CN106546740B - Detection of the CA2 in nasopharyngeal carcinoma, treatment application - Google Patents
Detection of the CA2 in nasopharyngeal carcinoma, treatment application Download PDFInfo
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Abstract
The invention discloses a kind of CA2 quantitative reagents to prepare the application in nasopharyngeal carcinoma diagnosis reagent.And disclosing expression quantity of the CA2 in nasopharyngeal carcinoma has the characteristics of apparent rising, this illustrates that CA2 has the potential as nasopharyngeal carcinoma auxiliary diagnosis marker.It also illustrates simultaneously, CA2 inhibitor great clinical value in the pharmaceutical composition of preparation treatment nasopharyngeal carcinoma, provides new thinking for effective treatment of nasopharyngeal carcinoma.
Description
Technical field
The application of the detection, treatment that the present invention relates to CA2 in nasopharyngeal carcinoma.
Background technique
Nasopharyngeal carcinoma (Nasopharyngeal carcinoma, NPC) is the malignant tumour that a kind of lesion is located at pharynx nasalis,
Once morbidity shows as rapid progress, and has the invasion and transfer characteristic of height, and with headache, tinnitus, hearing or eyesight
Decline.Meanwhile the great regionality of nasopharyngeal carcinoma morbidity, it is high-incidence southern in SOUTHERN CHINA area, Southeast Asia, the Middle East and Africa,
It is annual that the disease incidence in Guangdong Province is up to every 100,000 people of 10 ~ 30 people.With the development of the course of disease, 5 years survival rate dramatic decreases of patient,
If 5 years survival rates of clinical I phase patient are up to 90%, and the end-stage patients of clinic IV phase, 5 years survival rates then only remain 58%.Therefore, if
It does not find and treats early, nasopharyngeal carcinoma will cause great threat to patient vitals.
However, the early diagnosis of nasopharyngeal carcinoma is always problem, it is primarily due to that Nasopharyngeal Carcinoma Lesions are hidden, and illness is unknown
It is aobvious, it is difficult to find, most of the nasopharyngeal carcinoma of hospital's detection has been advanced stage.High sensitivity and specificity are taken into account further, since lacking
Good serodiagnosis marker makes nasopharyngeal carcinoma be difficult to become hospital's routine inspection project.Currently, the diagnostic method master of nasopharyngeal carcinoma
There is centainly invasive dependent on the tissue biopsy by conchoscope.In addition, due to nasopharyngeal carcinoma and Epstein-Barr virus
(Epstein-Barr virus, EBV) infection is highly relevant, and the nucleic acid or albumen for clinically also using Epstein-Barr virus are as auxiliary
Diagnosis index or screening indexes, such as EBV capsid antigen IgA (VCA-IgA), early antigen Ig A
(EA-IgA), nuclear antigen (EBNA1) IgA, circulating EBV DNA etc..But due to ebv infection
Relatively conventional in crowd, These parameters can not be as the diagnosis index of confirmation.Although in scientific research it has also been found that some
It is directed to the biomarker of patient itself and the potential diagnostic significance of tool, but is not applied to clinic, such as Bmi-1,
HMGB1, KLHDC4 etc..The treatment of nasopharyngeal carcinoma is based on conventional chemicotherapy, although obtaining certain effect, (3 years just after treatment
Not yet there is specific drug at present up to 80%) in normal survival rate.Moreover, it is widely different between various cancers, it is mainly reflected in difference
The intracellular biological process of cancer is different, and various expressing quantities are also different.The high expression in certain cancer of certain albumen,
Possible expression quantity is very low in another cancer.So the marker of a certain cancer or the effect of specific drug can not be other
It is predicted in cancer, can not be transplanted in another cancer and use.Therefore, it clinically needs to be directed to patient itself
, the good single creature marker for nasopharyngeal carcinoma diagnosis of specificity.
Human carbonic anhydrase 2(Carbonic anhydrase 2, CA2 disclosed by the invention) due to its in nasopharyngeal carcinoma it is special
The anisotropic high expression in ground, and expression quantity is lowered in other cancers, such as in colorectal cancer, clear-cell carcinoma, hepatocellular carcinoma, therefore
The marker that can be used as nasopharyngeal carcinoma specificity is applied to the detection of nasopharyngeal carcinoma, in treatment.
Summary of the invention
The application of the detection, treatment that the invention discloses CA2 in nasopharyngeal carcinoma.
The scheme that the present invention takes is:
CA2 quantitative reagent is preparing the application in nasopharyngeal carcinoma diagnosis reagent.
Preferably, the antibody containing CA2 albumen in the test sample of the diagnostic reagent.
Preferably, the sample that diagnostic reagent can be detected is tissue, blood, serum.
A kind of standard judging nasopharyngeal carcinoma, which is characterized in that when the expression of CA2 is more than 2.5 times of normal person or more,
Judgement suffers from nasopharyngeal carcinoma.
CA2 is as nasopharyngeal carcinoma detection, the application of therapy target.
A kind of oncotherapy medicament, which is characterized in that contain the antagonist of CA2 in medicament.
The beneficial effects of the present invention are:
The expression quantity that the invention discloses CA2 in nasopharyngeal carcinoma has the characteristics of apparent rising, this, which illustrates that CA2 has, becomes
The potential of nasopharyngeal carcinoma auxiliary diagnosis marker.It also illustrates simultaneously, pharmaceutical composition of the CA2 inhibitor in preparation treatment nasopharyngeal carcinoma
Upper great clinical value provides new thinking for effective treatment of nasopharyngeal carcinoma.
Detailed description of the invention
Fig. 1 is organization chip immunohistochemistry measurement result.
Fig. 2 is protein immunoblot measurement result.
Specific embodiment
By following specific experiment example, the present invention is described in further detail, is easy to understand technical side of the invention
Case, embodiments of the present invention are not limited thereto.
Experimental example 1:Super-SILAC MS
The nasopharyngeal tissue sample of 9 nasopharyngeal carcinoma and 9 normal controls is shredded respectively, 1% SDS lysate, liquid nitrogen is added
Grinding, ultrasonic treatment, 17,000 × g are centrifuged 30 min and go to precipitate.Each sample takes equivalent egg respectively in cancer group and normal group
White mixing.Two groups of mixed protein samples are mixed with super-SILAC labelled protein 1:1 in mass ratio respectively, carry out next step
Super filter tube enzymatic hydrolysis.Every group of sample is denaturalized in 8 M urea and 50 mM DTT, and 100 mM IAA alkylation is added, is then transferred to
It is washed in super filter tube with 8 M urea and 50 mM ammonium hydrogen carbonate.Then 1:30 in mass ratio is added pancreatin and is incubated for 8 h.Enzyme is collected by centrifugation
Peptide fragment after solution carries out shotgun mode mass spectral analysis with 5600 MS of TripleTOF (AB SCIEX).With
ProteinPilot (AB SCIEX) software obtains protein quantification knot after searching library (UniProt mankind's holoprotein database)
Fruit.Wherein, the ratio between cancer group CA2 albumen and labelled protein expression quantity are 27.58, and the ratio between normal group and labelled protein expression quantity are
10.31, CA2 relative expression quantity (cancer group/control group) is 2.67.
Experimental example 2:SWATH-MS
The nasopharyngeal tissue sample of 5 nasopharyngeal carcinoma and 6 normal controls is shredded respectively, 1% SDS lysate, liquid nitrogen is added
Grinding, ultrasonic treatment after 17,000 × g centrifugation, 30 min go precipitating, carry out the super filter tube enzymatic hydrolysis of next step.Every group of sample
Be denaturalized in 8 M urea and 50 mM DTT, 100 mM IAA alkylation be added, be then transferred in super filter tube with 8 M urea and
50 mM ammonium hydrogen carbonate are washed.Then 1:30 in mass ratio is added pancreatin and is incubated for 8 h.Peptide fragment after enzymatic hydrolysis is collected by centrifugation is used
5600 MS of TripleTOF (AB SCIEX) carries out the mass spectral analysis of SWATH mode.With ProteinPilot (AB
SCIEX) software obtains after searching library (UniProt mankind's holoprotein database) and the analysis of PeakView (AB SCIEX) software
To every sample protein label-free result.Wherein, cancer group quantitative values are respectively 390256.1,560929.5,
266646.4,1934358.4,78910.2, mean value 646220.1；Normal group quantitative values are respectively 135438.6,
245280.5,47071.3,123667.2,110478.4,16571.5, mean value 113084.6.The average relative expression quantity of CA2
(cancer group/control group) is 5.71.
Experimental example 3: immunohistochemistry
To the organization chip the containing 52 tissues of nasopharyngeal carcinoma slice samples and 13 normal control tissue slice samples
SuperPicture 3rd Gen IHC Detection Kit (ThermoFisher) carries out immunohistochemistry.With
2 antibody of Carbonic anhydrase (1:100 dilution, justice stick up Divine Land) is incubated for, and is then dyed with DAB, hematoxylin
(Hematoxylin) it redyes.Coloration result is with Histologic Score(staining power score value × staining cell proportion)
It indicates.Coloration result is analyzed, as a result as shown in Figure 1, intermediate value of the expression quantity of CA2 in cancer group is 44.38, just
The intermediate value of Chang Zuzhong is 0.The expression quantity of CA2 significant difference (Kolmogorov-Smirnov in cancer group and normal group
Test, two-tailed, P < 0.01), it is more significantly raised than in normal group in cancer group.Meanwhile CA2 is in receiver
Higher diagnosis effect (The Area Under Curve is presented in operating characteristic (ROC) analysis
(AUC)=0.94).
Experimental example 4: protein immunoblot
The nasopharyngeal tissue sample of nasopharyngeal carcinoma and normal control is shredded respectively, 1% SDS lysate of addition, liquid nitrogen grinding, from
After the heart goes precipitating, ultrasonic treatment measures protein concentration.Each sample respectively takes 10 μ g albumen that 10% SDS polyacrylamide gel is added
Middle carry out electrophoresis.The albumen separated is all transferred on pvdf membrane.Rabbit is added to the pvdf membrane after being closed with skim milk
Anti- 2 antibody of Carbonic anhydrase (1:2000 dilution, three hawk of Wuhan), 4 DEG C of overnight incubations.With containing 0.2% polysorbas20
TBS washes film 3 times, every time 5 min.Then goat-anti rabbit secondary antibody (1:2000 dilution, Bioworld) is added, is incubated at room temperature 1 h.With containing
The TBS of 0.2% polysorbas20 washes film 3 times, every time 5 min.Luminescent solution development is added later, takes pictures.Develop the color band intensity gray value
It indicates.The gray value of nasopharyngeal carcinoma group (NPC) is 41516.75, and the gray value of normal group (Normal) is 4121.78.Nasopharyngeal carcinoma group
The expression quantity of middle CA2 is 10.07 times in normal group, shows CA2 high expression (see figure 2) in cancer group.
Claims (2)
1.CA2 quantitative reagent is preparing the application in nasopharyngeal carcinoma diagnosis reagent；The diagnostic reagent is detection tissues of nasopharyngeal carcinoma
Reagent.
2. application according to claim 1, which is characterized in that the antibody containing CA2 albumen in the diagnostic reagent.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101326166A (en) * | 2005-12-09 | 2008-12-17 | 索尔瓦药物有限公司 | Novel N-sulfamoyl-piperidineamides for the prophylaxis or treatment of obesity and related conditions |
| CN102716124A (en) * | 2012-05-25 | 2012-10-10 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | New application of carbonic anhydrase inhibitor |
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- 2016-10-27 CN CN201610968723.XA patent/CN106546740B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101326166A (en) * | 2005-12-09 | 2008-12-17 | 索尔瓦药物有限公司 | Novel N-sulfamoyl-piperidineamides for the prophylaxis or treatment of obesity and related conditions |
| CN102716124A (en) * | 2012-05-25 | 2012-10-10 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | New application of carbonic anhydrase inhibitor |
Non-Patent Citations (2)
| Title |
|---|
| HIF-1α和碳酸酐酶Ⅸ在鼻咽癌中表达及其与患者预后关系;陈宇翰 等;《中华放射肿瘤学杂志》;20150131;第24卷(第1期);第46-47页 * |
| Identification of potential serum markers for nasopharyngeal carcinoma from a xenografted mouse model using Cy-dye labeling combined with three-dimensional fractionation;Chih-Ching Wu et al.;《Proteomics》;20080930;第8卷;摘要，第3606-3609页"2 Materials and methods"，第3609-3617页"3 Results and discussion" * |
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