CN108318688A - Application of the Smad3 albumen as molecular labeling, liver cancer detection kit in peripheral blood excretion body - Google Patents

Application of the Smad3 albumen as molecular labeling, liver cancer detection kit in peripheral blood excretion body Download PDF

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CN108318688A
CN108318688A CN201810023545.2A CN201810023545A CN108318688A CN 108318688 A CN108318688 A CN 108318688A CN 201810023545 A CN201810023545 A CN 201810023545A CN 108318688 A CN108318688 A CN 108318688A
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liver cancer
smad3
albumen
peripheral blood
excretion body
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CN108318688B (en
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梁廷波
白雪莉
章琦
傅琦涵
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Zhejiang University ZJU
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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
    • G01N33/57488—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 compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

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Abstract

The invention discloses Smad3 albumen in a kind of peripheral blood excretion body as application of the molecular labeling in preparing liver cancer detection kit, liver cancer detection kit.Present invention research finds that Smad3 protein contents significantly increase in liver cancer patient in the excretion body that human peripheral extracts, as area under tumor marker diagnosing liver cancer ROC curve up to 0.888, joint alpha-fetoprotein diagnosing liver cancer, area is up to 0.975 under ROC curve, so, alpha-fetoprotein can be used as the tumor marker of diagnosing liver cancer to Smad3 in peripheral blood alone or in combination in peripheral blood excretion body, it can also be used to judge liver cancer patient postoperative recurrence risk.

Description

Smad3 albumen detects examination as the application of molecular labeling, liver cancer in peripheral blood excretion body Agent box
Technical field
The present invention relates to biotechnology detection technique fields, more particularly to Smad3 albumen conducts in peripheral blood excretion body The application of molecular labeling, liver cancer detection kit.
Background technology
China's hepatocellular carcinoma number of the infected accounts for more than half of the whole world.Liver cancer grade malignancy is high, poor prognosis, survives within 5 years Rate only 16.8%.
One of the main reason for prognosis in hcc is poor is the concealment of liver cancer onset, and most of patients has been late period when finding, and late period Lack effective drug therapy.Therefore, developing new tumor marker help, to early diagnose liver cancer pre- for improving liver cancer patient It is most important afterwards.Alpha-fetoprotein (Alpha-fetal protein, AFP) is clinically widely used hepatic carcinoma marker. Although the sensibility and specificity of AFP diagnosing liver cancers is higher, have the following problems, on the one hand, active hepatitis, liver are hard The patient of the diseases such as change, carcinoma of testis also can behave as AFP raisings, and on the other hand, a large amount of liver cancer patient does not appear as AFP liters It is high.Therefore new tumor marker is developed, joint AFP diagnosing liver cancers have applications well foreground.
In addition, current liver cancer is based on the local therapeutic approaches such as perform the operation, postoperative recurrence is that its successive treatment is difficult, it is dead to cause The major reason died.The method of assessment liver cancer patient postoperative recurrence risk is not perfect at present, postoperative adjuvant therapy means shortcoming. Therefore effective recurring risk assessment index is developed, the high patient of identification risk of recurrence is developed further directed to high-risk patient The basis of corresponding auxiliary treatment strategy, is of great significance and potential applicability in clinical practice.
Excretion body is the vesica of the size about 100nm of various living cells secretions, contains abundant protein, nucleic acid and lipid Ingredient can be widely present in the body fluid of human body, such as blood, saliva, breast milk, urine.Due to the substance inside excretion body compared with Free substance is more stable, and obtains excretion body and have many characteristics, such as noninvasive, simple, easy to detect, therefore more and more grinds Study carefully tolerant index and Index for diagnosis index as diagnosing tumor in concern excretion body.
Invention content
Present invention research finds that Smad3 protein contents significantly rise in liver cancer patient in the excretion body that human peripheral extracts Height combines alpha-fetoprotein diagnosing liver cancer, ROC curve as area under tumor marker diagnosing liver cancer ROC curve up to 0.888 Lower area up to 0.975, so, alpha-fetoprotein can be used as diagnosis to Smad3 in peripheral blood alone or in combination in peripheral blood excretion body The tumor marker of liver cancer.
The present invention provides Smad3 albumen in peripheral blood excretion body as molecular labeling in preparing liver cancer detection kit Application.
Invention further provides a kind of liver cancer detection kits, including the reagent of extraction peripheral blood excretion body and detection The antibody of Smad3 albumen.
The liver cancer detection kit, application method are：
(1) it takes the peripheral blood sample of object to be detected and extracts excretion body therein；
(2) it extracts albumen in excretion body and utilizes the antibody of detection Smad3 albumen to Smad3 protein quantifications；
(3) be compared with the amount of Smad3 albumen in normal sample according to quantitative result, obtain object liver cancer to be detected into Exhibition degree.
Invention further provides AFP albumen in Smad3 albumen in peripheral blood excretion body and peripheral blood as dual molecule mark Remember the application in preparing liver cancer detection kit.
Invention further provides a kind of liver cancer detection kits, including the reagent of extraction peripheral blood excretion body, detection Smad3 The antibody of albumen and the antibody for detecting AFP albumen.
The liver cancer detection kit, application method are：
(1) it takes the peripheral blood sample of object to be detected and extracts excretion body therein；
(2) it extracts albumen in excretion body and utilizes the antibody of detection Smad3 albumen to Smad3 protein quantifications；
(3) AFP protein quantifications in the antibody human peripheral blood sample of detection AFP albumen are utilized；
(4) it is compared with the amount of Smad3 albumen and AFP albumen in normal sample according to the two quantitative result, obtains and wait for Detect object liver cancer progress extent.
Present invention research finds that Smad3 protein contents significantly rise in liver cancer patient in the excretion body that human peripheral extracts Height combines alpha-fetoprotein diagnosing liver cancer, ROC curve as area under tumor marker diagnosing liver cancer ROC curve up to 0.888 Lower area up to 0.975, so, alpha-fetoprotein can be used as diagnosis to Smad3 in peripheral blood alone or in combination in peripheral blood excretion body The tumor marker of liver cancer, it can also be used to judge liver cancer patient postoperative recurrence risk.
Description of the drawings
Fig. 1 is all blood excretion body Smad3 protein content comparing result figures of Healthy People and liver cancer patient.
Fig. 2 is variance analysis result figure of the peripheral blood excretion body Smad3 protein contents in liver cancer patient various disease by stages.
Fig. 3 is the variance analysis result figure that peripheral blood excretion body Smad3 protein contents are classified in liver cancer patient different pathological.
Fig. 4 is the sensibility and specificity analysis result figure that peripheral blood excretion body Smad3 albumen combines AFP diagnosing liver cancers.
Fig. 5 is the relation of the height and liver cancer patient life span and survival rate of peripheral blood excretion body Smad3 protein contents Analyse result figure.
Specific implementation mode
Sample source：Healthy People and liver cancer patient blood serum sample source are in 2nd Affiliated Hospital Zhejiang University School of Medicine.
Embodiment 1
The excretion body extraction of human peripheral source and identification.Method is as follows：
Human peripheral is acquired using anticoagulant blood-collecting pipe, after centrifugation (3000g × 5 minute) removes haemocyte, supernatant is in -80 DEG C Refrigerator freezes.When detection, taking-up freezes supernatant, melts at 4 DEG C.(10000g × 60 minute) are centrifuged to remove big cell fragment Equal substances.10 μ l supernatants are taken, 3 μ l Exoquick reagents (being purchased from System Biosciences companies) are added, 4 DEG C stand 30 Minute.Supernatant is abandoned after centrifugation (3000g × 30 minute), abandons supernatant after centrifuging (3000g × 5 minute) again, gained sediment is For excretion body.
Embodiment 2
Excretion body Smad3 contents in peripheral blood are detected using Enzyme-linked Immunosorbent Assay double antibody sandwich method (ELISA), it is specific to walk It is rapid as follows：Excretion body is extracted from peripheral blood in patients, and albumen is obtained after multigelation 3 times.According to Smad3 protein ELISA reagents The method and step of box (being purchased from Mlbio companies) detects Smad3 protein contents.AFP data come from patient clinical diagnosis and treatment process and produce Raw detection data.Method step of the clinical laboratory of the Zhe Yi Second Academys according to alpha-fetoprotein determination kit (being purchased from Architect companies) It is rapid to measure AFP contents.
Embodiment 3
Peripheral Blood of Patients with Hepatocellular Carcinoma excretion body Smad3 protein contents are detected using 2 the method for embodiment and are obtained corresponding AFP data, separately as a contrast with healthy human peripheral blood.Respective patient and the AFP data of Healthy People come from its clinic diagnosis process The detection data of middle generation.
It detects and is related to totally 17, healthy human peripheral blood sample altogether, benign tumor of liver patient 20, liver cancer patient 94, as a result Show that Smad3 protein levels are significantly higher than control group (Fig. 1) in Peripheral Blood of Patients with Hepatocellular Carcinoma.Further analysis it is more different by stages, The Smad3 protein levels of the liver cancer patient of different pathological classification.It is related to I phase patient 23, II phase patient 28, III phase patient altogether 34, it is related to I grade of Edmondson patients 5, II grade of Edmondson patients 46, III grade of Edmondson patients 32, The result shows that liver cancer peripheral blood excretion body Smad3 levels are unrelated with staging, pathological grading (Fig. 2, Fig. 3).
The receiver operating curves that further carry out analysis shows that, peripheral blood excretion body Smad3 albumen itself is used as liver cancer Diagnostic marker has greater advantage, and area under the curve 0.888, the tumor marker AFP's best with current diagnosis liver cancer examines Disconnected effect is close to (area under the curve 0.926).And Smad3 combines AFP diagnosing liver cancers, further increases sensibility and special Property, area is up to 0.975 (Fig. 4) under ROC curve.Therefore Smad3 is independent or Smad3 combines the tumour that AFP can be used as diagnosing liver cancer Marker.
The present embodiment totally 94 liver cancer patients, 4 non-row Liver Cancer under Radical Operation, 8 patients are lost to follow-up, and 82 conditions of patients can chase after Track.Position Smad3 is horizontal (3500ng/ml) according in, and patient is divided into 2 groups, respectively Smad3HIGH(Smad3 in excretion body> 3500ng/ml) and Smad3LOW(Smad3≤3500ng/ml in excretion body).The result shows that Smad3HIGHThe Patients on Recurrence of group Probability is higher than Smad3LOWAs a result group has significance,statistical (Fig. 5).The above result shows that Peripheral Blood of Patients with Hepatocellular Carcinoma excretion body Smad3 levels can be used for judging liver cancer patient postoperative recurrence risk.

Claims (6)

1. application of the Smad3 albumen as molecular labeling in preparing liver cancer detection kit in peripheral blood excretion body.
2. AFP albumen is preparing liver cancer detection as dual molecular labeling in Smad3 albumen and peripheral blood in peripheral blood excretion body Application in kit.
3. a kind of liver cancer detection kit, which is characterized in that reagent and detection Smad3 eggs including extracting peripheral blood excretion body White antibody.
4. a kind of liver cancer detection kit, which is characterized in that reagent, detection Smad3 albumen including extracting peripheral blood excretion body Antibody and detect AFP albumen antibody.
5. liver cancer detection kit as claimed in claim 3, which is characterized in that application method is：
(1) it takes the peripheral blood sample of object to be detected and extracts excretion body therein；
(2) it extracts albumen in excretion body and utilizes the antibody of detection Smad3 albumen to Smad3 protein quantifications；
(3) it is compared with the amount of Smad3 albumen in normal sample according to quantitative result, obtains object liver cancer progress journey to be detected Degree.
6. liver cancer detection kit as claimed in claim 4, which is characterized in that application method is：
(1) it takes the peripheral blood sample of object to be detected and extracts excretion body therein；
(2) it extracts albumen in excretion body and utilizes the antibody of detection Smad3 albumen to Smad3 protein quantifications；
(3) AFP protein quantifications in the antibody human peripheral blood sample of detection AFP albumen are utilized；
(4) it is compared, is obtained to be detected with the amount of Smad3 albumen and AFP albumen in normal sample according to the two quantitative result Object liver cancer progress extent.
CN201810023545.2A 2018-01-10 2018-01-10 Smad3 albumen is as the application of molecular labeling, liver cancer detection kit in peripheral blood excretion body Active CN108318688B (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111521826A (en) * 2020-05-21 2020-08-11 江海松 Application of Notch3 protein in peripheral blood exosome as molecular marker and detection kit

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111521826A (en) * 2020-05-21 2020-08-11 江海松 Application of Notch3 protein in peripheral blood exosome as molecular marker and detection kit

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