CN109988840A

CN109988840A - The biological diagnosis and treatment marker of cervical lesions disease

Info

Publication number: CN109988840A
Application number: CN201910254356.0A
Authority: CN
Inventors: 杨承刚; 孙耀兰; 黄露宁
Original assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2019-03-31
Filing date: 2019-03-31
Publication date: 2019-07-09

Abstract

The invention discloses the biological diagnosis and treatment marker of cervical lesions disease, specific biology diagnosis and treatment marker is BDKRB2.The invention discloses application of the BDKRB2 in diagnosis cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma, and the present invention discloses application of the BDKRB2 in treatment cervical squamous cell carcinoma and its transfer and invasion.

Description

The biological diagnosis and treatment marker of cervical lesions disease

Technical field

The invention belongs to biomedicine fields, are related to the biological diagnosis and treatment marker of cervical lesions disease, are specifically related to mark Will object is BDKRB2.

Background technique

Cervical carcinoma is the more typical malignant tumour of women, and disease incidence is located at the second of female tumor, accounts for female reproductive system The 73%~93% of system Cancer Mortality.It is more than 130,000 that China increases morbidity number newly every year, it is especially noted that due to Environmental pollution makes the cervical carcinoma for being mainly in 50 years old or so women originally plus the bad health habit in life, nowadays also more next More rejuvenation.Epidemiological study finds that the generation of cervical carcinoma and human papilloma virus (HPV) infection are in close relations, especially high Danger type HPV infection.But HPV infection is not independent risk factor.From HPV infection, cervical intraepithelial neoplasia (CIN) (CIN) to palace The distribution of this pyramid of neck cancer show cervical carcinoma forming process other than HPV infection there is also other necessary factors, The occurrence and development of cervical carcinoma should be host genetic factor and the coefficient result of HPV.

Recently as going deep into for oncomolecularbiology research, find gene in the initiation of cancer, precancerous cell and cancer It plays an important role in the existence growth of cell.Meaning of the gene in cervical lesions disease development is inquired into, it is specified Effect in Cervical Carcinogenesis each stage, can for clinical appraisal by stages, judging prognosis reference is provided, to treat, preventing to swell Tumor opens up new way.

Summary of the invention

In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a kind of and cervical diseases: in epithelium of cervix uteri Tumor-like lesion, the relevant gene marker of cervical squamous cell carcinoma occurrence and development, study the marker in cervical intraepithelial neoplasia (CIN), palace Effect in carcinoma of neck provides precision diagnosing and treating for disease.

To achieve the goals above, the present invention adopts the following technical scheme:

The first aspect of the present invention provides a kind of reagent, and the reagent is able to detect BDKRB2 gene or its expression product Level.

Further, the reagent includes probe, primer or the antibody for BDKRB2.

Further, for the primer sequence of BDKRB2 as shown in NO.1~2 SEQ ID.

The second aspect of the present invention provides a kind of kit, and the kit includes examination described in first aspect present invention Agent.

The third aspect of the present invention provides a kind of chip, and the chip includes reagent described in first aspect present invention.

The fourth aspect of the present invention provides a kind of nucleic acid film item, and the nucleic acid film item includes described in first aspect present invention Reagent.

The fifth aspect of the present invention provides a kind of composition, and the composition includes the promotor and/or medicine of BDKRB2 Acceptable carrier on.The promotor includes improving BDKRB2 gene or its expression product stability, up-regulation BDKRB2 base The expression of cause or its expression product increases BDKRB2 gene or the substance of its expression product effective acting time.

Further, the promotor is the carrier for being overexpressed BDKRB2.

The sixth aspect of the present invention provides a kind of method of the drug candidate of screening treatment cervical squamous cell carcinoma, the method packet It includes:

The cultivating system of expression or the albumen containing BDKRB2 gene or its coding is handled with substance to be screened；With detection institute State the expression or activity of the albumen of BDKRB2 gene or its coding in system；

Wherein, if the substance to be screened can promote the level or expression activity of BDKRB2 gene, show this wait sieve Selecting substance is the drug candidate for treating cervical squamous cell carcinoma.

In the present invention, the system includes but is not limited to: cell system, subcellular system, solution system, organizer System, organ systems or animal system.

In the present invention, the step further include: to the drug candidate of acquisition carry out further cell experiment and/or Animal experiment, with further selection can treat the drug of cervical squamous cell carcinoma from drug candidate.

The seventh aspect of the present invention provides following described in any item applications:

A. kit, third aspect present invention described in reagent, second aspect of the present invention described in first aspect present invention Nucleic acid film item described in the chip, fourth aspect present invention early diagnoses cervical intraepithelial neoplasia (CIN), uterine neck in preparation Application in the product of squamous carcinoma；

B. composition described in fifth aspect present invention preparation treatment cervical squamous cell carcinoma and its transfer, invasion drug in Using；

C.BDKRB2 preparation treatment cervical squamous cell carcinoma and its transfer, invasion drug in application.

Detailed description of the invention

Fig. 1 is the expression using QPCR detection BDKRB2 gene in cervical squamous cell carcinoma patient；

Fig. 2 is BDKRB2 gene expression dose figure；

Fig. 3 is the CCK-8 method detection active influence diagram of BDKRB2 cell proliferation；

Fig. 4 is using the influence diagram of the cell transwell detection BDKRB2 gene pairs cell migration invasion, wherein figure A is To the influence diagram of cell migration；Figure B is the influence diagram to cell invasion.

Specific embodiment

The present invention is by high throughput sequencing technologies and carries out high-flux sequence analysis, detection cervical intraepithelial neoplasia sample disease Become, the gene expression dose in cervical squamous cell carcinoma patient tissue, discovery wherein expresses the gene with notable difference, inquires into itself and palace Neck intraepithelial neoplasia, cervical squamous cell carcinoma generation between relationship, thus be cervical squamous cell carcinoma early detection and targeted therapy Find better approaches and methods.By screening present invention firstly discovers that BDKRB2 expresses downward in cervical squamous cell carcinoma patient, And BDKRB2 is further demonstrated by gene overexpression technology and participates in the proliferation of cervical cancer cell, invasive procedure, it prompts BDKRB2 can be used as diagnosing and treating of the molecular target of cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma applied to disease.

BDKRB2 gene

The gene I/D of BDKRB2 in the present invention be 624, in the present invention, BDKRB2 include wild type, saltant type or its Segment.One skilled in the art will appreciate that primitive sequencer result can be compared to the reference genome to people when carrying out sequencing analysis On, therefore the BDKRB2 in the selection result may include different transcripts, as long as can compare on reference genome BDKRB2 (gene I/D: 624).A kind of sequence of representative BDKRB2 is as shown in NM_000623.3.

BDKRB2 nucleotide full length sequence or its segment of the invention can usually use PCR amplification method, recombination method or artificial Synthetic method obtains.

The present invention can use any method known in the art measurement gene expression.Those skilled in the art should manage Solution, the means for measuring gene expression are not importances of the invention.Biology can be detected on transcriptional level or translation skill The expression of marker.

Terms used herein " differential expression " indicates to mark with one or more present invention biologies identical in second of sample The expression of will object compares, after measured the amount or level of mRNA, one or more biology marks of the invention in a sample The difference of one or more splice variant expressions of the RNA of the will object and/or biomarker mRNA." differential expression " it can also include compared with protein expression quantity in second of sample or sample group or level, to this in sample or sample group The measurement of the protein of invention biomarker coding.Differential expression can it is as described herein and those skilled in the art understand that Method determines.Given biomarker can in term " differential expression " or " variation of expression " expression and second of sample Measurement expression compares, after measured the amount of RNA and/or the amount of protein, and expression can be measured by giving biomarker in sample Horizontal increases or decreases.Term " differential expression " or " variation of expression " also may indicate that and life in second sample group The expression that measures of object marker compares, and biomarker is given in sample group can measure the increase or drop of expression It is low." differential expression " used herein can be with the expression of given biomarker relative to biomarker given in control The ratio of Average expression level be measured, wherein ratio is not equal to 1.0.Differential expression can also be measured with p value.Work as use When p value, when p value is less than 0.1, biomarker is accredited as the differential expression between the first and second groups.More preferable p value Less than 0.05.Even more preferably p value is less than 0.01.Even more preferably from p value less than 0.005.Most preferably p value is less than 0.001.When being based on When ratio determines differential expression, if the ratio of expression is more than or less than 1.0, RNA in the first and second of sample Or protein is differential expression.For example, the ratio greater than 1.2,1.5,1.7,2,3,4,10,20, or the ratio less than 1 Rate, such as 0.8,0.6,0.4,0.2,0.1,0.05.In another embodiment of the present invention, if the first group is averaged The ratio of expression and the Average expression level of the second group is more than or less than 1.0, then transcribed nucleic acid is originally differential expression. For example, the ratio greater than 1.2,1.5,1.7,2,3,4,10,20, or the ratio less than 1, for example, 0.8,0.6,0.4,0.2, 0.1,0.05.In another embodiment of the present invention, if put down in expression and second colony in first sample The ratio of equal expression is more than or less than 1.0, is greater than 1.2,1.5,1.7,2,3,4,10,20 or ratio for example including ratio Less than 1, such as 0.8,0.6,0.4,0.2,0.1,0.05, then transcribed nucleic acid is originally differential expression.

" differential expression increase " or " up-regulation " indicates gene relative in contrast, and gene expression is (with rna expression or albumen Matter expression measurement) display increase at least 10% or more, such as 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or more or 1.1 times, 1.2 times, 1.4 times, 1.6 times, 1.8 times or more.

" differential expression reduction " or " downward " indicates gene relative in contrast, and gene expression is (with rna expression or albumen Matter expression measurement) display reduce at least 10% or more, such as 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or less than 1.0 times, 0.8 times, 0.6 times, 0.4 times, 0.2 times, 0.1 times or less gene.For example, up-regulation gene include with The expression of the mRNA or protein that separate from normal individual are compared, from the individual separation characterized by with cervical lesions disease MRNA or the increased gene of protein expression level in tissue.For example, down-regulated gene includes the tissue separated with from normal individual It compares, the base that mRNA or protein expression level reduce from the tissue of the individual separation characterized by with cervical lesions disease Cause.

Gene and albumen of the invention is detected using multiple technologies known to persons of ordinary skill in the art, these skills Art includes but is not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technologies, protein immunization technology.

The nucleic acid amplification technologies be selected from polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), Amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA) and the amplification based on nucleic acid sequence of transcriptive intermediate (NASBA).Wherein, PCR is needed RNA reverse transcription before amplification into DNA (RT-PCR), TMA and the direct cloning RNA of NASBA.

Nucleic acid hybridization technique in the present invention include but is not limited in situ hybridization (ISH), microarray and Southern or Northern trace.In situ hybridization (ISH) be it is a kind of use label complementary DNA or RNA chain as probe with position tissue one Part or slice (original position) or if organize it is sufficiently small if for entirely organize (full organization embedding ISH) in specific DNA or The hybridization of RNA sequence.

Protein immunization technology of the invention includes sandwich immunoassay, such as sandwich ELISA, wherein using identification biology mark Two kinds of antibody of different epitopes carry out the detection of the biomarker on will object；Radiommunoassay (RIA), directly, indirectly or Comparison enzyme linked immunosorbent assay (ELISA) (ELISA), fluorescence immunoassay (FIA), immunoblotting, is exempted from enzyme immunoassay (EIA) (EIA) The epidemic disease precipitation method and immunoassays based on any particle are (as used gold particle, Argent grain or latex particle, magnetic-particle or quantum Point).Immunization for example can be implemented in the form of microtiter plate or item.

The reagent for detecting BDKRB2 albumen is the specific binding agent of BDKRB2 albumen.Specific binding agent is such as albumen The receptor of matter BDKRB2, conjugated protein BDKRB2 agglutinin, for PROTEIN B DKRB2 antibody, be directed to protein Peptide antibody (peptidebody), the agent of bispecific dual combination or the bispecific antibody form of BDKRB2.

The example of specific binding agent is peptide, peptide mimics, aptamer, spiegelmer, darpin, ankyrin repetition Albumen, Kunitz type domain, antibody, single domain antibody and monovalent antibody fragments.In a specific embodiment of the present invention, the specificity Bonding agent is BDKRB2 specific antibody.

Term " monoclonal antibody " refers to the antibody that the antibody from a group substantially homogeneity obtains as used herein, that is, constitutes Each antibody of group it is identical and/or combine same epitope, in addition to production monoclonal antibody during issuable possibility Outside variant, such variant is generally with indivisible presence.Such monoclonal antibody is typically include comprising the polypeptide sequence in conjunction with target The antibody of column, wherein target combination polypeptide sequence is by including selecting single target combination polypeptide sequence in the more peptide sequence of comforming What the process in being listed in obtained.For example, selection course can be comform polyclonal such as hybridoma clone, phage clone or again Unique clones are selected in the set of group DNA clone.It should be appreciated that selected target binding sequence can further change, for example, Improve the affinity to target, by target binding sequence humanization, improve its yield in cell culture, reduce its Intracorporal immunogenicity, creation multi-specificity antibody etc., and include the antibody of target binding sequence after changing be also this hair Bright monoclonal antibody.From the typical polyclonal antibody preparations comprising the different antibodies for different determinants (epitope) not Together, every kind of monoclonal antibody of monoclonal antibody preparations is for the single determinant on antigen.Other than their specificity, The advantage of monoclonal antibody preparations is that they are generally not affected by the pollution of other immunoglobulins.Modifier " monoclonal " refers to Show antibody basically homogeneity antibody population obtain feature, should not be construed as require generated by any ad hoc approach it is anti- Body.

" antibody fragment " includes a part of full length antibody, usually its antigen binding domain or variable region.Antibody fragment Example includes Fab, Fab ', F (ab ') 2 and Fv segment；Double antibody；Linear antibodies；Single-chain antibody molecules；And by antibody fragment shape At multi-specificity antibody.

" functional fragment " of antibody of the invention refers to that those retain and derive their complete full chain molecule with substantially Identical affinity combination polypeptide and active at least one measuring method (such as in mouse model, or inhibit in vitro Antibody fragment combine antigen biological activity) segment.

The present invention provides the product of the expression of detection BDKRB2, the product includes but is not limited to chip, examination Agent box, nucleic acid film item.Wherein chip includes: solid phase carrier；And the oligonucleotides being orderly fixed on the solid phase carrier is visited Needle or antibody, the oligonucleotide probe some or all of specifically correspond to shown in BDKRB2 sequence, the antibody The combination BDKRB2 albumen of specificity.

The solid phase carrier includes inorganic carrier and organic carrier, the inorganic carrier include but is not limited to have silicon carrier, Glass carrier, ceramic monolith etc.；The organic carrier includes polypropylene film, nylon membrane etc..

The present invention provides a kind of kit, the kit can be used for detecting the reagent of BDKRB2 gene or albumen.Choosing From one or more substances of the following group: container, operation instructions, positive control, negative control object, buffer, auxiliary agent or molten Agent.

In kit of the invention can also have kit operation instructions, be described how using kit into Row detection.

In certain embodiments, it provided herein is the protein levels for detecting one or more biomarkers Kit.In certain embodiments, the kit includes the coated examination of antibody with identification of protein biomarker Paper slip, washing solution, the reagent for carrying out the test, Separation of Proteins or tools for purification, detection instrument and the positive and feminine gender are right According to.In certain embodiments, the kit also includes the specification using the kit.The kit can customize confession Use at home, clinical use or research use.

The present invention provides a kind of nucleic acid film item, nucleic acid film item includes substrate and the oligonucleotides that is fixed in the substrate Probe；The substrate can be any substrate suitable for immobilized oligonucleotide probe, such as nylon membrane, nitrocellulose filter, poly- Propylene film, sheet glass, silica gel chip, miniature magnetic bead etc..

Promotor and pharmaceutical composition

The present invention provides a kind of drug (compositions), it contains the promotor of a effective amount of BDKRB2, and Pharmaceutically acceptable carrier.The composition can be used for treating cervical squamous cell carcinoma.

As a kind of preferred embodiment of the invention, the promotor of the BDKRB2 is the expression vector of BDKRB2 a kind of. The expression vector is usually also containing promoter, replication orgin and/or marker gene etc..

The example of pharmaceutically acceptable carrier or diluent is softened water or distilled water；Saline solution；Plant oil, such as flower Oil generation, safflower oil, olive oil, cottonseed oil, corn oil, sesame oil class, such as peanut oil (peanutoil), safflower oil, olive oil, cotton Seed oil, corn oil, sesame oil, peanut oil (arachisoil) or coconut oil；Silicone oil, including it is polysiloxane-based, such as methyl polysilicon Oxygen alkane, phenyl polysiloxane and methyl phenyl silicone；Volatile silicone class；Mineral oils, as atoleine, soft paraffin or Saualane；Cellulose derivative, such as methylcellulose, ethyl cellulose, carboxymethyl cellulose, sodium carboxymethylcellulose or hydroxypropyl Ylmethyl cellulose；Lower alkanols, such as ethyl alcohol or isopropanol；Rudimentary aralkyl alcohols (aralkanols)；Rudimentary poly- alkylene Glycols or rudimentary alkylene glycols, such as polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-BDO or glycerol； Fatty acid ester, such as isopropyl palmitate, isopropyl myristate or ethyl oleate；Polyvinylpyrrolidone；Agar；Astragalus tree Glue or Arabic gum and vaseline.General carrier or variety carrier form the 10wt% to 99.9wt% of composition.

Composition can be the dosage form of suitable drug administration by injection, be suitble to dosage form (such as capsule, tablet, caplet, the wine made of broomcorn millet of orally ingestible Agent), be suitble to local administration ointment, emulsifiable paste or lotion form, be suitable as eye drops delivering dosage form, fit through sucking to The aerosol form (as sucked by nasal inhalation or mouth) of medicine, the dosage form for being suitble to parenteral, i.e., subcutaneously, intramuscular or quiet Injection in arteries and veins.

Administration for Injectable solution or suspension, nontoxic parenteral acceptable diluent or carrier may include Lin Ge Family name's solution, isotonic saline solution, phosphate buffered saline (PBS), ethyl alcohol and 1,2 propylene glycol.

Some examples for oral suitable carrier, diluent, excipient and adjuvant include peanut oil, atoleine, Sodium carboxymethylcellulose, methylcellulose, sodium alginate, Arabic gum, adragant, dextrose, sucrose, sorbierite, sweet dew Alcohol, gelatin and lecithin.In addition, these peroral dosage forms can contain suitable flavoring agent and colorant.It is used when with capsule formulation When, capsule can be with that can delay the compound of disintegration to be coated with, such as glycerin monostearate or distearin.

Adjuvant typically comprises lubricant, emulsifier, thickener, preservative, fungicide and buffer.

The solid dosage forms of oral administration may include people and veterinary pharmaceutical practice in acceptable adhesive, sweetener, collapse Solve agent, diluent, flavoring agent, coating agent, preservative, lubricant and/or delay agent (timedelayagent).Suitable bonding Agent includes Arabic gum, gelatin, cornstarch, adragant, sodium alginate, carboxymethyl cellulose or polyethylene glycol.Suitably Sweetener includes sucrose, lactose, glucose, aspartame or saccharin.Suitable disintegrating agent includes cornstarch, Methyl cellulose Element, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar.Suitable diluent includes lactose, sorb Alcohol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicates or Dicalcium Phosphate.Suitable flavoring agent includes peppermint Oil, wintergreen, cherry, citrus or raspberry flavoring agent.Suitable coating agent include acrylic acid and/or methacrylic acid and/or it Esters polymer or copolymer, wax, fatty alcohol, zein, shellac or seitan.Suitable preservative includes benzoic acid Sodium, vitamin E, alpha-tocopherol, ascorbic acid, methyl p-hydroxybenzoate, propylparaben or sodium hydrogensulfite.It closes Suitable lubricant includes magnesium stearate, stearic acid, enuatrol, sodium chloride or talcum.Suitable delay agent includes that monostearate is sweet Grease or distearin.

Liquid formulation for oral administration also may include liquid-carrier in addition to the drugs that are set out above.Suitable liquid carries Body includes water, oils such as olive oil, peanut oil (peanutoil), sesame oil, sunflower oil, safflower oil, peanut oil (arachisoil), coconut oil, atoleine, ethylene glycol, propylene glycol, polyethylene glycol, ethyl alcohol, propyl alcohol, isopropanol, glycerol, rouge Or mixtures thereof fat alcohol, triglycerides.

Suspension for oral administration can further comprise dispersing agent and/or suspending agent.Suitable suspending agent includes carboxylic first Base sodium cellulosate, methylcellulose, hydroxypropyl methyl cellulose, kollidon, sodium alginate or acetyl alcohol.Properly Dispersing agent include lecithin, such as stearic acid etc fatty acid polyoxyethylene esters, polyoxyethylene sorbitol it is mono- or two- Oleate ,-stearate or-laurate, polyoxyethylene sorbitan be mono- or two-oleates ,-stearate or the-moon Cinnamic acid ester and analog.

Emulsion for oral administration can further comprise one or more emulsifying agents.Suitable emulsifier includes institute above The dispersing agent or natural gum enumerated, such as guar gum, Arabic gum or adragant.

Pharmaceutical composition of the invention can also be with the drug combination of other treatment cervical squamous cell carcinoma, other therapeutic compound It can be administered simultaneously with main active constituent, or even be administered simultaneously in same composition.It can also be with individual composition Or the dosage form different from main active constituent individually gives other therapeutic compounds.

Preferably, the means that gene therapy can be used carry out.For example, can be directly by the promotor of BDKRB2 by such as infusing It the methods of penetrates and to deliver medicine to subject；Alternatively, the ceneme that the promotion for carrying BDKRB2 is adjusted (can be compared by certain approach Such as expression vector or virus) it is delivered on target spot, concrete condition need to be depending on the type of the promotor, these are these Known to the technical staff of field.

Below with reference to specific embodiment further illustrate the present invention, the embodiment of the present invention for explaining only the invention, It is not intended to limit protection scope of the present invention.

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Embodiment 1 screens gene marker relevant to cervical squamous cell carcinoma

1, sample collection

Collect 32 cervical squamous cell carcinoma (CESC) tissues and 18 normal tissues (N), 24 cervical intraepithelial neoplasia (CIN)s (CIN) Tissue, all cases are preoperative not to do any immunosuppressant treatment, radiotherapy and chemotherapy, and all research objects being included in are in receipts Informed consent form is signed before collection sample.The acquirement of above-mentioned all samples passes through the agreement of the committee, organizational ethics.Every group takes 4 Example sample carries out the detection and analysis of gene expression profile, carries out the screening of difference expression gene, and in each group all cases sample Carry out confirmatory experiment.

2, the preparation of RNA sample

The total serum IgE in each group tissue, specific steps reference are extracted using the tissue RNA extracts kit of QIAGEN Book.

3, the quality analysis of RNA sample

The RNA of said extracted is subjected to agarose gel electrophoresis, using Nanodrop2000 to the concentration of mentioned RNA and pure Degree is detected, and agarose gel electrophoresis detects RNA integrality, and Agilent2100 measures RIN value.Single requirement for construction data base RNA is total 5 μ g are measured, concentration >=200ng/ μ L, OD260/280 is between 1.8~2.2.

4, the building of cDNA library

The building of cDNA library, concrete operations are carried out using Illumina TruseqTM RNA sample Prep Kit By specification carries out.

5, it is sequenced

CDNA library is sequenced using Illumina X-Ten microarray dataset, concrete operations by specification carries out.

6, high-throughput transcript profile sequencing data analysis

Bioinformatic analysis is carried out to sequencing result, is analyzed using metaMA packet, p value merges side used in meta analysis Method is inverse normal method, and the screening criteria of difference expression gene is FDR < 0.05.

7, result

RNA-seq is the results show that compared with normal control, and BDKRB2 gene is in cervical squamous cell cancer and on uterine neck Expression quantity in intraepithelial neoplasia tissue is significantly lowered, wherein the expression quantity in cervical squamous cell cancer and cervical intraepithelial neoplasia sample Pathological tissues are compared, and expression quantity is also significantly lowered, and difference has statistical significance, therefore carries out further full-page proof to BDKRB2 This verifying.

The differential expression of embodiment 2QPCR sequence verification BDKRB2 gene

1, large sample QPCR verifying is carried out to BDKRB2 gene differential expression.

2, RNA is extracted

3、QPCR

1) reverse transcription reaction

LncRNA reverse transcription is carried out using FastQ μ ant the first chain of cDNA synthetic agent box (article No.: KR106), is gone first Except genomic DNA reacts, 5 × gDNA B μ ffer, 2.0 μ l is added in test tube, 1 μ g of total serum IgE adds RNase Free ddH₂O Make total volume to 10 μ l, 42 DEG C of heating 3min. in water-bath

By 10 × Fast RT B μ, 2.0 μ l, RT Enzyme Mix of ffer, 1.0 μ l, FQ-RT Primer Mix, 2.0 μ L, RNase Free ddH₂5.0 μ l of O is added in above-mentioned test tube after mixing and is mixed together totally 20 μ l, 42 DEG C of heating in water-bath 15min, 95 DEG C of heating 3min.

2) design of primers

QPCR amplimer, You Bomai are designed according to the coded sequence of BDKRB2 gene and GAPDH gene in Genebank The synthesis of moral biotech firm.Specific primer sequence is as follows:

BDKRB2 gene:

Forward primer is 5 '-GAGTGTCATCACCTTCTG-3 ' (SEQ ID NO.1)；

Reverse primer is 5 '-TCTGGATCTCCTTGAACT-3 ' (SEQ ID NO.2).

GAPDH gene:

Forward primer is 5 '-AATCCCATCACCATCTTCCAG-3 ' (SEQ ID NO.3)；

Reverse primer is 5 '-GAGCCCCAGCCTTCTCCAT-3 ' (SEQ ID NO.4).

3) QPCR amplification is examined

It with SuperReal PreMix Plus (SYBR Green) (article No.: FP205), is expanded, experimental implementation is by production Product specification carries out.

Using 20 μ l reaction systems: 2 × SuperReal PreMix Plus 10 μ l, each 0.6 μ of forward and reverse primer (10 μM) L, 5 × ROX Reference Dye △ 2 μ l, 2 μ l of DNA profiling, 4.8 μ l of sterile purified water.3 parallel pipes are arranged in each sample, All amplified reactions are repeated three times the above reliability to guarantee result.

Amplification program are as follows: 95 ° of 15min, (95 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 32s) × 40 circulations, 95 DEG C of 15s, 60 DEG C 60s, 95 DEG C of 15s).

4) screening of cDNA template concentrations

After each sample cDNA is mixed, 10 times of gradients (10,100,1000,10000,100000 times) are carried out as template Dilution, sample respectively takes 2 μ l to make template after dilution, is expanded respectively with target gene primer and reference gene primer, while 60-95 DEG C of progress melt curve analysis analysis carries out the screening of template concentrations according to amplification efficiency height and the unimodal principle of solubility curve.

According to solubility curve, it can be seen that when 10 times of dilutions of carry out of cDNA, the amplification efficiency of PCR is higher, and dissolution is bent Line is unimodal relatively good.

5) sample RealTime PCR is detected

2 μ l will be taken to make template after 10 times of each sample cDNA dilutions, respectively with target gene primer and reference gene primer into Row amplification.Simultaneously in 60-95 DEG C of progresss solubility curve analysis, purpose band is determined by melt curve analysis analysis and electrophoresis, 2^-ΔΔCT Method carries out relative quantification.

4, result

As a result as shown in Figure 1, compared with normal tissue, in cervical squamous cell cancer and cervical intraepithelial neoplasia (CIN) tissue BDKRB2 expression quantity is significantly lowered, and wherein cervical intraepithelial neoplasia (CIN) tissue is presented conspicuousness and lowers compared with normal tissue, Cervical squamous cell cancer is presented conspicuousness and lowers, difference all has statistical significance compared with cervical intraepithelial neoplasia (CIN) tissue (P<0.05)。

The overexpression of 3 BDKRB2 gene of embodiment

Specific primer is designed according to BDKRB2 gene sequence information in NCBI, amplification is carried out and obtains corresponding gene, by base Because being transformed into DH5 α competent cell after being attached respectively with expression vector pEGFP-C1 after the recycling of segment glue, picking is positive Clone obtains pEGFP-C1-BDKRB2 recombinant plasmid after PCR and sequencing identification.

1, cell culture

Cervical squamous cell carcinoma cell (Hela) is with the RPIM-1640 culture medium containing 10% fetal calf serum and 1%P/S in 37 DEG C, 5% CO₂Incubator in cultivate, when cell it is long to 80%~90% when, trypsase conventional digestion of the use 0.25% containing EDTA passes Generation.

2, it transfects

Experiment is divided into 3 groups, respectively control group (Hela), blank control group (transfection pEGFP-C1), experimental group (transfection PEGFP-C1-BDKRB2), turned according to 2000 transfection reagent specification of the lipofectamine of invitrogen company Dye.Steps are as follows:

1) cell dissociation of logarithmic growth phase gently blows and beats into single cell suspension, the not antibiotic culture medium inoculated of use in 6 orifice plates, the cell densities of 6 orifice plates is up to 2 × 10⁵A/hole, when cell confluency carries out transfection assay up to 40%-60%；

2) next day transfects, and takes 10 μ l Lipofectamine 2000 to be diluted in Opti-MEMI of the 200 μ l without serum and trains Base is supported, 5min is stored at room temperature；

3) 4 μ g plasmids is taken to be dissolved in the Opti-MEM I culture medium of 200 μ l serum-frees；

4) above two dilution is uniformly mixed, is incubated at room temperature 20 minutes；The ratio of plasmid and transfection reagent is every hole 4 μg:10μl；

5) cell in 6 orifice plates is replaced into the fresh RPIM-1640 culture medium of 1.6ml.Above-mentioned suspension is added separately to 6 holes In plate, every hole culture solution final volume is 2ml；

6) the replacement fresh complete culture solution of 2ml continues to cultivate after transfecting 6 hours；

7) 72h collects cell, extracts RNA, carries out QPCR detection.

3, QPCR detects the transcriptional level of BDKRB2 gene

1) extraction of cell total rna

The extraction of cell total rna is carried out using the cell RNA extracts kit of QIAGEN, specific steps, which are detailed in kit, to be said Bright book

3.2 reverse transcription steps are the same as embodiment 2.

3.3QPCR amplification step is the same as embodiment 2.

4, statistical method

Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD, Using SPSS18.0 statistical software come difference for statistical analysis, being overexpressed between BDKRB2 gene expression panel and control group It is examined using t, it is believed that there is statistical significance as P < 0.05.

5, result

As a result such as Fig. 2 is shown, compared to the control group Hela and transfection zero load pEGFP-C1, experimental group pEGFP-C1-BDKRB2 The expression of BDKRB2 gene can be dramatically increased, difference has statistical significance.

4 CCK-8 method of embodiment detects cervical squamous cell carcinoma cell-proliferation activity

1, with 0.25% trypsin digestion and cell after transfecting 6 hours, list is made into the culture solution containing 10% fetal calf serum A cell suspension (1 × 10⁴/ hole), (100 hole μ l/) is inoculated in 96 well culture plates, and every group sets 6 multiple holes, while setting up nothing The culture solution blank control of cell.

2, it is separately added into cell Proliferation detection reagent CCK-8 in detection time point (0h, for 24 hours, 48h, 72h, 96h, 120h), Working concentration is 1:10, i.e. 10 μ l CCK-8 are added in 100 μ l culture solutions；

3, at 37 DEG C, 5%CO₂It after being incubated for 1h in incubator, is detected, microplate reader reads OD 450nm.

4, result judges: 0h after transfection, for 24 hours, 48h, 72h, 96h, 120h observe the proliferation of Hela cell using microplate reader Activity, absorption photometric value (OD value) of the cell at wavelength 450nm indicates the cell quantity for being in vegetative state, with cell-free Culture solution group OD value is control.

5, result

As a result it as shown in figure 3, the cell Proliferation of the experimental group of transfection pEGFP-C1-BDKRB2 substantially reduces, prompts to change The expression of BDKRB2 can change the proliferative capacity of cervical squamous cell carcinoma cell, illustrate the proliferation of BDKRB2 Yu cervical squamous cell carcinoma cell Correlation prompts BDKRB2 to can be used as the treatment that target is applied to cervical squamous cell carcinoma.

5 Transwell method of embodiment detects cervical squamous cell carcinoma cell migration and invasion

1, cell migration ability detects

1) migration the previous day, complete medium was added in orifice plate, is put into cell and is placed in incubator overnight；

2) cell after transfection 48h is subjected to Nature enemy, cell count is collected, with the serum free medium of 0.2%BSA It is made 1 × 10⁵Suspension.

3) take 200 μ l cell suspension inoculations in the small indoor culture of Transwell.

4) cell is taken out, remaining liq is blotted and is placed on the fixed 30min of 70% methanol, wipe the thin of upper chamber face with cotton swab Born of the same parents.

5) cell immerses 0.4% violet staining 10min, PBS twice of cleaning, randomly selects the visual field under microscope and counts film The cell number of bottom surface.

2, cell invasion ability detects

1) matrigel, each 24 hole suspension type cell bottom are diluted with 1640 1:8 of RPIM on ice on the day before Matrigel Portion film upper chamber face adds the 60 μ l dried for standby of matrigel after dilution.

2) aquation basilar memebrane: 50 serum-free mediums of the μ l containing 10g/LBSA are added in every hole, and 37 DEG C, 30min, culture is sucked out Residual liquid in plate.

3) cell after transfection 48h is subjected to Nature enemy, cell count is collected, with the serum free medium of 0.2%BSA It is made 1 × 10⁵Suspension.

3, data processing

Statistical analysis is carried out to data with SPSS18.0 software.Measurement data is indicated with mean ± standard deviation.Multiple samples This mean compares using one-way analysis of variance, and P < 0.05 is that difference is statistically significant.

4, result

As a result as shown in figure 4, the cell migration of experimental group and invasion number relatively transfect lacking for empty plasmid respectively, illustrated table Migration and the invasive ability that cervical squamous cell carcinoma cell is reduced up to BDKRB2 gene prompt BDKRB2 to can be used as molecular target applied to palace The treatment of carcinoma of neck transfer and invasion.

The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.

Sequence table

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Claims

1. a kind of reagent, which is characterized in that the reagent is able to detect BDKRB2 gene or the level of its expression product.

2. reagent according to claim 1, which is characterized in that probe, primer or antibody including being directed to BDKRB2.

3. reagent according to claim 2, which is characterized in that for primer sequence such as NO.1~2 SEQ ID of BDKRB2 It is shown.

4. a kind of kit, which is characterized in that the kit includes the described in any item reagents of claim 1-3.

5. a kind of chip, which is characterized in that the chip includes the described in any item reagents of claim 1-3.

6. a kind of nucleic acid film item, which is characterized in that the nucleic acid film item includes the described in any item reagents of claim 1-3.

7. a kind of composition, which is characterized in that the composition includes the promotor of BDKRB2 and/or pharmaceutically acceptable Carrier.

8. composition according to claim 7, which is characterized in that the promotor is the carrier containing BDKRB2.

9. a kind of method of the drug candidate of screening treatment cervical squamous cell carcinoma, which is characterized in that the described method includes:

The cultivating system of expression or the albumen containing BDKRB2 gene or its coding is handled with substance to be screened；With the detection body The expression or activity of the albumen of BDKRB2 gene or its coding in system；

Wherein, if the substance to be screened can promote the level or expression activity of BDKRB2 gene, show the object to be screened Matter is the drug candidate for treating cervical squamous cell carcinoma.

10. following described in any item applications:

A. the described in any item reagents of claim 1-3, kit as claimed in claim 4, chip described in claim 5, Nucleic acid film item as claimed in claim 6 answering in the product that preparation early diagnoses cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma With；

B. composition described in claim 7 or 8 preparation treatment cervical squamous cell carcinoma and its transfer, invasion drug in application；