CN106811532A - ACTA1 as Dendritic cell diagnosis and treatment mark purposes - Google Patents
ACTA1 as Dendritic cell diagnosis and treatment mark purposes Download PDFInfo
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Abstract
The invention discloses a kind of gene marker, the gene marker is ACTA1.ACTA1 can be used to judging whether subject has the risk of suffering from Dendritic cell or judge whether subject suffers from Dendritic cell.In addition, ACTA1 can be also used for preparing the medicine for the treatment of Dendritic cell.The present invention provides new diagnostic method for the clinical Dendritic cell of diagnosis on a molecular scale, while for the gene therapy of Dendritic cell provides new drug target.
Description
Technical field
The present invention relates to biological technical field, more particularly to use of the ACTA1 genes in the diagnosis, treatment of Dendritic cell
On the way.
Background technology
Tongue cancer is one of common malignant tumour of oromaxillo-facial region, most commonly seen with squamous carcinoma, is occupied first of carcinoma of mouth.Tongue cancer is more
/ 3rd lateral margins in tongue are betided, grade malignancy is higher, local infiltration is strong, often involves lingualis, makes tongue limitation of movement, shadow
Ring speech, feed and function of deglutition.Although achieving larger progress in terms for the treatment of, the survival rate of Patients With Tongue exists
It is not significantly improved in past recent decades.
Current study show that, smoking, chemical factor, people's nipple (shape) tumor virus (the human papilloma such as drink
Virus, HPV) and inherent cause change develop with the generation of tongue cancer it is closely related.Although researchers have had found near several successively
Ten tumor genes or CDKN2 take part in the occurrence and development of tongue cancer, but up to the present, the molecular mechanism of tongue cancer morbidity is also
Fail clearly, to be likely present the pathogenic process that other tumor-related genes take part in tongue cancer.Therefore, if tongue cancer can be searched out
Specific related gene, this will be helpful to illustrate the molecular mechanism of tongue cancer morbidity, will also be carried for the diagnosis of tongue cancer, treatment and prognosis
For molecular target.
The content of the invention
An object of the present invention is to provide one kind to diagnose tongue squama by detecting ACTA1 genes or protein expression difference
The method of cancer.
The second object of the present invention is to provide one kind to predict tongue squama by detecting ACTA1 genes or protein expression difference
The method of cancer prognosis.
The third object of the present invention is to provide one kind to treat Dendritic cell by suppressing ACTA1 genes or ACTA1 albumen
Method.
The fourth object of the present invention is to provide a kind of method for screening the medicine for the treatment of Dendritic cell.
The fifth object of the present invention is to provide a kind of medicine for treating Dendritic cell.
To achieve these goals, present invention employs following technical scheme：
The invention provides detection ACTA1 genes or the use of the product in Dendritic cell diagnostic tool is prepared of ACTA1 albumen
On the way.
Product present invention also offers detection ACTA1 genes or ACTA1 albumen is preparing prediction Dendritic cell prognostic tool
In purposes.
Further, the product of the detection ACTA1 genes or ACTA1 albumen includes detection ACTA1 genes or ACTA1 albumen
Expression product.The product includes combining the nucleic acid of ACTA1 genes or can combine the thing of ACTA1 albumen
Matter (such as antibody).The nucleic acid can detect the expression of ACTA1 genes；The material can detect ACTA1 albumen
Expression.
The product of detection ACTA1 genes of the invention can be based on playing its function using the known method of nucleic acid molecules：
As PCR, such as Southern hybridization, Northern hybridization, dot blot, FISH (FISH), DNA microarray, ASO methods,
High-flux sequence platform etc..Can qualitatively, quantitatively or semi-quantitatively implement analysis using the product.
The nucleic acid being included in the said goods can be obtained by chemical synthesis, or be contained by being prepared from biomaterial
Expect the gene of nucleic acid, then expand it to obtain using the primer for being designed for amplification expectation nucleic acid.
Further, the PCR method is known method, for example, ARMS (Amplification Refractory
Abruptly-changing system is not answered in Mutation System, amplification) method, RT-PCR (reverse transcriptase-PCR) method, nesting PCR methods etc..Amplification
Nucleic acid can be by using dot blotting hybridization method, Surface Plasmon Resonance (SPR methods), PCR-RFLP methods, original position RT-PCR
Method, PCR-SSO (sequence specific oligonucleotide) method, PCR-SSP methods, AMPFLP (amplifiable fragment length polymorphism) method,
MVR-PCR methods and PCR-SSCP (single-strand conformation polymorphism) methods are detected.
Nucleic acid recited above includes the primer of amplification ACTA1 genes, and the primer that product includes can be by by changing
Synthesis is learned prepare, by using those skilled in the art will know that method be suitably designed with reference to Given information, and pass through
It is prepared by chemical synthesis.
In specific embodiments of the present invention, the nucleic acid is the amplimer used during QPCR is tested, the primer
Sequence such as SEQ ID NO.1 (positive sequence) and SEQ ID NO.2 (reverse sequence) shown in.
Nucleic acid recited above may also include probe, and the probe can be prepared by chemical synthesis, by using this
The method that art personnel know appropriately is designed with reference to Given information, and is prepared by chemical synthesis, or can be led to
Cross and prepared containing the gene for expecting nucleotide sequence from biomaterial, and expect that the primer of nucleotide sequence expands using amplification is designed for
Increase it to prepare.
The product of detection ACTA1 albumen of the invention can be based on playing its function using the known method of antibody：For example,
Can be including ELISA, radioimmunoassay, immunohistochemical method, western blot etc..
The product of detection ACTA1 albumen of the invention includes the antibody or its fragment of specific binding ACTA1 albumen.Can be with
Using the antibody or its fragment of any structure, size, immunoglobulin class, origin etc., as long as it combines target protein.
The antibody or its fragment that detection product of the invention includes can be monoclonals or polyclonal.Antibody fragment refers to that reservation is anti-
Peptide of the body to an antibody part (Partial Fragment) of the binding activity of antigen or containing an antibody part.Antibody fragment can include
F(ab′)2, Fab ', Fab, scFv (scFv), the Fv (dsFv) of disulphide bonding or its polymer, dimerization V areas it is (dual anti-
Body) or peptide containing CDR.The product of detection ACTA1 albumen of the invention can include encoding antibody or Encoding Antibody Fragment
The nucleic acid of the separation of amino acid sequence, the carrier comprising the nucleic acid, and carry the cell of the carrier.
Antibody can be obtained by well known to a person skilled in the art method.For example, prepare retaining target all or in part
The polypeptide of protein integrates the mammalian cell expression vector for encoding their polynucleotides as antigen.Exempted from using antigen
After epidemic disease animal, from by immune animal adaptive immune cell and merging myeloma cell to obtain hybridoma.Then from hybridization
Knurl culture collects antibody.Finally can by using ACTA1 albumen for being used as antigen or part thereof to obtain antibody reality
Antigentic specificity purifying is applied to obtain the monoclonal antibody for ACTA1 albumen.Polyclonal antibody can as follows be prepared：With with it is upper
Literary identical antigen-immunized animal, blood sample is collected from by immune animal, and serum is isolated from blood, is then used
Above-mentioned antigen implements antigentic specificity purifying to serum.Can be by the antibody obtained with ferment treatment or resisting by using acquisition
The sequence information of body obtains antibody fragment.
The combination of label and antibody or its fragment can be implemented by method as commonly known in the art.For example, can
With following fluorescent marker protein or peptide：Clean protein or peptide with phosphate buffer, addition DMSO, buffer, etc. standard
Standby dyestuff, then mixed solution, places 10 minutes then at room temperature.In addition, mark can be used the labelling kit of commercialization, it is all
Such as biotin labeling reagent box, such as biotin labeling reagent box-NH2, biotin labeling reagent box-SH
(DojindoLaboratories)；Alkali phosphatase enzyme mark kit such as alkali phosphatase enzyme mark kit-NH2, alkaline phosphorus
Sour enzyme labelling kit-SH (Dojindo Laboratories)；Peroxidase labelling kit such as peroxidase mark
Note kit-NH2, peroxidase labelling kit-NH2 (Dojindo Laboratories)；Phycobniliprotein labelled reagent
Box such as phycobniliprotein labelling kit-NH2, phycobniliprotein labelling kit-SH, B- phycoerythrin labelling kit-NH2,
B- phycoerythrin labelling kits-SH, R-PE labelling kit-NH2, R-PE labelling kit SH
(DojindoLaboratories)；Fluorescent labeling reagent box such as fluorescein labelling kit-NH2, HiLyte Fluor (TM)
555 labelling kit-NH2, the labelling kit-NH2 of HiLyte Fluor (TM) 647 (Dojindo Laboratories)；And
DyLight 547 and DyLight647 (Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody
Labelling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation) and EZ- label protein marks
Note kit (Funakoshi Corporation).For correct labeling, it is possible to use suitable instrument come detect by mark
Antibody or its fragment.
As the sample according to detection product of the invention, it is possible to use the tissue sample for for example being obtained from biopsy subject
Or fluid.Sample is not particularly limited, as long as it is suitable to measure of the invention；For example, it can include tissue, blood, blood plasma,
Serum, lymph, urine, serous cavity liquid, spinal fluid, synovia, aqueous humor, tear, saliva or its fraction or treated material
Material.
In specific embodiments of the present invention, tissue of the sample from subject.
In the present invention, " prognosis " refers to tumor patient in the mistake after surgical procedure etc. suppresses or alleviates tumour growth
Journey or result.In this manual, prognosis can be by surgical procedure suppress or alleviate tumour growth after 1,2,3,4,5,6,
7th, 8,9,10,15,20 years or more long when life state.Prognosis can be by checking biomarker i.e. ACTA1 albumen or volume
The gene of code ACTA1 albumen is predicted.Prognosis prediction can be carried out so：According to biomarker with or without, or raise
Or reduce, the prognosis for determining patient is good or bad, or determines the probability of good prognosis or poor prognosis.
In the present invention, " prognosis bona " refer to by surgical procedure etc. be patient suppress or alleviate tumour growth it
Afterwards, patient's long-term (such as 3,5,6,7,8,9,10,15,20 years longer) is without critical condition.Or, good prognosis can anticipate
Refer to and survived in so long-time, sent out again without transfer, without recurrence or nothing.For example, prognosis bona can mean at least 3 years or outstanding
It is to survive at least 5 years, preferably without transfer or recurrence.The most preferred state of prognosis bona is the long-term survival without disease.Such as
Used herein, " prognosis bona " can also include any such state, wherein disease is can be found that as shifted, but
It is pernicious low and do not severely impact survival ability.
In the present invention, " prognosis mala " refers to patient short after surgical procedure etc. suppresses or alleviates tumour growth
There is fatal condition in period (such as 1,2,3,4,5 years shorter).Or, poor prognosis refer to dead in such short-term
Die, shift, recur or send out again.For example, poor prognosis can mean Preventive or dead at least 3 years or especially at least 5 years
Die.
Prediction prognosis refers to the process of prediction status of patient or result, is not meant to be predicted with 100% degree of accuracy
The process or result of status of patient.Prediction prognosis refers to whether the possibility for determining some processes or result increases, and simultaneously unexpectedly
Taste the possibility compared by situation about not occurring with some processes or result to determine some processes or result.Such as this
For invention, in the present invention in the elevated patient of level of ACTA1 genes or ACTA1 albumen, with the patient for not showing this feature
Compare, more likely observe particular procedure or result.
Further, the product of the detection ACTA1 genes or ACTA1 albumen can be detection ACTA1 genes or ACTA1 egg
White reagent, can also be the kit comprising the reagent, chip, test paper etc., or the high pass using the reagent
Amount microarray dataset.
Present invention also offers a kind of instrument for diagnosing Dendritic cell, the instrument can detect ACTA1 genes or ACTA1 eggs
White expression.The instrument includes combining the nucleic acid of ACTA1 genes or can combine the material of ACTA1 albumen
(such as antibody).The nucleic acid can detect the expression of ACTA1 genes；The material can detect the table of ACTA1 albumen
Up to level.
Further, the property of the nucleic acid and the material is with noted earlier.
Further, the instrument of the diagnosis Dendritic cell includes but is not limited to chip, kit, test paper or high-flux sequence
Platform；High-flux sequence platform is a kind of instrument of special diagnosis Dendritic cell, with the development of high throughput sequencing technologies, to one
The structure of personal gene expression profile will be as very easily working.By the gene table for contrasting Disease and normal population
Up to spectrum, the exception for easily analyzing which gene is related to disease.Therefore, the different of ACTA1 genes is known in high-flux sequence
The normal purposes for falling within ACTA1 genes related to Dendritic cell, equally within protection scope of the present invention.
Present invention also offers a kind of instrument for predicting Dendritic cell prognosis, the prediction Dendritic cell prognostic tool includes can
With reference to ACTA1 genes nucleic acid or can combine ACTA1 albumen material (such as antibody).The nucleic acid can be detected
The mRNA level in-site of ACTA1 genes；The material can detect the expression of ACTA1 albumen.
Further, the property of the nucleic acid and the material is with noted earlier.
Further, the instrument of the prediction Dendritic cell prognosis includes but is not limited to chip, kit, test paper or high flux
Microarray dataset；High-flux sequence platform is a kind of instrument of special diagnosis Dendritic cell, with the development of high throughput sequencing technologies,
Will be as very easily working to a structure for the gene expression profile of people.By the base for contrasting Disease and normal population
Because of express spectra, the exception for easily analyzing which gene is related to disease.Therefore, ACTA1 genes are known in high-flux sequence
The exception purposes for falling within ACTA1 genes related to Dendritic cell, equally within protection scope of the present invention.
The amino acid that the anti-ACTA1 antibody or its fragment used in detection product of the invention, diagnostic tool are recognized
Number is not particularly limited, as long as antibody can combine ACTA1.
Present invention also offers a kind of method for diagnosing Dendritic cell or prediction Dendritic cell prognosis, methods described includes following step
Suddenly：
(1) sample of subject is obtained；
(2) expression of ACTA1 genes or albumen in Samples subjects is detected；
(3) the ACTA1 genes or the expression of albumen that will be measured are associated with the whether ill of subject.
(4) compared with the control, the expression of ACTA1 genes or albumen is raised, then the subject is diagnosed as Dendritic cell,
Or the subject is confirmed as prognosis mala.
Inhibitor present invention also offers ACTA1 genes and/or its expression product is preparing the medicine for the treatment of Dendritic cell
In application.The inhibitor includes suppressing the reagent of ACTA1 gene expressions, and/or suppresses the examination of ACTA1 gene expression products
Agent.
Further, the reagent of the suppression ACTA1 gene expressions includes the reagent of suppressor transcription, suppressor translation
Reagent；The reagent of the suppression ACTA1 gene expression products includes suppressing the reagent of ACTA1 gene mRNAs, suppresses ACTA1 eggs
White reagent.The reagent of the suppression ACTA1 gene mRNAs includes suppressing the reagent of mRNA stability, suppresses mRNA translation activity
Reagent.The reagent of the suppression ACTA1 albumen includes suppressing the reagent of ACTA1 protein stabilities, suppresses ACTA1 protein actives
Reagent, suppress ACTA1 protein functions reagent.
Further, suppressing the reagent of ACTA1 gene mRNAs includes being directed to the double stranded RNA of ACTA1 gene mRNAs；Suppression
The reagent of ACTA1 protein functions processed includes the tumor vaccine of ACTA1 antigen proteins, suppresses the antibody of ACTA1 protein functions.It is described
Antibody can be polyclonal antibody, or monoclonal antibody.
In specific embodiments of the present invention, the double stranded RNA for ACTA1 gene mRNAs is siRNA.
In order to ensure ACTA1 genes can be rejected efficiently or silence, it is special that the mRNA sequence according to ACTA1 genes devises siRNA
Property fragment.The design of siRNA is according to general design principle (Elbashir et.al 2001, the Schwarz et.al for having delivered
2003, Khvorova et.al 2003, Reynolds et.al 2004, Hsieh et.al 2004, Ui-Tei et.al
2004), by online tool complete design, the online tool is：siRNASelectionProgram of Whitehead
Institute (BingbingYuan et.al 2004, http://jura.wi.mit.edu/bioc/siRNAext/) and
BLOCK-iTTM RNAi Designer ofINVITROGEN(winner of the 2004Frost&Sullivan
Excellence in Research Award, https://rnaidesigner.invitrogen.com/sirna/).In order to
Further improve the validity of siRNA segments, comprehensive two advantages of Photographing On-line instrument are designed for the siRNA pieces of screening
It is disconnected.Finally, siRNA sequence is filtered by sequence analysis (NCBI BLAST), to improve the specificity of siRNA segments and subtract
The effect of missing the target of few RNAi interference.
Preferably, the sequence of the siRNA is as shown in SEQ ID NO.5 and SEQ ID NO.6.
Present invention also offers a kind of pharmaceutical composition for treating Dendritic cell, described pharmaceutical composition includes institute above
The ACTA1 genes and/or the inhibitor of its expression product stated.
Pharmaceutical composition of the invention also includes pharmaceutically acceptable carrier, and the wherein carrier can be excipient, dilution
Agent, thickener, filler, bonding agent, disintegrant, lubricant, grease or non-grease base, surfactant, suspending agent, glue
Mixing more than solidifying agent, adjuvant, preservative, antioxidant, stabilizer, colouring agent or spices either or both of which.
Pharmaceutical composition of the invention can be used to manufacture the medicament for the treatment of Dendritic cell.
Pharmaceutical composition first-selection of the invention is applied to mammal, and wherein the mammal is preferably human patients.
Pharmaceutical composition of the invention for example can be given to human patients' body in modes such as oral, injections.
Pharmaceutical composition of the invention can also be with the drug combination of other treatment Dendritic cell, and multi-medicament is used in combination can be with
The success rate for the treatment of is mentioned significantly.
Present invention also offers a kind of screening technique of tumour medicine, can be by after testing drug be added to cancer cell
Or certain period measurement ACTA1 genes or the expression water of ACTA1 albumen after testing drug is applied to tumor model animal
Put down to determine the effect of tumour medicine improvement tumor prognosis.More specifically, when the expression of ACTA1 genes or ACTA1 albumen
When level is reduced in addition or after applying testing drug or recovers normal level, the medicine may be selected as improvement tumor prognosis
Medicine.
The advantages of the present invention：
Present invention firstly discovers that ACTA1 gene expressions are related to Dendritic cell, by detecting that subject organizes in ACTA1
Expression, it can be determined that whether subject suffers from Dendritic cell or judge subject with the presence or absence of the risk with Dendritic cell, so that
Clinician is instructed to provide prevention scheme or therapeutic scheme to subject.
The early diagnosis that carcinoma of mouth is carried out on gene level has become the development trend in carcinoma of mouth field, application number
For：201611136247.1、201511009921.5、201511009794.9、201610245087.8、
201610277716.5th, 201511009921.5,201610798012.2 patent documents disclose can be used for carcinoma of mouth or
The gene marker of person's Dendritic cell diagnosis, present invention finds a kind of new molecular marked compound-ACTA1 genes, can realize tongue
The early diagnosis of squamous carcinoma, so as to reduce the death rate of Dendritic cell.
Brief description of the drawings
Differential expression figure of Fig. 1 displays using RT-PCR detection ACTA1 genes in Dendritic cell tissue and normal structure；
Expression of Fig. 2 displays using Western blot detection ACTA1 albumen in Dendritic cell tissue and normal structure is poor
Different figure；
Fig. 3 displays detect inhibition figures of the siRNA to ACTA1 gene expressions using Western blot；
The influence figure that Fig. 4 display ACTA1 gene expressions are bred to Tca8113 cells；
The influence figure that Fig. 5 display ACTA1 gene expressions are migrated to Tca8113 cells；
Fig. 6 show ACTA1 gene expressions to Tca8113 cells into knurl influence figure.
Specific embodiment
The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this
Invention rather than limitation the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment, generally according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) described in condition, or according to the condition proposed by manufacturer.
The differential expression of the ACTA1 genes of embodiment 1
1st, experiment material
5 Dendritic cell tissue specimens take from Oral and Maxillofacial Surgery patient, wherein, differentiated squamous carcinoma 2, middle differentiation squama
Cancer 2, low differentiated squamous-cell carcinomas 1；Including male 2, women 3.Meanwhile, choose around each cancerous tissue cancerous swelling>At 5cm just
Often it is organized as own control.Without chemotherapy, radiotherapy, biological therapy and other controlling for tumour before all patient assessments
Treat.Materials rear portion tissue is stored for future use in being immediately placed in liquid nitrogen.
2nd, RNA is extracted, cDNA synthesizes
Trizol RNA reagents (Invitrogen companies) extracted total RNA, through ultraviolet specrophotometer (ND-1000,
NanoDrop companies) and agarose gel electrophoresis identification total serum IgE；According to Qiagen companies specification, through RNeasy MinElute
Cleanup Kit purifying obtains mRNA；MRNA synthesizes through Poly-ARNA Controlkit (Affymetrix companies) reverse transcription
Double-strand cDNA, and purify；
3rd, biotin labeling cRNA hybridization
With cDNA as template, using MessageAmpTM II-Biotin Arna Amplification Kit (Ambion
Company) in-vitro transcription synthesizing biotinylated mark cRNA, after purification by the eucaryote express spectra single-wheel core of Affymetrix companies
Piece amplification program adds 5 × fragmentation buffer to obtain fragmentation cRNA of the size distribution in 35~200nt；Target is prepared and completed
Afterwards, using eucaryote Hybridization Control Kit (Affymetrix companies) preparing hybrid liquid, hybridization solution is injected
Chip balance be positioned in hybrid heater, 45 DEG C, 60r/min rotation hybridization 16h (Hybridization Oven 640,
Affymetrix companies), then Affymetrix companies provide washing work station in (Fluidics Station 450,
Affymetrix companies) complete chip cleaning dyeing；
4th, scan and analyze
UsingScanner 3000 (Affymetrix companies) scanner scanning image.Scan image is first
WithOperating Software Version1.4 (GCOS 1.4, Affymetrix company) software carries out figure
As the conversion to signal value, raw data file is converted into.Then the invariant set in software dChip 2006 are used
Normalization methods and Model-base ExpressionIndex models are further counted to GCOS output results
According to analysis.According to the P values detected in each chip, as detected value Call values (the Absolute Call, Abs of data set
Call it is) during in the absence of A (Absent) or critical value M (Marginal), to be considered as not expressing, only Abs Call values are to deposit
Just it is used for further analysis in the data set of P (present).
5th, the screening of histological difference expressing gene
The screening of difference expression gene utilizes Significant Analysis of Microarray Software
(SAM) algorithm is carried out.
6th, result
Chip filters out 859 difference expression genes altogether.Compared with normal structure, the base of up-regulated in Dendritic cell tissue
Because 517, it is 342 to express the gene lowered.
Checking of the difference expression gene of embodiment 2 in large sample
1st, research object
Method according to embodiment 1 collects Dendritic cell tissue specimen 45, normal structure sample 50.
2nd, RNA is extracted and cDNA synthesis
Method according to embodiment 1 carries out RNA and extracts and cDNA synthesis.
3、RT-PCR
Primer is designed by primer-design software Primer 5.0, and Dalian treasured biotech firm and Shanghai Ying Jun companies synthesize.
ACTA1 genes and reference gene the primer sequence are as follows：
ACTA1 gene primer sequences
5 '-ATTCACGAGACCACCTAC-3 ' (SEQ ID NO.1),
5’-ATGACGTTGTTGGCATAC-3’(SEQ ID NO.2)；
GAPDH gene primer sequences
5 '-AAGGTCGGAGTCAACGGATTTG-3 ' (SEQ ID NO.3),
5’-CCATGGGTGGAATCATATTGGAA-3’(SEQ ID NO.4)。
Take 1 μ l cDNA products and enter performing PCR amplification, reaction condition is：95 DEG C of denaturation, 5min；95 DEG C of 30s, 60 DEG C of 30s, 72
DEG C 30s, 32 circulations；72 DEG C of extension 10min.PCR primer is expanded after electrophoresis on 1% Ago-Gel, is observed under uviol lamp,
Taken a picture in VDS gel imaging instruments.ACTA1 genes and GAPDH are drawn using gel image analysis software Bandleader analyses
PCR primer band absorbance value, then calculates the relative ratio of ACTA1 genes and GAPDH absorbance values, is used to represent mesh
Gene relative expression intensities.
4th, the extraction of total protein of cell
Specification according to the full cell extraction kits of EpiQuik carries out the operation of protein extraction.
5th, Western blot detections
Total protein Brandford standard measures, take to mix with sample buffer in right amount and boil 5min, cool down 5min；Take
30pg albumen is loaded to 15% polyacrylamide gel for preparing, and carries out electrophoresis, starts to be set to 80V constant pressures, sees Marker
After increase to 120V；Glue after electrophoresis is taken out, using the half-dried transferring systems of Bio.Rad in 100V transferase 45s 0min；Transferring film is finished
Afterwards, washed once with 1xPBS, immerse confining liquid, 40 DEG C overnight；Confining liquid is outwelled, Western cleaning solutions washing 5-10min is added,
Add primary antibody shaking table room temperature hybridization 2h；According to proper proportion with Western secondary antibody diluteds in Block buffer, be incubated
60min；Film washing liquid is washed 3 times, each 10min；Developed using ECL reagents, be fixed detection protein expression.
6th, statistical procedures
The gray value of protein band is analyzed using Image J softwares, with β-actin as internal reference, by ACTA1 albumen
The gray value of band is normalized.Result data is represented in the way of mean+SD, is used
SPSS13.0 statistical softwares carry out statistical analysis, and difference between the two is checked using t, it is believed that work as P<There is system when 0.05
Meter learns meaning.
7th, result
As depicted in figs. 1 and 2, compared with normal structure, the expression of ACTA1 significantly increases result in Dendritic cell tissue
Plus, difference has statistical significance (P<0.05).
Measure of the expression of the ACTA1 genes of embodiment 3 to Tca8113 cells multiplication capacity
1st, ACTA1 gene expressions are disturbed
1.1 siRNA synthesize
MRNA sequence according to ACTA1 genes designs and synthesizes siRNA sequence siRNA-ACTA1：
Positive-sense strand is 5 '-AUGAUCUUGAUCUUCAUGGUG-3 ' (SEQ ID NO.5)；
Antisense strand is 5 '-CCAUGAAGAUCAAGAUCAUCG-3 ' (SEQ ID NO.6),
Above siRNA sequence and negative control siRNA sequence (siRNA-NC) (negative control group siRNA and ACTA1 genes
Sequence without homology) by Shanghai JiMa pharmacy Technology Co., Ltd provide.
The culture of 1.2 Tca8113 cells and transfection
Human tongue cancer cell line strain HN4 is incubated at containing 10%FBS, 100U/m L penicillin and 100 μ g/m L streptomysins
In DMEM/F12 culture mediums.All cells are placed on containing 5%CO237 DEG C of cell culture incubators in.Before transfection 24h by cell with 2 ×
105/ hole is inoculated in 6 orifice plates, adds DMEM/F12 culture mediums, it is adherent overnight, transfected when cell confluency reaches 80-90%.
The DMEM/F12 culture mediums without serum are replaced by before transfection.SiRNA (final concentration of 33nM) is diluted with 250 μ l DMEM/F12,
Gently 3-5 mixing of pressure-vaccum.Gently overturn and mix transfection reagent, 5 μ l liposomes are diluted with 250 μ l DMEM/F12 culture mediums
2000, gently 3-5 mixing of pressure-vaccum, stands 5min at room temperature.Mixing transfection reagent and siRNA dilutions, gently pressure-vaccum 3-5 times
Mix, 20min is stood at room temperature.Transfection composite is added in 6 porocyte plates, front and rear jog cell plates are well mixed.Carefully
Born of the same parents' plate is placed in 37 DEG C, 5%CO2Cultivated in incubator.Transfection 6h changes the DMEM/F12 culture mediums containing serum.
The jamming effectiveness of 1.3Western blot experiment detections siRNA-ACTA1
Step is with embodiment 2.
Result is as shown in figure 3, compared with siRNA-NC groups are transfected, transfect ACTA1 albumen in the cell of siRNA-ACTA1
Content is substantially reduced, and difference has statistical significance (P<0.05).
2nd, MTT experiment analysis cell-proliferation activity
96 orifice plates, 1 × 10 are inoculated in after the cell 24h that siRNA will have been transfected3Individual/hole, respectively 24,48,72,96h
Time point adds 20 μ L MTT (5mg/m L) solution, 6 multiple holes of every group of setting；After incubator culture 4h, culture is carefully sucked
Liquid, 150 μ L DMSO are added per hole；Absorbance at ELIASA detection 590nm, experiment is in triplicate.
3rd, experimental result
Result is as shown in figure 4, compared with siRNA-NC groups are transfected, transfection siRNA-ACTA1 groups cell propagation is slow, difference
With statistical significance (P<0.05).It is above-mentioned test result indicate that, ACTA1 gene expressions promote the propagation of Tca8113 cells.
The expression of the ACTA1 genes of embodiment 4 is to Tca8113 cells migration and the measure of invasive ability
1st, Cell migration assay
To transfect after the cell 24h of siRNA with pancreatin digest piping and druming and it is fully resuspended for single cell suspension after, cell
Tally is counted, and cell number is diluted into 5 × 106Individual/mL, is inoculated in 6 orifice plates, is placed in 37 DEG C, 5%CO2Cell culture incubator
Middle culture, nutrient solution is discarded after cell grows up to individual layer, is marked in the center of 6 well culture plate bottom monolayer cells with sterilizing pipette tips
One cut, washes away dead cell, serum-free medium culture, is taken pictures counting respectively at 0,24,48h, and experiment is repeated 3 times.
2nd, cell invasion experiment
Matrigel is diluted by (1: 6) using DMEM/F12, is added to Transwell cells upper chamber, 100 μ L/ holes, cell
Incubator places 2h, and DMEM culture mediums of the 1m L containing 10%PBS is added in lower room, and 1 × 10 is added in upper chamber6The transfection of individual/ml
The μ L of cell suspension 200 of siRNA, after cellar culture 24h, take out Transwell cells, and upper chamber is softly wiped with cotton swab
Matrigel, PBS are carefully rinsed, and paraformaldehyde is fixed, violet staining, then wash away unnecessary crystal violet dye liquor with PBS, are dried in the air
Dry, optical microphotograph Microscopic observation is simultaneously taken pictures, and experiment is repeated 3 times.
3rd, experimental result
Migration experiment：As shown in figure 5, compared with siRNA-NC groups are transfected, transfecting siRNA-ACTA1 group cell migration numbers
Few, difference has statistical significance (P<0.05).
Matrigel：Counted under microscope is just being put after violet staining and is wearing theca cell number, finding transfection siRNA-NC groups
Theca cell number is worn for (67 ± 5) are individual, transfection siRNA-ACTA1 groups wear theca cell number (28 ± 3).
Measure of the expression of the ACTA1 genes of embodiment 5 to Tca8113 cells one-tenth knurl ability
Plate clone is tested
The single-layer culturing cell for having transfected siRNA is taken, is digested with pancreatin and is blown and beaten into individual cells, counted, make cell
1000/m L are diluted in the DMEM/F12 nutrient solutions containing 10%FBS and are inoculated in culture dish, be shaken gently for making cell
Uniformly, it is put into incubator 2~3 weeks.Often observation, as the clone of visible 30~50 cells under there is mirror in culture dish, eventually
Only cultivate.Abandoning supernatant, is carefully embathed 2 times with PBS.The paraformaldehyde for plus 4% fixes 15min.Removal fixer, plus in right amount
10~30min of violet staining, then with flowing water clean, be air-dried.Clone's number of 30 cells of counted under microscope ＞, meter
Calculate cloning efficiency, cloning efficiency (%)=clone's number/inoculating cell number × 100%.
Result is as shown in fig. 6, compared with siRNA-NC groups are transfected, transfection siRNA-ACTA1 group Cell colonies assays are bright
Aobvious to decline, difference has statistical significance (P<0.05).
The explanation of above-described embodiment is only intended to understand the method for the present invention and its core concept.It should be pointed out that for this
For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improvement can also be carried out to the present invention
And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.
SEQUENCE LISTING
<110>Beijing Yang Shen biology information technologies Co., Ltd
<120>ACTA1 as Dendritic cell diagnosis and treatment mark purposes
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence
<400> 1
attcacgaga ccacctac 18
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
atgacgttgt tggcatac 18
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence
<400> 3
aaggtcggag tcaacggatt tg 22
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence
<400> 4
ccatgggtgg aatcatattg gaa 23
<210> 5
<211> 21
<212> RNA
<213>Artificial sequence
<400> 5
augaucuuga ucuucauggu g 21
<210> 6
<211> 21
<212> RNA
<213>Artificial sequence
<400> 6
ccaugaagau caagaucauc g 21
Claims (10)
1. the product of detection ACTA1 genes or ACTA1 albumen is in the instrument for preparing diagnosis Dendritic cell or prediction Dendritic cell prognosis
Application.
2. application according to claim 1, it is characterised in that the product bag of the detection ACTA1 genes or ACTA1 albumen
Include the product of the expression of detection ACTA1 genes or ACTA1 albumen.
3. application according to claim 1 and 2, it is characterised in that the product includes combining the core of ACTA1 genes
Acid can be with reference to the material of ACTA1 albumen；The nucleic acid can detect the expression of ACTA1 genes；The material energy
Enough detect the expression of ACTA1 albumen.
4. application according to claim 3, it is characterised in that the nucleic acid is the special expansion used in real-time quantitative PCR
The primer of increasing ACTA1 genes is as shown in SEQ ID NO.1 and SEQ ID NO.2.
5. it is a kind of diagnose Dendritic cell or prediction Dendritic cell prognosis instrument, it is characterised in that the instrument include can detect
The instrument of the expression of ACTA1 genes or ACTA1 albumen.
6. instrument according to claim 5, it is characterised in that the instrument includes combining the nucleic acid of ACTA1 genes
Or the material of ACTA1 albumen can be combined；The nucleic acid can detect the expression of ACTA1 genes；The material can
Detect the expression of ACTA1 albumen.
7. instrument according to claim 6, it is characterised in that the nucleic acid is the special expansion used in real-time quantitative PCR
The primer of increasing ACTA1 genes is as shown in SEQ ID NO.1 and SEQ ID NO.2.
Application of the inhibitor of 8.ACTA1 genes and/or its expression product in the medicine for preparing treatment Dendritic cell.
9. application according to claim 8, it is characterised in that the inhibitor includes suppressing the examination of ACTA1 gene expressions
Agent, and/or the reagent of suppression ACTA1 gene expression products.
10. a kind of pharmaceutical composition for treating Dendritic cell, it is characterised in that described pharmaceutical composition includes claim 8
Or the inhibitor described in 9.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609644A (en) * | 2019-01-21 | 2019-04-12 | 上海市东方医院（同济大学附属东方医院） | Application of PAX3-FKHR and ACTA1 genes in preventing and treating rhabdomyosarcoma |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012088298A2 (en) * | 2010-12-21 | 2012-06-28 | Sloan-Kettering Institute For Cancer Research | Epigenomic markers of cancer metastasis |
| WO2012175562A2 (en) * | 2011-06-21 | 2012-12-27 | University Of Tartu | Methylation and microrna markers of early-stage non-small cell lung cancer |
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2017
- 2017-03-03 CN CN201710123994.XA patent/CN106811532B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012088298A2 (en) * | 2010-12-21 | 2012-06-28 | Sloan-Kettering Institute For Cancer Research | Epigenomic markers of cancer metastasis |
| WO2012175562A2 (en) * | 2011-06-21 | 2012-12-27 | University Of Tartu | Methylation and microrna markers of early-stage non-small cell lung cancer |
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| SAJNANI ET AL: "Identification of novel transcripts deregulated in buccal cancer by RNA-seq", 《GENE》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609644A (en) * | 2019-01-21 | 2019-04-12 | 上海市东方医院（同济大学附属东方医院） | Application of PAX3-FKHR and ACTA1 genes in preventing and treating rhabdomyosarcoma |
| CN109609644B (en) * | 2019-01-21 | 2022-04-01 | 上海市东方医院（同济大学附属东方医院） | Application of PAX3-FKHR and ACTA1 genes in preventing and treating rhabdomyosarcoma |
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