CN110501501B - Application of tumor marker for early diagnosis of lung cancer and kit - Google Patents
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Abstract
The invention provides an application of a tumor marker for early diagnosis of lung cancer and a kit. The invention relates to an application of two indexes of human serum total acid glycoprotein (alpha-1-acid glycoprotein, AGP/ORM) and transforming growth factor (TGF-beta) which are used as early lung cancer tumor diagnosis markers singly or jointly. Different from the prior report that the serum levels of ORM and TGF-beta are increased in the advanced detection of tumors, the tumor markers ORM and TGF-beta in the invention are proved to be remarkably reduced in the serum of a patient with early lung cancer, and the expression of ORM is regulated and controlled by the expression level of TGF-beta.
Description
Technical Field
The invention relates to the technical field of inspection medicine, in particular to application of a tumor marker for early diagnosis of lung cancer and a kit.
Background
Lung cancer is one of the most common primary malignancies worldwide. Only in 2012, 180 million new lung cancer patients are added all over the world, accounting for 13% of all cancer diagnosis patients, and are the cancers with the highest incidence and the highest cancer mortality rate in men. From the clinical treatment perspective, lung cancer can be divided into two broad categories by combining biological properties: small cell lung cancer and non-small cell lung cancer, wherein non-small cell lung cancer accounts for about 80% -85% of all lung cancers and small cell lung cancer accounts for 15% -20%. Despite the improvement in the therapeutic effect of lung cancer patients in recent years, the overall five-year survival rate of lung cancer patients in the past two decades has remained around 15%. The etiology of lung cancer is not completely clear up to now, and a large amount of data show that a large amount of smoking for a long time has a very close relationship with the occurrence of lung cancer. The five-year survival rate of the lung cancer patients successfully diagnosed at early stage can reach 70-80%. Therefore, lung cancer is also one of the most preventable cancers, and increasing the diagnosis rate of early lung cancer is one of the most effective ways to prevent and reduce lung cancer mortality.
The tumor marker is a substance specifically present in a malignant tumor cell, produced abnormally by a malignant tumor cell, or produced by a host in response to a stimulus to a tumor cell. The tumor marker can reflect the occurrence and development of tumors and monitor the response of tumors to treatment. Tumor markers common to physical examination at present include: 1) CEA: it is commonly found in gastrointestinal cancer, lung cancer, ovarian cancer and other tumors; 2) AFP: is the earliest found tumor marker, and is commonly seen in primary hepatocellular carcinoma; 3) PSA: is common in prostate cancer; 4) CA 199: is common in pancreatic cancer. However, the tumor markers clinically applied at present lack good sensitivity and specificity, so the development of novel high-specificity sensitive tumor markers is very critical, and the development of the novel high-specificity sensitive tumor markers is beneficial to early tumor discovery, patient prognosis, personalized treatment and tumor patient management and follow-up.
Serum total acid glycoprotein is an acute phase protein, and during acute inflammatory reaction, ORM in serum is mainly regulated and expressed by liver parenchymal cells. Recent studies have found that ORM is also expressed in some immune cells, such as neutrophils, macrophages, T cells, etc. Numerous studies have also shown that serum ORM1 or ORM1 in urine is highly expressed in the serum of patients with advanced bladder, kidney, and lung cancer.
Compared with normal people, the invention discovers for the first time that the content of total acid glycoprotein ORM and transforming growth factor (TGF-beta) in serum of a patient with early lung cancer is obviously reduced, and the diagnosis efficiency is higher than that of a conventional tumor marker-CEA index. The combined use of TGF-beta and ORM in serum has higher diagnosis efficiency on early lung cancer patients, so ORM, TGF-beta or ORM & TGF-beta is a reliable tumor diagnosis marker for early diagnosis of lung cancer.
Disclosure of Invention
Aiming at the problem that the existing molecular marker for early lung cancer diagnosis is low in diagnosis positive rate, sensitivity and specificity, the invention provides application of the tumor marker for early lung cancer diagnosis and a kit, namely novel application of ORM and TGF-beta protein.
In order to achieve the above object, an aspect of the present invention provides an application of a tumor marker for early diagnosis of lung cancer in the preparation of a reagent or a kit for early diagnosis of lung cancer, wherein the tumor marker is characterized in that: the tumor marker for early diagnosis of lung cancer is human serum total acid glycoprotein AGP1/ORM or/and human serum transforming growth factor TGF-beta.
Preferably, the total acid glycoprotein in human serum comprises two proteins of AGP1/ORM1 and/or AGP2/ORM 2.
Further, the early diagnosis of lung cancer comprises clinical diagnosis of stage IA and stage IB.
In another aspect, the present invention further provides a kit for early diagnosis of lung cancer, which is characterized in that: comprises an antibody for detecting ORM or a primary antibody with fluorescence, a primary antibody with chemiluminescence and a primary antibody with a label; or an antibody or a primary antibody with fluorescence, a primary antibody with chemiluminescence, or a primary antibody with a label for detecting TGF-beta; or antibodies or fluorescent primary antibodies, chemiluminescent primary antibodies, and labeled primary antibodies that detect ORM and TGF-beta.
As described above, the present invention provides the following technical solutions:
1) the application of the serum ORM and TGF-beta alone or ORM & TGF-beta protein combined diagnosis as a diagnosis marker of early lung cancer:
(1) the application of serum ORM protein as a serum marker for diagnosing early lung cancer;
(2) the application of serum TGF-beta protein as a serum marker for diagnosing early lung cancer;
(3) the serum ORM and TGF-beta combined diagnosis is used as a serum marker for lung cancer diagnosis.
2) The use of an antibody against ORM protein, an antibody against TGF-beta protein, alone or in combination, in the preparation of a test product for the diagnosis of lung cancer.
3) The diagnosis threshold value of the ORM detection kit is 0.65mg/mL, and the detection threshold value of the TGF-beta detection kit is 31.997 ng/mL.
4) The test product tests serum.
5) The products tested were used to differentiate between early lung cancer patients, including patients in stages IA and IB, and healthy, benign, and intermediate and advanced lung cancer patients.
The invention has the following advantages and beneficial effects:
the early lung cancer tumor marker is differentially expressed protein screened from peripheral blood leukocyte of an early lung cancer patient by using a transcriptome sequencing technology, wherein total acid glycoprotein ORM is obviously reduced in serum of the early lung cancer patient, has high specificity and high sensitivity, is combined with TGF-beta in the serum for diagnosis, further improves the sensitivity and specificity of early diagnosis, and can be used for early diagnosis of lung cancer. Different from the prior report that the serum levels of ORM and TGF-beta are increased in the advanced detection of tumors, the tumor markers ORM and TGF-beta in the invention are proved to be remarkably reduced in the serum of patients with early lung cancer, and the expression of ORM is regulated and controlled by the expression level of TGF-beta.
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FIG. 1 shows that serum ORM levels are specifically decreased in early lung cancer patients.
FIG. 2 shows the variation of ORM levels in serum in the tumor stages of lung cancer patients.
FIG. 3 shows the results of ROC curve analysis of ORM in the diagnosis of lung cancer patients at various tumor stages.
FIG. 4 is a graph showing that TGF-. beta.regulates the expression of ORM1 in human peripheral blood leukocytes.
FIG. 5 is a graph showing that TGF-. beta.regulates the expression of ORM1 in human peripheral blood leukocytes via the Smad2/3 signaling pathway.
FIG. 6 is a graph showing the specific decrease in serum TGF- β levels in early lung cancer patients.
FIG. 7 shows the ROC curve analysis of ORM, TGF-. beta.and the results of ORM and TGF-. beta.combination diagnosis in early (IA stage) lung cancer diagnosis.
FIG. 8 shows the performance of ROC curve analysis of ORM, TGF-. beta.and the combined diagnosis of ORM and TGF-. beta.in the diagnosis of early (stage IB) lung cancer.
Wherein: FIGS. 7 and 8 show that the combined diagnostic performance of ORM and TGF-beta is better than that of ORM and TGF-beta alone.
Detailed Description
The invention is explained in further detail below with reference to the figures and the specific embodiments.
The experimental method comprises the following steps:
1) patients and samples were included:
in this experiment, a total of 130 serum samples from lung cancer patients, 59 healthy and 21 benign patient controls were included.
Inclusion and exclusion criteria for tumor patients included: 1) the diagnosis is confirmed as corresponding tumor with definite pathological diagnosis, and the size and pathological stage of the tumor have accurate diagnosis results; 2) initial diagnosis of the tumor without any prior anti-cancer treatment; 3) there are no other acute diseases or systemic diseases such as autoimmune diseases, diabetes, coronary heart disease, etc.
TABLE I clinical information of serum taken into account in lung cancer patients and normal patients
2) Sample collection
Collecting blood into inert separation gel coagulation promoting blood collecting tube, standing at room temperature for 30min, centrifuging at 4 deg.C 400 Xg for 15min, subpackaging serum, and immediately freezing and storing at-80 deg.C for use, wherein each tube of serum can not be frozen and thawed for 2 times before use.
3) Immunoturbidimetry for detecting total acidic glycoprotein in serum
Thawing the frozen lung cancer patient serum sample at-80 deg.C on ice, centrifuging at 4 deg.C and 15000 Xg for 10min, sucking supernatant 10 μ L, manually diluting with sample diluent prepared by detection instrument for 6 times, and diluting with BN x II/BN ProSpec
Detecting, wherein the instrument is set to automatically dilute the sample by 5 times, and a reagent kit of NAntiserum to Human alpha 1-acid Glycoprotein (SIEMENS) is used as a detection reagent.
4) Isolation of human peripheral blood leukocytes
Using a catalyst containing K2Fresh whole blood sample is gathered to the heparin tube of EDTA anticoagulant, according to whole blood in superclean bench: erythrocyte lysate 1: 3, adding erythrocyte lysate (Tiangen), mixing, standing at room temperature for 5min, mixing again, dissolving erythrocyte completely, centrifuging at room temperature of 450 × g for 10min, white sediment at the bottom of the tube is white blood cells, the white blood cells are washed twice by PBS, the white blood cells are centrifuged for 5min at the room temperature of 450 Xg each time, and the PBS is used for re-suspending the white blood cells for later use.
5) Fluorescent quantitative PCR and western immunoblotting examined the effect of TGF- β on ORM expression in peripheral blood leukocytes.
Separating peripheral blood leukocyte of health sample at 2.5 × 107The density of each well was inoculated into 6-well plates, and after 3h of culture, peripheral blood leukocytes were treated with TGF-. beta.s (5, 10, 20, 30, 40, 60ng/mL) in combination with their inhibitors SB431542 and LY2109761 for 24h at 37 ℃ in a 5% CO2 incubator. Cells were harvested, cellular RNA was extracted, and the expression level of ORM1 was detected by fluorescent quantitative PCR. Or collecting cells, lysing the cells on ice by RIPA lysate for 0.5h, centrifuging at 12000 Xg for 15min, collecting supernatant, and detecting the expression of ORM1, Smad2, Smad3 and phosphorylation modified Smad2 and Smad3 by Western blotting.
6) Enzyme-linked immunosorbent assay serum TGF-beta
The TGF enzyme linked immunosorbent assay kit is purchased from Wuhan Ebotaic biotechnology limited, the operation steps are strictly executed according to the instructions provided by manufacturers, the light absorption value of each sample at the position of absorbance of 450nm is detected, and the concentration is calculated.
7) Statistical method
Statistical methods include t-tests, performed using SPSS19.0 for Windows (SPSS inc., Chicago, USA) and Graphpad prism software (version 5.0).
Results of the experiment
1. Serum total acid glycoprotein (ORM) content is significantly reduced in early lung cancer patients. (as shown in FIGS. 1 and 2)
2. The serum total acid glycoprotein (ORM) has good diagnosis effect on early stage (IA + IB stage) lung cancer patients. (as shown in FIG. 3)
The analysis of the points below the ROC curve shows that the ORM has good diagnosis effect on early lung cancer patients. For stage IA patients, the area under the ROC curve for ORM was 0.920, and the optimal threshold for ORM was 0.6525mg/mL, at which time sensitivity and specificity were 91.3% and 79.0%, respectively. For patients in stage IB, the ORM had an area under the ROC curve of 0.817% and sensitivity and specificity of 85.7% and 79.0%, respectively. The above results indicate that ORM has a very good diagnostic effect as early lung cancer.
3. TGF-. beta.was able to significantly regulate the expression of ORM1 in peripheral blood leukocytes (as shown in FIG. 4). The TGF-beta receptor inhibitor (SB431542 and LY2109761) can obviously inhibit the promoting effect of TGF-beta.
4. TGF-. beta.regulates the expression of ORM1 in human peripheral blood leukocytes through the Smad2/3 signaling pathway (as shown in FIG. 5). TGF- β promotes expression of Smad2 and Smad3 and its phosphorylation modifications in peripheral blood leukocytes.
5. Seroconversion growth factor (TGF-. beta.) levels are significantly reduced in early lung cancer patients. (as shown in FIG. 6)
ELISA results show that the TGF-beta content in the serum of early lung cancer patients is obviously lower than that of normal people (P <0.001), wherein the IA patients are most remarkable.
6. The combined diagnostic performance of the ORM and the TGF-beta is better than that of the ORM and the TGF-beta which are used independently to a certain extent. (as shown in FIG. 7 and FIG. 8)
Analysis of the area under the ROC curve shows that TGF-beta has good diagnosis effect on early lung cancer patients. For stage IA patients, the area under the ROC curve for TGF-. beta.is 0.932, and the optimal threshold for TGF-. beta.is 31.997ng/mL, at which time the sensitivity and specificity were 1.00 and 50.0%, respectively. The results of the ORM and TGF-beta combined diagnosis show that for the patients in IA stage, the area under the ROC curve of ORM & TGF-beta is 0.985, and the diagnosis effect is obviously better than that of ORM and TGF-beta which are respectively used independently (figure 7). The results show that ORM & TGF-beta has good diagnosis efficiency as an early lung cancer tumor marker and has great clinical application prospect.
Claims (1)
1. An application of a tumor marker for early diagnosis of lung cancer in preparing a reagent or a kit for early diagnosis of lung cancer is characterized in that: the tumor marker for early diagnosis of the lung cancer is human serum total acid glycoprotein AGP1/ORM or/and human serum transforming growth factor TGF-beta;
the early diagnosis of lung cancer is the clinical diagnosis of the IA stage.
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| WO2013152989A2 (en) * | 2012-04-10 | 2013-10-17 | Eth Zurich | Biomarker assay and uses thereof for diagnosis, therapy selection, and prognosis of cancer |
| CN103076456B (en) * | 2012-12-26 | 2015-02-04 | 潍坊三维生物工程集团有限公司 | Kit for detecting alpha 1-acidoglycoprotein by using immunity transmission turbidity method |
| WO2014193999A2 (en) * | 2013-05-28 | 2014-12-04 | Caris Science, Inc. | Biomarker methods and compositions |
| CN103529221A (en) * | 2013-10-22 | 2014-01-22 | 青岛金洋生物技术有限公司 | Kit for detecting acid glycoprotein content in serum |
| US20150147765A1 (en) * | 2013-11-28 | 2015-05-28 | Korea Basic Science Institute | Serological markers for cancer diagnosis using blood sample |
| CN105785057A (en) * | 2015-12-22 | 2016-07-20 | 山东博科生物产业有限公司 | Alpha 1-acid glycoprotein detection kit |
| CN106680511B (en) * | 2017-01-17 | 2019-04-26 | 南京弘泰德生物科技有限公司 | Serum molecules marker combines the application as pulmonary cancer diagnosis and curative effect monitoring marker |
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