CN106990245A - Detect application of the reagent of PITX1 expression quantity in stomach cancer prognosis evaluation reagent kit is prepared - Google Patents
Detect application of the reagent of PITX1 expression quantity in stomach cancer prognosis evaluation reagent kit is prepared Download PDFInfo
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- CN106990245A CN106990245A CN201710216328.0A CN201710216328A CN106990245A CN 106990245 A CN106990245 A CN 106990245A CN 201710216328 A CN201710216328 A CN 201710216328A CN 106990245 A CN106990245 A CN 106990245A
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- G01N33/57446—Specifically defined cancers of stomach or intestine
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Abstract
The present invention relates to application of the reagent of detection PITX1 expression quantity in stomach cancer prognosis evaluation reagent kit is prepared, belong to biological technical field, more particularly to area of medical diagnostics.Application of the reagent of detection PITX1 expression quantity in stomach cancer prognosis evaluation reagent kit is prepared is we disclosed in the present invention.It can be marked by assessing stomach cancer prognosis prediction and chemotherapy of gastric cancer resistance, and the expression of PITX1 genes is detected using SABC chemical technology, so as to obtain the situation of prediction stomach cancer prognosis and chemotherapy resistance, and the incorporation way of PITX1 genes is simple, whole forecast assessment is simple and easy to do, and feasibility is high.
Description
Technical field
The present invention relates to application of the reagent of detection PITX1 expression quantity in stomach cancer prognosis evaluation reagent kit is prepared, belong to
Biological technical field, more particularly to area of medical diagnostics.
Background technology
Stomach cancer is the disease for seriously endangering our people's health, up to the present there is no preferable targeted drug, causes to suffer from
Person's therapeutic effect is poor, prognosis mala.In addition, stomach cancer is a heterogeneity very high disease, the cause of disease of different patients is different, causes
Difference in diagnosis and treatment.Especially different hereditary basis, are likely to result in the curative effect difference of Chemotherapy in Patients medication.Current state
It is inside and outside still without can effectively to this chemotherapy medication curative effect difference carry out Index for diagnosis technological means.
Set up effective Postoperative determination predicting marker, excavation chemotherapy resistance correlation molecule postoperative for confirming which patient
Be likely to occur relapse and metastasis and the treatment of postoperative chemicotherapy need to be carried out in time, which patient can produce resistance for chemotherapy, it is necessary to
Select individualized treatment（Such as molecular targeted therapy）It is significant.
The content of the invention
It is an object of the invention to provide a kind of reliable prognosis evaluation reagent kit for stomach cancer, so as to monitor stomach cancer
The prognosis situation of patient, can go to a doctor in time；Prognosis evaluation result specified individual therapeutic scheme is utilized simultaneously, rationally should
With chemotherapeutics, it is to avoid invalid chemotherapy, effective cure rate is improved.
In order to realize this goal of the invention, the reagent for we disclosing detection PITX1 expression quantity is commented in preparation stomach cancer prognosis
Estimate the application in kit.
The therapeutic effect of the chemotherapeutics such as the expression of stomach cancer cell PITX1 genes and 5-FU, cis-platinum is relevant,
Curative effect can be improved by detecting that the expression of patient's gene instructs clinical chemotherapy of gastric cancer medication.Accordingly, it is proposed that
PITX1 genetic tests can apply to stomach cancer prognosis judgement and be assessed with chemotherapy resistance.So as to the stomach cancer prognosis that calculates to a nicety,
And can the prognosis based on prediction effectively set up optimal individual treatment scheme, it is dead to substantially reduce as caused by stomach cancer
Die.
Further, the reagent for detecting PITX1 expression quantity is we disclosed（Including but not limited to）It is selected from：It is right
PITX1, which has, detects specific probe, genetic chip or PCR primer.
More specifically, we disclose in the present invention and PITX1 is detected using ImmunohistochemistryMethods Methods.
The detection reagent that we are used in being detected using ImmunohistochemistryMethods Methods is mainly by PITX1 polyclonal antibodies, HRP
Secondary antibody and groupization kit DAB developers is marked to constitute.
Wherein polyclonal antibody specifically refers to 1:200 (Abcam, rabbit source property, ab72640), HRP mark secondary antibody be
Refer to DAKO, K5007 instant, groupization kit DAB developers refer to DAKO, K5007,1:100.
Include the reagent that PITX1 expression quantity is detected in detection biological sample, the biological sample for the detection in kit
Product be formalin fix and/or FFPE stomach organization.
Described detection kit, the Index for diagnosis of stomach cancer is used for by following steps：（a）In detection biological sample
PITX1 expression quantity,（b）Basis（a）In the PITX1 expression quantity that detects result is divided into low expression（Or not express）With
Two kinds of height expression,（1）PITX1 not/low expression patient：5-FU chemotherapy is aided with PITX1 gene therapies simultaneously（PITX1 introducing is simultaneously
Stomach normal epithelium cell toxicity will not be induced）, while because of its poor prognosis, close follow-up patient, corresponding tumour being checked in time
The expression of indication and PDCD5, the dose time of Reasonable adjustment chemotherapy；（2）PITX1 height expression patients：Though prognosis less/it is low
Express patient good, but it is to the easy resistances of 5-FU, therefore once find that control effect is not obvious, and other chemotherapeutics should be changed in time.
Low expression mentioned here refers to negative, false positive and weakly positive, and height expression refers to positive and strong positive.
The action effect of the chemotherapeutics such as expression and 5-FU, cis-platinum due to stomach cancer cell PITX1 genes has
Close, curative effect can be improved by detecting that the expression of patient's gene instructs the clinical chemotherapy regimen for formulating individuation.
After technical scheme disclosed by the invention, can by stomach cancer prognosis prediction and chemotherapy of gastric cancer resistance are assessed into
Line flag, and using the expression of SABC chemical technology detection PITX1 genes, so as to obtain prediction stomach cancer prognosis and change
The situation of resistance is treated, and the incorporation way of PITX1 genes is simple, whole forecast assessment is simple and easy to do, and feasibility is high.
Brief description of the drawings
Fig. 1 is Kaplan-Meier（KM）Analyze PITX1 low expressions and high expression group and the correlation of prognosis, result figure.
Embodiment
In order to be better understood from the present invention, below we the present invention is carried out with accompanying drawing in conjunction with specific embodiments it is further
Elaboration, it should be noted that the present invention implementation be not limited only to this.
Agents useful for same and raw material of the present invention are commercially available or can be prepared according to literature method.In the embodiment of the present invention
The test method of unreceipted actual conditions is according to normal condition, or according to the condition proposed by manufacturer.
The method for immunohistochemical detection of the PITX1 expression quantity of embodiment 1
1. the stomach cancer sample of surgery excision, neutral formalin is cut into the tissue block that thickness is 2-3 mm, paraffin bag after fixing
Bury, cut into slices, thickness is 3-4 mm；
2. roasting piece：65 DEG C, 1 ~ 2 hour；
3. section dewaxes to water：Section is inserted first 15 min are handled in xylene solution I, insert diformazan after taking-up again
Benzole soln II, and 15 min are stopped, section taking-up is then placed into 5 min in absolute ethyl alcohol I, anhydrous second is inserted in taking-up again
Alcohol II is stopped after 5 min, and taking-up is placed into 85% min of Ethanol Treatment 5, is then taken out and is inserted the min of 75% ethanol 5, finally will
Section taking-up, which is put into distilled water, cleans 5 min；
4. antigen retrieval：Histotomy is placed in the reparation box for the EDTA antigen retrieval buffer solutions for filling with pH 9.0 in micro-wave oven
Interior carry out antigen retrieval, should prevent from during this after buffer solution excessive vaporization, natural cooling slide being placed in pH 7.4 PBS
Washing 3 times is rocked on decolorization swinging table, every time 5 min；
5. endogenous peroxydase is blocked：Section is put into 3% hydrogenperoxide steam generator, and room temperature lucifuge is incubated 20 min, by slide
It is placed in pH 7.4 PBS and washing 3 times is rocked on decolorization swinging table, every time 5 min；
6. serum is closed：Section is drawn a circle after slightly drying with groupization pen around tissue（Prevent antibody from flowing away）, mountain is added dropwise in circle
Sheep blood serum, makes its uniform fold tissue, and room temperature closes 30 min；Here lowlenthal serum is according to 1 with PBS:20（Blood of goats
Clearly:PBS）Volume ratio dilution after lowlenthal serum；
7. primary antibody is incubated：Gently get rid of confining liquid, outer liquid wiped clean will be enclosed with toilet paper, then in circle dropwise addition according to
1:The 200 primary antibody PBS solutions configured, section lies against 4 °C of overnight incubations in wet box；
8. secondary antibody is incubated：Slide is placed in pH 7.4 PBS rocks washing 3 times, each 5min on decolorization swinging table.Section with
Toilet paper will enclose outer liquid wiped clean, then enclose the secondary antibody of kind corresponding to primary antibody in interior dropwise addition group kit（HRP is marked
Note, 1:2000）Tissue is covered, is incubated at room temperature 1 hour.
9. DAB develops the color：Slide is placed in pH 7.4 PBS rocks washing 3 times on decolorization swinging table, every time 5 min.Cut
Piece will enclose outer liquid wiped clean with toilet paper, then be controlled under the interior DAB nitrite ions that Fresh is added dropwise of circle, microscope aobvious
Color time, the positive is brown color, and running water rinses section color development stopping.
10. redye nucleus：Harris haematoxylins redye 3 min or so, originally wash, 1% hydrochloride alcohol differentiation number
Second, running water is rinsed, and ammoniacal liquor returns indigo plant, and flowing water is rinsed.
11. it is dehydrated mounting：Section is put into 75% ethanol first and handles 6 min, is taken out, and be put into 85% ethanol
After 6 min of middle processing, it is put into absolute ethyl alcohol and is handled twice after taking-up, every time 6 min, section is then removed and placed in two again
Handled in toluene twice, every time 5 min, to be dehydrated it is transparent after, section is taken out from dimethylbenzene and slightly dried, neutral gum mounting
Micro- Microscopic observation staining conditions and scored afterwards；
12. standards of grading：（1）Pigmented cells number：The visual field of multiplication factor 200 selects 5, averages.<5%---0 points, 5 ~
25%---1 points, 26 ~ 50%---2 points, 51 ~ 75%---3 points,>75%---4 points；（2）：Tinctorial strength：It is colourless --- it is 0 point, yellowish
Color --- 1 point, brown color --- 2 points, sepia --- 3 points.Both fractional multiplications, total is positive grade, specific as follows：It is cloudy
Property（-）--- 0 point, weakly positive（+）--- it is 1 ~ 4 point, positive（++）--- 5 ~ 8 points, strong positive（+++）--- 9 ~ 12 points.
The PITX1 expression quantity of embodiment 2 and the relation of stomach cancer prognosis
By the way of disclosed in the embodiment 1, the PITX1 of 174 patients with gastric cancer expression is detected, wherein cloudy
Property, false positive and weakly positive be low expression group, positive and strong positive is high expression group, according to Kaplan-Meier（KM）Analysis
PITX1 low expressions and high expression group and the correlation of prognosis, as a result such as Fig. 1.
As a result show, PITX1 has obvious correlation with prognosis, i.e. PITX1 expression is stronger, and patient's prognosis can be better,
Conversely, PITX1 expression is weaker, then participant is poorer.
Claims (5)
1. detect application of the reagent of PITX1 expression quantity in stomach cancer prognosis evaluation reagent kit is prepared.
2. the reagent of detection PITX1 expression quantity according to claim 1 answering in stomach cancer prognosis evaluation reagent kit is prepared
With, it is characterized in that, for detecting that the reagent of PITX1 expression quantity is selected from：Have to PITX1 and detect specific probe, gene core
Piece or PCR primer.
3. the reagent of detection PITX1 expression quantity according to claim 1 answering in stomach cancer prognosis evaluation reagent kit is prepared
With, it is characterized in that, PITX1 is detected using ImmunohistochemistryMethods Methods.
4. the reagent of detection PITX1 expression quantity according to claim 3 answering in stomach cancer prognosis evaluation reagent kit is prepared
With, it is characterized in that, detection reagent mainly marks secondary antibody and groupization kit DAB developers by PITX1 polyclonal antibodies, HRP
Composition.
5. the reagent of the detection PITX1 expression quantity according to claim 3 or 4 is in stomach cancer prognosis evaluation reagent kit is prepared
Using, it is characterized in that, the reagent that PITX1 expression quantity is detected in detection biological sample, described biological sample are included in kit
Product be formalin fix and/or FFPE stomach organization.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109709333A (en) * | 2018-08-01 | 2019-05-03 | 东南大学 | Application of the tri-methylated amount detection reagent of H4K20, H3K9 and H3K36 in cancer of the esophagus prognosis evaluation |
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| WO2007003397A2 (en) * | 2005-07-01 | 2007-01-11 | Epigenomics Ag | Method and nucleic acids for the improved treatment of cancers |
| US20090148848A1 (en) * | 1999-09-15 | 2009-06-11 | The Johns Hopkins University School Of Medicine | PITX2 Polynucleotide, Polypeptide and Methods of Use Therefor |
| CN105829547A (en) * | 2013-12-02 | 2016-08-03 | 昂科梅德制药有限公司 | Identification of predictive biomarkers associated with Wnt pathway inhibitors |
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| US20090148848A1 (en) * | 1999-09-15 | 2009-06-11 | The Johns Hopkins University School Of Medicine | PITX2 Polynucleotide, Polypeptide and Methods of Use Therefor |
| JP2006094733A (en) * | 2004-09-28 | 2006-04-13 | As One Corp | Method for detecting cancer |
| WO2007003397A2 (en) * | 2005-07-01 | 2007-01-11 | Epigenomics Ag | Method and nucleic acids for the improved treatment of cancers |
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Cited By (1)
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| CN109709333A (en) * | 2018-08-01 | 2019-05-03 | 东南大学 | Application of the tri-methylated amount detection reagent of H4K20, H3K9 and H3K36 in cancer of the esophagus prognosis evaluation |
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