CN106319070A - Application of NEURL1B in preparation of product for diagnosing prostatic carcinoma - Google Patents
Application of NEURL1B in preparation of product for diagnosing prostatic carcinoma Download PDFInfo
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Abstract
The invention discloses an application of NEURL1B or an expression product thereof in preparation of a product for diagnosing prostatic carcinoma. The invention discloses mRNA NEURL1B related to the prostatic carcinoma. The condition that the expression of the NEURL1B or the mRNA expression product in prostatic carcinoma tissue is up-regulated is further confirmed. The invention further discloses a diagnostic kit for detecting the prostatic carcinoma. The kit comprises specific-amplified prostatic carcinoma-related mRNA primers and a description, wherein the prostatic carcinoma-related mRNA is NEURL1B. The invention further discloses an application of an NEURL1B depressant in preparation of pharmaceutical compositions for preventing, relieving and/or treating the prostatic carcinoma. Through detecting the prostatic carcinoma by using the NEURL1B, early detection can be rapidly and effectively achieved, and therapy target points and important basis are provided for clinical applications such as gene therapy and drug therapy.
Description
Technical field
The present invention relates to biomedicine field, be specifically related to people NEURL1B answering in preparing prostate cancer diagnosis product
With.
Background technology
Carcinoma of prostate is one of common male reproductive system malignant tumor.In world wide, prostate-cancer incidence exists
Occupying second in all malignant tumor of male, the sickness rate in U.S.'s carcinoma of prostate alreadys more than pulmonary carcinoma, becomes first harm
The tumor of men's health.The sickness rate of Asia carcinoma of prostate is well below American-European countries, but presents ascendant trend in recent years, and increases
Long rapider than European and American developed countries.Patients with prostate cancer is mainly elderly men, typically at 50 years old with sequela, and 95%
Being born in the elderly men of more than 60 years old, incidence rate increases the most with age.Carcinoma of prostate is the most without any disease
Shape, even if uncomfortable, is also not enough to cause the attention of patient, when tumor increases urethra, the most often increases with prostate
Life is obscured mutually.First patient in China about 80% finds that metastasis focus just finds carcinoma of prostate.Now, pathological changes has reached late
Phase, prognosis mala.
The clinical diagnostic modalities of carcinoma of prostate currently mainly has serum PSA (PSA) detection, rectum to refer to
Inspection, the detection of endorectal ultrasonography ripple, living tissue pathologic finding etc..PSA (Prostate Specific Antigen) is prostata tissue
Specific antigen, is understood PSA by prostatic anatomical structure and enters in blood and urine by prostate duct, some cases or
Under physiological conditions, PSA can enter in blood, such as prostatitis, urine retention, prostate infection, prostatic hyperplasia and prostate
By detection Serum PSA level, cancers etc., therefore predict that carcinoma of prostate has certain false positive rate.From the beginning of 1991, inspection
Surveying PSA content in serum and be used for predicting carcinoma of prostate, it is positive that content is considered carcinoma of prostate higher than 4ng/mL, sensitivity 79%,
Specificity 20%-59%, average sensitivity about 33%, in concentration 4-10ng/mL gray area part, specificity is minimum, at this moment
Generally require and be determined by invasive aspiration biopsy, bring considerable distress and spirit and financial burden to patient.So
PSA can not be preferably as the mark of diagnosis of prostate cancer.Digital rectal examination is the method for practicality the simplest, most economical, main
The forefinger of doctor to be passed through touches prostate, in order to find a lot of asymptomatic patients with prostate cancer, it is possible to obtains and examines in early days
Chance that is disconnected and that effect a radical cure.But all there is limitation in above method.The limitation of such as digital rectal examination is mainly at 4 aspects: (1)
When patient's prostate lump is little, easy to cause missed diagnosis；(2) enlargement of some patients carcinoma of prostate is inconspicuous, but is already belonging to late period, is difficult to root
Control；(3) patient's rectum can not use this to detect when having illness；(4) may have during doctors experience deficiency fail to pinpoint a disease in diagnosis or mistaken diagnosis can
Energy.
Currently for Late-stage Prostate Cancer patient, the weak effect of operative treatment, great majority use Drug therapy.According to
The different state of an illness can use chemotherapy, fluconazole ear drops, radionuclide internal radiotherapy and various therapy
Integrated application etc..But, radiotherapy and chemotherapy medicine acts not only on tumor, it is also possible to act on the tissue of tumor adjacent healthy, thus
While killing tumor, also bring the biggest side effect to body, finally affect the therapeutic effect to tumor.
Therefore, this area is in the urgent need to developing the Specific marker of carly fruit drop carcinoma of prostate, and develops carcinoma of prostate
Targeted drug.
Summary of the invention
In order to realize the early discovery of carcinoma of prostate, early intervention, it is an object of the invention to provide a kind of new prostatitis
Adenocarcinoma is correlated with mRNA, and the expression product of this mRNA.
The present invention also aims to provide carcinoma of prostate to be correlated with mRNA or its expression product for preparing carcinoma of prostate
Application in diagnostic products.
For achieving the above object, present invention firstly provides NEURL1B or its expression product examining for preparing carcinoma of prostate
Application in pregnancy ceased product.
Preferably, the expression product of described NEURL1B or this mRNA raises at expression in prostate cancer.
Further, described diagnostic products includes detectable or test kit.
Preferably, described detectable includes the specific primer of NEURL1B, specific antibody, probe and/or chip.
Wherein, described detectable includes: the specific antibody of (1) anti-NEURL1B albumen；And/or (2) specific amplification
The primer of NEURL1B.Wherein, the specific antibody of described anti-NEURL1B albumen can use the antibody of commercially available product, it is possible to by known
Method manufacture.
Further, the invention provides a kind of diagnostic kit for detecting carcinoma of prostate, described test kit includes
Specific amplification carcinoma of prostate is correlated with the primer of mRNA and description, and the described carcinoma of prostate mRNA that is correlated with is NEURL1B.Preferably
Ground, described primer has the primer shown in SEQ ID NO:1 and SEQ ID NO:2.
Preferably, dated herein below in described description:
When NEURL1B expression E1 and normal prostate cell or tissue in the prostate gland cancer cell or tissue of detection object
Ratio >=2 of NEURL1B expression E2, then point out the probability of this detection object carcinoma of prostate higher than general population.Described E1
It is prostate gland cancer cell or the NEURL1B expression of tissue of detection object；Described E2 is the normal prostatic of normal population
The NEURL1B expression of cell or tissue.Described normal prostate cell or tissue include prostatic cell or the group on cancer side
Knit.Described expression is the relative expression quantity relative to crt gene (such as GAPDH).
Preferably, described test kit also includes 10 × Buffer, dNTP, MgCl2, Taq enzyme and SYBR Green fluorescence dye
Material.
Further, the invention provides a kind of NEURL1B inhibitor, described inhibitor include NEURL1B antibody,
The antisense RNA of NEURL1B nucleic acid, the activity inhibitor of microRNA, siRNA, shRNA and NEURL1B.
Preferably, the inhibitor of described treatment carcinoma of prostate contain following in one group or several groups of siRNA:SEQ ID
NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.More preferably
, it is SEQ ID NO.9 and SEQ ID NO.10 that the inhibitor of described treatment carcinoma of prostate contains siRNA sequence.
Further, above-mentioned inhibitor answering in preparation prevents, alleviates and/or treat the pharmaceutical composition of carcinoma of prostate
With.
Preferably, described pharmaceutical composition includes that NEURL1B inhibitor is as active component and pharmaceutically acceptable
Carrier.Described carrier includes (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
Preferably, described medicine is by being administered selected from the application method of lower group: oral, intravenous injection, muscle note
Penetrate, subcutaneous injection, sublingual administration, rectal perfusion, nasal spray, mouthspray, local skin or whole body transdermal administration.Described
The preparation of medicine is selected from lower group: tablet, capsule, injection, granule, spray.Described NEURL1B inhibitor with
The dosage of 0.01-20mg/kg body weight is applied to mammal at (each or every day).Described mammal includes people, mice, big
Mus, more preferably, for people.
Beneficial effects of the present invention is as follows:
The invention discloses a kind of NEURL1B relevant to carcinoma of prostate, and be further characterized by this NEURL1B or this mRNA
Expression product expression in prostate cancer raise.This mRNA detection carcinoma of prostate is utilized to do fast and effectively
To detection in early days, and provide therapy target and important evidence for the clinical practice such as gene therapy, Drug therapy.
Accompanying drawing explanation
NEURL1B protein expression situation in carcinoma of prostate forming process in Fig. 1 WB analysis Mice Body.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.If not specializing, embodiment
The conventional means that technological means used by is well known to those skilled in the art, reagent used can be commercially available.
The experimental technique of unreceipted actual conditions, usually this area conventional method in embodiment, as according to normal condition
Such as Sambrook et al., molecular cloning, the described condition in laboratory manual (third edition) (Science Press, 2002), or according to
Condition proposed by reagent manufacturing firm.
The present inventor carries out high flux transcript profile to the mRNA of prostate cancer tissue sample and cancer beside organism's sample
Order-checking, carries out genescreen by bioinformatics method, picks out candidate mRNA NEURL1B, in existing research not
The report that NEURL1B is relevant with carcinoma of prostate, further, inventor carried out molecular biology method checking it was confirmed
NEURL1B is up-regulated in prostate gland cancer cell.
The NEURL1B of the present invention is known mRNA before making the present invention, and its essential information is as follows: Genbank accession number:
NCBI reference sequences: NM_001308178.1, derives from human genome.
The present invention also use RT-PCR method and Western-Blot method detect above-mentioned mRNA at prostate cancer tissue and
The expression of Carcinoma side normal tissue, and demonstrate this mRNA up-regulated in carcinoma of prostate.
Terms used herein " up-regulated " refers to the sequence corresponding to expressed gene, wherein the measurement card of sequence amount
Bright, and from normal individual or from being determined by stages of prostate cancer the individuality identifying morbid state different with carcinoma of prostate
Same gene in the biological sample separated is compared, and described gene is from suffering from carcinoma of prostate or being determined by stages of prostate cancer
Carcinoma of prostate identified that the expression in the biological sample separated in the individuality of morbid state increases.According to the present invention,
" up-regulated " refer to by the inventive method intensity for hybridization measure at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,
8%, 9%, 10% or higher express and increase, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more
High or higher than 1 times, it is up to 2 times, 3 times, 4 times, 5 times, 10 times, 50 times, 100 times or higher.
Embodiment 1 high-flux sequence screening difference expression gene
1, sampling
In BJ Union Hospital's urological surgery, tissue specimen is obtained during taking in December, 2015 in October, 2012 to
27 examples, all specimen all confirm through pathological examination, and wherein cancer beside organism's sample 8 example, carcinoma of prostate specimen 19 example, after numbering
Put-80 DEG C of cryogenic refrigerators to preserve.
2, tissue samples is carried out Total RNAs extraction
UseReagent (invitrogen, article No. 15596-018) carries out sample rna extraction, experiment behaviour
Making to carry out by product description, concrete operations are as follows:
After collecting sample, frozen mortar tissue being put into pre-cooling after liquid nitrogen, taking-up is ground, sample to be organized
After this is powdered:
1. Trizol, room temperature preservation 5 minutes are added；
2. adding chloroform 0.2mL, use forced oscillation centrifuge tube, fully mix, ambient temperatare puts 5 minutes-10 minutes；
3. 12000rpm high speed centrifugation draws upper strata aqueous phase (inhale 70%) in another new centrifuge tube pipe after 15 minutes, notes
It is not drawn onto the protein substance between two-layer aqueous phase.Move into new pipe, add isopyknic-20 DEG C of pre-cold isopropanols, the most reverse
Mixing, is placed in 10 minutes on ice；
4. 12000rpm carefully discarded supernatant at a high speed after 15 minutes, added 75% in the ratio of 1mL/mL Trizol
Paint precipitation (4 DEG C of preservations) washed by DEPC ethanol, washes paint precipitate, vibration mixing, 12000rpm high speed centrifugation 5 minutes at 4 DEG C；
5. discard ethanol liquid, ambient temperatare put 5 minutes fully to dry precipitation, add the water dissolution that processed of DEPC and sink
Form sediment；
6. RNA purity and concentration are measured with Nanodrop2000 ultraviolet spectrophotometer, frozen in-80 DEG C.RNA mass is sentenced
Calibration standard: the OD260/OD280 value of RNA sample is between 1.7-2.2；Total serum IgE electrophoresis pattern has 28S, 18S band clearly；
70 DEG C of water bath heat preservation electrophoresis patterns after 1 hour and the collection of illustrative plates no significant difference before water bath heat preservation.
3, the quality analysis of RNA sample
RNA extract after agarose gel electrophoresis, from electrophoresis result can with preliminary judgement extract RNA sample up-to-standard with
No, if to may be used for further transcriptome analysis.And then by NanoDrop1000 spectrophotometer detection RNA sample
Extraction situation, the sample requirement of RNA-seq order-checking: OD260/OD280 is 1.8-2.2.
4, high-flux sequence
Order-checking platform is the HiSeq 2500 high-flux sequence platform of Illumina company, carries out the high flux transcript profile degree of depth
Order-checking, after order-checking, we use Fast-QC (http://www.bioinformatics.babraham.ac.uk/projects/
Fastqc/) software carries out total evaluation to the quality of sequencing data, and the mass value including base is distributed, and the position of mass value is divided
Cloth, G/C content, PCR duplication content, the frequency etc. of kmer.When differential genes expression analysis, according to obtaining
FPKM value, use internationally recognized algorithm EBSeq to carry out differential screening.Wherein, during screening, LOG2FC>1 or<-1, FDR<
0.05.In order to be better understood from the function of difference expression gene, we difference expression gene has been carried out Gene Ontology and
Signal path is analyzed, and difference expression gene carries out functional annotation and protein interaction analysis of network, in view of above number
According to the result analyzed, in conjunction with document, we have screened differential expression NEURL1B, and this mRNA expresses in prostate cancer tissue sample
Raise.
Embodiment 2 RT-PCR checking prostate cancer tissue and cancer beside organism's NEURL1B expression
1, material
Choose prostate cancer tissue sample 34 example and cancer beside organism's sample 8 example, it is grouped and numbers.All samples
All confirm through pathological examination.
2, method
2.1 pairs of tissue samples carry out Total RNAs extraction, with the extracting method of embodiment 1.
2.2 reverse transcription synthesis cDNA
UseIII Reverse Transcriptase (invitrogen, article No. 18080-044)
Carrying out cDNA reverse transcription, experimental implementation is carried out by product description, and concrete operations are as follows:
Use Reverse Transcriptase kit, with RT Buffer, l μ g total serum IgE is carried out converse record and synthesize cDNA.Use 25 μ L
Reaction system, each sample takes 1 μ g total serum IgE as template ribonucleic acid.It is saved in-20 DEG C after the cDNA diluted sample 10 times of acquisition
Refrigerator is standby.
2.3 Real-Time PCR
2.3.1 instrument and the method for analysis
With ABI 7500 type quantitative real time PCR Instrument, use 2-ΔΔCtMethod carries out data relative quantitative assay.
2.3.2 design of primers
Using primer premier5.0 primer-design software, gene order is with reference to NCBI:NM_001308178.1
(NEURL1B), interior participation in the election GAPDH, synthesized by invitrogen company after design of primers.Concrete primer sequence is as follows:
Table 1 primer sequence
Operating process is as follows:
Table 2 Real Time reaction system
(1) reaction system: use PowerGreen PCR Master Mix (invitrogen, article No.
4367659) expanding, experimental implementation is carried out by product description.
Amplification program is: 95 DEG C of 5min, (95 DEG C of 15sec, 60 DEG C of 45sec, 72 DEG C of 35sec) × 40 circulations.
(2) primer screening
After being mixed by each sample cDNA, carrying out 5 times of gradient dilutions as template, after dilution, sample respectively takes 2 μ L and makees template,
Expand with genes of interest primer and reference gene primer respectively, carry out melt curve analysis analysis at 60-95 DEG C, according to expansion simultaneously
Increasing Efficiency is high and the unimodal principle of solubility curve carries out primer screening.
(3) sample Real Time-PCR detection
Take 2 μ L after each sample cDNA 10 times being diluted and make template, enter with genes of interest primer and reference gene primer respectively
Row amplification.Carry out solubility curve analysis at 60-95 DEG C simultaneously.
3, experimental result
Real-time quantitative PCR amplification curve flex point understands, amplification curve entirety collimation is good, shows the amplification effect of each reaction tube
Rate is close, and the limit is flat and nothing raises up now, and exponent phase slope is relatively big, illustrates that amplification efficiency is higher；Sample amplified production is molten
Solution curve is all unimodal, illustrates that amplified production only has one, for specific amplification；Relative quantification formula according to qRT-PCR: 2-ΔΔCt× 100%, compare NEURL1B expression in prostate cancer tissue and cancer beside organism.Result shows: qRT-PCR
Stable amplification result, wherein NEURL1B suffers from expression is cancer beside organism 2 times in organizing, result above in carcinoma of prostate
Demonstrate high flux transcript profile and express confluence analysis NEURL1B result of up-regulated in patients with prostate cancer of data.
The structure of embodiment 3 carcinoma of prostate mouse model
The foundation of carcinoma of prostate mouse model has multiple method, such as spontaneous and induction model, xenogenesis implant cast, transgenic
Animal model, Gene Knock-Out Animal Model model etc..Correlational study shows, is noted by the cell suspension of PC-3 LNCaP
Enter subcutaneous with mouse armpit.People's tumour transplatation thing of nude mice is closest to the experimental model of human tumor self character, before being research
The preferable modeling method of row adenocarcinoma.
1, material
Human prostate tumor LNCaP cell strain be purchased from ATTC company of the U.S., SPF level nude mice, male, 3-5 week old, body weight
18g-23g, is provided by Concord Hospital's laboratory center.
2, method
According to random assortment table, laboratory animal is divided into 6 groups, and often group is 5, indicates that experimental group is Al, A2, A3 respectively,
Indicate that matched group is C1, C2, C3 respectively.
2.1 cells are cultivated
LNCaP cell is incubated in the F-12 culture medium (Gibco company) of the hyclone containing 12%, and incubator temperature is protected
Hold 37 DEG C, 5%CO2Culture environment, changes liquid for 1-2 days 1 time, when cell is in exponential phase and cell concentration is about 5x107Individual/
ML is standby.
2.2 cell transplantation
The LNCaP cell of exponential phase is collected with after 0.25% trypsinization, brine.Take a small amount of cell to enter
Row Trypan Blue detects its existence motility rate, and qualified then concentrating cells to its concentration is close to 1x109Individual/mL.It is anesthetized with ether
Nude mice, cuts off fur with eye scissors in oxter, both sides around, exposes skin.The concentrating cells of 300 μ L is drawn with 1mL syringe
At injection nude mice axillary fossa.Day by day transplantation site is observed to understand incubation period and the speed of growth of tumor growth.Al, A2, A3 group is noted
Entering cell and select liquor 0.3mL, C1, C2, C3 group injects the normal saline solution of equivalent.
Sampling and process: the 4th weekend (A1) after modeling, at the 6th weekend (A2), the 8th weekend (A3), de-neck was put to death little
Mus, dissects mice and separates prostate tumor tissue and normal prostate tissue, uses filter paper to wipe the blood taken out at specimen away, puts
In PBS solution, all specimen are inserted in-80 DEG C of refrigerators and preserve.
The protein expression level of NEURL1B in embodiment 4 WB method detection prostate cancer tissue
One, protein example is prepared and quantitative
1, RIPA lysate (Beyotime) carries out protein example and prepares, and operating procedure is as follows:
Tissue samples is taken out from refrigerator, by the structural PBS solution of filter paper wiping, weighs weight, and tissue is placed in
In mechanical tissue homogenizers, the lysate that 1:10 tissue adds respective volume than the bulking value ratio of lysate is homogenized,
10000-14000g is centrifuged 3-5 minute, takes supernatant, adds sample buffer strength mixing by 1:1 and is placed on the water-bath of 100 degree
Case heating in water bath 3-5 minute, 10000g is centrifuged 10 minutes, takes supernatant, proceeded in the test tube of another cleaning.
2, BCA determination of protein concentration test kit is utilized to carry out total protein quantitative
Using health is century trace BCA protein quantification test kit (article No.: CW2011), and concrete steps are shown in its description.
Two, SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
1, protein example degeneration:
A) according to BCA determination of protein concentration result, each gel well adds the total protein extraction of equal in quality
Thing.The ratio of 0.25 microlitre albumen sample-loading buffer, mixed protein sample and albumen loading is added according to every 1 microlitre protein sample
Buffer (5x).
B) 100 DEG C or boiling water bath heat 3-5 minute, with abundant Denatured protein.
C), after being cooled to room temperature, directly it is loaded in SDS-PAGE glue well.
2, prepared by offset plate:
The miniature vertical plate electrophoresis device using Bio-Rad company prepares the gel that 0.75mm is thick, and book installs as directed
After glass plate, first preparing the separation gel of 5mL 10% in small beaker, formula is as follows:
Table 3 separation gel formula
| Component | Consumption |
| 30% acrylamide solution | 1.7mL |
| Tris-HCl (1.5M, pH8.8) | 1.3mL |
| 10%SDS | 0.05mL |
| 10%AP | 0.05mL |
| TEMED | 0.002mL |
| Sterilizing ddH2O | It is supplemented to 5mL |
Encapsulating immediately after mixing, then adds 1mL distilled water and covers, and ambient temperatare puts about 30min after glue is polymerized, with distillation
Wash 2-3 time, then blot with filter paper.Then preparing the concentration glue of 2mL 5%, formula is as follows:
Table 4 concentrates glue formula
| Component | Consumption |
| 30% acrylamide solution | 0.33mL |
| Tris-HCl (1.0M, pH6.8) | 0.25mL |
| 10%SDS | 0.02mL |
| 10%AP | 0.02mL |
| TEMED | 0.002mL |
| Sterilizing ddH2O | It is supplemented to 2mL |
Encapsulating immediately after mixing, inserts sample comb, it is to avoid produce bubble, after gelling is solid, takes out sample comb, rear distillation
Water and 1x protein electrophoresis buffer successively lavage specimens sample wells.
Three, loading and electrophoresis
Being contained on electrophoretic apparatus by gel slab, fill it up with lx protein electrophoresis buffer in inside groove, in water jacket, lx protein electrophoresis delays
Rush liquid and should exceed platinum filament, in order loading.Protein quality standard protein gradient is added in end swimming lane.Blueness dye during electrophoresis
Expect to reach and electrophoresis near the bottom end of glue, can be stopped.
Four, Western blotting
1, PAGE gel electrophoretic separation albumen is the most first carried out.
2, NC film, filter paper, foam rubber cushion are soaked with transfer buffer in advance.SDS-PAGE takes out gel after terminating, and removes dense
Contracting glue, rinses the several seconds in Tris/ glycine buffer, is subsequently placed in transfer buffer and soaks 15-30min.Open electricity to turn
Print folder, what every side pad lastblock was special soaks saturating foam rubber cushion with transfer buffer, respectively puts one piece of saturating filter paper of transfer immersion,
Filter paper identical with foam rubber cushion size or with NC film, gel size is identical, is lain in by gel on cathode side filter paper, finally will
NC film lies on gel, removes bubble removing, clips electricity transfer folder.Fill it up with electricity transfer liquid at electrophoresis tank, insert electricity transfer folder, by electricity
Swimming groove puts into refrigerator (refrigerator pre-cooling to be put into before electricity transfer liquid), connects electrode, turn-on current, the NC film of transfer folder
The positive pole of reply electrophoresis tank.
3, close: rinse once with 1xTBS.Add the alipoidic milk power TBS Block buffer containing 5%, be placed in shaken cultivation
Case is closed；
4, an anti-hybridization: abandon confining liquid, adds with the one of an anti-diluted anti-(Anti-NEURL1B antibody
(ab156988)) hybridization solution, is placed in 4 DEG C of hybridized overnight, within second day, hybridizes in shaken cultivation case；
5, reclaim an anti-hybridization solution, wash film 3 times with TBST；
6, TBST is abandoned, two anti-(Goat Anti-Rabbit IgG, the HRP that addition dilutes with Block buffer
Conjugated (CW0103)) hybridization solution, it is placed in shaken cultivation case and hybridizes；
7, abandon two anti-solution, wash film 3 times with TBST；
8, ECL chemiluminescence and image acquisition and analysis: (health is century according to high sensitivity chemistry luminescence detection kit
Article No. CW0049B), concrete steps are with reference to description.
9, carrying out data normalization using β-Actin as internal reference, NEURL1B in cancer beside organism is as sample for reference, meter
Calculate the relative expression levels of NEURL1B albumen in experimental group.
Five, experimental result
Within the 4th week after modeling, put to death A1 group and C1 group respectively, within the 6th week, put to death A2 group and C2 group, the 8th week execution A3 group and C3
Group obtain sample detection result show, experimental group A1, A2, A3 group compared with matched group C1, C2 and C3 group, NEURL1B albumen
Expressing significantly raised (P < 0.01), in matched group C1, C2 and C3 group, NEURL1B protein expression is without significant difference, but experimental group A1,
In A2, A3 group, NEURL1B protein expression difference is notable, wherein A1 < A2 < A3 (P < 0.01).Referring specifically to shown in Fig. 1.
Embodiment 5 RNAi interference NEURL1B expression and the impact on prostate gland cancer cell
One, material
1, cell derived
Human Prostate Cancer PC-3 Cell Line strain is purchased from ATCC company of the U.S.
2, siRNA design and synthesis
According to Photographing On-line software siDirect version 2.0 (http://design.rnai.jp/), according to gene
Sequence, with reference to NCBI:NM_001308178.1 (NEURL1B), designs corresponding siRNA, and particular sequence is shown in Table 5.It is sent to after design
Synesis Company synthesizes.
Table 5 siRNA sequence list
Two, experimental technique
1, cell packet
C group: blank group；C1 group: transfection liposome group；C2 group: transfect nonspecific siRNA-NC group；S1, S2,
S3 group: transfect specific siRNA group.
2, transfection
According to LipofectamineTMThe step that 2000Transfection Reagent provides is carried out.
(1) during PC-3 cell is incubated at F-12 culture medium, in culture medium containing 10% new fetal calf serum (Gibco company),
1%L-glutamine, 100U.mL-1Penicillin, 100mg.L-1Streptomycin.In 37 DEG C, 5%CO2Incubator is cultivated, selects logarithm
Trophophase cell is tested.
(2) by 5x104Cell is seeded on 6 orifice plates, and these, should be able to be in 24 hours for the cell quantity of initial vaccination
Cell confluency is made to reach 70%；
(3) siRNA-Lipofectamine is preparedTM2000 complex:
A. dilute 5 μ L LipofectamineTM 2000 with 250 μ L Opti-MEM, mix gently, incubated at room temperature 5 points
Clock.
B. test each group to take 7.5 μ L siRNA respectively and add in 250 μ L Opti-MEMI and be diluted, and be shaken gently for by
Its mixing；
C. after hatching 5 minutes, by siRNA and Lipofectamine of dilutionTM20 are at room temperature hatched after 2000 mixing
Minute.
(4) each hole in culture plate adds cell, culture medium and siRNA-LipofectamineTM2000 is multiple
Compound.Then it is shaken gently for culture plate, makes them be sufficiently mixed；
(5) 37 DEG C it are placed on, CO2Hatch in incubator 48 hours, observation of cell transfection quantity, inspection under fluorescence microscope
Survey transfection efficiency；
(6) transfection efficiency is the ratio of cryptoscope and the cell quantity in the light microscopic visual field, and the transfection efficiency of cell all reaches
More than 90%, side can carry out follow-up experiment.Computing formula is as follows:
Cell quantity x100% under the quantity/same field of view of the transfection efficiency=cell that fluoresces
3, the change that before and after application Real-time PCR method detection transfection, NEURL1B expresses
(1) structure of standard curve: be chosen in 50mL culture bottle the prostate gland cancer cell 1 bottle of normal cultivation, extract
RNA, measures RNA concentration and purity, carries out reverse transcription reaction, and DNA profiling ten times dilution reaction generated is equivalent to
104-101The DNA profiling of copies/ μ L, is separately added into NEURL1B primer and internal reference primer, prepares 25 μ L reaction systems, uses
Real-time PCR amplification instrument, carries out pcr amplification reaction.Obtain the standard curve of NEURL1B and internal reference.
(2) change that before and after Real-time PCR method detection transfection, NEURL1B expresses: extract the RNA of each group of cell,
Measuring RNA concentration and purity, carry out reverse transcription reaction, often group DNA profiling enters the Real-time of NEURL1B and internal reference simultaneously
PCR reacts, and experiment is in triplicate.
(3) PCR primer is carried out agarose gel electrophoresis.
Three, experimental result
Transfecting first generation prostate gland cancer cell with the siRNA and comparison siRNA of 3 NEURL1B respectively, result shows greatly
Green fluorescence is found, it was demonstrated that have been obtained for the transfection of siRNA in prostate gland cancer cell, then glimmering in amount prostate gland cancer cell
Light microscopic and light Microscopic observation prostate gland cancer cell quantity, carry out the detection of transfection efficiency, and result display transfection efficiency all reaches 90%
Above.Real-time PCR result shows, transfects nonspecific siRNA-NC group to NEURL1B table in prostate gland cancer cell
Reach without obvious inhibiting effect, and blank group no difference of science of statistics, 3 NEURL1B-siRNA groups of transfection are all to prostate
In cancerous cell, NEURL1B expresses and plays certain inhibitory action, and NEURL1B-siRNA1 suppression ratio is 56%, NEURL1B-siRNA2
The most obvious with NEURL1B-siRNA3 inhibition, suppression ratio reaches 65% and 72%.
The Proliferation Ability situation of embodiment 6 mtt assay detection prostate gland cancer cell
One, mtt assay experimental procedure
1, in 96 orifice plates, about 1x10 is added according to every hole4Cell, then at 37 DEG C of 5%CO2Under conditions of cultivate 24 little
Time；
2, according to experiment packet (blank group, transfect non-specific siRNA-NC group, transfection NEURL1B-siRNA3
Group, often 6 repetitions of group) continue to cultivate until being suitable for detection；
3, then at 37 DEG C, 5%CO2And 100% continue to hatch appropriate time under conditions of humidity；
4, with Dilution Buffer, 5xMTT is diluted to 1xMTT simultaneously；
5, in every hole, add 50 μ L 1xMTT, and under the conditions of 37 DEG C, hatch 4 hours；
6, after sucking-off supernatant, in every hole, also need to add 150 μ L DMSO, and place it in and carry out on plate shaker
Shake up；
7, microplate reader wavelength is set as 570nm, detects the optical density in each hole.
8, the calculating of cell proliferation inhibition rate: cell proliferation inhibition rate %=(1-experimental port OD value/compared with control cells OD value)
X100%, compared with control cells OD value is the normal OD value cultivating cell hole.
Two, experimental result
Compared with the blank group of untransfected, transfection siRNA-AC cell proliferation has no significant effect (P > 0.05), turns
Dye NEURL1B-siRNA3 can substantially suppress prostate gland cancer cell to breed, and can be used for the preparation of carcinoma of prostate medicine, suppression ratio
Prolongation in time and increase, between group, comparing difference has significance (P < 0.05), the results are shown in Table shown in 6.
The proliferation inhibition rate (%) of prostatic cell respectively organized by table 6
Although, the present invention is described in detail the most with a general description of the specific embodiments, but
On the basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (10)
1.NEURL1B or the application in preparing prostate cancer diagnosis product of its expression product.
Apply the most as claimed in claim 1, it is characterised in that described NEURL1B or its expression product are at prostate cancer tissue
Middle up-regulated.
Apply the most as claimed in claim 1 or 2, it is characterised in that described diagnostic products includes detectable or test kit.
Apply the most as claimed in claim 3, it is characterised in that described detectable includes the specific primer of NEURL1B, spy
Heterogenetic antibody, probe and/or chip.
5. the diagnostic kit being used for detecting carcinoma of prostate, it is characterised in that before described test kit includes specific amplification
Row adenocarcinoma is correlated with the primer of mRNA and description, and the described carcinoma of prostate mRNA that is correlated with is NEURL1B.
6. test kit as claimed in claim 5, it is characterised in that described primer has SEQ ID NO:1 and SEQ ID NO:2
Shown primer.
7. the test kit as described in claim 5 or 6, it is characterised in that described test kit also include 10 × Buffer, dNTP,
MgCl2, Taq enzyme and SYBR Green fluorescent dye.
8. a NEURL1B inhibitor, it is characterised in that described inhibitor includes the antibody of NEURL1B, NEURL1B nucleic acid
Antisense RNA, the activity inhibitor of microRNA, siRNA, shRNA and NEURL1B.
9. inhibitor as claimed in claim 8 is in preparation prevents, alleviates and/or treat the pharmaceutical composition of carcinoma of prostate
Application.
Apply the most as claimed in claim 9, it is characterised in that described pharmaceutical composition includes NEURL1B inhibitor conduct
Active component, and pharmaceutically acceptable carrier.
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| CN201610862415.9A CN106319070A (en) | 2016-09-28 | 2016-09-28 | Application of NEURL1B in preparation of product for diagnosing prostatic carcinoma |
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| CN201610862415.9A CN106319070A (en) | 2016-09-28 | 2016-09-28 | Application of NEURL1B in preparation of product for diagnosing prostatic carcinoma |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102656457A (en) * | 2009-12-17 | 2012-09-05 | 细胞生物株式会社 | Kit for diagnosing prostate cancer and diagnosis method |
| CN105658810A (en) * | 2013-06-13 | 2016-06-08 | 南澳大学 | Methods for detecting prostate cancer |
-
2016
- 2016-09-28 CN CN201610862415.9A patent/CN106319070A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102656457A (en) * | 2009-12-17 | 2012-09-05 | 细胞生物株式会社 | Kit for diagnosing prostate cancer and diagnosis method |
| CN105658810A (en) * | 2013-06-13 | 2016-06-08 | 南澳大学 | Methods for detecting prostate cancer |
Non-Patent Citations (1)
| Title |
|---|
| MYERS JS ET AL.: "Differentally expressed genes and signature pathways of human prostate cancer", 《PLOS ONE》 * |
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