CN105734147A - Gene MUC21 and application of expression product of gene MUC21 in hypophysoma diagnosis and treatment - Google Patents

Gene MUC21 and application of expression product of gene MUC21 in hypophysoma diagnosis and treatment Download PDF

Info

Publication number
CN105734147A
CN105734147A CN201610201350.3A CN201610201350A CN105734147A CN 105734147 A CN105734147 A CN 105734147A CN 201610201350 A CN201610201350 A CN 201610201350A CN 105734147 A CN105734147 A CN 105734147A
Authority
CN
China
Prior art keywords
muc21
gene
expression
product
pituitary tumor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201610201350.3A
Other languages
Chinese (zh)
Other versions
CN105734147B (en
Inventor
宋宏涛
杨承刚
孙耀兰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qingdao Yangshen Biomedical Co Ltd
Original Assignee
Beijing Medintell Bioinformatic Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Medintell Bioinformatic Technology Co Ltd filed Critical Beijing Medintell Bioinformatic Technology Co Ltd
Priority to CN201610201350.3A priority Critical patent/CN105734147B/en
Publication of CN105734147A publication Critical patent/CN105734147A/en
Application granted granted Critical
Publication of CN105734147B publication Critical patent/CN105734147B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups

Abstract

The invention discloses a gene MUC21 and application of an expression product of the gene MUC21 in hypophysoma diagnosis and treatment.The gene MUC21 related to generation and development of hypophysoma is screened through a bioinformatics method, function verification is conducted through a fluorescent quantitative method and the like, and down-regulated expression of the gene MUC21 is conducted in hypophysoma patient tissue; by increasing the expression of the gene MUC21, the excessive proliferation of hypophysoma cells can be restrained.The invention further discloses a product for diagnosing hypophysoma and a medicine composition for treating hypophysoma.

Description

MUC21 gene and expression product purposes in pituitary tumor diagnosis and treatment thereof
Technical field
The invention belongs to biomedicine field, relate to MUC21 gene and expression product thereof the purposes in pituitary tumor diagnosis and treatment.
Background technology
Pituitary tumor is a kind of common intracranial tumor, accounts for the 10%～20% of intracranial tumor.It derives from gene expression, for the clonal expansion of tumor cell, presents the characteristic increased without hormone or hormone secretion.Most pituitary tumors are benign adenoma, and only a few is cancer.
Pituitary tumor is divided into without the non-functional chromophobe adenoma of granule by pathology and dyeing, has granule to have the oncocytoma of function and basophiloma and mixed type four kinds.Pituitary tumor is divided into prolactinoma, somatotropinoma, thyroliberin tumor, thyrotropin tumor, promoting sexual gland hormone tumor, many hormones adenoma and nonfunctional adenoma according to emiocytosis function in recent years.
Pituitary tumor often shows as the oncothlipsis symptoms such as headache, vision and visual field change and the symptom that the too much hormone of tumors secrete causes clinically.When adenoma is excessive, compressing, when destroying normal pituitary cell, it may appear that hypopituitarism, as weak, poor appetite, be afraid of cold, the symptom such as sexual hypofunction.
The diagnosis of pituitary tumor depends on mensuration and the imaging examination of each hormone of laboratory.Treatment for pituitary tumor at present has Drug therapy, operative treatment, radiotherapy and Comprehensive Treatment, operative treatment is the first-selection of other all kinds pituitary tumors at present, but traditional operation treatment is while tumor resection, causing a degree of damage, small part patient is possibly even collapsed owing to postoperative urine blind, permanent occur in location of operation or excision degree difference, the serious post-operative complication such as long-term high heat, stupor.And the Secondary cases Radioactive seeds implantation after radiotherapy is also still in current radiotherapy technology uncontrollable difficult point.
Along with molecular biological development, the study of incident mechanism aspect of pituitary tumor is also achieved considerable progress.Understanding and treatment pituitary tumor are had important effect by the pathogenesis exploring pituitary tumor, and treatment and the guidance of patient are had great significance by the index understanding pituitary tumor prognosis.
Summary of the invention
In order to make up the deficiencies in the prior art, it is an object of the invention to provide the molecular marked compound-MUC21 gene of pituitary tumor diagnosis and treatment.Gene marker is used to diagnose and treat pituitary tumor more timely, special.
To achieve these goals, the present invention adopts the following technical scheme that
The invention provides the application in the pharmaceutical composition of preparation treatment pituitary tumor of MUC21 gene and expression product thereof.
Further, described pharmaceutical composition includes increasing MUC21 gene expression, strengthening MUC21 expressive function and/or strengthen the reagent of MUC21 expression product activity.
Further, described reagent includes: containing can the reagent of nucleic acid of encoding function MUC21 albumen, the activator of MUC21 albumen, reagent containing MUC21 protein.
Wherein, the reagent of the described nucleic acid containing energy encoding function MUC21 albumen can be single-chain nucleic acid (such as mRNA) or the double-strandednucleic acid (such as DNA) of the MUC21 albumen translating into activity form under advantage, described nucleic acid can be connected on expression vector or restructuring is in host cell, as long as activity MUC21 albumen, the carrying mode of any MUC21 gene can be encoded into.Described MUC21 protein activator refer to stimulate MUC21 protein active, increase MUC21 protein active, promote MUC21 protein active, strengthen MUC21 protein activation, make MUC21 protein active sensitization or raise MUC21 protein active reagent, transcriptional activation agent as specific in demethylation reagent, MUC21 promoter and/or enhancer, MUC21 albumen agonist (as activate antibody) etc..
Further, aforementioned pharmaceutical compositions also includes pharmaceutically acceptable carrier, and described carrier can be one can also be multiple, and described carrier includes but not limited to diluent such as lactose, sodium chloride, glucose, carbamide, starch, water etc.；Binding agent such as starch, pregelatinized Starch, dextrin, maltodextrin, sucrose, arabic gum, gelatin, methylcellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, Polyethylene Glycol, PVP, alginic acid and alginate, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methyl cellulose etc.；Surfactant is polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulphate, glycerol monostearate, hexadecanol etc. such as；Humectant is glycerol, starch etc. such as；Absorption carrier such as starch, lactose, bentonite, silica gel, Kaolin and soap clay etc.；Lubricant such as zinc stearate, glyceryl monostearate, Polyethylene Glycol, Pulvis Talci, calcium stearate and magnesium, Polyethylene Glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, single Laurel sucrose acid ester, sodium laurylsulfate, magnesium laurylsulfate, Stepanol MG etc.；Filler such as mannitol (granular or powdery), xylitol, sorbitol, maltose, erythrose, microcrystalline Cellulose, polymerization sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and sodium bicarbonate etc.；Disintegrating agent is cross-linked ethylene ketopyrrolidine, carboxymethyl starch sodium, low-substituted hydroxypropyl ylmethyl, cross-linking sodium carboxymethyl cellulose, soybean polysaccharide etc. such as.
Described pharmaceutical composition can use different additives to be prepared, for instance stabilizer, antibacterial, buffer agent, isotonic agent, chelating agen, pH controlling agent and surfactant.
Stabilizer includes Human serum proteins, l-amino acid, sugar and cellulose derivative.L-amino acid can also include any one in glycine, cysteine and glutamic acid.Saccharide includes monosaccharide, for instance glucose, mannose, galactose, fructose etc.；Sugar alcohol, for instance mannitol, inositol, xylitol etc.；Disaccharide, for instance sucrose, maltose, lactose etc.；Polysaccharide, for instance glucosan, hydroxypropyl starch, sulfuration chrondroitin, hyaluronic acid etc. and their derivant.Cellulose derivative includes methylcellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose and Carboxymethyl cellulose sodium.
Surfactant includes ion or nonionic surfactant, for instance polyethylene glycol oxide Arrcostab, sorbitan monoacyl ester, fatty glyceride.
Additive buffer agent can include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid and corresponding salt (their alkali metal or alkaline rare earth metal salt, for instance sodium salt, potassium salt, calcium salt and magnesium salt).Isotonic agent includes potassium chloride, sodium chloride, sugar and glycerol.Chelating agen includes sodium ethylene diamine tetracetate and citric acid.
Present invention also offers a kind of pharmaceutical composition treating pituitary tumor, described pharmaceutical composition includes increasing MUC21 gene expression, strengthening MUC21 expressive function and/or strengthen the reagent of MUC21 expression product activity.
The invention provides the application in the product of preparation diagnosis pituitary tumor of MUC21 gene and expression product thereof.
Further, described product diagnoses pituitary tumor by detecting the expression of the MUC21 gene in pituitary tissue.
Further, the described product by the expression of the MUC21 gene detected in pituitary tissue includes: by the expression of RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip detection MUC21 gene and expression product thereof.
Further, the product of described RT-PCR diagnosis pituitary tumor at least includes the primer of a pair specific amplified MUC21 gene；The product of described real-time quantitative PCR diagnosis pituitary tumor at least includes the primer of a pair specific amplified MUC21 gene；The product of described immune detection diagnosis pituitary tumor includes: the antibody being combined with MUC21 protein-specific；The product of described in situ hybridization diagnosis pituitary tumor includes: with the probe of the nucleic acid array hybridizing of MUC21 gene；The product of described chip diagnosis pituitary tumor includes: protein chip and gene chip；Wherein, protein chip includes the antibody being combined with MUC21 protein-specific, and gene chip includes the probe of the nucleic acid array hybridizing with MUC21 gene.
Further, described gene chip can be used for detecting the expression of the multiple genes (such as, relevant to pituitary tumor multiple genes) including MUC21 gene.Described protein chip can be used for detecting the expression of the multiple protein (such as relevant to pituitary tumor multiple protein) including MUC21 albumen.By being detected by multiple marks with pituitary tumor simultaneously, it is greatly improved the accuracy rate of pituitary tumor diagnosis.
The invention provides a kind of product diagnosing pituitary tumor, described product can diagnose pituitary tumor by the expression of MUC21 gene in detection pituitary tissue.
Further, described product includes chip or test kit；Wherein, described chip includes gene chip, protein chip；Described test kit includes gene detecting kit, protein immunization detection kit.Described gene chip includes solid phase carrier and is fixed on the oligonucleotide probe of solid phase carrier, and described oligonucleotide probe includes the oligonucleotide probe for MUC21 gene for detecting MUC21 gene transcription level；Described protein chip includes solid phase carrier and is fixed on the specific antibody of MUC21 albumen of solid phase carrier；Described gene detecting kit includes the reagent for detecting MUC21 gene transcription level；Described protein immunization detection kit includes the specific antibody of MUC21 albumen.
Gene detecting kit of the present invention can be used for detecting the expression of the multiple genes (such as, relevant to pituitary tumor multiple genes) including MUC21 gene.Described protein immunization detection kit can be used for detecting the expression of the multiple protein (such as relevant to pituitary tumor multiple protein) including MUC21 albumen.Multiple marks of pituitary tumor are detected simultaneously, is greatly improved the accuracy rate of pituitary tumor diagnosis.
In the present invention, pharmaceutical composition may be used for supplementing disappearance or the deficiency of endogenic MUC21 albumen, by improving the expression of MUC21 albumen, thus treatment reduces, because of MUC21 albumen, the pituitary tumor caused.
The carrier carrying gene of the present invention is various carrier known in the art, such as commercially available carrier, includes plasmid, cosmid, phage, virus etc..
Further, in the present invention, the nucleic acid of MUC21 albumen or coding MUC21 albumen can be given by liposome, and the effect of described liposome is in specific tissue by drug targeting, and increases the half-life of medicine.Liposome includes emulsifying agent, foaming agent, liquid fatty substance, Solid lipid, insoluble monolayer, phospholipid dispersions, surfactant etc..Described liposome can also include be combined with the acceptor molecule in the cell of targeting or other treatment or immunogenic composition.
The pharmaceutical composition of the present invention can be formulated into any administration fashion, such as, utilize in syringe or the intradermal injection of other device, subcutaneous injection, intravenous injection, peritoneal injection, intrapleural injection, Intravesical administration, coronary artery or intra-tumoral injection, oral administration, rectally.
The medicine of the present invention imports the mode of tissue or cell can be divided into external or internal mode.Vitro formats includes in the medicine containing MUC21 gene or the medicine transfered cell containing MUC21 protein, then by cell transplantation or feed back to internal.Internal mode includes directly by the medicine containing MUC21 gene or the infusion of medicine in-vivo tissue containing MUC21 protein.
The medicine of the present invention also can with the drug combination of other treatment pituitary tumor, and other treatment compound can be administered with main active component (such as, the nucleic acid of MUC21 albumen or encoding said proteins) simultaneously, is administered even in same compositions simultaneously.Other therapeutic compound can also be individually given with independent compositions or the dosage form different from main active component.The Fractional of main component (nucleic acid such as MUC21 albumen or encoding said proteins) can be administered with other therapeutic compound simultaneously, and other dosage can be individually dosed.
In treatment of diseases, it is possible to the physiologic response according to the order of severity of symptom, the frequency of recurrence and therapeutic scheme, adjust the dosage of pharmaceutical composition of the present invention.
In the present invention, term " host cell " includes prokaryotic cell and eukaryotic cell.The example of conventional prokaryotic host cell includes escherichia coli, bacillus subtilis etc..Conventional eukaryotic host cell includes yeast cells, insect cell and mammalian cell.It is preferred that this host cell is eukaryotic cell, such as Chinese hamster ovary celI, COS cell etc..
The present invention can be DNA, RNA, DNA-RNA chimera, PNA or other derivant for the probe of MUC21 gene.The length of described probe does not limit, as long as completing specific hybrid and purpose nucleotide sequence is specific binding, any 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 can be grown to 60,80,100,150,300 base pairs or longer, even whole gene.Owing to hybridization efficiency, signal specificity are had different impacts, the length of described probe be typically at least 14 base pairs by different probe length, the longest it is usually no more than 30 base pairs, best with 15-25 base pair with the length of purpose nucleotide sequence complementary.Described probe self-complementary sequences is most preferably less than 4 base pairs, in order to avoid affecting hybridization efficiency.
The specific antibody of heretofore described MUC21 albumen includes monoclonal antibody, polyclonal antibody.The specific antibody of described MUC21 albumen includes complete antibody molecule, any fragment of antibody or modification, for instance, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc..As long as described fragment can retain and the binding ability of MUC21 albumen.For detect the antibody of protein level be prepared by well known to a person skilled in the art, and the present invention can use any method to prepare described antibody, and fragment can be passed through chemical method de novo synthesis or utilize recombinant DNA technology to synthesize as mentioned.
In the context of the present invention, " MUC21 gene " includes the polynucleotide of any function equivalent of people's MUC21 gene and people's MUC21 gene.MUC21 gene includes having more than 70% homology and coding identical function protein DNA sequence with MUC21 gene (NC_000006.12) DNA sequence in international common core sequence databank GeneBank at present；
Preferably, the coded sequence of MUC21 gene includes any one DNA molecular following:
(1) DNA sequence shown in SEQ ID NO.1；
(2) the DNA sequence hybridization limited with (1) under strict conditions and coding identical function protein DNA sequence；
(3) DNA sequence limited with (1) or (2) has 70%, preferably, more than 90% homology, and coding identical function protein DNA molecule.
In specific embodiments of the present invention, the coded sequence of described MUC21 gene is the DNA sequence shown in SEQIDNO.1.
In the context of the present invention, MUC21 gene expression product includes people's MUC21 albumen and the partial peptide of people's MUC21 albumen.The partial peptide of described MUC21 albumen contains the functional domain relevant to pituitary tumor.
" MUC21 albumen " includes any function equivalent of MUC21 albumen and MUC21 albumen.Described function equivalent includes MUC21 albumen conservative variation's protein or its active fragment, or its reactive derivative, allelic variant, natural mutation, induced mutants, can with the protein coded by the DNA of the DNA hybridization of people MUC21 under high or low stringent condition.
Preferably, MUC21 albumen is the protein with following amino acid sequences:
(1) protein that the aminoacid sequence shown in SEQ ID NO.2 forms；
(2) aminoacid sequence shown in SEQIDNO.2 had the protein that the aminoacid sequence shown in SEQIDNO.2 of identical function is derivative through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with the aminoacid sequence shown in SEQIDNO.2.The amino acid whose number replacing, lack or adding is generally 1-50, it is preferred that 1-30, and more preferably 1-20,1-10 is individual best.
(3) with the aminoacid sequence shown in SEQIDNO.2, there is at least 80% homology (being also called sequence iden), preferably, with the aminoacid sequence shown in SEQIDNO.2 at least about 90% to 95% homology, be often 96%, 97%, 98%, 99% homology aminoacid sequence constitute polypeptide.
In specific embodiments of the present invention, described MUC21 albumen is the protein with the aminoacid sequence shown in SEQIDNO.2.
Generally, in a protein one or more amino acid whose modifications without influence on the function of protein.Those skilled in the art can approve the aminoacid of change single amino acids or little percentage ratio or be conservative modifications to indivedual interpolations of aminoacid sequence, disappearance, insertion, replacement, and wherein the generation that changes of protein has the protein of identity function.It is well known in the art for providing intimate amino acid whose Conservative substitution tables.
By adding the fusion protein that the example of the protein of an aminoacid or multiple Modification of amino acid residues is MUC21 albumen.Peptide or protein with MUC21 protein fusion is not limited, as long as the fusion protein of gained retains the biologic activity of MUC21 albumen.
The MUC21 albumen of the present invention also includes the non-conservative modification to the aminoacid sequence shown in SEQIDNO.2, as long as the protein through modifying remains able to retain the biologic activity of MUC21 albumen.In this type of modifying protein, the amino acid number of sudden change is usually 10 or less, for instance 6 or less, for instance 3 or less.
In the context of the present invention, " treatment pituitary tumor " divides according to the state of disease, including the generation of any symptom and the recurrence that can eliminate, alleviate, alleviate, reverse or prevent or postpone disease, namely includes the therapeutic intervention to disease and Primary preventive intervention；Different according to the effect that medicine plays, it is possible to include cell growth inhibiting, promote apoptosis.
Advantages of the present invention and beneficial effect:
Present invention firstly discovers that the new molecular marked compound-MUC21 gene of a kind of pituitary tumor, by detecting the expression of MUC21 in experimenter's pituitary tissue, may determine that whether experimenter suffers from pituitary tumor, thus instructing clinicist to provide prevention scheme or therapeutic scheme to experimenter；Utilize molecular marked compound to realize diagnosing and treating of disease, compare traditional means, have more promptness, specificity, non-invasive.
Accompanying drawing explanation
Fig. 1 show utilize QPCR detect MUC21 gene expression in pituitary tumor tissue；
Fig. 2 show utilize QPCR detect MUC21 gene expression in pituitary tumor cell；
Fig. 3 shows that utilizing soft-agar cloning to form experiment detects the MUC21 gene expression impact on pituitary tumor cell multiplication capacity.
Specific embodiment
Below in conjunction with drawings and Examples, the present invention is further detailed explanation.Following example are merely to illustrate the present invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment, generally conventionally condition, such as Sambrook et al., molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to manufacturer it is proposed that condition.
The gene marker that embodiment 1 screening is relevant to pituitary tumor
1, sample collection
Each collection 6 example normal pituitary tissues and pituitary tumor tissue samples.The acquirement of above-mentioned all specimen is each through the agreement of committee of organizational ethics.
2, the preparation of RNA sample (utilizesMiRNAkit is operated)
The tissue of above-mentioned acquisition puts in liquid nitrogen after shredding and is ground to Powdered, extracts according to the description in test kit and separates RNA.Specific as follows:
1) separation of RNA:
A. tissue homogenate or cell addReagentII1ml；
B. room temperature places 3min, is aggressively shaken 15s after adding 0.2ml chloroform；
C. it is placed in and prevents 10min on ice；
D.12000g, 4 DEG C of centrifugal 15min；
E. the aqueous phase of transfer 80% enters in new 2mlEP pipe, adds the dehydrated alcohol of 1/2 amount, jolting；
F. the aforesaid liquid less than 700 μ l is transferred toRNAMinicolumn, the centrifugal 60s of 10000g room temperature after jolting.
2) RNA purification:
A. toRNAMinicolumn adds the centrifugal 30s of 500 μ lRWCWashBuffer, 10000g；
B. add the centrifugal 30s of 500 μ lRWBWashBuffer, 10000g, after repeating twice, take maximum centrifugal to be completely driedRNAMinicolumn；
C. adding 15 μ l to pillar and be preheated to the DEPC water of 70 DEG C, room temperature is centrifugal at full speed after placing 2min.
3, high flux transcript profile order-checking
1) RNA-seq reads section location
First low-quality reading section is removed and obtain cleaning reading section, then utilize TopHatv1.3.1 will clean fragment to mate with reference to genome (hg19) with UCSCH.sapiens, the index built in advance of H.sapiensUCSChg19 version is downloaded from TopHat homepage, and as with reference to genome, when utilizing TopHat to mate with genome, each reading section (defaulting to 20) is allowed to have multiple coupling site, maximum 2 mispairing.TopHat sets up possible shearing site storehouse according to exon region and GT-AG shear signal, will not navigate to genomic reading section according to these shearing site storehouses and navigate on genome.We use the system default parameter of TopHat method.
2) transcript abundance assessment
The reading segment file matched is by Cufflinksv1.0.3 process, and RNA-seq segment number is standardized calculating the relative abundance of transcript by Cufflinksv1.0.3.FPKM value refers to the segment number of the exon region matching specific gene 1kb length in each million order-checking fragments.The confidence interval of FPKM estimated value is calculated by Bayesian inference method.The GTF comment file of the reference that Cufflinks uses downloads (Homo_sapiens.GRCh37.63.gtf) from Ensembl data base.
3) detection of difference expression gene
The EnsemblGTF file of download and the original document that mated by TopHat are transferred to Cuffdiff, Cuffdiff uses original matching files again to estimate the gene expression abundance of the transcript listed in GTF file, detects differential expression.Only having q value < 0.01 in Cuffidff exports, test display is considered as successfully more just differential expression.
4, result
RNA-seq result shows, MUC21 gene expression in pituitary tumor tissue is substantially less than the expression in normal pituitary tissues.
The differential expression of embodiment 2QPCR sequence verification MUC21 gene
1, MUC21 gene differential expression is carried out large sample QPCR checking.Normal pituitary tissues and each 50 examples of pituitary tumor tissue are selected according to the sample collection mode in embodiment 1.
2, QPCR concrete operation step is as follows:
(1) RNA extracts
RNA extraction step is as described in Example 1.
(2) reverse transcription
A. in Microtube, configure mixed liquor: OligodT (50 μMs) 1 μ l, dNTP mixed liquor (10mM) 1 μ l, RNA template 5 μ g, add ddH2O to 10 μ l, mix homogeneously；
B. in PCR instrument, degeneration, annealing reaction are carried out according to following reaction condition: after 65 DEG C of 5min, place immediately on ice；
C. in above-mentioned Microtube pipe, following inverse transcription reaction liquid is prepared:
Reactant liquor 10 μ l after above-mentioned degeneration, annealing, 5 ×Buffer4 μ l, RNase inhibitor (40U/ μ l) 0.5 μ l,RTase (200U/ μ l) 1 μ l, ddH2O (RNase-free) 4.5 μ l, mix homogeneously；
D. in PCR instrument, reverse transcription reaction is carried out according to following reaction condition:
(30 DEG C of 10min) × 3, (42 DEG C of 45min) × 4, (95 DEG C of 5min) × 5, after process, be placed on ice；
E. PCR reactant liquor is prepared on ice by following formula:
PremixExTaqII (TliRNaseHPlus) (2 ×) 12.5 μ l, forward primer (10 μMs) 1 μ l, reverse primer (10 μMs) 1 μ l, DNA profiling (< 100ng) 2.0 μ l, ddH2O8.5μl；
The cycling condition of F.PCR is as follows:
94 DEG C of 5min, (94 DEG C of 30s, 58 DEG C of 40s, 72 DEG C of 40s) × 35, choose β-actin as internal reference.The primer sequence of PCR is as follows:
The primer sequence of MUC21:
Forward primer: 5 '-AATGTTCTCCTTATGTTTGGTCTA-3 ' (SEQIDNO.3),
Reverse primer: 5 '-GATCCAGTGTTGGCAGAG-3 ' (SEQIDNO.4)
The primer sequence of β-actin:
Forward primer: 5 '-CCTGGGCATGGAGTCCTGTG-3 ' (SEQIDNO.5)
Reverse primer: 5 '-TCCTTCTGCATCCTGTCG-3 ' (SEQIDNO.6)
Using SYBRGreen as fluorescent marker, in the enterprising performing PCR reaction of LightCycler fluorescence real-time quantitative PCR instrument, determining purpose band by melt curve analysis analysis and electrophoresis, Δ Δ CT method carries out relative quantification.
3, statistical method
Experiment all completes for 3 times according to repetition, and result data is all represent in the way of mean+SD, adopts SPSS13.0 statistical software to carry out statistical analysis, and difference between the two adopts t inspection, it is believed that when P < has statistical significance when 0.05.
4, result
Result is as it is shown in figure 1, compared with normal pituitary tissues, MUC21 gene is lowered in pituitary tumor tissue, and difference has statistical significance (P < 0.05), consistent with RNA-sep result.
The process LAN of embodiment 3MUC21 gene
1, cell is cultivated
People pituitary tumor cell strain GT1.1, with the culture medium 1640 containing 10% hyclone and 1%P/S at 37 DEG C, 5%CO2, relative humidity be 90% incubator in cultivate.Within 2-3 days, change liquid 1 time, use the 0.25% trypsin conventional digestion containing EDTA to go down to posterity.
2, the process LAN of MUC21 gene
The structure of 2.1MUC21 expression vector
Coded sequence (as shown in SEQIDNO.1) design amplimer according to MUC21 gene, primer sequence is as follows:
Forward primer: 5 '-CCGAAGCTTGCCACCATGAAGATGCAGA-3 ' (SEQIDNO.7)
Reverse primer: 5 '-CGGGCGGCCGCGGGCCCGCTGTTCCTCC-3 ' (SEQIDNO.8)
From cDNA library (the clontech company becoming Human fetal spleen, article No.: 638831) in the coded sequence of MUC21 gene of amplification total length, above-mentioned cDNA sequence is inserted in the eukaryotic expression vector pcDNA3.1 of restricted enzyme HindIII and NotI double digestion after restricted enzyme HindIII and NotI double digestion, connects the recombinant vector pcDNA3.1-MUC21 obtained for subsequent experimental.
2.2 transfections
Pituitary tumor cell is divided into two groups, respectively matched group (transfection pcDNA3.1 empty carrier) and MUC21 process LAN group (transfection pcDNA3.1-MUC21).Using liposome 2000 to carry out the transfection of carrier, the instruction to specifications of concrete transfection method carries out.The working concentration of pcDNA3.1 empty carrier and pcDNA3.1-MUC21 is 0.5 μ g/ml.
2.3RT-PCR detects
Concrete steps are with embodiment 2.
3, statistical method
Experiment all completes for 3 times according to repetition, and result data is all represent in the way of mean+SD, adopts SPSS13.0 statistical software to carry out statistical analysis, and difference between the two adopts t inspection, it is believed that when P < has statistical significance when 0.05.
4, result
As in figure 2 it is shown, compared with the cell of transfection pcDNA3.1 empty carrier, in the cell of transfection pcDNA3.1-MUC21, the content of MUC21 significantly raises, and difference has statistical significance (P < 0.05).
The impact of embodiment 4MUC21 gene pairs pituitary tumor cell apoptosis
Use the flow cytomery apoptotic impact of MUC21 gene pairs.
1, cell culture step is with embodiment 3.
2, cell transfecting step is with embodiment 3.
3, step
After cell transfecting 72h, use pre-cooling PBS washed cell, then use 0.25% trypsin digestion cell, stop digestion, use PBS resuspended in the cell of centrifugal collection, be 1 × 10 by cell quantification6Individual/ml, takes the 200 above-mentioned cell suspension of μ l and is placed in Eppendorf pipe, adds 10 μ lAnnexin-V-FITC mixings, and dyeing 15min is hatched in room temperature dark place, and before upper machine, 5min adds 10mg/L iodate the third ingot (PI) and dyes 5 μ l.The cell of untransfected siRNA is used for standard quantitative with Annexin-V-FITC and PI dyeing respectively.Two Colour Fluorescence cell cytometry, observing apoptosis cell percentages is carried out with FACS flow cytometer.
3, statistical method
Experiment all completes for 3 times according to repetition, result data is all represent in the way of mean+SD, SPSS13.0 statistical software is adopted to carry out statistical analysis, the t inspection that difference between the two adopts, it is believed that when P < has statistical significance when 0.05.
4, result:
The apoptosis rate of transfection pcDNA3.1-MUC21 group is (25.34 ± 0.013) %, the apoptosis rate of transfection pcDNA3.1 empty carrier group is (6.58 ± 0.009) %, above-mentioned difference has statistical significance (P < 0.05), the above results shows, the process LAN of MUC21 gene promotes the apoptosis of pituitary tumor cell.
Embodiment 5 soft-agar cloning forms experiment
1, be in the cell of exponential phase with 0.25% trypsinization, piping and druming makes single cell suspension gently, and centrifugal collecting cell precipitates.
2, resuspended with the DMEM complete medium containing 20% hyclone, suitably counting after dilution, adjusting cell concentration is 5 × 103Individual/ml.
3, two concentration of preparation respectively 1.2% and 0.7% LMP agar sugar liquid, after autoclaving, maintain in 40 DEG C of water-baths.
4,1.2% agarose and 2 × DMEM culture medium 1:1 mixing, add the calf serum of 2 × antibiotic and 20%, take 3ml mixed liquor and inject diameter 6cm plate is placed 5min cooled and solidified, be placed in CO as bottom-layer agar2In incubator standby.
5, in sterile test tube, 1:1 mixes agarose and 2 × DMEM culture medium of 0.7%, then addition 0.2ml concentration is 5 × 10 in pipe3The stable infection cell suspension of individual/ml, fully mixes, injects in above-mentioned plate, gradually forms double; two agar layer, and each experimental group repeats 4 samples.
6, after top-layer agar solidifies, 37 DEG C of 5%CO are inserted2Incubator is cultivated, within every 3 days, adds culture medium 1.5ml.
7, culture dish is taken out after cultivating 14 days, with the Gentian Violet dyeing 90min that 1ml concentration is 0.005%.Plate being placed under inverted microscope observe, often group cell randomly selects 10 low power fields, the number of cell clones that under mirror, technology is formed.
8, result
Result is as it is shown on figure 3, compared with other groups, the groups of cells single cell clone Colony forming number of transfection pcDNA3.1-MUC21 significantly reduces.
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 the person of ordinary skill of the art, under the premise without departing from the principles of the invention, it is also possible to the present invention carries 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 application in the pharmaceutical composition of preparation treatment pituitary tumor of 1.MUC21 gene and expression product thereof.
  2. 2. application according to claim 1, it is characterised in that described pharmaceutical composition includes increasing MUC21 gene expression, strengthening MUC21 expressive function and/or strengthen the reagent of MUC21 expression product activity.
  3. 3. application according to claim 2, it is characterised in that described reagent includes: containing can the reagent of nucleic acid of encoding function MUC21 albumen, the activator of MUC21 albumen, reagent containing MUC21 protein.
  4. 4. the application according to Claims 2 or 3, it is characterised in that described pharmaceutical composition also includes pharmaceutically acceptable carrier.
  5. 5. the pharmaceutical composition treating pituitary tumor, it is characterised in that described pharmaceutical composition includes increasing MUC21 gene expression, strengthening MUC21 expressive function and/or strengthen the reagent of MUC21 expression product activity.
  6. The application in the product of preparation diagnosis pituitary tumor of 6.MUC21 gene and expression product thereof.
  7. 7. application according to claim 6, it is characterised in that described product diagnoses pituitary tumor by detecting the expression of the MUC21 gene in pituitary tissue.
  8. 8. application according to claim 7, it is characterized in that, the expression of the described MUC21 gene by detecting in pituitary tissue includes: by the expression of RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip detection MUC21 gene and expression product thereof.
  9. 9. the product diagnosing pituitary tumor, it is characterised in that described product can diagnose pituitary tumor by the expression of MUC21 gene in detection pituitary tissue.
  10. 10. product according to claim 9, it is characterised in that described product includes chip or test kit.
CN201610201350.3A 2016-03-31 2016-03-31 The purposes of MUC21 gene and its expression product in hypophysoma diagnosis and treatment Active CN105734147B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610201350.3A CN105734147B (en) 2016-03-31 2016-03-31 The purposes of MUC21 gene and its expression product in hypophysoma diagnosis and treatment

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610201350.3A CN105734147B (en) 2016-03-31 2016-03-31 The purposes of MUC21 gene and its expression product in hypophysoma diagnosis and treatment

Publications (2)

Publication Number Publication Date
CN105734147A true CN105734147A (en) 2016-07-06
CN105734147B CN105734147B (en) 2019-10-11

Family

ID=56252637

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610201350.3A Active CN105734147B (en) 2016-03-31 2016-03-31 The purposes of MUC21 gene and its expression product in hypophysoma diagnosis and treatment

Country Status (1)

Country Link
CN (1) CN105734147B (en)

Cited By (5)

* Cited by examiner, † Cited by third party
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
CN108114283A (en) * 2018-01-12 2018-06-05 蚌埠医学院第附属医院 Applications of the biomarker MUC21 in osteosarcoma diagnose and treat
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
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
CN116492366A (en) * 2023-04-17 2023-07-28 中山大学附属第五医院 Application of biomarker MUC21 in pancreatic cancer diagnosis and treatment

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007034603A1 (en) * 2005-09-20 2007-03-29 Niigata University Marker gene for malignant brain tumor and use thereof
CN105132550A (en) * 2015-08-31 2015-12-09 北京泱深生物信息技术有限公司 Application of MUC21 gene expression detection reagent in preparation of osteoarthritis diagnosis and treatment products

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007034603A1 (en) * 2005-09-20 2007-03-29 Niigata University Marker gene for malignant brain tumor and use thereof
CN105132550A (en) * 2015-08-31 2015-12-09 北京泱深生物信息技术有限公司 Application of MUC21 gene expression detection reagent in preparation of osteoarthritis diagnosis and treatment products

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
宋怀东等: "垂体瘤发病相关基因的大规模筛选及鉴定", 《癌症》 *

Cited By (6)

* Cited by examiner, † Cited by third party
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
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
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
CN108114283A (en) * 2018-01-12 2018-06-05 蚌埠医学院第附属医院 Applications of the biomarker MUC21 in osteosarcoma diagnose and treat
CN116492366A (en) * 2023-04-17 2023-07-28 中山大学附属第五医院 Application of biomarker MUC21 in pancreatic cancer diagnosis and treatment
CN116492366B (en) * 2023-04-17 2023-09-29 中山大学附属第五医院 Application of biomarker MUC21 in pancreatic cancer diagnosis and treatment

Also Published As

Publication number Publication date
CN105734147B (en) 2019-10-11

Similar Documents

Publication Publication Date Title
CN105734147A (en) Gene MUC21 and application of expression product of gene MUC21 in hypophysoma diagnosis and treatment
CN105734159A (en) Molecular marker of esophageal squamous carcinoma
CN105695616A (en) Analysis marker for diagnosing thyroid cancer and application thereof
CN105779598B (en) A kind of molecular marker of diagnosis and treatment hypophysoma
CN105803069A (en) Application of SMR3B gene to hypophysoma diagnosis and treatment
CN105525018B (en) Application of the FAM176A gene in rectal adenocarcinoma diagnosing and treating
CN105624324B (en) Hypophysoma diagnosis and treatment marker
CN105861679B (en) Purposes of the PCSK9 in esophageal squamous cell carcinoma diagnosis and treatment
CN105734161A (en) Disease-causing gene of esophageal squamous carcinoma and application of disease-causing gene
CN105296656B (en) A kind of molecular marker of diagnosis and treatment nasopharyngeal carcinoma
CN105886629B (en) Application of the molecular marker in diagnosis and treatment esophageal squamous cell carcinoma
CN105296623B (en) A kind of molecular marker of diagnosis and treatment osteoarthritis
CN105624325B (en) The diagnosis and treatment marker of adenocarcinoma of lung
CN105112550B (en) MTUS1 genes as osteoporosis diagnosis and treatment target
CN104878104B (en) Cholangiocarcinoma diagnosis and treatment molecular marker and its application
CN105087821B (en) A kind of molecular marker of diagnosis and treatment osteoporosis
CN105463123B (en) A kind of molecular marked compound for rectal adenocarcinoma diagnosis and treatment
CN105803068A (en) Molecular marker for diagnosis and treatment of lung adenocarcinoma
CN105886656B (en) Application of the GIF gene in esophageal squamous cell carcinoma diagnosis and treatment
CN105331691B (en) Application of the PZP genes in cholangiocarcinoma diagnose and treat
CN105624313A (en) Molecular marker for diagnosing and treating adenocarcinoma of lungs
CN105969884B (en) Application of the CHIA in esophageal squamous cell carcinoma
CN105385776B (en) Application of the CCDC57 genes in Degenerative disc disease diagnosing and treating
CN105506170B (en) Purposes of the SAV1 gene as fibroid diagnosis and treatment marker
CN105861740B (en) Purposes of the ABLIM3 gene as cancer of the esophagus diagnosis and treatment marker

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
CB02 Change of applicant information

Address after: Room 1210, Building 3, Ronghua Xintai Building, 10 Ronghua South Road, Yizhuang Economic and Technological Development Zone, Daxing District, Beijing

Applicant after: Beijing Yang Shen biology information technology company limited

Address before: 100080 Beijing city Haidian District Shanyuan Street No. 1 cubic court building room 3103

Applicant before: Beijing Yang Shen biology information technology company limited

CB02 Change of applicant information
GR01 Patent grant
GR01 Patent grant
TR01 Transfer of patent right

Effective date of registration: 20211116

Address after: 266000 room 2503, Qianshan building, building D2, phase II, Qingdao International Innovation Park, No. 1, Keyuan Weiyi Road, Laoshan District, Qingdao, Shandong

Patentee after: Qingdao Yangshen biomedical Co., Ltd

Address before: Room 1210, building 3, Ronghua Xintai building, No. 10, Ronghua South Road, Yizhuang Economic and Technological Development Zone, Daxing District, Beijing 100176

Patentee before: Beijing Yangshen biological information technology Co., Ltd

TR01 Transfer of patent right