CN105838818B - Application of the NFIC gene in preparation hypophysoma diagnosis and treatment product - Google Patents
Application of the NFIC gene in preparation hypophysoma diagnosis and treatment product Download PDFInfo
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Abstract
The invention discloses the molecular markers that NFIC gene can be used as hypophysoma early diagnosis.The experiment proves that NFIC gene expression significantly increases in hypophysis tumor tissue compared with normal pituitary tissues, and prove that NFIC can influence the proliferation of pituitary tumor cell by RNA interference experiment.Research achievement according to the present invention can research and develop the drug for being able to suppress NFIC gene expression, to realize clinically for the prevention and treatment of hypophysoma.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to purposes of the NFIC gene in the diagnosis, treatment of hypophysoma.
Background technique
Hypophysoma (pituitary adenoma) is common intracranial tumors, and third is located in intracranial tumors incidence
Position, is only second to glioma and meningioma, accounts for about the 10-25% of intracranial tumors, although pituitary adenoma is a kind of benign tumour,
Being Partial tumors often involves optic nerve, sphenoid sinus, cavernous sinus, internal carotid C in invasive growth2It is important around section, hypothalamus etc.
Structure.The normal endocrine of patient and nervous function are influenced, such as amenorrhoea, lactation, infertile, sex dysfunction, acromegalia, skin
Matter alcohol increase disease, visual impairment, defect of visual field etc..For most of hypophysomas, Extracranial repair is still preferred controls
Treatment method, although performing the operation relative to traditional craniotomy, Extracranial repair complication is reduced, and operation can not be complete
The problem of releasing patient, while Postoperative recurrent rate is higher, the postoperative 5 years high recurrence rates of macroadenoma (diameter > lcm) reach.Invasion is hung down
The operation of body adenoma is difficult to cut off completely, and recurrence rate is higher, is a great problem in clinical treatment.
Therefore, the early diagnosis for improving the tumour explores effective treatment method with important economic value and society
Benefit.
Summary of the invention
The purpose of the present invention is to provide a kind of molecular markers that can be used for hypophysoma early diagnosis.Use genetic marker
Object has timeliness, specificity and sensitivity diagnose hypophysoma, to make patient that can know disease wind in disease early stage
Corresponding prevention and treatment measure is taken for risk height in danger.
To achieve the goals above, the present invention adopts the following technical scheme:
The present invention provides application of the product of detection NFIC gene expression in the tool of preparation diagnosis hypophysoma.
Further, it is described detection NFIC gene expression product include detect NFIC gene mRNA levels product, and/or
Detect the product of NFIC protein level.
Further, the product of the detection NFIC gene expression includes: by RT-PCR, real-time quantitative PCR, immune inspection
It surveys, in situ hybridization or chip detect NFIC gene expression to diagnose the product of hypophysoma.
Further, the product with RT-PCR diagnosis hypophysoma includes at least drawing for a pair of of specific amplified NFIC gene
Object；The product with real-time quantitative PCR diagnosis hypophysoma includes at least the primer of a pair of of specific amplified NFIC gene；The use
The product of immune detection diagnosis hypophysoma includes: the antibody in conjunction with NFIC protein-specific；Described diagnosed in situ hybridization is hung down
The product of body tumor includes: the probe with the nucleic acid array hybridizing of NFIC gene；It is described with chip diagnosis hypophysoma product include:
Protein chip and genetic chip；Wherein, protein chip includes the antibody in conjunction with NFIC protein-specific, genetic chip include with
The probe of the nucleic acid array hybridizing of NFIC gene.
The primer for a pair of of specific amplified NFIC gene that the product with real-time quantitative PCR diagnosis hypophysoma includes at least
As shown in SEQ ID NO.3 and SEQ ID NO.4.
The product of the detection NFIC gene expression can be the reagent of detection NFIC gene expression, be also possible to comprising institute
The kit, chip, test paper etc. for stating reagent, are also possible to the high-flux sequence platform using the reagent.
The tool of the diagnosis hypophysoma includes but is not limited to chip, kit, test paper or high-flux sequence platform；It is high
Flux microarray dataset is a kind of tool of special diagnosis hypophysoma, with the development of high throughput sequencing technologies, to people's
The building of gene expression profile will become very easily work.By comparing the gene expression profile of Disease and normal population,
The exception for being easy to analyze which gene is related to disease.Therefore, the exception of NFIC gene is known in high-flux sequence and is hung down
Body tumor correlation also belongs to the purposes of NFIC gene, equally within protection scope of the present invention.
The present invention also provides a kind of tool for diagnosing hypophysoma, the tool includes the examination for detecting NFIC gene expression
Agent；The reagent includes the primer and/or probe, the antibody for detecting NFIC albumen for detecting NFIC gene mRNA.
The tool includes but is not limited to chip, kit, test paper or high-flux sequence platform.
Wherein, the chip includes genetic chip, protein-chip；The genetic chip includes solid phase carrier and fixation
In the oligonucleotide probe of solid phase carrier, the oligonucleotide probe includes for detecting being directed to for NFIC gene transcription level
The oligonucleotide probe of NFIC gene；The protein-chip includes solid phase carrier and the NFIC albumen for being fixed on solid phase carrier
Specific antibody；The genetic chip can be used for detecting multiple genes including NFIC gene (for example, with hypophysoma phase
Close multiple genes) expression.The protein-chip can be used for detecting multiple protein including NFIC albumen
The expression of (such as multiple protein relevant to hypophysoma).By the way that multiple markers with hypophysoma are detected simultaneously,
It is greatly improved the accuracy rate of hypophysoma diagnosis.
Wherein, the kit includes gene detecting kit and protein immunization detection kit；The genetic test examination
Agent box includes the reagent for detecting NFIC gene transcription level；The protein immunization detection kit includes the spy of NFIC albumen
Heterogenetic antibody.Further, the reagent includes using RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip side
Method detects reagent needed for NFIC gene expression dose process.Preferably, the reagent includes the primer for NFIC gene
And/or probe.It is easy to design according to the nucleotide sequence information of NFIC gene and can be used for detecting NFIC gene expression dose
Primer and probe.
The test paper includes the reagent for detecting NFIC gene expression.
The high-flux sequence platform includes the reagent for detecting NFIC gene expression.
It can be DNA, RNA, DNA-RNA chimera with the probe of the nucleic acid array hybridizing of NFIC gene, PNA or other spreads out
Biology.There is no limit appoint as long as completing specific hybrid, specifically binding with purpose nucleotide sequence the length of the probe
What length is ok.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of the probe
Degree can be grown to 60,80,100,150,300 base-pairs or longer or even whole genes.Since different probe lengths is to hybridization
Efficiency, signal specificity have different influences, and the length of the probe is typically at least 14 base-pairs, and longest is usually no more than
30 base-pairs, the length complementary with purpose nucleotide sequence are best with 15-25 base-pair.The probe self-complementary sequences
Most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.
Further, the specific antibody of the NFIC albumen includes monoclonal antibody, polyclonal antibody.The NFIC albumen
Specific antibody include complete antibody molecule, antibody any segment or modification (for example, chimeric antibody, scFv, Fab, F
(ab ') 2, Fv etc..As long as the segment can retain the binding ability with NFIC albumen.Antibody for protein level
Preparation when well known to a person skilled in the art and the present invention may use any method to prepare the antibody.
In specific embodiments of the present invention, it is described detection NFIC gene mRNA primer include SEQ ID NO.3 and
Primer pair shown in SEQ ID NO.4.
The present invention also provides the inhibitor of NFIC gene and/or its expression product in the drug of preparation treatment hypophysoma
Application.The inhibitor includes the reagent for inhibiting NFIC gene expression, and/or the reagent for inhibiting NFIC gene expression product.
Further, the reagent for inhibiting NFIC gene expression includes the reagent of suppressor transcription, suppressor translation
Reagent；The reagent for inhibiting NFIC gene expression product includes the reagent for inhibiting NFIC gene mRNA, inhibits NFIC albumen
Reagent.The reagent for inhibiting NFIC gene mRNA includes the reagent for inhibiting mRNA stability, inhibits mRNA translation active
Reagent.The reagent for inhibiting NFIC albumen includes the reagent for inhibiting NFIC protein stability, the examination for inhibiting NFIC protein active
Agent, the reagent for inhibiting NFIC protein function.
Further, the reagent for inhibiting NFIC gene mRNA includes the double stranded RNA for being directed to NFIC gene mRNA；Inhibit
The reagent of NFIC protein function includes the tumor vaccine of NFIC antigen protein, the antibody for inhibiting NFIC protein function.The antibody
It can be polyclonal antibody or monoclonal antibody.
In specific embodiments of the present invention, the double stranded RNA for NFIC gene mRNA is siRNA.For
Ensuring NFIC gene can efficiently be rejected or silencing, according to the mRNA sequence of NFIC gene devise siRNA specificity piece
Section.The design of siRNA is according to general design principle (Elbashir et.al 2001, the Schwarz et.al delivered
2003, Khvorova et.al 2003, Reynolds et.al 2004, Hsieh et.al 2004, Ui-Tei et.al
2004), pass through online tool complete design, the online tool are as follows: 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
It further increases the validity of siRNA segment, the siRNA piece of screening is designed for the advantages of comprehensive two Photographing On-line tools
It is disconnected.Finally, siRNA sequence is filtered by sequence analysis (NCBI BLAST), to improve the specificity of siRNA segment and subtract
The undershooting-effect of few RNAi interference.
Preferably, the sequence of the siRNA is as shown in SEQ ID NO.9 and SEQ ID NO.10.
The present invention also provides a kind of for treating the pharmaceutical composition of hypophysoma, and described pharmaceutical composition includes institute above
The inhibitor of the NFIC gene and/or its expression product stated.
Pharmaceutical composition of the invention further includes pharmaceutically acceptable carrier, and wherein the carrier can be excipient, dilution
Agent, thickener, filler, bonding agent, disintegrating agent, lubricant, grease or non-grease base, surfactant, suspending agent, glue
Mixing more than solidifying agent, adjuvant, preservative, antioxidant, stabilizer, colorant or fragrance either or both of them.
Pharmaceutical composition of the invention can be used for manufacturing the medicament for the treatment of hypophysoma.
Pharmaceutical composition first choice of the invention is applied to mammal, and wherein the mammal is preferably human patients.
Pharmaceutical composition of the invention can be given in a manner of by oral, injection to the human patient.
Pharmaceutical composition of the invention can also can be with the drug combination of other treatment hypophysoma, a variety of Drug combinations
The success rate for the treatment of is mentioned significantly.
In the context of the present invention, " NFIC gene " includes any functional equivalent of NFIC gene and NFIC gene
Polynucleotides.NFIC gene includes and NFIC gene (NC_ in the public GenBank GeneBank in the current world
000019.10) DNA sequence dna has 70% or more homology, and encodes the DNA sequence dna of identical function protein.
Preferably, the coded sequence of NFIC gene includes following any DNA molecular:
(1) DNA sequence dna shown in SEQ ID NO.1 in sequence table；
(2) under strict conditions with 1) DNA sequence dna that limits hybridizes and encodes the DNA sequence dna of identical function protein；
(3) DNA sequence dna limited with (1) or (2) has 70%, preferably, 90% or more homology, and encodes identical function
The DNA molecular of energy protein.
In specific embodiments of the present invention, the coded sequence of the NFIC gene is DNA shown in SEQ ID NO.1
Sequence.
In the context of the present invention, NFIC gene expression product includes the partial peptide of NFIC albumen and NFIC albumen.
The partial peptide of the NFIC albumen contains functional domain relevant to hypophysoma.
" NFIC albumen " includes any functional equivalent of NFIC albumen and NFIC albumen.The functional equivalent includes
NFIC albumen conservative variation protein or its active fragment or its reactive derivative, allelic variant, lure at natural mutation
Lead mutant, can be with the encoded protein of DNA of the DNA hybridization of NFIC under high or low stringent condition.
Preferably, NFIC albumen is the protein with following amino acid sequences:
(1) protein that the amino acid sequence shown in SEQ ID NO.2 in sequence table forms；
(2) amino acid sequence shown in SEQ ID NO.2 by the substitution of one or several amino acid residues and/or is lacked
Lose and/or addition and with the ammonia with the same function as shown in SEQ ID NO.2 of amino acid sequence shown in SEQ ID NO.2
Protein derived from base acid sequence.The number for the amino acid for replacing, lacking or adding is usually 1-50, preferably 1-30
It is a, more preferably 1-20, most preferably 1-10.
(3) there is at least 80% homology (also known as sequence identity) with amino acid sequence shown in SEQ ID NO.2,
It is highly preferred that the homology with amino acid sequence at least about 90% to 95% shown in SEQ ID NO.2, Chang Wei 96%, 97%,
98%, the polypeptide that the amino acid sequence of 99% homology is constituted.
In specific embodiments of the present invention, the NFIC albumen is with amino acid sequence shown in SEQ ID NO.2
The protein of column.
It is known that, conventionally, the modification of one or more amino acid will not influence the function of protein in a protein.
Those skilled in the art can approve the amino acid for changing single amino acids or small percentage or to amino acid sequence it is individual add,
Missing, insertion, replacement are conservative modifications, and wherein the change of protein generates the protein with identity function.Function phase is provided
As the Conservative substitution tables of amino acid be well known in the art.
Example by one amino acid of addition or the protein of more amino acid modification is the fusion of NFIC albumen
Albumen.For the peptide or protein with NFIC protein fusion, there is no limit as long as resulting fusion protein retains NFIC albumen
Biological activity.
NFIC albumen of the invention also includes the non-conservative modification to amino acid sequence shown in SEQ ID NO.2, as long as
Protein by modification still is able to retain the biological activity of NFIC albumen.It is mutated in such modification protein
Amino acid number is usually 10 perhaps less such as 6 perhaps less such as 3 or less.
In the context of the present invention, " diagnosis hypophysoma " both included judge subject whether suffered from hypophysoma or
Including judging that subject whether there is the risk with hypophysoma.
In the context of the present invention, " treatment hypophysoma " divides from the state change of disease, may include the slow of disease
The complete healing of solution, disease, further includes the therapeutic effect for evaluating disease.
The advantages of the present invention:
Present invention firstly discovers that NFIC gene expression is related to hypophysoma, pass through the table of NFIC in detection subject's tissue
It reaches, it can be determined that whether subject suffers from hypophysoma or judge that subject whether there is the risk with hypophysoma, to refer to
It leads clinician and provides prevention scheme or therapeutic scheme to subject.
Present invention finds a kind of new molecular marked compound-NFIC genes, and compared to traditional detection means, gene diagnosis is more
In time, more special, sensitiveer, it can be realized the early diagnosis of hypophysoma, to reduce the death rate and recurrence rate of hypophysoma.
Detailed description of the invention
Fig. 1 shows the expression feelings using genechip detection NFIC gene in hypophysis tumor tissue and normal pituitary tissues
Condition；
Fig. 2 shows the jamming effectiveness using QPCR detection siRNA to NFIC gene；
Fig. 3 shows influence of the NFIC gene expression to pituitary tumor cell proliferative capacity.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.
The differential expression of 1 NFIC gene of embodiment
1, sample is collected
Hypophysoma patient: male 5 in 10 hypophysoma patients, age are 28-73 years old, and average age is 47 years old；Women 5
Example, age are 25-68 years old, and average age is 44 years old.It is included in standard: the excision of patients with benign's nose butterfly pituitary adenoma operation, it is preoperative to have cranium
The imaging datas such as Typical AVM, postoperative pathological slice turn out to be hypophysoma.Agree to by Hospital Ethical Committee, is signed through patient
It draws materials after informed consent form.
Normal pituitary tissues: it is derived from dissection teaching and research room, university, totally 5, excludes endocrine related disorders.Method of drawing material is the same as vertical
Body tumor patient.
After operation takes required tissue, physiological saline to clean, it is immediately placed in the jelly through crossing at coke acid diethyl vinegar (DEPC) water
It deposits in pipe, merging liquid nitrogen is spare.
2, RNA extraction and quality analysis
The total serum IgE that hypophysis tumor tissue and normal pituitary tissues are extracted using Trizol one-step method, passes through Nanodrop ND-
1000 read the purity of absorbance value (A) the measurement RNA solution at 260nm and 280nm.Through 1% denaturing formaldehyde Ago-Gel
Electrophoresis is observed under ultraviolet transmission light, detects the integrality of RNA.
3, gene chip hybridization and scanning
After the linearized amplification of total serum IgE, cy3-UTP is marked, and the cRNAs after fluorescent marker uses RNEASY Mini Kit
Purifying, carries out fragmentation processing to the cRNAs marked with the RNA Fragmentation Reagents of Amhion.Using beauty
People's full genome chip of expression spectrum (4x 44K gene) of Agilent company, state, 65 DEG C of hybridization 17h in chip hybridization furnace, then
Elution, dyeing, finally use Agilent DNA MicroarrayScanner scanner scanning.
4, chip data processing and analysis
Chip after hybridization imports data to analysis software, for two groups of ratios after chip scanner reads data point
Natural logrithm absolute value be greater than 2.0 or the gene less than 0.5 as difference expression gene.
5, statistical procedures
Data analysis is carried out using 13.0 statistical software of SPSS, group difference compares using one-way analysis of variance method, P <
0.05 difference has significant.
6, result
As a result as shown in Figure 1, compared with normal pituitary tissues, the mRNA level in-site of NFIC gene significantly increases in hypophysis tumor tissue
Add, difference has statistical significance (P < 0.05).
The differential expression of 2 qPCR experimental verification NFIC gene of embodiment
1, sample is collected
According to standard collection hypophysoma patient 40 of embodiment 1, its hypophysis tumor tissue is taken.Collect normal pituitary tissues 30
Example.
2, RNA is extracted
UsingReagent (invitrogen, article No. 15596-018) carries out sample rna extraction.Concrete operations
It is as follows:
It freezes to be put into the mortar being pre-chilled tissue after liquid nitrogen, taking-up after collection sample and be ground, sample to be organized
This is at powdered rear:
1. Trizol is added, room temperature preservation 5 minutes；
2. chlorination imitates 0.2ml, with forced oscillation centrifuge tube, mix well, places 5-10 minutes at room temperature；
3. drawing upper strata aqueous phase (inhaling 70%) after 12000rpm high speed centrifugation 15 minutes into another new centrifuge tube pipe, pay attention to
The protein substance that be not drawn between two layers of water phase.New pipe is moved into, the pre- cold isopropanol of isometric -20 DEG C is added, it is sufficiently reverse
It mixes, is placed in 10 minutes on ice；
4. 12000rpm high speed from 15 minutes after carefully discard supernatant, in 1ml/ml Trizol ratio be added 75%
DEPC ethyl alcohol washes paint precipitating (4 DEG C of preservations), washes paint sediment, oscillation mixes, 12000rpm high speed centrifugation 5 minutes at 4 DEG C；
5. discarding ethanol liquid, 5 minutes are placed at room temperature sufficiently to dry precipitating, it is heavy that the processed water dissolution of DEPC is added
It forms sediment；
6. measuring RNA purity and concentration with Nanodrop2000 ultraviolet specrophotometer, freeze in -70 DEG C.RNA mass is sentenced
Calibration is quasi-: the OD260/OD280 value of RNA sample is between 1.7-2.2；Total serum IgE electrophorogram has clearly 28S, 18S band；
70 DEG C of water-baths keep the temperature 1 hour after electrophorogram and the map no significant difference before water-bath heat preservation.
3, reverse transcription synthesizes cDNA
UsingIII Reverse Transcriptase (invitrogen, article No. 18080-044) into
Row cDNA reverse transcription, experimental implementation are carried out by product description, and concrete operations are as follows:
Using Reverse Transcriptase kit, converse record is carried out to l μ g total serum IgE with RT Buffer and synthesizes cDNA.Using 25 μ l
Reaction system, each sample take 1 μ g total serum IgE as template ribonucleic acid, following components are separately added into PCR pipe:
5 × RT Buffer, 5 μ l, 10mmol/l dNTP, 1.25 μ l, 0.1mmol/l DTT 2.5 μ l, 30 μm of mol/l
Aqua sterilisa is added to 25 μ l of total system in 2 μ l, 200U/ μ l MMLV of Oligo dT 1.25 μ l, 1 μ g of template ribonucleic acid.42 DEG C are incubated for 1
Hour, 72 DEG C 10 minutes, of short duration centrifugation.It is spare that -20 DEG C of refrigerators are put in cDNA preservation.
4、qPCR
4.1 instruments and analysis method
With 7500 type fluorescence quantitative PCR instrument of ABI, the relative quantitative assay of data is carried out using 2- △ △ CT method.
4.2 reaction systems:
Use PowerGreen PCR Master Mix (invitrogen, article No. 4367659) is expanded, real
Operation is tested to carry out by product description.Amplification program are as follows: 95 DEG C of 10min, (95 DEG C of 15sec, 60 DEG C of 60sec) × 45 circulations.
RealTime reaction system are as follows:
Wherein, primer sequence is as shown in table 1:
1 primer sequence of table
5, experimental result
Compared with normal pituitary tissues, the mRNA level in-site of NFIC gene is dramatically increased in hypophysis tumor tissue, according to qRT-PCR
Relative quantification formula: 2- △ △ ct, relative expression quantity is about 5.92, difference have statistical significance (P < 0.05), verifying knot
Chip results that fruit is homogenic.
Embodiment 3 inhibits NFIC gene expression
1, siRNA design synthesis
For the siRNA sequence of NFIC:
SiRNA1-NFIC:
Positive-sense strand is 5 '-UCUUCUUCUUCUUCUUCUGCC-3 ' (SEQ ID NO.7)；
Antisense strand is 5 '-CAGAAGAAGAAGAAGAAGAAA-3 ' (SEQ ID NO.8),
SiRNA2-NFIC:
Positive-sense strand is 5 '-AGAUGAAUAACUUAGGAAGAC-3 ' (SEQ ID NO.9)；
Antisense strand is 5 '-CUUCCUAAGUUAUUCAUCUCC-3 ' (SEQ ID NO.10),
SiRNA3-NFIC:
Positive-sense strand is 5 '-UAUUGUUUAAAGGUAAUACUU-3 ' (SEQ ID NO.11)；
Antisense strand is 5 '-GUAUUACCUUUAAACAAUAUC-3 ' (SEQ ID NO.12)
The above siRNA sequence and negative control siRNA sequence (siRNA-NC) are by the limited public affairs of Shanghai Ji Ma pharmaceutical technology
Department provides:
2, the culture and transfection of pituitary tumor cell
2.1 cell culture
Hypophysoma tissue block is taken to immerse RPMI-1640 culture medium, DPBS is shredded after cleaning 3 times, at room temperature 0.25% pancreas egg
White enzymic digestion 1h is added the RPMI-1640 culture medium containing 10%FCS in equal volume and terminates digestion, addition final concentration of 0.87%
NH4Cl is in 37 DEG C of effect 5mim；After 70 mesh cell screen clothes are filtered to remove fragment of tissue, 1000r/min is centrifuged 5min, with containing 10%
Cell precipitation is resuspended in the RPMI-1640 culture medium of FCS, 2mM paddy ammonia phthalein amine, 100U/mL penicillin and 100pg/mL streptomysin,
5% carbon dioxide, the carbon dioxide cell incubator culture of 37 DEG C and saturated humidity.3d is changed liquid 1 time, until cell forms list
Layer, with 0.25% trypsin digestion, renewed vaccination, secondary culture.
2.2 cell transfecting
By 2 × 105A pituitary tumor cell is inoculated into 25cm2In tissue culture plate, in 37 DEG C, 5%CO2Incubator culture, when
Cell confluency degree carries out cell transfecting when reaching 70%.Transfection (is purchased from Invitrogen according to lipofectamine 2000
Company) specification transfection, experiment be divided into negative control group (siRNA-NC) and experimental group (siRNA-NFIC), concentration is
The hole 20nM/, while transfecting respectively.
3, the jamming effectiveness of detection siRNA is tested using QPCR.
3.1 extraction cell total rnas are operated using conventional method.
3.2 reverse transcription
With embodiment 2.
3.3 QPCR
With embodiment 2.
3.4 statistical method
With embodiment 2.
4, result
As a result as shown in Fig. 2, compared with siRNA1-NFIC, siRNA3-NFIC, siRNA2-NFIC can more effectively press down
The expression of NFIC gene processed, difference have statistical significance (P < 0.05), carry out subsequent experiment using siRNA2-NFIC.
Measurement of the expression of 4 NFIC gene of embodiment to pituitary tumor cell proliferative capacity
It is proliferated using Cell Counting kit-8 (cck-8) kit for detecting pituitary tumor cell
1, step
The culture and transfection of pituitary tumor cell are carried out according to the method for preceding embodiment 3, cell is divided into two experimental groups:
Group 1: transfection siRNA-NC groups of cells；
Group 2: transfection siRNA2-NFIC groups of cells.
After transfection for 24 hours, with 2.5 × 105/ ml density is inoculated in 96 porocyte culture plates, and each experimental group design three is multiple
Hole, the CCK-8 solution of 10 μ l of every hole addition, 37 DEG C, 5%CO24h is incubated in incubator；Then illustrate according to kit, select
450nm wavelength measures each hole absorbance value (OD value) in microplate reader.
2, statistical method
Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD,
Using SPSS13.0 statistical software come for statistical analysis, difference between the two is examined using t, it is believed that is had as P < 0.05
It is statistically significant.
3, result
Shown in result figure 3, compared with transfecting siRNA-NC group, transfection siRNA2-NFIC groups of cells cell Proliferation is slow, poor
It is different that there is statistical significance (P < 0.05).It is above-mentioned the experimental results showed that, NFIC gene expression promotes the proliferation of pituitary tumor cell.
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.
Claims (9)
1. detecting application of the product of NFIC gene expression in the tool of preparation diagnosis hypophysoma.
2. application according to claim 1, which is characterized in that the product include: by RT-PCR, real-time quantitative PCR,
Immune detection, in situ hybridization, chip or high-flux sequence detection of platform NFIC gene expression are to diagnose the product of hypophysoma；It is described
The primer of a pair of of specific amplified NFIC gene is included at least with the product of RT-PCR diagnosis hypophysoma；It is described to use real-time quantitative PCR
The product for diagnosing hypophysoma includes at least the primer of a pair of of specific amplified NFIC gene；It is described to diagnose hypophysoma with immune detection
Product includes: the antibody in conjunction with NFIC protein-specific；The product in situ hybridization diagnosis hypophysoma includes: and NFIC
The probe of the nucleic acid array hybridizing of gene；The product with chip diagnosis hypophysoma includes: protein chip and genetic chip；Its
In, protein chip includes the antibody in conjunction with NFIC protein-specific, and genetic chip includes miscellaneous with the nucleic acid sequence of NFIC gene
The probe of friendship.
3. application according to claim 2, which is characterized in that the product with real-time quantitative PCR diagnosis hypophysoma is extremely
The primer for a pair of of the specific amplified NFIC gene for including less is as shown in SEQ ID NO.3 and SEQ ID NO.4.
4. application according to claim 1, which is characterized in that the tool includes the reagent for detecting NFIC gene expression；
The reagent includes the primer and/or probe, the antibody for detecting NFIC albumen for detecting NFIC gene mRNA.
5. application according to claim 4, which is characterized in that the primer of the detection NFIC gene mRNA includes SEQ ID
Primer pair shown in NO.3 and SEQ ID NO.4.
Application of the inhibitor of 6.NFIC gene and/or its expression product in the drug of preparation treatment hypophysoma.
7. application according to claim 6, which is characterized in that the inhibitor includes the examination for inhibiting NFIC gene expression
Agent, and/or the reagent for inhibiting NFIC gene expression product.
8. application according to claim 7, which is characterized in that the reagent for inhibiting NFIC gene expression product includes needle
To the antibody of the siRNA, and/or NFIC albumen of NFIC gene.
9. application according to claim 8, which is characterized in that such as SEQ ID of the siRNA sequence for NFIC gene
Shown in NO.7 and SEQ ID NO.8.
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| CN108220439A (en) * | 2018-01-04 | 2018-06-29 | 大连医科大学附属第二医院 | CircRAPGEF52 is applied in pituitary adenoma biomarker |
| CN108441556A (en) * | 2018-01-04 | 2018-08-24 | 大连医科大学附属第二医院 | CircZNF521 is applied in pituitary adenoma biomarker |
| CN108220437A (en) * | 2018-01-04 | 2018-06-29 | 大连医科大学附属第二医院 | Application and expression of the NR4A2 genes in pituitary adenoma biomarker |
| CN108220440A (en) * | 2018-01-04 | 2018-06-29 | 大连医科大学附属第二医院 | Application and expression of the NFKBIZ genes in pituitary adenoma biomarker |
| CN107937548A (en) * | 2018-01-04 | 2018-04-20 | 大连医科大学附属第二医院 | Application and expression of the OLFM3 genes in pituitary adenoma biomarker |
| CN112746103A (en) * | 2021-01-20 | 2021-05-04 | 河南省中医院（河南中医药大学第二附属医院） | Molecular marker NFIA for evaluating coronary heart disease prognosis, reverse transcription primer, amplification primer and application thereof |
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