CN101995475A - Method for diagnosing breast cancer by expression of SOX2 protein - Google Patents
Method for diagnosing breast cancer by expression of SOX2 protein Download PDFInfo
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- CN101995475A CN101995475A CN201010532712XA CN201010532712A CN101995475A CN 101995475 A CN101995475 A CN 101995475A CN 201010532712X A CN201010532712X A CN 201010532712XA CN 201010532712 A CN201010532712 A CN 201010532712A CN 101995475 A CN101995475 A CN 101995475A
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Abstract
The invention belongs to a method for medical diagnosis, and in particular relates to a method for judging whether a breast lump is benign or malignant through detecting the expression condition of an SOX2 protein in the breast tissue. The method is realized through an immunological detection technology and belongs to a clinical immunological diagnosis method. The breast cancer is one of the most common malignant tumors seriously influencing the physical and psychological health of women even endangering the life, and the incidence of the cancer accounts for 7-10 percent of various malignant tumors in the whole body. In recent years, the incidence of the breast cancer is in an escalating trend, but if the bread cancer is early found and treated, about 90 percent of patients suffering from the breast cancer can survive. Therefore, the early rapid diagnosis on the malignant breast cancer can generate a positive function on the treatment. Found through experiment researches, the SOX2 is highly expressed in most of tissues of the breast cancer, and is not expressed or lowly expressed in normal tissues or tissues near the breast cancer, which expresses that the SOX2 can become a screening protein for diagnosing the breast cancer, i.e. the higher the expression degree in the tissues of the breast cancer, the larger the possibility of the breast cancer. Through an immunohistochemistry reaction on the pathological tissues, the expression quantity of SOX2 molecules in the tissues is detected, and the breast cancer is judged according to a result of the immunohistochemistry reaction.
Description
Technical field
The invention belongs to the method that a kind of medical diagnosis is used, a kind of specifically expression by SOX2 albumen in the detection breast tissue judges that breast lump is optimum or pernicious method, this method realizes by immunoassay technology, belong to clinical immune diagnostic method, thereby particularly use the SABC method to detect the method that SOX2 albumen is judged breast cancer.
Background technology
Breast cancer is the important malignancy disease that threatens WomanHealth, its early stage cardinal symptom shows as: symptom and signs such as breast lump, mammary gland pain and nipple discharge and skin change, axillary gland enlargement, at present clinically all can make judgement early by means of the tumor marker inspection and the pathological diagnosis of modern imaging examination and broad sense as suspicious situation.Breast cancer incidence is on the rise in recent years, if but early find, early treatment, about 90% patient with breast cancer can survive.Therefore, the early stage quick diagnosis to malignant breast carcinomas can produce positive effect to treatment.
SOX2 (SRY-Related HMG-Box Gene 2) is a member of SOX family, it is one of significant gene of stem cell, exist in the nuclear of stem cell, it is a kind of important transcription factor of keeping the stem cell characteristic, participate in complicated transcriptional control network, playing crucial effects aspect the multipotency state property of keeping embryonic stem cell and cancer stem cell and the self ability.SOX2 embryo in early days takes place, suppresses to play key effect in the multiple important growth incidents such as first Neural Differentiation and crystalline lens growth, and simultaneously, the carciongenic potency of SOX2 and cancer stem cell is closely related.Therefore the SOX2 in the cancer stem cell is very likely to the close adjusting of carrying out of cancer, and might become tumorigenic marker protein.We are by finding SOX2 high expressed in most breast cancer tissue to the immunohistochemical staining analysis of 319 routine breast tissue chips (comprising normal and breast cancer); and in normal or cancer beside organism, do not express or low the expression, show that SOX2 can become the screening albumen of diagnosing mammary cancer.Promptly the expression degree is high more in breast tissue, is that the possibility of breast cancer is big more.(seeing Table 1)
Relation between table 1.SOX2 expression and breast cancer take place
This experiment accounts for the judgement of the number percent of whole tissue as feminine gender and positive findings with the SOX2 positive cell number, and promptly＜10% negative (-), and 〉=10% positive (+).The result shows SOX2 along with breast tissue is pernicious from normal direction of rotation, and expression obviously strengthens.χ
2Check analysis shows the significant correlativity that has of SOX2 and breast cancer.
Summary of the invention
At above-mentioned situation, the present invention judges whether it is breast cancer by the SOX2 molecule in the method detection pathological tissue of SABC with this.Be exactly concretely,, detect the expression quantity of SOX2 molecule in this tissue, judge breast cancer according to the result of immunohistochemical reaction by immunohistochemical reaction to pathological tissue.
Its technology contents comprises: steps such as immunohistochemical reaction and microscopic examination.
The present invention is achieved by the following technical solutions:
1. dewax and aquation:
Concrete steps are as follows:
1. histotomy places the dimethylbenzene I to soak 10 minutes,
2. histotomy places the dimethylbenzene II to soak 10 minutes,
3. histotomy places the absolute ethyl alcohol I to soak 10 minutes,
4. histotomy places the absolute ethyl alcohol II to soak 10 minutes,
5. histotomy places 95% ethanol to soak 10 minutes,
6. histotomy places 80% ethanol to soak 10 minutes,
7. histotomy places tap water to soak 1 minute,
8. histotomy places distilled water to soak 5 minutes.
2. antigen retrieval by micro-wave oven, heat the Citrate Buffer (pH 6.0) of 10mM 10 minutes, puts into the low fire of histotomy and heats the cold of at room temperature drying in the air 10-15 minute.
3. the histotomy of handling with ultrapure washing is 3 times, each five minutes.
4. 3% hydrogen peroxide solution is dripped on microslide, room temperature left standstill 10 minutes, got rid of unnecessary liquid.
5. with ultrapure washing 2 times, each is five minutes
6. drip confining liquid (normal goats serum, 5%BSA solution), at room temperature hatched one hour, get rid of unnecessary liquid.
7. drip anti-(1: 100) 50 μ L, room temperature left standstill 1 hour.
8.TBST damping fluid is washed 3 times, each 5 minutes.
9. drip two anti-(1: 100) 50 μ L, room temperature left standstill 0.5 hour.
10.TBST damping fluid is washed 3 times, each 5 minutes.
11. drip three anti-(1: 100) 50 μ L, room temperature left standstill 0.5 hour.
12.TBST damping fluid is washed 4 times, each 5 minutes.
13.DAB develop the color, grasp dye levels at microscopically, after reaching promising result, to put into clear water and stop dyeing, the back was washed 10 minutes with tap water.
14. haematoxylin redyeing 20 seconds, tap water flushing 10 minutes.
15 dehydrations, transparent:
Concrete steps are as follows:
1. histotomy places 80% ethanol to soak 30 seconds,
2. soaked 3 minutes in 95% ethanol,
3. II was soaked 5 minutes in the absolute ethyl alcohol,
4. the absolute ethyl alcohol I was soaked 5 minutes,
5. soaked 5 minutes in the dimethylbenzene II,
6. the dimethylbenzene I was soaked 5 minutes,
16. resin mounting, microscopy.
Description of drawings
Fig. 1 testing result in normal structure shows that SOX2 does not express or low the expression in mammary glandular cell.
Fig. 2 testing result in malignant tumor tissue shows mainly high expressed in breast tumor cell of SOX2.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is described in further detail.
Embodiment 1
Sample: the histotomy of certain mammary glands in women.
1. dewax and aquation:
Concrete steps are as follows:
1. histotomy places the dimethylbenzene I to soak 10 minutes,
2. histotomy places the dimethylbenzene II to soak 10 minutes,
3. histotomy places the absolute ethyl alcohol I to soak 10 minutes,
4. histotomy places the absolute ethyl alcohol II to soak 10 minutes,
5. histotomy places 95% ethanol to soak 10 minutes,
6. histotomy places 80% ethanol to soak 10 minutes,
7. histotomy places tap water to soak 1 minute,
8. histotomy places distilled water to soak 5 minutes.
2. antigen retrieval by micro-wave oven, heat the Citrate Buffer (pH 6.0) of 10mM 10 minutes, puts into the low fire of histotomy and heats the cold of at room temperature drying in the air 10-15 minute.
3. the histotomy of handling with ultrapure washing is 3 times, each five minutes.
4. 3% hydrogen peroxide solution is dripped on microslide, room temperature left standstill 10 minutes, got rid of unnecessary liquid.
5. with ultrapure washing 2 times, each is five minutes
6. drip confining liquid (normal goats serum, 5%BSA solution), at room temperature hatched one hour, get rid of unnecessary liquid.
7. drip anti-(1: 100) 50 μ L, room temperature left standstill 1 hour.
8.TBST damping fluid is washed 3 times, each 5 minutes.
9. drip two anti-(1: 100) 50 μ L, room temperature left standstill 0.5 hour.
10.TBST damping fluid is washed 3 times, each 5 minutes.
11. drip three anti-(1: 100) 50 μ L, room temperature left standstill 0.5 hour.
12.TBST damping fluid is washed 4 times, each 5 minutes.
13.DAB develop the color, grasp dye levels at microscopically, after reaching promising result, to put into clear water and stop dyeing, the back was washed 10 minutes with tap water.
14. haematoxylin redyeing 20 seconds, tap water flushing 10 minutes.
15 dehydrations, transparent:
Concrete steps are as follows:
1. histotomy places 80% ethanol to soak 30 seconds,
2. soaked 3 minutes in 95% ethanol,
3. II was soaked 5 minutes in the absolute ethyl alcohol,
4. the absolute ethyl alcohol I was soaked 5 minutes,
5. soaked 5 minutes in the dimethylbenzene II,
6. the dimethylbenzene I was soaked 5 minutes,
16. resin mounting, microscopy.
The microscopy result as shown in Figure 1.
As seen the expression degree of SOX2 is lower in this histotomy, does not have obvious brown yellow granule, for SOX2 detects negative findings, after the pathology detection result is a normal galactophore tissue.
Embodiment 2
Sample: the histotomy of certain mammary glands in women tumour.
1. dewax and aquation:
Concrete steps are as follows:
1. histotomy places the dimethylbenzene I to soak 10 minutes,
2. histotomy places the dimethylbenzene II to soak 10 minutes,
3. histotomy places the absolute ethyl alcohol I to soak 10 minutes,
4. histotomy places the absolute ethyl alcohol II to soak 10 minutes,
5. histotomy places 95% ethanol to soak 10 minutes,
6. histotomy places 80% ethanol to soak 10 minutes,
7. histotomy places tap water to soak 1 minute,
8. histotomy places distilled water to soak 5 minutes.
2. antigen retrieval by micro-wave oven, heat the Citrate Buffer (pH 6.0) of 10mM 10 minutes, puts into the low fire of histotomy and heats the cold of at room temperature drying in the air 10-15 minute.
3. the histotomy of handling with ultrapure washing is 3 times, each five minutes.
4. 3% hydrogen peroxide solution is dripped on microslide, room temperature left standstill 10 minutes, got rid of unnecessary liquid.
5. with ultrapure washing 2 times, each is five minutes.
6. drip confining liquid (normal goats serum, 5%BSA solution), at room temperature hatched one hour, get rid of unnecessary liquid.
7. drip anti-(1: 100) 50 μ L, room temperature left standstill 1 hour.
8.TBST damping fluid is washed 3 times, each 5 minutes.
9. drip two anti-(1: 100) 50 μ L, room temperature left standstill 0.5 hour.
10.TBST damping fluid is washed 3 times, each 5 minutes.
11. drip three anti-(1: 100) 50 μ L, room temperature left standstill 0.5 hour.
12.TBST damping fluid is washed 4 times, each 5 minutes.
13.DAB develop the color, grasp dye levels at microscopically, after reaching promising result, to put into clear water and stop dyeing, the back was washed 10 minutes with tap water.
14. haematoxylin redyeing 20 seconds, tap water flushing 10 minutes.
15 dehydrations, transparent:
Concrete steps are as follows:
1. histotomy places 80% ethanol to soak 30 seconds,
2. soaked 3 minutes in 95% ethanol,
3. II was soaked 5 minutes in the absolute ethyl alcohol,
4. the absolute ethyl alcohol I was soaked 5 minutes,
5. soaked 5 minutes in the dimethylbenzene II,
6. the dimethylbenzene I was soaked 5 minutes,
16. resin mounting, microscopy.
The microscopy result as shown in Figure 2.
As seen the expression degree height of SOX2 in this histotomy has obvious brown yellow granule, for SOX2 detects positive findings, after the pathology detection result is a malignant breast tumor.
Claims (3)
1. one kind with the method for SOX2 albumen as breast cancer diagnosis, it is characterized in that, handle histopathologic slide, antigen retrieval to the immunohistochemical reaction that carries out of pathological tissue, is handled histotomy dehydration, transparence, and resin mounting, microscopy detects the expression quantity of SOX2 molecule in this tissue thus, judges breast cancer according to the result of immunohistochemical reaction.
2. according to claim 1 a kind of with the method for SOX2 albumen as breast cancer diagnosis, it is characterized in that the antibody that immunohistochemical reaction dripped:
Drip anti-(1: 100) 50 μ L, room temperature left standstill 1 hour;
Drip two anti-(1: 100) 50 μ L, room temperature left standstill 0.5 hour;
Drip three anti-(1: 100) 50 μ L, room temperature left standstill 0.5 hour;
DAB develops the color, and grasps dye levels at microscopically, puts into clear water and stop dyeing after reaching promising result, and the back was washed 10 minutes with tap water.
3. according to claim 1ly a kind ofly it is characterized in that with the method for SOX2 albumen microscopic examination detects the expression quantity of SOX2 molecule in this tissue, thereby judge breast cancer as breast cancer diagnosis.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201010532712XA CN101995475A (en) | 2010-11-05 | 2010-11-05 | Method for diagnosing breast cancer by expression of SOX2 protein |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102353781A (en) * | 2011-06-10 | 2012-02-15 | 华中科技大学 | Breast cancer detection kit |
| CN102590512A (en) * | 2012-02-17 | 2012-07-18 | 博生吉医药科技(苏州)有限公司 | Kit for mammary cancer individual administration testing and using method thereof |
| CN107300496A (en) * | 2011-05-20 | 2017-10-27 | 独立行政法人理化学研究所 | Biomaterial is with transparence reagent and its utilizes |
-
2010
- 2010-11-05 CN CN201010532712XA patent/CN101995475A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107300496A (en) * | 2011-05-20 | 2017-10-27 | 独立行政法人理化学研究所 | Biomaterial is with transparence reagent and its utilizes |
| US10444124B2 (en) | 2011-05-20 | 2019-10-15 | Riken | Clarifying reagent for biological materials and use thereof |
| CN102353781A (en) * | 2011-06-10 | 2012-02-15 | 华中科技大学 | Breast cancer detection kit |
| CN102353781B (en) * | 2011-06-10 | 2014-04-09 | 华中科技大学 | Breast cancer detection kit |
| CN102590512A (en) * | 2012-02-17 | 2012-07-18 | 博生吉医药科技(苏州)有限公司 | Kit for mammary cancer individual administration testing and using method thereof |
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Application publication date: 20110330 |