CN108048564A - The new application of people's GLT8D1 genes - Google Patents
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Abstract
The invention discloses the new applications of people's GLT8D1 genes, i.e., apply people's GLT8D1 gene expression amount detection reagents in schizophrenia reagent for clinical diagnosis is prepared；And apply people's GLT8D1 gene low expressions in treatment of schizophrenia drug is screened, experimental result is shown：GLT8D1 has adjustment effect to the multiplication of neural stem cell, self-renewing and differentiation in vitro；After GLT8D1 Knockdowns, neurite lengths, number and the bifurcated number ratio control group of neuron significantly reduce；GLT8D1 genes strikes the low frequency for having substantially change miniature excitatory postsynaptic currents and the amplitude of miniature inhibitory postsynaptic current in neuron；This is that schizoid risk genes provide evidence for GLT8D1；It is schizoid potential risk gene the invention firstly discloses GLT8D1 genes, new biomarker is provided for schizoid clinical diagnosis.
Description
Technical field
The present invention relates to a kind of new applications of gene, the especially new application of people GLT8D1 genes.
Background technology
Schizophrenia is a kind of serious mental illness, and all one's life illness rate is about between 0.5 to 1% people.Mostly in person between twenty and fifty
(i.e. indifferently, slow or subacute onset clinically often shows as patient's positive symptom (i.e. vain hope and illusion), negative symptoms
Motivation is damaged, Social Withdrawal) and cognitive disorder (i.e. disturbance in thinking, working memory are damaged and perform function obstacle), it is related to perception
Many obstacles such as feel, thinking, emotion and behavior and cerebration it is uncoordinated.The general Clear consciousness of patient, intelligent base
This is normal.The course of disease is generally delayed, in recurrent exerbation, exacerbation or deterioration.Schizophrenia is one of most common mental illness, extremely
Rate height is died, long-term incidence is high.Schizophreniac's suicide risk is higher, and there are about 30% people's suicide attempts, and there are about 10% people is dead
In suicide.Therefore, schizophrenia is born especially big caused by government economy.Between only 1 year 2002, U.S. government spends
Expense on schizophrenia patients is about 62,700,000,000 dollars, and including direct cost, (such as outpatient service is hospitalized, drug and long-term care
Expense) and non-medical expense (such as law enforcement reduces workplace productivity and unemployment）.Although schizoid incidence
It is very high, it constitutes a serious threat to Global Health, but at present still without the effective therapeutic scheme that can cure this disease completely.It makes
The main reason for being stagnated into this treatment is that the schizoid cause of disease is unclear.Although at present to the understanding of its cause of disease still not
Clearly, but the undesirable element of inherent cause, the susceptible quality of individual mind and external social environment is to the occurrence and development of disease
Effect is known together by everybody.Either inherent cause, susceptible quality or external undesirable element may all pass through inherent biology
Factor collective effect and cause the generation of disease.Family, twins and adopt control case research in show schizophrenia
Disease has stronger Hereditary efficiency, and genetic force is about 0.8 or so.Existing result of study shows that schizophrenia is in nerve
The ratio highest that inherent cause in phrenoblabia accounts for, this shows that inherent cause rises mainly in schizoid pathogenesis
Effect.In order to illustrate schizoid hereditary basis, researchers have carried out substantial amounts of genetics research in different crowd.
Current many candidate genes are found by genetic linkage and association study, but so far, schizoid heredity
Power is still not very clear.
Although genome correlation research（GWAS）Huge success, but GWAS are achieved in schizoid research
It was found that most of schizophrenia risk variants be all located at non-coding region, without apparent follow-up function.Therefore, these are easy
How perception variation causes schizoid risk still largely not clear.In order to determine expression by spirit
The gene that the variation of Split disease risk influences, and latent effect of these genes in schizophrenic onset mechanism is inquired into, we
It is mutual including coming from extensive GWAS (PGC 2), brain eQTL, protein-protein by integrating the data from different levels
Effect（PPI）Genetic association, the coexpression pattern of schizophreniac and control group, spatial and temporal expression profile, differential gene
External functional study of relation and candidate gene of expression and human brain structure etc..Our comprehensive analysis shows GLT8D1 bases
Because may be schizoid risk genes.
The sugar chain of glycosyl transferase catalytic activation in vivo is connected to different acceptor molecules, such as albumen, nucleic acid, oligosaccharides
On fat, Substratspezifitaet of the glycosylated product with many biological functions and with height participates in work important in vivo
Property substance such as glycoprotein and glycolipid in sugar chain synthesis, effect is a corresponding active donor（Typically nucleoside diphosphate
NDP- sugar）Monosaccharide moieties be transferred to sugar, protein, lipid and nucleic acid etc., complete the glycosylation processing of the latter, and realize its phase
The biological function answered.8 structural domain 1 of GLT8D1, that is, glycosyl transferase, as a kind of than common glycosyl transferase of glycosyl transferase
Increasingly complex, C- end structures increase the protein that identification is not folded（Polypeptide）Structure.It is being present in the human body overwhelming majority just
Often tissue, including brain tissue, and GLT8D1 functions in brain tissue are unclear.
The content of the invention
It is an object of the invention to provide the new applications of people's GLT8D1 genes, i.e., detect people GLT8D1 gene expression amounts and try
Agent is applied in schizophrenia reagent for clinical diagnosis is prepared；People GLT8D1 genes are used as schizophrenia associated gene,
It is detected applied to schizophrenia, people's GLT8D1 gene expression amount detection reagents are to detect the examination of people's GLT8D1 gene low expression amounts
Agent, i.e., using people's GLT8D1 genes low expression as the schizoid mark of diagnosis.
Detection people GLT8D1 gene expression amounts can drawing using the mRNA of people GLT8D1 gene order designers GLT8D1
Object sequence, and pass through the level of the mRNA of real-time quantitative PCR method detection people GLT8D1；The primer sequence of the mRNA is：
SEQ ID NO:1 ︰ CGGAATGGAAACGACAGAAT；
SEQ ID NO:2 ︰ GCGGACATTCCACATAGGAT.
For the situation of schizophrenia correlation GLT8D1 low expressions, the present invention is another object is that low by people's GLT8D1 genes
Expression and Application is in treatment of schizophrenia drug is screened.
For the phenotype of GLT8D1 low expressions in schizophrenia patients, by the effect of neural stem cell in schizophrenia
Target spot is RNA interference effect targets.
The RNA interference effects target spot is selected from following nucleotide sequence：
SEQ ID NO:3 ︰ GATGATGATGTCATTGTACAA；
SEQ ID NO:4 ︰ ACACACTATGTGGGAAGGTAA.
The shRNA sequences for inhibiting GLT8D1 gene expressions are cloned into after slow virus carrier and obtain RNA interference slow virus,
As for screening the screening cell line for the treatment of of schizophrenia drug after RNA interference slow-virus infection neural stem cell；Expression
The sequence of shRNA includes the inverted repeats of two targeting GLT8D1 gene codes DNA, intermediate to be separated by a stem ring sequence；
Wherein, two inverted repeats are respectively the shRNA target sequences and its complementary series of GLT8D1 genes.
The sense strand sequence such as SEQ ID NO of the sequence of the expression shRNA:Shown in 5, antisense strand sequence such as SEQ
ID NO:Shown in 6；Or sense strand sequence such as SEQ ID NO:7 shown and antisense strand sequence such as SEQ ID NO:Shown in 8.
Forward oligo: GLT8D1 FO1（SEQ ID NO:5）
CCGGGATGATGATGTCATTGTACAACTCGAGTTGTACAATGACATCATCATCTTTTTG；
Reverse oligo: GLT8D1 RO1（SEQ ID NO:6）
AATTCAAAAAGATGATGATGTCATTGTACAACTCGAGTTGTACAATGACATCATCATC；
Or
Forward oligo: GLT8D1 FO2（SEQ ID NO:7）
CCGGACACACTATGTGGGAAGGTAACTCGAGTTACCTTCCCACATAGTGTGTTTTTTG；
Reverse oligo: GLT8D1 RO2（SEQ ID NO:8）
AATTCAAAAAACACACTATGTGGGAAGGTAACTCGAGTTACCTTCCCACATAGTGTGT；
We are by integrating the data from different levels, including coming from extensive GWAS (PGC 2), brain eQTL, protein-egg
The genetic association of white matter interaction (PPI), the coexpression pattern of schizophreniac and control group, spatial and temporal expression profile,
It is schizoid risk genes that relation of differential gene expression and human brain structure etc., which finds people's GLT8D1 genes,.Therefore, I
By ncbi database, find the sequence of the GLT8D1 of people, the nucleotide sequence of people's GLT8D1 genes is shown in base in genebank
Because accession number is ID：76485, Chromosome 14 shown in 31001347..31012441, and mRNA sequence is shown in NM_
029626.2, CDS region sequence is wherein shown in 604-1719.
The shRNAs for targeting GLT8D1 is transfected neural stem cell by us（NSCs）, observe its multiplication, self-renewing and divide
Change ability.We firstly evaluated the poor efficiency of striking of designed shRNA, real-time quantitative PCR and immunoblotting analysis the results show that
ShRNAs reduces GLT8D1mRNA expression and protein expression, and compared with the control, difference has conspicuousness (P<0.05).It is interesting that
The proliferation rate (BrdU positive NSCs quantity) of the neural stem cell of GLT8D1 Knockdowns is also significantly greater than control group, and right
The expression pattern of stem cell dryness related gene after GLT8D1 Knockdowns is analyzed, it is found that these marker gene exist
GLT8D1 strikes low rear consistent up-regulation；These are the result shows that GLT8D1 plays important adjusting work in the multiplication of NSCs and growth
With.
In order to verify GLT8D1 to neural stem cell differentiating effect, We conducted neural stem cell differentiating experiment,
GLT8D1 strikes low group compared with the control group, finds TUJ1（Mark differentiation early stage neuron）、MAP2（Mark mature neuron）、
GFAP（Spongiocyte label）And O4（Oligopoly structure marker）Positive cell and the adjusting nerve cord of key are thin
The expression of the mRNA of born of the same parents' differentiation, such as NeuroD1, GFAP, oligo1 and oligo2 are decreased obviously, and show neural stem cell
Differentiation capability is impaired.Further detection GLT8D1 strikes the index of correlation of low group of neuron and finds, the neural process number of neuron,
Length and bifurcated number ratio control group significantly reduce.These statistics indicate that, GLT8D1 is thin by regulating and controlling nerve cord in neurodevelopment
The multiplication of born of the same parents and differentiation play a significant role.Prove for the first time GLT8D1 by adjust the multiplication of neural stem cell, self-renewing and
Break up and play a positive role in nervous system.
And low GLT8D1 is struck in neuron and has substantially change the frequency of miniature excitatory postsynaptic currents (mEPSC) and micro-
The amplitude of type inhibitory postsynaptic current (mIPSC) prompts normal synaptic transmission to need the participation of GLT8D1, shows that GLT8D1 exists
Dendron grows and plays an important roll in cynapse transmittance process；It is schizoid potential risk base that GLT8D1 is determined for the first time
Cause.
In short, experimental result is shown：GLT8D1 has adjusting to the multiplication of neural stem cell, self-renewing and differentiation in vitro
Effect；After GLT8D1 Knockdowns, neurite lengths, number and the bifurcated number ratio control group of neuron significantly reduce；It demonstrate,proves for the first time
Bright GLT8D1 is played a positive role by adjusting multiplication, self-renewing and the differentiation of neural stem cell in nervous system.I
It has also been found that：GLT8D1's strikes the low frequency for having substantially change miniature excitatory postsynaptic currents (mEPSC) and micro- in neuron
The amplitude of type inhibitory postsynaptic current (mIPSC).Prompting normal synaptic transmission needs the participation of GLT8D1.Due to GLT8D1's
ESNPs is also related to cognitive function, and GLT8D1 may transfer to have an impact cognitive function by adjusting cynapse.These results
Show that GLT8D1 plays an important role in terms of adjusting the development of neural process and adjusting cynapse transmission, this is spirit point for GLT8D1
The risk genes for splitting disease provide evidence.It is schizoid potential risk base the invention firstly discloses GLT8D1 genes
Cause provides new biomarker for schizoid clinical diagnosis.
The present invention specifies the expression of GLT8D1 and schizoid correlation；And it establishes available for screening spirit point
The cell line of disease medicine is split, there is larger application value and prospect.
Description of the drawings
Fig. 1 behaviours GLT8D1 is substantially less than normal population in the expression of schizophrenia patients hippocampus；A figures are hippocampus, b
Figure is prefrontal cortex, and c figures are corpus straitum.
Fig. 2 is that GLT8D1 strikes the low steady identification schematic diagram for turning strain；A figures are real-time quantitative PCR as a result, b figures are albumen inspection
Survey result；
Fig. 3 behaviours GLT8D1 strikes low rear promotion nerve stem cell proliferation result schematic diagram；A figures are immunofluorescence dyeing as a result, b schemes
For the quantitative result of immunofluorescence in a figures；
Fig. 4 behaviours GLT8D1 strikes low rear promotion neural stem cell dryness correlation marker expression of results schematic diagram；Have detected Klf4
（a）, Sox2 (b), Nanog (c) and Nestin (d) expression；
Fig. 5 behaviours GLT8D1 strikes the low rear neural stem cell differentiating result schematic diagram of inhibition；A figures are Tuj1 (differentiation early stage neurons
Label) immunofluorescence results, b figures are the quantitative result of immunofluorescence in a figures；C figures are O4（Oligopoly structure label）
Immunofluorescence results, d figures are the quantitative result of immunofluorescence in c figures；E figures are GFAP（Astrocyte marker object）It is immune glimmering
Light is as a result, f figures are the quantitative result of immunofluorescence in e figures；
Fig. 6 behaviours GLT8D1 strikes the low rear neural stem cell differentiating correlation marker mRNA level in-site detects schematic diagram of inhibition；It has detected
Neurod1(a）、Gfap(b)、 Oligo1(c）With Oligo2 (d) mRNA level in-sites；
It is neural stem cell differentiating for neuron result schematic diagram that Fig. 7 behaviours GLT8D1 strikes low rear inhibition；A figures are MAP2 (differentiation evenings
Phase neuron marker) immunofluorescence results, b figures are the quantitative result of immunofluorescence in a figures.C figures are MAP2 immunofluorescence knots
Fruit enlarged drawing, d, e, f figures are respectively immunofluorescence neuron dendron quantity in c figures（d）, length（e）And bifurcated（f）Quantitative knot
Fruit；
Fig. 8 behaviours GLT8D1 strikes low rear influence postsynaptic currents result schematic diagram；A figures are miniature excitatory postsynaptic currents
(mEPSC) frequency and amplitude schematic diagram and quantitative result；B figures be miniature inhibitory postsynaptic current (mIPSC) frequency and
Amplitude schematic diagram and quantitative result；
Fig. 9 is the flow diagrams that finder's GLT8D1 genes are schizoid risk genes；
Figure 10 behaviour GLT8D1 primers are used to detect the expression of people GLT8D1.
Specific embodiment
The present invention is described in further detail below by embodiment, but protection scope of the present invention be not limited to it is described
Content, method, is conventional commercial unless otherwise specified using conventional method using reagent unless otherwise specified in embodiment
Reagent or the reagent using conventional method configuration.
Embodiment 1：In order to determine that the expression of gene variation may assign schizoid risk, we use pattra leaves
This statistical framework（sherlock）Systematically incorporate gene in up to the present maximum schizoid GWAS (PGC2)
Correlation signal, the data of brain expression quantitative trait loci (eQTL)；We determined that 10 potential schizophrenia susceptibilities
Gene, expression may cause schizophrenia.In order to further screen most probable candidate gene, We conducted comprehensive
Close analysis, including spatial and temporal expression profile analysis, protein-protein interaction (PPI) analysis, co expression analysis,
Differential expression analysis etc.；We demonstrate our hair using independent brain eQTL data and comprehensive analysis method (SMR)
It is existing；Idiographic flow is shown in Fig. 9, the result is shown in Figure 1 after comprehensive analysis, and people GLT8D1 is notable in the expression of schizophrenia patients hippocampus
Less than normal population.
Embodiment 2：The separation and culture of mouse neural stem cells
Neural stem cell is separated from 14 age in days tire murine brain of embryonic period, embryonic phase, the training in serum free growth medium (DMEM/F12)
It supports, epidermal growth factor (EGF) 20ng/mL, fibroblast growth factor (BFGF) 20ng/ is contained in serum free growth medium
ML, penicillin/Streptavidin 1%, 1XN2,1XB27 and 10 μ g/mL of heparin.
Embodiment 3：The steady foundation for turning to strike low cell line
The shRNA sequences for inhibiting GLT8D1 gene expressions are cloned into after slow virus carrier and obtain RNA interference slow virus, RNA is done
It disturbs after slow-virus infection neural stem cell as screening the screening cell line for the treatment of of schizophrenia drug；Express shRNA
Sequence include the inverted repeats of two targeting GLT8D1 gene codes DNA, it is intermediate to be separated by a stem ring sequence；Wherein,
Two inverted repeats are respectively the shRNA target sequences and its complementary series of GLT8D1 genes.
The sense strand sequence such as SEQ ID NO of the sequence of the expression shRNA:Shown in 5, antisense strand sequence such as SEQ
ID NO:Shown in 6；Or sense strand sequence such as SEQ ID NO:7 shown and antisense strand sequence such as SEQ ID NO:Shown in 8.
Forward oligo: GLT8D1 FO1（SEQ ID NO:5）
CCGGGATGATGATGTCATTGTACAACTCGAGTTGTACAATGACATCATCATCTTTTTG；
Reverse oligo: GLT8D1 RO1（SEQ ID NO:6）
AATTCAAAAAGATGATGATGTCATTGTACAACTCGAGTTGTACAATGACATCATCATC；
Or
Forward oligo: GLT8D1 FO2（SEQ ID NO:7）
CCGGACACACTATGTGGGAAGGTAACTCGAGTTACCTTCCCACATAGTGTGTTTTTTG；
Reverse oligo: GLT8D1 RO2（SEQ ID NO:8）
AATTCAAAAAACACACTATGTGGGAAGGTAACTCGAGTTACCTTCCCACATAGTGTGT；
We utilize pLKO.1 vector constructions for GLT8D1 RNA interferings (shRNAs), and have carried out sequence verification to it.Target
The length of sequence is 21 bp：GLT8D1-shRNA#1, GATGATGATGTCATTGTACAA；GLT8D1-shRNA#2,
ACACACTATGTGGGAAGGTAA；The shRNA sequences of control are：GCACTACCAGAGCTAACTCAG；It is transfected according to specification
Pack slow virus；After virus infection 72h, NSCs is handled with puromycin (1 μ g/mL), screens and stablizes expression instruction shRNA's
NSCs。
Embodiment 4：Poor efficiency is struck in real-time quantitative PCR detection
1st, cell total rna extracts
（1）When Neural Stem Cells ' Growth is in good condition, remove supernatant, PBS removes serum, adds in 1mL Trizol, stands 5 points
Clock ensures the abundant cell lysis of Trizol, cell is blown and beaten from culture dish, liquid is transferred in centrifuge tube, is blown repeatedly
It beats and is precipitated until without apparent bulk；It is stored at room temperature 5min；
（2）4 DEG C of centrifuges are opened in advance, 12,000g centrifugation 5min, supernatant is transferred in new 1.5mL centrifuge tubes；
（3）200 μ L chloroforms are added in, the vibration of whirlpool instrument centrifuges 15min in 4 DEG C of centrifuges with 12,000g, and hereafter liquid is divided into
Three layers；
（4）Draw upper strata aqueous phase（It is careful not to encounter intermediate protein layer）, it is transferred to the centrifuge tube of new RNAase free
In；
（5）Isometric isopropanol is added in, gently turns upside down 5 times, is stored at room temperature 10min；
（6）, there is white precipitate at this time in 12,000rpm rotating speeds, 4 DEG C of centrifugation 10min；
（7）Supernatant is abandoned, adds in 75% ethyl alcohol that 1mL is handled through DEPC, turn upside down washing precipitation for several times；7500g is centrifuged 5 minutes,
It abandons supernatant and retains precipitation；
（8）RNA dries 10min at room temperature, then adds in the water dissolution of no RNase；
（9）OD values are measured, to determine the concentration and quality of RNA, are subsequently placed in -80 DEG C of preservations.
2nd, reverse transcription reaction（Reverse Transcription, RT)
The RNA of said extracted takes 1 μ g to carry out reverse transcription with TAKARA kit, specific as follows：Genomic DNA reaction is removed by such as
Then lower ingredient is dispensed into each reaction tube in preparing reaction mixture on ice, is eventually adding RNA sample again；Soft mixing,
According to 42 DEG C of 2min（Or room temperature 5min）It is reacted；
；
Sample is placed on ice after reaction, and according to the form below prepares mixing Mix, then dispenses 10 μ L again into each reaction tube；
Reverse transcription reaction is carried out immediately after soft mixing：37 DEG C of 15 min, 85 DEG C of 5s, 4 DEG C.
3、 qPCR
Each sample sets three repeating holes, is prepared by following component：
,
Mixing more than component, is added to each hole of 96 orifice plates, and after sealer, centrifugation makes liquid accumulation to tube bottom；According to the following conditions into
Row PCR reacts, and thermal circulation parameters are as follows：50 DEG C, 2 min；95 DEG C, 2 min；95 DEG C, 10 min；95 DEG C, 15 s, 60 DEG C 1
Min, 40X.
The primer see the table below：
Wherein, GAPDH is as internal reference.
The result is shown in Fig. 2 a, Fig. 4, Fig. 6；The mRNA level in-site that GLT8D1 strikes GLT8D1 in low cell line significantly reduces（Fig. 2 a）,
Neural stem cell dryness correlation marker significantly rises（Fig. 4）, neural stem cell differentiating correlation marker significantly reduces（Figure
6）.
The mRNA level in-site of GLT8D1 is detected in the brain cell line of people source simultaneously, it was demonstrated that SEQ ID NO:1 and SEQ ID NO:2
Primer is effective；The result is shown in Figure 10.
Embodiment 5：Protein immunoblot（Western Blot）Detect GLT8D1 protein expression situations
1、WB（Western Blot）Detection
The extraction of 1.1 neural stem cell total proteins
According to particular experiment treated neural stem cell, supernatant discarding culture medium is washed 1 time with PBS；According to cell precipitation
Amount adds in corresponding cell pyrolysis liquid, and multigelation cracks 2 times；4 DEG C, 12000 rpm, 10 min of centrifugation, take supernatant to abandon precipitation and use
In subsequent experimental.
1.2 protein concentrations detect and denaturation treatment
Protein concentration detection kit be green skies BCA determination of protein concentration kit (enhanced), article No.：P0010S；Method
It is as follows：
Standard items are added to by 0,1,2,4,8,12,16,20 μ L in the standard sample wells of 96 orifice plates, standard dilutions is added to supply
To 20 μ L so that standard concentration is respectively 0,0.025,0.05,0.1,0.2,0.3,0.4,0.5 mg/mL；Add proper volume
Protein sample is into the sample well of 96 orifice plates；Each hole adds in 200 μ L BCA working solutions, 37 DEG C of placement 20min；It is surveyed with microplate reader
Determine the absorbance of A562 wavelength；The protein concentration of sample is calculated according to standard curve and the sample volume used.
Suitable protein sample is taken, 5 × sample-loading buffer is added in, is placed in 100 DEG C of xeothermic instrument of metal and boils 5 min；It takes
Go out postcooling, dispense, -80 DEG C preserve or directly carry out SDS-PAGE (polyacrylamide gel) electrophoresis.
1.3 SDS- polyacrylamide gel electrophoresises
（1）It cleans electrophoresis and prepares gel glass plate, drying is fixed on on glue shelf；As required, it is different to prepare 8%-12%
The separation gel of concentration adds in concentration glue by formula after glue fully solidifies, is inserted into sample cell comb；After gelling is solid, glue is taken out,
Extract comb, distilled water cleaning surface glue；Glass plate and gel are fixed on together on electrophoresis frame, the Tris- glycine of addition
Electrophoretic buffer makes electrophoretic buffer in about 0.5 cm of short glass plate；Protein content 30-50 μ g are added in per hole, first
Electrophoresis runs through concentration glue under 80V voltages, then with 120V electrophoresis；Transferring film frame is placed in transferring film liquid, black plastic plate face to
Under, white puts one block of foam-rubber cushion above black side, a filter paper is put above foam-rubber cushion upper；Gently gel from electrophoresis glass
It is put into after being taken off in glass plate on filter paper, PVDF films is put into above gel, then put a filter paper, one piece of sea is put above filter paper
Silk floss pad, two pieces of plastic plate fixations of black and white, is put into pre-cooled transferring film buffer solution；Under condition of ice bath, 83 V voltages
3 h of transferring film；Pvdf membrane is put into the TBST containing 5% skimmed milk power, is slowly shaken on shaking table, room temperature closing 2h；It adds in
With the diluted primary antibodies of TBST containing 5% skimmed milk power, the antibody for detecting protein expression is: Rabbit anti-GLT8D1
(1:500, Absin abs128289a) 4 DEG C slowly shake in be incubated overnight；TBST washes film 10min × 3 time；Add in HRP
The secondary antibody of mark（Anti-rabbit 1:2000 dilutions）, it is incubated at room temperature 2h；TBST washes 10 min × 3 time of film；Add under the conditions of being protected from light
Enter ECL reagents, it is developed, taking pictures after in PVDF film transfers to luminescent screen.
The result is shown in Fig. 2 b, the expression of GLT8D1 albumen in low cell line is struck；Cell lysate detect respectively GLT8D1 and
The expression of GAPDH, GAPDH is as control；Experimental result shown compared with control cell, is struck and low is surely turned GLT8D1 eggs in cell
White expression is substantially lowered.
Embodiment 6：BrdU incorporation experiments
When stablizing the Neural Stem Cells ' Growth for striking drop GLT8D1 to 80% coverage, removing supernatant, PBS removes serum, and 0.05%
Pancreatin 1mL digests 5 minutes, and culture medium terminates, and blows and beats into single cell suspension, is counted with Countstar cell counters,
According to every 500 μ L 5 × 10 of hole4Cell total amount, plant respectively in 8 orifice plates, be placed in 37 DEG C, 5% CO2In CO2gas incubator
Overnight, 24 it is small when after, add in BrdU（10 μM of final concentration）It is incubated, is taken out after 20 min from incubator in the incubator, go to train
Base is supported, PBS is rinsed once；Add 200 μ L, 4% paraformaldehydes（PFA）Fixed, after twenty minutes, PBS is washed once, adds in 200 μ L 2N
HCL-0.5%TrionX-100, incubation at room temperature add in 1M NaHCO after 30 minutes3Rinse once, then discards, and adds in 200 μ L
+ 0.1% tween 20 of PBS are rinsed twice, and 200 μ L, 10% Normal Goat Serums are added in after removing residual liquid（NGS uses PBS
+ 0.1% tween 20 dilutes）, when room temperature shaker 1 is small, by BrdU primary antibodies 20+NGS (final concentrations of PBS+0.1% tween
5%) (antibody concentration is 1 according to antibody specification for dilution:1000) 200 μ L, are added in per hole, 4 DEG C of overnight incubations add in 200 per hole
μ L PBS+0.1% tween 20 are washed three times, 10 minutes every time, then with primary antibody diluted secondary antibody CY3-goat anti
Mouse (kind is according to primary antibody), according to 1:500 ratio and DAPI is according to 1:100 ratio adds in together, room temperature shaker 2
Hour, 200 μ L PBS+0.1% tween 20 are added in per hole and are washed three times, 10 minutes every time, are placed in PBS+NaN3In, 4 DEG C of preservations,
Fluorescence microscope is taken pictures and is counted.It is examined with Image J softwares and t, is carried out so that double-blind fashion is horizontal to the multiplication of cell
Quantitative analysis. ***p<0.001
The result shows that the incorporation that the GLT8D1 surely turned strikes BrdU in low cell line substantially increases compared with control group（See Fig. 3 a, b）.
Embodiment 7：GLT8D1 strikes low rear neural stem cell spontaneous differentiation experiment
Using differential medium i.e. containing 1% penicillin/Streptavidin, addition 1XN-2 (Gibco）, 1XB27 (Gibco) and 10 μ
Cell is fixed after when the DMEM/F12 cultures 72 of g/mL heparin (10 μ g/mL) are small.Immunocytochemical stain is as previously described.
With 30 min of PBS closing cell containing 5% part of Normal Goat Serum and 0.1% Tween-20, respectively with Tuj 1 (1：1000,
Sigma, T 8578), GFAP (1：1000、DAKO、Z 0334)、O4(1：1000, R＆D system, MAB1326), MAP2
(1：1000, Millibus, AB 5622) it is incubated jointly.With sheep anti-mouse antibody or goat-anti rabbit Cy3 antibody incubations (1：500,
Abclonal), then with fluorecyte core dyestuff 4 ', bis- meter promises -2 ' of 6--phenyl ring indoles dihydrochloride (DAPI, Sigma, D
3571) dye.With ImageJ softwares to Tuj1, GFAP, O4, MAP2 cell numbers quantified, with t inspection carry out statistics credit
Analysis.With 6.0 analysis measurement dendron length of Image-pro plus, quantity and dendron complexity（Bifurcated）.It measures respectively pair
According to 60 neurons of group and experimental group；All experiments are independent experiment three times, and every group at least repeats three times.Data are with flat
± SEM is represented.When P values are less than 0.05, difference is statistically significant.*p<0.05, **p<0.01,***p<0.001,t-
test；
It was found from experimental result, GLT8D1 strikes low stable cell strain, neural stem cell differentiating ability and cellular control unit phase
Than significantly reducing.Either break up toward neuron direction（Fig. 5 a, b；Fig. 7 a, b）Or toward spongiocyte differentiation (Fig. 5 e, f) or
It is to break up toward oligopoly structure（Fig. 5 c, d）It is all fewer than cellular control unit.And the neuronal cell that differentiation obtains, neural process
Quantity（Fig. 7 c, d）, length (Fig. 7 c, e) and complexity (Fig. 7 c, f) are also significantly lower than cellular control unit.
Embodiment 8：
We record the electro physiology of neuron, and primary hippocampal neurons are from newborn mice in 0-24h.Use phosphoric acid
Calcium method transfects DIV (cultured in vitro) 10 generation cell, every diameter 8mm in 48 orifice plates, 0.5 μ g plasmids (GFP-pLKO.1 shRNA)
With 0.992 μ l 2M CaCl2Mixing, by the DNA/CaCl of premixing2Solution is added in the 8 μ l of 2 × HBS.By DNA/
CaCl2/ HBS mixed liquors are incubated at room temperature 30 min, add in the hippocampal neuron of culture, then continue in the incubator
It is incubated 30 minutes (5%CO2, 37 DEG C).Before electrophysiological recording, with containing 10 mM MgCl2Neuronal cultured solution rinse 15 min,
Culture 4 days.The record of whole-cell patch-clamp is as previously described.With containing 145 mM KCl, 1 mM MgCl2、5 mM NaCl、5
mM EGTA、0.3 mM Na2GTP, 4 mM MgATP, the solution filling pipette of 5 mM QX-314 and 10 mM HEPES.God
Through member containing 4 mM KCl, 150 mM NaCl, 2 mM CaCl2、1 mM MgCl2, 10 mM HEPES and 10 mM glucose
Solution in maintain.Using Multiclamp 700 B and pCLAMP 10.0 (Molecular Devices, Sunnyvale,
CA, USA) signal is amplified and gathered.Using Clampfit 9.02 (Molecular Devices, Sunnyvale,
CA, USA), 5 (GraphPad of Igor 4.0 (WaveMetrics, Portland, US) and GraphPad Prism
Software, La Jolla, CA, USA) data are analyzed.*p<0.05, **p<0.01,***p<0.001,t-test.
The result is shown in Fig. 8, as we know from the figure GLT8D1 strike it is low after, the frequency of miniature excitatory postsynaptic currents (mEPSC) and
The amplitude of miniature inhibitory postsynaptic current (mIPSC) has notable rise compared with the control group.
Sequence table
<110>Kunming Institute of Zoology, Chinese Academy of Sciences
<120>The new application of people's GLT8D1 genes
<160> 28
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial)
<400> 1
cggaatggaa acgacagaat 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Artificial)
<400> 2
gcggacattc cacataggat 20
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence (Artificial)
<400> 3
gatgatgatg tcattgtaca a 21
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence (Artificial)
<400> 4
acacactatg tgggaaggta a 21
<210> 5
<211> 58
<212> DNA
<213>Artificial sequence (Artificial)
<400> 5
ccgggatgat gatgtcattg tacaactcga gttgtacaat gacatcatca tctttttg 58
<210> 6
<211> 58
<212> DNA
<213>Artificial sequence (Artificial)
<400> 6
aattcaaaaa gatgatgatg tcattgtaca actcgagttg tacaatgaca tcatcatc 58
<210> 7
<211> 58
<212> DNA
<213>Artificial sequence (Artificial)
<400> 7
ccggacacac tatgtgggaa ggtaactcga gttaccttcc cacatagtgt gttttttg 58
<210> 8
<211> 58
<212> DNA
<213>Artificial sequence (Artificial)
<400> 8
aattcaaaaa acacactatg tgggaaggta actcgagtta ccttcccaca tagtgtgt 58
<210> 9
<211> 19
<212> DNA
<213>Artificial sequence (Artificial)
<400> 9
cacagatgca accgatgca 19
<210> 10
<211> 18
<212> DNA
<213>Artificial sequence (Artificial)
<400> 10
ggtgccctgc tgcgagta 18
<210> 11
<211> 22
<212> DNA
<213>Artificial sequence (Artificial)
<400> 11
cacacaggcg agaaacctta cc 22
<210> 12
<211> 16
<212> DNA
<213>Artificial sequence (Artificial)
<400> 12
cggagcgggc gaattt 16
<210> 13
<211> 20
<212> DNA
<213>Artificial sequence (Artificial)
<400> 13
aggatgaagt gcaagcggtg 20
<210> 14
<211> 21
<212> DNA
<213>Artificial sequence (Artificial)
<400> 14
tgctgagccc ttctgaatca g 21
<210> 15
<211> 20
<212> DNA
<213>Artificial sequence (Artificial)
<400> 15
ccagagctgg actggaactc 20
<210> 16
<211> 20
<212> DNA
<213>Artificial sequence (Artificial)
<400> 16
acctgcctct tttggttcct 20
<210> 17
<211> 20
<212> DNA
<213>Artificial sequence (Artificial)
<400> 17
accaaatccg tgtcagaagg 20
<210> 18
<211> 20
<212> DNA
<213>Artificial sequence (Artificial)
<400> 18
cagaaggaag ggaagtgctg 20
<210> 19
<211> 20
<212> DNA
<213>Artificial sequence (Artificial)
<400> 19
gcaactacat cctgctgctg 20
<210> 20
<211> 20
<212> DNA
<213>Artificial sequence (Artificial)
<400> 20
caccagctgg gagagagaac 20
<210> 21
<211> 20
<212> DNA
<213>Artificial sequence (Artificial)
<400> 21
ctggtgtcta gtcgcccatc 20
<210> 22
<211> 20
<212> DNA
<213>Artificial sequence (Artificial)
<400> 22
aggaggtgct ggaggaagat 20
<210> 23
<211> 20
<212> DNA
<213>Artificial sequence (Artificial)
<400> 23
caaagccacg gatcaatctt 20
<210> 24
<211> 20
<212> DNA
<213>Artificial sequence (Artificial)
<400> 24
cccgggaata gtgaaactga 20
<210> 25
<211> 20
<212> DNA
<213>Artificial sequence (Artificial)
<400> 25
gttcccagtg caaagaaagc 20
<210> 26
<211> 20
<212> DNA
<213>Artificial sequence (Artificial)
<400> 26
tccccggatg atgactttag 20
<210> 27
<211> 26
<212> DNA
<213>Artificial sequence (Artificial)
<400> 27
ctcaactaca tggtctacat gttcca 26
<210> 28
<211> 20
<212> DNA
<213>Artificial sequence (Artificial)
<400> 28
ccattctcgg ccttgactgt 20
Claims (6)
1. application of people's GLT8D1 gene expression amounts detection reagent in schizophrenia reagent for clinical diagnosis is prepared.
2. application according to claim 1, it is characterised in that：People's GLT8D1 gene expression amount detection reagents are detection people
The reagent of GLT8D1 gene low expression amounts.
3. application according to claim 2, it is characterised in that：Detection people's GLT8D1 gene expression amounts are to utilize people GLT8D1
The primer sequence of the mRNA of gene order designer GLT8D1, and pass through the mRNA of real-time quantitative PCR method detection people GLT8D1
It is horizontal；The primer sequence of the mRNA is：
SEQ ID NO:1 ︰ CGGAATGGAAACGACAGAAT；
SEQ ID NO:2 ︰ GCGGACATTCCACATAGGAT.
Application of the 4.GLT8D1 genes low expression in treatment of schizophrenia drug is screened.
5. application according to claim 4, it is characterised in that：The shRNA sequences for inhibiting GLT8D1 gene expressions are cloned
RNA interference slow virus is obtained after entering slow virus carrier, is used as to screen spirit after RNA interference slow-virus infection neural stem cell
The screening cell line of Split disease medicine；The sequence for expressing shRNA targets the anti-of GLT8D1 gene codes DNA including two
It is intermediate to be separated by a stem ring sequence to repetitive sequence；Wherein, two inverted repeats are respectively the shRNA of GLT8D1 genes
Target sequence and its complementary series；The shRNA target spots nucleotide sequence of the GLT8D1 genes is selected from following nucleotide sequence：
SEQ ID NO:3 ︰ GATGATGATGTCATTGTACAA；
SEQ ID NO:4 ︰ ACACACTATGTGGGAAGGTAA.
6. application according to claim 5, it is characterised in that：Express the sense strand sequence such as SEQ of the sequence of shRNA
ID NO:Shown in 5, antisense strand sequence such as SEQ ID NO:Shown in 6；Or sense strand sequence such as SEQ ID NO:Shown in 7,
Antisense strand sequence such as SEQ ID NO:Shown in 8.
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| CN103502473A (en) * | 2011-03-01 | 2014-01-08 | 耶鲁大学 | Predicting gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) |
| CN107208132A (en) * | 2014-09-15 | 2017-09-26 | 克里夫顿生命科学有限责任公司 | Composition, method and kit for diagnosing stomach and intestine pancreas neuroendocrine tumors |
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| CN103502473A (en) * | 2011-03-01 | 2014-01-08 | 耶鲁大学 | Predicting gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) |
| CN107208132A (en) * | 2014-09-15 | 2017-09-26 | 克里夫顿生命科学有限责任公司 | Composition, method and kit for diagnosing stomach and intestine pancreas neuroendocrine tumors |
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