P=0.000), and threshold value is 0.455; Susceptibility is 0.78, and specific degree is 0.65.The ROC curve display of miR-26b in schizophrenia and generalized anxiety disorder, the ROC area under curve of miRNA-26b is 0.802 (P=0.000), and threshold value is 0.492; Susceptibility is 0.667, and specific degree is 0.825.The ROC curve display of miRNA-26b in schizophrenia and dysthymia disorders, the ROC area under curve of miRNA-26b is 0.629 (P=0.013), and threshold value is 0.212; Susceptibility is 0.537, and specific degree is 0.675.ROC area under curve is larger, shows that the diagnostic region calibration of miRNA to dysthymia disorders and anxiety disorder is larger.
4, compare with before medication, miRNA-21-3p, miRNA-3916 expression level is significantly lowered.The results are shown in Table 3:
Before and after the medication of table 3 case group, miRNA relative expression levels change is compared (Δ CT)
Often kind of miRNA separately as the biomarker of prediction antipsychotics curative effect, susceptibility and specific degree data as follows: under miRNA-21-3p line, area is 0.622, and susceptibility is 0.512, and specific degree is 0.598; Under miRNA-3916 line, area is 0.625, and susceptibility is 0.523, and specific degree is 0.645.
For predicting that the test kit of thymoleptic curative effect comprises the primed probe as shown in SEQ ID NO.9 and SEQ ID NO.10, can be buied by Life Technologies company; The common agents that can have needed for corresponding round pcr can also be had, as: Taq enzyme, reversed transcriptive enzyme, damping fluid, dNTPs, MgCl
2with DEPC water etc., these are all well known to those skilled in the art; Standard substance and/or reference substance can also be contained in addition.
All documents that the present invention mentions are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.
Claims (10)
1. schizophrenia diagnosis biomarker, described marker comprises miRNA-1273d, miRNA-1303, miRNA-3064-5p, miRNA-3131, miRNA-3687, miRNA-4428, miRNA-4725-3p and miRNA-5096.
2. the screening method of a schizophrenia diagnosis biomarker as claimed in claim 1, comprise the following steps: schizophreniac and the normal healthy controls group of choosing equivalent are respectively several, blood sampling, therefrom extract total serum IgE, the total serum IgE that extraction obtains is through Ploly A tailing, use biotin labeling further again, RNA and the RNA chip hybridization marked also goes out the miRNA of differential expression by the examination of RNA chip; Choose some routine schizophreniacs, be set to case group; Choose some routine normal healthy controls persons, be set to control group; Blood sample collection respectively, reverse transcription and the fluorescence quantitative PCR detection of miRNA is carried out after extracting the total serum IgE in peripheral blood lymphocytes, the data obtained Using statistics method is analyzed, the differential expression miRNA obtained by the examination of RNA chip is verified, obtains the miRNA kind in significant difference between case group and control group.
3. a schizophrenia diagnosis test kit, it contains the primed probe of miRNA-1273d, miRNA-1303, miRNA-3064-5p, miRNA-3131, miRNA-3687, miRNA-4428, miRNA-4725-3p and miRNA-5096, and described probe sequence is as shown in SEQ ID NO.1, SEQ ID NO.2, SEQID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ IDNO.8.
4. the primed probe of sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 is preparing the application in schizophrenia diagnosis test kit.
5. predict the biomarker of antipsychotic drug curative effect, described marker comprises miRNA-21-3p and miRNA-3916.
6. predict the screening method of the biomarker of antipsychotic drug curative effect as claimed in claim 5 for one kind, comprise the following steps: choose some routine schizophreniacs and carry out clinical medicine intervention follow-up observation, clinical conventional antipsychotic drug is imposed respectively to the patient chosen, difference blood sample collection before and after medication, reverse transcription and the fluorescence quantitative PCR detection of miRNA is carried out after extracting the total serum IgE in peripheral blood lymphocytes, the data obtained Using statistics method is analyzed, and obtains the miRNA that expression level before and after medication has significant difference.
7. predict a test kit for antipsychotic drug curative effect, it can measure the content of miRNA-21-3p and miRNA-3916 in blood.
8. test kit as claimed in claim 7, it is characterized in that it contains the primed probe of miRNA-21-3p and miRNA-3916, described probe sequence is as shown in SEQ ID NO.9 and SEQ ID NO.10.
9. differentiate the test kit of schizophrenia and generalized anxiety disorder, dysthymia disorders, mental retardation, described test kit contains the primed probe of miRNA-26b, and described probe sequence is as shown in SEQ ID NO.11.
10. the primed probe of sequence as shown in SEQ ID NO.11 differentiates the application in the test kit of schizophrenia and generalized anxiety disorder, dysthymia disorders, mental retardation in preparation.
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| CN106222243A (en) * | 2016-05-24 | 2016-12-14 | 张理义 | A kind of circRNA mark, test kit and gene chip for schizophrenia diagnosis |
| CN108148904A (en) * | 2018-02-13 | 2018-06-12 | 广州市番禺区中心医院(广州市番禺区人民医院、广州市番禺区心血管疾病研究所) | Applications of the ELP5 in the diagnosis, treatment and prognosis of depression |
| CN110241204A (en) * | 2019-07-05 | 2019-09-17 | 北京太东生物科技有限公司 | Application of the SOX11 as schizophrenia diagnosis marker |
| CN110317866A (en) * | 2019-05-07 | 2019-10-11 | 中国人民解放军联勤保障部队第九0四医院 | Schizophrenia disease mouse model hippocampus circRNA sequencing analysis and kit |
| CN110931129A (en) * | 2019-12-10 | 2020-03-27 | 上海市精神卫生中心（上海市心理咨询培训中心） | Painting and drawing computer analysis method for evaluating schizophrenia mental state |
| CN112813155A (en) * | 2021-01-20 | 2021-05-18 | 武汉大学 | DNA methylation marker for predicting therapeutic effect of antipsychotic drug, screening method and application |
| WO2022052678A1 (en) * | 2020-09-09 | 2022-03-17 | 上海交通大学 | Application of mirna marker in preparing product for therapeutic effect assessment of olanzapine in treating schizophrenia, and test kit |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106222243A (en) * | 2016-05-24 | 2016-12-14 | 张理义 | A kind of circRNA mark, test kit and gene chip for schizophrenia diagnosis |
| CN106222243B (en) * | 2016-05-24 | 2021-04-23 | 张理义 | circRNA marker, kit and gene chip for schizophrenia diagnosis |
| CN108148904A (en) * | 2018-02-13 | 2018-06-12 | 广州市番禺区中心医院(广州市番禺区人民医院、广州市番禺区心血管疾病研究所) | Applications of the ELP5 in the diagnosis, treatment and prognosis of depression |
| CN110317866A (en) * | 2019-05-07 | 2019-10-11 | 中国人民解放军联勤保障部队第九0四医院 | Schizophrenia disease mouse model hippocampus circRNA sequencing analysis and kit |
| CN110241204A (en) * | 2019-07-05 | 2019-09-17 | 北京太东生物科技有限公司 | Application of the SOX11 as schizophrenia diagnosis marker |
| CN110931129A (en) * | 2019-12-10 | 2020-03-27 | 上海市精神卫生中心（上海市心理咨询培训中心） | Painting and drawing computer analysis method for evaluating schizophrenia mental state |
| WO2022052678A1 (en) * | 2020-09-09 | 2022-03-17 | 上海交通大学 | Application of mirna marker in preparing product for therapeutic effect assessment of olanzapine in treating schizophrenia, and test kit |
| CN112813155A (en) * | 2021-01-20 | 2021-05-18 | 武汉大学 | DNA methylation marker for predicting therapeutic effect of antipsychotic drug, screening method and application |
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