CN105624324B - Hypophysoma diagnosis and treatment marker - Google Patents
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Abstract
The invention discloses a kind of hypophysoma diagnosis and treatment markers.Hypophysoma is the common benign tumour of encephalic, but also some hypophysomas have the characteristics that surrounding tissue infiltrative growth.It is demonstrated experimentally that TRIM15 gene expresses up-regulation in hypophysis tumor tissue, the overexpression of TRIM15 gene can promote the growth of pituicyte, inhibit the expression of TRIM15 gene that can inhibit the proliferation of pituitary tumor cell.The present invention provides a kind of diagnostic methods of hypophysoma, also disclose a kind of Molecular tools for the treatment of hypophysoma.
Description
Technical field
The invention belongs to biomedicine fields, are related to a kind of diagnosis and treatment marker of hypophysoma, the more specific marker
For TRIM15 gene.
Background technique
Hypophysoma is common one of the neuroendocrine that pituitary hormone secretion can be caused unbalance, disease incidence row
After glioma and meningioma, the third position of intracranial tumors disease incidence is occupied.In recent years, mentioning due to people's living standard
Height, the improvement of inspection method, the development of iconography and each subject technology, the discovery rate of hypophysoma is in the trend risen.Generally
In the case of, disease incidence gradually increases non-functional hypophysoma with advancing age, and pituitary tumor,functioning is generally in young man
In it is common.Hypophysoma is not common in children, only accounts for 2% or so of the primary intracranial tumors of children.It reports in recent years both at home and abroad
Hypophysoma has the tendency that increased significantly.
Hypophysoma histologically belongs to benign tumour, but there are also hypophysomas to have to surrounding tissue infiltrative growth
The characteristics of, referred to as invasive pituitary adenomas.Though invasive pituitary adenomas is pathologically belonging to benign tumour, have to surrounding normal knot
Structure such as skull, cavernous sinus, endocranium, sphenoid sinus, three ventricles of the brain, on saddle, by saddle, slope even nasopharyngeal cavity position be in invasive growth
The characteristics of.Surgery alone treatment is difficult to complete resection, so as to cause to recur.Even if postoperative be aided with radiotherapy, recurrence rate is still
It can be very high.
With the progress of medical technology and the development of molecular biology, work of the gene played in tumor development is studied
With having great importance.It was found that and illustrate the mechanism of action of related gene, tumour-specific can be reacted by not only facilitating discovery
The marker of biological behaviour is used for clinical diagnosis and follow-up investigation, it is often more important that provides molecule for the treatment and research of tumour
Means and theoretical basis.
Summary of the invention
In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a kind of diagnosis and treatment markers-of hypophysoma
TRIM15 gene.
To achieve the goals above, the present invention adopts the following technical scheme:
The present invention provides a kind of application of gene and its expression product in the product of preparation diagnosis hypophysoma, wherein
The gene is TRIM15.
Further, the product includes by RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip detection
The expression of TRIM15 gene and its expression product is to diagnose the product of hypophysoma.
Wherein, the product with RT-PCR diagnosis hypophysoma includes at least drawing for a pair of of specific amplified TRIM15 gene
Object；The product with real-time quantitative PCR diagnosis hypophysoma includes at least the primer of a pair of of specific amplified TRIM15 gene；It is described
Product with immune detection diagnosis hypophysoma includes: the antibody in conjunction with TRIM15 protein-specific；It is described to be examined in situ hybridization
The product of disconnected hypophysoma includes: the probe with the nucleic acid array hybridizing of TRIM15 gene；The production with chip diagnosis hypophysoma
Product include: protein chip and genetic chip；Wherein, protein chip includes the antibody in conjunction with TRIM15 protein-specific, gene
Chip includes the probe with the nucleic acid array hybridizing of TRIM15 gene.
Further, the genetic chip can be used for detecting multiple genes including TRIM15 gene (for example, and hypophysis
The relevant multiple genes of tumor) expression.The protein-chip can be used for detecting multiple including TRIM15 albumen
The expression of protein (such as multiple protein relevant to hypophysoma).By the mark for detecting multiple hypophysomas simultaneously
Object is greatly improved the accuracy rate of hypophysoma diagnosis.
The present invention provides a kind of product for diagnosing hypophysoma, the product can pass through TRIM15 in detection pituitary tissue
The expression of gene diagnoses hypophysoma.
Further, the product includes chip or kit；Wherein, the chip includes genetic chip, protein core
Piece；The genetic chip includes solid phase carrier and the oligonucleotide probe for being fixed on solid phase carrier, the oligonucleotide probe
Including the oligonucleotide probe for TRIM15 gene for detecting TRIM15 gene transcription level；The protein-chip packet
It includes solid phase carrier and is fixed on the specific antibody of the TRIM15 albumen of solid phase carrier；The kit includes genetic test examination
Agent box and protein immunization detection kit；The gene detecting kit includes the examination for detecting TRIM15 gene transcription level
Agent；The protein immunization detection kit includes the specific antibody of TRIM15 albumen.
Further, the reagent includes the primer and/or probe for TRIM15 gene.
Gene detecting kit of the present invention can be used for detecting multiple gene (examples including TRIM15 gene
Such as, multiple genes relevant to hypophysoma) expression.The protein immunization detection kit can be used for detecting
The expression of multiple protein (such as multiple protein relevant to hypophysoma) including TRIM15 albumen.By hypophysoma
Multiple markers are detected simultaneously, are greatly improved the accuracy rate of hypophysoma diagnosis.
The answering in the pharmaceutical composition of preparation treatment hypophysoma the present invention provides TRIM15 gene and its expression product
With.
Further, described pharmaceutical composition includes TRIM15 gene and/or the inhibitor of its expression product.The inhibitor
Substance, the substance for inhibiting TRIM15 gene expression product stability, and/or inhibition including inhibiting TRIM15 gene expression
The active substance of TRIM15 gene expression product.
Preferably, the inhibitor is the siRNA for TRIM15 gene.
The present invention also provides a kind of pharmaceutical composition for treating hypophysoma, the drug include TRIM15 gene and/or
Its expression product inhibitor.The inhibitor includes the substance for inhibiting TRIM15 gene expression, TRIM15 gene expression is inhibited to produce
The substance, and/or the inhibition active substance of TRIM15 gene expression product of object stability.
Pharmaceutical composition of the invention can also can be with the drug combination of other treatment hypophysoma, a variety of Drug combinations
Improve the success rate for the treatment of.
Further, above-mentioned pharmaceutical composition further includes pharmaceutically acceptable carrier, and this kind of carrier includes (but and unlimited
In): diluent, excipient such as lactose, sodium chloride, glucose, urea, starch, water etc., filler such as starch, sucrose etc.；Bonding
Agent such as simple syrup, glucose solution, starch solution, cellulose derivative, alginates, gelatin and polyvinylpyrrolidone；It is wet
Agent such as glycerol；Disintegrating agent such as dried starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and sodium bicarbonate；It absorbs
Promotor quaternary ammonium compound, lauryl sodium sulfate etc.；Surfactant such as polyoxyethylene sorbitan aliphatic ester, 12
Sodium alkyl sulfate, glyceryl monostearate, hexadecanol etc.；Humectant such as glycerol, starch etc.；Absorption carrier for example starch, lactose,
Bentonite, silica gel, kaolin and soap clay etc.；Lubricant such as talcum powder, calcium stearate and magnesium, polyethylene glycol, boric acid powder etc..
Pharmaceutical composition of the invention can be used different additives and be prepared, such as stabilizer, fungicide, buffering
Agent, isotonic agent, chelating agent, pH controlling agent and surfactant.
Stabilizer includes Human serum proteins, l-amino acid, sugar and cellulose derivative.L-amino acid can also include sweet
Any one in propylhomoserin, cysteine and glutamic acid.Carbohydrate includes monosaccharide, such as glucose, mannose, galactolipin, fructose
Deng；Sugar alcohol, such as mannitol, inositol, xylitol etc.；Disaccharides, such as sucrose, maltose, lactose etc.；Polysaccharide, such as Portugal
Glycan, hydroxypropul starch, vulcanization chondroitin, hyaluronic acid etc. and their derivative.Cellulose derivative includes Methyl cellulose
Element, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hypromellose and sodium cellulose glycolate.
Buffer may include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid and corresponding salt (their alkali metal or alkali
Property rare earth metal salt, such as sodium salt, sylvite, calcium salt and magnesium salts).Isotonic agent includes potassium chloride, sodium chloride, sugar and glycerol.Chelating
Agent includes sodium ethylene diamine tetracetate and citric acid.
Surfactant includes ion or nonionic surfactant, such as polyethylene glycol oxide Arrcostab, sorbitan list
Acyl ester, fatty glyceride.
Drug of the invention may also include pharmaceutically acceptable coating material, fast decoupled coating
Material, coloring agent, enteric polymer, plasticizer, water-soluble polymer, insoluble polymer, dyestuff, pigment, other collapse
Powder.Common fast decoupled coating material includes OPADRY；Enteric polymer includes methylacrylic acid polymer, phosphorus hydroxypropyl first
Base cellulose phthalate, hypromellose acetic acid esters, hypromellose succinate, hydroxyl first ethyl cellulose
Element, cellulose acetophthalate；Plasticizer includes polyethylene glycol (PEG), propylene glycol etc..
The unit dosage forms of drug of the present invention can make diversified forms, and representative dosage form includes solid dosage forms such as pill, powder
Agent, tablet, dry powder doses, particle, capsule etc.；Liquid forms such as suspension, solution, emulsion, elixir, syrup etc..Institute of the present invention
Receptor can be given by any approach by stating drug, can be by oral or non-oral a variety of as long as can reach destination organization
Approach, such as oral administration, feeding drug into pulmones, drop rectum with drug, intranasal administration, subcutaneous administration, intradermal administration, intraperitoneal administration, flesh
Interior administration, intravenous administration.
The carrier of the present invention for carrying gene is various carriers known in the art, such as commercially available carrier including plasmid,
Clay, bacteriophage, virus etc..
In the present invention for TRIM15 gene oligonucleotide probe can be DNA, RNA, DNA-RNA chimera, PNA or
Other derivatives.There is no limit as long as complete specific hybrid and purpose nucleotide sequence specificity knot for the length of the probe
It closes, any length is ok.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the probe
Length can grow to 60,80,100,150,300 base-pairs or longer or even whole genes.Due to different probe lengths pair
Hybridization efficiency, signal specificity have different influences, and the length of the probe is typically at least 14 base-pairs, and longest is generally not
More than 30 base-pairs, the length complementary with purpose nucleotide sequence are best with 15-25 base-pair.The probe self-complementary
Sequence is most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.
The specific antibody of heretofore described TRIM15 albumen includes monoclonal antibody, polyclonal antibody.It is described
The specific antibody of TRIM15 albumen includes any segment or modification of complete antibody molecule, antibody, for example, chimeric antibody,
ScFv, Fab, F (ab ') 2, Fv etc..As long as the segment can retain the binding ability with TRIM15 albumen.For detecting
The preparation of the antibody of protein level is well known to those skilled in the art, and the present invention may use any method to prepare
The antibody, segment as mentioned can be synthesized by chemical method de novo formation or using recombinant DNA technology.
In the present invention, the solid phase carrier includes plastic products, microparticle, membrane carrier etc..The plastic products can lead to
It crosses non-covalent or physical absorption mechanism to combine with antibody or proteantigen, most common plastic products are made of polystyrene
Small test tube, globule and micro-reaction plate；The microparticle is the microballoon or particle aggregated by high polymer monomer, and diameter is mostly
Micron easily can form chemical coupling with antibody (antigen), binding capacity is big with the functional group in conjunction with protein due to having；Institute
Stating membrane carrier includes the miillpore filters such as nitrocellulose filter, glass fibre element film and nylon membrane.
In the present invention, the RNA interference (RNAinterference, RNAi), which refers to, is highly conserved during evolution
, being induced by double-stranded RNA (double-stranded RNA, dsRNA), homologous mRNA efficient selective degradation the phenomenon that.Make
With RNAi technology can with specific depletion or close specific gene expression, the technology have been widely used for explore gene function and
The field of gene of communicable disease and malignant tumour.RNAi based on cell is screened in terms of functional gene research
With many advantages, RNAi method can be used by being mainly manifested in most cell types, and be easier to lower or sink relatively
It writes from memory the expression of any target gene.
In the present invention, term " host cell " includes prokaryotic cell and eukaryocyte.Common prokaryotic host cell
Example includes Escherichia coli, hay bacillus etc..Common eukaryotic host cell includes yeast cells, insect cell and mammal
Cell.Preferably, the host cell is eukaryocyte, such as Chinese hamster ovary celI, COS cell.
In the context of the present invention, " TRIM15 gene " includes any of people TRIM15 gene and people's TRIM15 gene
The polynucleotides of functional equivalent.TRIM15 gene include in the public GenBank GeneBank in the current world
TRIM15 gene (NC_000006.12) DNA sequence dna has 70% or more homology, and encodes the DNA sequence of identical function protein
Column；
Preferably, the coded sequence of TRIM15 gene includes following any DNA molecular:
(1) DNA sequence dna shown in SEQ ID NO.1 in sequence table；
(2) hybridize under strict conditions with the DNA sequence dna that (1) limits and encode 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 TRIM15 gene is shown in SEQ ID NO.1
DNA sequence dna.
In the context of the present invention, TRIM15 gene expression product includes people TRIM15 albumen and people's TRIM15 albumen
Partial peptide.The partial peptide of the TRIM15 albumen contains functional domain relevant to hypophysoma.
" TRIM15 albumen " includes any functional equivalent of TRIM15 albumen and TRIM15 albumen.The function is equivalent
Object includes TRIM15 albumen conservative variation protein or its active fragment or its reactive derivative or its mutant.Mutant
Pass through missing, substitution, increase and/or insertion including allelic variant, natural mutation, induced mutants, its amino acid sequence
The mutant that morphs, can be with the encoded albumen of DNA of the DNA hybridization of people TRIM15 under high or low stringent condition
Matter.
Preferably, TRIM15 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,
Preferably, with the homology of 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 TRIM15 albumen is with amino acid shown in SEQ ID NO.2
The protein of sequence.
In general, the modification of one or more amino acid will not influence the function of protein in a protein.This field skill
Art personnel can approve the amino acid for changing single amino acids or small percentage or individual additions to amino acid sequence, missing, slotting
Entering, replacing is conservative modification, and wherein the change of protein generates the protein with identity function.Intimate amino is provided
The Conservative substitution tables of acid are well known in the art.
The modification of amino acid sequence is modified after being originated from spontaneous mutation or heredity, can also be produced with artificial induction's natural gene
It is raw.Example by one amino acid of addition or the protein of more amino acid modification is the fusion egg of TRIM15 albumen
It is white.For the peptide or protein with TRIM15 protein fusion, there is no limit as long as resulting fusion protein retains TRIM15 egg
White biological activity.
TRIM15 albumen of the invention also includes the non-conservative modification to amino acid sequence shown in SEQ ID NO.2, only
It still to be able to retain the biological activity of TRIM15 albumen by the protein of modification.It dashes forward in such modification protein
The amino acid number of change is usually 10 perhaps less such as 6 perhaps less such as 3 or less.
In the context of the present invention, " treatment hypophysoma " may include disease according to the state change of disease come point
Alleviate, the complete healing of disease；The effect played according to drug is different, may include inhibiting cell growth, promoting Apoptosis.
The advantages of the present invention:
Present invention firstly discovers that molecular marker-TRIM15 relevant to hypophysoma occurrence and development, tested by detecting
The expression of TRIM15 in person's pituitary tissue, it can be determined that whether subject suffers from hypophysoma, while the present invention provides one kind to control
The Molecular tools for treating hypophysoma are had specific, non-invasive using gene marker treatment disease.
Detailed description of the invention
Fig. 1 shows the expression using QPCR detection TRIM15 gene in hypophysis tumor tissue；
Fig. 2 shows the influence using QPCR detection siRNA to TRIM15 gene expression；
Fig. 3 shows that soft-agar cloning forms the influence of experiment detection siRNA cell proliferation；
Fig. 4 shows the influence of mtt assay detection TRIM15 gene pairs people hypophysis tumor proliferation.
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.
Embodiment 1 screens gene marker relevant to hypophysoma
1, sample collection
Respectively collect 6 normal pituitary tissues and hypophysoma tissue samples.The acquirement of above-mentioned all samples passes through tissue human relations
The agreement of the reason committee.
2, the preparation of RNA sample (utilizesMiRNA kit is operated)
The tissue of above-mentioned acquisition shred after put into liquid nitrogen in and be ground to it is powdered, according in kit specification extract
Separate RNA.It is specific as follows:
1) separation of RNA:
A. it is added in tissue homogenate or cellReagent II 1ml；
B. it is placed at room temperature for 3min, is aggressively shaken 15s after 0.2ml chloroform is added；
C. be placed in prevents 10min on ice；
D.12000g, 4 DEG C of centrifugation 15min；
E. the water phase of transfer 80% enters in new 2ml EP pipe, and the dehydrated alcohol of 1/2 amount, shaking is added；
F. the aforesaid liquid less than 700 μ l is transferred toRNA Mini column, 10000g room temperature after shaking
It is centrifuged 60s.
2) RNA is purified:
A. toRNA Mini column is added 500 μ l RWC Wash Buffer, 10000g and is centrifuged 30s；
B. 500 μ l RWB Wash Buffer, 10000g centrifugation 30s are added, take maximum centrifugal complete after being repeated twice
It is dryRNA Mini column；
C. the DEPC water that 15 μ l are preheated to 70 DEG C is added to pillar, is centrifuged at full speed after being placed at room temperature for 2min.
3, high-throughput transcript profile sequencing
1) RNA-seq read positions
Low-quality read is removed to obtain cleaning read first, then using TopHat v1.3.1 will clean segment and
UCSC H.sapiens is matched with reference to genome (hg19), the H.sapiens UCSC hg19 editions indexes constructed in advance
It is downloaded from TopHat homepage, and as reference genome, when matching using TopHat with genome, allows each read (default
To 20) having multiple matching sites, most 2 mispairing.TopHat establishes possible according to exon region and GT-AG shear signal
Shearing site library navigates to the read for not navigating to genome on genome according to these shearing site libraries.We use
The system default parameter of TopHat method.
2) transcript abundance is assessed
The read file matched is handled by Cufflinks v1.0.3, and Cufflinks v1.0.3 is by RNA-seq piece
Number of segment mesh is standardized the relative abundance for calculating transcript.FPKM value refers to being matched in every 1,000,000 sequencing fragment specific
The segment number of the exon region of gene 1kb long.The confidence interval of FPKM estimated value is calculated by Bayesian inference method.
The GTF comment file for the reference that Cufflinks is used downloads (Homo_ from Ensembl database
sapiens.GRCh37.63.gtf)。
3) detection of difference expression gene
It is transferred to Cuffdiff by the Ensembl GTF file of downloading and by the matched original document of TopHat,
Cuffdiff re-evaluates the gene expression abundance for the transcript listed in GTF file using original matching files, detects difference table
It reaches.The only q value < 0.01 in Cuffidff output, test display is more just considered as successfully differential expression.
4, result
RNA-seq is the results show that expression quantity of the TRIM15 gene in hypophysis tumor tissue is significantly higher than normal pituitary tissues
In expression quantity.
The differential expression of embodiment 2QPCR sequence verification TRIM15 gene
1, large sample QPCR verifying is carried out to TRIM15 gene differential expression.According to the sample collection mode in embodiment 1
Select normal pituitary tissues and hypophysis tumor tissue each 50.
2, RNA extraction step is as described in Example 1.
3, reverse transcription:
1) reaction system:
| Reagent | Volume |
| MgCl2 | 2μl |
| 10×RT Buffer | 1μl |
| Without Rnase water | 3.75μl |
| DNTP mixed liquor | 1μl |
| Rnase inhibitor | 0.25μl |
| AMV reverse transcriptase | 0.5μl |
| Oligomerization dT aptamer primer | 0.5μl |
| Laboratory sample | 1μl |
2) reverse transcription reaction condition
It is carried out according to reverse transcription reaction condition in RNA PCR Kit (AMV) Ver.3.0.
42 DEG C~55 DEG C 60min, 99 DEG C of 2min, 5 DEG C of 5min.
3) polymerase chain reaction
1) design of primers
QPCR amplimer, You Bomai are designed according to the coded sequence of TRIM15 gene and GAPDH gene in Genebank
The synthesis of moral biotech firm.Specific primer sequence is as follows:
TRIM15 gene:
Forward primer is 5 '-AGAGATGAGATTGAGGATGTAA-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-TTCCACCTGATGCTTCTT-3 ' (SEQ ID NO.4).
β-actin gene:
Forward primer is 5 '-CTGGGACGACATGGAGAAAA-3 ' (SEQ ID NO.5)；
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.6).
2) PCR reaction system is prepared according to table 1:
1 PCR reaction system of table
| Reagent | Volume |
| Forward primer | 0.5μl |
| Reverse primer | 0.5μl |
| Takara Ex Taq HS | 12.5μl |
| Template | 10μl |
| Deionized water | Supply 25 μ l |
3) PCR reaction condition: 94 DEG C of 5min, (94 DEG C of 30s, 58 DEG C of 40s, 72 DEG C of 40s) × 35 circulations, 72 DEG C of 5min.
Using SYBR Green as fluorescent marker, PCR reaction is carried out on Light Cycler fluorescence quantitative PCR instrument, by melting
Tracing analysis and electrophoresis determine that purpose band, Δ Δ CT method carry out relative quantification.
5, 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.
6, result
As a result as shown in Figure 1, compared with normal pituitary tissues, TRIM15 gene expresses up-regulation in hypophysis tumor tissue, poor
It is different that there is statistical significance (P < 0.05), it is consistent with RNA-sep result.
The overexpression of embodiment 3TRIM15 gene
1, cell culture
People pituitary tumor cell strain GT1.1, with the culture medium 1640 containing 10% fetal calf serum and 1%P/S in 37 DEG C, 5%
CO2, relative humidity be 90% incubator in cultivate.It changes within 2-3 days liquid 1 time, trypsase of the use 0.25% containing EDTA is conventional
Had digestive transfer culture.
2, siRNA is designed
For the siRNA sequence of TRIM15 gene:
Negative control siRNA sequence (siRNA-NC):
Positive-sense strand is 5 '-UUCUCCGAACGUGUCACGU-3 ' (SEQ ID NO.7),
Antisense strand is 5 '-ACGUGACACGUUCGGAGAA-3 ' (SEQ ID NO.8)；
SiRNA1-PLD5:
Positive-sense strand is 5 '-AGAAGAAGUAGAUCUUCUCGC-3 ' (SEQ ID NO.9),
Antisense strand is 5 '-GAGAAGAUCUACUUCUUCUGC-3 ' (SEQ ID NO.10)；
SiRNA2-PLD5:
Positive-sense strand is 5 '-UCACAAAAGUCUUCAUCUCAC-3 ' (SEQ ID NO.11),
Antisense strand is 5 '-GAGAUGAAGACUUUUGUGAGU-3 ' (SEQ ID NO.12)；
Cell is pressed 5 × 105/ hole is inoculated into six porocyte culture plates, in 37 DEG C, 5%CO2Cell culture in incubator
For 24 hours, in DMEM culture medium without double antibody, containing 10%FBS, transfection (is purchased from according to lipofectamine 2000
Invitrogen company) specification transfection, experiment be divided into control group (GT1.1) negative control group (siRNA-NC) and experimental group
(20nM) (siRNA1-TRIM15, siRNA2-TRIM15), wherein the sequence of negative control group siRNA and TRIM15 gene without
Homology, concentration is the hole 20nM/, while being transfected respectively.
3, QPCR detects the transcriptional level of TRIM15 gene
The extraction of 3.1 cell total rnas
Using TRIzol Reagent (Invitrogen Cat.No.15596-018) total RNA extraction reagent, by specification
The total serum IgE of providing method extraction GT1.1 cell.
1) cell is taken, is rinsed 3 times with the PBS that concentration is 0.01M.
2) appropriate TRIzol reagent is added, is placed at room temperature for 5min lytic cell, piping and druming is uniform.
3) it is dispensed with 1ml/ pipe into 1.5ml EP pipe.0.2ml chloroform is added in every pipe, acutely shakes 15s, is placed at room temperature for 2-
3min。
4) 4 DEG C, 12000rpm centrifugation 15min.
5) upper strata aqueous phase is moved in clean EP pipe, 0.5ml isopropanol is added, mixes gently, is placed at room temperature for 10min.
6) 4 DEG C, 7500rpm centrifugation 10min.
7) supernatant, 75% ethanol washing RNA precipitate are abandoned, 7500rpm is centrifuged 5min.
8) drying at room temperature RNA precipitate is dissolved in appropriate DEPC water after 5-10min.
9) agarose gel electrophoresis that mass fraction is 1.0% detects the integrality of RNA sample, using Bio-
Photometer quantitative determines the RNA of extraction.
3.2 reverse transcription steps are the same as embodiment 2.
3.3QPCR amplification step is the same as embodiment 2.
4, statistical method
Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD,
Using SPSS13.0 statistical software come for statistical analysis, the difference between TRIM15 gene expression panel and control group is interfered to adopt
It is examined with t, it is believed that there is statistical significance as P < 0.05.
5, result
As a result such as Fig. 2 is shown, compared to GT1.1, transfection zero load siRNA-NC, siRNA2-TRIM15 group, siRNA1-
TRIM15 group can significantly reduce the expression of TRIM15 gene, and difference has statistical significance (P < 0.05).
4 soft-agar cloning of embodiment forms experiment
1, the cell of logarithmic growth phase is in 0.25% trypsin digestion, gently piping and druming makes unicellular outstanding
Cell precipitation is collected by centrifugation in liquid.
2, it is resuspended with the DMEM complete medium containing 20% fetal calf serum, is counted after appropriate dilution, adjustment cell concentration is 5
×103A/ml.
3, the low melting point agar liquid glucose that two concentration are respectively 1.2% and 0.7% is prepared, after high pressure sterilization, maintains 40
In DEG C water-bath.
4,1.2% agarose and 2 × DMEM culture medium 1:1 mixing, are added the calf serum of 2 × antibiotic and 20%,
It takes in 3ml mixed liquor injection diameter 6cm plate and places 5min cooled and solidified, be placed in CO as bottom-layer agar2It is spare in incubator.
5, in sterile test tube 1:1 mixing 0.7% agarose and 2 × DMEM culture medium, then into pipe be added 0.2ml it is dense
Degree is 5 × 103A/ml's stablizes infection cell suspension, mixes well, injects in above-mentioned plate, gradually forms double agar layers, often
A experimental group repeats 4 samples.
6, after top-layer agar solidification, 37 DEG C of 5%CO are placed in2It is cultivated in incubator, every 3 days plus culture medium 1.5ml.
7, culture dish is taken out after cultivating 14 days, dyes 90min with the gentian violet that 1ml concentration is 0.005%.Plate is placed
It is observed under inverted microscope, every group of cell randomly selects 10 low-power fields, the number of cell clones that technology is formed under mirror.
8, result
As a result as shown in figure 3, compared with other groups, siRNA1-TRIM15 group single cell clone Colony forming digital display writes drop
It is low.
The influence of embodiment 5TRIM15 gene pairs people hypophysis tumor proliferation
It is influenced using MTT experiment detection TRIM15 gene pairs pituitary tumor cell proliferative capacity.
1, cell culture and transfection procedure are the same as embodiment 3.
2, step: pancreatin digests after group of cells transfects 12h, single cell suspension is made, with 6000, every hole cell inoculation
In 96 well culture plates, at 7 time points of every component, each time point sets 6 multiple holes.After cell is adherent, the 1st detection is carried out:
The 20 μ l of MTT liquid of 5g/L is added in every hole, continues after cultivating 4h, sucks culture medium, and 150 μ l of DMSO is added, and carefulness piping and druming makes purple
Blue precipitate sufficiently dissolves, and surveys absorbance value (A value) in 490nm wavelength with microplate reader.Then every 12h is detected 1 time, continuous to detect
72h, totally 7 times.This experiment is repeated 3 times.
3, statistical method
Experiment is completed according to being repeated 3 times, using SPSS13.0 statistical software come for statistical analysis, the two it
Between difference using t examine, it is believed that as P < 0.05 have statistical significance.
4, result
It is shown in Fig. 4 as the result is shown: the vitro growth rates of siRNA1-TRIM15 group significantly lower than transfection siRNA-NC group
Vitro growth rates, difference have statistical significance (P < 0.05).The above results show that TRIM15 expression is conducive to hypophysoma
The growth of cell, by inhibiting the expression of TRIM15 gene that can inhibit 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 (6)
1. the substance for detecting gene and its expression product is preparing the application in the product for diagnosing hypophysoma, which is characterized in that institute
Stating gene is TRIM15.
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 or chip detect the expression of TRIM15 gene and its expression product to diagnose the production of hypophysoma
Product.
3. application according to claim 1, which is characterized in that the product includes chip or kit；Wherein, described
Chip includes genetic chip, protein-chip；The genetic chip includes solid phase carrier and the few core for being fixed on solid phase carrier
Thuja acid probe, the oligonucleotide probe include the few core for TRIM15 gene for detecting TRIM15 gene transcription level
Thuja acid probe；The protein-chip include solid phase carrier and be fixed on solid phase carrier TRIM15 albumen specificity it is anti-
Body；The kit includes gene detecting kit and protein immunization detection kit；The gene detecting kit includes using
In the reagent of detection TRIM15 gene transcription level；The protein immunization detection kit includes that the specificity of TRIM15 albumen is anti-
Body.
4. application according to claim 3, which is characterized in that the reagent include for TRIM15 gene primer and/
Or probe.
Application of the inhibitor of 5.TRIM15 gene and its expression product in the pharmaceutical composition of preparation treatment hypophysoma, it is special
Sign is that the inhibitor is the substance for inhibiting TRIM15 gene expression.
6. application according to claim 5, which is characterized in that the inhibitor is the siRNA for TRIM15.
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| 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 |
| CN108220441A (en) * | 2018-01-04 | 2018-06-29 | 大连医科大学附属第二医院 | Application and expression of the TBX19 genes in pituitary adenoma biomarker |
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