CN108148902A - Applications of the TFCP2L1 in the diagnosis, treatment and prognosis of depression - Google Patents

Abstract

The invention discloses TFCP2L1 as depression detection, treatment, the application of prognosis target spot；And it discloses a kind of depression detection kit and quantitatively detects the sequence of TFCP2L1.TFCP2L1 has obvious differential expression in depression crowd and non-depressed crowd, and the clinical assistant diagnosis technology currently used for depression is not perfect, therefore TFCP2L1 has the potential as the relevant biomarker of depression.

Description

Applications of the TFCP2L1 in the diagnosis, treatment and prognosis of depression

Technical field

The invention belongs to biotechnology, more particularly to TFCP2L1 answering in the diagnosis, treatment and prognosis of depression With.

Background technology

The core symptom of depression is depressed, interest declines, energy missing, has more than 3.5 hundred million in the world People suffers from depression.World Health Organization's prediction, the second that will occupy world's Disease Spectrum to the year two thousand twenty, depression.

Depression significant burden caused by society, reason have high incidence, morbidity rejuvenation, in chronic progression, damage Its social function of evil and cognitive ability and unnatural death, the ratio of committed suicide and attempted suicide is much in patients with depression Higher than the ratio in general population.Therefore it studies depression mechanism and the biological marker of its severity can be embodied Object, and it is to treat disease, the key factor of mitigation burden on society for the cause of disease intervene and treat in time.

About depression pathogenesis and treatment principle be still not clear at present, previously think inherent cause, biology because Element, psychosocial factor have a certain impact to depression, and later academia has carried out to be divided in neural biochemical, nerve It secretes, many-sided research such as neural plasticity, Electrophysiology and neuroimaging, it is proposed that Different types of etiopathogenises hypothesis, Ke Nengyou The endocrine axis of pass, the variation of brain area structure and function, variation of brain area loop and signal path etc. achieve corresponding Progress, but still can not explain the pathogenic factor of depression completely at present.Modern molecular science of heredity think many social environments or The factors such as itself experience can cause the variation of gene expression, can cause certain specific genes that epigenetic occurs and change, these Variation can cause the plasticity and dysfunction of neuron, and these Novel presentations can be handed down by heredity, i.e. epigenetic machine System, the research of pathogenesis and biomarker based on gene level gradually cause the concern of scholars.Therefore one kind is found The biomarker of depression is to having a very important significance.

Invention content

It is an object of the invention to disclose a kind of marker of new depression.

The technical solution used in the present invention is：

TFCP2L1 is detected as depression, is treated, the application of prognosis target spot.

A kind of depression detection kit, containing the reagent that detects TFCP2L1 genes can be quantified in kit.

Preferably, the primer sequence containing real time fluorescent quantitative detection TFCP2L1 genes in the kit.

Preferably, quantitatively the primer sequence of detection TFCP2L1 genes is：

F：5’-AGCCCCTTAGGCTGTCTGTC-3’(SEQ ID NO：3)；

R：5’-TTTAGCCCACCATCATTTGG-3’(SEQ ID NO：4).

Application of the reagent of quantitative TFCP2L1 in depression detection reagent is prepared.

The beneficial effects of the invention are as follows：TFCP2L1 disclosed in the present patent application is in depression crowd and non-depressed crowd In have obvious differential expression, the clinical assistant diagnosis technology currently used for depression is not perfect, thus should TFCP2L1 has the potential as the relevant biomarker of depression.

Specific embodiment

The present invention is further elaborated for specific examples below, but not as a limitation of the invention.

1 material of experimental example and processing：

The collection of sample

Fresh anticoagulation 1ml is taken, with whole blood and tissue homogenate dilution liquid 1:After 1 mixing, it is carefully added on the cell point of 2ml On the liquid level of chaotropic, with 1500-2000 revs/min of centrifugation (radius 15cm horizontal rotors) 15 minutes, at this time in centrifuge tube by up to Four layers of lower cell point.First layer；For plasma layer.The second layer；For cyclic annular milky lymphocyte.Third layer；For transparent separating liquid Layer.4th layer；For red blood cell layer, collect the second confluent monolayer cells and be put into 4-5 containing cell washing solution milliliters of test tube, abundant mixing Afterwards, it is centrifuged 10-30 minutes with 1500-2000 revs/min.Precipitation through washing 2 times up to required cell repeatedly.

2) preparation and assessment of sample

It is prepared by RNA sample

Every group takes 2ml samples, with 1mL TRIzol reagents and pipette by cell pressure-vaccum lysate several times, and suspension cell It is then TRIzol reagents to be added in the precipitation of centrifugation and pressure-vaccum with lytic cell, is added in TRIzol reagents repeatedly with pipette Before not wash cell in case increase mRNA degradation possibility.

In order to which nucleic acid-protein complex will be completely dissociated, 5min is placed in 15-30 DEG C the sample of TRIzol has been added；Often The chloroform of 0.2mL is added in the sample of the TRIzol reagents homogenate of 1mL, covers tightly pipe lid；Manually acutely after oscillation tube body 15s, 15- 30 DEG C of incubation 2-3min；12,000 × g centrifuges 15mim at 4 DEG C；Mixing liquid is classified into the red phenol chloroform of lower floor after centrifugation Phase, the colourless water phase on middle layer core upper strata；RNA is all distributed in water phase；The volume of water phase about makes to add in during homogenate TRIzol reagents 60%.

Water phase is transferred in new centrifuge tube；Water phase is mixed with isopropanol makes RNA precipitate therein (add in the amount of isopropanol Add the isopropanol of 0.5mL when adding in 1mL TRIzol reagents for each sample homogenization)；After mixing after 15-30 DEG C of incubation 10min, 10min is centrifuged in 4 DEG C of 12,000 × g；RNA precipitate will form gelatinous precipitate block in bottom of the tube and side wall.

Supernatant is removed, at least 75% ethyl alcohol 1mL is added in the sample per the homogenate of 1mL TRIzol reagents for cleaning RNA precipitate；After oscillation, 4 DEG C 7,500 × g centrifugations 5min.

Finally, in order not to reduce the solubility of RNA, endless white drying RNA precipitate 5-10min in air.Dissolve RNA When, the water for first adding in no RNA enzyme is blown and beaten several times repeatedly with rifle, then 55-60 DEG C of incubation 10min.The RNA solution of acquisition preserves In -70 DEG C.

Total serum IgE purifies and quality inspection

RNA amounts are assessed using NanoDrop ND-1000 and quality, absorbance measure at 260 and 280mn, A260/ The ratio of A280 needs access to 2.0 (between 1.8-2.1), and A260/A230 ratios are greater than 1.8.RNA integralities and Purity is assessed by standard denaturating gel electrophoresis.Specific experiment process is as follows.

Be separately added into microcentrifugal tube μ L of the RNA that re-dissolves≤85,10 × reaction buffer, 10 μ L, 5 μ L of Baseline-ZERO DNA enzymatics, the water without RNA enzyme to 100 μ L；37 DEG C of incubation 30min；350 μ L buffer solution RLT are added in, are mixed It is even；250 μ L ethyl alcohol (96-100%) are added in, mixing is beaten in suction；The sample of above-mentioned 700 μ L is added on 2mL collecting pipes The small-sized centrifugal columns of RNeasy in, carefully close the lid, centrifuge 15s on the centrifuge of >=8000 × g, discard fluid； 500 μ L buffer solutions RPE are added in RNeasy pillars, and (RPE buffer solutions are for the first time before use, by 4 times of volumes of addition described on bottle 96-100% ethyl alcohol is made into working solution), >=8000 × g that carefully closes the lid centrifugation 15s wash RNeasy films, discard stream Body；500 μ L buffer solution RPE are added in again, are closed the lid, and >=8000 × g centrifugation 2min wash RNeasy films；RNeasy columns It is placed in the collecting pipe of a new 2mL, old collecting pipe is removed with filtration method, centrifuges 1min at full speed；RNeasy columns are transferred to one In the centrifuge tube of new 1.5mL, draw the water without RNA enzyme in right amount and be added directly into RNeasy films；Lid upper tube cap, >=8000 × G centrifuges 1min, elution；Finally carry out that RNA is quantitative and quality control.

2 real-time fluorescence quantitative PCR of experimental example detects

Design of primers

The random TFCP2L1 for selecting verification carries out fluorescent quantitation verification.With 2^-ΔΔCtMethod calculates fold differences, passes through RT- PCR is detected.PCR primer sequence is as follows：

1 RT-PCR primer list of table

RT-PCR is verified

It is verified using RT-PCR.8 μ L systems are established, add in 2 × Master Mix, 5 μ L, forward and reverse primer each 0.5 μ L, 2 μ L of distilled water.8 μ L mixed liquors are added in the corresponding each hole of 384-PCR plates, add in corresponding 2 μ L cDNA, it is careful viscous Upper Sealing Film sealed membranes, and of short duration centrifugation mixing.Above-mentioned 384-PCR plates are placed in Realtime PCR instruments and are carried out PCR reacts.Response procedures are as follows：95 DEG C, 10min；40 PCR cycles (95 DEG C, 10s；60 DEG C, 60s collects fluorescence).In order to build The melting curve of vertical PCR product, after amplified reaction, by (95 DEG C, 10s；60 DEG C, 60s；95 DEG C, 15s)；It is and slow from 60 DEG C It is slow to be heated to 99 DEG C (instrument carries out-Ramp Rate as 0.05 DEG C/s automatically).Due to by RNA concentration quantitatives error and RNA reverses The influence of efficiency error etc. is recorded, its content of the cDNA of 2 μ L volumes of each sample is not fully identical, to correct this difference, I Use Gapdh (different sample room expression quantity substantially constants) as internal reference, in the value of sample testing gene divided by this sample The value of ginseng, the ratio finally obtained are the testing gene relative amount of sample.

Experimental example 3RT-PCR Analysis of test results

Choose Fanyu central hospital depression 105 (man 54, female 51) and physical examination normal population 132 (man 69, Female 63) data, it is analyzed, it is as a result as follows

The △ Ct values of 2 patients with depression of table and normal population

	Average Ct	ΔCt	2-ΔCt
				Depression crowd	29.92201067	11.806658	2.79E-04
Normal population	30.79637367	12.103317	2.27E-04

The result shows that relative to normal population, the gene expression amount of the TFCP2L1 of depression crowd has significant raising, Therefore, detection expression TFCP2L1 genes can be as the marker of auxiliary diagnosis depression.

SEQUENCE LISTING

<110>Panyu District of Guangzhou City central hospital

<120>Applications of the TFCP2L1 in the diagnosis, treatment and prognosis of depression

<130>

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 21

<212> DNA

<213>Artificial sequence

<400> 1

tgacttcaac agcgacaccc a 21

<210> 2

<211> 21

<212> DNA

<213>Artificial sequence

<400> 2

caccctgttg ctgtagccaa a 21

<210> 3

<211> 20

<212> DNA

<213>Artificial sequence

<400> 3

agccccttag gctgtctgtc 20

<210> 4

<211> 20

<212> DNA

<213>Artificial sequence

<400> 4

tttagcccac catcatttgg 20

Claims (5)

1.TFCP2L1 is detected as depression, is treated, the application of prognosis target spot.

2. a kind of depression detection kit, which is characterized in that detection TFCP2L1 genes can be quantified by containing in the kit Reagent.

3. depression detection kit according to claim 2, which is characterized in that determine in the kit containing real-time fluorescence The primer sequence of amount detection TFCP2L1 genes.

4. depression detection kit according to claim 3, which is characterized in that described quantitatively to detect TFCP2L1 genes Primer sequence be：

F：5’-AGCCCCTTAGGCTGTCTGTC-3’(SEQ ID NO：3)；

R：5’-TTTAGCCCACCATCATTTGG-3’(SEQ ID NO：4).

5. application of the reagent of quantitative TFCP2L1 in depression detection reagent is prepared.