CN105886625A - Application of CHKA gene to preparation of esophageal cancer diagnosis and treatment product - Google Patents
Application of CHKA gene to preparation of esophageal cancer diagnosis and treatment product Download PDFInfo
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Abstract
The invention discloses a CHKA gene that can function as a molecular marker for early diagnosis of esophageal cancer. Experiments prove that compared with normal esophageal tissues, the CHKA gene in esophageal cancer tissues has significantly increased expression, and RNA interference experiments prove that CHKA can affect proliferation of esophageal cancer cells. According to study results herein, it is possible to develop medicine to inhibit CHKA gene expression so as to clinically prevent and treat the esophageal cancer.
Description
Technical field
The present invention relates to biological technical field, more particularly to CHKA gene in the diagnosis of the esophageal carcinoma, treatment
In purposes.
Background technology
The esophageal carcinoma is as a kind of alimentary system malignant tumour, and serious threat people are healthy, affect quality of life.
Showing according to Epidemiological study, China is the area occurred frequently of the whole world esophageal carcinoma, reaches because of death from esophageal carcinoma number every year
More than 150000 people, occupy first of world's death from esophageal carcinoma number.The generation of the esophageal carcinoma and dietary habit, cause
The factors such as cancer material, disease, heredity, environment are the most closely related.At present, the clinical method master of the esophageal carcinoma is treated
Operative therapy to be had, X-ray therapy, chemotherapeutics therapy, non-operative treatment etc. including endoscopic therapy.
But esophageal carcinoma early diagnosis is difficult, and grade malignancy is high, and one after diagnosing, to late period, so the esophageal carcinoma at present
Therapeutic effect and prognosis the most very poor, within 5 years, survival rate is still below 30%.Along with molecular biology is oncosis
Because of the development in terms of research, people have progressively had new understanding to the pathogenic factor of the esophageal carcinoma.Generally recognize at present
Relate to many factors, several genes for the esophageal carcinoma, and play a role in the multistage, utilize at molecule
Biological means to its early diagnosis, treat and prevent significant.
Summary of the invention
It is an object of the invention to provide a kind of molecular marker that can be used for esophageal carcinoma early diagnosis.Compare existing
The diagnostic method of the esophageal carcinoma, use what gene marker carried out diagnosis of esophageal cancer to have promptness, specificity and spirit
Quick property, so that patient just can know disease risks in early days in disease, for risk height, takes corresponding pre-
Prevent and remedy measures.
To achieve these goals, the present invention adopts the following technical scheme that
The invention provides product the answering in the instrument preparing diagnosis of esophageal cancer of detection CHKA gene expression
With.
Further, the product of described detection CHKA gene expression includes detecting CHKA gene mRNA levels
Product and/or detection CHKA protein level product.
Further, the product of described detection CHKA gene expression includes: by RT-PCR, real-time quantitative
PCR, immune detection, in situ hybridization or the gene expression of chip detection CHKA are with the product of diagnosis of esophageal cancer.
Further, the product of described RT-PCR diagnosis of esophageal cancer at least includes a pair specific amplified CHKA base
The primer of cause；The product of described real-time quantitative PCR diagnosis of esophageal cancer at least includes a pair specific amplified CHKA
The primer of gene；The product of described immune detection diagnosis of esophageal cancer includes: be combined with CHKA protein-specific
Antibody；The product of described in situ hybridization diagnosis of esophageal cancer includes: with the nucleic acid array hybridizing of CHKA gene
Probe；The product of described chip diagnosis of esophageal cancer includes: protein chip and gene chip；Wherein, albumen
Chip includes the antibody being combined with CHKA protein-specific, and gene chip includes the nucleic acid sequence with CHKA gene
The probe of row hybridization.
A pair specific amplified CHKA base that the product of described real-time quantitative PCR diagnosis of esophageal cancer at least includes
The primer of cause is as shown in SEQ ID NO.3 and SEQ ID NO.4.
The product of described detection CHKA gene expression can be the reagent of detection CHKA gene expression, also may be used
Being to comprise the test kit of described reagent, chip, reagent paper etc., it is also possible to be that the high pass using described reagent measures
Sequence platform.
The instrument of described diagnosis of esophageal cancer includes but not limited to that chip, test kit, reagent paper or high-flux sequence are flat
Platform；High-flux sequence platform is the instrument of a kind of special diagnosis of esophageal cancer, along with sending out of high throughput sequencing technologies
Exhibition, will become the structure of the gene expression profile of a people and work the most easily.By contrast Disease and
The gene expression profile of normal population, the exception easily analyzing which gene is relevant to disease.Therefore, in high pass
Measure the exception purposes that fall within CHKA gene relevant to the esophageal carcinoma knowing CHKA gene in sequence, equally
Within protection scope of the present invention.
Present invention also offers the instrument of a kind of diagnosis of esophageal cancer, described instrument includes detecting CHKA gene expression
Reagent；Described reagent includes primer and/or probe, the detection CHKA albumen detecting CHKA gene mRNA
Antibody.
Described instrument includes but not limited to chip, test kit, reagent paper or high-flux sequence platform.
Wherein, described chip includes gene chip, protein chip；Described gene chip include solid phase carrier with
And it being fixed on the oligonucleotide probe of solid phase carrier, described oligonucleotide probe includes for detecting CHKA gene
The oligonucleotide probe for CHKA gene of transcriptional level；Described protein chip include solid phase carrier and
It is fixed on the specific antibody of the CHKA albumen of solid phase carrier；Described gene chip can be used for detection and includes
CHKA gene is at the expression of interior multiple genes (such as, relevant to the esophageal carcinoma multiple genes).Institute
State multiple protein that protein chip can be used for detecting including CHKA albumen (such as relevant to the esophageal carcinoma
Multiple protein) expression.By multiple marks with the esophageal carcinoma are detected simultaneously, can significantly carry
The accuracy rate of high esophagus cancer diagnosis.
Wherein, described test kit includes gene detecting kit and protein immunization detection kit；Described gene is examined
Test agent box includes the reagent for detecting CHKA gene transcription level；Described protein immunization detection kit bag
Include the specific antibody of CHKA albumen.Further, described reagent include use RT-PCR, real-time quantitative PCR,
Reagent required during immune detection, in situ hybridization or chip method detection CHKA gene expression dose.Excellent
Degree of choosing, described reagent includes the primer for CHKA gene and/or probe.Nucleotide according to CHKA gene
Sequence information easily designs the primer and probe that may be used for detecting CHKA gene expression dose.
Described reagent paper includes the reagent detecting CHKA gene expression.
Described high-flux sequence platform includes the reagent detecting CHKA gene expression.
Can be that DNA, RNA, DNA-RNA are chimeric with the probe of the nucleic acid array hybridizing of CHKA gene
Body, PNA or other derivant.The length of described probe does not limit, if complete specific hybrid and
Purpose nucleotide sequence is specific binding, and any length can.The length of described probe can be as short as 25,20,
15,13 or 10 bases longs.Equally, the length of described probe can be grown to 60,80,100,150,300
Individual base pair or longer, the most whole gene.Owing to different probe length is to hybridization efficiency, signal specificity
There are different impacts, the length of described probe to be typically at least 14 base pairs, the longest are usually no more than 30
Base pair, optimal with 15-25 base pair with the length of purpose nucleotide sequence complementary.Described probe self is mutual
Complementary series is most preferably less than 4 base pairs, in order to avoid affecting hybridization efficiency.
Further, the specific antibody of described CHKA albumen includes monoclonal antibody, polyclonal antibody.Described
The specific antibody of CHKA albumen include complete antibody molecule, any fragment of antibody or modify (such as,
Chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc..As long as described fragment can retain and CHKA albumen
Binding ability.Well known to a person skilled in the art when the preparation of the antibody of protein level, and this
Invention can use any method to prepare described antibody.
In specific embodiments of the present invention, the primer of described detection CHKA gene mRNA includes SEQ ID
Primer pair shown in NO.3 and SEQ ID NO.4.
Present invention also offers the inhibitor of CHKA gene and/or its expression product in the preparation treatment esophageal carcinoma
Application in medicine.Described inhibitor includes reagent and/or the suppression CHKA suppressing CHKA gene expression
The reagent of gene expression product.
Further, the reagent of described suppression CHKA gene expression includes reagent, the suppression that suppressor gene transcribes
The reagent of gene translation；The reagent of described suppression CHKA gene expression product includes suppressing CHKA gene
The reagent of mRNA, the reagent of suppression CHKA albumen.The reagent of described suppression CHKA gene mRNA
Reagent, the reagent of suppression mRNA translation activity including suppression mRNA stability.Described suppression CHKA
The reagent of albumen include suppress CHKA protein stability reagent, suppression CHKA protein active reagent,
The reagent of suppression CHKA protein function.
Further, what the reagent of suppression CHKA gene mRNA included for CHKA gene mRNA is double
Chain ribonucleic acid；The reagent of suppression CHKA protein function includes the tumor vaccine of CHKA antigen protein, presses down
The antibody of CHKA protein function processed.Described antibody can be polyclonal antibody, or monoclonal antibody.
In specific embodiments of the present invention, the described double stranded RNA for CHKA gene mRNA is
siRNA.Can efficiently be rejected in order to ensure CHKA gene or reticent, according to the mRNA of CHKA gene
Sequential design siRNA specific fragment.The design of siRNA is according to the general design principle delivered
(Elbashir et.al 2001, Schwarz et.al 2003, Khvorova et.al 2003, Reynolds et.al 2004,
Hsieh et.al 2004, Ui-Tei et.al 2004), by online tool complete design, this 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 improve the effectiveness of siRNA segment further,
The advantage of comprehensive two Photographing On-line instruments is designed for the siRNA segment of screening.Finally, homology is passed through
Property comparison (NCBI BLAST) filters siRNA sequence, to improve the specificity of siRNA segment and to reduce
The effect of missing the target of RNAi interference.
Preferably, the sequence of described siRNA is as shown in SEQ ID NO.7 and SEQ ID NO.8.
Present invention also offers a kind of pharmaceutical composition for treating the esophageal carcinoma, described pharmaceutical composition includes
CHKA gene described in face and/or the inhibitor of its expression product.
The pharmaceutical composition of the present invention also includes pharmaceutically acceptable carrier, wherein this carrier can be excipient,
Diluent, thickening agent, filler, bonding agent, disintegrating agent, lubricant, oils and fats or non-grease base, table
Face activating agent, suspending agent, gellant, adjuvant, preservative, antioxidant, stabilizer, coloring agent or perfume (or spice)
Mixing more than material either or both of which.
The pharmaceutical composition of the present invention can be used for manufacturing the medicament of the treatment esophageal carcinoma.
The pharmaceutical composition first-selection of the present invention is applied to mammal, and wherein this mammal is preferably human patients.
The pharmaceutical composition of the present invention can such as give to this human patients's body in modes such as oral, injections.
The pharmaceutical composition of the present invention also can be used in combination with the drug combination of the other treatment esophageal carcinoma, multi-medicament
Can significantly mention the success rate for the treatment of.
In the context of the present invention, " CHKA gene " includes CHKA gene and CHKA gene
The polynucleotide of any function equivalent.CHKA gene includes and the most international common core sequence databank
In GeneBank, CHKA gene (NC_000011.10) DNA sequence has more than 70% homology, and coding
Identical function protein DNA sequence；
Preferably, the coded sequence of CHKA gene includes any DNA molecular following:
(1) DNA sequence shown in SEQ ID NO.1 in sequence table；
(2) under strict conditions with 1) the DNA sequence hybridization that limits and coding identical function protein
DNA sequence；
(3) DNA sequence limited with (1) or (2) has 70%, preferably, more than 90% with
Source property, and coding identical function protein DNA molecule.
In specific embodiments of the present invention, the coded sequence of described CHKA gene is SEQ ID NO.1
Shown DNA sequence.
In the context of the present invention, CHKA gene expression product includes CHKA albumen and CHKA egg
White partial peptide.The partial peptide of described CHKA albumen contains the functional domain relevant to the esophageal carcinoma.
" CHKA albumen " includes any function equivalent of CHKA albumen and CHKA albumen.Described
Function equivalent includes CHKA albumen conservative variation's protein or its active fragment, or its activity is derivative
Thing, allelic variant, natural mutation, induced mutants, energy and CHKA under high or low stringent condition
The protein coded by DNA of DNA hybridization.
Preferably, CHKA albumen is the protein with following amino acid sequences:
(1) protein being made up of the aminoacid sequence shown in SEQ ID NO.2 in sequence table；
(2) aminoacid sequence shown in SEQ ID NO.2 is passed through the replacement of one or several amino acid residue
And/or disappearance and/or add and with the aminoacid sequence shown in SEQ ID NO.2 have identical function by
The protein that aminoacid sequence shown in SEQ ID NO.2 is derivative.Replace, lack or add is amino acid whose
Number is usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10.
(3) with the aminoacid sequence shown in SEQ ID NO.2, there is at least 80% homology and (be also called sequence
Homogeneity), it is highly preferred that same with the aminoacid sequence at least about 90% to 95% shown in SEQ ID NO.2
Source property, is often the polypeptide of the aminoacid sequence composition of 96%, 97%, 98%, 99% homology.
In specific embodiments of the present invention, described CHKA albumen has shown in SEQ ID NO.2
The protein of aminoacid sequence.
It is known that, conventionally, in a protein, one or more amino acid whose modifications do not interfere with protein
Function.Those skilled in the art can approve change single amino acids or the aminoacid of little percentage ratio or to aminoacid sequence
Adding individually, lacking, insert, replace of row is conservative modification, and wherein the change generation of protein has similar
The protein of function.It is well known in the art for providing intimate amino acid whose Conservative substitution tables.
It is CHKA albumen by adding the example of the protein of an aminoacid or multiple Modification of amino acid residues
Fusion protein.Peptide or protein with CHKA protein fusion is not limited, if the melting of gained
Hop protein retains the biologic activity of CHKA albumen.
The CHKA albumen of the present invention also includes repairing the non-conservative of aminoacid sequence shown in SEQ ID NO.2
Decorations, as long as the protein through modifying remains able to retain the biologic activity of CHKA albumen.At this
In class modifying protein, the amino acid number of sudden change is typically 10 or less, such as 6 or less,
Such as 3 or less.
In the context of the present invention, " diagnosis of esophageal cancer " both includes judging that experimenter has suffered from esophagus
Cancer, also include judging whether experimenter exists the risk suffering from the esophageal carcinoma.
In the context of the present invention, " the treatment esophageal carcinoma " divides from the state change of disease, can include disease
The alleviation of disease, the healing completely of disease, also include the therapeutic effect for evaluating disease.
Advantages of the present invention and beneficial effect:
Present invention firstly discovers that CHKA gene expression is relevant to the esophageal carcinoma, in being organized by detection experimenter
The expression of CHKA, it can be determined that whether experimenter suffers from the esophageal carcinoma or judge whether experimenter exists and suffer from
The risk of the esophageal carcinoma, thus instruct clinicist to provide prevention scheme or therapeutic scheme to experimenter.
Present invention finds a kind of new molecular marked compound-CHKA gene, compare traditional detection means, base
Because of diagnosis more in time, more special, sensitiveer, it is possible to realize the early diagnosis of the esophageal carcinoma, thus reduce the esophageal carcinoma
Mortality rate.
Accompanying drawing explanation
Fig. 1 shows the expression feelings utilizing genechip detection CHKA gene in human esophageal carcinoma with normal esophageal tissue
Condition；
Fig. 2 shows the expression utilizing Western blot detection CHKA albumen in human esophageal carcinoma with normal esophageal tissue
Situation；
Fig. 3 show utilize QPCR detect the siRNA jamming effectiveness to CHKA gene；
Fig. 4 show utilize Western blot detect the siRNA impact on CHKA protein expression；
Fig. 5 shows the suppression CHKA protein function impact on esophagus carcinoma proliferation.
Specific embodiment
The present invention is further detailed explanation with embodiment below in conjunction with the accompanying drawings.Following example are only used for
The bright present invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment is logical
Often according to normal condition, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold
Spring HarborLaboratory Press, 1989) condition described in, or according to the bar proposed by manufacturer
Part.
The differential expression of embodiment 1CHKA gene
1, experiment material:
Human esophageal carcinoma is the specimen of hospital's thoracic surgery excision, and normal esophageal is organized as under Dndoscope Laboratory mirror taking out
Specimen, draw materials immediately after the esophageal neoplasm tissue of excision is in vitro, when drawing materials, all wear disposable sterilized hands
Set, applies autoclaved knife blade to cut.
All cases are all through definitive pathological diagnosis.Including human esophageal carcinoma 40 example, normal esophageal organizes 30 examples.Wherein
Esophageal squamous cell carcinoma 34 example, adenocarcinoma of esophagus 6 example.With normal esophageal setup action matched group.Before all operation in patients all
Proved by pathology, the treatment such as the most non-preoperative row radiotherapy, chemotherapy, and without the tumor etc. at other positions.Institute is the most equal
Within half an hour of performing the operation after Liquid nitrogen storage, move into-80 degree refrigerators frozen.
2, the acquisition of the RNA of human esophageal carcinoma and normal esophageal tissue
Use Trizol one-step method to extract human esophageal carcinoma and the total serum IgE of normal esophageal tissue, pass through Nanodrop
ND-1000 reads the absorbance (A) at 260nm and 280nm and measures the purity of RNA solution.Through 1%
Denaturing formaldehyde agarose gel electrophoresis, observes under ultraviolet transmission light, the integrity of detection RNA.
3, gene chip hybridization and scanning
After the linearized amplification of total serum IgE, cy3-UTP labelling, the cRNAs after fluorescent labeling uses RNEASY
Mini Kit purification, enters the cRNAs that labelling is good with the RNA Fragmentation Reagents of Amhion
Row fragmentation processes.Use people's full genome chip of expression spectrum (4x 44K gene) of Agilent company of the U.S.,
In chip hybridization stove, 65 DEG C of hybridization 17h, then eluting, dyeing, finally uses Agilent DNA
MicroarrayScanner scanner scanning.
4, chip data processes and analyzes
Chip after hybridization, after chip scanner reads data point, imports data to analyze software, for two groups
The natural logrithm absolute value of the ratio gene more than 2.0 or less than 0.5 is as difference expression gene.
5, statistical procedures
Using SPSS 13.0 statistical software to carry out data analysis, group difference compares employing one factor analysis of variance
Method, P < 0.05 difference has significant.
6, result
Result shows (as shown in Figure 1), compared with normal esophageal tissue, and CHKA gene in human esophageal carcinoma
MRNA level in-site dramatically increase, difference has statistical significance (P < 0.05).
The differential expression of embodiment 2CHKA albumen
1, object of study is with embodiment 1.
2, tissue total protein is extracted
The operation of protein extraction is carried out according to the description of EpiQuik tissue/cell total protein extraction test kit.
3, Western blot detection
The protein quantification of extraction is carried out SDS-PAGE electrophoresis, carry out afterwards transferring film, closing, one anti-hatch,
Two anti-hatch, develop the color.
4, statistical procedures
Image J software is used to be analyzed, with β-actin as internal reference, by CHKA the gray value of protein band
The gray value of protein band is normalized.Result data is all to carry out table in the way of mean+SD
Showing, using SPSS13.0 statistical software to carry out statistical analysis, difference between the two uses t inspection,
Think when P < has statistical significance when 0.05.
5, result
Result as in figure 2 it is shown, compared with normal esophageal tissue, the expression of CHKA albumen in human esophageal carcinoma
Level dramatically increases, and difference has statistical significance (P < 0.05).
Embodiment 3 suppresses CHKA gene expression
1, siRNA design synthesis
SiRNA sequence for CHKA:
SiRNA1-CHKA:
Positive-sense strand is 5 '-AUUCUUCAGUAUCUAAUCGCC-3 ' (SEQ ID NO.7)；
Antisense strand is 5 '-CGAUUAGAUACUGAAGAAUUA-3 ' (SEQ ID NO.8),
SiRNA2-CHKA:
Positive-sense strand is 5 '-AUGAAAUGUAGCCAUUUUCUC-3 ' (SEQ ID NO.9)；
Antisense strand is 5 '-GAAAAUGGCUACAUUUCAUGG-3 ' (SEQ ID NO.10),
SiRNA3-CHKA:
Positive-sense strand is 5 '-AAGAUAUUACCUUCUUGACAG-3 ' (SEQ ID NO.11)；
Antisense strand is 5 '-GUCAAGAAGGUAAUAUCUUGU-3 ' (SEQ ID NO.12)
Above siRNA sequence and negative control siRNA sequence (siRNA-NC) are by Shanghai Ji agate pharmacy skill
Art company limited provides:
2, the cultivation of esophageal cancer cell and transfection
2.1 cells are cultivated
Esophageal carcinoma cell line ECA109 is inoculated in the DMEM culture fluid containing 10% hyclone, by it
It is positioned over 37 DEG C, 5%CO2Cultivate in incubator, when cell reaches 80% fusion, use 0.25% trypsin
Had digestive transfer culture.
2.2 cell transfecting
By esophageal cancer cell by 1 × 104/ hole is inoculated in 24 porocyte culture plates, at 37 DEG C, 5%CO2Training
Supporting cell in case and cultivate 24h, transfection is according to lipofectamine 2000 (purchased from Invitrogen company)
Description transfection, experiment is divided into, negative control group and experimental group (20nM), wherein, negative control group
The sequence of siRNA Yu CHKA gene is 20nM/ hole without homology, concentration, transfects the most respectively.
2, the jamming effectiveness of QPCR experiment detection siRNA is utilized.
2.1 extract cell total rna utilizes conventional method to operate.
2.2 reverse transcription
The Reverse Transcriptase kit utilizing TAKARA company carries out the reverse transcription of RNA.
2.3QPCR
(1) design of primers
Draw according to the coded sequence design QPCR amplification of CHKA gene and GAPDH gene in Genbank
Thing, is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Concrete primer sequence is as follows:
CHKA gene:
Forward primer is 5 '-TTGCTTGAATCTACTCCAT-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-CTGTAATTGTAACTGCTGTAT-3 ' (SEQ ID NO.4),
GAPDH gene:
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.5)；
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.6).
(2) PCR reaction system is prepared according to table 1:
Wherein, SYBR Green polymerase chain reaction system is purchased from Invitrogen company.
Table 1PCR reaction system
| Reagent | Volume |
| Forward primer | 1μl |
| Reverse primer | 1μl |
| SYBR Green polymerase chain reaction system | 12.5μl |
| Template | 2μl |
| Deionized water | Supply 25 μ l |
(3) PCR reaction condition: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 40s) * 40 circulations.With
SYBR Green, as fluorescent marker, reacts at the Light enterprising performing PCR of Cycler quantitative real time PCR Instrument,
Determine that purpose band, Δ Δ CT method carry out relative quantification by melt curve analysis analysis and electrophoresis.
2.4 statistical method
Experiment is all according to being repeated 3 times, and result data is all to come in the way of mean+SD
Representing, using SPSS13.0 statistical software to carry out statistical analysis, interference CHKA gene expression group is with right
T inspection is used, it is believed that when P < has statistical significance when 0.05 according to the difference between group.
2.5 result
Result as it is shown on figure 3, compared with siRNA2-CHKA, siRNA3-CHKA, siRNA1-CHKA
Can more effectively suppress the expression of CHKA gene, difference has statistical significance (P < 0.05), uses
SiRNA1-CHKA carries out follow-up experiment.
3, the jamming effectiveness of Western blot experiment detection siRNA1-CHKA
Step is with embodiment 2.
Result as shown in Figure 4, compared with transfection siRNA-NC group, transfects in the cell of siRNA1-CHKA
The content of CHKA albumen substantially reduces, and difference has statistical significance (P < 0.05).
The expression of the embodiment 4CHKA gene mensuration to esophagus carcinoma proliferation ability
Cell Counting kit-8 (cck-8) test kit is used to be used for detecting esophagus carcinoma proliferation
1, step
Carry out cultivation and the transfection of esophageal cancer cell according to the method for preceding embodiment, cell is divided into two experimental grouies:
Group 1: transfection siRNA-NC groups of cells；
Group 2: transfection siRNA1-CHKA groups of cells.
After transfection 24h, with 2.5 × 105/ ml density is inoculated in 96 porocyte culture plates, and each experimental group sets
Meter three wells, every hole adds the CCK-8 solution of 10 μ l, 37 DEG C, 5%CO2Incubator is hatched 4h；So
After illustrate according to test kit, select 450nm wavelength, microplate reader measures each hole absorbance (OD value).
2, statistical method
Experiment is all according to being repeated 3 times, and result data is all to come in the way of mean+SD
Representing, using SPSS13.0 statistical software to carry out statistical analysis, difference between the two uses t inspection,
Think when P < has statistical significance when 0.05.
3, result
Result is as shown in table 2, and compared with transfection siRNA-NC group, transfection siRNA1-CHKA groups of cells is thin
Born of the same parents breed slowly, and difference has statistical significance (P < 0.05).Above-mentioned test result indicate that, CHKA gene
Express the propagation promoting esophageal cancer cell.
Table 2 esophageal cancer cell OD value
| Experimental group | OD value (optical density) |
| siRNA-NC | 1.517±0.101 |
| siRNA-CHKA | 0.751±0.051 |
In embodiment 5 esophageal cancer cell antibody and experiment
1, step:
Esophageal cancer cell is inoculated in 96 porocyte culture plates, every hole 2*103Individual cells/well/200 μ l, cell
It is handled as follows after adherent:
Experimental group 1 (matched group): add unrelated monoclonal antibody (1:50) in esophageal cancer cell；
Experimental group 2: add anti-human CHKA monoclonal antibody (1:50) in esophageal cancer cell.
By cell at 37 DEG C, 5%CO2After incubator hatches 24 hours, add3H-TdR (1 μ Ci/ hole), then
Cultivate 24 hours, collect cell, add liquid scintillation solution, β calculating instrument detection cpm value.
2, statistical method
Experiment is all according to being repeated 3 times, and result data is all to come in the way of mean+SD
Representing, using SPSS13.0 statistical software to carry out statistical analysis, difference between the two uses t inspection,
Think when P < has statistical significance when 0.05.
3, result
Result is as it is shown in figure 5, compared to matched group, add the groups of cells cell proliferation of anti-human CHKA monoclonal antibody
Slow down.Above-mentioned test result indicate that, the function of suppression CHKA albumen can suppress esophagus carcinoma proliferation.
The explanation of above-described embodiment is only intended to understand the method for the present invention and core concept thereof.It is right to it should be pointed out that,
For those of ordinary skill in the art, under the premise without departing from the principles of the invention, it is also possible to the present invention
Carrying out some improvement and modification, these improve and modify also by the protection domain falling into the claims in the present invention.
Claims (10)
1. the product of detection CHKA gene expression application in the instrument preparing diagnosis of esophageal cancer.
Application the most according to claim 1, it is characterised in that described product includes: by RT-PCR,
Real-time quantitative PCR, immune detection, in situ hybridization, chip or high-flux sequence detection of platform CHKA gene
Express the product with diagnosis of esophageal cancer；The product of described RT-PCR diagnosis of esophageal cancer at least include a pair special
The primer of amplification CHKA gene；The product of described real-time quantitative PCR diagnosis of esophageal cancer at least includes a pair
The primer of specific amplified CHKA gene；The product of described immune detection diagnosis of esophageal cancer includes: with CHKA
The antibody that protein-specific combines；The product of described in situ hybridization diagnosis of esophageal cancer includes: with CHKA base
The probe of the nucleic acid array hybridizing of cause；The product of described chip diagnosis of esophageal cancer includes: protein chip and gene
Chip；Wherein, protein chip includes the antibody being combined with CHKA protein-specific, gene chip include with
The probe of the nucleic acid array hybridizing of CHKA gene.
Application the most according to claim 2, it is characterised in that described real-time quantitative PCR diagnoses esophagus
The primer such as SEQ ID NO.3 and SEQ ID of a pair specific amplified CHKA gene that the product of cancer at least includes
Shown in NO.4.
4. the instrument of a diagnosis of esophageal cancer, it is characterised in that described instrument includes detecting CHKA gene table
The reagent reached；Described reagent includes primer and/or probe, the detection CHKA egg detecting CHKA gene mRNA
White antibody.
Instrument the most according to claim 4, it is characterised in that described detection CHKA gene mRNA
Primer includes the primer pair shown in SEQ ID NO.3 and SEQ ID NO.4.
The application in the medicine of the preparation treatment esophageal carcinoma of the inhibitor of 6.CHKA gene and/or its expression product.
Application the most according to claim 6, it is characterised in that described inhibitor includes suppressing CHKA
The reagent of gene expression and/or the reagent of suppression CHKA gene expression product.
Application the most according to claim 7, it is characterised in that described suppression CHKA gene expression product
Reagent include suppressing the antibody of siRNA and/or CHKA albumen for CHKA gene.
Application the most according to claim 8, it is characterised in that the described siRNA for CHKA gene
Sequence is as shown in SEQ ID NO.7 and SEQ ID NO.8.
10. the pharmaceutical composition being used for treating the esophageal carcinoma, it is characterised in that described pharmaceutical composition includes
Inhibitor according to any one of claim 6-9.
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| CN106520925A (en) * | 2016-10-14 | 2017-03-22 | 浙江大学 | Primer group for detecting esophagus cancer, and detection method |
| CN113391069A (en) * | 2021-05-31 | 2021-09-14 | 浙江大学 | Application of CHK alpha-based non-metabolic function as target for cancer treatment, diagnosis and prognosis prediction |
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| CN106290895A (en) * | 2016-07-26 | 2017-01-04 | 王红阳 | CHKA albumen application, test kit and method in preparing prognosis in hcc assessment test kit |
| CN106520925A (en) * | 2016-10-14 | 2017-03-22 | 浙江大学 | Primer group for detecting esophagus cancer, and detection method |
| CN113391069A (en) * | 2021-05-31 | 2021-09-14 | 浙江大学 | Application of CHK alpha-based non-metabolic function as target for cancer treatment, diagnosis and prognosis prediction |
| CN113391069B (en) * | 2021-05-31 | 2023-10-27 | 浙江大学 | Use of chkα -based non-metabolic function as a target for cancer treatment, diagnosis and prognosis prediction |
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