CN103954768A - Application of SAMSN1 protein in preparation of glioblastoma prognosis evaluation reagent or kit - Google Patents
Application of SAMSN1 protein in preparation of glioblastoma prognosis evaluation reagent or kit Download PDFInfo
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- CN103954768A CN103954768A CN201410140083.4A CN201410140083A CN103954768A CN 103954768 A CN103954768 A CN 103954768A CN 201410140083 A CN201410140083 A CN 201410140083A CN 103954768 A CN103954768 A CN 103954768A
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Abstract
The invention relates to the field of biotechnology, and provides a new application of SAMSN1 protein, especially an application of SAMSN1 protein in preparation of a glioblastoma prognosis evaluation reagent or kit. According to the invention, an immunohistochemical method is adopted to detect the relative expression level of SAMSN1 protein in glioblastoma tissue, so as to determine the survival prognosis of glioblastoma patients. Based on the correlation of the relative expression level of SAMSN1 protein with glioblastoma, the protein can be used as a molecular marker, and detection of the expression level of the protein can guide the prognosis evaluation of glioblastoma.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of new purposes of SAMSN1 albumen, specifically, relate to the application of SAMSN1 albumen in preparation glioblastoma prognosis evaluation reagent or kit.
Background technology
Glioma is the most common primary tumo(u)r of central nervous system, and glioblastoma (Gliobalstoma multiforme, GBM) is the most common and type that grade malignancy is the highest wherein.At present, classical Pathomorphology method is still the main method of glioblastoma diagnosis, classification, has many deficiencies.Such as, be diagnosed as equally in the case of GBM, although most of patients life cycle is about 1 year, also there are some survival of patients phases can reach more than 3 years.Under the roughly the same prerequisite of the therapeutic scheme of accepting, the otherness of glioblastoma patient's prognosis points out this tumour may have some hypotypes in genetic background and pathomechanism, distinguishes GBM hypotype and accurate judging prognosis but still lack at present effective index.
This area is in the urgent need to finding related gene and/or the albumen that can distinguish glioblastoma molecular isoform and accurate judging prognosis, and the research of this respect is significant to clinical treatment glioblastoma and prevention tumor recurrence.
SAMSN1 albumen (SAM domain, SH3 domain and nuclear localization signal 1, GeneID:64092) function is still not fully aware of at present, infer and there is adaptor protein function, can be combined with other molecules, in signal transduction cascade reaction, play a role, and may participate in mediating the cell nuclear localization of correlation molecule.Studies show that, SAMSN1 participates in activation and the atomization of immunocyte; In normal brain tissue, there is a certain amount of low expression level in SAMSN1; Relevant to the pathomechanism of marrow series leukemia, Huppert's disease; But the effect of SAMSN1 is still not clear in entity tumor.(referring to document: von HM, Gohla A, Janssen KP, Iritani BM, Beer-Hammer S.Immunoinhibitory adapter protein Src homology domain3lymphocyte protein2 (SLy2) regulates actin dynamics and B cell spreading.J Biol Chem.2011.286 (15): 13489-501.; Claudio JO, Zhu YX, Benn SJ, et al.HACS1encodes a novel SH3-SAM adaptor protein differentially expressed in normal andmalignant hematopoietic cells.Oncogene.2001.20 (38): 5373-7.)
Up to now, the molecular mechanism of the generation of glioblastoma is still unclear, and effectively diagnosis, somatotype, treatment and prognosis judge that target spot is also very limited.
There is no at present bibliographical information SAMSN1 judges relevant to glioblastoma pathomechanism or prognosis.
Summary of the invention
The object of the present invention is to provide the application in preparation glioblastoma prognosis evaluation reagent or kit of the new purposes of SAMSN1 albumen, particularly SAMSN1 albumen.
The inventor, through extensive and deep research, finds first, adopts ImmunohistochemistryMethods Methods to detect the relative expression quantity of SAMSN1 albumen in glioblast tumor tissue, can judge glioblastoma patient's existence prognosis.This correlativity of the relative expression quantity based on SAMSN1 albumen and glioblastoma, detects the prognosis judgement that can be used for instructing glioblastoma using this albumen as molecular labeling to its expression.
The invention provides the application of SAMSN1 albumen in preparation glioblastoma prognosis evaluation reagent or kit.
Described application, is the antibody of preparing SAMSN1 albumen with conventional method, sets up and detects the quantivative approach of SAMSN1 albumen and supporting reagent or kit.
The antibody of described SAMSN1 albumen is monoclonal antibody or polyclonal antibody.
Described application, refers to using SAMSN1 albumen as molecular labeling, utilizes SAMSN1 monoclonal antibody or polyclonal antibody, and immunohistochemical experiment reagent, analyzes the relative expression quantity of SAMSN1 albumen in glioblast tumor tissue.
Utilize the antibody of the anti-SAMSN1 albumen of specificity, comprise monoclonal antibody and polyclonal antibody, for the preparation of preparation or the kit of determining glioblastoma operation prognosis, this it will be apparent to those skilled in the art that.
Described SAMSN1 polyclonal antibody is commercialization antibody, preparation method is referring to document Yokoyama, W.M., Christensen, M., Santos, G.D.and Miller, D.2006.Production of Monoclonal Antibodies.Current Protocols in Immunology.74:2.5.1 – 2.5.25.
Described application, supporting reagent or kit also comprise immunohistochemical experiment reagent, i.e. dimethylbenzene, ethanol, 3%H
2o
2methanol solution, 1%BSA confining liquid, DAB chromogenic reagent, haematoxylin and horseradish peroxidase.
Further, the present invention also provides and has utilized above-mentioned supporting reagent or kit, the method for the expression of vitro detection SAMSN1 albumen in glioblast tumor tissue, and the method comprises the following steps:
(a) utilize immunohistochemical experiment reagent (dimethylbenzene, ethanol, the 3%H in this kit
2o
2methanol solution, 1%BSA confining liquid, DAB chromogenic reagent, haematoxylin and horseradish peroxidase) glioblastoma histotomy carries out immunohistochemical staining by mark goat anti-rabbit igg;
(b) the female small cell tumor tissue dyeing of light Microscopic observation colloid situation, carries out integration to SAMSN1 antibody staining situation in tumour cell endochylema;
(c) judge SAMSN1 high expressed or low expression in glioblastoma according to scoring.
Described the method concrete steps are as follows:
(1) prepare glioblastoma tissue paraffin section de, 60 DEG C of baking boxs spend the night;
(2) section is de-cured to water;
(dimethylbenzene is 2. 3. 10min → 100% ethanol 5min → 95% ethanol 5min → 85% ethanol 5min → 75% ethanol 5min → distilled water 5min of 10min → dimethylbenzene of 10min → dimethylbenzene 1.)
(3) 3%H
2o
2methanol solution, room temperature is placed 20min;
(4) distilled water is washed 5min × 3;
(5) antigen retrieval: section is put into 0.01M citrate buffer (pH6.0) and boiled 5min, truce 10min, then boil 5min;
(6) naturally cool to room temperature, distilled water is washed 5min × 3;
(7) 1%BSA sealing 30min, 37 DEG C;
(8) get rid of serum deprivation confining liquid, drip primary antibodie (the anti-human SAMSN1 polyclonal antibody of goat), 37 degrees Celsius continue one hour;
(9) 4 DEG C of taking-ups, room temperature rewarming 15min, then 0.01M PBS washes 5min × 4;
(10) drip two anti-(the anti-kits of Envision bis-), hatch according to two anti-kit instructionss.
(11) 0.01M PBS washes 5min × 4, the DAB 2-10min that develops the color, Microscopic observation;
(12) distilled water color development stopping, haematoxylin is redyed 10 seconds;
(13) after differentiation, tap water returns indigo plant, distilled water immersion;
(14) dewater transparent, cover glass cover;
(15) micro-Microscopic observation positive staining, random 5 high power lens visuals field, 100 cells of each visual field counting, the calculating positive cell place percent selected of each sample.Positive there is yellow dyeing in cytoplasm, according to the depth of dyeing, be divided into Three Estate, be wherein light yellowly designated as 1 point, brown color is designated as 2 points, and sepia is designated as 3 points; There is the positive cell of cell of yellow dyeing in cytoplasm, calculate the quantity of the positive cell in 100 cells, score value standards of grading: ratio≤10% of positive cell is designated as 0 point ,≤60% is designated as 1 point, ≤ 90% is designated as 2 points, and > 90% is designated as 3 points.Be multiplied by positive rate integration with dyeing integration, be the whole integration of SAMSN1 expression in corresponding cellular component in this sample point.
(16) SAMSN1 is low expression in intracytoplasmic integration≤5, and > 5 is high expressed.
The existence or the death that are found to be after prediction glioblastoma risk of recurrence and operation in patients of SAMSN1 albumen of the present invention and glioblastoma correlativity provide a brand-new approach, to judging that glioblastoma patient prognosis has vital role, also has important directive significance for the postoperative monitoring of glioblastoma patient and sequential therapy.Mark higher than 5 timesharing when SABC, glioblastoma is prone to recurrence, and patient's shorter survival is short, easily dead.
The present invention utilizes immunohistochemistry technique, system scoring to measure the expression of SAMSN1 albumen in glioblastoma tumor tissues, and in conjunction with Follow-up After information, determine that the rear glioblastoma patient prognosis of SAMSN1 protein expression level and operation exists correlativity, SAMSN1 albumen can, for the preparation of the protein molecular marker that judges glioblastoma patient prognosis, also have important directive significance for the postoperative monitoring of molecule parting, patient and the sequential therapy of glioblastoma.
Brief description of the drawings
Fig. 1 is the immunohistochemical staining result figure of SAMSN1 in glioblast tumor tissue (A) and normal cerebral tissue (B); In tumor tissues, visible SAMSN1 strong positive is painted, and normal cerebral tissue is weak painted.
Fig. 2 is the expression of results figure of SAMSN1 in glioblast tumor tissue and normal cerebral tissue; Show that the expression integrated value of SAMSN1 in normal cerebral tissue and glioblast tumor tissue is respectively 1.69 ± 1.30 and 5.86 ± 3.02, difference between the two has significant statistical significance (p<0.01).Fig. 3 is SAMSN1 immunohistochemical staining result figure in glioblast tumor tissue; In visible tumour cell endochylema, SAMSN1 is strong positive painted (integration >5) widely, SAMSN1 high expressed in tumor tissues.
Fig. 4 is SAMSN1 immunohistochemical staining result figure in glioblast tumor tissue; In visible tumour cell endochylema, SAMSN1 is the positive being dispersed in painted (integration <5), the low expression of SAMSN1 in tumor tissues.
Fig. 5 is that SAMSN1 expresses the influence curve figure to 113 routine glioblastoma life cycles; Wherein A is the difference of overall life cycle (OS) between SAMSN1 high expressed and two groups of patients of low expression; B is the difference of Progression free survival phase (PFS) between SAMSN1 high expressed and two groups of patients of low expression.
Embodiment
Below in conjunction with drawings and Examples of the present invention, enforcement of the present invention is elaborated; following examples are to implement under taking technical solution of the present invention as prerequisite; provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
In following examples, glioblastoma patient's tumor tissues sample, all from Shanghai Long March Hospital, is clearly glioblastoma by 2 Pathologis.
Embodiment 1:
(tumor tissue section, all from Shanghai Long March Hospital, is diagnosed as glioblastoma by 2 Pathologis because operation needs the paraffin section of normal cerebral tissue of excision to choose at random tumor tissues and 16 examples after 113 routine glioblastoma operation in patients; Normal cerebral tissue need to go the brain tissue of interior decompression excision from patients with cerebral injury because of the state of an illness, obtaining all of sample examined by The 2nd Army Medical College ethics), the present invention adopts ImmunohistochemistryMethods Methods to detect expression the scoring of SAMSN1 albumen in glioblast tumor tissue and normal cerebral tissue, and concrete steps are:
(1) prepare glioblastoma tissue paraffin section de, 60 DEG C of baking boxs spend the night;
(2) section is de-cured to water;
(dimethylbenzene is 2. 3. 10min → 100% ethanol 5min → 95% ethanol 5min → 85% ethanol 5min → 75% ethanol 5min → distilled water 5min of 10min → dimethylbenzene of 10min → dimethylbenzene 1.)
(3) 3%H
2o
2methanol solution, room temperature is placed 20min;
(4) distilled water is washed 5min × 3;
(5) antigen retrieval: section is put into 0.01M citrate buffer (pH6.0) and boiled 5min, truce 10min, then boil 5min;
(6) naturally cool to room temperature, distilled water is washed 5min × 3;
(7) 1%BSA sealing 30min, 37 DEG C;
(8) get rid of serum deprivation confining liquid, drip primary antibodie (the anti-human SAMSN1 polyclonal antibody of goat, purchased from Abgent company, model AF1955a), 37 degrees Celsius continue one hour;
(9) 4 DEG C of taking-ups, room temperature rewarming 15min, then 0.01M PBS washes 5min × 4;
(10) drip two anti-(the anti-kit of Envision bis-, purchased from DAKO company of Denmark, model GK500705), hatch according to two anti-kit instructionss.
(11) 0.01M PBS washes 5min × 4, the DAB 2-10min that develops the color, Microscopic observation;
(12) distilled water color development stopping, haematoxylin is redyed 10 seconds;
(13) after differentiation, tap water returns indigo plant, distilled water immersion;
(14) dewater transparent, cover glass cover;
(15) micro-Microscopic observation positive staining, random 5 high power lens visuals field, 100 cells of each visual field counting, the calculating positive cell place percent selected of each sample.Positive there is yellow dyeing in cytoplasm, according to the depth of dyeing, be divided into Three Estate, be wherein light yellowly designated as 1 point, brown color is designated as 2 points, and sepia is designated as 3 points; There is the positive cell of cell of yellow dyeing in cytoplasm, calculate the quantity of the positive cell in 100 cells, score value standards of grading: ratio≤10% of positive cell is designated as 0 point ,≤60% is designated as 1 point, ≤ 90% is designated as 2 points, and > 90% is designated as 3 points.Be multiplied by positive rate integration with dyeing integration, be the whole integration of SAMSN1 expression in corresponding cellular component in this sample point.
(16) as shown in Figure 1, in tumour cell endochylema, visible SAMSN1 is strong positive dyeing (Figure 1A) widely, presents remarkable high expressed.The normal cerebral tissue weak painted (Figure 1B) of contrast, through the experimental verification of many groups, as shown in Figure 2, the expression integrated value of SAMSN1 in glioblast tumor tissue and normal cerebral tissue is respectively 1.69 ± 1.30 and 5.86 ± 3.02, and difference between the two has significant statistical significance (p<0.01).
Embodiment 2:
Get at random the tumor specimen of glioblastoma corrective surgery excision, the histotomy of preparation glioblastoma patient tumors, adopt the test procedure of above ImmunohistochemistryMethods Methods to detect the relative expression quantity of SAMSN1 albumen in glioblast tumor tissue, calculate SABC scoring.Detect altogether 113 routine glioblast tumor tissues, find that the expression integration of SAMSN1 in 77 examples (68.14%) glioblast tumor tissue is higher than 5 points.Express height (when SABC scoring is divided into high expressed higher than 5, being divided into low expression lower than 5) according to SAMSN1 and, by glioblastoma patient grouping, draw overall survivorship curve figure (Fig. 5 A) and Progression free survival curve map (Fig. 5 B).Result shows that SAMSN1 in tumour is Overall survival and Progression free survival time after the corrective surgery of high expressed and is all significantly shorter than low expresser.
Embodiment 3:
Sample: the histotomy of certain glioblast cancer patient tumors, adopts the test procedure of above ImmunohistochemistryMethods Methods to detect the relative expression quantity of SAMSN1 albumen in glioblast tumor tissue.The displaing micro picture of glioblast tumor tissue as shown in Figure 3.As calculated, the scoring of the SABC of this tissue is 9.
Know 6 months tumor recurrences dead after this operation in patients, only 6 months life cycle through Follow-up After.
Embodiment 4:
Sample: the histotomy of certain glioblastoma patient tumors, adopts the test procedure of above ImmunohistochemistryMethods Methods to detect the relative expression quantity of SAMSN1 albumen in glioblast tumor tissue.The displaing micro picture of glioblast tumor tissue as shown in Figure 4.As calculated, the scoring of the SABC of this tissue is 0.
Know through Follow-up After, within after this operation in patients 37 months, be still still living and in good health, and without tumor recurrence.
From above test findings, can predict Progression free survival time and the Overall survival after glioblastoma operation in patients by adopting the method for SABC to detect SAMSN1 protein molecular relative expression quantity.When SABC scoring is higher than 5 time, glioblastoma is prone to recurrence, easily dead after operation in patients.Obviously, SAMSN1 albumen and glioblastoma have correlativity, therefore, using SAMSN1 albumen as protein molecular marker, its expression are detected to events such as can predicting glioblastoma recurrence after operation judging prognosis.Accordingly, the antibody of the anti-SAMSN1 albumen of specificity, comprises monoclonal antibody and polyclonal antibody, can be for the preparation of preparation or the kit of determining glioblastoma operation prognosis, and this it will be apparent to those skilled in the art that.
Below the preferred embodiment of the invention is illustrated, but the invention is not limited to described embodiment, those of ordinary skill in the art also can make all modification being equal to or replacement under the prerequisite without prejudice to the invention spirit, and the modification that these are equal to or replacement are all included in the application's claim limited range.
Claims (6)
- The application of 1.SAMSN1 albumen in preparation glioblastoma prognosis evaluation reagent or kit.
- 2. the application of SAMSN1 albumen according to claim 1 in preparation glioblastoma prognosis evaluation reagent or kit, it is characterized in that, this application is the antibody of preparing SAMSN1 albumen with conventional method, sets up and detects the quantivative approach of SAMSN1 albumen and supporting reagent or kit.
- 3. the application of SAMSN1 albumen according to claim 2 in preparation glioblastoma prognosis evaluation reagent or kit, is characterized in that the antibody of described SAMSN1 albumen is monoclonal antibody or polyclonal antibody.
- 4. the application of SAMSN1 albumen according to claim 2 in preparation glioblastoma prognosis evaluation reagent or kit, it is characterized in that, this application refers to using SAMSN1 albumen as molecular labeling, utilize SAMSN1 monoclonal antibody or polyclonal antibody, and immunohistochemical experiment reagent, analyze the relative expression quantity of SAMSN1 albumen in glioblast tumor tissue.
- 5. the application in preparation glioblastoma prognosis evaluation reagent or kit according to the arbitrary described SAMSN1 albumen of claim 2 to 4, is characterized in that, described supporting reagent or immunohistochemical experiment reagent comprise: dimethylbenzene, ethanol, 3%H 2o 2methanol solution, 1%BSA confining liquid, DAB chromogenic reagent, haematoxylin and horseradish peroxidase.
- 6. the application in preparation glioblastoma prognosis evaluation reagent or kit according to the arbitrary described SAMSN1 albumen of claim 2 to 4, it is characterized in that, this application is to utilize this reagent or kit, the method of the expression of vitro detection SAMSN1 albumen in glioblast tumor tissue, the method comprises the following steps:(a) utilize the immunohistochemical experiment reagent mark goat anti-rabbit igg in this kit that glioblastoma histotomy is carried out to immunohistochemical staining;(b) the female small cell tumor tissue dyeing of light Microscopic observation colloid situation, carries out integration to SAMSN1 antibody staining situation in tumour cell endochylema;(c) judge SAMSN1 high expressed or low expression in glioblastoma according to scoring.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107460251A (en) * | 2017-09-28 | 2017-12-12 | 郑州大学第附属医院 | A kind of the glioblastoma auxiliary diagnosis based on FUCA1 genes, prognostic evaluation kit and its application method |
| CN107574247A (en) * | 2017-09-28 | 2018-01-12 | 郑州大学第附属医院 | A kind of the glioblastoma auxiliary diagnosis based on CLCF1 genes, prognostic evaluation kit and its application method |
| CN110885886A (en) * | 2018-09-07 | 2020-03-17 | 华中科技大学同济医学院附属协和医院 | Method for differential diagnosis of glioblastoma and typing of survival prognosis of glioma |
| CN117074692A (en) * | 2023-08-15 | 2023-11-17 | 复旦大学附属华山医院 | Identification method for tumor cell HER2 antigen expression biological information in cerebrospinal fluid |
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| US20110081648A1 (en) * | 2005-02-28 | 2011-04-07 | Norman Latov | Methods for diagnosing peripheral neuropathy |
Non-Patent Citations (2)
| Title |
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| YONG YAN: "SAMSN1 Is Highly Expressed and Associated with a Poor Survival in Glioblastoma Multiforme", 《PLOS ONE》 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107460251A (en) * | 2017-09-28 | 2017-12-12 | 郑州大学第附属医院 | A kind of the glioblastoma auxiliary diagnosis based on FUCA1 genes, prognostic evaluation kit and its application method |
| CN107574247A (en) * | 2017-09-28 | 2018-01-12 | 郑州大学第附属医院 | A kind of the glioblastoma auxiliary diagnosis based on CLCF1 genes, prognostic evaluation kit and its application method |
| CN110885886A (en) * | 2018-09-07 | 2020-03-17 | 华中科技大学同济医学院附属协和医院 | Method for differential diagnosis of glioblastoma and typing of survival prognosis of glioma |
| CN110885886B (en) * | 2018-09-07 | 2023-04-07 | 华中科技大学同济医学院附属协和医院 | Method for differential diagnosis of glioblastoma and typing of survival prognosis of glioma |
| CN117074692A (en) * | 2023-08-15 | 2023-11-17 | 复旦大学附属华山医院 | Identification method for tumor cell HER2 antigen expression biological information in cerebrospinal fluid |
| CN117074692B (en) * | 2023-08-15 | 2024-04-09 | 复旦大学附属华山医院 | Identification method for tumor cell HER2 antigen expression biological information in cerebrospinal fluid |
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Application publication date: 20140730 |