CN106011288A - Multiple myeloma diagnosis and treatment marker and application thereof - Google Patents
Multiple myeloma diagnosis and treatment marker and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of medicines, and discloses an ERI3 gene and new use of a code thereof. Studies prove that the ERI3 gene and encoded protein thereof not only can be taken as a molecular marker for diagnosing the multiple myeloma, but also can be used as a molecular target for treating the multiple myeloma. Based on the study results, the invention provides a new diagnosis method and treatment means for clinically.
Description
Technical field
The present invention relates to diagnosing tumor, treat, predict prognosis field, more particularly it relates to detection
ERI3 is abnormal is the diagnosing tumor of means, prediction method of prognosis；And suppress ERI3 gene or the tumor of protein
Therapeutic agent.
Background technology
Multiple myeloma (Multiple myeloma, MM) is to assemble bone marrow and at blood with malignant plasma cell
Or urine occurs a kind of malignant disease that m protein (M albumen) is characterized.Clinical characters includes easily
Send out infection, impaired renal function, anemia, hypercalcemia and osteolytic lesion.MM accounts for all hematologic
Sick 10%, is apt to occur in old people, and along with China's aged tendency of population, MM number of the infected has the trend of increase.
It is now recognized that MM is a process being progressively in progress: a lot of patients occur the list of interrogatory in early days
Clone ball albumen disease (MGUS), then progresses to MM, and some patients finally develops into marrow dermoskeleton myeloma (slurry
Chronic myeloid leukemia), whole during Bone Marrow of Patients plasma cell with the sudden change of several genes.Clinically, according to
MM is carried out by stages by some clinical parameters such as M albumen of MM, β 2-MG, to take different controlling
Treatment measure.Conventional staging scale has Durie-Salmon (DS) Staging System and staging system system (ISS).
ISS is the most simple and easy to do, according to β 2-MG and serum albumin two indices, MM is divided into I phase, II
Phase, III phase, III phase conditions of patients is worst with prognosis.Present clinical treats the Main Means of each phase MM to be had
Hormone, alkyls medicine, immunomodulator, protease inhibitor etc., these methods can make MM delay
Solve, but final unavoidable recurrence, therefore there are new Clinics and Practices strategies more to be developed.
Summary of the invention
An object of the present invention is that providing a kind of is diagnosed by detection ERI3 gene or protein expression difference
The method of multiple myeloma.
The two of the purpose of the present invention are that providing a kind of is predicted by detection ERI3 gene or protein expression difference
The method of multiple myeloma prognosis.
The three of the purpose of the present invention are that providing a kind of treats many by suppression ERI3 gene or ERI3 albumen
The method of the property sent out myeloma.
A kind of method that the four of the purpose of the present invention are to provide medicine for screening treatment multiple myeloma.
The five of the purpose of the present invention are to provide a kind of medicine for treating multiple myeloma.
To achieve these goals, present invention employs following technical scheme:
The product that the invention provides detection ERI3 gene or ERI3 albumen is preparing Diagnosis of Multiple Myeloma
Purposes in instrument.
The product that present invention also offers detection ERI3 gene or ERI3 albumen predicts multiple bone marrow in preparation
Purposes in tumor prognostic tool.
Further, the product of described detection ERI3 gene or ERI3 albumen includes detecting ERI3 gene or ERI3
The product of the expression of albumen.Described product includes can be in conjunction with the nucleic acid of ERI3 gene or can be in conjunction with
The material (such as antibody) of ERI3 albumen.Described nucleic acid can detect the expression of ERI3 gene；Described
Material can detect the expression of ERI3 albumen.
The product of the detection ERI3 gene of the present invention can play its merit based on the known method using nucleic acid molecules
Can: as PCR, as Southern hybridization, Northern hybridization, dot blot, fluorescence in situ hybridization (FISH),
DNA microarray, ASO method, high-flux sequence platform etc..Use this product can qualitatively, quantitatively,
Or semi-quantitatively implement to analyze.
The nucleic acid being included in the said goods can be obtained by chemosynthesis, or by preparing from biomaterial
Containing expectation nucleic acid gene, then use be designed for amplification expectation nucleic acid primer amplification it obtain.
Further, described PCR method is known method, such as, and ARMS (Amplification Refractory
Mutation System, amplification do not answer abruptly-changing system) method, RT-PCR (reverse transcriptase-PCR) method, nesting
PCR method etc..The nucleic acid of amplification can be by using dot blotting hybridization method, Surface Plasmon Resonance (SPR
Method), PCR-RFLP method, B by means of in situ RT PCR, PCR-SSO (sequence specific oligonucleotide) method,
PCR-SSP method, AMPFLP (amplifiable fragment length polymorphism) method, MVR-PCR method and
PCR-SSCP (single strand conformation polymorphism) method detects.
Nucleic acid recited above includes the primer expanding ERI3 gene, and the primer that product includes can be by logical
Crossing chemosynthesis to prepare, the method that be those skilled in the art will know that by use is come suitably with reference to Given information
Design, and prepared by chemosynthesis.
In specific embodiments of the present invention, described nucleic acid is the amplimer used in QPCR experiment, institute
State shown in sequence such as SEQ ID NO.1 (forward sequence) and SEQ ID NO.2 (reverse sequence) of primer.
Nucleic acid recited above may also include probe, and described probe can be prepared by chemosynthesis, by making
Appropriately design with reference to Given information by the method that those skilled in the art will know that, and prepared by chemosynthesis,
Or the gene containing expectation nucleotide sequence from biomaterial preparation can be passed through, and use is designed for the amplification phase
Hope nucleotide sequence primer amplification it prepare.
The product of the detection ERI3 albumen of the present invention can play its function based on the known method using antibody:
For example, it is possible to include ELISA, radioimmunoassay, immunohistochemical method, western blot etc..
The product of the detection ERI3 albumen of the present invention includes antibody or its fragment of specific binding ERI3 albumen.
Antibody or its fragment of any structure, size, immunoglobulin class, origin etc. can be used, if its knot
Conjunction target protein.Antibody or its fragment that the detection product of the present invention includes can be monoclonal or many
Clone's.Antibody fragment refers to that retaining antibody to an antibody part (Partial Fragment) of the combination activity of antigen or contains
There is the peptide of an antibody part.Antibody fragment can include F (ab ')2, Fab ', Fab, scFv (scFv),
The Fv (dsFv) of disulphide bonding or its polymer, dimerization V district (double antibody) or containing CDR
Peptide.The product of the detection ERI3 albumen of the present invention can include the aminoacid of encoding antibody or Encoding Antibody Fragment
The nucleic acid of the separation of sequence, comprises the carrier of this nucleic acid, and carries the cell of this carrier.
Antibody can be by well known to a person skilled in the art that method obtains.Such as, preparation retains whole or portion
The polypeptide dividing target protein or the mammalian cell expression vector integrating their polynucleotide of coding are as anti-
Former.Use after antigen-immunized animal, from through the animal adaptive immune cell of immunity fused bone myeloma cells with
Obtain hybridoma.Then antibody is collected from Hybridoma culture.Finally can be used as antigen by use
ERI3 albumen or its part antibody to obtaining are implemented antigenic specificity purification and are obtained the list for ERI3 albumen
Clonal antibody.Polyclonal antibody can be prepared as follows: with antigen-immunized animal same as above, from through exempting from
The animal of epidemic disease collects blood sample, isolates serum from blood, then uses above-mentioned antigen to implement serum anti-
Former specificity purification.Can be by the antibody obtained with ferment treatment or the sequence information of the antibody obtained by use
Obtain antibody fragment.
The combination of label and antibody or its fragment can be implemented by method as commonly known in the art.Such as,
Can fluorescent marker protein or peptide as follows: clean protein or peptide with phosphate buffer, add with DMSO,
Buffer agent, etc. preparation dyestuff, then mixed solution, then at room temperature place 10 minutes.It addition, labelling can
The labelling kit of commodity in use, such as biotin labeling reagent box, as biotin labeling reagent box-NH2,
Biotin labeling reagent box-SH (DojindoLaboratories)；Alkali phosphatase enzyme mark test kit such as alkalescence phosphorus
Acid enzyme labelling test kit-NH2, alkali phosphatase enzyme mark test kit-SH (Dojindo Laboratories)；Peroxide
Compound enzyme labelling test kit such as peroxidase labelling test kit-NH2, peroxidase labelling test kit
-NH2(Dojindo Laboratories)；Phycobniliprotein labelling kit such as phycobniliprotein labelling kit
-NH2, phycobniliprotein labelling kit-SH, B-phycoerythrin labelling kit-NH2, B-phycoerythrin
Labelling kit-SH, R-PE labelling kit-NH2, R-PE labelling kit
SH(DojindoLaboratories)；Fluorescent labeling reagent box such as fluorescein labelling kit-NH2, HiLyte
Fluor (TM) 555 labelling kit-NH2, HiLyte Fluor (TM) 647 labelling kit-NH2 (Dojindo
Laboratories)；And DyLight 547 and DyLight647 (Techno Chemical Corp.), Zenon (TM),
Alexa Fluor (TM) antibody labeling test kit, Qdot (TM) antibody labeling test kit (Invitrogen
And EZ-label Protein Labeling Kit (Funakoshi Corporation) Corporation).In order to correctly
Labelling, it is possible to use suitable instrument detects the antibody through labelling or its fragment.
Sample as the detection product according to the present invention, it is possible to use the tissue such as obtained from biopsy experimenter
Sample or fluid.Sample is not particularly limited, as long as it is suitable to the mensuration of the present invention；Such as, it can include
Tissue, blood, blood plasma, serum, lymph fluid, urine, serous cavity liquid, spinal fluid, synovial fluid, aqueous humor, tear
Liquid, saliva or its fraction or treated material.
In specific embodiments of the present invention, described sample is from the tissue of experimenter.
In the present invention, " prognosis " refer to that tumor patient is by the suppression such as surgical procedure or alleviate tumor growth
After process or result.In this manual, prognosis can be to be suppressed by surgical procedure or alleviate tumor growth
Life state when latter 1,2,3,4,5,6,7,8,9,10,15,20 years or more long.Prognosis can
With by checking that the gene of biomarker i.e. ERI3 albumen or coding ERI3 albumen is predicted.Prognosis prediction
Can be performed such that according to biomarker with or without, or be raised and lowered, determine that the prognosis of patient is
Good or bad, or determine the probability of good prognosis or poor prognosis.
In the present invention, " prognosis bona " refers to suppressed or alleviation tumor life for patient by surgical procedure etc.
After length, patient (such as 3,5,6,7,8,9,10,15,20 years or longer) over a long time not danger
Anxious situation.Or, good prognosis can mean to survive in the most long-time, without transfer, without recurrence or without again
Send out.Such as, prognosis bona can mean at least 3 years or survival in especially at least 5 years, preferably without transfer
Or recurrence.The most preferred state of prognosis bona is the long-term survival without disease.As used herein, " pre-
Rear good " can also include any such state, wherein it appeared that disease such as transfer, but pernicious low and
Do not severely impact survival ability.
In the present invention, " prognosis mala " refers to that patient is by the suppression such as surgical procedure or alleviation tumor growth
After short-term (such as 1,2,3,4,5 years or shorter) in occur fatal condition.Or, poor prognosis is
Refer to death in such short-term, shift, recur or send out again.Such as, poor prognosis can mean at least 3
Year or Preventive or death in especially at least 5 years.
Prediction prognosis refers to predict process or the result of status of patient, and being not meant to can be with the accuracy of 100%
The process of prediction status of patient or result.Prediction prognosis refers to whether the probability determining some process or result increases
Add, and be not meant to be compared by situation about not occurring with some process or result determine generation some process
Or the probability of result.For the present invention, in the present invention, the level of ERI3 gene or ERI3 albumen raises
Patient in, compared with the patient not showing this feature, more likely observe particular procedure or result.
Further, the product of described detection ERI3 gene or ERI3 albumen can be detection ERI3 gene or
The reagent of ERI3 albumen, can also be to comprise the test kit of described reagent, chip, reagent paper etc., it is also possible to be
Use the high-flux sequence platform of described reagent.
Present invention also offers a kind of instrument diagnosing multiple myeloma, described instrument can detect ERI3 base
Cause or the expression of ERI3 albumen.Described instrument includes can be in conjunction with the nucleic acid of ERI3 gene or can
Material (such as antibody) in conjunction with ERI3 albumen.Described nucleic acid can detect the expression of ERI3 gene；
Described material can detect the expression of ERI3 albumen.
Further, the character of described nucleic acid and described material is with noted earlier.
Further, the instrument of described diagnosis multiple myeloma include but not limited to chip, test kit, reagent paper,
Or high-flux sequence platform；High-flux sequence platform is the instrument of a kind of special diagnosis multiple myeloma, with
The development of high throughput sequencing technologies, the structure of the gene expression profile of a people will be become and work the most easily.
By contrast Disease and the gene expression profile of normal population, easily analyze exception and the disease of which gene
Relevant.Therefore, in high-flux sequence, know that the exception of ERI3 gene is relevant to multiple myeloma fall within
The purposes of ERI3 gene, equally within protection scope of the present invention.
Present invention also offers a kind of instrument predicting multiple myeloma prognosis, described prediction multiple myeloma
Prognostic tool includes can be in conjunction with the nucleic acid of ERI3 gene or can in conjunction with the material of ERI3 albumen (such as
Antibody).Described nucleic acid can detect the mRNA level in-site of ERI3 gene；Described material can detect ERI3
The expression of albumen.
Further, the character of described nucleic acid and described material is with noted earlier.
Further, the instrument of described prediction multiple myeloma prognosis includes but not limited to chip, test kit, examination
Paper or high-flux sequence platform；High-flux sequence platform is the instrument of a kind of special diagnosis multiple myeloma,
Along with the development of high throughput sequencing technologies, the structure of the gene expression profile of a people will be become work the most easily
Make.By contrast Disease and the gene expression profile of normal population, easily analyze the exception of which gene with
Disease is correlated with.Therefore, in high-flux sequence, know that the exception of ERI3 gene is relevant to multiple myeloma also
Belong to the purposes of ERI3 gene, equally within protection scope of the present invention.
The amino that the anti-ERI3 antibody used in detection product, the diagnostic tool of the present invention or its fragment are identified
The number of acid is not particularly limited, as long as antibody can be in conjunction with ERI3.When antibody is as medicine,
Preferably it is capable of identify that aminoacid as much as possible, as long as it can suppress ERI3 function.Antibody or its sheet
The amino acid whose number that section identifies is at least one, more preferably at least three.The immunoglobulin class of antibody is not
Restricted, can be IgG, IgM, IgA, IgE, IgD or IgY.
Present invention also offers a kind of method diagnosing multiple myeloma or prediction multiple myeloma prognosis, institute
The method of stating comprises the steps:
(1) sample of experimenter is obtained；
(2) ERI3 gene or the expression of albumen in detection Samples subjects；
(3) the ERI3 gene recorded or the expression of albumen are associated with the whether ill of experimenter.
(4) compared with the control, the expression of ERI3 gene or albumen raises, then this experimenter is diagnosed as
Multiple myeloma, or this experimenter is confirmed as prognosis mala.
Present invention also offers the Therapeutic Method of a kind of multiple myeloma, described method includes suppressing ERI3 base
Cause or ERI3 albumen.
Further, described method include suppress ERI3 gene expression, or suppression ERI3 albumen expression or
The activity of suppression ERI3 albumen.
Present invention also offers the screening technique of a kind of tumour medicine, can be by tumor cell be being added test
Certain period after medicine or after tumor model animal is used testing drug measures ERI3 gene or ERI3
The expression of albumen measures tumour medicine and improves the effect of tumor prognosis.More specifically, when ERI3 base
Because of or the expression of ERI3 albumen when adding or reduce after using testing drug or recover normal level
Time, this medicine optional is as the medicine improving tumor prognosis.
Present invention also offers a kind of containing ERI3 gene or the medicine of the inhibitor of ERI3 albumen.
Present invention also offers the application in the medicine of preparation treatment multiple myeloma of the above-mentioned inhibitor.
The ERI3 gene of the present invention or the inhibitor of ERI3 albumen are unrestricted, as long as ERI3 can be suppressed
Or relate to expression or the activity of the material of ERI3 upstream or downstream pathway, and for the treatment effective medicine of tumor
?.
Further, described inhibitor includes antisensenucleic acids, dsRNA, ribozyme, fit, ERI3 associated proteins
Fragment or antibody or its fragment.
" antisensenucleic acids " refers to containing the nucleic acid with the sequence of the mRNA complementation of coding ERI3.Antisensenucleic acids can
With by DNA, RNA or the two form.It is complementary that antisensenucleic acids need not the mRNA100% with target ERI3.
Antisensenucleic acids can contain Non-complementary bases, as long as it can specific hybrid under strict conditions.When by anti-
Phosphorothioate odn introduce cell time, it combine target polynucleotide and suppression is transcribed, RNA processing, translation or stability.
In addition to antisense polynucleotides, antisensenucleic acids also includes polynucleotide analogies, it contain through modification main chain,
With 3 ' and 5 ' end portion.Such antisensenucleic acids can appropriately design according to ERI3 sequence information and use
The method of well known to a person skilled in the art generates.
" dsRNA " refers to, containing duplex-RNA constructs, carry out inhibition of gene expression by RNA interference (RNAi)
RNA, including siRNA (short interfering rna) and shRNA (short hairpin RNA).DsRNA need not
With the homology that target-gene sequence has 100%, as long as it can suppress expression of target gene.For stabilisation
Or other purpose, a part of DNA of dsRNA can be substituted.Preferably, siRNA is 21-23
The double-stranded RNA of individual base.SiRNA can be by well known to a person skilled in the art prepared by method, such as
By chemosynthesis or as the analog naturally occurring RNA.ShRNA is to have hair clip corner (hairpin
Turn) Short interfering RNA of structure.ShRNA can by well known to a person skilled in the art prepared by method,
Such as by chemosynthesis or by the DNA of coding shRNA is introduced cell expressible dna.
" ribozyme " refers to the RNA with catalysis activity, and it can cut, pastes, insert and transfer RNA.
The structure of ribozyme can include tup, hair clip etc..
" fit " refers to combine the nucleic acid of something such as protein.Fit can be RNA or DNA.Nucleic acid
Form can be double-strand or strand.Fit infinite in length system, if it can specific binding target molecule,
Can by such as 10 to 200 nucleotide, preferably 10 to 100 nucleotide, more preferably 15 to 80
Nucleotide, even more preferably 15 to 50 nucleotide compositions.Fit can use those skilled in the art
Known method selects.It is for instance possible to use SELEX (part carried out by exponential form enrichment be
System is evolved).
" the protein-bonded fragment of ERI3 " refers to combine ERI3 and suppression ERI3 implements the protein of original function
Fragment.
The medicine of the present invention can be administered alone as medicine or use together with other medicines.Can be with the present invention
The other medicines used together of medicine unrestricted, as long as it does not damage the therapeutic of the present invention or preventative medicine
The effect of thing, it is preferred that the medicine for treatment or prophylaxis of tumours can include such as alkylating agent, all
As ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, procarbazine,
Melphalan and Ranimustine；Antimetabolite, such as enocitabine, capecitabine, carmofur, cladribine,
Gemcitabine, cytosine arabinoside, cytosine arabinoside octadecyl phosphate (cytarabine ocfosfate), ftorafur,
UFT, ftorafur gimeracil oteracil potassium, doxifluridine, hydroxyurea, fluorouracil,
Fludarabine, pemetrexed, pentostatin, mercaptopurine and methotrexate；Plant alkaloid, such as Yi Li
For health, etoposide, sobuzoxane, docetaxel, nogitecan, Pa Litasai, vinorelbine, Changchun
Pungent and the vinblastine in ground；Antitumor antibiotic, such as actinomycin D, aclarubicin, amrubicin, Yi Da ratio
Star, epirubicin, zinostatin stimalamer, daunorubicin, doxorubicin, pirarubicin, rich come mould
Element, peplomycin, ametycin and mitoxantrone；Medicine based on platinum, such as oxaliplatin, carboplatin,
Cisplatin and nedaplatin；Hormonal medicaments, such as Anastrozole, exemestane, estramustine, ethinylestradiol, chlorine
Ground progesterone, goserelin, tamoxifen, dexamethasone, toremifene, bicalutamide, flutamide, bold and vigorous Buddhist nun
Song Long, fostestrol, mitotane, methyltestosterone, medroxyprogesterone, mepitiostane, leuprorelin and letrozole；Raw
Thing reaction dressing agent, such as interferon-ALPHA, interferon beta, interferon gamma, interleukin, ubenimex, dry BCG,
And lentinan；And molecular targeted agents, such as imatinib (imatinib), gefitinib (gefitinib), Ji
Nurse monoclonal antibody, ozogamicin, Tamibarotene, trastuzumab, tretinoin, bortezomib (bortezomib),
With Rituximab etc..
The medicine of the present invention can be prepared as various dosage form as required.Include but not limited to, percutaneous, mucosa, nose,
Buccal, Sublingual or the tablet of per os use, solution, granule, patch, unguentum, capsule, aerosol
Or suppository.
The route of administration of the medicine of the present invention is unrestricted, as long as it can play desired therapeutic effect or pre-preventive effect
Fruit, includes but not limited to intravenous, intraperitoneal, ophthalmic, intra-arterial, in lung, is administered orally, in vesicle,
Intramuscular, tracheal strips, subcutaneous, by skin, by pleura, local, suck, by mucosa, skin
Skin, the intestines and stomach, intraarticular, in ventricle, rectum, vagina, in skull, in urethra, in liver, in tumor.At certain
In the case of Xie, can systematically be administered.It is to be administered partly in some cases.
The dosage of the medicine of the present invention is unrestricted, as long as obtaining desired therapeutic effect or preventive effect,
Appropriate determination can be carried out according to symptom, sex, age etc..The medicine of the present invention or the agent of prophylactic agent
Amount can use such as therapeutic effect or preventive effect to disease to determine as index.
In the context of the present invention, " diagnosis multiple myeloma " both includes judging that experimenter has suffered from
There is multiple myeloma, also include judging whether experimenter exists the risk suffering from multiple myeloma.
" treatment " used herein contains treatment phase in the mammal such as mankind suffering from relevant disease or disease
The disease closed or morbid state, and include:
(1) prevention disease or morbid state occur in mammal, especially susceptible in institute when this mammal
State morbid state, but be not yet diagnosed when suffering from this morbid state；
(2) suppression disease or morbid state, i.e. stop it to occur；Or
(3) disease or morbid state are alleviated, even if disease or morbid state disappear.
Term " is treated " and is usually directed to treat the mankind or animal (such as, applied by veterinary), wherein can reach certain
Some intended therapeutic effect, such as, the development (including reducing development speed, making development stop) of suppression disease,
Improve disease and cure disease.Also include the treatment as preventive measure (such as prevention).To the most not developing into
Disease but have develops into the purposes of the dangerous patient of this disease, is also included within during term " treats ".
Advantages of the present invention and beneficial effect:
The present invention is found that a kind of molecular marker diagnosing multiple myeloma, uses this molecular marker can
Can be used as judging multiple myeloma occurs early stage, it is provided that the survival rate of patient.
It addition, by the prognosis predicting patient, the present invention can provide significant information to determine to control for patient
Treat scheme policies.
The medicine of the inhibitor including ERI3 gene or albumen of the present invention can be used as new multiple bone marrow
The medicine of tumor.
Accompanying drawing explanation
Fig. 1 shows the impact that multiple myeloma cells is bred by suppression ERI3 gene expression；
Fig. 2 shows the impact that multiple myeloma cells is bred by suppression ERI3 protein function；
Fig. 3 shows the suppression ERI3 gene expression impact on multiple myeloma cells apoptosis；
Fig. 4 shows the impact that multiple myeloma cells is attacked by suppression ERI3 gene expression；
Fig. 5 shows the impact that multiple myeloma cells is migrated by suppression ERI3 gene expression.
Specific embodiment
The present invention is further detailed explanation with embodiment below in conjunction with the accompanying drawings.Following example are only used for
The bright present invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment is logical
Often according to normal condition, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold
Spring HarborLaboratory Press, 1989) condition described in, or according to the bar proposed by manufacturer
Part.
Embodiment 1 gene chip screening difference expression gene
1, draw materials:
Multiple myeloma tissue: collecting MM Bone Marrow of Patients biopsy specimen totally 10 example made a definite diagnosis, MM examines
Disconnected standard is refering to " China's multiple myeloma diagnosis and treatment guide 2011 revised edition ".Male 5 examples in patient, female 5
Example, the median age 59 years old.
Normal marrow tissue: collect same time malnutritional anemia Bone Marrow of Patients biopsy specimen 10 example and make
For matched group, the maleest 5 examples, female 5 example, the median age 53 years old.
2, the acquisition of RNA is organized
Trizol one-step method is used to extract total tissue RNA.
3, RNA purity and the mensuration of concentration
Take RNA solution 1 μ l, Instrument measuring OD260, OD280, RNA concentration be OD260 value ×
Extension rate × 40/1000, calculates OD260/OD280, and it is high that ratio represents RNA solution purity at 1.7-2.0,
Few containing impurity such as protein ,-20 DEG C of preservations.
4, RNA integrity detection
(1) 2 μ l RNA sample row 1.5% agarose gel electrophoresis (80v, 15min) are taken；
(2) after separating zone, genefinder dyes, and observes electrophoresis zone under blue light；
(3) as 28s/18s about 2:1, illustrate that RNA is stable without degraded.
5, gene chip hybridization and scanning
After the linearized amplification of total serum IgE, cy3-UTP labelling, the cRNAs after fluorescent labeling uses RNEASY
Mini Kit purification, enters the cRNAs that labelling is good with the RNAFragmentation Reagents of Amhion
Row fragmentation processes.Use people's full genome chip of expression spectrum (4x 44K gene) of Agilent company of the U.S.,
In chip hybridization stove, 65 DEG C of hybridization 17h, then eluting, dyeing, finally uses Agilent DNA
MicroarrayScanner scanner scanning.
6, chip data processes and analyzes
Chip after hybridization, after chip scanner reads data point, imports data to analyze software, for two groups
The natural logrithm absolute value of the ratio gene more than 2.0 or less than 0.5 is as difference expression gene.
7, statistical procedures
Using SPSS 13.0 statistical software to carry out data analysis, group difference compares employing one factor analysis of variance
Method, P < 0.05 difference has significant.
8, result
Chip results shows, filters out 489 differences between multiple myeloma tissue and normal marrow tissue altogether
Expressing gene, the gene 215 that wherein expression raises, the gene 274 that expression is lowered.
The difference expression gene filtered out verified by embodiment 2 large sample
Consider prior art yet there are no and carry out, about this gene and multiple myeloma dependency, the gene studied
As candidate gene, considering the result of gene sequencing, (it is expressed at multiple bone to select ERI3 gene simultaneously
Myeloma tissue raises) verify.
1, sample collection
Collecting multiple myeloma according to the method for embodiment 1 and organize 50 examples, normal marrow organizes 60 examples.
2, verify in mRNA level in-site
2.1 extract tissue RNA
Step is with embodiment 1.
2.2 reverse transcription
Reverse transcription system totally 20 μ L, including cell total rna 2 μ g/2 μ L, 50U/ μ L Rnasin 1 μ L, 5 ×
Reverse transcription reaction buffer 4 μ L, 10m M d NTP 2 μ l, 50 μ g/mL random primer 2 μ L (promega),
200U/ μ L M-MLV reverse transcriptase 1 μ L, DEPC 8 μ L.
37 DEG C are reacted 60 minutes, and 95 DEG C terminate reaction in 5 minutes.CDNA preserves at-80 DEG C or carries out PCR
Amplification.
2.3PCR
Reaction system according to Real Master Mix (SYBR Green) test kit (purchased from sky root biochemical technology
(Beijing) company limited) configuration, SYBR reaction system totally 25 μ L, 2.5 × Real Master Mix 10 μ L,
20 × SYBR solution 1.25 μ L, forward primer 0.5 μ L, downstream primer 0.5 μ L, deionized water 10.75 μ L,
cDNA2μL.Reaction condition is 94 DEG C of 5min, 94 DEG C of 45s, 60 DEG C of 1min, 30 circulations, if empty
White comparison.
The fluorescence signal of front 10 circulations of PCR reaction is located to suitable as autofluorescent background signal, regulation baseline,
Each fluorescence curve is Ct value with the period in baseline cross point.According to Δ C (t)=C (t)Genes of interest-C(t)β-actin, Δ
Δ C (t)=2-ΔC(t), calculate genes of interest and β-actin relative expression quantity.
PCR primer sequence is as follows:
ERI3 gene primer sequence is as follows:
Forward primer: 5 '-GTATTTACTTTCAAGAGCAAGA-3 ' (SEQ ID NO.1)；
Downstream primer: 5 '-TGGATATGGAGCAGAACT-3 ' (SEQ ID NO.2).
β-actin gene primer sequence is as follows:
Forward primer: 5 '-CTGGCACCACACCTTCTACAAT-3 ' (SEQ ID NO.3)；
Downstream primer: 5 '-AATGTCACGCACGATTTCCCGC-3 ' (SEQ ID NO.4)
2.4 result
Result shows, compared with normal marrow tissue, and the mRNA of ERI3 gene in multiple myeloma tissue
Level substantially raises, and relative expression quantity is 7.24 ± 0.94, and difference has statistical significance (P < 0.05).
3, verify on protein level
3.1 extract tissue total protein
The operation of protein extraction is carried out according to the description of EpiQuik tissue/cell total protein extraction test kit.
3.2Western blot detects
The protein quantification of extraction is carried out SDS-PAGE electrophoresis, carry out afterwards transferring film, closing, one anti-hatch,
Two anti-hatch, develop the color.
3.3 statistical procedures
Image J software is used to be analyzed, with β-actin as internal reference, by ERI3 the gray value of protein band
The gray value of protein band is normalized.Result data is all to carry out table in the way of mean+SD
Showing, using SPSS13.0 statistical software to carry out statistical analysis, difference between the two uses t inspection,
Think when P < has statistical significance when 0.05.
3.4 result
Result shows, compared with normal marrow tissue, in multiple myeloma tissue, ERI3 protein level is notable
Increasing, relative expression quantity is 3.74 ± 0.63, and difference has statistical significance (P < 0.05).
Embodiment 3 suppresses ERI3 gene expression
1, siRNA design synthesis
SiRNA sequence for ERI3:
SiRNA-ERI3:
Positive-sense strand is 5 '-UCUAGAAGUCCAUUCUUGGGC-3 ' (SEQ ID NO.5)
Antisense strand is 5 '-CCAAGAAUGGACUUCUAGACA-3 ' (SEQ ID NO.6)；
Above siRNA sequence and negative control siRNA sequence (siRNA-NC) are by Shanghai Ji agate pharmacy skill
Art company limited provides.
2, the cultivation of multiple myeloma cells and transfection
2.1 cells are cultivated
RPMI8226 cell uses containing 15% hyclone (FBS), penicillin 100U/ml, streptomycin
RPMI 1640 culture medium of 100 μ g/ml is placed in 37 DEG C, 5%CO2, cultivate under saturated humidity environment.
2.2 cell transfecting
(1) first 24 hours are transfected, at 500 μ l nonreactive inoculation of medium 0.5-2*105Individual cell, during transfection
Cell degrees of fusion is 30-50%.Cell dissociation is mixed completely during bed board, it is to avoid cell accumulation grows.
(2) siRNA (the final concentration of 33nM of transfectional cell, pressure-vaccum gently are diluted with 50 μ l Opti-MEM
3-5 mixing.
(3) overturn mixing transfection reagent gently, dilute 1 μ l Lipofectamine2000 with 50 μ l Opti-MEM
3-5 mixing of pressure-vaccum, left at room temperature 5min gently.
(4) mixing transfection reagent and siRNA diluent, gently 3-5 mixing of pressure-vaccum, left at room temperature 20min.
(5) during transfection composite joins 24 porocyte plates, 100 μ l/ hole, front and back jog cell plates mix homogeneously.
(6) cell plates are placed in 37 DEG C, 5%CO2Incubator is cultivated 18-48 hour.After transfecting 4-6 hour
Interchangeable fresh culture medium.
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
Step is with embodiment 2.
3.3QPCR
Step is with embodiment 2.
3.4 result
Result shows, compared with siRNA-NC group, siRNA-ERI3 can effectively suppress ERI3 gene
Expressing, relative expression quantity is 0.36 ± 0.07, and difference has statistical significance (P < 0.05).
The expression of the embodiment 4ERI3 gene mensuration to multiple myeloma cells multiplication capacity
1, step:
By the RPMI8226 cell of transfection 48h according to 2*103Cells/well is inoculated in 96 orifice plates,
According to the description of Brd U cell proliferation reagent box (Chemicon International), measure cell
Multiplication rate.
2, result
Result is as it is shown in figure 1, compared with transfection siRNA-NC group, transfect siRNA-ERI3 group cell proliferation
Slowly, difference has statistical significance (P < 0.05).Above-mentioned test result indicate that, ERI3 gene expression promotees
Enter the propagation of multiple myeloma cells.
In embodiment 5 multiple myeloma cells antibody and experiment
1, step:
Multiple myeloma cells RPMI8226 is inoculated in 96 porocyte culture plates, every hole 2*103Individual
Cells/well/200 μ l, is handled as follows after cell attachment:
Experimental group 1 (matched group): add unrelated monoclonal antibody (1:50) in multiple myeloma cells；
Experimental group 2: add anti-human ERI3 monoclonal antibody (1:50) in multiple myeloma cells.
By cell at 37 DEG C, 5%CO2After incubator hatches 24 hours, according to Brd U cell proliferation reagent
The description of box (Chemicon International), measures cell proliferation rate.
2, statistical method
Experiment is all according to being repeated 3 times, and result data is all to come in the way of mean+SD
Representing, using SPSS13.0 statistical software to carry out statistical analysis, difference between the two uses t inspection,
Think when P < has statistical significance when 0.05.
3, result
Result is as in figure 2 it is shown, compared to matched group, add the group cells proliferation slowed down of anti-human ERI3 monoclonal antibody.
Above-mentioned test result indicate that, the function of suppression ERI3 albumen can suppress multiple myeloma cells to breed.
Embodiment 6 measures ERI3 gene expression to apoptotic impact
1, TUNEL method detection apoptosis
(1) cell transfecting is carried out according to the step of embodiment 3；
(2) according to In situ cell apoptosis detection kit (Chemicon International) description, utilize
The quantitative apoptotic cell of Laser Scanning Confocal Microscope；
(3) to TUNEL Positive Cell Counts.
2, statistical method:
Experiment is all according to being repeated 3 times, and result data is all to come in the way of mean+SD
Representing, using SPSS13.0 statistical software to carry out statistical analysis, difference between the two uses t inspection,
Think when P < has statistical significance when 0.05.
3, result:
Result is as it is shown on figure 3, compare siRNA-NC groups of cells, and transfection siRNA-ERI3 group apoptosis is bright
Aobvious aggravation, difference has statistical significance (P < 0.05), and the above results shows ERI3 gene expression inhibition
Apoptosis.
Embodiment 7 detects ERI3 gene expression cell migration, the impact of invasion and attack
1, Matrigel
1.1 experimental procedures:
(1) upper room Matrigel (artificial basement membrane) precoats；
(2) cell after planting transfection in upper room, inoculum density is 5*104/ 100 μ l cells, at serum-free
Culture medium is cultivated 18h；
(3) the RPMI-1640 culture medium of the FBS that cell is joined 0.1% is cultivated 18h；
(4) 50 μ l Matrigel are drawn on ice；
(5) add in 150 μ l serum-free RPMI-1640 culture medium of pre-cooling and fully mix；
(6) take the Matrigel 50 μ l of mixing in (5) respectively, join room on transwell, cover whole
Individual film；
(7) 37 DEG C, to overnight, make Matrigel be polymerized plastic；
(8) cell serum-free RPMI-1640 culture medium is washed 2 times, adds the RPMI-1640 of serum-free
In culture medium, to cumulative volume 100 μ l；
(9) (8) are uniformly added on Transwell in room, between lower floor's culture fluid and cell, it is to avoid bubble；
(10) the 500-600 μ l of the room addition downwards RPMI-1640 containing 5%FBS cultivates, 37 DEG C, 5%CO2；
(11) exhaust liquid after 48h, wipe the cell not penetrated, move to the cell of lower surface of film with 100%
Methanol is fixed 30min, PBS and is washed 2 times；
In (12) 0.2% violet staining, room 30min, PBS wash away uncalled crystal purple；
(13) count under inverted microscope.
1.2 results:
Experimental result such as Fig. 4 shows, compared with transfection siRNA-NC group, transfects siRNA-ERI3 group cell
Invasion and attack number significantly reduces, and difference has statistical significance (P < 0.05).
2, experiment is migrated
2.1 experimental procedure
(1) upper room need not precoat Matrigel artificial basement membrane.Cell after planting transfection in upper room,
Inoculum density is (1*105)/100 l cell of μ, cultivates 18h in serum-free medium；
(2) the serum-free RPMI-1640 culture medium of the FBS that cell is joined 0.1% is cultivated 18h；
(3) 50 μ l Matrigel are drawn on ice with the rifle head of pre-cooling；
(4) add in 150 μ l serum-free RPMI-1640 culture medium of pre-cooling and fully mix；
(5) take the Matrigel 50 μ l of mixing in (4) respectively and join room on Transwell, cover whole
Film；
(6) 37 DEG C, to overnight, make Matrigel be polymerized plastic；
(7) cell serum-free RPMI-1640 culture medium is washed 2 times, adds the RPMI-1640 of serum-free
In culture medium, to cumulative volume 100 μ l；
(8) (7) are uniformly added on Transwell in room, between lower floor's culture fluid and cell, it is to avoid bubble,
The 500-600 μ l of the room addition downwards RPMI-1640 containing 10%FBS cultivates, 37 DEG C, 5%CO2；
(9) exhaust liquid after 48h, wipe the cell not penetrated, move to the cell of lower surface of film with 100%
Methanol is fixed 30min, PBS and is washed 2 times；
In (10) 0.2% violet staining, room 30min, PBS wash away uncalled crystal purple；
(11) count under inverted microscope.
2.2 result
Experimental result such as Fig. 5 shows, compared with transfection siRNA-NC group, transfects siRNA-ERI3 groups of cells
Cell migration number significantly reduces, and difference has statistical significance (P < 0.05).
The above results shows, ERI3 gene expression is conducive to migration and the invasion and attack of myeloma cell.
The explanation of above-described embodiment is only intended to understand the method for the present invention and core concept thereof.It is right to it should be pointed out that,
For those of ordinary skill in the art, under the premise without departing from the principles of the invention, it is also possible to the present invention
Carrying 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 ERI3 gene or ERI3 albumen in preparation diagnosis multiple myeloma or is predicted multiple
Application in the instrument of property myeloma prognosis.
Application the most according to claim 1, it is characterised in that described detection ERI3 gene or ERI3 egg
White product includes the product detecting the expression of ERI3 gene or ERI3 albumen.
Application the most according to claim 1 and 2, it is characterised in that described product includes can be in conjunction with ERI3
The nucleic acid of gene or can be in conjunction with the material of ERI3 albumen；Described nucleic acid can detect the expression of ERI3 gene
Level；Described material can detect the expression of ERI3 albumen.
Application the most according to claim 3, it is characterised in that described nucleic acid is to make in real-time quantitative PCR
The primer of specific amplified ERI3 gene as shown in SEQ ID NO.1 and SEQ ID NO.2.
5. the instrument diagnosing multiple myeloma or prediction multiple myeloma prognosis, it is characterised in that
Described instrument includes the instrument that can detect the expression of ERI3 gene or ERI3 albumen.
Instrument the most according to claim 5, it is characterised in that described instrument includes can be in conjunction with ERI3
The nucleic acid of gene or can be in conjunction with the material of ERI3 albumen；Described nucleic acid can detect the expression of ERI3 gene
Level；Described material can detect the expression of ERI3 albumen.
Instrument the most according to claim 6, it is characterised in that described nucleic acid is to make in real-time quantitative PCR
The primer of specific amplified ERI3 gene as shown in SEQ ID NO.1 and SEQ ID NO.2.
8. the medicine treating multiple myeloma, it is characterised in that described pharmaceutical pack containing ERI3 gene or
The inhibitor of ERI3 albumen.
Medicine the most according to claim 8, it is characterised in that described inhibitor can suppress ERI3 or relate to
And the expression of the material of ERI3 upstream or downstream pathway or activity.
10. the application in the medicine of preparation treatment multiple myeloma of the inhibitor described in claim 8 or 9.
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| CN106868197A (en) * | 2017-04-24 | 2017-06-20 | 刘志国 | Application of the gene marker in Huppert's disease |
| CN106868197B (en) * | 2017-04-24 | 2019-07-05 | 刘志国 | Application of the gene marker in Huppert's disease |
| CN114317745A (en) * | 2020-03-30 | 2022-04-12 | 中国医学科学院肿瘤医院 | Kit, device and method for lung cancer diagnosis |
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