CN103472226A - Use of serum DKK1 in preparation of diagnostic reagent for early hepatocellular carcinoma or small hepatocellular carcinoma - Google Patents
Use of serum DKK1 in preparation of diagnostic reagent for early hepatocellular carcinoma or small hepatocellular carcinoma Download PDFInfo
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Abstract
The present invention relates to a use of serum DKK1 in preparation of a diagnostic reagent for early hepatocellular carcinoma or small hepatocellular carcinoma. According to the present invention, a large number of hepatocellular carcinoma samples are analyzed, expression conditions of DKK1 and AFP are analyzed, the serum DKK1 provides high sensitivity and specificity for diagnosis of early hepatocellular carcinoma or small hepatocellular carcinoma, and particularly clinical diagnosis accuracy can be substantially increased with the DKK1 and AFP combined diagnosis.
Description
Technical field
The invention belongs to biomedicine field; More specifically, the present invention relates to the purposes of serum DKK1 in the diagnostic reagent for preparing early hepatocyte cancer or Small Hepatocellular Carcinoma.
Background technology
The liver primary tumor comprises other Hepatic Sarcomas such as hepatocellular carcinoma (hepatocellular carcinoma, HCC), intrahepatic cholangiocarcinoma, Combination hepatocellular carcinoma, bile duct cystadenoma, hepatoblastoma, hemangioma, carcinoid, lymph cancer, EH, squamous cell carcinoma, teratoma and leiomyosarcoma, fibrosarcoma, malignant fibrous histiocytoma, rhabdomyosarcoma.Hepatocellular carcinoma (HCC) is a kind of histological subtypes wherein.At present, the cause of disease of hepatocellular carcinoma is not yet fully clear and definite, it is a process that relates to multifactor, multistage and polygenes accumulation variation, and main factor has that absorption, alcoholism, the water of virus infections, aflatoxin pollute, liver pylori and cirrhosis etc.It is the main pathogenic of hepatocellular carcinoma that hepatitis type B virus (hepatitis B virus, HBV) infects, especially in Asia and Africa; Hepatitis C virus (hepatitis C virus, HCV) infects and to be worldwide distribution, is the American-European principal element that waits western countries' hepatocellular carcinoma incidence of disease rising.Cirrhosis is that the most dangerous factor occurs hepatocellular carcinoma, is also the main cause of the death of patient with liver cirrhosis.Belonged to middle and advanced stage while going to a doctor due to most of patients with hepatocellular carcinomas, lost best occasion for the treatment, only had the patients with hepatocellular carcinoma of 30-40% can implement effective operative treatment at present, five year survival rate only has 3-5%.So diagnosing hepatocellular carcinoma is one of key improved survival timely and effectively.
At present, the most frequently used method of examination and diagnosing hepatocellular carcinoma is that iconography and serum alpha-fetoprotein (alpha-fetoprotein, AFP) detect.Image comprises ultrasonic, CT, MRI etc., but image detects both expensive, is difficult to widespread use, and be subject to the impact of operator's experience, and be difficult to distinguish liver cancer and non-malignant hyperplasia.AFP is the most widely used hepatocellular carcinoma mark in the current whole world.When its dividing value is 20ng/mL, the sensitivity of AFP diagnosing hepatocellular carcinoma is only 55-60%, produces approximately 40% false negative rate; And the ability of its diagnosis of small hepatic cell carcinoma is lower, and sensitivity is reduced to 25-52%, most of early hepatocyte cancer patient AFP is all without obviously raising.The negative hepatocellular carcinoma patient of this part AFP is difficult to diagnosis clinically, is difficult to assess its result for the treatment of and disease process.Therefore find new reliable mark that can the early diagnosis hepatocellular carcinoma, can greatly improve and improve the clinical complex treatment effect, contribute to the prolongation of patients with hepatocellular carcinoma life cycle and the raising of life quality.Desirable tumor markers need to have higher specificity, the differences such as hepatocellular carcinoma and hepatitis, cirrhosis, liver regeneration tubercle can be come, also need higher sensitivity simultaneously, can diagnose the early hepatocyte cancer, and there is easy detection, can repeat, the characteristics of Noninvasive.
Completing of the Human Genome Project, the development of high flux gene expression detection method and the raising of large-scale data analysis ability, make people can more in depth study the change of gene expression in the hepatocellular carcinoma carcinogenesis of human, understand better the pathogenesis of hepatocellular carcinoma, screening is expected to be applied to clinical biomarker and drug target.The difference of Gene Expression Profile of Liver by cDNA chip of expression spectrum (Affymetrix GeneChip Human Genome U133Plus 2.0Array) technical Analysis has been passed through in earlier stage in this laboratory patients with hepatocellular carcinoma cancerous tissue and corresponding cancer.Further find that DKK1 does not express in the Various Tissues of normal adult, only express in placenta tissue, there is tumour-specific.
DKK1 is the inhibiting factor of Wnt signal path, is cloned in the earliest 1998, finds that it can suppress the copying of axle that Wnt induces and participate in the formation of head in the Africa xenopus early development.The Wnt path is a signal path that embryonic development is played to important regulating action, participates in the processes such as cell proliferation, differentiation, apoptosis, cell polarity and motion, and in its path, the sudden change of signaling molecule or unconventionality expression are relevant to various diseases and cancer.The genesis of this signal path and hepatocellular carcinoma is also closely related.The Wnt path is subject to the regulation and control of several secretory proteins, comprises DKK, WIF (Wnt-inhibitor factor) and SFRP (secreted frizzled related protein) etc.DKK family comprises 4 members (DKK1-4) relatively conservative in evolution.The DKK1 secreting type glycoprotein of encoding, contain two conservative regions (Cys1, Cys2) that are rich in halfcystine, can with LDH receptor related protein 5/6 (low-density lipoprotein receptor-related protein 5/6, LRP5/6) combination, and under memebrane protein Kremen1/2 participates in by causing the LRP5/6 endocytosis, suppress the formation of Wnt-Frizzled-LRP5/6 complex, and suppress classical Wnt signal path.
The inventor adopts the ELISA method subsequently, detect first the DKK1 albumen of high concentration in 10 kinds of dissimilar human tumor cells culture supernatant, prompting various human tumor cell secretion and high expressed DKK1, DKK1 can be used for the clinical serodiagnosis of human malignancies.So the inventor had declared national inventing patent (CN200510110298.2) and International PCT patent (PCT/CN2006/000382) in 2005.DKK1 is the inventor for the Chinese patent of cancer serodiagnosis and international monopoly and files an application at home and in the world first.
But this area is at present to the clinical meaning of serum DKK1, particularly also unclear to early hepatocyte cancer or Small Hepatocellular Carcinoma patient's diagnosis capability.
Summary of the invention
The object of the present invention is to provide the purposes of serum DKK1 in the diagnostic reagent for preparing early hepatocyte cancer or Small Hepatocellular Carcinoma.
In a first aspect of the present invention, provide DKK1 albumen or the purposes of its encoding gene in the diagnostic reagent for preparing early hepatocyte cancer or Small Hepatocellular Carcinoma.
In another aspect of this invention, provide DKK1 albumen or the purposes of its encoding gene in the diagnostic kit for preparing early hepatocyte cancer or Small Hepatocellular Carcinoma.
In a preference, described diagnostic reagent is selected from (but being not limited to):
The primer of the encoding gene of specific amplification DKK1 albumen; Or
The encoding gene of specific recognition DKK1 albumen or the probe of its transcript; Or
The antibody of the anti-DKK1 albumen of specificity.
In another preference, described early hepatocyte cancer is the hepatocellular carcinoma of BCLC 0+A level; And/or described Small Hepatocellular Carcinoma is the hepatocellular carcinoma that single diameter of tumor is less than 2cm.
In another aspect of this invention, provide a kind of for measuring the kit of early hepatocyte cancer or Small Hepatocellular Carcinoma, contain in described kit:
(1) detect alpha-fetoprotein or the expression of its encoding gene (preferably, being the alpha-fetoprotein in serum or its encoding gene) or the diagnostic reagent of expression; With
(2) detect DKK1 albumen or the expression of its encoding gene (preferably, being the DKK1 albumen in serum or its encoding gene) or the diagnostic reagent of expression.
In a preference, in described kit, use respectively the middle alpha-fetoprotein of diagnostic reagent detection testing sample (preferably, being serum) of (1) or expression or the expression of its encoding gene; And use the diagnostic reagent of (2) to detect testing sample (preferably, for serum) in DKK1 albumen or expression or the expression of its encoding gene, can detect early hepatocyte cancer or Small Hepatocellular Carcinoma (DKK1 is expressed as the positive in hepatocellular carcinoma or Small Hepatocellular Carcinoma in early days, but AFP is positive or negative).
In a preference, in described kit, the expression of described detection alpha-fetoprotein or its encoding gene or the diagnostic reagent of expression are selected from (but being not limited to):
The primer of the encoding gene of specific amplification alpha-fetoprotein; Or
The encoding gene of specific recognition alpha-fetoprotein or the probe of its transcript; Or
The antibody of specificity anti-alpha-fetoprotein.
In a preference, in described kit, described detection DKK1 albumen or the expression of its encoding gene or the diagnostic reagent of expression are selected from:
The primer of the encoding gene of specific amplification DKK1 albumen; Or
The encoding gene of specific recognition DKK1 albumen or the probe of its transcript; Or
The antibody of the anti-DKK1 albumen of specificity.
In a preference, in described kit, also comprise:
The nucleic acid extraction agent; And/or
Pcr reagent; And/or
Protein immunoblot reagent; And/or
Enzyme chain immune response reagent.
In another preference, in described kit, also comprise: the operation instructions how diagnostic reagent is used are described.
In another aspect of this invention, provide a kind of method of measuring early hepatocyte cancer or Small Hepatocellular Carcinoma, described method comprises:
(a) expression or the expression of DKK1 albumen or its encoding gene in the detection testing sample; With
(b) alpha-fetoprotein in the detection testing sample or expression or the expression of its encoding gene;
DKK1 is expressed as the positive in hepatocellular carcinoma or Small Hepatocellular Carcinoma in early days, but AFP is positive or negative.
In a preference, adopt described kit to be measured.
Other side of the present invention, due to the disclosure of this paper, is apparent to those skilled in the art.
The accompanying drawing explanation
Fig. 1, assessment serum DKK1 albumen research and design to the diagnosis capability of early hepatocyte cancer (HCC).
Normal person (healthy controls, HC); Chronic hepatitis B (chronic hepatitis B, CHB); Cirrhosis (liver cirrhosis, LC); Hepatocellular carcinoma (hepatocellular carcinoma, HCC); Enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA); Experimenter's operating characteristic (receive operating characteristics, ROC); DKK1:dickkopf-1.
Fig. 2, test set and checking collect the concentration of serum DKK1 albumen and Serum AFP albumen in each group.
Figure A and C are Normal group (healthy controls, HC), chronic hepatitis B group (chronic hepatitis B, CHB), liver cirrhosis group (liver cirrhosis, LC) concentration of serum DKK1 albumen and in early hepatocyte cancer group (early-stage hepatocellular carcinoma, Early-HCC).Figure B and D are the concentration of Serum AFP albumen in these 4 groups, and when serum concentration of AFP, during higher than 1210ng/ml, its concentration is calculated with 1210ng/ml.*，P<0.001。
Fig. 3, the serum DKK1 diagnosis capability to the early hepatocyte cancer.
Figure A and C be for take the early hepatocyte cancer as patient's group, take normal healthy people (HC), chronic hepatitis B (chronic hepatitis B, CHB) and cirrhosis (liver cirrhosis, LC) the ROC curve as the control group making.Figure B and D are DKK1 and the AFP positive rate in patients with hepatocellular carcinoma in early days respectively, and DKK1 is at AFP negative (AFP-, Serum AFP≤20ng/ml) or the positive (AFP of AFP
+, Serum AFP>and 20ng/ml) positive rate in early hepatocyte cancer (Early-HCC) patient.
Fig. 4, serum DKK1 are less than the diagnosis capability of the patients with hepatocellular carcinoma of 2cm to single tumour.
Figure A and D be serum DKK1 at single tumor size≤2cm, 2 and≤5cm, 5 and≤10cm and concentration in the 10cm patients with hepatocellular carcinoma.Figure B and E are patient's group for take the small liver cancer patient that single tumour is less than 2cm, take the ROC curve that normal person, Chronic Hepatitis B and liver cirrhosis patient draw as control group.Figure C and F are patient's group for take the small liver cancer patient that single tumour is less than 2cm, take the ROC curve that Chronic Hepatitis B and liver cirrhosis patient draw as control group.
Embodiment
The inventor, by analyzing a large amount of hepatocellular carcinoma samples, has therefrom analyzed the expression of DKK1 and AFP, finds that first serum DKK1 diagnosis early hepatocyte cancer or Small Hepatocellular Carcinoma have higher sensitivity and specificity; More particularly, DKK1 associating AFP diagnosis can improve the accuracy of clinical diagnosis greatly.
In the present invention, the inventor has designed large sample multicenter cohort study, (comprise 213 routine normal persons by detecting the test set queue, 98 routine chronic hepatitis B patients, 96 routine liver cirrhosis patients and 285 routine early hepatocyte cancer patients) DKK1 protein concentration in serum, find that first serum DKK1 diagnosis early hepatocyte cancer or Small Hepatocellular Carcinoma have higher sensitivity and specificity, and (comprise 99 routine normal persons at the checking collection, 73 routine chronic hepatitis B patients, 72 routine liver cirrhosis patients and 65 routine early hepatocyte cancer patients) in verified the diagnostic value of serum DKK1 to early hepatocyte cancer and Small Hepatocellular Carcinoma.
As used herein, " early hepatocyte cancer " is the hepatocellular carcinoma according to the clinical liver cancer standard in Barcelona (Barcelona Clinic Liver Cancer, BCLC) 0+A level.
As used herein, " Small Hepatocellular Carcinoma " refers to that single diameter of tumor is less than the hepatocellular carcinoma of 2cm.
In the present invention, the amino acid sequence of term " DKK1 albumen " is substantially the same with the protein sequence that GenBank accession number AAQ89364 provides, and also comprises the homologous protein of DKK1 albumen.The variant of substantially the same or its degeneracy of the nucleotide sequence that the nucleotide sequence of " encoding gene of DKK1 albumen " provides with GenBank accession number NM_012242.2, also comprise the homologous gene of DKK1 gene.The amino acid sequence of term " AFP albumen " is substantially the same with the protein sequence that GenBank accession number AAH27881.1 provides.The variant of substantially the same or its degeneracy of the nucleotide sequence that the nucleotide sequence of " encoding gene of AFP albumen " provides with GenBank accession number NM_001134.1.
At present clinically, liver cancer mainly is divided into following a few class: (1) hepatocellular carcinoma, originate from hepatocellular malignant tumour; (2) cholangiocarcinoma, originate from the epithelial malignant tumour of extrahepatic duct, accounts for 20% of primary carcinoma of liver, and the incidence of disease raises year by year in recent years; (3) Combination primary carcinoma of liver, in same liver cancer lump, hepatocellular carcinoma and cholangiocellular carcinoma are parallel; (4) hepatic hemangioma, comparatively common liver benign tumour, account for the 5-20% of benign tumor of liver; (5) adenoma of liver: in the liver benign tumour, its incidence of disease is only second to hepatic hemangioma, and pathogenesis may be relevant with the property endocrine disturbance, main sending out in the women; (6) Liver Focal nodular hyperplasia, hepatic benign lesions, pathogenic factor wouldn't be clear; (7) carcinoid of liver, malignant tumour Primary Hepatic carcinoid is more rare, and the Secondary cases carcinoid of liver mainly contains due to the class metastasis of cancer of the internal organs such as alimentary canal, more common; (8) hepatoblastoma: a kind of pernicious Embryo tumour with multiple differentiation mode is modal liver tumour in children; (9) metastatic hepatic carcinoma: primary tumor, at other position of health, is transferred to the malignant tumour of liver growth.In addition, also have chronic liver disease (for example hepatitis, cirrhosis) also to show as liver's discomfort, be easy to be diagnosed as liver cancer.Known according to the characteristics of above liver cancer and hepatopathy, there is complicacy in liver cancer clinically, how distinguishing identification, improving the clinical diagnosis accuracy is people's problem demanding prompt solutions, be starved of clinically can Accurate Diagnosis hepatocellular carcinoma (the particularly conventional negative hepatocellular carcinoma of AFP, early hepatocyte cancer or the Small Hepatocellular Carcinoma that is difficult to Accurate Diagnosis) mark.And although AFP has been used as a kind of liver cancer marker of classics, it can not solve clinically a difficult problem of accurately segmenting, diagnosing fully, and misdiagnosis rate is high.And this mark of DKK1, although the inventor formerly has been associated it with cancer, yet whether it can to segment the type of liver cancer be unknown in research in the past.
New discovery based on the inventor, can using DKK1 albumen or its encoding gene as the mark (label) of measuring hepatocellular carcinoma, particularly early hepatocyte cancer or Small Hepatocellular Carcinoma.By analyzing the expression of DKK1 albumen in testing sample (sample) or its encoding gene, thereby learn experimenter's disease state, for diagnosis or the prognosis of disease provides foundation.Described testing sample or sample to be tested are patient's body fluid, are preferably serum.
Can adopt various technology to detect the expression of DKK1, these technology all comprise in the present invention.Detect prior art that nucleic acid can use as (but being not limited to): the methods such as biochip technology, Probe Hybridization technology, polymerase chain reaction (PCR), Northern Blot.Detecting albumen can be by means of mass spectrometer etc., maybe can be by methods such as Western Blot or ELISA.
As a kind of selection mode of the present invention, come expression and the expression of DKK1 gene in analytic sample by quantitative or semiquantitative polymerase chain reaction (PCR) method, thereby can judge.Preferably, by real-time quantitative Realtime-PCR, realize detecting.
Based on new discovery of the present invention, the present invention also provides the reagent of specific recognition DKK1 albumen or its encoding gene.The reagent of any DKK1 of identification albumen or its encoding gene all comprises in the present invention, as the mark that detects early hepatocyte cancer or Small Hepatocellular Carcinoma.The reagent of described specific recognition DKK1 albumen or its encoding gene is for example: the primer of the encoding gene of specific amplification DKK1 albumen; Or the encoding gene of specific recognition DKK1 albumen or the probe of its transcript; Or the antibody of the anti-DKK1 albumen of specificity.
The present invention also provides the purposes of the reagent of a kind of specific recognition DKK1 albumen or its encoding gene, for detection of early hepatocyte cancer or Small Hepatocellular Carcinoma people at highest risk.
As one embodiment of the present invention, described reagent is the antibody of anti-DKK1; For example more particularly monoclonal antibody or polyclonal antibody.
Antibody of the present invention can be known by those skilled in that art various technology be prepared.For example, the antigen of purifying can be applied to animal to induce the generation of polyclonal antibody, and described animal is as rabbit, mouse, rat etc.Multiple adjuvant can be used for strengthening immune response, includes but not limited to Freunds adjuvant etc.
Antibody of the present invention can be also monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare.
Described antibody can be used in immunochemical technique, detects the DKK1 level in sample, thereby for diagnosing early hepatocyte cancer or Small Hepatocellular Carcinoma or judging ill risk (neurological susceptibility).
As another embodiment of the invention, described reagent is the primer of specific amplification DKK1 gene, and after the nucleotide sequence that obtains cicada DKK1, people are easy to design primer based on this.As more preferably mode of the present invention, described primer is primer pair, has the nucleotide sequence shown in SEQ ID NO:1 and SEQ ID NO:2.Described primer pair can increase and obtain the amplified production of appropriate length, and the specificity of detection is good.
Also can utilize biochip technology to carry out the detection of DKK1.After the nucleotide sequence that obtains cicada DKK1, people are easy to design probe based on this.For example, if what solid phase carrier adopted is to modify slide or silicon chip, 5 ' end of probe contains amido modified poly-dT string, oligonucleotide probe can be mixed with to solution, then point sample instrument is being modified slide or silicon chip by its point, be arranged in predetermined sequence or array, then by placement, spend the night and fix, just can obtain genetic chip of the present invention.If oligonucleotide probe is not containing amido modified, its preparation method also can be with reference to known technology.
Utilize described antibody, probe or primer, can detect the level of DKK1 in body fluid, thereby can be used for detecting early hepatocyte cancer or Small Hepatocellular Carcinoma, can be used for predicting the generation of early hepatocyte cancer or Small Hepatocellular Carcinoma, perhaps for the preparation of the preparation that detects early hepatocyte cancer or Small Hepatocellular Carcinoma or kit etc., for the preparation of the preparation of the generation of prediction early hepatocyte cancer or Small Hepatocellular Carcinoma or kit etc.
The present invention also provides for diagnosing the kit of early hepatocyte cancer or Small Hepatocellular Carcinoma, and this kit comprises: the reagent of specific recognition DKK1 albumen or its encoding gene.More preferably, described kit also comprises: the reagent of specific recognition AFP albumen or its encoding gene; Thereby can be by the expression (hepatocellular carcinoma is high-risk as the positive) of AFP in the joint-detection testing sample and DKK1, make the detection of hepatocellular carcinoma more accurate, and can, in time more early by Accurate Diagnosis, can greatly reduce Misdiagnosis and treat and gain time for hepatocellular carcinoma.Described reagent is for example: the reagent of described specific recognition DKK1 albumen or its encoding gene for example: the primer of the encoding gene of specific amplification DKK1 albumen; Or the encoding gene of specific recognition DKK1 albumen or the probe of its transcript; Or the antibody of the anti-DKK1 albumen of specificity (monoclonal antibody or polyclonal antibody).The reagent of described specific recognition AFP albumen or its encoding gene is for example: the primer of the encoding gene of specific amplification AFP albumen; Or the encoding gene of specific recognition AFP albumen or the probe of its transcript; Or the antibody of the anti-AFP albumen of specificity (monoclonal antibody or polyclonal antibody).
In described kit, also can contain: nucleic acid extraction agent (as the nucleic acid extract); And/or pcr reagent (as dNTP, the Taq enzyme); And/or protein immunoblot reagent; And/or enzyme chain immune response reagent (as nitrite ion or hybridization solution).
As a kind of optimal way, in described kit, also can contain: for the reagent of immunochemical analyses, described reagent such as second antibody, coloring agent, developer etc.In addition, also can comprise operation instructions etc. in described kit.More specifically, described kit can be a kind of kit based on enzyme linked immunoassay (ELISA) technology, for detection of the generation of early hepatocyte cancer or Small Hepatocellular Carcinoma or prediction early hepatocyte cancer or Small Hepatocellular Carcinoma.Elisa technique and the detection reagent based on this technology are apparent for a person skilled in the art.
As another kind of optimal way, in described kit, also can contain: (A) various PCR reaction reagent, such as but not limited to: Taq enzyme, PCR damping fluid, dNTP, archaeal dna polymerase etc.; Or (B) various extraction DNA or the required reagent of RNA (preparing the PCR reaction template), such as but not limited to: phenol, chloroform, isoamylol, NaCl etc.; Or (C) extract the kit of DNA or RNA.
The reagent of the reagent of described specific recognition DKK1 albumen or its encoding gene or specific recognition AFP1 albumen or its encoding gene also can be fixed on test paper, is prepared into immune colloid gold test paper or similar test material.
Operation instructions and/or the standard operating procedure (SOP) that in described kit, also can contain in addition, kit of the present invention.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only are not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, write molecular cloning experiment guide, Science Press, the condition described in 2002, or the condition of advising according to manufacturer usually as J. Pehanorm Brooker etc. according to normal condition.
I, materials and methods
1, serum specimen is collected
(1) serum specimen
The test set serum specimen is totally 692 examples, 213 routine normal person (healthy controls wherein, HC) serum, 98 routine chronic hepatitis patients (chronic hepatitis B, CHB) serum and 96 liver cirrhosis patients (liver cirrhosis, LC) serum is from the first Infectious Disease of affiliated hospital of University Of Suzhou, and 285 routine patients with hepatocellular carcinoma serum are from Zhongshan Hospital Attached to Fudan Univ's liver surgery; Checking collection serum specimen is totally 309 examples, comprises 99 routine normal human serums, 73 routine chronic hepatitis patient serum and 72 liver cirrhosis patients, 65 routine patients with hepatocellular carcinoma serum, all from attached east hospital of liver and gall surgical department of The 2nd Army Medical College.Serum is collected when clinical diagnosis, and the time of collecting is from year June in October, 2008 to 2011.Serum is collected in short solidifying pipe, 2,000-3, and the centrifugal 18-20min of 000rpm, draw supernatant in centrifuge tube, and-80 ℃ of packing are preserved.Two set of queue patients with hepatocellular carcinoma, hepatitis, liver cirrhosis patient clinical pathologic characteristic are as shown in table 1-3.
Test set and checking concentrate early stage (BCLC 0+A) patients with hepatocellular carcinoma Clinical symptoms as table 1.
Table 1
Abbreviation: BCLC:Barcelona Clinic Liver Cancer, the clinical liver cancer standard in Barcelona; AFP:alpha-fetoprotein, alpha-fetoprotein; Hb sAg:hepatitis B surface antigen, hepatitis B virus surface antigen; HbeAg:hepatitis B e antigen, hepatitis B virus core antigen; ALT:alanine aminotransferase, alanine aminotransferase; GGT:gamma glutamyl transpeptidase, γ-paddy acyl transpeptidase.
Test set and checking concentrate the chronic hepatitis B patient Clinical symptoms as table 2.
Table 2
Abbreviation: HBV:hepatitis B virus, hepatitis type B virus; AFP:alpha-fetoprotein, alpha-fetoprotein; HbsAg:hepatitis B surface antigen, hepatitis B virus surface antigen; HbsAb:hepatitis B surface antibody, the hepatitis B surface antibody; HbeAg:hepatitis B e antigen, HBeAg; HBeAb, hepatitis B e antibody hepatitis B e antibody; HbcAb:hepatitisB core antibody, hepatitis B virus core antibody.
a, in the routine chronic hepatitis B patient of test set 98,57 examples are not included calculating in without AFP value person.
b, in checking collection 73 routine chronic hepatitis B patients, 18 examples are not included calculating in without AFP value person.
Test set and checking concentrate the liver cirrhosis patient Clinical symptoms as table 3.
Table 3
Abbreviation: HBV:hepatitis B virus, hepatitis type B virus; AFP:alpha-fetoprotein, alpha-fetoprotein; HbsAg:hepatitis B surface antigen, hepatitis B virus surface antigen; HbsAb:hepatitis B surface antibody, the hepatitis B surface antibody; HbeAg:hepatitis B e antigen, HBeAg; HBeAb, hepatitis B e antibody hepatitis B e antibody; HbcAb:hepatitis B core antibody, hepatitis B virus core antibody.
*, in the routine liver cirrhosis patient of test set 96,22 examples are not included calculating in without related data person.
(2) inclusion criteria
Normal human serum is voluntary blood donor's serum, and liver function is normal, without hepatitis history.Chronic hepatitis is made a definite diagnosis standard according to chronic hepatitis B practice guidelines (Lok AS, McMahon BJ.Chronic hepatitis B:update 2009.Hepatology 2009; 50:661-2).Liver cirrhosis diagnosis criterion referenced " practical clinical practice " the 12nd edition (Chen Haozhu chief editor), comprise that imaging diagnosis, clinical indices detect, pathology is made a definite diagnosis etc.The diagnosis of hepatoma standard is carried out (Bruix J, Sherman M.Management of hepatocellular carcinoma.Hepatology 2005 according to AASLD (American Association for the Study of Liver Diseases) policy; 42:1208-36), comprise that imaging diagnosis, clinical indices detect, pathology is made a definite diagnosis etc.
(3) every clinical indices determines
(a) the AFP positive: detect Serum AFP content with radioimmunology or enzyme immunoassay, positive to be greater than 20ng/ml, be less than 20ng/ml negative.
The grade of liver function standard is according to Child-Pugh stage division (Van Deusen MA; Abdalla EK; Vauthey JN, Roh MS.Staging classifications for hepatocellular carcinoma.Expert Rev Mol Diagn 2005; 5:377-83).
(b) neoplasm staging is according to Barcelona Clinic Cancer (BCLC) grade scale (Llovet JM; Di Bisceglie AM; Bruix J, et al.Design and endpoints of clinical trials in hepatocellular carcinoma.J Natl Cancer Inst 2008; 100:698-711).BCLC stage 0+A is early stage, and BCLC stage B+C+D is late period.
(c) the Tumor Differentiation degree is according to the diagnosis of the tissue specimen of each case and further consultation result, and with reference to the Edmondson standard (Wittekind C.[Pitfalls in the classification of liver tumors] .Pathologe 2006; 27:289-93), Edmondson I-II level is for to break up, and III-IV level is poor for differentiation.
(d) cancer embolus carries out further consultation according to the tissue pathological slice to each case, it is cancer embolus under mirror that cancer embolus person under mirror is arranged, preoperative CT/MRI prompting TTPV is arranged and in art the person of being confirmed be the naked eyes cancer embolus, both be cancer embolus positive group, all the other are diagnosed as the cancer embolus feminine gender.
2, ELISA detects serum DKK1 concentration
DKK1 ELISA detection method is according to R& D company (article No. DY1906) instructions operation, step is briefly as follows: 100 μ l mouse-anti people DKK1 monoclonal coated antibodies (4 μ g/ml) are coated with elisa plate, ambient temperature overnight; Next day, with the PBS solution containing 1%BSA, in 37 ℃, seal 1.5h; Add the standard items (maximum concentration 4ng/ml, totally 7 dilutabilitys) of gradient dilution and the serum (100 μ l/ hole) diluted with the PBS containing 10% calf serum, hatch 1.5h for 37 ℃; Then add the anti-human DKK1 of biotin labeled rabbit to detect antibody (50ng/ml), hatch 1.5h for 37 ℃; The IgG-HRP that adds again streptavidin (streptavidin) coupling, hatch 20min for 37 ℃; Finally add substrate hydrogen peroxide (H
2o
2) and 3,3,5,5-tetramethyl benzidine (Tetramethyl benzidine, TMB), room temperature 20min; 1M sulfuric acid cessation reaction, and measure 450nm absorbing wavelength and 570nm reference wavelength in microplate reader.Wash three times and pat dry with the PBS solution containing 5 ‰ Tween-20 between every step.With four parametric regression method matching typical curves and calculate DKK1 concentration.
3, ELISA detects serum concentration of AFP
AFP ELISA detects serological method according to the operation of the Shanghai section China bioengineering AFP of company limited kit instructions, and concise and to the point step is as follows: add standard items and serum sample (50 μ l/ hole) in pre-coated plate, to hatch 20min for 37 ℃; Add the detection antibody (50 μ l/ hole) of peroxidase coupling, hatch 20min for 37 ℃; Add substrate hydrogen peroxide (H
2o
2) and 3,3,5,5-tetramethyl benzidine (TMB), room temperature 10min; 1M sulfuric acid cessation reaction, and measure 450nm absorbing wavelength and 570nm reference wavelength in microplate reader.Wash three times and pat dry with the PBS solution containing 5 ‰ Tween-20 between every step.The matching typical curve also calculates serum concentration of AFP.
4, statistical analysis
Data analysis adopts SPSS 15.0 softwares (SPSS Inc., USA).Continuous variable adopts median (Median) and mean+SD (Mean ± SD) to mean; The measurement data group difference relatively adopts Mann-Whitney U test (nonparametric statistics) or T-Test; Enumeration data relatively adopts Chi-square Test or Fisher rigorous examination.By drawing experimenter's operating characteristic (Receiver operating characteristics, ROC) curve and calculating corresponding area under curve (areas under the curves, AUC) and estimate diagnosis capability.Best Cutoff value is chosen for sensitivity and the specificity sum is maximum, error [(1-sensitivity)
2+ (1-specificity)
2square root] minimum corresponding value.Adopt MedCale10.4.7.0 comparison area under curve AUC otherness.AFP is usingd clinical 20ng/ml commonly used as the cutoff value.P<0.05 (bilateral) is for there being significant difference.Adopt R software analysis power of test and sample size, usefulness >=0.8 is for having power of test.
II. embodiment
The inventor assesses the diagnosis capability of serum DKK1 albumen to early hepatocyte cancer (HCC) according to the research and design flow process of Fig. 1.
At first the inventor has analyzed serum DKK1 and the expression of AFP in each group of test set.As shown in Figure 2 A, early hepatocyte cancer (early-stage hepatocellular carcinoma, Early-HCC) group serum DKK1 protein level is significantly higher than normal controls group (healthy controls, HC), chronic hepatitis B (chronic hepatitis B, CHB) control group and cirrhosis (liver cirrhosis, LC) control group (P<0.001), its median (mean+SD) is 2.99 (3.49 ± 2.26) ng/ml, between three control groups without significant difference.Prompting serum DKK1 can be used as the haemocyanin mark of diagnosing hepatocellular carcinoma.Hepatocellular carcinoma group serum afp is also higher than other three groups (P<0.001), but its level in chronic hepatitis B (CHB) and cirrhosis (LC) group is apparently higher than Normal group (HC) (P<0.001) (Fig. 2 B).Serum DKK1 and the AFP concentration in each group is specifically in Table 4.
Serum DKK1 albumen and the concentration of Serum AFP albumen in each group are concentrated in table 4, test set and checking
Abbreviation: DKK1:dickkopf-1; AFP:alpha-fetoprotein, alpha-fetoprotein; HC:healthy controls, normal control; CHB:chronic hepatitis B, chronic hepatitis B; LC:liver cirrhosis, cirrhosis; Early-HCC:early-stage hepatocellular carcinoma, the early hepatocyte cancer.
The inventor be take early hepatocyte cancer (Early-HCC) as patient's group subsequently, take normal person (HC), chronic hepatitis B (CHB) and cirrhosis (LC) patient as control group making ROC curve, determine cutoff value, area under curve (aera under the curve, AUC), susceptibility (sensitivity) and the specificity (specificity) of serum DKK1 antidiastole hepatocellular carcinoma and chronic liver disease.As shown in Fig. 3 A and table 5, DKK1 is best, and the cutoff value is 2.153ng/ml, and AUC is 0.865 (95%CI:0.835-0.895), and susceptibility is 70.9%, and specificity is 90.5%.Take 20ng/ml during as AFP cutoff value, and the AUC of AFP is 0.819 (95%CI:0.788-0.851), and susceptibility is 54.4%, and specificity is 87.9%.The ability of DKK1 diagnosis early hepatocyte cancer is better than AFP (P=0.04).Both combine diagnosis can improve AUC to 0.905 (95%CI:0.880-0.929), susceptibility to 87.4%.In patients with hepatocellular carcinoma (Early-HCC), the AFP positive rate is only 54.4% (155/285) in early days, and the DKK1 positive rate is 70.9% (202/285) (Fig. 3 B).In the early hepatocyte cancer patient of AFP negative (AFP-, Serum AFP≤20ng/ml), the DKK1 positive rate reaches 73.1% (95/130), at the positive (AFP of AFP
+, Serum AFP>20ng/ml) early hepatocyte cancer patient in, its positive rate is 69.0% (107/155) (Fig. 3 B).
Table 5, serum DKK1, AFP and both combine the diagnostic value to the early hepatocyte cancer
*
Abbreviation: DKK1:dickkopf-1; AFP:alpha-fetoprotein; AUC:area under the curve, area under curve; 95%CI:95%confidence interval, 95% fiducial interval; PPV:positive predictive value, positive predictive value; NPV:negative predictive value, negative predictive value; + LR:positive likelihood ratio, positive likelihood ratio;-LR:negative likelihood ratio, negative likelihood; Early-HCC:early-stage hepatocellular carcinoma, the early hepatocyte cancer; HC:healthy controls, normal healthy controls; CHB:chronic hepatitis B, chronic hepatitis B; LC:liver cirrhosis, cirrhosis; Vs.:versus, than.
*, the cutoff value of serum DKK1 is 2.153ng/ml, and the cutoff value of Serum AFP is 20ng/ml.
The patients with hepatocellular carcinoma that embodiment 3, serum DKK1 are less than 2cm to single diameter of tumor has diagnosis capability
In current clinical practice, diagnosis Small Hepatocellular Carcinoma (single diameter of tumor is less than the hepatocellular carcinoma of 2cm) is extremely difficult.Therefore the inventor's subsequent analysis serum DKK1 single tumor size is less than to the Small Hepatocellular Carcinoma patient's of 2cm diagnosis capability.As shown in Figure 4 A, the level of serum DKK1 and tumor size are proportionate.The small liver cancer patient that the single tumour of take is less than 2cm is patient's group, take all non-liver cancer patients (normal person, Chronic Hepatitis B and liver cirrhosis patient) as control group drafting ROC curve, the AUC of DKK1 is 0.805 (95%CI:0.740-0.869), susceptibility is 58.5%, specificity is 84.7%, than AFP, does not improve significantly (P=0.564) (Fig. 4 B).The small liver cancer patient that the single tumour of take is less than 2cm is patient's group, take Chronic Hepatitis B and liver cirrhosis patient as control group drafting ROC curve, the AUC of serum DKK1 is 0.794 (95%CI:0.727-0.861), obviously be greater than AFP AUC (0.669,95%CI:0.589-0.748) (P=0.019) (Fig. 4 C).DKK1 combines with AFP can significantly improve the diagnosis capability (Fig. 4 B-C) that single tumour is less than to the Small Hepatocellular Carcinoma of 2cm.
Embodiment 4, the serum DKK1 checking to early hepatocyte cancer and Small Hepatocellular Carcinoma diagnosis capability
In order to verify the diagnosis capability of serum DKK1 to early hepatocyte cancer or Small Hepatocellular Carcinoma, the inventor has detected another independent sample queue (checking collection, the serum DKK1 concentration (Fig. 1) in n=309).As shown in Fig. 2 C-D, identical with the test group, in the checking group, with other three control groups, compare, serum DKK1 significantly raises in hepatocellular carcinoma in early days.Serum DKK1 diagnosis early hepatocyte cancer patient's ability is better than AFP equally, the AUC of DKK1 is 0.896 (95%CI:0.846-0.947), susceptibility is 73.8%, specificity is 87.2%, the AUC of AFP is 0.775 (95%CI:0.711-0.839), susceptibility is 72.3%, and specificity is 63.7% (Fig. 3 C).The definite 2.153ng/ml in test set of take is serum DKK1 cutoff value, and the positive rate of DKK1 in checking collection early hepatocyte cancer is 75.4% (49/65) (Fig. 3 D).Two blood serum designated objects are combined use and can be improved diagnosis capability (Fig. 3 C-D).
It is similar with test set that fruit is assembled in checking, illustrates that serum DKK1 has diagnostic value to the early hepatocyte cancer.Checking is concentrated, serum DKK1 level same relevant to single tumor size (Fig. 4 D), DKK1 is less than the Small Hepatocellular Carcinoma of 2cm diagnosis capability to single diameter of tumor all is better than AFP, and both join and can obviously improve the diagnosis capability (Fig. 4 E-F) to Small Hepatocellular Carcinoma.
Conclusion
As a novel tumor haemocyanin mark, serum DKK1 has diagnostic value to early stage (BCLC 0+A stage) hepatocellular carcinoma and Small Hepatocellular Carcinoma (diameter is less than 2cm).
All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned instruction content of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1.DKK1 albumen or the purposes of its encoding gene in the diagnostic reagent for preparing early hepatocyte cancer or Small Hepatocellular Carcinoma.
2.DKK1 albumen or the purposes of its encoding gene in the diagnostic kit for preparing early hepatocyte cancer or Small Hepatocellular Carcinoma.
3. purposes as claimed in claim 1 or 2, is characterized in that, described diagnostic reagent is selected from:
The primer of the encoding gene of specific amplification DKK1 albumen; Or
The encoding gene of specific recognition DKK1 albumen or the probe of its transcript; Or
The antibody of the anti-DKK1 albumen of specificity.
4. purposes as claimed in claim 3, is characterized in that, described early hepatocyte cancer is the hepatocellular carcinoma of BCLC0+A level; And/or
Described Small Hepatocellular Carcinoma is the hepatocellular carcinoma that single diameter of tumor is less than 2cm.
5. one kind for measuring the kit of early hepatocyte cancer or Small Hepatocellular Carcinoma, it is characterized in that, in described kit, contains:
(1) detect alpha-fetoprotein or the expression of its encoding gene or the diagnostic reagent of expression; With
(2) detect DKK1 albumen or the expression of its encoding gene or the diagnostic reagent of expression.
6. kit as claimed in claim 5, is characterized in that, the expression of described detection alpha-fetoprotein or its encoding gene or the diagnostic reagent of expression are selected from:
The primer of the encoding gene of specific amplification alpha-fetoprotein; Or
The encoding gene of specific recognition alpha-fetoprotein or the probe of its transcript; Or
The antibody of specificity anti-alpha-fetoprotein.
7. kit as claimed in claim 5, is characterized in that, described detection DKK1 albumen or the expression of its encoding gene or the diagnostic reagent of expression are selected from:
The primer of the encoding gene of specific amplification DKK1 albumen; Or
The encoding gene of specific recognition DKK1 albumen or the probe of its transcript; Or
The antibody of the anti-DKK1 albumen of specificity.
8. kit as claimed in claim 5, is characterized in that, wherein also comprises:
The nucleic acid extraction agent; And/or
Pcr reagent; And/or
Protein immunoblot reagent; And/or
Enzyme chain immune response reagent.
9. a method of measuring early hepatocyte cancer or Small Hepatocellular Carcinoma, is characterized in that, described method comprises:
(a) expression or the expression of DKK1 albumen or its encoding gene in the detection testing sample; With
(b) alpha-fetoprotein in the detection testing sample or expression or the expression of its encoding gene;
DKK1 is expressed as the positive in hepatocellular carcinoma or Small Hepatocellular Carcinoma in early days, but AFP is positive or negative.
10. method as claimed in claim 9, is characterized in that, right to use requires 5 described kits to be measured.
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