CN105969904B - Huppert's disease biomarker - Google Patents
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Abstract
Purposes the invention discloses CTSH as the diagnosis and treatment marker of Huppert's disease.CTSH can be used to develop accordingly the product of diagnosis Huppert's disease, the drug of exploitation treatment Huppert's disease.Research achievement of the invention provides fundamental basis for clinician's formulation personalized therapy program and can provide new drug target for the exploitation of Huppert's disease drug.
Description
Technical field
The present invention relates to diagnosing tumor, treatment, prediction prognosis fields, more particularly it relates to different to detect CTSH
It is often the diagnosing tumor of means, prediction method of prognosis；And the tumor therapeutic agent of activation CTSH gene or protein.
Background technique
Huppert's disease (multiple myeloma, MM) is a kind of hematological system of thick liquid cell paraplasm in marrow
Malignant neoplastic disease accounts for about hematologic malignancies 10%, is apt to occur in the elderly, with China human mortality aging, MM morbidity people
Number increased.It is mainly characterized by Clonal thick liquid cell paraplasm and invades marrow, generates Clonal immunoglobulin.Face
Bed shows as myeloma cell's hyperplasia, infiltration and bone marrow microenvironment are destroyed, cause anaemia, bleeding, ostalgia or fracture, high calcium disease,
Renal insufficiency and immunologic hypofunction etc..β 2-MG is the common clinical indices of multiple myeloma patients, and as the world
One of the index of staging scale (ISS), β 2-MG level with patients with malignant myeloma tumor load, there are correlations for prognosis.Treatment at present
Myeloma mainly applies hormone, proteasome inhibitor, immunomodulator etc., and the treatment of these drugs can be such that MM alleviates, but
Final inevitably recurrence, it is therefore desirable to find new diagnosing and treating strategy.
Summary of the invention
It is multiple to diagnose by detection CTSH gene or protein expression difference that one of the objects of the present invention is to provide one kind
The method of property myeloma.
The second object of the present invention is to provide a kind of multiple to predict by detection CTSH gene or protein expression difference
The method of property myeloma prognosis.
The third object of the present invention is to provide one kind by activation CTSH gene or CTSH albumen to treat multiple bone
The method of myeloma.
The fourth object of the present invention is to provide a kind of method for screening the drug for the treatment of Huppert's disease.
The fifth object of the present invention is to provide a kind of for treating the drug of Huppert's disease.
To achieve the goals above, present invention employs following technical solutions:
The present invention provides the products of detection CTSH gene or CTSH albumen in preparing Diagnosis of Multiple Myeloma tool
Purposes.
The present invention also provides the products of detection CTSH gene or CTSH albumen to predict Huppert's disease prognosis in preparation
Purposes in tool.
Further, the product of the detection CTSH gene or CTSH albumen includes the table for detecting CTSH gene or CTSH albumen
Up to horizontal product.The product includes the nucleic acid that can combine CTSH gene or the substance (example that can combine CTSH albumen
Such as antibody).The nucleic acid is able to detect the expression of CTSH gene；The substance is able to detect the expression water of CTSH albumen
It is flat.
The product of detection CTSH gene of the invention can play its function based on the known method of nucleic acid molecules is used: such as
PCR, such as Southern hybridization, Northern hybridization, dot blot, fluorescence in situ hybridization (FISH), DNA microarray, ASO method, height
Flux microarray dataset etc..It can qualitatively, quantitatively or semi-quantitatively implement analysis using the product.
Include that nucleic acid in the said goods can be obtained by chemical synthesis, or by containing from biomaterial preparation
It is expected that the gene of nucleic acid, then using primer amplification designed for amplification expectation nucleic acid, it is obtained.
Further, the PCR method is known method, for example, ARMS (Amplification Refractory
Abruptly-changing system is not answered in Mutation System, amplification) method, RT-PCR (reverse transcriptase-PCR) method, nesting PCR method etc..Amplification
Nucleic acid can be by using dot blotting hybridization method, Surface Plasmon Resonance (SPR method), PCR-RFLP method, original position RT-PCR
Method, 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 detect.
Nucleic acid recited above includes the primer for expanding CTSH gene, and the primer for including in product can be by passing through chemistry
Synthesis to prepare, by using those skilled in the art will know that method be suitably designed with reference to Given information, and passing through
Synthesis is learned to prepare.
In specific embodiments of the present invention, the nucleic acid is amplimer used in QPCR experiment, the primer
Sequence such as SEQ ID NO.1 (positive sequence) and SEQ ID NO.2 (reverse sequence) shown in.
Nucleic acid recited above may also include probe, and the probe can be prepared by chemical synthesis, by using this
The method that field technical staff knows appropriately is designed with reference to Given information, and is prepared by chemical synthesis, or can lead to
The gene for containing desired nucleic acid sequence from biomaterial preparation is crossed, and is expanded using the primer designed for amplification expectation nucleic acid sequence
Increase it to prepare.
The product of detection CTSH albumen of the invention can play its function based on the known method of antibody is used: for example,
It may include ELISA, radioimmunoassay, immunohistochemical method, western blot etc..
The product of detection CTSH albumen of the invention includes the antibody or its segment for specifically binding CTSH albumen.It can make
With the antibody or its segment of any structure and size, immunoglobulin class, origin etc., as long as it combines target protein.This
The antibody or its segment for including in the testing product of invention can be monoclonal or polyclonal.Antibody fragment refers to reservation antibody
Peptide to the active antibody of the combination of antigen a part of (Partial Fragment) or containing antibody a part.Antibody fragment may include F
(ab′)2, Fab ', Fab, scFv (scFv), the Fv (dsFv) of disulphide bonding or its polymer, the area dimerization V it is (dual anti-
Body) or peptide containing CDR.The product of detection CTSH albumen of the invention may include encoding antibody or Encoding Antibody Fragment
The isolated nucleic acid of amino acid sequence, the carrier comprising the nucleic acid, and carry the cell of the carrier.
Antibody can be obtained by the way that well known to a person skilled in the art methods.For example, preparation retains target all or in part
The mammalian cell expression vector of their polynucleotides of the polypeptide or integration coding of protein is as antigen.Exempted from using antigen
After epidemic disease animal, from the immune animal adaptive immune cell of process and myeloma cell is merged to obtain hybridoma.Then from hybridization
Tumor culture collects antibody.It can finally be implemented by using antibody of the CTSH albumen for being used as antigen or part thereof to acquisition
Antigentic specificity purifies to obtain the monoclonal antibody for CTSH albumen.Polyclonal antibody can be prepared as follows: with it is above
Identical antigen-immunized animal collects blood sample from by immune animal, serum is isolated from blood, then using upper
It states antigen and antigentic specificity purifying is implemented to serum.It can be by the antibody that is obtained with enzymatic treatment or by using the antibody of acquisition
Sequence information obtain antibody fragment.
The combination of marker and antibody or its segment can be implemented by method as commonly known in the art.For example, can
With following fluorescent marker protein or peptide: clean protein or peptide with phosphate buffer, addition DMSO, buffer, etc. standards
Standby dyestuff, then mixed solution, then at being placed at room temperature for 10 minutes.In addition, the labelling kit of commercialization can be used in label, it is all
Such as biotin labeling reagent box, such as biotin labeling reagent box-NH2, biotin labeling reagent box-SH
(DojindoLaboratories)；Alkali phosphatase enzyme mark kit such as alkali phosphatase enzyme mark kit-NH2, alkaline phosphorus
Sour enzyme labelling kit-SH (Dojindo Laboratories)；Peroxidase labelling kit such as peroxidase mark
Remember kit-NH2, peroxidase labelling kit-NH2 (Dojindo Laboratories)；Phycobniliprotein labelled reagent
Box 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-NH2,647 labelling kit-NH2 of HiLyte Fluor (TM) (Dojindo Laboratories)；And
DyLight 547 and DyLight647 (Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody
Labelling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation) and EZ- marker protein mark
Remember kit (Funakoshi Corporation).For correct labeling, suitable instrument can be used to detect by label
Antibody or its segment.
As the sample according to testing product of the invention, the tissue sample for example obtained from biopsy subject can be used
Or fluid.Sample is not particularly limited, as long as it is suitable for measurement of the invention；For example, it may include tissue, blood, blood plasma,
Serum, lymph, urine, serous cavity liquid, spinal fluid, synovia, aqueous humor, tear, saliva or its fraction or treated material
Material.
In specific embodiments of the present invention, tissue of the sample from subject.
In the present invention, " prognosis " refers to mistake of the tumor patient after inhibiting by surgical procedure etc. or alleviating tumour growth
Journey or result.In the present specification, prognosis can be by surgical procedure inhibit or alleviate tumour growth after 1,2,3,4,5,6,
7,8,9,10,15,20 years or more long when life state.Prognosis can be by checking biomarker, that is, CTSH albumen or coding
The gene of CTSH albumen is predicted.Prognosis prediction can be performed such that according to biomarker with or without or increasing or drop
It is low, 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 " refer to inhibit or alleviate for patient by surgical procedure etc. tumour growth it
Afterwards, patient's long-term (such as 3,5,6,7,8,9,10,15,20 years or longer) does not have critical condition.Alternatively, good prognosis can anticipate
Refer to and survives in such long-time, sent out again without transfer, without recurrence or nothing.For example, prognosis bona can mean at least 3 years or outstanding
It is to survive at least 5 years, preferably without transfer or recurrence.The most preferred state of prognosis bona is survival for a long time without disease.Such as
Used herein, " prognosis bona " can also include any such state, wherein it can be found that disease such as shifts, still
It is pernicious low and do not severely impact survival ability.
In the present invention, " prognosis mala " refers to that patient is short after inhibiting or alleviating tumour growth by surgical procedure etc.
Fatal condition occurs in period (such as 1,2,3,4,5 year or shorter).Alternatively, poor prognosis refers in such short-term extremely
It dies, shift, recur or sends out again.For example, poor prognosis can mean Preventive or dead at least 3 years or especially at least 5 years
It dies.
Prediction prognosis is referred to the process of prediction status of patient or as a result, is not meant to be predicted with 100% accuracy
The process or result of status of patient.Prediction prognosis refers to whether a possibility that determining certain processes or result increases, and simultaneously unexpectedly
Taste by determining a possibility that certain processes or result occurs or not compared with certain processes or result.Such as this
For invention, in the present invention in the horizontal patient reduced of CTSH gene or CTSH albumen, with the patient's phase for not showing this feature
Than more likely observing particular procedure or result.
Further, the product of the detection CTSH gene or CTSH albumen can be detection CTSH gene or CTSH albumen
Reagent is also possible to include kit, chip, test paper of the reagent etc., is also possible to measure using the high pass of the reagent
Sequence platform.
The present invention also provides it is a kind of diagnose Huppert's disease tool, the tool be able to detect CTSH gene or
The expression of CTSH albumen.The tool includes the nucleic acid that can combine CTSH gene or the object that can combine CTSH albumen
Matter (such as antibody).The nucleic acid is able to detect the expression of CTSH gene；The substance is able to detect the table of CTSH albumen
Up to level.
Further, the property of the nucleic acid and the substance is the same as noted earlier.
Further, the tool of the diagnosis Huppert's disease includes but is not limited to chip, kit, test paper or high pass
Measure microarray dataset；High-flux sequence platform is a kind of tool of special diagnosis Huppert's disease, with high-flux sequence skill
The development of art will become very easily work to the building of the gene expression profile of a people.By comparison Disease and just
The gene expression profile of ordinary person group, the exception for being easy to analyze which gene are related to disease.Therefore, know in high-flux sequence
The exception of the CTSH gene purposes for also belonging to CTSH gene related to Huppert's disease, equally protection scope of the present invention it
It is interior.
The present invention also provides a kind of tool for predicting Huppert's disease prognosis, the prediction Huppert's disease prognosis
Tool includes the nucleic acid that can combine CTSH gene or the substance (such as antibody) that can combine CTSH albumen.The nucleic acid energy
Enough detect the mRNA level in-site of CTSH gene；The substance is able to detect the expression of CTSH albumen.
Further, the property of the nucleic acid and the substance is the same as noted earlier.
Further, it is described prediction Huppert's disease prognosis tool include but is not limited to chip, kit, test paper or
High-flux sequence platform；High-flux sequence platform is a kind of tool of special diagnosis Huppert's disease, as high pass measures
The development of sequence technology will become very easily work to the building of the gene expression profile of a people.By comparing Disease
With the gene expression profile of normal population, the exception for being easy to analyze which gene is related to disease.Therefore, in high-flux sequence
The exception of the CTSH gene purposes for also belonging to CTSH gene related to Huppert's disease is known, equally in protection model of the invention
Within enclosing.
The number for the amino acid that anti-CTSH antibody used in testing product of the invention, diagnostic tool or its segment are identified
Mesh is not particularly limited, as long as antibody can combine CTSH.
The present invention also provides a kind of diagnosis Huppert's disease or the method for predicting Huppert's disease prognosis, the sides
Method includes the following steps:
(1) sample of subject is obtained；
(2) expression of CTSH gene or albumen in Samples subjects is detected；
(3) it associates whether by the expression of the CTSH gene or albumen that measure with the illness of subject.
(4) compared with the control, the expression of CTSH gene or albumen reduces, then the subject is diagnosed as multiple bone
Myeloma or the subject are confirmed as prognosis mala.
The present invention also provides a kind for the treatment of method of Huppert's disease, the method includes activation CTSH gene or
CTSH albumen.
Further, the method includes promoting the expression of CTSH gene, or expression or the enhanced CT SH of promotion CTSH albumen
The activity of albumen.
The present invention also provides a kind of screening techniques of tumour medicine, can be by after adding testing drug to cancer cell
Or the expression of some period measurement CTSH gene or CTSH albumen after applying testing drug to tumor model animal
Improve the effect of tumor prognosis to measure tumour medicine.More specifically, when CTSH gene or the expression of CTSH albumen
When increasing after adding or applying testing drug or when restoring normal level, the drug may be selected as improvement tumor prognosis
Therapeutic agent.
The present invention also provides a kind of drugs of activator containing CTSH gene or CTSH albumen.
The present invention also provides application of the above-mentioned activator in the drug of preparation treatment Huppert's disease.
The activator of CTSH gene or CTSH albumen of the invention is unrestricted, as long as can promote or enhanced CT SH
Or it is related to the expression or activity of the substance of the upstream CTSH or downstream pathway, and for treating the effective drug of tumour.
Further, the activator include CTSH gene, CTSH albumen, promoted type miRNA, promoted type transcriptional control because
Son or promoted type target small molecule compound.
The activator further includes carrier or host cell comprising carrying CTSH gene.
On the one hand activator of the invention can be used for supplementing the missing or deficiency of endogenic CTSH albumen, pass through raising
The expression of CTSH albumen, thus Huppert's disease caused by treating because of CTSH hypoproteinosis.On the other hand can be used for enhancing
The activity of CTSH albumen, to treat Huppert's disease.
Drug of the invention can be used as medicine and be administered alone or apply together with other medicines.It can be with medicine of the invention
The other medicines that object is applied together are unrestricted, as long as it does not damage therapeutic or preventive medicine effect of the invention i.e.
It can, it is preferred that the drug for treating or preventing tumour may include such as alkylating agent, such as ifosfamide, ring phosphinylidyne
Amine, Dacarbazine, Temozolomide, Nimustine, busulfan, procarbazine, melphalan and Ranimustine；Antimetabolite, such as
Enocitabine, capecitabine, Carmofur, Cladribine, gemcitabine, cytarabine, cytarabine octadecyl phosphate
(cytarabine ocfosfate), Tegafur, tegafur-Uracil, Tegafur gimeracil oteracil potassium, deoxidation fluorine urine
Glycosides, hydroxycarbamide, fluorouracil, fludarabine, pemetrexed, Pentostatin, mercaptopurine and methotrexate (MTX)；Plant alkaloid, it is all
As Irinotecan, Etoposide, Sobuzoxane, docetaxel, nogitecan, Palmer altruism, vinorelbine, eldisine and
Vincaleukoblastinum；Antitumor antibiotic, such as actinomycin D, Aclarubicin, Amrubicin, idarubicin, epirubicin, Zinostatin
Stimalamer, daunorubicin, Doxorubicin, pirarubicin, bleomycin, Peplomycin, mitomycin C and mitoxantrone；
Drug based on platinum, such as oxaliplatin, carboplatin, cis-platinum and Nedaplatin；Hormonal medicaments, such as Anastrozole, Exemestane,
Estramustine, ethinyloestradiol, chlormadinone, Goserelin, tamoxifen, dexamethasone, Toremifene, Bicalutamide, Flutamide,
Prednisolone, Fosfestrol, mitotane, methyltestosterone, Medroxyprogesterone, Mepitiostane, Leuprorelin and Letrozole；Biological respinse modification
Agent, such as interferon-' alpha ', interferon beta, interferon gamma, interleukin, ubenimex, dry BCG and lentinan；With molecular targeted medicine
Object, such as Imatinib (imatinib), Gefitinib (gefitinib), gemtuzumab, ozogamicin, Tamibarotene, song
Appropriate monoclonal antibody, Tretinoin, bortezomib (bortezomib) and Rituximab etc..
Drug of the invention can be prepared into various dosage forms as needed.Including but not limited to, percutaneous, mucous membrane, nose, buccal,
Tablet, solution, granule, patch, paste, capsule, aerosol or suppository sublingual or orally use.
The administration method of drug of the invention is unrestricted, as long as it can play desired therapeutic effect or preventive effect i.e.
Can, including but not limited to intravenously, in peritonaeum, intraocularly, intra-arterial, intrapulmonary is taken orally, in vesicle, intramuscular, intratracheally, subcutaneously
, local by pleura by skin, sucking, by mucous membrane, skin, stomach is intra-articular, intra-ventricle, rectum, vagina,
In skull, in urethra, in liver, in tumor.In some cases, it can systematically be administered.It is locally to be administered in some cases.
The dosage of drug of the invention is unrestricted, can as long as obtaining desired therapeutic effect or preventive effect
To carry out appropriate determination according to symptom, gender, age etc..Example can be used in the dosage of therapeutic agent or prophylactic agent of the invention
Such as the therapeutic effect of disease or preventive effect are determined as index.
In the context of the present invention, " diagnosis Huppert's disease " is both multiple including judging whether subject has suffered from
Property myeloma, also include judge subject with the presence or absence of suffer from Huppert's disease risk.
" treatment " used herein is covered treatment-related in such as mankind of the mammal with related disease or illness
Disease or morbid state, and include:
(1) prevent disease or morbid state occurs in mammals, especially when the mammal is susceptible in the disease
Diseased state, but when being not yet diagnosed with this morbid state；
(2) inhibit disease or morbid state, that is, prevent its generation；Or
(3) alleviate disease or morbid state, even if disease or morbid state subside.
Term " treatment " is usually directed to treatment mankind or animal (for example, being applied by animal doctor), wherein can reach certain pre-
The therapeutic effect of phase, for example, inhibiting the development (including reduce development speed, stop development) of illness, improving illness and healing
Illness.It further include the treatment as precautionary measures (such as prevention).To not yet development be illness but have development be the illness endanger
The purposes of the patient of danger, is also included in term " treatment ".
The advantages of the present invention:
It is of the invention to have found a kind of molecular marker for diagnosing Huppert's disease, it can be using the molecular marker
Huppert's disease occur early stage can be used as judging, provide the survival rate of patient.
In addition, the present invention is capable of providing significant information to determine treatment side for patient by the prognosis of prediction patient
Case strategy.
The therapeutic agent of activator including CTSH gene or albumen of the invention can be used as new Huppert's disease
Therapeutic agent.
Detailed description of the invention
Fig. 1 shows the influence that CTSH gene overexpression is proliferated multiple myeloma cells；
Fig. 2 shows the influence that CTSH gene overexpression invades multiple myeloma cells；
Fig. 3 shows the influence that CTSH gene overexpression migrates multiple myeloma cells.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:ColdSpring HarborLaboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.
1 genetic chip of embodiment screens difference expression gene
1, it draws materials:
Huppert's disease tissue: collecting MM Bone Marrow of Patients biopsy specimen totally 10 made a definite diagnosis, MM diagnostic criteria refering to " in
2011 revised edition of state's Huppert's disease diagnosis and treatment guide ".Male 5, female 5, the median age 59 years old in patient.
Normal bone marrow tissue: same time malnutritional anemia Bone Marrow of Patients biopsy specimen 10 are collected as control
Group, wherein male 5, female 5, the median age 53 years old.
2, the acquisition of RNA is organized
Total tissue RNA is extracted using Trizol one-step method.
3, the measurement of RNA purity and concentration
1 μ l of RNA solution, Instrument measuring OD260, OD280 are taken, RNA concentration is OD260 value × extension rate × 40/1000,
OD260/OD280 is calculated, ratio represents RNA solution purity is high in 1.7-2.0, -20 DEG C preservations few containing impurity such as protein.
4, RNA integrity detection
(1) 2 μ l RNA sample row, 1.5% agarose gel electrophoresis (80v, 15min) is taken；
(2) after separating zone, genefinder is dyed, and Zone electophoresis band is observed under blue light；
(3) as 28s/18s about 2:1, illustrate that RNA stablizes without degradation.
5, gene chip hybridization and scanning
After the linearized amplification of total serum IgE, cy3-UTP is marked, and the cRNAs after fluorescent marker uses RNEASY Mini Kit
Purifying, carries out fragmentation processing to the cRNAs marked with the RNA Fragmentation Reagents of Amhion.Using beauty
People's full genome chip of expression spectrum (4x 44K gene) of Agilent company, state, 65 DEG C of hybridization 17h in chip hybridization furnace, then
Elution, dyeing, finally use Agilent DNA MicroarrayScanner scanner scanning.
6, chip data processing and analysis
Chip after hybridization imports data to analysis software, for two groups of ratios after chip scanner reads data point
Natural logrithm absolute value be greater than 2.0 or the gene less than 0.5 as difference expression gene.
7, statistical procedures
Data analysis is carried out using 13.0 statistical software of SPSS, group difference compares using one-way analysis of variance method, P <
0.05 difference has significant.
8, result
Chip results are shown, filter out 489 differential expressions between Huppert's disease tissue and normal bone marrow tissue altogether
Gene, wherein expression up-regulation gene 215, expression lower gene 274.
2 large sample of embodiment verifies the difference expression gene filtered out
Consideration yet there are no the gene conduct studied about the gene with Huppert's disease correlation in the prior art
Candidate gene, at the same consider gene sequencing as a result, selection CTSH gene (its expression is lowered in Huppert's disease tissue)
It is verified.
1, sample collection
50, Huppert's disease tissue, 60, normal bone marrow tissue are collected according to the method for embodiment 1.
2, it is verified in mRNA level in-site
2.1 extract tissue RNA
Step is the same as embodiment 1.
2.2 reverse transcription
Reverse transcription system totally 20 μ L, including 2 μ g/2 μ L, 50U/ μ L Rnasin of cell total rna, 1 μ L, 5 × reverse transcription are anti-
Answer 4 μ L, 10m M d NTP of buffer, 2 μ l, 50 μ g/mL random primer 2 μ L (promega), 200U/ μ L M-MLV reverse transcriptase
18 μ L of μ L, DEPC.
37 DEG C are reacted 60 minutes, 95 DEG C of reactions of termination in 5 minutes.CDNA saves or carries out PCR amplification at -80 DEG C.
2.3PCR
Reaction system (is purchased from Tiangeng biochemical technology (Beijing) according to Real Master Mix (SYBR Green) kit
Co., Ltd) configuration, SYBR reaction system 10 μ L, 20 × SYBR solution of totally 25 μ L, 2.5 × Real Master Mix
1.25 μ L, 0.5 μ L of upstream primer, 0.5 μ L of downstream primer, 10.75 2 μ L of μ L, cDNA of deionized water.Reaction condition is 94 DEG C
5min, 94 DEG C of 45s, 60 DEG C of 1min, 30 circulations, if blank control.
The fluorescence signal of preceding 10 circulations of PCR reaction adjusts baseline to suitable place, each fluorescence is bent as autofluorescent background signal
The recurring number in line and baseline crosspoint is Ct value.According to Δ C (t)=C (t)Target gene-C(t)β-actin, Δ Δ C (t)=2-ΔC(t), calculate target gene and β-actin relative expression quantity.
PCR primer sequence is as follows:
CTSH gene primer sequence is as follows:
Upstream 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:
Upstream primer: 5 '-CTGGCACCACACCTTCTACAAT-3 ' (SEQ ID NO.3)；
Downstream primer: 5 '-AATGTCACGCACGATTTCCCGC-3 ' (SEQ ID NO.4)
2.4 result
The results show that the mRNA level in-site of CTSH gene is obvious in Huppert's disease tissue compared with normal bone marrow tissue
It lowers, relative expression quantity is 0.31 ± 0.05, and difference has statistical significance (P < 0.05).
3, it is verified on protein level
3.1 extract tissue total protein
The operation of protein extraction is carried out according to the specification of EpiQuik tissue/cell total protein extraction kit.
3.2Western blot detection
The protein quantification of extraction is subjected to SDS-PAGE electrophoresis, carry out later transferring film, closing, primary antibody is incubated for, secondary antibody is incubated for,
Colour developing.
3.3 statistical procedures
The gray value of protein band is analyzed using Image J software, using β-actin as internal reference, by CTSH albumen
The gray value of band is normalized.Result data is indicated in a manner of mean+SD, is used
SPSS13.0 statistical software is next for statistical analysis, and difference between the two is examined using t, it is believed that has system as P < 0.05
Meter learns meaning.
3.4 result
The results show that CTSH protein level significantly reduces in Huppert's disease tissue, phase compared with normal bone marrow tissue
It is 0.42 ± 0.07 to expression quantity, difference has statistical significance (P < 0.05).
Embodiment 3CTSH gene overexpression
1, plasmid construction
Amplimer is designed according to the coded sequence of CTSH gene, the design of primer is well known to those skilled in the art.
From at Human fetal spleen cDNA library (clontech company, article No.: 638831) in amplification overall length CTSH gene coded sequence,
Above-mentioned cDNA sequence is inserted into eukaryotic expression vector pcDNA3.1, connects the recombinant vector pcDNA3.1-CTSH of acquisition
For subsequent experimental.
2, the culture and transfection of multiple myeloma cells
2.1 cell culture
RPMI8226 cell is used containing 15% fetal calf serum (FBS), penicillin 100U/ml, streptomysin 100 μ g/ml
1640 culture medium of RPMI 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*105A tumour cell is suspended in the not antibiotic culture medium of 500 μ l, inoculation
To 24 well culture plates.
(2) transfection same day cell density should reach 80%-90%, prepare following compound A: 1 μ g Plasmid DNA is diluted in nothing
In blood serum medium, mix gently；Compound B: taking 4 μ l Lipofectamine2000 to be diluted in serum free medium, mixes
It is even.
(3) compound A and B are mixed, is mixed gently, is incubated at room temperature.
(4) 100 μ l liposome compounds are added in tumour cell, mix gently up and down, cell is put into 37 DEG C
Containing 5%CO2Incubator is incubated for 5-7 hours.
(5) it is small to continue culture cell 18-24 for the growth medium for adding 1ml to contain 2 times of normal serums and antibiotic concentration
When.
3, the overexpression situation of detection pcDNA3.1-CTSH is tested using QPCR
3.1 extraction cell total rnas are operated using conventional method.
3.2 reverse transcription
Step is the same as embodiment 2.
3.3QPCR
Step is the same as embodiment 2.
3.4 result
The results show that pcDNA3.1-CTSH can be successfully overexpressed, relative expression quantity is 7.13 ± 0.35, and difference has
Statistical significance (P < 0.05).
3, Western blot experiment detection pcDNA3.1-CTSH is overexpressed situation
Step is the same as embodiment 2.
The results show that transfecting the content of CTSH albumen in the cell of pcDNA3.1-CTSH compared with transfecting pcDNA3.1 group
It obviously increases, relative expression quantity is 4.21 ± 0.52, and difference has statistical significance (P < 0.05).
Measurement of the expression of embodiment 4CTSH gene to multiple myeloma cells proliferative capacity
1, step:
(1) by cell inoculation in 96 orifice plates, each concentration does 3 multiple holes；
(2) solution of MTT working concentration is added, with 37 DEG C of cell common incubation 4h；
(3) culture medium is removed, 200 μ l dimethyl sulfoxides are added to dissolve first a ceremonial jade-ladle, used in libation crystal；
(4) continuous 5d surveys OD570, and all experiments are repeated three times above, averaged；
2, result:
As a result as shown in Figure 1, compared with transfecting pcDNA3.1 group, transfection pcDNA3.1-CTSH group cell proliferation rate is bright
Aobvious to slow down, difference has statistical significance (P < 0.05).It is above-mentioned the experimental results showed that, CTSH gene expression inhibition multiple bone
The proliferation of myeloma cells.
Embodiment 7 detects influence of the CTSH gene expression to cell migration, invasion
1, Matrigel
1.1 experimental procedures:
(1) upper chamber is precoated with Matrigel (artificial basement membrane)；
(2) cell after planting transfection in upper chamber, inoculum density 5*104/ 100 μ l cells, in serum free medium
Middle culture 18h；
(3) cell is added in the RPMI-1640 culture medium of 0.1% FBS and cultivates 18h；
(4) 50 μ l Matrigel are drawn on ice；
(5) it is added in 150 μ l serum-free RPMI-1640 culture mediums of pre-cooling and mixes well；
(6) the 50 μ l of Matrigel mixed in (5) is taken respectively, is added to transwell upper chamber, is covered entire film；
(7) 37 DEG C, to overnight, make Matrigel polymerize plastic；
(8) cell is washed 2 times with serum-free RPMI-1640 culture medium, is added in the RPMI-1640 culture medium of serum-free,
To 100 μ l of total volume；
(9) (8) are uniformly added into Transwell upper chamber, between lower layer's culture solution and cell, avoid bubble；
(10) RPMI-1640 culture of the 500-600 μ l of room addition downwards containing 5%FBS, 37 DEG C, 5%CO2；
(11) liquid is exhausted after 48h, wipes the cell not penetrated, move to 100% methanol of cell of the lower surface of film
Fixed 30min, PBS are washed 2 times；
(12) 0.2% violet staining upper chamber 30min, PBS wash away uncalled crystal purple；
(13) it is counted under inverted microscope.
1.2 results:
Experimental result such as Fig. 2 is shown, compared with transfecting pcDNA3.1 group, transfects pcDNA3.1-CTSH group cell invasion number
It significantly reduces, difference has statistical significance (P < 0.05).
2, migration experiment
2.1 experimental procedure
(1) precoating Matrigel artificial basement membrane is not needed in upper chamber.Cell after planting transfection in upper chamber is inoculated with dense
Degree is (1*105The μ l cell of)/100, cultivates 18h in serum free medium；
(2) cell is added in the serum-free RPMI-1640 culture medium of 0.1% FBS and cultivates 18h；
(3) 50 μ l Matrigel are drawn with the pipette tips of pre-cooling on ice；
(4) it is added in 150 μ l serum-free RPMI-1640 culture mediums of pre-cooling and mixes well；
(5) it takes the 50 μ l of Matrigel mixed in (4) to be added to Transwell upper chamber respectively, covers entire film；
(6) 37 DEG C, to overnight, make Matrigel polymerize plastic；
(7) cell is washed 2 times with serum-free RPMI-1640 culture medium, is added in the RPMI-1640 culture medium of serum-free,
To 100 μ l of total volume；
(8) (7) are uniformly added into Transwell upper chamber, between lower layer's culture solution and cell, avoid bubble, downward room adds
Enter RPMI-1640 of the 500-600 μ l containing 10%FBS to cultivate, 37 DEG C, 5%CO2；
(9) liquid is exhausted after 48h, wipes the cell not penetrated, the cell for moving to the lower surface of film is solid with 100% methanol
Determine 30min, PBS is washed 2 times；
(10) 0.2% violet staining upper chamber 30min, PBS wash away uncalled crystal purple；
(11) it is counted under inverted microscope.
2.2 result
Experimental result such as Fig. 3 is shown, compared with transfecting pcDNA3.1 group, transfects pcDNA3.1-CTSH group cell migration number
It significantly reduces, difference has statistical significance (P < 0.05).
The above results show that CTSH gene expression is unfavorable for the migration and invasion of myeloma cell.
The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.
Claims (8)
1. detecting the product of CTSH gene or CTSH albumen in preparation diagnosis Huppert's disease or prediction Huppert's disease
Application in the tool of prognosis.
2. application according to claim 1, which is characterized in that the product packet of the detection CTSH gene or CTSH albumen
Include the product of the expression of detection CTSH gene or CTSH albumen.
3. application according to claim 1 or 2, which is characterized in that the product includes can be in conjunction with the core of CTSH gene
Acid can be in conjunction with the substance of CTSH albumen；The nucleic acid is able to detect the expression of CTSH gene；The substance energy
Enough detect the expression of CTSH albumen.
4. application according to claim 3, which is characterized in that the nucleic acid is specifically expanded used in real-time quantitative PCR
Increase the primer of CTSH gene as shown in SEQ ID NO.1 and SEQ ID NO.2.
5. application according to claim 1, which is characterized in that the tool includes being able to detect CTSH gene or CTSH
The tool of the expression of albumen.
6. application according to claim 5, which is characterized in that the tool includes can be in conjunction with the nucleic acid of CTSH gene
Or it can be in conjunction with the substance of CTSH albumen；The nucleic acid is able to detect the expression of CTSH gene；The substance can
Detect the expression of CTSH albumen.
7. application according to claim 6, which is characterized in that the nucleic acid is specifically expanded used in real-time quantitative PCR
Increase the primer of CTSH gene as shown in SEQ ID NO.1 and SEQ ID NO.2.
8. promoting application of the reagent of CTSH gene expression in the drug of preparation treatment Huppert's disease.
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| CN104321439A (en) * | 2012-03-15 | 2015-01-28 | 凯杰科技有限公司 | Thyroid cancer biomarker |
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