CN108318688B - Smad3 albumen is as the application of molecular labeling, liver cancer detection kit in peripheral blood excretion body - Google Patents

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

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CN108318688B
CN108318688B CN201810023545.2A CN201810023545A CN108318688B CN 108318688 B CN108318688 B CN 108318688B CN 201810023545 A CN201810023545 A CN 201810023545A CN 108318688 B CN108318688 B CN 108318688B
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liver cancer
smad3
excretion body
peripheral blood
albumen
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CN108318688A (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 molecular labeling preparation liver cancer detection kit in application, liver cancer detection kit.The research of the invention finds that Smad3 protein content significantly increases 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

Application, liver cancer detection examination of the Smad3 albumen as molecular labeling in peripheral blood excretion body Agent box
Technical field
The present invention relates to biotechnology detection technique fields, more particularly to Smad3 albumen conduct in peripheral blood excretion body The application of molecular labeling, liver cancer detection kit.
Background technique
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%.
The main reason for prognosis in hcc is poor first is that liver cancer onset is hidden, most of patients has been advanced stage when finding, and advanced stage 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 cancer 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 raising, and on the other hand, a large amount of liver cancer patient does not appear as AFP liter It is high.Therefore new tumor marker is developed, joint AFP diagnosing liver cancer has applications well prospect.
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 adjuvant 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, protein, nucleic acid and lipid rich in 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.
Summary of the invention
The research of the invention finds that Smad3 protein content significantly rises 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 preparation liver cancer detection kit in Application.
Invention further provides a kind of liver cancer detection kits, reagent and detection including extracting peripheral blood excretion body The antibody of Smad3 albumen.
The liver cancer detection kit, application method are as follows:
(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 quantification；
(3) be compared according to quantitative result with the amount of Smad3 albumen in normal sample, 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 preparation liver cancer detection kit.
Invention further provides a kind of liver cancer detection kits, reagent, detection Smad3 including extracting peripheral blood excretion body The antibody of albumen and the antibody for detecting AFP albumen.
The liver cancer detection kit, application method are as follows:
(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 quantification；
(3) AFP protein quantification in the antibody human peripheral blood sample of detection AFP albumen is utilized；
(4) be compared according to the two quantitative result with the amount of Smad3 albumen and AFP albumen in normal sample, obtain to Test object liver cancer progress extent.
The research of the invention finds that Smad3 protein content significantly rises 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.
Detailed description of the invention
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 content in liver cancer patient various disease by stages.
Fig. 3 is the variance analysis result figure that peripheral blood excretion body Smad3 protein content is 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 cancer.
Fig. 5 is the height and the relation of liver cancer patient life span and survival rate of peripheral blood excretion body Smad3 protein content Analyse result figure.
Specific embodiment
Sample source: Healthy People and liver cancer patient blood serum sample source are in 2nd Affiliated Hospital Zhejiang University School of Medicine.
Embodiment 1
Human peripheral source excretion body extracts 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 company) are added, 4 DEG C stand 30 Minute.Supernatant is abandoned in centrifugation (3000g × 30 minute) afterwards, is centrifuged (3000g × 5 minute) again and is abandoned supernatant afterwards, gained sediment is For excretion body.
Embodiment 2
It is specific to walk using excretion body Smad3 content in Enzyme-linked Immunosorbent Assay double antibody sandwich method (ELISA) detection peripheral blood It is rapid as follows: to extract excretion body from peripheral blood in patients, obtain albumen after multigelation 3 times.According to Smad3 protein ELISA reagent The method and step of box (being purchased from Mlbio company) detects Smad3 protein content.AFP data produce in patient clinical diagnosis and treatment process Raw detection data.Method step of the clinical laboratory, the Zhe Yi Second Academy according to alpha-fetoprotein determination kit (being purchased from Architect company) Rapid measurement AFP content.
Embodiment 3
Peripheral Blood of Patients with Hepatocellular Carcinoma excretion body Smad3 protein content is detected using 2 the method for embodiment and is obtained corresponding AFP data, separately using healthy human peripheral blood as control.Respective patient and the AFP data of Healthy People come from its clinic diagnosis process The detection data of middle generation.
Detection is related to totally 17, healthy human peripheral blood sample altogether, and benign tumor of liver patient 20, liver cancer patient 94, as a result Show that Smad3 protein level is 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 level 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 patient 5, II grade of Edmondson patient 46, III grade of Edmondson patient 32, The result shows that liver cancer peripheral blood excretion body Smad3 level is unrelated with staging, pathological grading (Fig. 2, Fig. 3).
The receiver operating curves of further progress 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 cancer, 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 level can be used for judging liver cancer patient postoperative recurrence risk.

Claims (2)

1. extracting the reagent of peripheral blood excretion body and detecting antibody the answering in preparation liver cancer detection kit of Smad3 albumen With.
2. extracting the reagent of peripheral blood excretion body, detecting the antibody of Smad3 albumen and detecting the antibody of AFP albumen in preparation liver Application in cancer detection kit.
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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