CN107907685A - Application of the combination of DNAJB6, Hsp70 and Hsp90 α in II phase colon cancer Index for diagnosis - Google Patents
Application of the combination of DNAJB6, Hsp70 and Hsp90 α in II phase colon cancer Index for diagnosis Download PDFInfo
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Abstract
The invention belongs to molecular biology, clinical detection technique field, combined more particularly to the protein marker comprising DNAJB6, Hsp70 and Hsp90 α, and the ligand combination thing that can be specifically bound with it, and the application of protein marker combination or ligand combination in II phase colon cancer Index for diagnosis kit.
Description
Technical field
The invention belongs to molecular biology, clinical detection technique field, and in particular to by DNAJB6, Hsp70 and Hsp90 α
The marker combination of composition, and marker combination are preparing II phase colon cancer Index for diagnosis and prediction relapse and metastasis risk
Application in marker kit.
Background technology
Colorectal cancer is common alimentary system malignant tumour, and the whole world there are about 1,300,000 new cases and is diagnosed every year.
European and American developed countries, colorectal cancer are the malignant tumours that morbidity and mortality occupy front three.In recent years, China's colorectal cancer
Incidence in ascendant trend year by year.The update that whole nation treatment and prevention of tumour office of the Ministry of Public Health provides shows that colorectal cancer is divided
China's Cancer Mortality and the 3rd of the death rate and the 5th are not occupied, its incidence is in urban area up to second
Position.
The treatment of colon cancer at present is the complex treatment based on operation.Compared with surgery alone, to III phase colorectal cancer
Patient, which carries out adjuvant chemotherapy of patients, can reduce by 30%~40% recurrence rate and the death rate, and overall 5 years survival rates can be corresponding
Improve more than 10%.For II phase colorectal cancer patients, individually 5 years survival rates total after operation are between 70%-80%.
Although most of II phase colorectal cancer patients prognosis is relatively preferable, but still has the patient of about 25%-30% is postoperative can go out
Now recurrence is simultaneously therefore dead.In addition, clinical common First-line chemotherapy scheme such as FOLFOX, FOLFIRI or CAPeOX, not only give
Patient brings measurable toxicity, and such as leucocyte, blood platelet reduce, and often with can not accurately measure and substantially
Reduce the symptom of patients ' life quality, such as weak, anorexia and pharmacological dependence.
Although neoplasm staging, differentiation degree, lymph node detects number, state is repaired in mispairing, whether chemotherapy is tied for the II phases
Patients with bowel cancer Index for diagnosis has suggesting effect, but also there are larger dispute.And molecular changes are colon cancer occurrence and development
This quality factor, it is possible to more accurate judging prognosis, but can be used for Accurate Prediction II phase colon cancers due to there is no so far
The molecular marker of patient's prognosis, in the result of study reported, II phase colorectal cancer patients are improved without knurl by auxiliary treatment
Survival rate or the effect of overall survival still have dispute.
Therefore, there is an urgent need in the art to develop to can be used for II phase of Accurate Prediction colon cancer risk of recurrence or accurate judgement to suffer from
Molecular marker or the molecular marker combination of person's prognosis, suffer from so as to identify the II phases colon cancer that can benefit from auxiliary treatment
Person, formulates rational individualized treatment scheme for it, improves this some patientss survival rate to the full extent so as to reach and change
It is apt to the purpose of its life quality.
The content of the invention
In the present invention, inventor screens from substantial amounts of cancer markers albumen by unremitting effort and obtains diving
In the protein marker combination for II phase colon cancer Index for diagnosis.The protein marker combination includes following three kinds of albumen,
DNAJB6, Hsp70 and Hsp90 α.Surprisingly, it was found that can be efficiently used for colon cancer special for marker combination
It is II phase colon cancer Index for diagnosis, such as the judgement of life cycle or risk of recurrence.Thus provide following inventions：
One aspect of the present invention is related to (1) or (2) chosen from the followings and is particularly II phase colon for colon cancer in preparation
The diagnosis of cancer, recurring risk assessment, Index for diagnosis or auxiliary treatment medicine in purposes,
(1) combination of DNAJB6, Hsp70 and Hsp90 α；
(2) reagent of DNAJB6, Hsp70 and Hsp90 α are detected.
In one embodiment of the invention, the medicine or reagent are the antibody of DNAJB6, Hsp70 and Hsp90 α,
Or include the pharmaceutical composition of the antibody；Preferably, the antibody is monoclonal antibody.
In one embodiment of the invention, the antibody is also associated with detectable label, such as radioactivity is same
Position element, fluorescent material, luminescent substance, coloring matter or enzyme.
Another aspect of the present invention is related to (1) or (2) chosen from the followings and is particularly II phase colon cancer in preparation colon cancer
Diagnosis, recurring risk assessment, Index for diagnosis or auxiliary treatment medicine in purposes,
(1) combination of the nucleic acid of coding DNA JB6, Hsp70 and Hsp90 α；
(2) reagent of the nucleic acid of coding DNA JB6, Hsp70 and Hsp90 α is detected.
In one embodiment of the invention, the medicine or reagent for specific detection coding DNA JB6, Hsp70 and
The primer of the nucleotide sequence of Hsp90 α either probe or the pharmaceutical composition comprising the primer or probe.
In one embodiment of the invention, the probe is also associated with detectable label, such as fluorophor；
It is preferably selected from least one of cy3, cy5, Texas Red, 6-FAMTM, AF532, AF647 and AF688.
Another aspect of the invention is related to a kind of protein marker combination, and it includes DNAJB6, Hsp70 and Hsp90 α.
The invention further relates to a kind of detection agent, and it includes the antibody of DNAJB6, Hsp70 and Hsp90 α；Preferably, it is described anti-
Body is monoclonal antibody；Preferably, the antibody is also associated with detectable label, such as radio isotope, fluorescence
Matter, luminescent substance, coloring matter or enzyme.
The invention further relates to a kind of detection agent, and it includes the nucleic acid of specific detection coding DNA JB6, Hsp70 and Hsp90 α
The primer or probe of sequence；The probe is also associated with detectable label, such as fluorophor；Be preferably selected from cy3,
At least one of cy5, Texas Red, 6-FAMTM, AF532, AF647 and AF688.
The invention further relates to a kind of kit, and it includes the detection agent described in claim 8 or 9.
In one embodiment of the invention, detection agent or the targeted sample of kit are II phase colorectal cancer patients
Tissue samples.
The invention further relates to the side that a kind of diagnosis, recurring risk assessment or Index for diagnosis colon cancer are particularly II phase colon cancer
Method, including the detection whether all positive steps of DNAJB6, Hsp70 and Hsp90 α, such as pass through immunohistochemistry (IHC)
Method be detected.In one embodiment of the invention, targeted sample is the tissue sample of II phase colorectal cancer patients
This.When the positive is presented in the antigen-antibody reaction of three kinds of markers of the present invention, judge to survey II phase colon cancer trouble
Person's prognosis is poor.
The method of detection protein marker is directed to use with its specific antibody and protein markers interaction of molecules simultaneously
It is detected.Such as can as described herein use for protein marker immunohistochemical assay (IHC) reagent (wherein
Antibody comprising the albumen).Antibody can be prepared using standard technique well known to those skilled in the art, or is used commercially
Antibody.Polyclonal antibody, but preferred monoclonal antibody can be used.
The method of immunoassays can be used quantitatively to detect the presence of protein marker.The immunoassays generally include by
Biological sample is incubated together with antibody, and detects binding antibody by known technology IHC.
In embodiments of the invention, it is as follows that Sample Method, IHC methods and positive criterion are prepared：
(1) sample is prepared：
Stayed overnight fixed in the bench technique tissue input neutral formalin fixer within 30min, flowing water rinses, warp
Dehydration, transparent and waxdip are crossed, prepares paraffin embedded tissues.It is judged as the case of II phase colon cancers with histopathology.Each case choosing
3 typical cancerous tissue points and 2 normal cancer beside organism points are taken, micro-array tissue is made and prepares 4 μm of sections.
(2)IHC：The specific detection method of albumen is as follows：
Organization chip front is placed in 65 DEG C of baking boxes windward and toasts 40min, attaches sample more preferable, immerses dimethylbenzene
Wash in cylinder I, II, III, be respectively placed in 10min in 25 DEG C of constant water bath box, be transferred to each 3min in 100%, 85%, 75% ethanol
Gradient aquation, is then transferred in PBS buffer I, II, III, each 5min, is placed in 3% hydrogen peroxide and washes 15min in cylinder, closing
Intrinsic oversxidase.Immerse in the sodium citrate buffer after heating, low fire 20min in micro-wave oven.Immersion PBS buffer I,
In II, III, each 3min.Its scope is drawn along organization chip edge with PAP Pen immunohistochemistry paraffin pens, after dilution
Primary antibody solution or antibody working solution with the tissue array surface that 200 μ L micro sample adding appliances are paved with drawn scope be placed in wet box it
In, 2h or 4 DEG C of refrigerator overnight is incubated in 37 DEG C of constant incubators.Unnecessary primary antibody is removed on paper handkerchief, by organization chip successively
Immerse in PBS buffer I, II, III, each 3min.Add the reagent II in two step method immunologic combined detection reagent kit, it is ensured that paving
Tissue array surface in full drawn scope is placed among wet box, and 15min is incubated in 37 DEG C of constant incubators.Get in paper handkerchief
Except Excess reagents, organization chip is immersed in PBS buffer I, II, III successively, each 3min.Two step method immunohistochemistry is added to examine
Reagent III (Polyperoxidase-anti-mouse/rabbit IgG) in test agent box, it is ensured that be paved with drawn scope
Tissue array surface is placed among wet box, and 30min is incubated in 37 DEG C of constant incubators.Excess reagents are removed on paper handkerchief, will
Organization chip immerses in PBS buffer I, II, III successively, each 3min.DAB kits are taken to take appropriate reagent, by 1mL DAB
Dilution adds the ratio of 50 μ LDAB concentrates to mix；The DAB solution after dilution is paved with drawn model with 200 μ L micro sample adding appliances
Tissue array surface in enclosing is developed the color, and same chip keeps developing time consistent；It is in Microscopic observation color developing effect until same
Formed between one chip internal and different chip and remove unnecessary DAB solution on paper handkerchief after more significant contrast and be placed in distilled water
In；Developing time is 60s, developing time difference between different antibodies.Haematoxylin dye liquor is fast with 1000 μ L micro sample adding appliances
Speed is paved with the tissue array surface in drawn scope, is washed away at once with slow flowing water after 10s；Chip after dyeing is placed in
In 1% ammonium hydroxide.Organization chip is immersed to each 3min serial dehydrations in 75%, 85%, 100% ethanol successively.Dimethylbenzene is immersed to wash
In cylinder, 10min, sloughs the person's handwriting that PAP Pen immunohistochemistry paraffin strokes go out, organization chip is placed in ventilation and is dried, with fast
Dry mountant mounting.
(3) criterion of positive or negative：
The independent positive criterion of DNAJB6, Hsp70 and Hsp90 α albumen：
The cell of the immunohistochemical reaction coloring of 20 high power fields is counted, staining power integration is：Dye-free 1 divides, weak
2 points of dyeing, moderate stain 3 is divided, strong 4 points of dyeing；Stained area integrates：Color range≤10% as 0 point,>10%-25%
For 1 point,>25%-50% is 2 points,>50%-75% is 3 points,>75% is 4 points.Judge if both integrated product >=6 point
For for the positive, less than 6 points then for feminine gender.
Marker combination is positive, II phase colon cancer poor prognosis.
When tri- protein expression testing results of DNAJB6, Hsp70 and Hsp90 α are all positive, judge to indicate in the sample
Thing combine detection result is the positive；The positive or only any two albumen are expressed as when having no or only one in three albumen
When being expressed as the positive, judge that marker combine detection result is feminine gender in the sample.
Here is the explanation to the part term involved in the present invention.
DNAJB6 belongs to Hsp40 families a member, can be combined with anti-apoptotic chaperone Hsp70, activate Hsp70's
Atpase activity, rise is expressed in colorectal cancer, can promote the invasion and attack of cell.In one embodiment of the invention,
The amino acid sequence of DNAJB6 refers to GenBank accession number NP_490647.1, its nucleic acid sequence encoding refers to NM_
058246.3。
Hsp70 is a kind of chaperone of the induced expression under stressed condition, intracellular nascent protein can be helped to fold, egg
Transport, albumen assembling and degraded, rise is expressed in the malignant tumours such as colorectal cancer, lung cancer, stomach cancer, can promote to swell in leucocyte
Knurl is grown.In one embodiment of the invention, the amino acid sequence of Hsp70 refers to GenBank accession number NP_
005336.3, its nucleic acid sequence encoding refers to NM_005345.5.
Hsp90 α can be combined with the auxiliary molecular chaperones of some intracellular, formed a variety of multiprotein complexes, passed through
Maintain the stabilization and activity of its receptor protein, a plurality of letter related with cell Proliferation, differentiation, survival and apoptosis in regulating cell
Number Signal Transduction Pathways.The high expression in colorectal cancer, lung cancer, liver cancer and breast cancer of Hsp90 α albumen, and it is related to patient's prognosis.
In one embodiment of the invention, the amino acid sequence of Hsp90 α refers to GenBank accession number NP_001017963.2, its
Nucleic acid sequence encoding refers to NM_001017963.2.
In the present invention, if not otherwise specified, the marker combination, refers to that DNAJB6, Hsp70 and Hsp90 α are formed
Marker combination.
I phase colon cancers：Tumour cell infringement colonic mucosa lower floor and muscularis propria, and the colon without regional lymph node metastasis
Cancer
II phase colon cancer：Tumour cell penetrates colonic muscularis propria and reaches subserosa, or invades the knot without peritonaeum covering
Tissue by intestines, or peritonaeum viscerale or other organs or structure are directly invaded or are adhered to thoroughly, the colon of no regional lymph node metastasis
Cancer.
III phase colon cancers：Tumour cell invades colonic mucosa lower floor and muscularis propria, or penetrates colonic muscularis propria arrival
Subserosa, or tissue by the colon without peritonaeum covering is invaded, or peritonaeum viscerale or directly invade or be adhered to other organs thoroughly
Or structure, and the colon cancer of domain of the existence lymphatic metastasis.
In the present invention, term " prognosis " has the implication that those skilled in the art know.In one embodiment of the present invention
In case, prognosis referred to the disease-free survival time and/or without the relapse and metastasis time.Wherein, if object is a colony (2 or 2
Above patient), prognosis refers to middle position disease-free survival time and/or middle position without the relapse and metastasis time.
In one embodiment of the invention, the disease-free survival time or middle position disease-free survival time are 45 months.
In one embodiment of the invention, the no relapse and metastasis time or middle position are 60 months without the relapse and metastasis time.At this
In one embodiment of invention, the disease-free survival time or middle position disease-free survival time are 72 months.The one of the present invention
In a embodiment, the no relapse and metastasis time or middle position are 73 months without the relapse and metastasis time.
When the marker of DNAJB6, Hsp70 and Hsp90 α combine positive, the disease-free survival time of II phase colorectal cancer patients
Or the middle position disease-free survival time is 45 months, and/or, no relapse and metastasis time or middle position are 60 months without the relapse and metastasis time.
When the marker of DNAJB6, Hsp70 and Hsp90 α combine positive, disease-free survival time of II phase colorectal cancer patients or middle position without
Sick life span is 72 months, and/or, no relapse and metastasis time or middle position are 73 months without the relapse and metastasis time.
Advantageous effect of the invention
The present invention utilizes the expression feelings of DNAJB6, Hsp70 and Hsp90 α albumen in specific antibody joint-detection sample
Condition, can be directed to II phase colorectal cancer patients (personal or colony) and carry out accurate Index for diagnosis, its accuracy is significantly high
In the testing result using single protein marker.Meanwhile it is II phase colon cancer that testing result of the present invention, which contributes to clinician,
Patient formulates more rational therapeutic scheme, including determines the frequency of follow-up, selects suitable therapeutic modality and medicine, because
And there is potential using value in terms of improving the therapeutic effect of patient and improving the survival rate of patient.
Brief description of the drawings
Fig. 1：Table of DNAJB6, Hsp70 and Hsp90 α albumen in II phases colon cancer and Carcinoma side normal tissue (Normal)
Up to situation (example).
Fig. 2A：DNAJB6 expresses the relation always survived with II phase colorectal cancer patients.It is directed to II phase colon cancer wax stone group
Sample is knitted, ordinate is II phase colorectal cancer patients survival rate.
Fig. 2 B：Hsp70 expresses the relation always survived with II phase colorectal cancer patients.It is directed to II phase colon cancer wax stone tissue
Sample, ordinate are II phase colorectal cancer patients survival rate.
Fig. 2 C：The relation that Hsp90 alpha expressions and II phase colorectal cancer patients are always survived.It is directed to II phase colon cancer wax stone group
Sample is knitted, ordinate is II phase colorectal cancer patients survival rate.
Fig. 2 D：The relation that marker combinational expression and II phase colorectal cancer patients are always survived.It is directed to II phase colon cancer wax
Block tissue samples, ordinate are II phase colorectal cancer patients survival rate.
Fig. 3 A：DNAJB6 expresses the relation with II phase colorectal cancer patients relapse and metastasis.It is directed to II phase colon cancer wax stone
Tissue samples, ordinate are II phase colorectal cancer patients recurrence and metastatic rate.
Fig. 3 B：Hsp70 expresses the relation with II phase colorectal cancer patients relapse and metastasis.It is directed to II phase colon cancer wax stone group
Sample is knitted, ordinate is II phase colorectal cancer patients recurrence and metastatic rate.
Fig. 3 C：The relation of Hsp90 alpha expressions and II phase colorectal cancer patients relapse and metastasis, is directed to II phase colon cancer wax stone
Tissue samples, ordinate are II phase colorectal cancer patients recurrence and metastatic rate.
Fig. 3 D：The relation of marker combinational expression and II phase colorectal cancer patients relapse and metastasis.It is directed to II phase colon cancer
Wax stone tissue samples, ordinate are II phase colorectal cancer patients recurrence and metastatic rate.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific
Condition person, the condition suggested according to normal condition or manufacturer carry out.Reagents or instruments used without specified manufacturer, is
Can be with conventional products that are commercially available.
In the present invention,
Antibody purchase and the test agent for detecting DNAJB6 are customized in Abcam (Britain) company, article No. ab198995；
The antibody that detection Hsp70 and Hsp90 α are used is purchased from Proteintech Group, Inc (China) company, article No. point
Wei not 10995-1-AP and 60318-1-Ig；
Immunohistochemistry secondary antibody kit is purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge, article No. PV-9000.
In the present invention, IHC methods and positive criterion are as follows：
(1)IHC：The specific detection method of each albumen is as follows：
Organization chip front is placed in 65 DEG C of baking boxes windward and toasts 40min, attaches sample more preferable, immerses dimethylbenzene
Wash in cylinder I, II, III, be respectively placed in 10min in 25 DEG C of constant water bath box, be transferred to each 3min in 100%, 85%, 75% ethanol
Gradient aquation, is then transferred in PBS buffer I, II, III, each 5min, is placed in 3% hydrogen peroxide and washes 15min in cylinder, closing
Intrinsic oversxidase.Immerse in the sodium citrate buffer after heating, low fire 20min in micro-wave oven.Immersion PBS buffer I,
In II, III, each 3min.Its scope is drawn along organization chip edge with PAP Pen immunohistochemistry paraffin pens, after dilution
Primary antibody solution or antibody working solution with the tissue array surface that 200 μ L micro sample adding appliances are paved with drawn scope be placed in wet box it
In, 2h or 4 DEG C of refrigerator overnight is incubated in 37 DEG C of constant incubators.Unnecessary primary antibody is removed on paper handkerchief, by organization chip successively
Immerse in PBS buffer I, II, III, each 3min.Add the reagent II in two step method immunologic combined detection reagent kit, it is ensured that paving
Tissue array surface in full drawn scope is placed among wet box, and 15min is incubated in 37 DEG C of constant incubators.Get in paper handkerchief
Except Excess reagents, organization chip is immersed in PBS buffer I, II, III successively, each 3min.Two step method immunohistochemistry is added to examine
Reagent III in test agent box, it is ensured that the tissue array surface being paved with drawn scope is placed among wet box, 37 DEG C of constant temperature trainings
Support in case and be incubated 30min.Excess reagents are removed on paper handkerchief, organization chip are immersed in PBS buffer I, II, III successively, often
Secondary 3min.DAB kits are taken to take appropriate reagent, the ratio for adding 50 μ LDAB concentrates in 1m LDAB dilutions mixes；With 200 μ L
Micro sample adding appliance develops the color the tissue array surface that the DAB solution after dilution is paved with drawn scope, and same chip is kept
Developing time is consistent；In Microscopic observation color developing effect until forming more significant contrast inside same chip and between different chips
Unnecessary DAB solution is removed on paper handkerchief afterwards to be placed in distilled water；Developing time is 60s, and developing time has been between different antibodies
Difference.Tissue array surface haematoxylin dye liquor being paved with rapidly with 1000 μ L micro sample adding appliances in drawn scope, with slow after 10s
Slow flowing water washes away at once；Chip after dyeing is placed in 1% ammonium hydroxide.Organization chip immerses to 75% successively, 85%,
Each 3min serial dehydrations in 100% ethanol.Immerse dimethylbenzene to wash in cylinder, 10min, sloughs PAP Pen immunohistochemistry paraffin strokes
The person's handwriting gone out, is placed in ventilation by organization chip and dries, with quick-drying mountant mounting.
(2) criterion of positive or negative：Count the cell of the immunohistochemical reaction coloring of 20 high power fields, dyeing
Intensity integrates：Dye-free 1 divides, and weak 2 points of dyeing, moderate stain 3 is divided, strong 4 points of dyeing；Stained area integrates：Color range
≤ 10% is 0 point,>10%-25% is 1 point,>25%-50% is 2 points,>50%-75% is 3 points,>75% is 4 points.If
Both integrated product >=6 point are then the positive, are then feminine gender less than 6 points.
Embodiment 1：Research of the protein marker combinational expression level in II phase colorectal cancer patients Overall survival is judged
(1) the tumor tissues sample for the 102 II phase colorectal cancer patients made a definite diagnosis by pathology detection is directed to, use is above-mentioned
IHC methods detection DNAJB6, Hsp70 and Hsp90 tri- albumen of α expression, and judge positive or negative.Detection knot
The related data of fruit and patient are as shown in Table 1 below.
Table 1：Expressions of DNAJB6, Hsp70 and Hsp90 α in II phase colon cancers
In table 1 above：
P values are marked with italic.
Pathological grading can be carried out to tumour according to colon tumor cell differentiation degree：I grade is to break up, and grade malignancy is low；
II grade is medium, the grade malignancy moderate of differentiation；III grade poor to break up, and grade malignancy is high.The colorectal cancer that 95% glandular tubes of ＞ are formed
For differentiated colorectal cancer, the colorectal cancer that 50%-95% glandular tubes are formed is middle differentiation colorectal cancer, and 0-49% glandular tubes are formed
Colorectal cancer be low differentiation colorectal cancer.
TNM stage：T：Tumor (Topography), represents the scope of primary tumo(u)r；N：Lymph Node, represent region
The presence or absence of lymphatic metastasis and scope；M：Metastasis, represents the presence or absence of DISTANT METASTASES IN.After three capitalizations
The situation of original site, lymphatic metastasis and DISTANT METASTASES IN can be expressed by connecing numeral or lowercase respectively.According to
AJCC is carried out by stages for the 8th edition by stages.
US National synthesis cancer network (NCCN) guide recommends colorectal cancer patients at least to detect 12 lymph nodes, to N0
Phase colorectal cancer patients, detection lymph node number are considered as high risk factor less than 12 persons.Lymph node in operation is cut off, Fu Er
Malin preserves, and send to pathology department and is embedded, HE dyeing.
Colon is divided into left hemicolon and left hemicolon two parts by positioning, and wherein left hemicolon includes sigmoid colon, drop knot
Intestines, transverse colon splenic flexure, left half transverse colon, right hemicolon include the colon ascendens, transverse colon right side.
The IHC testing results of some cases are as shown in Figure 1.It will be seen from figure 1 that DNAJB6, Hsp70 and Hsp90 α tri-
Level of the albumen in II phase colon cancer tissue is higher than Carcinoma side normal tissue.
(2) data in table 1 and the Overall survival data (table 2) of patient are combined, carry out single factor test and multifactor variance
Analysis.
Table 2：The Overall survival data and relapse and metastasis data of patient
The specific method of Kaplan-Meier method single factor test survival analysises：Calculating the time-to-live first exceedes the regular period
Patient live through the probability (i.e. survival probability) in next period again, then survival probability will be multiplied one by one, and be the corresponding period
Survival rate.
The specific method of Analyzed by Cox Model：Purpose is the multifactor survival analysis data of processing, and finding influences existence
The hazards of situation.It is recommended that the variable for including Cox regression models has：1) the statistically significant change of single factor analysis difference
Amount；2) during single factor analysis, do not find differences statistically significant, but clinically think with dependent variable it is in close relations from
Variable.
The COX Regression Analysis Results of Overall survival are shown in Table 3.Overall survival refers to since randomization to drawing for any reason
Play the dead time.
Table 3：The single factor test and multiplicity that II phase colon cancers are always survived
Table 3 shows the pass between DNAJB6, Hsp70 and Hsp90 α protein expressions and II each clinical parameter of phase colon cancer
System.
The results of univariate logistic analysis shows that when Hsp90 α high are expressed, patient's prognosis Overall survival is shorter (P=0.040), and
When the combination of DNAJB6, Hsp70 and Hsp90 α markers is positive, life cycle shorter patient preferably can be distinguished into (P=
0.01, table 3 and Fig. 2A-Fig. 2 D).
Multinomial logistic regression the results show that the positive expression of single albumen is not the independence of II phase colorectal cancer patients
Prognostic factor, but they at the same time positive expression can be as the independent prognostic factor (P for judging II phase colorectal cancer patients Overall survivals
=0.006), its P value less than clinically common T by stages, differentiation degree, mispairing repair the Index for diagnosis standards such as state, chemotherapy.
From table 3 it can be seen that in II phase colon cancer, positive expression and the pathological grading of DNAJB6 are proportionate, and with
Other clinicopathologic features non-correlations；And the expression height of Hsp70 and Hsp90 α is unrelated with clinicopathologic features.
Embodiment 2：Protein marker combines research of the positive expression in II phase colorectal cancer patients relapse and metastasis is judged
With reference to the data in the table 1 in preceding embodiment 1 and the relapse and metastasis data (table 2) of patient, single factor test is carried out
And the multifactor analysis of variance.
The specific method of Kaplan-Meier method single factor test survival analysises：Calculating the time-to-live first exceedes the regular period
Patient live through the probability (i.e. survival probability) in next period again, then survival probability will be multiplied one by one, and be the corresponding period
Survival rate.
The specific method of Analyzed by Cox Model：Purpose is the multifactor survival analysis data of processing, and finding influences existence
The hazards of situation.It is recommended that the variable for including Cox regression models has：1) the statistically significant change of single factor analysis difference
Amount；2) during single factor analysis, do not find differences statistically significant, but clinically think with dependent variable it is in close relations from
Variable.
The COX Regression Analysis Results of relapse and metastasis are shown in Table 4.
Table 4：The single factor test and multiplicity of II phase colon cancer relapse and metastasis
The results of univariate logistic analysis shows that DNAJB6 high expression instruction Patients on Recurrence transfer times are shorter (P=0.026).And
The expression of Conjoint Analysis DNAJB6, Hsp70 and Hsp90 α markers combination, then preferably can divide into recurrence by patient
Transfer time grows and two groups of short (P<0.001) (Fig. 3 A- Fig. 3 D and table 4), judges relapse and metastasis time length.
Multinomial logistic regression is not the results show that be to judge relapse and metastasis time length when three albumen are expressed alone
Independent prognostic factor, and during conduct marker combination coexpression, can be as the independent prognostic factor (P of colorectal cancer patients<
0.001), its P value less than clinically common T by stages, differentiation degree, mispairing repair the Index for diagnosis standards such as state, chemicotherapy.
Although the embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.Root
According to disclosed all teachings, various modifications and replacement can be carried out to those details, these change in the guarantor of the present invention
Within the scope of shield.The four corner of the present invention is provided by appended claims and its any equivalent.
Claims (10)
- (1) 1. chosen from the followings or (2) prepare be used for colon cancer be particularly the diagnosis of II phase colon cancer, recurring risk assessment, Purposes in Index for diagnosis or the medicine of auxiliary treatment,(1) combination of DNAJB6, Hsp70 and Hsp90 α；(2) reagent of DNAJB6, Hsp70 and Hsp90 α are detected.
- 2. purposes according to claim 1, wherein, the medicine or reagent are the anti-of DNAJB6, Hsp70 and Hsp90 α Body, or include the pharmaceutical composition of the antibody；Preferably, the antibody is monoclonal antibody.
- 3. purposes according to claim 2, wherein, the antibody is also associated with detectable label, such as radioactivity Isotope, fluorescent material, luminescent substance, coloring matter or enzyme.
- (1) 4. chosen from the followings or (2) is particularly diagnosis, recurring risk assessment, the prognosis of II phase colon cancer preparing colon cancer Purposes in the medicine of judgement or auxiliary treatment,(1) combination of the nucleic acid of coding DNA JB6, Hsp70 and Hsp90 α；(2) reagent of the nucleic acid of coding DNA JB6, Hsp70 and Hsp90 α is detected.
- 5. purposes according to claim 4, wherein, the medicine or reagent are specific detection coding DNA JB6, Hsp70 With the primer of the nucleotide sequence of Hsp90 α either probe or pharmaceutical composition comprising the primer or probe.
- 6. purposes according to claim 5, wherein, the probe is also associated with detectable label, such as fluorescent base Group；It is preferably selected from least one of cy3, cy5, Texas Red, 6-FAMTM, AF532, AF647 and AF688.
- 7. a kind of protein marker combination, it includes DNAJB6, Hsp70 and Hsp90 α.
- 8. a kind of detection agent, it includes the antibody of DNAJB6, Hsp70 and Hsp90 α；Preferably, the antibody resists for monoclonal Body；Preferably, the antibody is also associated with detectable label, for example, radio isotope, fluorescent material, luminescent substance, Coloring matter or enzyme.
- 9. a kind of detection agent, it includes the nucleotide sequence of specific detection coding DNA JB6, Hsp70 and Hsp90 α primer or Probe；The probe is also associated with detectable label, such as fluorophor；Be preferably selected from cy3, cy5, Texas Red, At least one of 6-FAMTM, AF532, AF647 and AF688.
- 10. a kind of kit, it includes the detection agent described in claim 8 or 9.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110865186A (en) * | 2018-08-28 | 2020-03-06 | 中国医学科学院肿瘤医院 | Application of protein marker or combination thereof in colorectal cancer prognosis |
| CN112067809A (en) * | 2020-08-28 | 2020-12-11 | 中国医学科学院肿瘤医院 | Application of DNAJB6 b-targeted reagent in preparation of drugs for treating tumors |
| CN113025715A (en) * | 2021-03-23 | 2021-06-25 | 中山大学附属第一医院 | Application of HOP in prediction of gastric cancer prognosis |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013152989A3 (en) * | 2012-04-10 | 2014-02-27 | Eth Zurich | Biomarker assay and uses thereof for diagnosis, therapy selection, and prognosis of cancer |
| CN103694332A (en) * | 2009-07-07 | 2014-04-02 | 清华大学 | New tumor marker |
| WO2014062218A1 (en) * | 2012-10-16 | 2014-04-24 | University Of Southern California | Colorectal cancer dna methylation markers |
-
2017
- 2017-11-07 CN CN201711083883.7A patent/CN107907685B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103694332A (en) * | 2009-07-07 | 2014-04-02 | 清华大学 | New tumor marker |
| WO2013152989A3 (en) * | 2012-04-10 | 2014-02-27 | Eth Zurich | Biomarker assay and uses thereof for diagnosis, therapy selection, and prognosis of cancer |
| WO2014062218A1 (en) * | 2012-10-16 | 2014-04-24 | University Of Southern California | Colorectal cancer dna methylation markers |
Non-Patent Citations (4)
| Title |
|---|
| DANIEL R. CIOCCA ET AL.,: "Heat shock proteins in cancer:diagnostic, prognostic, predictive,and treatment implications", 《CELL STRESS & CHAPERONES》 * |
| KARINA BAUER ET AL.,: "High HSP27 and HSP70 expression levels are independent adverse prognostic factors in primary resected colon cancer", 《CELLULAR ONCOLOGY》 * |
| ZHANG ET AL.,: "Overexpression of DNAJB6 promotes colorectal cancer cell invasion through an IQGAP1/ERK-dependent signaling pathway", 《MOLECULAR CARCINOGENESIS》 * |
| 李海军 等: "HSP90α蛋白在人结肠癌中的表达", 《世界华人消化杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110865186A (en) * | 2018-08-28 | 2020-03-06 | 中国医学科学院肿瘤医院 | Application of protein marker or combination thereof in colorectal cancer prognosis |
| CN110865186B (en) * | 2018-08-28 | 2023-07-04 | 中国医学科学院肿瘤医院 | Application of protein marker or combination thereof in colorectal cancer prognosis judgment |
| CN112067809A (en) * | 2020-08-28 | 2020-12-11 | 中国医学科学院肿瘤医院 | Application of DNAJB6 b-targeted reagent in preparation of drugs for treating tumors |
| CN113025715A (en) * | 2021-03-23 | 2021-06-25 | 中山大学附属第一医院 | Application of HOP in prediction of gastric cancer prognosis |
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