CN110501501A - Lung cancer early diagnoses application and the kit of tumor markers - Google Patents
Lung cancer early diagnoses application and the kit of tumor markers Download PDFInfo
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- CN110501501A CN110501501A CN201910664259.9A CN201910664259A CN110501501A CN 110501501 A CN110501501 A CN 110501501A CN 201910664259 A CN201910664259 A CN 201910664259A CN 110501501 A CN110501501 A CN 110501501A
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
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Abstract
The present invention provides the application of lung cancer early diagnosis tumor markers and kits.The present invention relates to two kinds of human serum total acidic glycoprotein (alpha-1-acid glycoprotein, AGP/ORM), transforming growth factor (TGF-β) indexs, and the purposes as early stage of lung cancer diagnosing tumor marker is used alone or in combination.It is different from ORM, the raising of TGF-β serum levels in previously reported advanced tumor detection, tumor markers ORM in the present invention, TGF-β are proved to be remarkably decreased in early stage of lung cancer patients serum, and the expression of ORM is regulated and controled by TGF-β expression, a kind of new discovery of system of the present invention, ORM, the serum expression quantity of TGF-β reduces the diagnosis marker that can be used as the early stage of lung cancer, show best sensibility and specificity, it can be used as the tumor markers of mass survey, the effect that ORM&TGF- β is used in combination is better than exclusive use.
Description
Technical field
The present invention relates to the application of laboratory medicine technical field, in particular to lung cancer early diagnosis tumor markers and reagents
Box.
Background technique
Lung cancer is one of most common primary malignant tumor in world wide.It is only just newly-increased in global range in 2012
1800000 patients with lung cancer, Zhan Suoyou cancer diagnosis patient 13% are incidence highest and the highest cancer of male cancer lethality.
From clinical treatment angle, lung cancer can be divided into two major classes in conjunction with biological characteristics: Small Cell Lung Cancer and non-small cell lung cancer,
Wherein non-small cell lung cancer accounts for about the 80%-85% of all lung cancer, and Small Cell Lung Cancer accounts for 15%-20%.Although when each in recent years
The therapeutic effect of the patients with lung cancer of phase all increases, but 20 years patients with lung cancer totality five year survival rates still maintain in the past
15% or so.The cause of disease of lung cancer is not completely clear so far, and great mass of data is shown, long-term a large amount of smokings have with lung cancer
Unusual close relationship.The patients with lung cancer that success early diagnoses, five year survival rate can achieve 70%-80%.Therefore, lung cancer
It is also one of most preventible cancer, the diagnosis for improving the early stage of lung cancer is prevention and the reduction most effective approach of lung cancer mortality
One of.
Tumor markers refer to that specificity is present in malignant cell, or the object generated extremely by malignant cell
The substance that matter or host generate the stimulate the reaction of tumour cell.Tumor markers are able to reflect tumorigenesis,
Tumour is monitored to a substance of therapeutic response.The common tumor markers of physical examination at present include: 1) CEA: be common in human primary gastrointestinal cancers,
In the tumours such as lung cancer, oophoroma；2) AFP: it is the tumour marker found earliest, is common in primary hepatocyte hepatocarcinoma；3)
PSA: it is common in prostate cancer；4) CA199: it is common in cancer of pancreas.But the tumor markers of current clinical application all lack very well
Sensibility and specificity, it is therefore, very crucial for the exploitation of novel high specific sensibility tumor marker, be conducive to me
Early detection tumour, patient's prognosis, carry out personalized treatment, and tumor patient is managed and follow-up.
Serum total acidic glycoprotein is a kind of acute phase protein, ORM master during acute inflammatory reaction, in serum
If by hepatic parenchymal cells regulating and expressing.It is nearest the study found that ORM also has an expression in partial immunity cell, such as in
Property granulocyte, macrophage, T cell etc..Numerous studies also indicate that, the ORM1 in serum ORM1 or urine is in bladder cancer, kidney
High expression is presented in end-stage patients' serum such as cancer, lung cancer.
Present invention firstly discovers that being compared with normal people, total acidic glycoprotein ORM, conversion in early stage of lung cancer patients serum
The content of growth factor (TGF-β) is remarkably decreased, and more conventional tumor markers-CEA index has higher diagnosis efficiency.It will
, therefore ORM, TGF-β or ORM& higher to the diagnosis efficiency of early stage of lung cancer patient is used in combination in TGF-β and ORM in serum
TGF-β is the reliable diagnosing tumor marker of lung cancer early diagnosis.
Summary of the invention
It is lower for the diagnosis positive rate for the molecular marker for being currently used for early stage of lung cancer diagnosis, sensitivity and specificity
The problem of, it to be a kind of ORM and TGF-β egg that the present invention provides the application of lung cancer early diagnosis tumor markers and kits
White new opplication.
To achieve the above object, the lung cancer early diagnosis tumor markers that one aspect of the present invention provides are in preparation lung cancer early stage
Application in diagnostic reagent or kit, it is characterised in that: the lung cancer early diagnosis tumor markers are human serum total acidic
Glycoprotein A GP1/ORM or/and human serum transforming growth factor TGF-β.
Preferably, total acidic glycoprotein includes AGP1/ORM1 and/or two kinds of AGP2/ORM2 in the human serum
Albumen.
Further, the lung cancer early diagnosis, the clinical diagnosis comprising IA phase and IB phase.
Another aspect of the present invention also provides a kind of lung cancer early diagnosis kit, it is characterised in that: includes the anti-of detection ORM
Body or the primary antibody with fluorescence, the primary antibody with chemiluminescent primary antibody, tape label；Or comprising the antibody for detecting TGF-β or with glimmering
The primary antibody of light, the primary antibody with chemiluminescent primary antibody, tape label；Or comprising the antibody for detecting ORM and TGF-β or with fluorescence
Primary antibody, the primary antibody with chemiluminescent primary antibody, tape label.
As described above, the present invention provides following technical schemes:
1) serum ORM, TGF-β be individually or diagnosis marker as the early stage of lung cancer of ORM&TGF- β albumen Combining diagnosis
Using:
(1) application of the serum ORM albumen as the diagnostic serum marker of the early stage of lung cancer；
(2) application of the serum TGF-β albumen as the diagnostic serum marker of the early stage of lung cancer；
(3) application of serum ORM and TGF-β Combining diagnosis as pulmonary cancer diagnosis blood serum designated object.
2) antibody of ORM albumen, the antibody of TGF-β albumen are used alone or in combination in the testing product for preparing diagnosing
In application.
3) the diagnostic threshold 0.65mg/mL of the ORM detection kit, the detection threshold value of TGF-β detection kit are
31.997ng/mL。
4) testing product detects serum.
5) product detected is used to distinguish the pulmonary carcinosis of early stage of lung cancer patient and Healthy People, benign patient and middle and advanced stage
People, wherein early stage of lung cancer patient includes the patient in IA phase and IB phase.
The invention has the advantages that and the utility model has the advantages that
Early stage of lung cancer tumor markers of the invention be using transcript profile sequencing technologies from early stage of lung cancer peripheral blood in patients
The albumen of the differential expression screened in leucocyte, wherein total acidic glycoprotein ORM is significant in early stage of lung cancer patients serum
There is TGF-β Combining diagnosis in high specific and high sensitivity, with serum to further improve the sensitivity of early diagnosis for decline
And specificity, it can be used for the early diagnosis of lung cancer.It is increased with ORM, TGF-β serum levels in previously reported advanced tumor detection
Tumor markers ORM in the present invention, difference TGF-β, is proved to be remarkably decreased in early stage of lung cancer patients serum, and ORM
Expression is regulated and controled by TGF-β expression, and the serum expression quantity reduction of a kind of new discovery of system of the present invention, ORM, TGF-β can be made
For the diagnosis marker of the early stage of lung cancer, best sensibility and specificity is shown, can be used as the tumor markers of mass survey,
The effect that ORM&TGF- β is used in combination is better than exclusive use.
Detailed description of the invention
Fig. 1 is the specificity decline in early stage of lung cancer patient of serum ORM content.
Fig. 2 is serum ORM content interim situation of change in each tumour of lung cancer patient.
Fig. 3 is that ROC curve analyzes performance of the ORM in each tumour period diagnosis of lung cancer patient.
Fig. 4 is the expression that TGF-β adjusts ORM1 in human peripheral leucocytes.
Fig. 5 is the expression that TGF-β regulates and controls ORM1 in human peripheral leucocytes by Smad2/3 signal path.
Fig. 6 is the specificity decline in early stage of lung cancer patient of serum TGF-β content.
Fig. 7 is that ROC curve analyzes ORM, TGF-β and ORM and TGF-β Combining diagnosis in early stage (IA phase) pulmonary cancer diagnosis
Performance.
Fig. 8 is that ROC curve analyzes ORM, TGF-β and ORM and TGF-β Combining diagnosis in early stage (IB phase) pulmonary cancer diagnosis
Performance.
Wherein: Fig. 7, Fig. 8 are that ORM and TGF-β Combining diagnosis performance are used alone better than ORM and TGF-β.
Specific embodiment
In the following with reference to the drawings and specific embodiments, the present invention is further elaborated.
Experimental method:
1) patient and sample are included in:
In this experiment, 130 Serum of Patients with Lung Cancer samples, 59 Healthy Peoples and 21 benign patients couple are incorporated altogether
According to.
The selected and exclusion criteria of tumor patient includes: 1) there is specific pathological diagnosis to be diagnosed as corresponding tumour, tumour
Size and pathological staging have accurate diagnostic result；2) first visit tumour did not carry out any anticancer therapy before；3)
Without other acute illness or systemic disease such as autoimmune disease, diabetes, coronary heart disease etc..
Table one is included in lung cancer patient and normal human serum clinical information
2) sample collection
It acquires blood to inertia separation gel to promote in solidifying heparin tube, is stored at room temperature 30min, then 4 DEG C of 400 × g are centrifuged 15min,
It is immediately placed on -80 DEG C after serum is dispensed and freezes spare, every pipe serum is using being preceding unable to freeze thawing 2 times.
3) immunoturbidimetry detects serum total acidic glycoprotein
It will freeze and be placed on ice to melt in -80 DEG C of lung cancer patient serum sample, through 4 DEG C, 15000 × g is centrifuged 10min,
10 μ L of supernatant is drawn, after diluting 6 times with the sample diluting liquid that detecting instrument is equipped with manually, uses II/BN of BN* ProSpecDetection, instrument are set as automatic 5 times of dilute samples, and detection reagent uses NAntiserum to Human α 1-acid
Glycoprotein (SIEMENS) kit.
4) separation of human peripheral leucocytes
Using containing K2The heparin tube of EDTA anti-coagulants acquires fresh whole blood sample, in superclean bench according to complete
Blood: erythrocyte cracked liquid (Tiangeng) is added in erythrocyte cracked liquid=1:3 ratio, and cracking is placed at room temperature for after being gently mixed by inversion
5min, turning upside down halfway, it is primary to mix, erythrocyte splitting completely after solution take on a red color transparence, 450 × g of room temperature centrifugation
10min, tube bottom white precipitate are leucocyte, and iuntercellular PBS is washed twice, and each 450 × g of room temperature is centrifuged 5min, and PBS is resuspended white
It is spare after cell.
5) quantitative fluorescent PCR and protein immunoblotting detect the influence of ORM expression in TGF-β Number of Peripheral Blood Leucocyte.
By the healthy sample peripheral white blood cells of separation with 2.5 × 107The density in a/hole is inoculated into 6 orifice plates, cultivates 3h
Afterwards, it carries out outside TGF-β (5,10,20,30,40,60ng/mL) and its inhibitor SB431542 and LY2109761 Combined Treatment
All blood leukocytes, 37 DEG C, in 5%CO2 incubator for 24 hours.Cell is collected, cell RNA is extracted, fluorescence quantitative PCR detection ORM1's
Expression.Or cell is collected, after RIPA lysate on ice lytic cell 0.5h, 12000 × g is centrifuged 15min, in collection
Clear sample preparation, protein immunoblotting detect ORM1, the expression feelings of the Smad2 of Smad2, Smad3 and phosphorylation modification, Smad3
Condition.
6) MBP enzyme linked immuno-adsorbent assay serum TGF-β
TGF enzyme-linked immuno sorbent assay kit is bought in the Wuhan Tyke Ai Bo Biotechnology Co., Ltd, operating procedure
Specification is provided in strict accordance with producer to execute, detects each sample light absorption value at absorbance 450nm, calculates concentration.
7) statistical method
Statistical method includes that t is examined, using SPSS19.0 for Windows (SPSS Inc., Chicago, USA) and
Graphpad prism software (version 5.0) software carries out.
Experimental result
1, serum total acidic glycoprotein (ORM) content is remarkably decreased in early stage of lung cancer patient.(as shown in Figure 1, 2)
2, serum total acidic glycoprotein (ORM) has good diagnosis effect to early stage (IA+IB phase) lung cancer patient.(such as
Shown in Fig. 3)
It is shown through areal analysis under ROC curve, ORM has good diagnosis effect for early stage of lung cancer patient.For IA
Phase patient, the optimal threshold that area is 0.920, ORM under the ROC curve of ORM are 0.6525mg/mL, sensitivity at this time and special
Property is respectively 91.3% and 79.0%.For IB phase patient, area is 0.817 under the ROC curve of ORM, sensitivity and specificity
Respectively 85.7% and 79.0%.The above result shows that ORM has good diagnosis effect as the early stage of lung cancer.
3, TGF-β can show the expression (as shown in Figure 4) adjusted into ORM1 in peripheral white blood cells.TGF-β receptor is added
The facilitation effect of TGF-β can be significantly inhibited after inhibitor (SB431542 and LY2109761).
4, TGF-β regulates and controls the expression (as shown in Figure 5) of ORM1 in human peripheral leucocytes by Smad2/3 signal path.
TGF-β promotes the expression of Smad2 and Smad3 and its phosphorylation modification in peripheral white blood cells.
5, seroconversion growth factor (TGF-β) content is remarkably decreased in early stage of lung cancer patient.(as shown in Figure 6)
ELISA the results show that TGF-β content in early stage of lung cancer patients serum is significantly lower than normal person (P < 0.001),
Middle IA phase patient is the most significant.
6, ORM and TGF-β Combining diagnosis performance are better than ORM to a certain extent and TGF-β is used alone.(such as Fig. 7, Fig. 8 institute
Show)
It is shown through areal analysis under ROC curve, TGF-β has good diagnosis effect for early stage of lung cancer patient.For
IA phase patient, area is 0.932 under the ROC curve of TGF-β, and the optimal threshold of TGF-β is 31.997ng/mL, at this time sensitivity
It is respectively 1.00 and 50.0% with specificity.And ORM and TGF-β Combining diagnosis the results show that for IA phase patient, ORM&
Area is 0.985 under the ROC curve of TGF-β, and diagnosis effect is substantially better than ORM and TGF-β uses individually (Fig. 7).More than
The result shows that ORM&TGF- β has good diagnostic as early stage of lung cancer tumor markers, there is very big clinical application
Prospect.
Claims (4)
1. a kind of application of lung cancer early diagnosis tumor markers in preparation lung cancer early diagnosis reagent or kit, feature
Be: the lung cancer early diagnosis tumor markers are human serum total acidic glycoprotein A GP1/ORM or/and human serum conversion life
Long factor TGF-β.
2. lung cancer early diagnosis tumor markers according to claim 1 are in preparation lung cancer early diagnosis reagent or kit
In application, it is characterised in that: in the human serum total acidic glycoprotein include two hatching egg of AGP1/ORM1 and/or AGP2/ORM2
It is white.
3. lung cancer early diagnosis tumor markers according to claim 1 or 2 are in preparation lung cancer early diagnosis reagent or examination
Application in agent box, it is characterised in that: the lung cancer early diagnosis, the clinical diagnosis comprising IA phase and IB phase.
4. a kind of lung cancer early diagnosis kit, it is characterised in that:
Antibody or the primary antibody with fluorescence, the primary antibody with chemiluminescent primary antibody, tape label comprising detecting ORM；
Or antibody or the primary antibody with fluorescence, the primary antibody with chemiluminescent primary antibody, tape label comprising detecting TGF-β；
Or antibody or the primary antibody with fluorescence, the primary antibody with chemiluminescent primary antibody, tape label comprising detecting ORM and TGF-β.
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