CN105838818A - Application of NFIC gene in preparation of diagnostic and therapeutic products for pituitary adenomas - Google Patents
Application of NFIC gene in preparation of diagnostic and therapeutic products for pituitary adenomas Download PDFInfo
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Abstract
The invention discloses NFIC gene capable of serving as a molecular marker for early diagnosis of pituitary adenomas. Experiments of the invention prove that NFIC gene has significantly improved expression in pituitary adenomas tissues when compared to normal pituitary tissues; RNA interference experiments prove that NFIC can affect the proliferation of pituitary adenomas cells. According to the study results of the invention, it is possible to develop drugs capable of inhibiting NFIC gene expression, thus preventing and treating pituitary adenomas clinically.
Description
Technical field
The present invention relates to biological technical field, more particularly to NFIC gene purposes in the diagnosis, treatment of hypophysoma.
Background technology
Hypophysoma (pituitary adenoma) is common ICT, is positioned at the 3rd in ICT incidence
Position, is only second to glioma and meningioma, accounts for the 10-25% of ICT, although pituitary adenoma is a kind of benign tumour, but
Be Partial tumors be that invasive growth often involves optic nerve, sphenoid sinus, cavernous sinus, internal carotid C2Section, hypothalamus etc. are the most important
Structure.Affect the normal endocrine of patient and nervous function, 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 controlling of first-selection
Treatment method, although performing the operation relative to traditional craniotomy, Extracranial repair complication has reduced, but operation can not be complete
The problem releasing patient, Postoperative recurrent rate is higher simultaneously, macroadenoma (diameter > lcm) postoperative 5 years recurrence rates are up to.Aggressive is hung down
The operation of body adenoma is difficult to excise completely, and recurrence rate is higher, is a great problem in clinical treatment.
Therefore, improve the early diagnosis of this tumour, explore effective methods for the treatment of and there is important economic worth and society
Benefit.
Summary of the invention
It is an object of the invention to provide a kind of molecular marker that can be used for hypophysoma early diagnosis.Use genetic marker
What thing diagnosed hypophysoma has promptness, specific and sensitivity, so that patient just can know disease wind in early days in disease
Danger, for risk height, takes to prevent accordingly and remedy measures.
To achieve these goals, the present invention adopts the following technical scheme that
The invention provides the product detecting NFIC gene expression application in the instrument of preparation diagnosis hypophysoma.
Further, the product of described detection NFIC gene expression include detect NFIC gene mRNA levels product and/or
The product of detection NFIC protein level.
Further, the product of described detection NFIC gene expression includes: by RT-PCR, real-time quantitative PCR, immunity inspection
Survey, in situ hybridization or the gene expression of chip detection NFIC are to diagnose the product of hypophysoma.
Further, the product of described RT-PCR diagnosis hypophysoma at least includes drawing of a pair specific amplified NFIC gene
Thing；The product of described real-time quantitative PCR diagnosis hypophysoma at least includes the primer of a pair specific amplified NFIC gene；Described use
The product of immune detection diagnosis hypophysoma includes: the antibody being combined with NFIC protein-specific；Described in situ hybridization diagnosis is hung down
The product of body knurl includes: with the probe of the nucleic acid array hybridizing of NFIC gene；The product of described chip diagnosis hypophysoma includes:
Protein chip and genetic chip；Wherein, protein chip includes the antibody being combined with NFIC protein-specific, genetic chip include with
The probe of the nucleic acid array hybridizing of NFIC gene.
The primer of a pair specific amplified NFIC gene that the product of described real-time quantitative PCR diagnosis hypophysoma at least includes
As shown in SEQ ID NO.3 and SEQ ID NO.4.
The product of described detection NFIC gene expression can be the reagent of detection NFIC gene expression, can also be to comprise institute
State the kit of reagent, chip, test paper etc., it is also possible to be the high-flux sequence platform using described reagent.
The instrument of described diagnosis hypophysoma includes but not limited to chip, kit, test paper or high-flux sequence platform；High
Flux order-checking platform is the instrument of a kind of special diagnosis hypophysoma, along with the development of high throughput sequencing technologies, to a people's
The structure of gene expression profile will become and work the most easily.By contrast Disease and the gene expression profile of normal population,
The exception easily analyzing which gene is relevant to disease.Therefore, in high-flux sequence, know that the exception of NFIC gene is with vertical
Body knurl is correlated with and is fallen within the purposes of NFIC gene, equally within protection scope of the present invention.
Present invention also offers a kind of instrument diagnosing hypophysoma, described instrument includes the examination detecting NFIC gene expression
Agent；Described reagent includes primer and/or probe, the antibody of detection NFIC albumen detecting NFIC gene mRNA.
Described instrument includes but not limited to chip, kit, test paper or high-flux sequence platform.
Wherein, described chip includes genetic chip, protein-chip；Described genetic chip includes solid phase carrier and fixes
At the oligonucleotide probe of solid phase carrier, described oligonucleotide probe include for detect NFIC gene transcription level for
The oligonucleotide probe of NFIC gene；Described protein-chip includes solid phase carrier and is fixed on the NFIC albumen of solid phase carrier
Specific antibody；Multiple genes that described genetic chip can be used for detecting including NFIC gene are (such as, with hypophysoma phase
Close multiple genes) expression.Described protein-chip can be used for the multiple protein detecting including NFIC albumen
The expression of (such as relevant to hypophysoma multiple protein).By multiple marks with hypophysoma are detected simultaneously,
It is greatly improved the accuracy rate of hypophysoma diagnosis.
Wherein, described kit includes gene detecting kit and protein immunization detection kit；Described genetic test tries
Agent box includes the reagent for detecting NFIC gene transcription level；Described protein immunization detection kit includes the spy of NFIC albumen
Heterogenetic antibody.Further, described reagent includes using RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip side
Reagent required during method detection NFIC gene expression dose.Preferably, described reagent includes the primer for NFIC gene
And/or probe.Nucleotide sequence information according to NFIC gene is easily designed and be may be used for detecting NFIC gene expression dose
Primer and probe.
Described test paper includes the reagent detecting NFIC gene expression.
Described high-flux sequence platform includes the reagent detecting NFIC gene expression.
With the probe of the nucleic acid array hybridizing of NFIC gene can be DNA, RNA, DNA-RNA chimera, PNA or other spread out
Biological.The length of described probe does not limit, as long as it is specific binding with purpose nucleotide sequence to complete specific hybrid, appoints
What length can.The length of described probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of described probe
Degree can be grown to 60,80,100,150,300 base-pairs or longer, the most whole gene.Owing to different probe length is to hybridization
Efficiency, signal specificity have different impacts, the length of described probe to be typically at least 14 base-pairs, the longest are usually no more than
30 base-pairs, optimal with 15-25 base-pair with the length of purpose nucleotide sequence complementary.Described probe self-complementary sequences
Most preferably less than 4 base-pairs, in order to avoid affecting hybridization efficiency.
Further, the specific antibody of described NFIC albumen includes monoclonal antibody, polyclonal antibody.Described NFIC albumen
Specific antibody include complete antibody molecule, any fragment of antibody or modify (such as, chimeric antibody, scFv, Fab, F
(ab ') 2, Fv etc..As long as described fragment can retain and the binding ability of NFIC albumen.Antibody for protein level
Preparation time well known to a person skilled in the art, and the present invention can use any method to prepare described antibody.
In specific embodiments of the present invention, the primer of described detection NFIC gene mRNA include SEQ ID NO.3 and
Primer pair shown in SEQ ID NO.4.
Present invention also offers the inhibitor of NFIC gene and/or its expression product in the medicine of preparation treatment hypophysoma
Application.Described inhibitor includes reagent and/or the reagent of suppression NFIC gene expression product suppressing NFIC gene expression.
Further, the reagent of described suppression NFIC gene expression includes reagent, the suppressor translation that suppressor transcribes
Reagent；The reagent of described suppression NFIC gene expression product includes suppressing the reagent of NFIC gene mRNA, suppression NFIC albumen
Reagent.The reagent of described suppression NFIC gene mRNA includes suppressing the reagent of mRNA stability, suppression mRNA to translate activity
Reagent.The reagent of described suppression NFIC albumen includes suppressing the reagent of NFIC protein stability, the examination of suppression NFIC protein active
Agent, the reagent of suppression NFIC protein function.
Further, the reagent of suppression NFIC gene mRNA includes the double stranded RNA for NFIC gene mRNA；Suppression
The reagent of NFIC protein function includes the tumor vaccine of NFIC antigen protein, the antibody of suppression NFIC protein function.Described antibody
Can be polyclonal antibody, or monoclonal antibody.
In specific embodiments of the present invention, the described double stranded RNA for NFIC gene mRNA is siRNA.For
Guarantee that NFIC gene can efficiently be rejected or reticent, the specific sheet of siRNA according to the mRNA sequences Design of NFIC gene
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), by online tool complete design, this online tool is: siRNASelectionProgram of Whitehead
Institute (BingbingYuan et.al 2004, http://jura.wi.mit.edu/bioc/siRNAext/) and
BLOCK-iTTM RNAi Designer ofINVITROGEN(winner of the 2004Frost&Sullivan
Excellence in Research Award, https: //rnaidesigner.invitrogen.com/sirna/).In order to
Improving the validity of siRNA segment further, the advantage of comprehensive two Photographing On-line instruments is designed for the siRNA sheet of screening
Disconnected.Finally, filter siRNA sequence by sequence analysis (NCBI BLAST), to improve the specific of siRNA segment and to subtract
The effect of missing the target of few RNAi interference.
Preferably, the sequence of described siRNA is as shown in SEQ ID NO.9 and SEQ ID NO.10.
Present invention also offers a kind of pharmaceutical composition for treating hypophysoma, described pharmaceutical composition includes institute above
The NFIC gene stated and/or the inhibitor of its expression product.
The pharmaceutical composition of the present invention also includes pharmaceutically acceptable carrier, and wherein this carrier can be excipient, dilution
Agent, thickener, filler, bonding agent, disintegrant, lubricant, grease or non-grease base, surfactant, suspending agent, glue
Mixing more than solidifying agent, adjuvant, preservative, antioxidant, stabilizer, colouring agent or spices either or both of which.
The pharmaceutical composition of the present invention can be used for manufacturing the medicament for the treatment of hypophysoma.
The pharmaceutical composition first-selection of the present invention is applied to mammal, and wherein this mammal is preferably human patients.
The pharmaceutical composition of the present invention can such as give to this human patients's body in modes such as oral, injections.
The pharmaceutical composition of the present invention also can be used in combination permissible with the drug combination of other treatment hypophysoma, multi-medicament
Significantly mention the success rate for the treatment of.
In the context of the present invention, " NFIC gene " includes any function equivalent of NFIC gene and NFIC gene
Polynucleotides.NFIC gene includes and NFIC gene (NC_ in the most international common core sequence databank GeneBank
000019.10) DNA sequence dna has more than 70% homology, and coding identical function protein DNA sequence.
Preferably, the coded sequence of NFIC gene includes any DNA molecular following:
(1) DNA sequence dna shown in SEQ ID NO.1 in sequence table；
(2) under strict conditions with 1) the DNA sequence dna hybridization that limits and coding identical function protein DNA sequence；
(3) DNA sequence dna limited with (1) or (2) has 70%, preferably, more than 90% homology, and encodes identical merit
Can protein DNA molecule.
In specific embodiments of the present invention, the coded sequence of described NFIC gene is the DNA shown in SEQ ID NO.1
Sequence.
In the context of the present invention, NFIC gene expression product includes NFIC albumen and the partial peptide of NFIC albumen.
The partial peptide of described NFIC albumen contains the functional domain relevant to hypophysoma.
" NFIC albumen " includes any function equivalent of NFIC albumen and NFIC albumen.Described function equivalent includes
NFIC albumen conservative variation's protein or its active fragment, or its reactive derivative, allelic variant, natural mutation, lure
Lead mutant, can be with the protein coded by the 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 being made up of the amino acid sequence shown in SEQ ID NO.2 in sequence table；
(2) amino acid sequence shown in SEQ ID NO.2 through the replacement of one or several amino acid residue and/or is lacked
Lose and/or add and with the amino acid sequence shown in SEQ ID NO.2 have identical function by the ammonia shown in SEQ ID NO.2
The protein that base acid sequence is derivative.The amino acid whose number replacing, lack or adding is usually 1-50, preferably 1-30
Individual, more preferably 1-20, most preferably 1-10.
(3) with the amino acid sequence shown in SEQ ID NO.2, there is at least 80% homology (being also called sequence iden),
It is highly preferred that with the homology of the amino acid sequence at least about 90% to 95% shown in SEQ ID NO.2, be often 96%, 97%,
98%, the polypeptide that the amino acid sequence of 99% homology is constituted.
In specific embodiments of the present invention, described NFIC albumen is to have the amino acid sequence shown in SEQ ID NO.2
The protein of row.
It is known that, conventionally, in a protein, one or more amino acid whose modifications do not interfere with the function of protein.
Those skilled in the art can approve change single amino acids or the amino acid of little percentage or amino acid sequence indivedual are added,
Lacking, inserting, replace is conservative modification, and wherein changing of protein produces the protein with identity function.Function phase is provided
As amino acid whose Conservative substitution tables be well known in the art.
By adding the fusion that the example of the protein of an amino acid or multiple Modification of amino acid residues is NFIC albumen
Albumen.Peptide or protein with NFIC protein fusion is not limited, as long as the fusion protein of gained retains NFIC albumen
BA.
The NFIC albumen of the present invention also includes the non-conservative modification to the amino acid sequence shown in SEQ ID NO.2, as long as
Protein through modifying remains able to retain the BA of NFIC albumen.Sudden change in this type of modifying protein
Amino acid number is typically 10 or less, such as 6 or less, such as 3 or less.
In the context of the present invention, " diagnosis hypophysoma " both includes judging that experimenter has suffered from hypophysoma, also
Including judging whether experimenter exists and suffer from the risk of hypophysoma.
In the context of the present invention, " treatment hypophysoma " divides from the state change of disease, can include the slow of disease
Solution, the healing completely of disease, also include the result for the treatment of for evaluating disease.
Advantages of the present invention and beneficial effect:
Present invention firstly discovers that NFIC gene expression is relevant to hypophysoma, the table of NFIC in being organized by detection experimenter
Reach, it can be determined that whether experimenter suffers from hypophysoma or judge whether experimenter exists the risk suffering from hypophysoma, thus refers to
Lead clinician and provide prevention scheme or therapeutic scheme to experimenter.
Present invention finds a kind of new molecular marked compound-NFIC gene, compare traditional detection means, gene diagnosis is more
In time, more special, sensitiveer, it is possible to realize the early diagnosis of hypophysoma, thus reduce the death rate and the recurrence rate of hypophysoma.
Accompanying drawing explanation
Fig. 1 shows the expression feelings utilizing genechip detection NFIC gene in hypophysoma tissue with normal pituitary tissues
Condition；
Fig. 2 show utilize QPCR detect the siRNA jamming effectiveness to NFIC gene；
Fig. 3 shows the NFIC gene expression impact on pituitary tumor cell multiplication capacity.
Specific embodiment
The present invention is further detailed explanation with embodiment below in conjunction with the accompanying drawings.Following example are merely to illustrate this
Invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment, generally according to conventional strip
Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in, or according to the condition proposed by manufacturer.
The differential expression of embodiment 1 NFIC gene
1, sample is collected
Hypophysoma patient: the male sex 5 example in 10 example hypophysoma patients, the age is 28-73 year, and the mean age is 47 years old；Women 5
Example, the age is 25-68 year, and the mean age is 44 years old.Inclusive criteria: patients with benign's nose butterfly pituitary adenoma operation excises, and preoperative have cranium
The imaging datas such as Typical AVM, postoperative pathological section turns out to be hypophysoma.Agree to through Hospital Ethical Committee, sign through patient
Draw materials after Informed Consent Form.
Normal pituitary tissues: take from university and dissect teaching and research room, totally 5 example, get rid of endocrine related disorders.Method of drawing material is with hanging down
Body knurl patient.
Operation takes required tissue, after physiological saline is cleaned, is immediately placed in freezing of crossing at coke acid diethyl vinegar (DEPC) water
Deposit in pipe, insert liquid nitrogen standby.
2, RNA extracts and quality analysis
Trizol one-step method is used to extract hypophysoma tissue and the total serum IgE of normal pituitary tissues, by Nanodrop ND-
1000 read the absorbance (A) at 260nm and 280nm measures the purity of RNA solution.Through 1% denaturing formaldehyde Ago-Gel
Electrophoresis, observes under ultraviolet transmission light, the integrality of detection RNA.
3, gene chip hybridization and scanning
After the linearized amplification of total serum IgE, cy3-UTP marks, and the cRNAs after fluorescence labeling uses RNEASY Mini Kit
Purify, carry out fragmentation process with the RNA Fragmentation Reagents of the Amhion cRNAs to having marked.Use U.S.
People's full genome chip of expression spectrum (4x 44K gene) of Agilent company of state, 65 DEG C of hybridization 17h in chip hybridization stove, then
Wash-out, dyeing, finally by Agilent DNA MicroarrayScanner scanner scanning.
4, chip data processes and analyzes
Chip after hybridization, after chip scanner reads data point, imports data to analyze software, for two groups of ratios
Natural logrithm absolute value more than 2.0 or less than 0.5 gene as difference expression gene.
5, statistical procedures
Use SPSS 13.0 statistical software carry out data analysis, group difference compare employing one-way analysis of variance method, P <
0.05 difference has significant.
6, result
Result is as it is shown in figure 1, compared with normal pituitary tissues, in hypophysoma tissue, the mRNA level in-site of NFIC gene significantly increases
Adding, difference has statistical significance (P < 0.05).
The differential expression of embodiment 2 qPCR experimental verification NFIC gene
1, sample is collected
According to standard collection hypophysoma patient 40 example of embodiment 1, take its hypophysoma tissue.Collect normal pituitary tissues 30
Example.
2, RNA extracts
UseReagent (invitrogen, article No. 15596-018) carries out sample rna extraction.Concrete operations
As follows:
After collecting sample, frozen mortar tissue being put into precooling 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 5-10 minute；
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 sediment, 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 that processed of DEPC dissolve heavy
Form sediment；
6. RNA purity and concentration are measured with Nanodrop2000 ultraviolet specrophotometer, frozen in-70 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, reverse transcription synthesis cDNA
UseIII Reverse Transcriptase (invitrogen, article No. 18080-044) enters
Row 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, is separately added into following components in 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 mol/l
Oligo dT 2 μ l, 200U/ μ l MMLV 1.25 μ l, template ribonucleic acid 1 μ g, addition aqua sterilisa to total system 25 μ l.Hatch 1 for 42 DEG C
Hour, 72 DEG C 10 minutes, of short duration centrifugal.It is standby that-20 DEG C of refrigerators are put in cDNA preservation.
4、qPCR
4.1 instruments and the method for analysis
With ABI 7500 type quantitative real time PCR Instrument, 2-△ △ CT method is used to carry out the relative quantitative assay of data.
4.2 reaction systems:
Use PowerGreen PCR Master Mix (invitrogen, article No. 4367659) expands, real
Test operation to carry out by product description.Amplification program is: 95 DEG C of 10min, (95 DEG C of 15sec, 60 DEG C of 60sec) × 45 circulations.
RealTime reaction system is:
Wherein, primer sequence is as shown in table 1:
Table 1 primer sequence
5, experimental result
Compared with normal pituitary tissues, in hypophysoma tissue, the mRNA level in-site of NFIC gene dramatically increases, according to qRT-PCR
Relative quantification formula: 2-△ △ ct, relative expression quantity is about 5.92, and difference has a statistical significance (P < 0.05), checking knot
The most homogenic chip results.
Embodiment 3 suppresses NFIC gene expression
1, siRNA design synthesis
SiRNA sequence for 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)
Above siRNA sequence and negative control siRNA sequence (siRNA-NC) are by the Shanghai Ji limited public affairs of agate pharmaceutical technology
Department provides:
2, the cultivation of pituitary tumor cell and transfection
2.1 cells are cultivated
Taking hypophysoma tissue block and immerse RPMI-1640 culture medium, DPBS shreds after cleaning 3 times, and room temperature descends 0.25% pancreas egg
White enzymic digestion 1h, adds the equal-volume RPMI-1640 culture medium containing 10%FCS and terminates digestion, add final concentration of 0.87%
NH4Cl acts on 5mim in 37 DEG C；After 70 mesh cell screen filtrations remove fragment of tissue, 1000r/min is centrifuged 5min, with containing 10%
The RPMI-1640 culture medium re-suspended cell precipitation of FCS, 2mM paddy ammonia phthalein amine, 100U/mL penicillin and 100pg/mL streptomysin,
5% carbon dioxide, 37 DEG C and the carbon dioxide cell incubator cultivation of saturated humidity.3d changes liquid 1 time, until cell forms list
Layer, uses 0.25% Trypsin Induced, renewed vaccination, Secondary Culture.
2.2 cell transfecting
By 2 × 105Individual pituitary tumor cell is inoculated into 25cm2In Tissue Culture Plate, at 37 DEG C, 5%CO2Incubator is cultivated, when
Cell confluency degree reaches to carry out cell transfecting when 70%.Transfect according to lipofectamine 2000 (purchased from Invitrogen
Company) specification transfection, experiment be divided into negative control group (siRNA-NC) and experimental group (siRNA-NFIC), concentration is
20nM/ hole, transfects the most respectively.
3, the jamming effectiveness of QPCR experiment detection siRNA is utilized.
3.1 extract cell total rna utilizes conventional method to operate.
3.2 reverse transcription
With embodiment 2.
3.3 QPCR
With embodiment 2.
3.4 statistical method
With embodiment 2.
4, result
Result is as in figure 2 it is shown, compared with siRNA1-NFIC, siRNA3-NFIC, siRNA2-NFIC can more effectively press down
The expression of NFIC gene processed, difference has statistical significance (P < 0.05), uses siRNA2-NFIC to carry out follow-up experiment.
The expression of the embodiment 4 NFIC gene mensuration to pituitary tumor cell multiplication capacity
Cell Counting kit-8 (cck-8) kit is used to be used for detecting pituitary tumor cell propagation
1, step
Carry out cultivation and the transfection of pituitary tumor cell according to the method for preceding embodiment 3, cell is divided into two experimental group:
Group 1: transfection siRNA-NC groups of cells；
Group 2: transfection siRNA2-NFIC groups of cells.
After transfection 24h, with 2.5 × 105/ ml density is inoculated in 96 porocyte culture plates, and each experimental group design three is multiple
Hole, every hole adds the CCK-8 solution of 10 μ l, 37 DEG C, 5%CO2Incubator is hatched 4h；Then according to kit explanation, select
450nm wavelength, measures each hole absorbance (OD value) on ELIASA.
2, statistical method
Experiment is all according to being repeated 3 times, and result data is all to represent in the way of mean+SD,
Using SPSS13.0 statistical software to carry out statistical analysis, difference between the two uses t inspection, it is believed that when P < has when 0.05
Statistically significant.
3, result
Shown in result Fig. 3, compared with transfection siRNA-NC group, transfection siRNA2-NFIC groups of cells cell proliferation is slow, poor
Different have statistical significance (P < 0.05).Above-mentioned test result indicate that, NFIC gene expression promotes the propagation of pituitary tumor cell.
The explanation of above-described embodiment is only intended to understand the method for the present invention and core concept thereof.It should be pointed out that, for this
For the those of ordinary skill in field, under the premise without departing from the principles of the invention, it is also possible to the present invention is carried out some improvement
And modification, these improve and modify also by the protection domain falling into the claims in the present invention.
Claims (10)
1. the product of detection NFIC gene expression application in the instrument of preparation diagnosis hypophysoma.
Application the most according to claim 1, it is characterised in that described product includes: by RT-PCR, real-time quantitative PCR,
Immune detection, in situ hybridization, chip or high-flux sequence detection of platform NFIC gene expression are to diagnose the product of hypophysoma；Described
The primer of a pair specific amplified NFIC gene is at least included with the product of RT-PCR diagnosis hypophysoma；Described real-time quantitative PCR
The product of diagnosis hypophysoma at least includes the primer of a pair specific amplified NFIC gene；Described immune detection diagnosis hypophysoma
Product includes: the antibody being combined with NFIC protein-specific；The product of described in situ hybridization diagnosis hypophysoma includes: with NFIC
The probe of the nucleic acid array hybridizing of gene；The product of described chip diagnosis hypophysoma includes: protein chip and genetic chip；Its
In, protein chip includes the antibody being combined with NFIC protein-specific, and genetic chip includes miscellaneous with the nucleotide sequence of NFIC gene
The probe handed over.
Application the most according to claim 2, it is characterised in that the product of described real-time quantitative PCR diagnosis hypophysoma is extremely
The primer of a pair specific amplified NFIC gene included less is as shown in SEQ ID NO.3 and SEQ ID NO.4.
4. the instrument diagnosing hypophysoma, it is characterised in that described instrument includes the reagent detecting NFIC gene expression；Described
Reagent includes primer and/or probe, the antibody of detection NFIC albumen detecting NFIC gene mRNA.
Instrument the most according to claim 4, it is characterised in that the primer of described detection NFIC gene mRNA includes SEQ ID
Primer pair shown in NO.3 and SEQ ID NO.4.
The application in the medicine of preparation treatment hypophysoma of the inhibitor of 6.NFIC gene and/or its expression product.
Application the most according to claim 6, it is characterised in that described inhibitor includes the examination suppressing NFIC gene expression
Agent and/or the reagent of suppression NFIC gene expression product.
Application the most according to claim 7, it is characterised in that the reagent of described suppression NFIC gene expression product includes pressing down
Make the antibody of siRNA and/or the NFIC albumen for NFIC gene.
Application the most according to claim 8, it is characterised in that the described siRNA sequence for NFIC gene such as SEQ ID
Shown in NO.7 and SEQ ID NO.8.
10. the pharmaceutical composition being used for treating hypophysoma, it is characterised in that described pharmaceutical composition includes claim 6-
Inhibitor according to any one of 9.
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Cited By (6)
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| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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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