CN107385096A - The purposes of LRRTM1 genes and its expression product in gastric gland metastasis of cancer diagnosis and treatment instrument is prepared - Google Patents
The purposes of LRRTM1 genes and its expression product in gastric gland metastasis of cancer diagnosis and treatment instrument is prepared Download PDFInfo
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Abstract
The invention discloses the application of LRRTM1 genes and its expression product in sdenocarcinoma of stomach diagnosis and treatment.Experiment proves that LRRTM1 genes and its expression product have differences expression in normal structure and gastric adenocarcinoma tissue, accordingly can be using LRRTM1 genes and its expression product as the biomarker for diagnosing sdenocarcinoma of stomach.In addition, In vitro cell experiment of the present invention proves to be overexpressed LRRTM1 genes, Gastric Adenocarcinoma Cell Line and migration can be suppressed, the medicine for the treatment of sdenocarcinoma of stomach can be developed accordingly.
Description
Technical field
The present invention relates to diagnosing tumor, therapy field, more particularly it relates to abnormal for means to detect LRRTM1
Diagnosing tumor method；And promote the tumor therapeutic agent of LRRTM1 genes or protein.
Background technology
Stomach cancer is derived from the malignant tumour of gastric epithelial cell, predominantly gland cancer.The whole world is newly diagnosed to be stomach cancer within 2008
Nearly 1,000,000, number of dying of illness 740,000, be the whole world incidence of disease the 4th, the cancer of fatal rate second, is a kind of clinically common
Malignant tumour.Although stomach cancer whole world total incidence has declined, the gastric cancer cases for having nearly 2/3rds concentrate on economic owe
Developed countries and regions, China and other East Asian countries are occurred frequently.According to《Life Times》Latest edition " the Cancer in China of issue in 2014
Map ".It is about 3120000 that tumor cases are newly sent out in China every year, average daily 8550, per minute to have 6 people to be diagnosed as cancer
Disease, there are 5 people to die from cancer.And shown according to the investigation of the World Health Organization, China belongs to high incidence area of gastric cancer, Liaoning, Shandong, sweet
It is even more serious that respectful, Jiangsu, Fujian etc. save situation.Clinically, although the treatment of early carcinoma of stomach oneself through achieving major progress, often
Surgically excision plus regional lymph nodes are cleaned and postoperative chemicotherapy is supported, but the long-term survival rate of late gastric cancer is still very
It is low.Many documents and research show that invasion and attack, the transfer of tumour result in the death of most of patients with gastric cancer and in the bad of stomach cancer
Play the part of pivotal player in prognosis.The invasion and attack of stomach cancer, transfer, which are a polygenes regulation and control, multiple-factor participates in, multi-step is carried out answers
Miscellaneous biological process, it is considered that the motion of cancer cell and invasive ability are the essential conditions for producing transfer.But for late period
Stomach cancer, it is attacked and shifts potential molecular mechanism and is still not clear.The mark sex factor of reliable tumor prognosis is thus found, and
The molecular target of metastases and invasion and attack can effectively be suppressed by finding, and improved the prognosis of tumour, be the focus of Recent study,
It is an emphasis and the difficult point in tumor research.Therefore, urgent need differentiates the important molecule of stomach cancer progressive stage, researches and develops new
Drug target, the prognosis for the treatment of and patient for tumour provide valuable help.
The content of the invention
An object of the present invention is that provide one kind realizes that sdenocarcinoma of stomach turns by detecting LRRTM1 gene expression differences
Move diagnosis, sdenocarcinoma of stomach prediction prognosis, the method for sdenocarcinoma of stomach prognosis evaluation.
The second object of the present invention is to provide a kind of treats gastric gland metastasis of cancer by promoting LRRTM1 gene expressions
Method.
The third object of the present invention is to provide a kind of method for screening gastric gland metastasis of cancer medicine.
The fourth object of the present invention is to provide a kind of medicine for being used to treat gastric gland metastasis of cancer.
To achieve these goals, present invention employs following technical scheme：
The invention provides detection LRRTM1 reagent to prepare gastric gland metastasis of cancer diagnosis, sdenocarcinoma of stomach prediction prognosis, gastric gland
Application in cancer prognosis evaluation instrument.
Further, the reagent of the detection LRRTM1 includes the reagent of detection LRRTM1 gene expression amounts.
Further, the reagent of the detection LRRTM1 includes the reagent that can quantify LRRTM1 gene mRNAs, and/or energy
Enough quantify the reagent of LRRTM1 albumen.
The reagent of the quantitative LRRTM1 gene mRNAs of the present invention can be based on playing it using the known method of nucleic acid molecules
Function：As PCR, such as Southern hybridization, Northern hybridization, dot blot, FISH (FISH), DNA microarray,
ASO methods, high-flux sequence platform etc..It can qualitatively, quantitatively or semi-quantitatively implement analysis using the reagent.
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, nested PCR methods etc..Amplification
Nucleic acid can be by using dot blotting hybridization method, Surface Plasmon Resonance (SPR methods), PCR-RFLP methods, RT-PCR in situ
Method, PCR-SSO (sequence specific oligonucleotide) method, PCR-SSP methods, AMPFLP (amplifiable fragment length polymorphism) method,
MVR-PCR methods and PCR-SSCP (single-strand conformation polymorphism) methods detect.
The reagent that LRRTM1 gene mRNAs can be quantified can be the specific primer of LRRTM1 genes or transcript,
It can also be the specific recognition probe of LRRTM1 genes or transcript, or include primer and probe simultaneously.
The specific primer of LRRTM1 genes or transcript recited above includes the special expansion used in real-time quantitative PCR
Increase the primer of LRRTM1 genes.In the specific embodiment of the present invention, the primer sequence such as SEQ ID NO.1 and SEQ ID
Shown in NO.2.
Primer can be prepared by chemical synthesis, by using those skilled in the art will know that method reference known believe
Breath is prepared to be suitably designed by chemical synthesis.
Probe can be prepared by chemical synthesis, by using those skilled in the art will know that method reference known believe
Breath appropriately designs, and is prepared by chemical synthesis, or can be by being prepared from biomaterial containing expectation nucleotide sequence
Gene, and using designed for amplification it is expected nucleotide sequence primer expand it to prepare.
The reagent of the quantitative LRRTM1 albumen of the present invention can be based on playing its function using the known method of antibody：Example
Such as, ELISA, radioimmunoassay, immunohistochemical method, western blot etc. can be included.
The reagent of the quantitative LRRTM1 albumen of the present invention includes the antibody or its fragment of specific binding LRRTM1 albumen.Can
To use the antibody of any structure and size, immunoglobulin class, origin etc. or its fragment, as long as it is with reference to target protein
Can.The antibody or its fragment that the detection product of the present invention includes can be monoclonals or polyclonal.Antibody fragment refers to guarantor
Stay peptide of the antibody to the antibody a part of (Partial Fragment) of the binding activity of antigen or containing an antibody part.Antibody fragment can be with
Including F (ab ')2, Fab ', Fab, scFv (scFv), disulphide bonding Fv (dsFv) or its polymer, dimerization V areas
(double antibody) or the peptide containing CDR.The reagent of the quantitative LRRTM1 albumen of the present invention can include encoding antibody or encoding antibody
The nucleic acid of the separation of the amino acid sequence of fragment, the carrier comprising the nucleic acid, and carry the cell of the carrier.
Antibody can obtain by the way that well known to a person skilled in the art method.Retain target all or in part for example, preparing
The polypeptide of protein integrates the mammalian cell expression vector for encoding their polynucleotides 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
Knurl culture collects antibody.Finally can be real to the antibody of acquisition by using LRRTM1 albumen for being used as antigen or part thereof
Antigentic specificity purifying is applied to obtain the monoclonal antibody for LRRTM1 albumen.Polyclonal antibody can be prepared as follows：With with
Identical antigen-immunized animal above, blood sample is collected from by immune animal, serum is isolated from blood, is then made
Implement antigentic specificity to serum with above-mentioned antigen to purify.Can be by using the antibody of ferment treatment acquisition or by using acquisition
The sequence information of antibody obtains antibody fragment.
The combination of label and antibody or its fragment 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. standard
Standby dyestuff, then mixed solution, is placed 10 minutes then at room temperature.In addition, the labelling kit of commercialization can be used in mark, 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 kits-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, the labelling kit-NH2 of HiLyte Fluor (TM) 647 (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- label protein marks
Remember kit (Funakoshi Corporation).For correct labeling, can be detected using suitable instrument by mark
Antibody or its fragment.
The acquisition of the sample for being used to detect the detection of LRRTM1 gene expression amounts of the present invention is the ordinary skill in the art, excellent
Noninvasive may be selected in choosing or the method with minimally-invasive property obtains.
Described sample can be (but are not limited to)：Peripheral blood, marrow, lymph node, abdominal cavity cleaning fluid, parietal cell or stomach
Liquid.In specific embodiments of the present invention, tissue of the sample from subject.
As well known to those skilled in the art, the cell of tumor tissues can be shed in body fluid, and these cells to come off are referred to as
Circulating tumor cell, the property of circulating tumor cell is identical with the property of tumour cell in tumor tissues, therefore detects in body fluid
The property can of circulating tumor cell represents the property of tumor tissues especially in blood.For the present invention, detection is passed through
LRRTM1 gene expressions can be used for diagnosing sdenocarcinoma of stomach transfer in tumor tissues, and can easily draw can also be circulated by detecting
LRRTM1 gene expressions are shifted to diagnose sdenocarcinoma of stomach in tumour cell.
Present invention also offers a kind of for gastric gland metastasis of cancer diagnosis, sdenocarcinoma of stomach prediction prognosis, sdenocarcinoma of stomach prognosis evaluation
Instrument, the instrument can detect LRRTM1 gene expression amounts.
Further, the instrument includes the reagent that can quantify LRRTM1 gene mRNAs, and/or can quantify LRRTM1 eggs
White reagent.
Generally, described reagent is present in appropriate container.Diluent such as deionized water can be used described to draw every kind of
Thing or probe are adjusted to the concentration of at least one requirement, are sub-packed in container.
Further, the reagent that can quantify LRRTM1 gene mRNAs includes the special expansion used in real-time quantitative PCR
Increase the primer of LRRTM1 genes, the primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
Further, it is described to include for gastric gland metastasis of cancer diagnosis, sdenocarcinoma of stomach prediction prognosis, the instrument of sdenocarcinoma of stomach prognosis evaluation
But it is not limited to chip, kit, test paper or high-flux sequence platform；High-flux sequence platform is a kind of special tool(s), with height
The development of flux sequencing technologies, the structure of the gene expression profile of a people will be turned into and very easily worked.By contrasting disease
Patient and the gene expression profile of normal population, the exception for easily analyzing which gene are related to disease.Therefore, in high flux
Know that the exception of LRRTM1 genes is related to gastric gland metastasis of cancer in sequencing and fall within the new application for having used the LRRTM1 of the present invention,
Equally within protection scope of the present invention.
The reagent for extracting nucleic acid is may also include in the kit of the present invention, for PCR reagent, for dyeing or showing
Reagent of color etc..For example, these reagents include but is not limited to：Extract, amplification liquid, hybridization solution, nitrite ion, washing lotion etc..
In addition, the specification of method of description detection gastric gland metastasis of cancer is may also include in described kit etc..
Kit of the present invention, which can include, is suitable to a variety of different of practical (being such as directed to different detection methods)
Reagent, however it is not limited to cited reagent at present, as long as sdenocarcinoma of stomach is judged based on the detection of LRRTM1 genes or transcript
The reagent of transfer is all contained in the scope of the invention.
Present invention also offers the side of a kind of gastric gland metastasis of cancer diagnosis, sdenocarcinoma of stomach prediction prognosis or sdenocarcinoma of stomach prognosis evaluation
Method, methods described comprise the following steps：
(1) sample of subject is obtained；
(2) LRRTM1 gene expression doses in Samples subjects are detected；
(3) the LRRTM1 gene expression doses measured are associated with the disease associated of subject.
(4) compared with normal control, LRRTM1 gene expression doses statistically reduce, and show that subject is judged stomach
Gland cancer has occurred and that subject's prognosis mala of transfer or prediction gastric gland metastasis of cancer or assesses the tested of gastric gland metastasis of cancer
Person has been recurred.
Present invention also offers a kind for the treatment of method of gastric gland metastasis of cancer, methods described includes promoting the table of LRRTM1 genes
Reach, or promote the expression of LRRTM1 albumen or strengthen the activity of LRRTM1 albumen..
, can be by after testing drug be added to cancer cell present invention also offers a kind of screening technique of tumour medicine
Or some period after testing drug is applied to tumor model animal measures the expression of LRRTM1 genes or LRRTM1 albumen
Level improves the effect of tumor prognosis to determine tumour medicine.More specifically, when LRRTM1 genes or LRRTM1 albumen
It is swollen as improving that the medicine may be selected when being raised after adding or applying testing drug or when recovering normal level in expression
The medicine of knurl prognosis.
Present invention also offers a kind of medicine for treating gastric gland metastasis of cancer, the medicine includes LRRTM1 activator.
The LRRTM1 genes of the present invention or the activator of LRRTM1 albumen are unrestricted, as long as it can promote or strengthen
LRRTM1 or be related to LRRTM1 upstreams or downstream pathway material expression or activity, and for treatment the effective medicine of tumour be
Can.
The activator is also included comprising the carrier or host cell for carrying LRRTM1 genes.
On the one hand the activator of the present invention can be used for the missing or deficiency for supplementing endogenic LRRTM1 albumen, by carrying
The expression of high LRRTM1 albumen, so as to treat the sdenocarcinoma of stomach caused by LRRTM1 hypoproteinosis.On the other hand can be used for strengthening
The activity of LRRTM1 albumen, so as to treat sdenocarcinoma of stomach.
Present invention also offers application of the above-mentioned activator in the medicine for preparing treatment gastric gland metastasis of cancer.
The activator of the present invention can be used by any of mode compounding pharmaceutical composition in this area.This group
Compound includes active component, plus one or more pharmaceutically acceptable carrier, diluent, filler, bonding agent and other taxes
Shape agent, this depends on administering mode and designed dosage form.The known treatment of this area branch art personnel it is inert inorganic or
Organic carrier includes but is not limited to lactose, cornstarch or derivatives thereof, talcum, vegetable oil, wax, fat, polyhydroxylated
Compound such as polyethylene glycol, water, sucrose, ethanol, glycerine, such, various preservatives, lubricant, dispersant, flavouring.
Moisturizing is cut to pieces, antioxidant, sweetener, colouring agent, stabilizer, salt, buffer solution is such can also be added thereto, these material roots
The stability of formula is used to help according to needs or is favorably improved activity or its biological effectiveness or is produced in the case of oral
Raw acceptable mouthfeel or smell, the preparation that can be used in such a composition can be the form of its original chemical in itself,
Or the form of its pharmaceutically acceptable salt is optionally used, activator of the invention can be administered alone, or with various combinations
Administration, and combining form is administered together with other healing potions.This area may be selected in the composition so prepared as needed
Any appropriate mode is administered activator known to technical staff.It is by safe and effective amount during using pharmaceutical composition
Inhibitor of the invention be applied to people, wherein oral safe and effective amount typically at least about 100 micrograms/kg body weight.Certainly, have
Body dosage is also contemplated that the factors such as method of administration, patient health situation, within the scope of these are all skilled practitioners technical ability.
The medicine of the present invention can be prepared into various formulations 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 route of administration of the medicine of the present invention is unrestricted, as long as it can play desired therapeutic effect or preventive effect i.e.
Can, include but is not limited to intravenously, intraperitoneal, intraocular, intra-arterial, intrapulmonary, orally, in vesicle, intramuscular, tracheal strips, subcutaneously
, local by pleura by skin, suction, intra-ventricle intra-articular by mucous membrane, skin, stomach, rectum, vagina,
In skull, in urethra, in liver, in knurl.In some cases, can systematically be administered.It is to be partly administered in some cases.
The dosage of the medicine of the present invention is unrestricted, can as long as obtaining desired therapeutic effect or preventive effect
To carry out appropriate determination according to symptom, sex, age etc..The medicine of the present invention or the dosage of prophylactic agent can be with use examples
Therapeutic effect or preventive effect such as to disease determine as index.
(NC_000002.12 (80301878..80304738) sequence can be with NCBI data for " the LRRTM1 genes " of the present invention
Inquired about in storehouse.
In the context of the present invention, " gastric gland metastasis of cancer diagnosis " includes judging whether subject has occurred and that sdenocarcinoma of stomach turns
Move, judge that whether subject whether there is the risk of gastric gland metastasis of cancer, judges gastric gland metastasis of cancer patient relapse and metastasis.
" treatment " used herein is covered treatment-related in such as mankind of the mammal with relevant disease or illness
Disease or morbid state, and including：
(1) prevention disease or morbid state occur in mammal, especially when the mammal is susceptible in the disease
Diseased state, but when being not yet diagnosed with this morbid state；
(2) suppress disease or morbid state, that is, prevent its generation；Or
(3) disease or morbid state are alleviated, even if disease or morbid state disappear.
Term " treatment " is usually directed to the treatment mankind or animal (for example, being applied by animal doctor), wherein can reach some pre-
The therapeutic effect of phase, for example, suppressing the development (including reduce development speed, stop development) of illness, improving illness and healing
Illness.Also include the treatment as precautionary measures (such as prevention).Pair illness is not developed into also but develop into illness danger
The purposes of the patient of danger, is also included within term " treatment ".
The advantages of the present invention：
Present invention firstly discovers that and confirm the Close relation of LRRTM1 gene expressions and gastric adenocarcinoma tissue transfer, checking
Sample size is more, as a result accurately.The it is proposed of the correlation provides new approach for the Clinics and Practices of gastric gland metastasis of cancer.
The present invention have developed the reagent or kit for being suitable for carrying out gastric gland metastasis of cancer detection, and detection sensitivity is good.
Brief description of the drawings
Fig. 1 shows the difference in metastatic gastric adenocarcinoma tissue and normal control tissue using QPCR detection LRRTM1 genes
Expression；
Fig. 2 is shown using Western blot experiment detection LRRTM1 albumen in metastatic gastric adenocarcinoma tissue and normal control
Differential expression in tissue；
Fig. 3 is shown is overexpressed efficiency using Western blot experiment detection LRRTM1 gene expressions.
Specific embodiment
The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this
Invention rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in embodiment, generally according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in, or according to the condition proposed by manufacturer.
The LRRTM1 gene differential expressions of embodiment 1
1st, sample is collected
Metastatic gastric adenocarcinoma tissue and its Carcinoma side normal tissue 50 are collected, collects non-metastatic gastric adenocarcinoma tissue and its cancer
Other normal structure 45.Tissue samples are helped to sample by Pathologis, and it is as follows to collect sample canonical：
(1) primary sdenocarcinoma of stomach, no other diseases, patient are preoperative without carcinosis radiotherapy and chemotherapy；(2) sample is diagnosed by pathology department
For sdenocarcinoma of stomach, and Carcinoma side normal tissue and it is not detected by tumour cell；(3) in order to avoid unnecessary intersection is stained, during sampling
Using two sets of equipment, first take 5cm's minimum from cancerous tissue to visually observe the Carcinoma side normal tissue sample without obvious lesion, after take it is complete
The gastric adenocarcinoma tissue sample of layer, sample are dispensed and are put into the EP pipes added with 1ml RNAlater immediately after collecting, and 4 DEG C are overnight, and
It is placed in -70 DEG C of refrigerator and preserves for a long time afterwards.And it is numbered and marks on each EP pipes.Simultaneously in sample record sheet
Carry out the remarks of relevant information.
2nd, the RNA of extraction pairing sdenocarcinoma of stomach and cancer beside organism's sample
Using RNA extracts kits (the Trans Zol purchased from Beijing Quanshijin Biotechnology Co., LtdTM Up Plus
RNA Kit) extraction tissue samples RNA.Comprise the following steps that：
(1) it after the sample weighing of superfreeze, will be transferred quickly in the mortar with Liquid nitrogen precooler, fully ground with pestle
The sample being ground into powder is transferred in centrifuge tube up to being ground into powder, 1ml is added per 50-100mg samples by mill
Trans Zol TMUp carries out homogenized with Syrup-homogenizing instrument, or pressure-vaccum mixes repeatedly with rifle.It is stored at room temperature 5 minutes.
(2) 1ml Trans Zol are often usedTMUp, add 0.2ml chloroforms, acutely vibration 30 seconds, be incubated at room temperature 3 minutes.
(3) 4 DEG C of 10000 × g are centrifuged 15 minutes.Now sample is divided into three layers, colourless aqueous phase (upper strata), and intermediate layer is pink
Color organic phase (lower floor).RNA draws the μ l liquid of colourless aqueous phase layer 500 in colourless aqueous phase.
(4) aqueous phase colourless 500 μ l of transfer absorption adds 500 μ l absolute ethyl alcohol, gently run in new centrifuge tube
Mix.(hereafter centrifuging can be carried out at room temperature)
(5) solution for obtaining 700 μ l and precipitation are added in centrifugal column together, and 12000 × g room temperatures centrifuge 30 seconds, discard
Efflux (such as fruit volume is more than centrifugation column capacity, can divide and complete several times).
(6) 500 μ l CB9,12000 × g of room temperature room temperatures are added to centrifuge 30 seconds, discards efflux.
(7) repeat step (6) is once.
(8) 500 μ l WB9 (please first checked whether before use and add absolute ethyl alcohol), the centrifugation of 12000 × g of room temperature room temperatures are added
30 seconds, discard efflux
(9) repeat step (8) is once.
(10) 12000 × g of room temperature room temperatures centrifuge 2 minutes, thoroughly remove residual ethanol, be stored at room temperature several minutes it is thorough
Dry centrifugal column.
(11) centrifugal column is put into RNase-free Tube (kit has been matched somebody with somebody), adds 30 μ l RNase-free Water
In the center of centrifugal column, it is stored at room temperature 1 minute.
(12) 12000 × g of room temperature room temperatures centrifuge 1 minute, eluted rna.
(13) RNA is placed in into -80 DEG C of refrigerators to preserve.
3rd, the RNA concentration and the measure of purity extracted
RNA concentration and purity are detected using the ND-1000 instruments of Nano Drop companies of the U.S..
Using UV detector Nano Drop 1000, the Nano Drop icons on computer screen are double-clicked, into system
System menu；According to system prompt after selection nucleic acid measurement option, 2 μ l distilled waters are added in well；Click on and determine, with initial
Change system；2 μ l distilled waters are added in well, select RNA items, click on Blank with measuring system background.Cleaned with rear lens wiping paper
RNA sample to be measured is clicked and entered after distilled water, clicks on Measure.Reading numerical values simultaneously record, and RNA concentration (ng/ μ l), must now need
It is noted that A values 260/280, if numerical value between 1.8-2.0, illustrates that RNA sample quality is preferable.By the RNA of each sample
Frozen after concentration markers are complete in -70 DEG C of refrigerators.
4th, the RNA integrity mensuration's extracted
Ago-Gel detects RNA sample operations steps：
1) electrophoresis tank, the cleaning of glue apparatus：Detergent wash clean (general soaked overnight), water are used after rinsing, 3%H2O2
Electrophoresis tank is filled, room temperature places 10min, is rinsed, dried standby with 0.1% (V/V) DEPC water.
2) glue：0.5g agarose powders are weighed, addition is placed with the 45ml conical flask of DEPC water, and heating makes agarose
It is completely dissolved.5ml 10 × TAE electrophoretic buffers and final concentration of 0.5 μ g/ml Ethidum Eremide are added after slightly cooling down.Then
The Casting of gels in glue groove, it is plugged comb, horizontal positioned rear use to be solidified.
3) it is loaded：After sample mixes with 6 × electrophoretic buffer, it is loaded onto in gel loading wells.
4) electrophoresis：Open electrophoresis apparatus, voltage stabilizing 80V electrophoresis.
5) observe after electrophoresis terminates (about 30 minutes), under uviol lamp, and taken pictures preservation with gel imaging system.
Integrity mensuration' is detected by agarose gel electrophoresis, if 28s rRNA, 18s rRNA, 5s can be clearly apparent
RRNA three bands, and 28s rRNA brightness should be twice of 18s rRNA.Illustrate that the total serum IgE integrality of extraction is preferable, RNA
Satisfactory quality.
5th, the design and preparation of primer
The primer sequence of qRT-PCR detection LRRTM1 gene expressions and qRT-PCR amplification internal references GAPDH primer sequence
Design and synthesize by Sangon Biotech (Shanghai) Co., Ltd., and through the retrieval of UCSC databases, its sequence is believed
Typing NCBI software Design primers are ceased, and the blast in gene pool confirm correctly.
LRRTM1 gene primers：
Sense primer：5’-TCTGCCCAGGAATACTAC-3’(SEQ ID NO.1)；
- the AGCCATACTCGTTGATGA-3 ' of anti-sense primer 5 ' (SEQ ID NO.2),
GAPDH gene primers：
Sense primer：5’-GGGAGCCAAAAGGGTCA-3’(SEQ ID NO.3)；
Anti-sense primer：5’-GAGTCCTTCCACGATACCAA-3’(SEQ ID NO.4).
6th, real time fluorescent quantitative cDNA reverse transcriptions detecting step
Reverse transcription synthesis cDNA is carried out to l μ g total serum IgEs with RT Buffer.Using 25 μ l reaction systems, each sample
1 μ g total serum IgEs are taken to be separately added into following components in PCR pipe as template ribonucleic acid：DEPC water, 5 × RT Buffer,
10mmol/L dNTP, 0.1mmol/l DTT, 30 μm of mol/l Oligo dT, 200U/ μ l M-MLV, template ribonucleic acid.42 DEG C of incubations
1h, 72 DEG C of 10min, of short duration centrifugation.
7、PCR
(1) Bio-RAD real-time fluorescence quantitative PCR instruments are applied, reaction system is prepared by table 1.
The PCR reaction systems of table 1
(2) carried out using following qPCR response parameters：95 DEG C of pre-degeneration 10min, then, 95 DEG C of denaturation 15s, 57 DEG C are moved back
Fire, extension 1min, totally 40 circulation；Then, the collection of fluorescence signal and the making of product solubility curve, each 3, sample are carried out
Repeat, take its average value.Using 2-△△CtRelative quantification method analyzes LRRTM1 expression, and Ct is that thermal cycler detects instead
Answer the intensity level of fluorescence signal in system.Computational methods are：Δ Δ Ct=(Ct target gene-Ct reference genes) tumor tissues are real
Test group-(Ct target gene-Ct reference genes) normal structure control group, 2-△△CtWhat is represented is the expression of experimental group target gene
Relative to the change multiple of control group, analysis of experimental data is completed by Bio-RAD analysis softwares.
5th, statistical analysis carries out data analysis using statistics software SPSS19.0, judges gastric gland using paired-samples T-test
LRRTM1 expression is all double with the presence or absence of all statistical checks of difference of statistical significance in cancerous tissue and cancer beside organism's sample
Side is examined, and P ＜ 0.05 are that difference is statistically significant.
6th, result
As a result as shown in figure 1, in transfer group, gastric adenocarcinoma tissue is compared with cancer beside organism, LRRTM1 gene mRNA expression water
Flat to reduce, difference has statistical significance；And at non-diverting group, gastric adenocarcinoma tissue is compared with cancer beside organism, LRRTM1 genes
MRNA expressions do not have the change occurred on statistical significance, are detected simultaneously by transfer group cancer beside organism and non-diverting group of cancer
LRRTM1 mrna expressions are identicals in the tissue of side.The above results show that the low expression of LRRTM1 genes take part in
The transfer of gastric adenocarcinoma tissue.
The differential expression of the LRRTM1 albumen of embodiment 2
1st, the total protein of tissue samples in embodiment 1 is extracted
The operation of protein extraction is carried out according to the specification of EpiQuik tissue/cell total protein extraction kits.
2、Western blot
Using β-actin as internal reference.50 μ g total proteins are after SDS-PAGE points, electrotransfer to pvdf membrane, with containing 5% defatted milk
1 × TBST room temperatures jog closing 1h of powder；It is separately added into rabbit-anti people LRRTM1 monoclonal antibodies (1: 800 dilution) and the anti-human β-actin of mouse
More anti-(1: 3 000 dilutions), 4 DEG C overnight；1 × TBST washes film 4 times, adds goat-anti rabbit and sheep anti mouse Ig G (1: 2 000 dilution),
It is incubated at room temperature 1h；After 1 × TBST washes film 4 times, it is placed in Super Signal chemical illuminating reagents and reacts 2min, make X in darkroom
Mating plate exposes, conventional method developing fixing.
3rd, statistical procedures
The gray value of protein band is analyzed using Image J softwares, using β-actin as internal reference, by LRRTM1 eggs
The gray value of informal voucher band is normalized.Result data is represented in a manner of mean+SD, is used
SPSS13.0 statistical softwares carry out statistical analysis, and difference between the two is examined using t, it is believed that works as P<There is system when 0.05
Meter learns meaning.
4th, result
As a result as shown in Fig. 2 in transfer group, compared with cancer beside organism, LRRTM1 protein levels reduce gastric adenocarcinoma tissue,
Difference has statistical significance；And at non-diverting group, compared with cancer beside organism, LRRTM1 protein levels do not have gastric adenocarcinoma tissue
The change on statistical significance occurs, is detected simultaneously by LRRTM1 albumen in transfer group cancer beside organism and non-diverting group of cancer beside organism
Level is identical.The above results show that LRRTM1 albumen low expressions take part in the transfer of gastric adenocarcinoma tissue.
The LRRTM1 gene overexpressions of embodiment 3
1st, plasmid construction
According to the coded sequence of LRRTM1 genes design amplimer, primer be designed as those skilled in the art institute it is ripe
Know.From cDNA library (clontech companies, the article No. into Human fetal spleen：638831) coding of the LRRTM1 genes of amplification total length in
Sequence, above-mentioned cDNA sequence are inserted into eukaryotic expression vector pcDNA3.1, connect the recombinant vector pcDNA3.1- of acquisition
LRRTM1 is used for subsequent experimental.
2nd, the culture of human gastric adenocarcinoma
People's gastric adenocarcinoma cell line SGC 7901 uses the RPMI1640 culture mediums containing 10% hyclone to add penicillin 100units/
Ml, the μ g/ml of streptomysin 100, put 37 DEG C, 5%CO2Incubator in cultivate, change 1 nutrient solution every 24h, 48h is passed on 1 time.
Take the logarithm growth period cell carry out subsequent experimental.
3rd, cell transfecting
Liposome Lipofectamine2000 is used as transfection reagent.2 groups of experiment point：Control group (transfection pcDNA3.1)；
Experimental group (transfection pcDNA3.1-LRRTM1).Take the logarithm the SGC7901 cells in growth period, be inoculated in 6 hole cell culture
Plate.Tissue Culture Plate coverage rate is about 70%-80% after 24h.Rotaring transfecting mode enters with reference to Lipofectamine2000 specifications
OK.
4th, Western blot experiments detection pcDNA3.1-LRRTM1 overexpression efficiency
Step is the same as embodiment 2.
5th, result
As shown in figure 3, compared with control group, LRRTM1 expressing quantities significantly raise in experimental group cell, and difference has
Statistical significance (P<0.05).
The LRRTM1 gene overexpressions of embodiment 4 are to Gastric Adenocarcinoma Cell Line, the influence of transfer ability
1st, the detection of the external transfer ability of gastric adenocarcinoma cells
48h after transfection, by control group (transfection pcDNA3.1), turn of two groups of experimental group (transfection pcDNA3.1-LRRTM1)
Dye SGC7901 cells are prepared into single cell suspension with without dual anti-RPMI1640 nutrient solutions, with 106It is individual/400 μ l density add
In the cell of Transwell upper stratas, 600 μ l Apoptosis NIH3T3 conditioned mediums are added in bottom chamber, are put
In 37 DEG C, 5%CO2In incubator, after cultivating 24h, upper indoor cell is wiped with wet cotton swab, 15min, HE are fixed through absolute ethyl alcohol
Dyeing, light Microscopic observation, and upper and lower, left and right and middle each 5 visuals field are randomly selected, theca cell is worn in counting, and is averaged,
To wear the external transfer ability that the number of theca cell represents SGC7901 cells.
As a result：Experimental group SGC7901 cells wear theca cell number (154.5 ± 11.8) compared with control group (369.4 ± 10.4)
It is obvious to reduce, no significant difference (P ＞ 0.05).It is above-mentioned test result indicates that, promote LRRTM1 gene expressions can press down
Gastric adenocarcinoma cells migration processed.
2nd, the detection of gastric adenocarcinoma cells vitro invasion ability
After ECM matrigels are diluted in 1: 7 ratio with serum-free RP-MI1640 nutrient solutions, 30 μ l are taken equably to be coated in
The upper chamber face of Tran-swell cells bottom film, ultraviolet irradiation overnight, make ECM matrigels aggregate into gel naturally.By each group
Single cell suspension is made with without dual anti-RPMI1640 nutrient solutions in SGC7901 cells, with 105It is individual/400 μ l density add
In the cell of Transwell upper stratas, remaining step represents the external of SGC7901 cells with experiment is migrated to wear the number of theca cell
Invasive ability.
As a result：Experimental group SGC7901 cells wear theca cell number (29.4 ± 2.5) compared with negative control group (93.6 ± 6.4)
It is obvious to reduce.It is above-mentioned test result indicates that, promote LRRTM1 gene expressions can suppress Gastric Adenocarcinoma Cell Line.
The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this
For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improvement can also be carried out to the present invention
And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.
Sequence table
<110>Beijing Yang Shen biology information technologies Co., Ltd
<120>The purposes of LRRTM1 genes and its expression product in gastric gland metastasis of cancer diagnosis and treatment instrument is prepared
<160> 4
<170> SIPOSequenceListing 1.0
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<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
tctgcccagg aatactac 18
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
agccatactc gttgatga 18
<210> 3
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gggagccaaa agggtca 17
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
gagtccttcc acgataccaa 20
Claims (10)
1. detect LRRTM1 reagent prepare gastric gland metastasis of cancer diagnosis, sdenocarcinoma of stomach prediction prognosis or sdenocarcinoma of stomach transfer and relapse comment
Estimate the application in instrument.
2. application according to claim 1, it is characterised in that the reagent of the detection LRRTM1 includes detection LRRTM1 bases
Because of the reagent of expression quantity.
3. application according to claim 1 or 2, it is characterised in that the reagent of the detection LRRTM1 includes quantifying
The reagent of LRRTM1 gene mRNAs, and/or the reagent of LRRTM1 albumen can be quantified.
4. application according to claim 3, it is characterised in that the reagent that can quantify LRRTM1 gene mRNAs includes
The primer of the specific amplified LRRTM1 genes used in real-time quantitative PCR；The reagent that LRRTM1 albumen can be quantified includes
Specifically bind the antibody of LRRTM1 albumen.
5. a kind of instrument assessed for gastric gland metastasis of cancer diagnosis, the prognosis of sdenocarcinoma of stomach branch prediction or sdenocarcinoma of stomach transfer and relapse, its
It is characterised by, the instrument includes the instrument that can detect LRRTM1 gene expression amounts；Preferably, the instrument includes determining
The reagent of LRRTM1 gene mRNAs is measured, and/or the reagent of LRRTM1 albumen can be quantified.
6. instrument according to claim 5, it is characterised in that the reagent that can quantify LRRTM1 gene mRNAs includes
The primer of the specific amplified LRRTM1 genes used in real-time quantitative PCR, the primer sequence such as SEQ ID NO.1 and SEQ ID
Shown in NO.2；The reagent that LRRTM1 albumen can be quantified includes the antibody of specific binding LRRTM1 albumen.
7. the instrument according to claim 5 or 6, it is characterised in that the instrument includes kit, chip, test paper, high pass
Measure microarray dataset.
8. a kind of medicine for being used to treat gastric gland metastasis of cancer, it is characterised in that the medicine includes LRRTM1 activator.
9. medicine according to claim 8, it is characterised in that the activator includes to promote LRRTM1 or being related to
The expression of the material of LRRTM1 upstreams or downstream pathway or the activator of activity.
10. application of the activator in the medicine for preparing treatment gastric gland metastasis of cancer described in claim 8 or 9.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2007050798A2 (en) * | 2005-10-26 | 2007-05-03 | Five Prime Therapeutics, Inc. | Lrrtm1 compositions and methods of their use for the diagnosis and treatment of cancer |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007050798A2 (en) * | 2005-10-26 | 2007-05-03 | Five Prime Therapeutics, Inc. | Lrrtm1 compositions and methods of their use for the diagnosis and treatment of cancer |
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| Title |
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| HEEJEI YOON 等: "Identification of genes concordantly expressed with Atoh1 during inner ear development", 《ANAT CELL BIOL》 * |
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