CN103175973B - Application of galanin in preparation of female depression detection tool - Google Patents
Application of galanin in preparation of female depression detection tool Download PDFInfo
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- CN103175973B CN103175973B CN201310059245.7A CN201310059245A CN103175973B CN 103175973 B CN103175973 B CN 103175973B CN 201310059245 A CN201310059245 A CN 201310059245A CN 103175973 B CN103175973 B CN 103175973B
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses application of galanin in preparation of a female depression detection tool. According to the invention, a result shows that the concentration of galanin in female blood is positively associated with the concentration of the female depression by researching the correlation between the depression and the galanin in blood of lots of clinical depression patients. The invention further discloses a kit for detecting the female depression and a use method of the kit.
Description
Technical field
The present invention relates to the new opplication of a kind of biochemical in disease detection.Specifically, the present invention relates to detection method and the application of galanin in Depression in women detects of galanin in human plasma.
Background technology
Depression, also known as depressive disorder, is that what to be caused by a variety of causes take depression as mood disorder or the affective disorder of cardinal symptom, relates to the many factors such as heredity, biochemistry, psychology and society environment.Low for principal character with persistent mood, main manifestations is depressed, depression, is easy to cry, can with feeling oneself inferior, feeling guilty, and patient feels that energy is tired out, mental retardation, prospects are dim, pessimism etc.Depression belongs to the sick category of Mental, to be dispersed in Gu doctor nationality melancholia, demented, hysteria, lily disease, palpitation with fear, palpitation, to be insomnia, the disease such as up-rushing gas syndrome.Along with the development of society, increasing of stressor, the incidence of disease of depression is in the trend increased year by year.Research display, the lifetime prevalence of unipolar depression is more than 10%, and women is about 2 times [Moldin SO, Reich T, the Rice JP.Current perspectives on the genetics ofunipolar depression.Behav Genet1991 of the male sex; 21 (3): 211-242].
The incidence of disease of depression, recurrence rate, suicide disability rate are higher, are but faced with that recall rate is low, treatment rate is low and the feature such as cure rate is low.The World Health Organization (WHO) predicts, developing country will be become to the year two thousand twenty depression and be only second to cardiopathic second largest disease, become the most serious Disease Spectrum [Kessler RC, Berglund P, Demler O.The epidemiology of major depressive disorder:results from the nationalcomorbidity survey replication (NCS-R) .JAMA2003,89 (2): 3095].
Genetic research is thought, depression has certain genetic predisposition.Twin study shows, and the heredity grade of depression is at 40% ~ 50% [Torgersen S.Genetic factors in moderately severe and mildaffective disorders.Arch Gen Psychiatry1986; 43 (3): 222-226].Adoptee study also points out heredity in depression, have vital role [Wender PH, Kety SS, Rosenthal D, Schulsinger F, Ortmann J, Lunde I.Psychiatric disorders in the biological andadoptive families of adopted individuals with affective disorders.Arch GenPsychiatry1986; 43 (10): 923-929].Depression propositus first degree relative is compared with general population, and its relative risk is 2 ~ 3, and Recurrent depression and early a depressed relative risk are at least 4 ~ 5.But up to now, the hereditary pattern of depression is also very unclear.
Come from MAOI [Richelson E.Synaptic effects of antidepressants.JClin Psychopharmacol1996; 16 (3Suppl2): 1S-7S] and tricyclic antidepressant [Tran PV, Bymaster FP, McNamara RK, Potter WZ.Dual monoamine modulation for improvedtreatment of major depressive disorder.J Clin Psychopharmacol2003; 23 (1): 78-86] the monoamine neurotransmitter hypothesis being played antidepressant effects by increase synaptic cleft norepinephrine (norepinephrine) and five hydroxytryptamine (serotonin) concentration is the etiologic etiological main flow hypothesis of depression [Chopra K, Kumar B, Kuhad A.Pathobiological targets of depression.Expert Opin Ther Targets2011; 15 (4): 379-400].Hypothesis is thought, depression is caused by central norepinephrine and serotonin insufficiency of function or disappearance.A large amount of evidence shows: the mechanism of action of central serotonin and norepinephrine functional disturbance and depression and antidepressants is closely related.But the behind mechanism of mediator imbalance is fuzzy.
At present, still there are problems in this hypothesis.Most antidepressants, mainly for monoamine transporter target spot, increase norepinephrine and the serotonin level of synaptic cleft, play antidepressant effect.But, synaptic cleft neurotransmitter can be increased in the antidepressants short time, and antidepression curative effect will postpone 2 ~ 4 weeks display [Davis LL, Wisniewski SR, Howland RH.Does comorbid substance usedisorder impair recovery from major depression with SSRI treatment? An analysis ofthe STAR*D level one treatment outcomes.Drug Alcohol Depend2010; 107 (2-3): 161-170]; The total effective rate of antidepressants is only 60% ~ 80%, still have quite a few patient lack curative effect or due to toxicity abandon treat [Wong ML, Licinio J.From monoamines togenomic targets:a paradigm shift for drug discovery in depression.Nature ReviewsDrug Discovery2004; 3:136-151]; Be that the novel antidepressant of action target spot successfully researches and develops listing with epiphysin, further illustrate the complicacy of depression etiology and pathology, illustrate that current monoamine hypothesis can not annotate the pathomechanism of depression, impel researcher to inquire into its concrete pathogenesis further.Meanwhile, because depression is a kind of paroxysmal disease, there is certain self-healing tendency.This prompting, some endogenic protection factor [Zhang JM is there is at internal body, Tonelli L, Regenold WT.Effects of neonatal flutamidetreatment on hippocampal neurogenesis and synaptogenesis correlate withdepression-like behaviors in preadolescent male rats.Neuroscience2010; 169 (1): 544-554].For these endogenous protection factors seek perhaps can to understanding depression mechanism, to lapse to and treatment exerts far reaching influence.
Neuromodulator, especially relevant to mood disorder neuropeptide, causes the attention of researcher gradually.In brain, these neuropeptides are normal to coexist with classical neurotransmitter such as norepinephrine, serotonin, dopamine etc.These modified key characters are activated exactly under stress situation or neuron are in high activity state.They regulate the neuronic function of coexpression and activity by multiple receptor subtype.
Galanin all has and distributes comparatively widely in brain, participates in the adjustment of the multiple senior physiological function of human body, as cognition, affective behavior, neurotrosis etc.Serotonergic neurons and the galanin coexistence of transmitters of 95% is had, the noradrenergic neuron of locus coeruleus more than 80% and galanin coexistence of transmitters at nucleus raphes dorsalis; It is no matter the existence all having norepinephrine or serotonin mediator in all galanin energy positive neuron of locus coeruleus or nucleus raphes dorsalis.The acceptor of galanin all has at locus coeruleus and nucleus raphes dorsalis position expresses and has its Fiber Projections [Kehr J, Yoshitake T, Wang FH.Galanin is a potent in vivo modulator ofmesencephalic serotonergic neurotransmission.Neuropsychopharmacology2002; 27 (3): 341-356].Galanin and nucleus raphes dorsalis major part serotonergic neurons and TPH coexpression.Therefore, galanin systematic influence serotonin energy and noradrenergic neuron function.
Based on above-mentioned research, the present invention, mainly through galanin in patients with depression body and the relevance between sex hormone and depression, seeks the relation between neuropeptide and sex hormone associated hormone, and the detection for Depression in women provides new method.
Summary of the invention
The present invention, for detecting prevention and therapy depressive illness, have studied the relevance between neuropeptide and depression in patients with depression blood plasma, and result of study shows, and in blood plasma, galanin concentration and the Depression in women order of severity are correlativity.
The present invention needs to set sample collection standard according to research, and the successful acquisition blood sample of 700 patients with depression, has carried out statistics and the layering of the aspects such as sex, age, Degree of Depression to sample.Age, age of onset, body mass index, HAMD, HAMA and gender differences thereof there are no significant difference.
The present invention adopts the method for radio-immunity to measure the concentration of the neuropeptides such as galanin in sample blood plasma, statistical research finds that in overall sample, galanin concentration and the depression order of severity do not exist correlativity, related coefficient (r) between galanin and HAMD mark is: 0.233 (P=0.084), HAMD are Hamilton depressive scale.
After sex layering, female patient blood plasma galanin concentration and HAMD mark, there is correlativity between galanin and testosterone, correlation coefficient r is respectively: 0.324 (P=0.046), 0.421 (P=0.014), and male patient's blood plasma galanin concentration and HAMD mark without significant correlation, r=-0.68 (P=0.694).
The correlation analysis Late Cambrian of the present invention by marking to patients with depression blood plasma galanin, sex hormone associated hormone and HAMD, the plasma concentration of galanin marks into positive correlation with the HAMD of Depression in women patient, and related coefficient is 0.324 (P=0.046).We can be inferred by the plasma concentration measuring Depression in women patient galanin or be judged the seriousness of depressive symptom, and then provide biological index for the clinical assessment of depression.The present invention is by right
The invention also discloses a kind of kit detecting Depression in women, kit comprises galanin antibody, and described galanin antibody can pass through the mammals such as immune rat, mouse, rabbit, obtains galanin polyclonal antibody; Also the hybridoma technology by classics obtains galanin monoclonal antibody.
The invention discloses a kind of radioimmunological kit detecting Depression in women, comprise following component: containing Galanin antibody rat blood serum,
125i-Galanin (
125i mark Galanin), damping fluid, separating agent, normal rabbit serum, Galanin standard items are 5,10,20,40,80,160pg/mL; Described damping fluid is 200mMpH5.0 phosphate buffer; Described separating agent is rabbit Chinese People's Anti-Japanese Military and Political College mouse IgG serum.
The invention also discloses a kind of method detecting galanin, comprise the following steps:
1, making sample be placed in before measuring, room temperature or cold water are multiple melts, then 3000 revs/min centrifugal 5 minutes, gets supernatant and measure.
2, polystyrene tube numbering is got, T, NSB, S
0~ S
6with testing sample pipe etc.
3, be wherein numbered NSB pipe with damping fluid 300ul, be numbered S
0pipe is with damping fluid 200 μ l and the rat blood serum 100 μ l containing Galanin antibody; Be numbered S
1~ S
6pipe first adds 100 μ l damping fluids and the 100 μ l rat blood serum containing Galanin antibody, then add respectively 100 μ l concentration be 5,10,20,40,80, the Galanin standard items of 160pg/mL; Sample hose adds cleer and peaceful 100 μ l on 200ul sample to be tested and contains the rat blood serum of Galanin antibody; Abundant mixing after above-mentioned each pipe application of sample completes, in 4 DEG C, places 24 hours.
4, above-mentioned each pipe adds 100ul
125i-Galanin fully mixes, and in 4 DEG C, places 24 hours.
5, above-mentioned each pipe adds 100ul normal rabbit serum, and fully after mixing, room temperature leaves standstill 20 minutes.
6, except the test tube being labeled as T, other each pipes add 500ul rabbit Chinese People's Anti-Japanese Military and Political College mouse IgG serum, mixing, and room temperature places 20 minutes, 4 DEG C centrifugal 3500 revs/min, 25 minutes, Aspirate supernatant immediately, measure each pipe precipitation cpm number.
7, experimental result process is as follows:
(1) survey the cpm value of every part of mark QC and calculate average;
(2) each standard pipe is calculated by following formula, the B/130% of Quality Control and patient specimen
B/B
0=(B-NSB)/(B
0-NSB)×100%
B=is sample hose value cpm, B
0=standard pipe B
0cpm value; The cpm value of NSB=non-specific binding pipe;
(3) with each standard pipe B/130 for the longitudinal axis, reference material concentration (ng/ml) is transverse axis drawing standard curve on logit-log coordinate paper;
(4) on typical curve, find the galanin concentration of quality controlled serum and each sample.
The present invention, by said method, have detected the blood plasma galanin concentration of 376 women, and result display is when Depression in women depression in patients degree is comparatively serious, and its blood plasma galanin concentration is more than 34.54pg/ml; When Depression in women depression in patients degree is modest depression, its blood plasma galanin concentration is 29.73 ~ 34.54pg/ml; When Depression in women depression in patients degree is minor depressive, it is 23.55 ~ 29.73pg/ml that its blood plasma galanin concentration reaches; The blood plasma galanin concentration of normal person is less than 21.02pg/ml.
Embodiment
Embodiment 1 blood plasma galanin concentration and depression
One, research object and method
1, research object
The Capital University of Medical Sciences's attached Beijing Anding Hospital's outpatient service or patient 700 example of being in hospital, meet DSM-I V criteria for depression, and meet the patient of following standard, as research object.
Patients with depression enters group standard: (1) is in hospital or out-patient; (2) Han nationality; (3) 18 ~ 60 years old age; (4) DSM-IV criteria for depression is met; (5) sufferers themselves or its legal guardian sign Informed Consent Form; .
Patients with depression exclusion standard: (1) spiritedness Split disease, alcohol and pharmacological dependence medical history; (2) brain organic disease and endocrine system disease history is had; (3) gestational period and nursing women; (4) manic or hypomanic episode history is had; (5) electro-shock therapy person.
2, research method
2.1 clinical datas are collected
For above-mentioned research object all by 2 above doctor's confirmations of middle rank, and do SCID scanning, data collection comprises: generally comprise age, sex, nationality, schooling, occupation, previously health status, habits of smoking and alcohol drinking, family history etc., sign Informed Consent Form to meeting into set condition person.
2.2 depressed evaluation instrument and methods
The doctor in charge of clinical position and Measuring scale assessing experience is for many years had to enter group personnel and carry out to all the order of severity that the understanding of generalized case and mini-mental state examination, 17 Hamilton depressive scales (HAMD) and Hamilton anxiety scale (HAMA) evaluate depression in patients disease by Hospital psychiatric department 5.
2.3 collections of specimens and disposal route
To the patient met into set condition, extract ulnar vein blood 5ml (EDTA anticoagulant tube) on an empty stomach between morning 6:00 to 9:00, slow inverted several times, the separation of blood plasma adopt 3000 revs/min centrifugal 5 minutes, Aspirate supernatant ,-80 DEG C of preservations.
2.4 plasma biochemical indexes detect
Blood plasma galanin, neuropeptide tyrosine measure and adopt radioimmunological kit, and Song301Fang Mian research institute entrusts and measures.Plasma estrogens, androgen, progesterone, testosterone etc. adopt chemiluminescence immunoassay labelling method to measure.
2.4.1 the mensuration of galanin concentration
2.4.1.1 experiment material and instrument:
Galanin radioimmunological kit, high speed freezing centrifuge (Beckman company of the U.S.).Described galanin radioimmunological kit comprises following component: containing Galanin antibody rat blood serum,
125i-Galanin (
125i mark Galanin), damping fluid, separating agent, normal rabbit serum, Galanin standard items are 5,10,20,40,80,160pg/mL; Described damping fluid is 200mM pH5.0 phosphate buffer; Described separating agent is rabbit Chinese People's Anti-Japanese Military and Political College mouse IgG serum.
The principle that kit of the present invention detects galanin is: application competition mechanism principle, Galanin in standard items or sample and adding
125i-Galanin is common with the immune response of a certain amount of anti-competing property of Galanin antibody.
125in the binding capacity of I-Galanin and antibody and standard items or sample, the content of Galanin is certain funtcional relationship.After bound fraction (B) being separated with free fraction (F) with immune separation agent (PR), measuring the radioactive intensity of bound fraction, and calculate corresponding Percentage bound B/B
0.Map with corresponding Percentage bound with known standard Galanin content, the standard that obtains suppresses curve.The content of Galanin in the testing sample of corresponding Percentage bound is detected from typical curve.
2.4.1.2 experimental procedure:
Making sample be placed in before mensuration, room temperature or cold water are multiple melts, then 3000 revs/min centrifugal 5 minutes, gets supernatant and measure.Measure and adopt imbalance method, get polystyrene tube numbering, operate by table 1.
Table 1 radiommunoassay liquid feeding program
Note: unit is that μ L, X represent each mensuration thing, and X represents Galanin herein.
Calculate NSB, S0 in conjunction with percent with B/T, calculate standard and testing sample in conjunction with percent with B/BO, drawing standard curve on semi-logarithmic scale paper, and find sample value or directly read the concentration of sample galanin by automatic gamma counter.
2.4.2 the mensuration of neuropeptide tyrosine concentration
2.4.2.1 experiment material and instrument:
Neuropeptide tyrosine radioimmunological kit (ALPCO company of the U.S.), high speed freezing centrifuge (Beckman company of the U.S.).
2.4.2.2 experimental procedure:
NSB, S0 is calculated in conjunction with percent with B/T, calculate standard and testing sample in conjunction with percent with B/BO, by automatic gamma counter preprogramming, directly provide relevant parameters, typical curve and sample concentration, after drawing concentration, draw actual concentrations divided by cycles of concentration 4.
Making sample be placed in before mensuration, room temperature or cold water are multiple melts, then 3000 revs/min centrifugal 5 minutes, gets supernatant and measure.Measure and adopt imbalance method, get polystyrene tube numbering, operate by table 1.
2.4.3 estrogenic concentration determination
2.4.3.1 experiment material and instrument:
Human estrin ELISA detection kit (RD company of the U.S.), distilled water, liquid-transfering gun, oscillator
2.4.3.2 experimental procedure:
A) all for kit reagent is at room temperature fully mixed, avoid producing foam.
B) standard items are diluted to 400,200,100,50,25,12.5,0pg/ml, 50 × damping fluid is diluted to 1 ×.
C) standard items arrange 2 multiple holes, and sample arranges 10 multiple holes.Sample sample diluent is added 50ul in reacting hole by after 1: 1 dilution.
D) add dilution good after standard items 50ul in reacting hole, add testing sample 50ul in reacting hole.Add the biotin labeled antibody of 50ul immediately.Cover lamina membranacea, mixing of vibrating gently, 37 DEG C of incubations 1 hour.
E) get rid of liquid in hole, cleansing solution is filled it up with in every hole, vibrates 30 seconds, gets rid of cleansing solution, pat dry with thieving paper.Repeat this operation 3 times.
F) every hole adds the affine chain enzyme-HRP of 80ul, mixing of vibrating gently, 37 DEG C of incubations 30 minutes.
G) get rid of liquid in hole, cleansing solution is filled it up with in every hole, vibrates 30 seconds, gets rid of cleansing solution, pat dry with thieving paper.Repeat this operation 3 times.
H) every hole adds substrate A, each 50ul of B, mixing of vibrating gently, 37 DEG C of incubations 10 minutes.Avoid illumination.
I) take out ELISA Plate, add rapidly 50ul stop buffer, measure the OD value in each hole immediately at 450nm wavelength place.
J) calculate: with absorbance OD value for ordinate (Y), corresponding estrogen standard concentration is horizontal ordinate (X), do to obtain corresponding curve, the estrogen content of sample can converse corresponding concentration according to its OD value by typical curve.
2.5 data analysis
Adopt SPSS16.0 statistical analysis software.The gender differences of galanin concentration adopt t inspection; Depressive symptom seriousness and neuropeptide concentration, the depression order of severity and hormone concentration adopt correlation analysis.All investigation values represent with average ± standard deviation, and P < 0.05 has statistical significance for difference, and P < 0.01 has conspicuousness for difference.
Two, experimental result
1, demographic data
This part collects Beijing Anding Hospital patients with depression 700 example altogether, wherein women 376 example, the male sex 324 example.Patient age, age of onset, body mass index, HAMD, HAMA and gender gap's differnce table 2 thereof.Result display age, age of onset, body mass index, HAMD, HAMA and gender differences thereof there are no significant difference.
Table 2 patients with depression demographic data and gender differences thereof
Note: Values were mean ± SD; HAMD: Hamilton depressive scale; HAMA: Hamilton anxiety scale; Galanin: galanin; BMI: body mass index
2, the correlation analysis result display between the neuropeptide concentration of blood plasma level and HAMD, related coefficient (r) between galanin and HAMD mark is: 0.241 (P=0.084), there is not correlativity between (see table 3) i.e. galanin and HAMD mark.
After sex layering female patient blood plasma galanin concentration and HAMD mark, significant difference between galanin and testosterone, r is respectively: 0.324 (P=0.046), 0.421 (P=0.014) (see table 5), and male patient's blood plasma galanin concentration and HAMD mark without significant correlation, r=-0.68 (P=0.694) (see table 4).
Correlation analysis between table 3 patients with depression Plasma Biochemical mediator and HAMD
NPY: neuropeptide tyrosine; SP: sensory neuropeptide substance P; GAL: galanin; COR: cortin; TES: testosterone; PRL: prolactin; PGN: progesterone; E2: estrogen
*.Correlation is significant at the0.05level(2-tailed).
**.Correlation is significant at the0.01level(2-tailed).
Correlation analysis between the biochemical transmitters of table 4 male patient blood plasma level and HAMD mark
*.Correlation is significant at the0.05level(2-tailed).
**.Correlation is significant at the0.01level(2-tailed).
Correlation analysis between the horizontal biochemical transmitters of table 5 Depression in women patients blood plasma and HAMD
*.Correlation is significant at the0.05level(2-tailed).
**.Correlation is significant at the0.01level(2-tailed).
The correlation analysis Late Cambrian of the present invention by marking to a large amount of patients with depression blood plasma galanin, sex hormone associated hormone and HAMD, the plasma concentration of galanin marks into positive correlation with the HAMD of Depression in women patient, and related coefficient is 0.324 (P=0.046).This result points out gender gap's opposite sex of galanin function further from another aspect.
3, the content of galanin in its blood plasma of female patient of different Degree of Depression
By carrying out 17 Hamilton depressive scale (HAMD) marking evaluations to 376 female patients, and detect the content of galanin in its blood plasma, the results are shown in Table 6:
The concentration of table 6 Depression in women patients blood plasma galanin
The display of above experimental result when Depression in women patients blood plasma galanin concentration reach at least 34.54pg/ml time, its Degree of Depression is comparatively serious; When blood plasma galanin concentration reaches at 29.73 ~ 34.54pg/ml, its Degree of Depression is modest depression; When blood plasma galanin concentration reaches at 23.55 ~ 29.73pg/ml, it is minor depressive; The blood plasma galanin concentration of normal person is less than 21.02pg/ml.
We can be inferred by the plasma concentration measuring Depression in women patient galanin or be judged the seriousness of depressive symptom, and then provide biological index for the clinical assessment of depression.
Claims (6)
1. galanin detects the application in the kit of Depression in women in preparation, it is characterized in that, by the concentration detecting galanin in female subjects blood plasma, described kit judges whether experimenter suffers from depression and judge that experimenter suffers from the order of severity of depression.
2. application according to claim 1, is characterized in that, described kit comprises the antibody of Galanin.
3. application according to claim 1, is characterized in that, described kit comprises following component: containing Galanin antibody rat blood serum,
125i-Galanin, damping fluid, separating agent, normal rabbit serum, Galanin standard items; The concentration of described standard items is 5,10,20,40,80,160pg/mL; Described damping fluid is 200mM pH5.0 phosphate buffer; Described separating agent is rabbit Chinese People's Anti-Japanese Military and Political College mouse IgG serum.
4. according to the application in Claims 2 or 3 described in any one, it is characterized in that, described antibody is monoclonal antibody.
5. according to the application in Claims 2 or 3 described in any one, it is characterized in that, described antibody is polyclonal antibody.
6. application according to claim 4, described monoclonal antibody is prepared by the method for hybridoma.
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CN102105059A (en) * | 2008-05-27 | 2011-06-22 | 细胞内治疗公司 | Methods and compositions for sleep disorders and other disorders |
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