CN105652013B - Colorectal cancer Index for diagnosis marker, expression appraisal procedure, kit and application - Google Patents
Colorectal cancer Index for diagnosis marker, expression appraisal procedure, kit and application Download PDFInfo
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- CN105652013B CN105652013B CN201510055483.XA CN201510055483A CN105652013B CN 105652013 B CN105652013 B CN 105652013B CN 201510055483 A CN201510055483 A CN 201510055483A CN 105652013 B CN105652013 B CN 105652013B
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- colorectal cancer
- expression
- slc12a5
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- diagnosis marker
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Abstract
The invention discloses a kind of colorectal cancer Index for diagnosis markers.The amino acid sequence of colorectal cancer marker SLC12A5 is as shown in SEQ NO:1.Application, a kind of expression appraisal procedure of colorectal cancer Index for diagnosis marker, a kind of kit and a kind of application of kit that the invention also discloses a kind of colorectal cancer Index for diagnosis markers in reagent preparation box.The high-caliber expression of the colorectal cancer Index for diagnosis marker SLC12A5 of the invention and prognosis of patient is poor is positively correlated, facilitates the prognosis situation for judging colorectal cancer patients.The height that may determine that the horizontal expression of knot SLC12A5 by expressing appraisal procedure.The SLC12A5 is applied to preparation for the kit that the expression of colorectal cancer Index for diagnosis marker detects, to be convenient to the expression of detection SLC12A5.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of colorectal cancer Index for diagnosis marker, the marker exist
The expression appraisal procedure of application, the marker in reagent preparation box, comprising the kit of the marker and the kit with
Application in the detection of colorectal cancer Index for diagnosis marker representation level.
Background technique
Colorectal cancer includes colon cancer and the carcinoma of the rectum, is malignant tumour common in worldwide, and case fatality rate occupies
The second of the global malignant tumour cause of the death, Advanced colorectal cancer prognosis are poor.Tissue public affairs in 2005 are studied according to international cancer
The data of cloth show that the disease incidence of colorectal cancer is in obvious ascendant trend, wherein most of is colon cancer.The mark of China's colon cancer
Quasi- disease incidence is 13.29/10 ten thousand, and disease incidence is in obvious ascendant trend.The generation of colorectal cancer and diet, environment, heredity etc. are close
Cut phase is closed, and is the result of multifactor synergistic effect.The study on prevention of colorectal cancer causes the great attention of people, biomarker
Discovery and detection, to prediction colon cancer generation, development, therapeutic effect and Index for diagnosis have certain Clinical significance of MG.
TNM stage is currently the most important Clinical criteria for judging colorectal cancer patients prognosis.However, many patients
Even if TNM stage belongs to early stage, tumour is it is possible to recurrence.Therefore it needs to study the prognostic marker for being preferably used for risk assessment
Object.
Summary of the invention
Present invention aims to overcome that the above-mentioned deficiency of the prior art, provides a kind of colorectal cancer Index for diagnosis marker
SLC12A5, high-caliber expression with the prognosis of patient is poor is positively correlated.
Another object of the present invention is to overcome the above-mentioned deficiency of the prior art, provides a kind of colorectal cancer Index for diagnosis label
Application of the object in reagent preparation box, the kit can be used for the expression water of colorectal cancer Index for diagnosis marker SLC12A5
Flat detection.
Yet another object of that present invention is to the above-mentioned deficiencies for overcoming the prior art, provide a kind of colorectal cancer Index for diagnosis label
The expression appraisal procedure of object, it can be estimated that the expression of colorectal cancer Index for diagnosis marker SLC12A5.
Still a further object of the present invention is to overcome the above-mentioned deficiency of the prior art, provides a kind of kit, can be used for detecting knot
The expression of carcinoma of the rectum Index for diagnosis marker SLC12A5.
A further purpose of the present invention is to overcome the above-mentioned deficiency of the prior art, a kind of kit is provided for Colon and rectum
Application in the detection of cancer Index for diagnosis marker SLC12A5 expression, it is straight by being convenient to detection knot using the kit
The expression of intestinal cancer Index for diagnosis marker SLC12A5.
In order to achieve the above-mentioned object of the invention, technical solution of the present invention is as follows:
A kind of colorectal cancer Index for diagnosis marker, the amino acid of the colorectal cancer Index for diagnosis marker SLC12A5
Sequence is as shown in SEQ NO:1.
A kind of application of colorectal cancer Index for diagnosis marker in reagent preparation box, the kit are used for amino acid sequence
Arrange the detection of the colorectal cancer Index for diagnosis marker SLC12A5 expression as shown in SEQ NO:1.
A kind of expression appraisal procedure of colorectal cancer Index for diagnosis marker, comprising:
Obtain sample to be measured;
The wax stone for embedding the sample to be measured is cut into slice;
Immunohistochemistry detection is carried out to the slice;
According to the positive cell of amino acid sequence colorectal cancer Index for diagnosis marker SLC12A5 as shown in SEQ NO:1
The expression of the percentage assessment colorectal cancer Index for diagnosis marker SLC12A5 of dyeing.
A kind of kit, the expression for colorectal cancer Index for diagnosis marker detects, including primary antibody, the primary antibody
For the antibody of amino acid sequence colorectal cancer Index for diagnosis marker SLC12A5 as shown in SEQ NO:1.
And a kind of mentioned reagent box is for the application in the detection of colorectal cancer Index for diagnosis marker representation level.
Colorectal cancer Index for diagnosis marker SLC12A5 provided by the invention, rises emphatically in colorectal cancer occurrence and development
The facilitation wanted, SLC12A5 expression silencing in normal colonic mucosa tissue, but the expression height in colorectal cancer
Different, the high-caliber expression of the analysis of clinical of the large sample discovery SLC12A5 and prognosis of patient is poor is positively correlated mentions
Show that SLC12A5 is expected to the biomarker as important colorectal cancer patients prognosis.
Using colorectal cancer Index for diagnosis marker of the invention, can prepare for amino acid sequence such as SEQ NO:1 institute
The kit of the expression detection of the colorectal cancer Index for diagnosis marker SLC12A5 shown.
The expression appraisal procedure of colorectal cancer Index for diagnosis marker provided by the invention, can detecte colorectal cancer prognosis
The expression of judge mark object facilitates the prognosis situation for judging colorectal cancer patients.
Kit provided by the invention, it is convenient to detect the expression of colorectal cancer Index for diagnosis marker
Using kit provided by the invention, the expression of detection colorectal cancer Index for diagnosis marker can be facilitated.
Detailed description of the invention
Fig. 1 is expression result of the SLC12A5 of the embodiment of the present invention 1 in colorectal cancer and normal colonic mucosa;
Fig. 2 is relative expression quantity knot of the SLC12A5 in pairs of colorectal cancer and cancer beside organism in the embodiment of the present invention 1
Fruit;
Fig. 3 is low expression and highly expressed schematic diagram of the SLC12A5 in colorectal cancer in the embodiment of the present invention 1;
Fig. 4 is the tumor growth curve figure of nude mice in the embodiment of the present invention 2;
Fig. 5 is the histogram of the tumor weight of nude mice in the embodiment of the present invention 2;
Fig. 6 is SLC12A5 difference expression in the embodiment of the present invention 3 to colorectal cancer patients Index for diagnosis
Kaplan-Meier analysis graph.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, right below in conjunction with drawings and examples
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
The present invention provides a kind of colorectal cancer Index for diagnosis markers.The colorectal cancer Index for diagnosis marker
The amino acid sequence of SLC12A5 is as shown in SEQ NO:1.
Colorectal cancer Index for diagnosis marker SLC12A5 is sent out by genome sequencing and large sample colorectal cancer research
It is existing, play important facilitation in colorectal cancer occurrence and development, SLC12A5 expression silencing in normal colonic mucosa tissue,
But the expression height in colorectal cancer is different, the analysis of clinical discovery SLC12A5's of large sample is high-caliber
Expression with the prognosis of patient is poor is positively correlated, prompt SLC12A5 to be expected to become the biology of important colorectal cancer patients prognosis
Marker.
Following embodiments 1 characterize colorectal cancer Index for diagnosis marker SLC12A5 in normal tissue and colorectal cancer
In expression.The embodiment demonstrates SLC12A5 expression silencing in normal colonic mucosa tissue, but in colorectal cancer
Expression height it is different, so as to as colorectal cancer Index for diagnosis marker.Embodiment 2 characterize strike it is low
The expression of SLC12A5 plays emphatically nude mouse tumor growth effect to demonstrate the SLC12A5 in colorectal cancer occurrence and development
The facilitation wanted shows that it can be used as colorectal cancer Index for diagnosis marker.
Embodiment 1SLC12A5 expresses up-regulation test in colorectal cancer
SLC12A5 is detected using the ImmunohistochemistryMethods Methods of the embodiment of the present invention in colorectal cancer and normal colonic mucosa respectively
Expression, find SLC12A5 expression silencing in normal colonic mucosa, and in colorectal cancer express up-regulation.Its result is such as
Shown in Fig. 1, Fig. 1 is expression result of the SLC12A5 of the embodiment of the present invention 1 in colorectal cancer and normal colonic mucosa.Its
In, figure (a) indicates Colorectal Carcinoma (CRC tumor tissues), and figure (b) indicates Carcinoma side normal tissue (Adjacent
normal tissues)。
Based on SLC12A5 in the up-regulation phenomenon of colorectal cancer, 15 pairs of Colorectal Carcinoma samples are chosen, and utilize immune group
Change the expression for analyzing its SLC12A5.As a result as shown in Fig. 2, Fig. 2 is SLC12A5 in the embodiment of the present invention 1 in pairs of knot
Relative expression quantity in the carcinoma of the rectum and cancer beside organism is as a result, wherein T indicates that Colorectal Carcinoma, AN indicate cancer beside organism.From Fig. 2
As can be seen that SLC12A5 is significantly higher than the expression quantity in cancer beside organism in the expression quantity of colorectal cancer.According to embodiments of the present invention
Colorectal cancer Index for diagnosis marker expression appraisal procedure standards of grading assessment colorectal cancer marker expression,
Its result is as shown in Figure 3.Fig. 3 is low expression and highly expressed signal of the SLC12A5 in colorectal cancer in the embodiment of the present invention 1
Figure, wherein figure (a) indicates low expression, and figure (b) indicates high expression.
Embodiment 2 strikes in vivo studies of the expression of low SLC12A5 to nude mouse tumor growth effect
Control cell strain HCT116/control or HCT116/shSLC12A5 are injected into the back side of nude mice at random, and right
The growth pattern of tumour compares.Measurement in gross tumor volume every 3 days is primary, continues for about three weeks.By measuring longest
Gross tumor volume (mm is calculated with shortest diameter of tumor length (cubature formula=0.5 × length × 2 × wide)3).It is real 22 days after inoculation
Termination is tested, mouse is condemned to death.The tumor growth curve of nude mice is as shown in Figure 4, wherein C indicates injection HCT116/control's
Tumor growth curve, S indicate the tumor growth curve of injection HCT116/shSLC12A5.Inject HCT116/shSLC12A5 nude mice
Average tumor size be substantially less than inject HCT116/control nude mice of control group (P < 0.001, variance analysis, n=5/
Group).Tumour is separated, weight is measured.As shown in figure 5, histogram respectively indicates HCT116/shSLC12A5 and HCT116/
The tumor weight that two groups of control (P value is examined less than 0.05, t), wherein C indicates the tumour weight of injection HCT116/control
Amount, S indicate the tumor weight of injection HCT116/shSLC12A5.Tumor tissues ratio under the separation of HCT116/shSLC12A5 group
Control group is light (P < 0.05).
The characteristic that can be used as colorectal cancer Index for diagnosis marker for the SLC12A5, the present invention also provides the knots
Application of the carcinoma of the rectum Index for diagnosis marker SLC12A5 in reagent preparation box, the kit are used for amino acid sequence such as SEQ
The detection of colorectal cancer Index for diagnosis marker SLC12A5 expression shown in NO:1.
It can be used as the characteristic of colorectal cancer Index for diagnosis marker for the SLC12A5, the present invention also provides one kind
The expression appraisal procedure of colorectal cancer Index for diagnosis marker can assess colorectal cancer Index for diagnosis marker by this method
Expression, to judge the prognosis situation of colorectal cancer patients.This method comprises the following steps that
Step S10: sample to be measured is obtained.
Sample to be measured can be colorectal cancer fresh sample or wax stone sample.
Step S20: the wax stone for embedding sample to be measured is cut into slice.
The purpose of the step is embedding tumor tissues.Wherein, slice with a thickness of 5 microns.
Step S30: immunohistochemistry detection is carried out to slice.
The detailed process of the immunohistochemistry:
Step S31: the glass slide for being loaded with slice successively dewaxing: is put into dimethylbenzene, -100% alcohol of dimethylbenzene, 100%
Dewaxing is impregnated in alcohol, 95% alcohol, 90% alcohol, 80% alcohol and 70% alcohol, is generally impregnated in every kind of reagent
10min。
Step S32: antigen retrieval: the slice after dewaxing rinses a period of time in clear water, and 3%H is added2O2It impregnates
Then 10min outwells H2O2, it is washed in clear water twice, adds citrate buffer solution, be put into moderate heat boiling 3min in micro-wave oven,
It is general just to arrive boiling, it is cooled to room temperature, then boiling is primary again, is cooled to room temperature.The purpose of boiling is to make antigen
Site be exposed.
Step S33: nonspecific site closing: after glass slide is cooled to room temperature, citrate buffer solution is outwelled, water
It washes 2 times, and the glass slide for being loaded with slice is placed in PBS and is washed 2 times, each 5min, in addition blocking agent animal blood serum, makes some non-
The site of specificity is closed, and then glass slide is put into 37 DEG C of incubators and keeps the temperature half an hour.
Step S34: primary antibody is incubated for: the glass slide in incubator being taken out, reverse side and the front of glass slide are dried with blotting paper
Serum around organizing, adds primary antibody, and primary antibody is amino acid sequence colorectal cancer Index for diagnosis marker as shown in SEQ NO:1
The antibody of SLC12A5;In the tissue plus PBS of negative control.It is saved overnight in 4 DEG C of refrigerators after adding primary antibody.
Step S35: secondary antibody is incubated for: glass slide being taken out from refrigerator, is put into PBS and washes 3 times, each 5min dries group
Secondary antibody is added after knitting the PBS of surrounding, is subsequently placed in 37 DEG C of incubators and keeps the temperature half an hour.Secondary antibody is peroxidase labelling with one
Resist corresponding secondary antibody.
Step S36: dyeing: wherein coloring agent is AEC coloring agent.
Step S37: after the glass slide after redying is placed in water flushing, glass slide successively dehydration, mounting: is put into 70%
Alcohol, 80% alcohol, 90% alcohol, 95% alcohol, 100% alcohol, 100% alcohol-dimethylbenzene and dimethylbenzene.In every kind of reagent
2min is placed, is finally immersed in dimethylbenzene, moves in vent cabinet.With neutral gum drop beside tissue, then with coverslip lid
On.Lid coverslip Shi Yaoxian is laid flat side, then gently puts down the other side, in order to avoid generating bubble, is set after sealing glass slide
It is dried in vent cabinet.
Step S40: it is commented according to the percentage of the positive cell dyeing of amino acid sequence SLC12A5 as shown in SEQ NO:1
Estimate the expression of colorectal cancer Index for diagnosis marker SLC12A5.
The standard of the evaluation is as follows:
It is scored according to the percentage of the positive cell dyeing of SLC12A5 to assess the degree of the dyeing of SLC12A5.Its
In, dye-free counts 0 point.< 20% positive cell dyeing counts 1 point.The positive cell dyeing of 20-50% counts 2 points.> 50%
Positive cell dyeing counts 3 points.The expression for the definition colorectal cancer marker SLC12A5 that dyeing scoring is 0,1 or 2 point
For low expression, the expression to score up to 3 points of definition colorectal cancer marker SLC12A5 is expressed to be high.
Show that the prognosis of patient is poor when the expression of colorectal cancer marker SLC12A5 is high expression.Therefore, originally
Inventive embodiments facilitate the prognosis situation for judging colorectal cancer patients by the expression of detection SLC12A5.
Expression assessment side of following embodiments 3 to further illustrate colorectal cancer Index for diagnosis marker of the invention
Method, the expression of SLC12A5 can be assessed by verifying method of the invention.
The assessment of the expression of 3 Colorectal Carcinoma SLC12A5 of embodiment
In embodiment 3, Colon and rectum is assessed using the expression appraisal procedure of colorectal cancer Index for diagnosis marker of the invention
The expression height of cancerous tissue SLC12A5.The method that the expression of SLC12A5 is detected in this method is immunohistochemistry side
Method.The process specifically assessed is as follows:
S10: 195 colorectal cancer wax stone samples are obtained.
S20: embedded wax stone is cut into 5 microns of slabs.
S30: immunohistochemistry detection is carried out to slice.The detailed process of step step S31~S37 institute as mentioned before
Show.
S40 assesses the expression of colorectal cancer marker according to the percentage of the positive cell dyeing of SLC12A5.It adopts
With standards of grading of the invention, the horizontal of SLC12A5 is carried out by immunohistochemistry to 195 colorectal cancer patients tissue samples and is examined
It surveys.As a result, it has been found that having 70 in the Colorectal Carcinoma sample of 195 clinical patients is high expression (i.e. scoring is 3 points).
On this basis, clinic of the expression of SLC12A5 in colorectal cancer Index for diagnosis is further had evaluated to answer
With analyzing the expression of SLC12A5 and the correlation of Clinical symptoms, including patient age, gender, tumor stage, tumour
Differentiation and Survival data find the high patient of the expression of the tumor tissues SLC12A5 patient poor prognosis lower than expression.
As shown in fig. 6, being to analyze curve (P < 0.01) to the Kaplan-Meier of colorectal cancer patients prognosis.From fig. 6 it can be seen that
The survival rate of the high patient of the expression of the tumor tissues SLC12A5 patient prognosis lower than expression is low, wherein L is indicated
Low expression, H indicate high expression.
Above-described embodiment shows that the expression appraisal procedure of the colorectal cancer Index for diagnosis marker of the invention can be convenient
Ground detects and assesses the expression of colorectal cancer Index for diagnosis marker SLC12A5, so as to be judged according to the expression
The prognosis situation of colorectal cancer patients.
For the expression appraisal procedure of above-mentioned colorectal cancer Index for diagnosis marker, the present invention provides a kind of kit,
It can be used for the expression appraisal procedure.
Above-mentioned kit includes primary antibody.Wherein primary antibody is amino acid sequence colorectal cancer prognosis as shown in SEQ NO:1
The antibody of judge mark object SLC12A5.For example, the antibody of the SLC12A5 can be the product that article No. is HPA004942SIGMA.
The kit further includes secondary antibody, enzyme, coloring agent and blocking agent.Wherein secondary antibody is secondary antibody corresponding with primary antibody.For example, the secondary antibody is
Anti-rabbit HRP tagged polymers.The secondary antibody is by peroxidase labelling.Enzyme is Avidin-biotin compound.Coloring agent
For AEC coloring agent.Blocking agent is animal blood serum.
The kit further includes dimethylbenzene used in expression detection process, alcohol, buffer and neutral gum.It should
Buffer includes citrate buffer solution and PBS buffer solution.
The kit further includes the calculator calculated the percentage of the cell dyed in sample to be tested, to obtain
The expression of the percentage assessment SLC12A5 of positive cell dyeing.
For example, the antibody of the SLC12A5 of the kit, dosage is that (antibody of 5 microlitres of SLC12A5 is added to for 1:100 dilution
In 500 microlitres of PBS buffer solution);Anti-rabbit HRP tagged polymers, dosage are that 1:2000 dilutes (1 microlitre of anti-rabbit HRP tagged polymers
It is added in 2000 microlitres of PBS buffer solution).
For mentioned reagent box, the present invention also provides the kits to be used for colorectal cancer Index for diagnosis marker representation
Application in level detection.The expression of detection colorectal cancer Index for diagnosis marker can be facilitated using the kit.
In conclusion the high-caliber expression of the colorectal cancer Index for diagnosis marker SLC12A5 of the embodiment of the present invention with
The prognosis of patient is poor to be positively correlated, and the prognosis situation for judging colorectal cancer patients is facilitated, and can be used for preparing detection should
The kit of the expression of SLC12A5.It is when corresponding by ImmunohistochemistryMethods Methods and according to the percentage of positive cell dyeing
Scoring criteria is scored, and judges the height of the expression of colorectal cancer Index for diagnosis marker SLC12A5.The colorectal cancer
Index for diagnosis marker SLC12A5 is applied to the reagent of expression detection of the preparation for colorectal cancer Index for diagnosis marker
Box, so as to easily detect the expression of SLC12A5.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc. within mind and principle should all include within protection scope of the present invention.
Claims (6)
1. existing for amino acid sequence antibody of colorectal cancer Index for diagnosis marker SLC12A5 as shown in SEQ ID NO:1
Preparation is for the purposes in the kit of colorectal cancer Index for diagnosis, wherein the antibody is used to detect the expression water of SLC12A5
It is flat.
2. purposes as described in claim 1, it is characterised in that: the kit further includes and is directed to amino acid sequence such as SEQ
The corresponding secondary antibody of antibody of colorectal cancer Index for diagnosis marker SLC12A5 shown in ID NO:1, the secondary antibody is by peroxide
Enzyme label.
3. purposes as described in claim 1, it is characterised in that: the kit further includes enzyme, and the enzyme is avidin 9
White-biotin composite.
4. purposes as described in claim 1, it is characterised in that: the kit further includes coloring agent, and the coloring agent is AEC
Coloring agent.
5. purposes as described in claim 1, it is characterised in that: the kit further includes blocking agent, and the blocking agent is
Object serum.
6. purposes as described in claim 1, which is characterized in that the kit further include: dimethylbenzene, alcohol, buffer and
Neutral gum.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
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CN201510055483.XA CN105652013B (en) | 2015-01-30 | 2015-01-30 | Colorectal cancer Index for diagnosis marker, expression appraisal procedure, kit and application |
HK16112158.5A HK1224013A1 (en) | 2015-01-30 | 2016-10-22 | A marker, an evaluation method of expression, a kit and its application for the prognostic of colorectal cancer |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510055483.XA CN105652013B (en) | 2015-01-30 | 2015-01-30 | Colorectal cancer Index for diagnosis marker, expression appraisal procedure, kit and application |
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CN105652013A CN105652013A (en) | 2016-06-08 |
CN105652013B true CN105652013B (en) | 2019-05-10 |
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HK (1) | HK1224013A1 (en) |
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CN113476607B (en) * | 2021-08-04 | 2022-06-03 | 中南大学湘雅二医院 | SLC12A5 and application of inhibitor thereof |
CN114242175A (en) * | 2021-12-22 | 2022-03-25 | 香港中文大学深圳研究院 | Method and system for evaluating brain white matter high signal volume |
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CN203350254U (en) * | 2013-07-02 | 2013-12-18 | 武汉三鹰生物技术有限公司 | Immunohistochemical kit |
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