CN101493464A - Molecular marker for breast carcinoma diagnosis - Google Patents
Molecular marker for breast carcinoma diagnosis Download PDFInfo
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- CN101493464A CN101493464A CNA2009100941682A CN200910094168A CN101493464A CN 101493464 A CN101493464 A CN 101493464A CN A2009100941682 A CNA2009100941682 A CN A2009100941682A CN 200910094168 A CN200910094168 A CN 200910094168A CN 101493464 A CN101493464 A CN 101493464A
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- thioredoxin
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- breast cancer
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Abstract
The invention discloses a molecular marker for diagnosing breast cancer, which is used for detecting expression of the thioredoxin (TRX) and the thioredoxin binding protein 2 (TRX-binding protein-2, TBP-2) in surrounding normal breast tissue and mass tissue. A protein immunoblot experiment is used for detecting the expression amount of the thioredoxin and the thioredoxin binding protein 2; in breast mass tissue, the expression of the thioredoxin is increased and the expression of the thioredoxin binding protein 2 is decreased; as significant difference exists between the breast mass tissue and the normal breast tissue, the thioredoxin and the thioredoxin binding protein 2 can be used as a new molecular marker of breast cancer and used for the clinical diagnosis of breast cancer.
Description
Technical field
The invention belongs to biological technical field, particularly, the protein immunoblotting method that relates to a kind of diagnosing mammary cancer, and thioredoxin and thioredoxin in conjunction with albumen 2 as the protein molecular marker that detects breast cancer, the application in diagnosing mammary cancer clinical.
Background technology
Breast cancer is one of principal disease of harm women's health, and the whole world has more than 100 ten thousand women to suffer from breast cancer every year approximately.The male sex also can suffer from breast cancer, but the probability that the male sex suffers from breast cancer is lacked nearly 100 times than the women.The period of disease majority of breast cancer is in 40-60 year, and its occurrence cause does not still have final conclusion, it is generally acknowledged relevantly with the variation of sex hormone, and has certain familial.Early-stage breast cancer can not have any subjective symptoms, and pathology breast lump can occur late period, and the lump quality is tough firmly, and the border is very not clear, no coating sense, and the movability that pushes away is little, most no obvious pain.Up to the present, it is relevant with breast cancer generation development that more existing gene unconventionality expressions are determined, but the unconventionality expression rate of the breast cancer related gene that is determined in breast cancer is not high, and the early diagnosing mammary cancer rate still remains to be improved.In addition, traditional mammary cancer surgery add chemotherapy and the several genes methods of treatment prognosis effect that is used in recent years relatively poor, do not significantly improve patient with breast cancer's survival rate, thereby especially breast cancer unconventionality expression gene is significant for the pathogenesis of inquiring into breast cancer to seek new breast cancer related gene, presses for and seeks new expression is changed significantly in breast cancer gene or albumen.
(thioredoxin TRX) claims the interleukin 1 like cell factor, adult T-cell leukemia the develop factor, early pregnancy factor etc. again to thioredoxin.The molecular weight that the Trx is made up of 104 amino acid is the polypeptide of 12kDa.Thioredoxin plays important regulatory role in cell proliferation, existence and inhibition apoptotic process.As a kind of growth factor, thioredoxin can be secreted by lymphocyte, liver cell, fibroblast and many cancerous cell lines, and induces himself growth.A series of people's primary tumo(u)r results of study are shown, tumor tissues is compared with normal structure, thioredoxin overexpression in the tumor tissues, its expression and cell proliferation are remarkable positive correlation.
(TRX-binding protein-2 TBP-2) is called vitamin D again to thioredoxin in conjunction with albumen 2
3Upregulated protein 1 (vitamin D
3Up-regulated protein 1, VDUP1), molecular weight is about 46kDa.Thioredoxin extensively is distributed in conjunction with albumen 2 in the multiple tissue of animal and comprises: adrenal gland, heart, lung tissue, thymus gland, spleen, testis, skeletal muscle, kidney, liver, mammary gland, ovary etc., wherein its expression in lung tissue is higher relatively, and the expression in brain tissue and liver organization is relatively low.Thioredoxin can interact with thioredoxin in conjunction with albumen 2.In some tumor tissues, thioredoxin is in conjunction with albumen 2 expression deletions, if with thioredoxin in conjunction with albumen 2 transfections in cancer cell, cross the thioredoxin of expressing in conjunction with albumen 2 cell growth inhibiting, impel Apoptosis.Thioredoxin also suppresses the cell cycle in conjunction with albumen 2, makes the cell cycle be trapped in the GO phase, and this shows that thioredoxin is closely related in conjunction with albumen 2 and tumor growth.
Therefore, thioredoxin and thioredoxin are the new target sites of research breast cancer in conjunction with albumen 2.But up to the present, do not see in the prior art that thioredoxin and thioredoxin are in conjunction with the report of albumen 2 with the correlativity of breast cancer.
Summary of the invention
The present invention aims to provide a kind of diagnosing mammary cancer molecular marked compound.The application has found and a kind ofly there are differences expressed protein in breast cancer enclosed mass tissue and normal perienchyma.Immunoblot experiment confirms that thioredoxin and thioredoxin exist notable difference to express in conjunction with albumen 2 really in breast cancer enclosed mass tissue and normal perienchyma.
, its expression is detected as a protein molecular mark with these two kinds of albumen and can be used to detect breast cancer in conjunction with this correlativity of albumen 2 based on thioredoxin and thioredoxin with breast cancer.
The object of the present invention is to provide thioredoxin and thioredoxin in a kind of vitro detection mammary glandular cell in conjunction with the whether unusual method of the expression of albumen 2;
Therefore, another object of the present invention is to provide a kind of thioredoxin and thioredoxin in conjunction with the application of albumen 2 as the protein molecular marker that detects breast cancer.
In view of up to the present, also there are not thioredoxin and thioredoxin in conjunction with the report of albumen 2 with the correlativity of breast cancer, therefore, this discovery of the present invention will provide a new auxiliary approach for the diagnosis and/or the treatment of breast cancer.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
The method of diagnosing mammary cancer comprises obtaining mammary gland nodules tissue and normal perienchyma sample that from sample thioredoxin and thioredoxin are in conjunction with the expression of albumen 2 in mensuration and the comparative sample.
Wherein the expression of albumen is measured by detected by Western blot.
Wherein when the thioredoxin expression is higher than, when thioredoxin is lower than the expression of NBT in conjunction with albumen 2 expressions, be judged as breast cancer.
Relatively the expression that thioredoxin and thioredoxin combine albumen 2 in enclosed mass tissue and the normal perienchyma be with thioredoxin in the enclosed mass tissue that relatively obtains in conjunction with the antibody test of albumen 2 with the anti-thioredoxin/thioredoxin of specificity, thioredoxin in conjunction with the expression of albumen 2 and normally these two kinds of protein contents of perienchyma compare, as the enclosed mass that records organizes the thioredoxin expression to be higher than normal perienchyma, when thioredoxin is lower than normal perienchyma in conjunction with albumen 2 expressions, be diagnosed as breast cancer.
This method may further comprise the steps:
With the anti-thioredoxin/thioredoxin of specificity in conjunction with thioredoxin and thioredoxin in the antibody test of albumen 2 mammary glandular cell to be measured in conjunction with the expression of albumen 2;
Thioredoxin that steps A is recorded and thioredoxin combine albumen 2 expressions in conjunction with thioredoxin in albumen 2 expressions and the normal galactophore tissue and thioredoxin and compare, as the thioredoxin expression that records is higher than normal value, thioredoxin is lower than normal value in conjunction with albumen 2 expressions, represents that then thioredoxin and thioredoxin are in conjunction with the abnormal expression of albumen 2 in the detected tissue.
Method of the present invention is used for diagnosing mammary cancer.
Description of drawings:
Fig. 1 is the immunoblotting assay result schematic diagram of thioredoxin at normal perienchyma of breast cancer and enclosed mass tissue, and the demonstration thioredoxin is expressed in patient with breast cancer's enclosed mass tissue obviously and raised;
Fig. 2 be thioredoxin in conjunction with the immunoblotting assay result schematic diagram of albumen 2 at normal perienchyma of breast cancer and enclosed mass tissue, show that thioredoxin expresses obvious the downward modulation in conjunction with albumen 2 in patient with breast cancer's enclosed mass tissue.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.
The experimental technique of unreceipted actual conditions in the following enforcement, the condition of advising usually according to normal condition, laboratory manual or manufacturer.
All enclosed mass tissues of present embodiment and normal perienchyma sample are all taken from same patient with breast cancer's paired samples, and 30 routine sample ages were at 28～71 years old.
Embodiment 1:
The preparation of normal perienchyma of breast cancer and enclosed mass histone quality sample:
Breast tissue: sample is all from attached first hospital of Yunnan Province unming Medical College, and all tumours have all obtained pathology and made a definite diagnosis.
Normal perienchyma of breast cancer and enclosed mass tissue protein sample preparation methods, specific as follows:
1, the flesh tissue piece of excision is stored in-80 ℃ of refrigerators.
When 2, preparing protein, cut the piece of tissue of 5 millimeter, grind to form cell precipitation on ice with mill, add 120 microlitre cell pyrolysis liquids (sodium chloride, NP-40, Tris, PMSF, aprotimin, NaN3/100 milliliter), place and act on 50 minutes on ice, 12000r/min afterwards, 4 ℃ centrifugal 15 minutes, get supernatant and be stored in-80 ℃ of refrigerators.
Embodiment 2:
Thioredoxin and thioredoxin are verified in conjunction with albumen 2 detected by Western blot:
For confirming thioredoxin and thioredoxin differential expression in conjunction with albumen 2, get 30 routine patient with breast cancers' enclosed mass tissue and corresponding normal perienchyma protein example, carry out immunoblotting assay with anti-thioredoxin, thioredoxin respectively in conjunction with the antibody of albumen 2, detailed process is summarized as follows:
Every routine sample is got the total applied sample amount of 7ug protein, separates with 15%SDS-PAGE, shifts on the matter pvdf membrane (available from Millipore company), and skimmed milk is hatched pvdf membrane for 4 ℃ and spent the night.With TPBS washing three times, each 5 minutes.The one anti-mouse anti human thioredoxin antibody (professor of the molten department of Kyoto Univ Japan's shallow lake well provides) that uses was hatched under the room temperature 1 hour, with TPBS washing three times, each 5 minutes.(affinity purifiedantibody peroxidase labled goat anti-mouse lgG (H+L) HSA liquid conjugate is available from U.S. Kirkegaard ﹠amp for two anti-mouse; Laboratories), incubated at room 1 hour, again with TPBS washing 5 times, each 10 minutes of first three time, twice of back is each 5 minutes.Use chemical luminous substrate (available from crystalline substance U.S. biotech firm) to spray pvdf membrane reagent at last, with X-mating plate (available from Lekai company) exposure tests, testing result as shown in Figure 1, thioredoxin expression in breast cancer enclosed mass tissue is higher than in corresponding normal perienchyma.
Every routine sample is got the total applied sample amount of 30ug protein, separates with 10%SDS-PAGE, shifts on the matter pvdf membrane (available from Millipore company), and skimmed milk is hatched pvdf membrane for 4 ℃ and spent the night.With TPBS washing three times, each 5 minutes.The anti-Trx of one anti-use rabbit was hatched under the room temperature 2 hours in conjunction with albumen 2 antibody (santa cruz Biotechnology), with TPBS washing three times, each 5 minutes.Two anti-(affinity purifiedantibody peroxidase labled goat anti-rabbit lgG (H+L) HSA liquid conjugate is available from U.S. Kirkegaard ﹠amp for anti-rabbit; Laboratories), incubated at room 1 hour is again with TPBS washing 6 times, each 10 minutes.Last chemical luminous substrate (available from crystalline substance U.S. biotech firm) sprays pvdf membrane reagent, with X-mating plate (available from Lekai company) exposure tests, testing result as shown in Figure 2, thioredoxin is lower than in corresponding normal perienchyma in conjunction with albumen 2 expression in breast cancer enclosed mass tissue.
The Western blotting result shows, substantially all present such phenomenon in the 30 pairs of enclosed mass tissues and the normal perienchyma: the band concentration of thioredoxin is apparently higher than corresponding normal perienchyma in the enclosed mass tissue, and thioredoxin is starkly lower than accordingly normally perienchyma in conjunction with the band concentration of albumen 2.As seen there is high expressed in thioredoxin in breast cancer enclosed mass tissue, and there be low the expression in thioredoxin in conjunction with albumen 2.
In sum, thioredoxin and thioredoxin there are differences expression in conjunction with albumen 2 in the enclosed mass tissue of breast cancer and normal perienchyma, and obviously the generation development with breast cancer has closely related property.Therefore, as the protein molecular mark its expression is detected in conjunction with albumen 2 with thioredoxin and thioredoxin and can be used to detect breast cancer.
Though, be sure as the label that detects breast cancer with it about thioredoxin and thioredoxin are still waiting further research in conjunction with albumen 2 dynamic biological functions and tumour related mechanism.Thioredoxin and thioredoxin can be used as the potential sign of breast cancer in conjunction with albumen 2, and its biological function prompting thioredoxin and thioredoxin in born of the same parents may be as the prognosis molecule sign of breast cancer and the target molecule of clinical treatment in conjunction with albumen 2.
Claims (6)
1, the molecular marked compound of diagnosing mammary cancer, this method comprise acquisition mammary gland nodules tissue and normal perienchyma sample from sample, and thioredoxin and thioredoxin are in conjunction with the expression of albumen 2 in mensuration and the comparative sample.
2, method as claimed in claim 1, wherein the expression of albumen is measured by detected by Western blot.
3, method as claimed in claim 1 is wherein when the thioredoxin expression is higher than, when thioredoxin is lower than the expression of NBT in conjunction with albumen 2 expressions, be judged as breast cancer.
4, method as claimed in claim 1, relatively the expression that thioredoxin and thioredoxin combine albumen 2 in enclosed mass tissue and the normal perienchyma be the enclosed mass tissue that obtains in conjunction with the antibody test of albumen 2 with the anti-thioredoxin/thioredoxin of specificity thioredoxin, thioredoxin in conjunction with the expression of albumen 2 and normally in the perienchyma expression of these two kinds of albumen compare, as the enclosed mass that records organizes the thioredoxin expression to be higher than normal perienchyma, when thioredoxin is lower than normal perienchyma in conjunction with albumen 2 expressions, be diagnosed as breast cancer.
5, thioredoxin and thioredoxin are in conjunction with the protein molecular marker of albumen 2 as detection breast cancer.
6, thioredoxin and thioredoxin are used for clinical diagnosis breast cancer in conjunction with albumen 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009100941682A CN101493464A (en) | 2009-03-09 | 2009-03-09 | Molecular marker for breast carcinoma diagnosis |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009100941682A CN101493464A (en) | 2009-03-09 | 2009-03-09 | Molecular marker for breast carcinoma diagnosis |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012067525A1 (en) | 2010-11-18 | 2012-05-24 | Pomorski Uniwersytet Medyczny | Genotypes and selenium level as a markers of breast/ovarian cancer risk in brca1 mutation carriers |
| WO2012125051A1 (en) | 2011-03-14 | 2012-09-20 | Pomorski Uniwersytet Medyczny | Selenoprotein genotypes and serum selenium level as markers of cancer risk |
| CN104991010A (en) * | 2015-07-29 | 2015-10-21 | 中国药科大学 | Breast cancer subtype biomarker distinguishing composition |
| WO2018052153A1 (en) * | 2016-09-13 | 2018-03-22 | 주식회사 이앤에스헬스케어 | Monoclonal antibody specifically binding to thioredoxin 1, and use thereof |
| CN109371130A (en) * | 2018-11-19 | 2019-02-22 | 北京大学深圳医院（北京大学深圳临床医学院） | RIPOR3 is in preparation for the application in the biological products of breast cancer detection and treatment |
-
2009
- 2009-03-09 CN CNA2009100941682A patent/CN101493464A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012067525A1 (en) | 2010-11-18 | 2012-05-24 | Pomorski Uniwersytet Medyczny | Genotypes and selenium level as a markers of breast/ovarian cancer risk in brca1 mutation carriers |
| WO2012125051A1 (en) | 2011-03-14 | 2012-09-20 | Pomorski Uniwersytet Medyczny | Selenoprotein genotypes and serum selenium level as markers of cancer risk |
| CN104991010A (en) * | 2015-07-29 | 2015-10-21 | 中国药科大学 | Breast cancer subtype biomarker distinguishing composition |
| WO2018052153A1 (en) * | 2016-09-13 | 2018-03-22 | 주식회사 이앤에스헬스케어 | Monoclonal antibody specifically binding to thioredoxin 1, and use thereof |
| CN109863176A (en) * | 2016-09-13 | 2019-06-07 | E&S医疗保健有限公司 | The monoclonal antibody and application thereof specifically bound with thioredoxin 1 |
| US11002739B2 (en) | 2016-09-13 | 2021-05-11 | E&S Healthcare Co., Ltd. | Monoclonal antibody specifically binding to thioredoxin 1, and use thereof |
| CN109863176B (en) * | 2016-09-13 | 2023-04-21 | E&S医疗保健有限公司 | Monoclonal antibody specifically binding to thioredoxin 1 and use thereof |
| CN109371130A (en) * | 2018-11-19 | 2019-02-22 | 北京大学深圳医院（北京大学深圳临床医学院） | RIPOR3 is in preparation for the application in the biological products of breast cancer detection and treatment |
| CN109371130B (en) * | 2018-11-19 | 2021-08-13 | 北京大学深圳医院（北京大学深圳临床医学院） | Application of RIPOR3 in preparation of biological products for breast cancer detection and treatment |
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