CN110488015A - Application of the Chemokines CC XCL14 in prediction colon cancer prognosis - Google Patents
Application of the Chemokines CC XCL14 in prediction colon cancer prognosis Download PDFInfo
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Abstract
The invention discloses application of the Chemokines CC XCL14 in prediction colon cancer prognosis, including detecting application of the reagent of Chemokines CC XCL14 in the tool of preparation prediction colorectal cancer prognosis；A kind of expression quantity of CXCL14 albumen in the detection colorectal carcinoma quantitative by immunological method is more specifically disclosed, with this come the method that carries out the prediction of colon cancer prognosis.The prognosis of this method life span total for III-IV colorectal cancer patients has significant predictive value, so as to targetedly improve undesirable treatment prognosis, improves medical level, improves the quality of living.
Description
Technical field
The present invention relates to the prediction fields of cancer prognosis, specifically, disclose Chemokines CC XCL14 in prediction colon cancer
Application in prognosis.
Background technique
Colorectal cancer is current relatively common a kind of malignant tumour, and not only incidence is higher, and the corresponding death rate also compares
It is higher.At present its whole world disease incidence and lethality is all high occupies the forefront, only neopathy number in 2018 reaches nearly 2,000,000, and
Lethal number nearly 900,000 people.In terms of the situation on current clinical treatment, the disease incidence of the colorectal cancer in city is normally at 2-3
Position, the ratio that 40 years old young man below suffers from colorectal cancer accounts for about the 20% of colorectal cancer total number of persons, and further rises
Trend.
Although the diagnostic techniques of colorectal cancer is horizontal and treatment level is also being continuously improved, its survival rates is also
Wait improve, according to different patients by stages, postoperative 5 years overall survivals are 10%-90% or so, the survival rate of early stage patient compared with
Height, but 5 years survival rates of patient that advanced stage especially shifts only have 10% or so.Therefore the early detection of colorectal cancer, it is early
Phase excision, and in the therapeutic process of the carcinoma of the rectum, surgical operation is particularly important component part, colon Total mesentery excision is early
It is just suggested in nineteen eighty-two, and was constantly developed and optimized in recent years.In surgical procedures, for tumor group
The judgement knitted is extremely crucial factor, and awaiting the honour of your presence sometimes can not judge whether tumour infects health tissues with naked eyes, therefore not cut
Cause to recur except clean, and the expanded resection of blindness sometimes, cause to have cut off unnecessary organ, causes disease life quality
The decline of amount.The tumor marker of currently used colorectal cancer has CEA, CA19-9, CA72-4 and CA242, but these are deposited
It is that the false positive rate of the marker in serum is also higher, or accurate less than the pathological section as goldstandard.
Therefore, finding in colorectal carcinoma, there are the tumor markers of index meaning to have for the prognosis of colorectal cancer
It is significant.
Summary of the invention
In view of the above-mentioned problems in the prior art, the answering in prediction colon cancer prognosis the invention discloses Chemokines CC XCL14
With, CXCL14 is a newcomer of Chemokines CC XC family, earliest from human mammary and renal tissue separation clone and
Come.CXCL14 participates in many biological processes, such as inflammatory and immune response in vivo.
Technical solution of the present invention is described below:
A kind of diagnostic kit for predicting colon cancer prognosis, comprising: obtain sample for carrying out pretreatment to sample to be tested
Reagent, for detection reagent, label or the operation instructions that CXCL14 expressing quantity is detected in sample, the mark
Label or operation instructions indicate the kit for predicting the prognosis of colorectal cancer.The reagent of the quantitative CXCL14 albumen of invention
Its function can be played based on the known method of antibody is used: for example, can wrap ELISA, radioimmunoassay, immuning tissue
Chemical method, western blot etc..
Further, the diagnostic kit of above-mentioned prediction colon cancer prognosis, the sample to be tested are colorectal carcinoma slice.
Further, the diagnostic kit of above-mentioned prediction colon cancer prognosis, the detection reagent include: endogenous peroxidating
Object enzyme blocking agent, animal non-immune serum, anti-CXCL14 polyclonal antibody, the secondary antibody of biotin labeling, streptomycete antibiotin
Albumen-peroxidase, DAB colour reagent, phosphate buffered saline solution.Antibody of the present invention can pass through art technology
Method well known to personnel obtains.For example, showing the anti-of library based on mouse/rabbit hybridoma technology and based on phage antibody
Body screening technique etc..
Further, the diagnostic kit of above-mentioned prediction colon cancer prognosis, it is described for every clinical sample histotomy
The dosage of detection reagent are as follows: endogenous peroxydase blocking agent 50ul, animal non-immune serum 50ul, anti-CXCL14 are polyclonal
Antibody 1ul, the secondary antibody 50ul of biotin labeling, streptavidin peroxidase 50ul, DAB colour reagent
100ul, phosphate buffered saline solution 200ul.Diagnostic kit of the present invention refers to using immunology, microbiology, molecule
The principles such as biology or method preparation, in vitro be used for examining to the diagnosis, detection and epidemiological survey of human diseases etc.
The organic combination of disconnected reagent.
Further, the application method of the diagnostic kit of above-mentioned prediction colon cancer prognosis, comprising the following steps:
(1) serial section is carried out after clinical tissue being carried out paraffin embedding, with a thickness of 2 μm；
(2) piece cut is placed into 2h in 55 DEG C of constant temperature roasters；
(3) slice after above-mentioned drying is subjected to dewaxing treatment；
(4) histotomy after dewaxing treatment is impregnated with PBS solution；
(5) every slice plus 50ul endogenous peroxydase blocking agent, are incubated at room temperature 20 minutes；
(6) PBS rinses or impregnates 3 × 3min；
(7) every histotomy adds the non-immunity animal blood serum of 50ul to close non-specific binding；
(8) serum, every slice plus the anti-CXCL14 polyclonal antibody in first antibody rabbit source diluted, 4 DEG C of constant temperature are dried
Overnight；
(9) PBS rinses or impregnates 3 × 3min；
(10) PBS liquid, every slice plus 50ul biotin labeling secondary antibody are got rid of, is incubated for 30min at room temperature；
(11) PBS rinses or impregnates 3 × 10min；
(12) PBS liquid, every slice plus 50ul streptomycete antibiotin-Peroxidase Solution are got rid of, is incubated at room temperature
25min；
(13) PBS rinses or impregnates 3 × 3min；
(14) PBS liquid, the DAB developing solution of every slice plus 100ul Fresh, microscopically observation, it is seen that yellow are got rid of
Slice is immersed immediately after dye and stops to dye in distilled water；
(15) haematoxylin is carried out to redye and mounting；
(16) mean absorbance values are calculated with PaintShop, and for statistical analysis.
Further, the diagnostic kit of above-mentioned prediction colon cancer prognosis, also comprising organized in the diagnostic kit
Microarray.Micro-array tissue (tissue microarray, TMA), is that tens of to thousands of a cells are fitly arranged in one
It opens on glass slide and manufactured histotomy, the detection in conventional reagents box in experimental procedure to single histotomy is evolved
Large batch of detection is carried out in Cheng Yi organization chips, it can high-throughput in a short time, quick, Parallel testing high-volume group
Multiple experimental index in sample are knitted, conventional efficient is greatly improved.
Further, the diagnostic kit of above-mentioned prediction colon cancer prognosis, the micro-array tissue are made of following steps:
(1) mold wax stone is made, size is 45mm × 20mm, and it is 0.1mm that spacing is got on mold wax stone, and diameter is
The aperture of 0.6mm, each aperture are accurately positioned with coordinate；
(2) sample wax stone is dyed according to HE and is observed, and is selected and is kept in mind respectively in HE slice and corresponding Paraffin tissue block subscript
The site of sample tissue is punctured with the stainless pin of internal diameter 0.6mm, and 2~3mm of depth is fixed in the small of mold wax stone
In hole；
(3) mold wax stone is sliced with 4 μ m thicks, is applied on the solid phase carriers such as glass slide or nylon membrane, is just made
Micro-array tissue.
Further, application of the diagnostic kit of above-mentioned prediction colon cancer prognosis in terms of colonic tissue pathological examination.
Further, the diagnostic kit of above-mentioned prediction colon cancer prognosis is when predicting advanced colorectal cancer patients overall survival
Between aspect application.
Further, the diagnostic kit of above-mentioned prediction colon cancer prognosis is in prediction colorectal cancer patients recurrence and metastasis after resection
The application of aspect.
The invention has the advantages that:
The prognosis of the detection of Chemokines CC XCL14 life span total for III-IV colorectal cancer patients has significant
Predictive value improves medical level, improves the quality of living to targetedly improve undesirable treatment prognosis；
The detection of Chemokines CC XCL14 has predictive value to the transfer of patient's postoperative recurrence, so as to be potential knot
The relapse and metastasis of the carcinoma of the rectum takes preventive measures；
The detection of Chemokines CC XCL14 to pathology can complete incidence graph also there is predictive value, it is corresponding so as to formulate
Therapeutic scheme.
Detailed description of the invention
Fig. 1 is CXCL14 protein staining intensity percent distribution in colorectal carcinoma tissue and itself cancer beside organism
Figure；
Fig. 2 is the Kaplan-Meier survivorship curve analysis chart for showing colorectal cancer patients；
Fig. 3 is CXCL14 protein staining intensity percentage in colorectal carcinoma tissue and corresponding lymphatic metastasis tumor tissue
Compare distribution map.
Specific embodiment
The technical scheme of the invention is further explained by means of specific implementation.Those skilled in the art should be bright
, the described embodiments are merely helpful in understanding the present invention, should not be regarded as a specific limitation of the invention；Made in following embodiments
Experimental method is conventional method unless otherwise specified；The materials, reagents and the like used in the following examples, such as without special
Illustrate, is commercially available.
Prediction prognosis described herein refers to the process of prediction status of patient or as a result, be not meant to can be with 100% standard
The process or result of exactness prediction status of patient.Prediction prognosis refers to whether a possibility that determining certain processes or result increases,
And be not meant to by with certain processes or result not there is a situation where compared with come determine occur certain processes or result can
It can property.For the present invention, in the present invention in the patient of the horizontal decline of CXCL14 albumen, with the people's phase for not showing this feature
Than more likely observing particular procedure or result.As used herein, term " antibody ", it is intended that be specifically bound to specific anti-
Any antigen-binding molecule or molecular complex former or with specific antigen interaction.As used herein, term " antibody " is gone back
Antigen-binding fragment including entire antibody molecule.
Embodiment 1
Detection Chemokines CC XCL14 is used to predict the application method of the diagnostic kit of colorectal cancer prognosis, including following
Step:
(1) serial section is carried out after clinical tissue being carried out paraffin embedding, with a thickness of 2 μm；
(2) piece cut is placed into 2h in 55 DEG C of constant temperature roasters；
(3) slice after above-mentioned drying is subjected to dewaxing treatment；
(4) histotomy after dewaxing treatment is impregnated with PBS solution；
(5) every slice plus 50ul endogenous peroxydase blocking agent, are incubated at room temperature 20 minutes；
(6) PBS rinses or impregnates 3 × 3min；
(7) every histotomy adds the non-immunity animal blood serum of 50ul to close non-specific binding；
(8) serum, every slice plus the anti-CXCL14 polyclonal antibody in first antibody rabbit source diluted, 4 DEG C of constant temperature are dried
Overnight；
(9) PBS rinses or impregnates 3 × 3min；
(10) PBS liquid, every slice plus 50ul biotin labeling secondary antibody are got rid of, is incubated for 30min at room temperature；
(11) PBS rinses or impregnates 3 × 10min；
(12) PBS liquid, every slice plus 50ul streptomycete antibiotin-Peroxidase Solution are got rid of, is incubated at room temperature
25min；
(13) PBS rinses or impregnates 3 × 3min；
(14) PBS liquid, the DAB developing solution of every slice plus 100ul Fresh, microscopically observation, it is seen that yellow are got rid of
Slice is immersed immediately after dye and stops to dye in distilled water；
(15) haematoxylin is carried out to redye and mounting；
(16) mean absorbance values are calculated with PaintShop Image-Pro, and for statistical analysis.
Embodiment 2
The preparation of micro-array tissue (tissue microarray, TMA), comprising the following steps:
(1) mold wax stone is made, size is 45mm × 20mm, and it is 0.1mm that spacing is got on mold wax stone, and diameter is
The aperture of 0.6mm, each aperture are accurately positioned with coordinate.
(2) sample wax stone is dyed according to HE and is observed, and is selected and is kept in mind respectively in HE slice and corresponding Paraffin tissue block subscript
The site of sample tissue is punctured with the stainless pin of internal diameter 0.6mm, and 2~3mm of depth is fixed in the small of mold wax stone
In hole.
(3) mold wax stone is sliced with 4 μ m thicks, is applied on the solid phase carriers such as glass slide or nylon membrane, is just made
Micro-array tissue can carry out immunohistochemical staining.
Embodiment 3
The expression of Chemokines CC XCL14 albumen and colorectal cancer shift and the related experiment of survival rate
1. micro-array tissue
The preparation of micro-array tissue is carried out referring to embodiment 2.
The micro-array tissue (TMA) for being prepared with 50 Colorectal Carcinomas and 40 cancer beside organisms carries out the immune of CXCL14
Histochemical analysis, to determine the correlation of CXCL14 protein expression level with prognosis.
Prepare another micro-array tissue of 50 colorectal cancer primary lesions and corresponding lymph gland transferring focus
(TMA) immunohistochemical analysis of CXCL14 is carried out, to compare the CXCL14 protein expression difference of primary lesion and metastatic lesion, into
And determine the correlation of CXCL14 protein expression level with metastases.
2. immunohistochemical method
Referring to the method for embodiment 1, step (1) is replaced with into above-mentioned micro-array tissue, is carried out according to step (2)-(15)
Dyeing, is then scanned TMA chip, and analyzed as follows: four grades are divided into according to staining power, 1 point represents yin
Property, 2 points represent weakly positive, 3 points represent in it is positive, 4 points represent strong positive.Chip differentiates by three observer's independent evaluations, divides
Value is 1~2 is considered as CXCL14 low expression, and score value is 3~4 to be considered CXCL14 high expression.
3. experimental result
(1) relationship that the expression of CXCL14 albumen and colorectal cancer occur
CXCL14 albumen in 50 colorectal carcinoma tissues and 40 cancer beside organisms is analyzed using micro-array tissue method
Expression, discovery.It is worth noting that, the Colorectal Carcinoma display of 78% (39/50) is negative or weakly positive
CXCL14 expression, and only having 27.5% (11/40) in cancer beside organism is negative or weakly positive.As shown in Figure 1.This tables of data
Bright, the expression of CXCL14 albumen and the morbidity of colorectal cancer are negatively correlated.We are it may be speculated that CXCL14 albumen is a suppression accordingly
Make tumorigenic albumen.
(2) relationship of CXCL14 protein expression and colorectal cancer clinicopathological parameters
Surgical resection primary colorectal cancer sample 50 are collected, according in December, 2016, UICC (International Contre connection
Alliance) TNM stage of the 8th edition malignant tumour of publication sorts out patient data, and Detailed clinical information is shown in Table 1.It adopts
Sample is divided into CXCL14 low expression group and CXCL14 by the detection for carrying out CXCL14 expression to each sample with the method for embodiment 1
High expression group analyzes the relationship between CXCL14 protein expression and colorectal cancer clinicopathological parameters.The results show that CXCL14
Low expression and lymphatic metastasis (P=0.004) and far-end transfer (P=0.014) are significant related.
Relationship between 1 CXCL14 protein expression of table and colorectal cancer clinicopathological parameters
(3) relationship of CXCL14 protein expression and post operative colo-rectal cancer survival rate
The data that according to hospital above-mentioned 50 colorectal cancer patients are carried out with Follow-up After, carries out life cycle statistics, is made
Life cycle analysis method is Kaplan-Meier survival analysis.The highly expressed patient of CXCL14 (median survival interval 30.5
Month), there is the longer time-to-live than the patient of CXCL14 low expression (median survival interval is 18 months).Log-rank analysis is aobvious
Show, the low expression of CXCL14 is significant related (Log-rank=5.324, P=0.022, Fig. 2) to poor prognosis
(4) CXCL14 protein expression difference in colorectal cancer primary lesion and lymph gland transferring focus
The micro-array tissue for including 50 colorectal cancer primary focus and corresponding lymph gland transferring focus is carried out
The expression analysis of CXCL14 albumen.Data show that the CXCL14 expression of most of metastasis (metastases)s is significantly lower than corresponding
Primary focus (Fig. 3), it was confirmed that above research achievement, the i.e. transfer of the low expression and tumour of CXCL14 albumen and bad pre-
Significant correlation afterwards, and CXCL14 can be used as diagnosis of colorectal carcinoma and predict an index of prognosis.
Certainly, the above description is not a limitation of the present invention, and the present invention is also not limited to the example above, this technology neck
The variations, modifications, additions or substitutions that the technical staff in domain is made within the essential scope of the present invention also should belong to of the invention
Protection scope.
Claims (10)
1. a kind of diagnostic kit for predicting colon cancer prognosis, which is characterized in that the diagnostic kit includes for to be measured
Sample carry out pretreatment obtain sample reagent and for the detection reagent that CXCL14 expressing quantity is detected in sample,
Label or operation instructions；The label or operation instructions indicate the kit for predicting the prognosis of colorectal cancer.
2. a kind of diagnostic kit for predicting colon cancer prognosis according to claim 1, which is characterized in that described to test sample
Product are colorectal carcinoma slice.
3. a kind of diagnostic kit for predicting colon cancer prognosis according to claim 2, which is characterized in that the detection examination
Agent include endogenous peroxydase blocking agent, animal non-immune serum, anti-CXCL14 polyclonal antibody, biotin labeling two
Anti-, streptavidin peroxidase, DAB colour reagent and phosphate buffered saline solution.
4. a kind of diagnostic kit for predicting colon cancer prognosis according to claim 3, which is characterized in that every is faced
Bed sample tissue slice, the dosage of the detection reagent are endogenous peroxydase blocking agent 50ul, animal non-immune serum
50ul, anti-CXCL14 polyclonal antibody 1ul, biotin labeling secondary antibody 50ul, streptavidin peroxidase
50ul, DAB colour reagent 100ul, phosphate buffered saline solution 200ul.
5. a kind of application method of the diagnostic kit of the described in any item prediction colon cancer prognosis of claim 1-4, feature
It is, comprising the following steps:
(1) serial section is carried out after clinical tissue being carried out paraffin embedding, with a thickness of 2 μm；
(2) piece cut is placed into 2h in 55 DEG C of constant temperature roasters；
(3) slice after above-mentioned drying is subjected to dewaxing treatment；
(4) histotomy after dewaxing treatment is impregnated with PBS solution；
(5) every slice plus 50ul endogenous peroxydase blocking agent, are incubated at room temperature 20 minutes；
(6) PBS rinses or impregnates 3 × 3min；
(7) every histotomy adds the non-immunity animal blood serum of 50ul to close non-specific binding；
(8) serum, every slice plus the anti-CXCL14 polyclonal antibody in first antibody rabbit source diluted are dried, 4 DEG C of constant temperature are stayed overnight；
(9) PBS rinses or impregnates 3 × 3min；
(10) PBS liquid, every slice plus 50ul biotin labeling secondary antibody are got rid of, is incubated for 30min at room temperature；
(11) PBS rinses or impregnates 3 × 10min；
(12) PBS liquid, every slice plus 50ul streptomycete antibiotin-Peroxidase Solution are got rid of, is incubated at room temperature
25min；
(13) PBS rinses or impregnates 3 × 3min；
(14) get rid of PBS liquid, the DAB developing solution of every slice plus 100ul Fresh, microscopically observation, it is seen that xanthochromia it
Slice is immersed immediately afterwards and stops to dye in distilled water；
(15) haematoxylin is carried out to redye and mounting；
(16) mean absorbance values are calculated with PaintShop, and for statistical analysis.
6. a kind of diagnostic kit for predicting colon cancer prognosis according to claim 1-4, which is characterized in that institute
Stating diagnostic kit further includes micro-array tissue.
7. a kind of diagnostic kit for predicting colon cancer prognosis according to claim 6, which is characterized in that the tissue is micro-
Array is made of following steps:
(1) mold wax stone is made, size is 45mm × 20mm, and it is 0.1mm, diameter 0.6mm that spacing is got on mold wax stone
Aperture, each aperture is accurately positioned with coordinate；
(2) sample wax stone is dyed according to HE and is observed, and is selected and is kept sample group in mind in HE slice and corresponding Paraffin tissue block subscript respectively
The site knitted is punctured with the stainless pin of internal diameter 0.6mm, and 2~3mm of depth is fixed in the aperture of mold wax stone；
(3) mold wax stone is sliced with 4 μ m thicks, is applied on the solid phase carriers such as glass slide or nylon membrane, tissue has just been made
Microarray.
8. a kind of diagnostic kit for predicting colon cancer prognosis according to claim 1-4 is in colonic tissue pathology
The application of context of detection.
9. a kind of diagnostic kit for predicting colon cancer prognosis according to claim 1-4 is straight in prediction advanced stage knot
Application in terms of the total life span of patients with bowel cancer.
10. a kind of diagnostic kit for predicting colon cancer prognosis according to claim 1-4 is in prediction Colon and rectum
Application in terms of cancer patient's recurrence and metastasis after resection.
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Citations (3)
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|---|---|---|---|---|
| US20090215053A1 (en) * | 2005-10-19 | 2009-08-27 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Vitro Method for the Prognosis of Progression of a Cancer and of the Outcome in a Patient and Means for Performing Said Method |
| CN103278650A (en) * | 2013-05-29 | 2013-09-04 | 上海交通大学医学院附属新华医院 | Method for evaluating prognosis of patients with colorectal cancers through positive expression of colorectal cancer immunological index CD45RO |
| CN107085106A (en) * | 2017-04-11 | 2017-08-22 | 广州市第人民医院 | Detection reagent and kit for prostate cancer progress prognosis |
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| US20090215053A1 (en) * | 2005-10-19 | 2009-08-27 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Vitro Method for the Prognosis of Progression of a Cancer and of the Outcome in a Patient and Means for Performing Said Method |
| CN103278650A (en) * | 2013-05-29 | 2013-09-04 | 上海交通大学医学院附属新华医院 | Method for evaluating prognosis of patients with colorectal cancers through positive expression of colorectal cancer immunological index CD45RO |
| CN107085106A (en) * | 2017-04-11 | 2017-08-22 | 广州市第人民医院 | Detection reagent and kit for prostate cancer progress prognosis |
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Application publication date: 20191122 |