CN105969904A - Multiple myeloma biomarker - Google Patents
Multiple myeloma biomarker Download PDFInfo
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- CN105969904A CN105969904A CN201610601437.XA CN201610601437A CN105969904A CN 105969904 A CN105969904 A CN 105969904A CN 201610601437 A CN201610601437 A CN 201610601437A CN 105969904 A CN105969904 A CN 105969904A
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- ctsh
- gene
- albumen
- multiple myeloma
- expression
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- 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
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- 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
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- 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
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- 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
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- 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
- C12Q2600/118—Prognosis of disease development
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- 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
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- 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 application of CTSH serving as a diagnosis and treatment marker of multiple myeloma. Accordingly, the CTSH can be used for developing products for diagnosing the multiple myeloma and drugs for treating the multiple myeloma. According to research results, a theoretical basis is provided for clinicians to establish individualized treatment plans, and a novel drug target can be provided for development of multiple myeloma drugs.
Description
Technical field
The present invention relates to diagnosing tumor, treat, predict prognosis field, more particularly it relates to detection
CTSH is abnormal is the diagnosing tumor of means, prediction method of prognosis；And activate CTSH gene or protein
Tumor therapeutic agent.
Background technology
Multiple myeloma (multiple myeloma, MM) is the paraplasm blood of plasma cell in a kind of bone marrow
System Malignant Tumor disease, accounts for hematologic malignancies 10%, is apt to occur in old people, along with China's population is old
In age, MM number of the infected increased.It is mainly characterized by Clonal plasma cell paraplasm and invades bone
Marrow, produces Clonal immunoglobulin.Clinical manifestation is that myeloma cell's hypertrophy, infiltration and bone marrow microenvironment are broken
Bad, cause anemia, hemorrhage, osteodynia or fracture, high calcium disease, renal insufficiency and immunologic hypofunction etc..β2-MG
It is the conventional clinical indices of multiple myeloma patients, is also one of index as staging system standard (ISS),
There is dependency in the level of β 2-MG and patients with malignant myeloma tumor load, prognosis.Treatment myeloma mainly should at present
With hormone, proteasome inhibitor, immunomodulator etc., the treatment of these medicines can make MM alleviate,
But final unavoidable recurrence, it is therefore desirable to find new diagnosis and therapeutic strategy.
Summary of the invention
An object of the present invention is that providing a kind of is examined by detection CTSH gene or protein expression difference
The method of disconnected multiple myeloma.
The two of the purpose of the present invention are that providing a kind of comes pre-by detection CTSH gene or protein expression difference
The method surveying multiple myeloma prognosis.
The three of the purpose of the present invention are that providing a kind of is treated by activation CTSH gene or CTSH albumen
The method of multiple 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 CTSH gene or CTSH albumen is examined in preparation multiple myeloma
Purposes in disconnected instrument.
Present invention also offers the product of detection CTSH gene or CTSH albumen at preparation prediction multiple bone
Purposes in myeloma prognostic tool.
Further, the product of described detection CTSH gene or CTSH albumen include detect CTSH gene or
The product of the expression of CTSH albumen.Described product include can in conjunction with the nucleic acid of CTSH gene or
Can be in conjunction with the material (such as antibody) of CTSH albumen.Described nucleic acid can detect the expression of CTSH gene
Level；Described material can detect the expression of CTSH albumen.
The product of the detection CTSH gene of the present invention can play it based on the known method using nucleic acid molecules
Function: 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 CTSH gene, and the primer that product includes can pass through
Being prepared by chemosynthesis, the method that be those skilled in the art will know that by use is come suitably with reference to Given information
Ground 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 CTSH 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 CTSH albumen of the present invention includes antibody or its sheet of specific binding CTSH albumen
Section.Antibody or its fragment of any structure, size, immunoglobulin class, origin etc. can be used, as long as
It combines target protein.Antibody or its fragment that the detection product of the present invention includes can be monoclonal
Or it is polyclonal.Antibody fragment refers to an antibody part (Partial Fragment) retaining antibody to the combination activity of antigen
Or the peptide containing 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 CTSH albumen of the present invention can include the amino of encoding antibody or Encoding Antibody Fragment
The nucleic acid of the separation of acid 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
CTSH albumen or its part antibody to obtaining are implemented antigenic specificity purification and are obtained for CTSH albumen
Monoclonal antibody.Polyclonal antibody can be prepared as follows: with antigen-immunized animal same as above, from warp
The animal crossing immunity collects blood sample, isolates serum from blood, then uses above-mentioned antigen real to serum
Execute antigenic specificity purification.Can be by the antibody obtained with ferment treatment or the sequence of the antibody obtained by use
Information obtains 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. CTSH albumen or coding CTSH albumen is predicted.Prognosis is pre-
Survey can be performed such that according to biomarker with or without, or be raised and lowered, determine the prognosis of patient
It is good or bad, or determines 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, the level fall of CTSH gene or CTSH albumen in the present invention
In low patient, compared with the patient not showing this feature, more likely observe particular procedure or result.
Further, the product of described detection CTSH gene or CTSH albumen can be detection CTSH gene
Or the reagent of CTSH albumen, can also be to comprise the test kit of described reagent, chip, reagent paper etc., it is possible to
To be the high-flux sequence platform using described reagent.
Present invention also offers a kind of instrument diagnosing multiple myeloma, described instrument can detect CTSH
Gene or the expression of CTSH albumen.Described instrument include can in conjunction with the nucleic acid of CTSH gene or
Can be in conjunction with the material (such as antibody) of CTSH albumen.Described nucleic acid can detect the expression of CTSH gene
Level；Described material can detect the expression of CTSH 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 CTSH gene is relevant to multiple myeloma also belong to
In the purposes of CTSH 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 CTSH gene or can be in conjunction with the material (example of CTSH albumen
Such as antibody).Described nucleic acid can detect the mRNA level in-site of CTSH gene；Described material can detect CTSH
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 CTSH gene is relevant to multiple myeloma
Fall within the purposes of CTSH gene, equally within protection scope of the present invention.
The ammonia that the anti-CTSH antibody used in detection product, the diagnostic tool of the present invention or its fragment are identified
The number of base acid is not particularly limited, as long as antibody can be in conjunction with CTSH.
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) CTSH gene or the expression of albumen in detection Samples subjects；
(3) the CTSH gene recorded or the expression of albumen are associated with the whether ill of experimenter.
(4) compared with the control, the expression of CTSH gene or albumen reduces, then this experimenter is diagnosed
For 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 activating CTSH
Gene or CTSH albumen.
Further, described method includes the expression promoting CTSH gene, or promotes the expression of CTSH albumen
Or the activity of enhanced CT SH albumen.
Present invention also offers the screening technique of a kind of tumour medicine, can be by cancerous cell being added test medicine
Measure certain period after thing or after tumor model animal is used testing drug CTSH gene or
The expression of CTSH albumen measures tumour medicine and improves the effect of tumor prognosis.More specifically, when
The expression of CTSH gene or CTSH albumen is when adding or raise after using testing drug or extensive
During multiple normal level, this medicine optional is as the medicine improving tumor prognosis.
Present invention also offers a kind of containing CTSH gene or the medicine of the activator of CTSH albumen.
Present invention also offers the application in the medicine of preparation treatment multiple myeloma of the above-mentioned activator.
The CTSH gene of the present invention or the activator of CTSH albumen are unrestricted, as long as can promote or
Person's enhanced CT SH or relate to the expression of material or the activity of CTSH upstream or downstream pathway, and for treatment
The effective medicine of tumor.
Further, described activator includes CTSH gene, CTSH albumen, promoted type miRNA, promotion
Type transcription regulatory factor or promoted type targeting micromolecular compound.
Described activator also includes comprising carrier or the host cell carrying CTSH gene.
On the one hand the activator of the present invention may be used for supplementing disappearance or the deficiency of endogenic CTSH albumen, logical
Cross the expression improving CTSH albumen, thus treat the multiple myeloma caused because of CTSH hypoproteinosis.Separately
On the one hand may be used for the activity of enhanced CT SH albumen, thus treat multiple myeloma.
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 activator including CTSH gene or albumen of the present invention can be used as new multiple bone
The medicine of myeloma.
Accompanying drawing explanation
Fig. 1 shows the impact that multiple myeloma cells is bred by CTSH gene overexpression；
Fig. 2 shows the impact that multiple myeloma cells is attacked by CTSH gene overexpression；
Fig. 3 shows the impact that multiple myeloma cells is migrated by CTSH gene overexpression.
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 RNA Fragmentation 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 multiple to select CTSH gene simultaneously
Myeloma tissue is lowered) 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,
cDNA 2μL.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:
CTSH gene primer sequence is as follows:
Forward primer: 5 '-TCCTTCTTAACAGACTCA-3 ' (SEQ ID NO.1)；
Downstream primer: 5 '-ATTGAACAGCGAATACAG-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 CTSH gene in multiple myeloma tissue
Level is substantially lowered, and relative expression quantity is 0.31 ± 0.05, 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 CTSH 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, CTSH protein level is notable
Reducing, relative expression quantity is 0.42 ± 0.07, and difference has statistical significance (P < 0.05).
Embodiment 3CTSH gene overexpression
1, plasmid construction
According to CTSH gene coded sequence design amplimer, primer be designed as those skilled in the art
Known.Amplification total length from the cDNA library (clontech company, article No.: 638831) becoming Human fetal spleen
The coded sequence of CTSH gene, above-mentioned cDNA sequence is inserted into eukaryotic expression vector pcDNA3.1
In, connect the recombinant vector pcDNA3.1-CTSH obtained for subsequent experimental.
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) day before transfection is by 0.5-2*105Individual tumor cell is suspended in the 500 μ l culture medium without antibiotic,
It is seeded to 24 well culture plates.
(2) transfection cell density on the same day should reach 80%-90%, prepares following complex A: by 1 μ g plasmid DNA
It is diluted in serum-free medium, mixes gently；Complex B: take 4 μ l Lipofectamine2000 and be diluted in
In serum-free medium, mixing.
(3) complex A and B is mixed, mix gently, incubated at room.
(4) 100 μ l liposome compounds are added in tumor cell, mix the most gently, cell is put
Enter 37 DEG C containing 5%CO2Incubator hatches 5-7 hour.
(5) add 1ml and contain 2 times of normal serums and the growth medium of antibiotic concentration, continue to cultivate cell
18-24 hour.
3, the process LAN situation of QPCR experiment detection pcDNA3.1-CTSH 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, pcDNA3.1-CTSH can success process LAN, relative expression quantity is 7.13 ± 0.35,
Difference has statistical significance (P < 0.05).
3, Western blot experiment detection pcDNA3.1-CTSH process LAN situation
Step is with embodiment 2.
Result shows, compared with transfection pcDNA3.1 group, and CTSH in the cell of transfection pcDNA3.1-CTSH
The content of albumen substantially increases, and relative expression quantity is 4.21 ± 0.52, and difference has statistical significance (P < 0.05).
The expression of the embodiment 4CTSH gene mensuration to multiple myeloma cells multiplication capacity
1, step:
(1) being inoculated in by cell in 96 orifice plates, each concentration does 3 multiple holes；
(2) add the solution of MTT working concentration, jointly hatch 4h with 37 DEG C of cell；
(3) remove culture medium, add 200 μ l dimethyl sulfoxide to dissolve first a ceremonial jade-ladle, used in libation crystal；
(4) 5d surveys OD570, more than all experiments the most in triplicate, averaged continuously；
2, result:
Result is as it is shown in figure 1, compared with transfection pcDNA3.1 group, transfect pcDNA3.1-CTSH group cell
Growth rate substantially slows down, and difference has statistical significance (P < 0.05).Above-mentioned test result indicate that, CTSH
The gene expression inhibition propagation of multiple myeloma cells.
Embodiment 7 detects CTSH 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. 2 shows, compared with transfection pcDNA3.1 group, transfects pcDNA3.1-CTSH group
Cell invasion 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. 3 shows, compared with transfection pcDNA3.1 group, transfects pcDNA3.1-CTSH group
Cell migration number significantly reduces, and difference has statistical significance (P < 0.05).
The above results shows, CTSH gene expression is unfavorable for 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 CTSH gene or CTSH albumen is many in preparation diagnosis multiple myeloma or prediction
Application in the instrument of the property sent out myeloma prognosis.
Application the most according to claim 1, it is characterised in that described detection CTSH gene or CTSH egg
White product includes the product detecting the expression of CTSH gene or CTSH albumen.
Application the most according to claim 1 and 2, it is characterised in that described product includes can be in conjunction with CTSH
The nucleic acid of gene or can be in conjunction with the material of CTSH albumen；Described nucleic acid can detect the table of CTSH gene
Reach level；Described material can detect the expression of CTSH 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 CTSH 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 CTSH gene or CTSH albumen.
Instrument the most according to claim 5, it is characterised in that described instrument includes can be in conjunction with CTSH base
The nucleic acid of cause or can be in conjunction with the material of CTSH albumen；Described nucleic acid can detect the expression of CTSH gene
Level；Described material can detect the expression of CTSH 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 CTSH 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 gene Han CTSH
Or the activator of CTSH albumen.
Medicine the most according to claim 8, it is characterised in that described activator can promote or strengthen
CTSH or relate to the expression of material or the activity of CTSH upstream or downstream pathway.
10. the application in the medicine of preparation treatment multiple myeloma of the activator 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 |
| CN109295208A (en) * | 2018-10-26 | 2019-02-01 | 德阳市人民医院 | Application of the PI15 as osteoarthritis marker |
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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 |
| CN109295208A (en) * | 2018-10-26 | 2019-02-01 | 德阳市人民医院 | Application of the PI15 as osteoarthritis marker |
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