CN105891372B - Intervention of hepatocellular carcinoma with bile duct thrombi biomarker and application thereof - Google Patents
Intervention of hepatocellular carcinoma with bile duct thrombi biomarker and application thereof Download PDFInfo
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Abstract
The present invention provides a kind of marker for distinguishing intervention of hepatocellular carcinoma with bile duct thrombi and biliary tract disease, with 5, sensitivity and specificity when five 6- dihydrouracil, D-Asp, stearic acid, behenic acid and tetracosanoic acid index joints are effective when being diagnosed compared with single index, have respectively reached 99.06% and 0%.
Description
Technical field
The present invention relates to a kind of intervention of hepatocellular carcinoma with bile duct thrombi markers, particularly, are further specifically related to G01N33/
68。
Background technology
Intervention of hepatocellular carcinoma with bile duct thrombi (BDTT), which is shifted with hepatocellular carcinoma into biliary system, causes obstructive jaundice for spy
Point is the most common secondary tumors of biliary system.But other than it can obscure with the compressing of hepatocellular carcinoma complicated bile duct,
It is easiest to occur mistaken diagnosis disease be biliary tract malignant disease, meanwhile, also can with the benign disease of biliary system, such as bile duct knot
Stone, the diseases such as cyst of bile duct are obscured.According to statistics, overall preoperative rate of clinical misdiagnosis is 16.6% (65/392), later stage preoperative clinic
Misdiagnosis rate (9.8%, 26/264) is less than early period (30.5%, 39/128).Often the disease of easily mistaken diagnosis includes：Liver cancer is with Hilar courage
Pipe is oppressed, and Hilar common hepatic duct adenoma/cancer is with Bile duct tumor thrombus, calculus of bile duct.Forward and backward phase mistaken diagnosis is that liver cancer is oppressed with hilar bile duct
Respectively 9.4% and 1.5%, mistaken diagnosis is that calculus of bile duct is respectively 7.8% and 1.9%, the statistically significant (P of difference<0.01).
In view of the above-mentioned problems, the present invention provides carry out screening in the method for human sample and identify BDTT and cholangiocarcinoma, courage
Pipe calculus, the marker of cyst of bile duct antidiastole and its application.
Invention content
Marker for distinguishing intervention of hepatocellular carcinoma with bile duct thrombi and biliary tract disease comprising：D-Asp,
One or more compositions in 5,6- dihydrouracil, behenic acid, threonic acid, tetracosanoic acid and stearic acid.
As one embodiment of the present invention, the p value of the marker is less than 0.05.
As one embodiment of the present invention, the biliary tract disease be cholangiocarcinoma, primary cholelithiasis,
It is one or more in congenital biliary duct cyst.
As one embodiment of the present invention, the marker is for distinguishing intervention of hepatocellular carcinoma with bile duct thrombi and primary
Property liver cancer with bile duct oppress.
The method for obtaining above-mentioned marker, step be,
Samples subjects are obtained, and carry out pre-treatment；
It is detected using GC-MS/LC-MS；
Polytomy variable statistical analysis is carried out, marker is obtained.
The method for distinguishing intervention of hepatocellular carcinoma with bile duct thrombi and biliary tract disease with above-mentioned marker, the method packet
It includes：
The level of above-mentioned one or two kinds of markers is measured in the tissue samples of subject；
By the level of one or more markers in one or both of Samples subjects marker and control sample
It compares, wherein compared with the level of one or more markers in control sample, it is one or more in Samples subjects
The horizontal difference of marker indicates that the subject has intervention of hepatocellular carcinoma with bile duct thrombi.
For determine with intervention of hepatocellular carcinoma with bile duct thrombi subject whether the method that will have reaction to therapy, it is described
Method includes：The level of above-mentioned one or more markers is measured in the sample of subject；By one in Samples subjects
Kind or the level of multiple markers are compared with the level of one or more markers in control sample, wherein with control sample
In the levels of one or more markers compare, the horizontal difference instruction of one or more markers in Samples subjects
The subject will have reaction to the therapy.
Method for monitoring therapeutic efficiency in the subject with intervention of hepatocellular carcinoma with bile duct thrombi, the method packet
It includes：Before starting the treatment, the level of above-mentioned one or more markers is measured in the first sample from subject；
After at least part for having given the treatment, above-mentioned one or more marks are measured in the second sample from subject
The level of will object；By the level of one or more markers in the first sample and one or more markers in the second sample
Level compare, wherein compared with the level of one or more markers in the second sample, one kind in the first sample or
The horizontal difference of multiple markers indicates that the subject will have reaction to the treatment.
As one embodiment of the present invention, wherein Samples subjects are fluid sample or tissue sample.
For determining whether subject has or will develop the kit of intervention of hepatocellular carcinoma with bile duct thrombi, the kit
It include the horizontal reagent for measuring above-mentioned one or more markers in Samples subjects.
Figure of description
Fig. 1：Embodiment 1, with 5,6- dihydrouracil be marker when ROC scheme；Fig. 2：Embodiment 20, with stearic acid,
ROC figures when tetracosanoic acid is marker；Fig. 3：Embodiment 40, using threonic acid, behenic acid and tetracosanoic acid as marker
When ROC figure；Fig. 4：Embodiment 60, with 5,6- dihydrouracil, D-Asp, stearic acid, behenic acid and 24
ROC figures when acid is marker；
Specific implementation mode
The detailed description for preferred implementation method of the invention below of participating in the election of and including embodiment this hair can be more easily understood
Bright content.Unless otherwise defined, all technologies used herein and scientific terminology have common with fields of the present invention
The normally understood identical meaning of technical staff.When there is a conflict, the definition in this specification shall prevail.Equivalent, concentration or
When a series of Range Representation that the other values of person or parameter are limited with range, preferred scope or upper limit preferred values and lower preferable values,
This, which should be understood as to specifically disclose, is matched by any range limit or preferred value with any range lower limit or any of preferred value
To being formed by all ranges, regardless of whether the range separately discloses.For example, when disclosing range " 1 to 5 ", retouched
The range stated should be interpreted as including range " 1 to 4 ", " 1 to 3 ", " 1 to 2 ", " 1 to 2 and 4 to 5 ", " 1 to 3 and 5 " etc..Work as number
When value range is described herein, unless otherwise stated, otherwise the range is intended to include its end value and institute in the range
There are integer and score.Singulative includes that plural number discusses object, unless the context clearly dictates otherwise." optional " or
" any one " refers to that the item described thereafter or event may or may not occur, and the description includes the feelings that event occurs
The situation that shape and event do not occur.
Approximate term in specification and claims is used for modifying quantity, and it is specific to indicate that the present invention is not limited to this
Quantity further includes the modified part of the acceptable change without lead to related basic function close to the quantity.Phase
It answers, modifies a numerical value with " about ", " about " etc., mean that the present invention is not limited to the exact numericals.In some examples, approximate
Term likely corresponds to the precision of the instrument of measured value.In present specification and claims, range limits can be with
Combination and/or exchange, these ranges include all subranges contained therebetween if not stated otherwise.
In addition, indefinite article "an" before element of the present invention or component and "one" quantitative requirement to element or component
(i.e. occurrence number) unrestriction.Therefore "one" or "an" should be read as including one or at least one, and odd number
The element or component of form also include plural form, unless the apparent purport of the quantity refers to singulative.
Various aspects of the invention are described in greater detail in following segment：
As used herein, each of following term have with it in this part relevant meaning.Article " one " and "
It is a kind of " one herein for referring to the article or more than one (i.e. at least one) grammar object.For example, " element " means
One element or more than one element.
" marker " or " biomarker " is to be derived from one compared with another phenotypic status (such as not suffering from disease)
Organic biomolecules existing for difference in the sample of the subject of kind phenotypic status (such as with disease).If in different groups
Average or Median levels, such as expression of biomarker are calculated as statistically significant, then biomarker is not
Difference exists in same phenotypic status.The common inspection of significance,statistical particularly including t- inspections, ANOVA, Kruskal-
Wallis, Wilcoxon, Mann-Whitney and odds ratio.Biomarker alone or in combination provides subject and belongs to a kind of
The measurement of the relative risk of phenotypic status or another phenotypic status.Therefore, they are used as marker, for example, (pre- for disease
Afterwards and diagnosis), the marker of the therapeutic efficiency of drug (treatment diagnostics) and drug toxicity.
In some embodiments, receiver can be passed through for the accuracy of the marker of the compositions and methods of the invention
Performance curve (" ROC curve ") characterizes.ROC is the true positive rate phase for the possible cutpoint of difference of diagnosis marker
To the curve of false positive rate.ROC curve shows the relationship between sensitivity and specificity.I other words the increase of sensitivity is by companion
With the reduction of specificity.Curve is closer to the left axle in the spaces ROC, and then apical margin, marker are more accurate.On the contrary, curve more leans on
45 degree of diagonal lines of nearly ROC figures, marker are more inaccurate.Area under ROC is the measurement of marker accuracy.The standard of marker
True property depends on how group to be tested is suitably divided into group with the disease and without the disease by marker
Group.Area under the curve (being known as " AUC ") is the 1 perfect marker of expression, and area is the poor mark of 0.5 expression serviceability
Object.Therefore, in some embodiments, biomarker of the invention and the AUC of method be greater than about 0.50, greater than about 0.60 or
Greater than about 0.70.
" intervention of hepatocellular carcinoma with bile duct thrombi " is also known as " BDTT " herein, main clinical manifestation：Can have simultaneously primary
Property liver cancer and two kinds of obstructive jaundice performance.
Primary carcinoma of liver：It is with respect to for secondary carcinoma of liver including hepatocellular carcinoma and intrahepatic cholangiocellular carcinoma.Primary
Hepatocellular carcinoma often easily invades portal vein and vena hepatica, forms cancer embolus.
Cancer embolus is one of tumour common complication, refers to cancer cell in growth, breeding, transfer process, invasion or is heaped
Blood vessel and lymphatic system, or cause the disturbance of blood coagulation of blood, lead to vascular function and blood operation obstacle, abnormal blood coagulation, thrombus
It is formed, generates a series of tumor complication of pathophysiological changes.Intervention of hepatocellular carcinoma with bile duct thrombi Forming Mechanism is complicated, and more
Kind of pathologic process is related, it is considered that forming feature is：1, liver cancer cells directly invade bile duct by bile duct wall, and form cancer embolus
Continue to grow downwards and block biliary tract；2, cancer cell first invades lymphatic vessel or portal vein, then enters courage by invading bile duct wall
Pipe；3, the portal vein tumor thrombus that primary carcinoma of liver is formed directly invades neighbouring bile duct；4, HCC invades bile duct by neural intervaginal spaces
Form cancer embolus；5, cancer cell is by invading the vasa vasorum in bile duct wall, into lumen of bile duct.
Common intervention of hepatocellular carcinoma with bile duct thrombi diagnosis relies primarily on：It is diagnosed in imaging diagnosis and art.
The therapeutic modality of common intervention of hepatocellular carcinoma with bile duct thrombi is mainly surgical operation therapy, biliary drainage art, liver
Alinjection, local external radiotherapy treatment and expectant treatment etc. in arterial embolization chemotherapy, percutaneous transhepatic puncture bile duct.
Subject, such as the subject with development intervention of hepatocellular carcinoma with bile duct thrombi risk.
" level of marker " or " level of biomarker " refers to the marker present in sample to be tested
Amount.The level of marker can be abswolute level or amount (such as μ g/ml) or relative level or amount (such as relative signal intensity).
" higher level " of marker or " level increases " refers to the standard error for being more than the measuring method for assessing marker levels
Marker levels in test specimen, and marker levels preferably in control sample." the more low-level " of marker or " horizontal
Reduction " refers to the level of the marker in the test specimen for the standard error for being less than the measuring method for assessing marker levels,
Preferably marker levels in control sample.Term " known standard level " or " control level " refer to generally acknowledging for marker
Or scheduled level, be used to compare the level of the marker in the sample from subject.In one embodiment, it marks
Level of the control level of will object based on the marker in the sample from the subject with normal health.In another embodiment party
In case, the control level of marker is based on the sample from one or more subjects with intervention of hepatocellular carcinoma with bile duct thrombi
In marker level.In another embodiment, the control level of marker is based on coming within past 18 months
It is diagnosed as the level of the marker in the sample of intervention of hepatocellular carcinoma with bile duct thrombi.In one embodiment, subject is come from
Sample in marker control level be before in the sample of subject fixed marker level.
In yet another embodiment, the control level of marker is based on giving to intervention of hepatocellular carcinoma with bile duct thrombi
The level of marker before therapy in the sample from subject.In another embodiment, the control level base of marker
Marker in the sample from the subject with intervention of hepatocellular carcinoma with bile duct thrombi not contacted with test compound
It is horizontal.In another embodiment, the control level of marker based on from contacted with test compound have normal health
Subject sample in marker level.In one embodiment, the control level of marker is based on from primary
Property liver cancer with Bile duct tumor thrombus animal model sample in marker expression；From intervention of hepatocellular carcinoma with bile duct thrombi
The cell of animal model or the marker in the sample of cell line expression.
Alternatively, and especially when because methods described herein it is conventional implement caused by further information become available when,
The community average of " control " level of marker expression can be used.In other embodiments, " control " of marker is horizontal
Can by measure be obtained from subject suspect intervention of hepatocellular carcinoma with bile duct thrombi breaking-out before subject Samples subjects,
The level of the marker of Samples subjects obtained from archive etc. determines.
As used herein, term " patient " or " subject " refer to people and non-human animal, such as veterinary science patient.Term
" non-human animal " includes all vertebrates, such as mammal and nonmammalian, for example, non-human primate, mouse,
Rabbit, sheep, dog, cat, horse, milk cow, chicken, amphibian and reptile.In one embodiment, the subject is people.
In some embodiments, the body mass index (BMI) of subject be less than about 40kg/m2 (for example, about 40,39,38,
37,36,35,34,33,32,31,30,29,28,27,26,25,24,23,22,21,20,19 or about 18kg/m2).In other realities
It applies in scheme, the body mass index (BMI) of subject is greater than about 40kg/m2(for example, about 41,42,43,44,45,46,47,48,
49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、
74,75,76,77,78,79 or about 80kg/m2)。
The term as used herein " sample " refers to the similar cell detached from subject or the gleanings of tissue, and by
Existing tissue, cell and fluid in examination person.Term " sample " include from subject any body fluid (such as blood, lymph,
Gynaecology's fluid, cyst fluid, urine, tears and the fluid that collection is rinsed by bronchial lavage and/or peritonaeum) or cell.At one
In embodiment, the tissue or cell are taken out from subject.In another embodiment, described or cell be present in it is described by
In examination person.Other Samples subjects include tear, serum, cerebrospinal fluid, excrement, phlegm and cell extract.In an embodiment
In, biological sample includes the protein molecular for carrying out self-test subject.In another embodiment, biological sample may include coming
The mRNA molecules of self-test subject or the genomic DNA molecule for carrying out self-test subject.
Term " measurement " means to include marker in the existence or non-existence for detecting marker in sample, quantitative determination sample
Amount, and/or determine biomarker type method.Measurement can further be retouched by methods known in the art and herein
Method those of is stated to realize.
As used herein, term " adjusting " various forms intention include stimulation (such as increase or increment adjusting it is specific
Reaction or activity) and inhibit (such as reduce or down-regulation specifically reaction or activity).
Kit is at least one reagent such as probe, primer or the antibody of the marker comprising the specific detection present invention
Any product (such as packaging or container), the product be used as carry out a unit of method of the invention promote,
Distribution or sale.In certain embodiments, kit may include matrix, such as include one or more markers of the present invention
Capture agent and/or the matrix of capture agent that is combined with one or more markers of the present invention.In some embodiments
In, such kit includes for the horizontal operation instructions using mass spectroscopy marker.
First aspect：Marker
The first aspect of the invention provides a kind of for distinguishing intervention of hepatocellular carcinoma with bile duct thrombi and biliary tract disease
Marker comprising：D-Asp, 5,6- dihydrouracil, behenic acid, threonic acid, tetracosanoic acid and stearic acid
In one or more compositions.
Threonic acid (Threonic acid)：I.e. 2,3,4- trihydroxy-butyric acids are widely present in nature and animal body
It is interior.The content of threonic acid and some other organic acid in human urine with the age, diet, the difference of health and it is different.Soviet Union
Saccharic acid is also one of the catabolite of vitamin (L-AA), and can promote ascorbic absorption and utilization, and makes
Metal ion be easy to amino acid or protein in conjunction with and absorb.Vitamin C is a kind of strength free radical scavenger, can be inhibited
Growth of tumour cell reduces tumor cell proliferation ability.Secondly vitamin C content highest in pituitary gland, kidney is
The positions such as eyeball, brain, liver.Content of the vitamin C in liver cirrhosis patient blood plasma is significantly lower than normal group, with liver dysfunction
It is closely related with active oxygen metabolism disorder.Moreover, Vitamin C content is below other hepatopathys and normal person in liver cancer patient body,
Illustrate that Vitamin C content is low closely related with liver cancer generation.In BDTT patients blood plasmas threonic acid compared with disease of biliary tract each group patient with
And normally obviously increase per capita, Vitamin C content significantly reduces in liver cancer and liver cirrhosis patient body, and threonic acid is vitamin C
Catabolite, ascorbate degradation speed may significantly be speeded in reasoning BDTT liver cancer patient bodies of being not difficult, and then lead to its degradation
Product threonic acid dramatically increases, this may be related to the quickening of the ascorbate degradation of BDTT liver cancer patient the whole bodies, it is also possible to
The vitamin C metabolic disorder of BDTT tumor cells of hepatocellular carcinoma itself is related, and specific mechanism still needs further to pass through experimental study
It confirms.
Stearic acid (Stearic acid)：That is octadecanoid acid is generated by grease hydrolysis.Stearic acid is in human hepatocyte
Normal vital ingredient, contains stearic acid in normal hepatocytes and liver cancer, but the work of the fatty acid synthetase in tumour cell
Property increase, and then the synthesis of aliphatic acid is caused to increase.Therefore stearic content can increase than the content in normal hepatocytes in liver cancer
Add.Stearic accumulation, which can destroy endoplasmic reticulum, leads to the apoptosis of liver cell.Its content in liver cancer and liver cirrhosis patient with
Normal group is compared to significant difference.Aliphatic acid is mainly metabolized by two kinds of forms in liver, and one is pass through esterification
Effect generates the lipid materials such as triglycerides, phosphatide, and second is to generate CO by beta oxidation2Or ketoboidies.
D-Asp (D-aspartic acid), 5,6- dihydrouracil (5,6-dihydrouracil) docosane
Sour (behenic acid), tetracosanoic acid (lignoceric acid).
In certain aspects of the invention, single marker method and composition for use in the present invention.For example, in a reality
It applies in scheme, the marker of the method and composition for the present invention is D-Asp.In one embodiment, marker
It is 5,6- dihydrouracil.In one embodiment, marker is behenic acid.In one embodiment, marker
It is threonic acid.In one embodiment, marker is tetracosanoic acid.In one embodiment, marker is stearic acid.?
The other aspects of the present invention, are more than a kind of marker, such as multiple markers, such as 2,3,4,5,6,7,8,9,10,11 or more
Multiple markers, method and composition for use in the present invention.For example, in one embodiment, the method for the present invention
Marker with composition includes D-Asp and 5,6- dihydrouracil.In one embodiment, marker includes D-
Aspartic acid and behenic acid.In one embodiment, marker includes D-Asp and threonic acid.Implement at one
In scheme, marker includes D-Asp and tetracosanoic acid.In one embodiment, marker include D-Asp and
Stearic acid.In one embodiment, marker includes 5,6- dihydrouracil and behenic acid.In an embodiment
In, marker includes 5,6- dihydrouracil and threonic acid.In one embodiment, marker includes 5,6- dihydrouracil
And tetracosanoic acid.In one embodiment, marker includes 5,6- dihydrouracil and stearic acid.In an embodiment
In, marker includes behenic acid and threonic acid.In one embodiment, marker includes behenic acid and 24
Acid.In one embodiment, marker includes behenic acid and stearic acid.In one embodiment, marker includes
Threonic acid and tetracosanoic acid.In one embodiment, marker includes threonic acid and stearic acid.In one embodiment,
Marker includes tetracosanoic acid and stearic acid.In one embodiment, marker include D-Asp, 5,6- dihydros urine it is phonetic
Pyridine and behenic acid.In one embodiment, marker includes D-Asp, 5,6- dihydrouracil and threonic acid.
In one embodiment, marker includes D-Asp, 5,6- dihydrouracil and tetracosanoic acid.In an embodiment
In, marker includes D-Asp, 5,6- dihydrouracil and stearic acid.In one embodiment, marker includes D-
Aspartic acid, behenic acid and threonic acid.In one embodiment, marker includes D-Asp, behenic acid
And tetracosanoic acid.In one embodiment, marker includes D-Asp, behenic acid and stearic acid.In a reality
It applies in scheme, marker includes D-Asp, threonic acid and tetracosanoic acid.In one embodiment, marker includes D-
Aspartic acid, threonic acid and stearic acid.In one embodiment, marker includes D-Asp, tetracosanoic acid and tristearin
Acid.In one embodiment, marker includes 5,6- dihydrouracil, behenic acid and threonic acid.In an embodiment party
In case, marker includes 5,6- dihydrouracil, behenic acid and tetracosanoic acid.In one embodiment, marker packet
Include 5,6- dihydrouracil, behenic acid and stearic acid.In one embodiment, marker includes that 5,6- dihydros urine is phonetic
Pyridine, threonic acid and tetracosanoic acid.In one embodiment, marker includes 5,6- dihydrouracil, threonic acid and stearic acid.
In one embodiment, marker includes behenic acid, threonic acid and stearic acid.In one embodiment, marker
Including behenic acid, threonic acid and tetracosanoic acid.In one embodiment, marker includes stearic acid, threonic acid and two
Tetradecylic acid.In one embodiment, marker includes D-Asp, 5,6- dihydrouracil, behenic acid and threose
Acid.In one embodiment, marker includes D-Asp, 5,6- dihydrouracil, behenic acid and tetracosanoic acid.
In one embodiment, marker includes D-Asp, 5,6- dihydrouracil, behenic acid and stearic acid.One
In a embodiment, marker includes 5,6- dihydrouracil, behenic acid, threonic acid and tetracosanoic acid.Implement at one
In scheme, marker includes 5,6- dihydrouracil, behenic acid, threonic acid and stearic acid.In one embodiment, it marks
Will object includes D-Asp, 5,6- dihydrouracil, behenic acid, threonic acid and tetracosanoic acid.In an embodiment
In, marker includes D-Asp, 5,6- dihydrouracil, behenic acid, threonic acid and stearic acid.In an embodiment party
In case, marker includes stearic acid, 5,6- dihydrouracil, behenic acid, threonic acid and tetracosanoic acid.In an embodiment party
In case, marker includes D-Asp, stearic acid, 5,6- dihydrouracil, behenic acid, threonic acid and tetracosanoic acid.
Liver is that triglycerides synthesizes best place, and when liver damage, triglycerides cannot be synthesized normally, so can
To lead to stearic accumulation.Therefore, present inventor speculates that stearic acid content increases in BDTT patients blood plasmas, a side
Face increase to fatty acid enzyme activity caused by tumour cell it is related, on the other hand may lipid-metabolism caused by the damage with liver
Reduce related, when liver damage, stearic content increases, and hepar damnification and apoptosis has been further aggravated, and forms vicious circle.
The most medical histories for previously having chronic hepatitis B or other hepatitis of BDTT patient, biliary obstruction caused by BDTT is into one
Step has aggravated the hepar damnification of such patient, and this kind of patient has dual pathological factor, therefore its blood plasma stearic acid content is wanted
It is significantly higher than only that (EHCC most patients do not have liver by a kind of cholangiocarcinoma that obstruction of biliary tract impairment factor is influenced (EHCC) patient
Scorching virus infection medical history).Present study also prompts stearic acid level in EHCC patients blood plasmas to be significantly higher than biliary benign disease
Group and normal person, such gradient also from reflecting stearic acid and liver damage degree is closely related to a certain degree.
Marker：That is biomarker, can changing with tagging system, organ, tissue, cell and subcellular structure or function
The biochemical indicator of change or the change that may occur has very extensive purposes.Biomarker can be used for medical diagnosis on disease, judge
Staging or for evaluating the safety and efficacy of new drug or new treatment in target group.Biomarker is biology
It is different because being influenced by environmental contaminants in different biological horizontal (molecule, cell, individual etc.) before body is severely damaged
The signal index of normalizing.It can injure serious toxicity and provide early warning.This signal index can be cellular elements knot
The variation of structure and function can be the variation of a certain biochemical metabolism process or generate abnormal metabolite or its content, can be with
It is the Novel presentation of a certain physiological activity or a certain physiological activator, can is the abnormal phenomenon that individual is shown, Ke Yishi
The anomalous variation of population or group can be the anomalous variation of the ecosystem.
Biliary tract disease：Biliary system is originating from bile capillaries in liver, along the dissection leaflet of liver, gradually converge for
Gan Nei branches at different levels drain left and right half liver, finally summarize outside liver for ductuli hepaticus communis respectively until Hilar becomes Left And Right Hepatic Duct.
Gall-bladder Via bile duct is communicated with ductuli hepaticus communis, enters ductuli hepaticus communis from cystic duct hereinafter, becoming choledochus.In general, by left and right common hepatic duct
Above bile duct is known as stones in intrahepatic bile duct, from ductuli hepaticus communis hereinafter referred to as extrahepatic bile ducts.There are many biliary tract disease type, are broadly divided into
4 major class, 35 kinds of diseases.The first kind is gall-bladder inflammatory lesion, including acute cholecystitis, chronic cholecystitis, cholecystolithiasis, yellow meat
The swollen property cholecystitis of bud, perforation of gallbladder, acute emphysematous cholecystitis.Second class be bile duct inflammatory lesion, including sclerosing cholangitis,
Eosinophilic cholangitis, pyogenic cholangitis.Third class is biliary tract neoplasms, and type is various, including tumour, hepatic bile tumor, courage
Tumour, choledochus adenomyoma, the small hamartoma of bile duct, Gallbladder polypoid disease, BILIARY PAPILLOMATOSIS, bile duct particle around pipe
Property cytoma, choledochus villous adenoma, adenoma papilliforum, bile duct class cancer and gall-bladder gland endocrine cell cancer, liver and gall in bile duct
Pipe primary melanoma, gallbladder cancer, gall-bladder malignant fibrous histiocytoma, smooth muscle of bile vesica tumor, smooth muscle of bile vesica sarcoma, courage
Capsule carcinosarcoma, hilar cholangiocarcinoma, cholangiocarcinoma.4th class includes that hematobilia, postcholecystectomy syndrome, gall-bladder are soft
Change spot disease, biliary tract variation, bile duct traumatic neuroma, bacterial biliary duct injury.
In the present invention, the biliary tract disease is preferably primary cholelithiasis, congenital biliary duct cyst, extrahepatic bile ducts
It is one or more in cancer.
Second aspect：Screening and appraisal mark object
Using the method for blood plasma metabolism group come screening and appraisal mark object in the present invention.
Metabolism group is the new branch of science to emerge rapidly after genomics, transcription group, proteomics,
As the important component of systems biology.Metabolism group is that research biosystem is generated after by outside stimulus or disturbance
The science of endogenous metabolism object entirety and its changing rule, be verify biosystem metabolic pathway research method it is many into the cell
Vital movement betides metabolism level, such as cell signal release, energy transmission, cell-cell communication, in body any physiology or
Pathological change all be unable to do without the variation of metabolic process and its component.
The research sample of metabolism group can be biological fluid (such as blood plasma, urine, saliva), cell extract, cell
Culture solution and tissue etc..Its central task includes detection, quantifies and edit and record entirety and its variation of biological endogenous property metabolite
Rule, and probe by this changing rule the essence of biological event occurred or process.The research pair of metabolism group
Relative molecular mass 1000Da small-molecule substances below are liked, including：Carbohydrate, amino acid, lipid, organic acid, carnitine etc..Generation
It thanks to group research learned, generally comprises acquisition and preparation, the acquisition of metabolome data, pretreatment, the multivariate statistics point of sample
Analysis and metabolic pathway analysis：Obtain biological sample, sample pre-treatments, the analyzing processing for carrying out analysis and testing technology, target
To or non-targeted analysis, the determination of data prediction, multivariate analysis and possible biomarker.
The acquisition and preparation of sample are one of the step of metabolism group grinds the most initial in making internal disorder or usurp and most important step.
It can be applied to metabolism group and grind the body fluid to make internal disorder or usurp in addition to common blood (serum, blood plasma) and urine, further include：Cerebrospinal fluid, ascites,
Amniotic fluid, sperm, digestive juice, pulmonary aspiration etc..
Currently, common analysis and testing technology be with nuclear magnetic resoance spectrum (NMR), mass spectrum (MS) be the analytical technology of core with
And chromatography and wave spectrum joint technology, including:NMR, LC-NMR or LC-NMR-MS, LC-MS and GC-MS etc..NMR technology has nothing
The features such as skewed popularity, not damaged sample, but the sensitivity is low, detection narrow dynamic range.MS and MS/MS technologies have higher
Sensitivity and specificity, quickly detection and quantitative analysis while may be implemented multiple compounds.
Data prediction and analysis：In metabolism group research, pretreatment, analysis and the management of mass data need specially
Mathematics, statistics and bioinformatics tools.Wherein, spectrum signal is divided into many junior units by H NMR spectroscopy data needs first
(bins), baseline correction and integration are then carried out, the cross-relaxation effect of variation and urea that water depression effect is brought is reduced；Base
It is on the basis of carrying out full scan to certain mass-to-charge ratio (zw/z) range, if the data of acquisition are divided into the analysis method of MS
Small window is done, computer subregion is facilitated to carry out peak match.
Since Metabolite components complexity, content difference are big in biological sample, high-content compound is easy to cover low content
The variation for closing object, often prevents some contents relatively low but the Metabolite components with important biomolecule meaning are from being identified.Therefore,
Need to data carry out it is upscaled, to eliminate different metabolic impersonal language difference.
Multivariate analysis：Also referred to as multi-variables analysis or multi-variate statistical analysis, mainly study multiple variables set it
Between relationship and individual with these variables between relationship.Briefly, it exactly studies between n observation object set
The method of feature, wherein observing p factor (variable) to each object.The set of observation object can entirely be gathered, and also may be used
To be taken from one or more subsets of larger set；It can be fixed, can also be random；Can be categorical variable,
It can also be quantitative variation；Quantitative variation can be continuous, and can also be discrete；And variables set itself can be taken from
One or more subsets of larger variables collection.
The second aspect of the present invention provides the method for obtaining the marker, and specific steps are：
1, Samples subjects are obtained, and carry out pre-treatment；2, by the Samples subjects of pre-treatment carry out GC-MS or
LC-MS is detected, and collects data；3, data analysis is carried out, feasible marker is obtained.
In above-mentioned steps, the Samples subjects can be solid, liquid, can also be cell, microorganism etc..
As an implementation, when the Samples subjects are solid, the method for the specific pre-treatment is：It will
100mg solids are dissolved in 400 μ L extracts reagents, and steel ball is added and is ground, and the condition of the grinding is 30Hz, 5 minutes；Then into
Row centrifugation, takes supernatant.To carry out GC-MS tests, drying can be concentrated.The step of concentrate drying is that will add in supernatant
The methoxamine for entering 20mg/ml, the dry 20min at 80 DEG C, is then added under reagent BSTFA after performing the derivatization processing 1 hour,
It is tested.To carry out LC-MS tests, then supernatant need to be only concentrated and dried, 100 μ L acetonitriles are added and are redissolved,
Through filtering plug, LC-MS tests can be directly carried out.As an implementation, when the Samples subjects are liquid, such as blood
Clearly, urine etc..The method of the specific pre-treatment is：100 μ L liquid be added 350 μ L methanol in, carry out mediation mixing, at a high speed from
The heart takes supernatant, is tested.To carry out GC-MS tests, drying can be concentrated.The step of concentrate drying is will
The methoxamine of 20mg/ml is added in supernatant, then the dry 20min at 80 DEG C is added under reagent BSTFA and performs the derivatization processing
After 1 hour, tested.To carry out LC-MS tests, then only supernatant need to be concentrated and dried, be added 100 μ L acetonitriles into
Row redissolves, and through filtering plug, can directly carry out LC-MS tests.
As an implementation, when the Samples subjects are microorganism, the processing side of the processing mode and cell
Formula is the same.
Threshold value (P values)：Amount statistically represents result conspicuousness.P values<0.05, which represents random error, generates this knot
The probability of fruit is less than 0.05.
The third aspect：The method for distinguishing intervention of hepatocellular carcinoma with bile duct thrombi and biliary tract disease
In some aspects, the present invention, which provides, distinguishes intervention of hepatocellular carcinoma with bile duct thrombi and biliary tract disease method.For example,
In one aspect, the present invention is provided to determine whether subject has the method for intervention of hepatocellular carcinoma with bile duct thrombi.The side
Method include measure in Samples subjects the present invention one or more markers level in the control sample described in
The level of one or more markers.Compared with the level of one or more markers in control sample, in Samples subjects
The level difference of one or more markers indicates that the subject has intervention of hepatocellular carcinoma with bile duct thrombi.
The method for distinguishing intervention of hepatocellular carcinoma with bile duct thrombi and biliary tract disease with the marker, the method packet
It includes：
The level of heretofore described one or two kinds of markers is measured in the tissue samples of subject；
By the level of one or more markers in one or both of Samples subjects marker and control sample
It compares, wherein compared with the level of one or more markers in control sample, it is one or more in Samples subjects
The horizontal difference of marker indicates that the subject has intervention of hepatocellular carcinoma with bile duct thrombi.
The control sample can be the sample of health volunteer.
Fourth aspect：Determine whether subject has therapy the method for reaction
For determine with intervention of hepatocellular carcinoma with bile duct thrombi subject whether the method that will have reaction to therapy.
The method includes measure in Samples subjects the present invention one or more markers level with right
The level of one or more markers in product in the same old way.In the horizontal phase with one or more markers in control sample
Than the level difference of one or more markers in Samples subjects indicates that the subject will have reaction to treatment.
5th aspect：The method for monitoring therapeutic efficiency
Method for monitoring therapeutic efficiency in the subject with intervention of hepatocellular carcinoma with bile duct thrombi.
The present invention also provides for detecting the development that can be used for inhibiting intervention of hepatocellular carcinoma with bile duct thrombi in subject；Drop
It is low or slow down development of the normal person to intervention of hepatocellular carcinoma with bile duct thrombi；And/or it reduces or inhibits relevant simultaneously with the disease
The method of the effect of therapy or therapeutic scheme of the development of hair disease or any other therapy.In these methods, assessment exists
Sheet in a pair of of sample (the first sample without therapeutic scheme and at least part of second sample by therapeutic scheme)
The level of one or more markers of invention.Relative to the second sample, the expression of one or more markers in the first sample
Therapy effectively inhibits intervention of hepatocellular carcinoma with bile duct thrombi in subject described in horizontal adjusting surface；Reduce or slow down normal person
Body to intervention of hepatocellular carcinoma with bile duct thrombi development；And/or reduce or inhibit the development with the relevant complication of shown disease.
6th aspect：
For determining whether subject has or will develop the kit of intervention of hepatocellular carcinoma with bile duct thrombi.
In any method and kit of the present invention, the level for being obtained from marker of the invention in the sample of subject can
It is measured by any in various widely-known techniques and method, the marker of the present invention in sample is converted to can
Detection and quantitative part.The non-limiting examples of such method include using for detect protein immunological method,
Method of purifying protein, protein function or activation measurement, nucleic acid hybridization, nucleic acid reverse-transcription method and nucleic acid amplification side
Method, Western blot but newspaper blotting RNA blottings, electron microscopy, mass spectrography, are immunoprecipitated, immunofluorescence, are immunized
Histochemistry, enzyme-linked immunosorbent assay, the quantitative determination process based on blood, the quantitative determination process based on urine, streaming are thin
Born of the same parents' art, DNA hybridization, display analysis etc. and a combination thereof or sub-combination analyze sample.
If term " marker activity " used interchangeably herein and " bioactivity of marker " include marker protein
To the activity that marker reacting cells or tissue are applied, or to work that marker nucleic acid molecule or albumen target molecule are applied
Property, as measured in vivo and/or in vitro according to standard technique.Marker activity can be direct activity, for example, with marker-
Target molecule associates.Alternatively, marker activity is indirect activity, such as by marker protein and marker-target molecule or it is related to
Other interactions of molecules in the signal transduction pathway of the marker and the downstream biological event mediated.
Another aspect of the present invention is related to the expression of adjustment marks object and/or active method in cell.The present invention's
Adjusting method includes that cell is made to be contacted with the expression of adjustment marks object and/or active reagent so that marker in cell
Expression and/or activity are regulated.In order to make the marker in cell expression and/or activity it is regulated, the cell with
It is enough the expression of adjustment marks object and/or the conditioning agent contact of active amount.
The conjunction object or molecule of " instrumentality " or " conditioning agent " being adjustment effect, and can be such as agonist, antagonism
Agent, activator, stimulant, co-inhibitor or inhibitor.Term " conditioning agent " as used herein refers to the activity of adjustment marks object
Any part, including adjustment marks object expression or adjustment marks object function part.Conditioning agent can be by adjusting in cell
The activity of marker polypeptide works, such as by making cell be contacted with reagent, the reagent such as interference indicator object and its phase
The combination of the molecule of interaction changes the binding specificity of marker or the expression (example of posttranslational modification marker or marker
Such as pass through the transcription of adjustment marks object gene or the translation of marker mRNA).Therefore, the present invention is characterized in that for adjust by
The method for one or more biological respinses that marker is adjusted, that is, pass through the expression for making cell and marker and/or active tune
Save agent contact so that biological respinse is regulated.
Representative conditioning agent is described below, including but not limited to albumen, nucleic acid molecules, antibody, nucleic acid (such as antisense
Molecule, such as ribozyme and rnai agent), immunoconjugates (such as with therapeutic agent be conjugated antibody), small molecule, fusion protein,
Aptamer, lipocalin (lipocalin) and the derivative peptide compounds of marker-.
Term " contact " (such as cell is made to be contacted with conditioning agent) as used herein is intended to include incubated in vitro tune together
Section agent and cell (such as in addition conditioning agent to cell of culture) give conditioning agent to subject and make conditioning agent and tested
The cell of person contacts in vivo.Term " contact " is not intended to include the reagent for exposing cells to be naturally occurring in subject
(that is, the exposure that may occur because of native physiological process).
Term " patient " as used herein or " subject " are intended to include people and veterinary science patient.In specific embodiment party
In case, subject is people.Term " non-human animal " includes all vertebrates, such as mammal and nonmammalian, such as
Non-human primate, mouse, rabbit, sheep, dog, milk cow, chicken, amphibian and reptile.
The present invention also provides for determining whether subject has the kit of intervention of hepatocellular carcinoma with bile duct thrombi.
These kits include that the tool for the level for measuring one or more markers of the present invention and kit are used and said
Bright book.The kit of the present invention can optionally include the other component for carrying out the method for the present invention.For example, kit may include using
In the reagent, control sample, one or more sample compartments, the intervention of hepatocellular carcinoma with bile duct thrombi that obtain biological sample from subject
Therapeutic agent, description carry out the guiding material and non-tissue specific control/standard items of the method for the present invention.It is one or more for measuring
The horizontal reagent of marker may include that such as buffer solution or the level for evaluating one or more markers (such as express water
Flat (such as on mRNA or protein level)) measuring method other reagents.Specification can be for example for carrying out evaluation originally
The specification of the printing of the horizontal measuring method of one or more markers of invention.For detaching biological sample from subject
Reagent may include the one or more reagents that can be used for obtaining fluid or tissue from subject, such as obtaining saliva or blood
Tool.The kit of the present invention can further comprise the reagent for cultivating the sample for being obtained from subject.It is preferred that kit passes through
Designed for human experimenter.
The present invention is described further by following embodiment, and embodiment should not be construed as further limiting.The application is complete
The content and attached drawing of all bibliography, patent and disclosed patent application cited in text, explicitly by reference with it
It is incorporated integrally into herein.
Grouping：Be divided into normal group, primary cholelithiasis group, congenital biliary duct cyst group, cholangiocarcinoma group and primary
Property liver cancer with Bile duct tumor thrombus group；
Choose 1250 subjects, wherein 220 people of normal healthy people；200 people of primary cholelithiasis；Congenital biliary capsule
Swollen 230 people；300 people of cholangiocarcinoma, 300 people of intervention of hepatocellular carcinoma with bile duct thrombi.
One, marker is obtained
Step：1, subject's blood is obtained, and carries out pre-treatment：100 μ L liquid are added in 350 μ L methanol, mediate
Mixing, high speed centrifugation take supernatant, are concentrated and dried, and 100 μ L acetonitriles are added and are redissolved, through filtering plug, can directly carry out
UHPLC-QQQ-MS is tested；UHPLC-QQQ-MS data are collected, polytomy variable statistical analysis is carried out.
Using BDTT groups are found after UHPLC-QQQ-MS quantitative verifications, with normal person's group, cholangiocarcinoma, primary biliary
Calculus group and congenital biliary duct cyst group are compared, and special high expression substance has：D-Asp, 5,6- dihydrouracil, two
Dodecanoic acid, threonic acid, tetracosanoic acid and stearic acid.T inspections are carried out to data, with the p value (threshold value of T-test<0.05) come true
Determine differential expression metabolin, the results are shown in Table 1：
1 BDTT groups of table comparison other groups (including normal group, primary cholelithiasis group, congenital biliary duct cyst group and liver
Outside lining pipe cancer group) special high expression substance
Two, BDTT is diagnosed with the single marker screened
Embodiment 1 is that single marker diagnoses BDTT with 5,6- dihydrouracil
It being tested by ROC curve (Receiver operating curve), it is known that AUC (area under ROC curve) is 0.8536,
Diagnostic sensitivity is 69.81%, specificity 0%.
Embodiment 2 is that single marker diagnoses BDTT with D-Asp
It being tested by ROC curve (Receiver operating curve), it is known that AUC (area under ROC curve) is 0.9669,
Diagnostic sensitivity is 95.28%, specificity 10.81%.
Embodiment 3 is that single marker diagnoses BDTT with threonic acid
It being tested by ROC curve (Receiver operating curve), it is known that AUC (area under ROC curve) is 0.4598,
Diagnostic sensitivity is 39.62%, specificity 29.73%.
Embodiment 4 is that single marker diagnoses BDTT with stearic acid
It being tested by ROC curve (Receiver operating curve), it is known that AUC (area under ROC curve) is 0.1466,
Diagnostic sensitivity is 100%, specificity 100%.
Embodiment 5 is that single marker diagnoses BDTT with behenic acid
It being tested by ROC curve (Receiver operating curve), it is known that AUC (area under ROC curve) is 0.8252,
Diagnostic sensitivity is 70.75%, specificity 13.51%.
Embodiment 6 is that single marker diagnoses BDTT with tetracosanoic acid
It being tested by ROC curve (Receiver operating curve), it is known that AUC (area under ROC curve) is 0.216,
Diagnostic sensitivity is 4.717%, specificity 2.703%.
Three, BDTT is diagnosed with two kinds of markers
Embodiment 7 is with 5,6- dihydrouracil and D-Asp Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil and D-Asp, which are combined, examines
When disconnected BDTT, AUC (area under ROC curve) is 0.9824；Diagnostic sensitivity is 97.17%, specificity 10.81%.
Embodiment 8 is with 5,6- dihydrouracil and threonic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil and threonic acid Combining diagnosis
When BDTT, AUC (area under ROC curve) is 0.86；Diagnostic sensitivity is 72.64%, specificity 2.703%.
Embodiment 9 is with 5,6- dihydrouracil and stearic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil and stearic acid Combining diagnosis
When BDTT, AUC (area under ROC curve) is 0.9592；Diagnostic sensitivity is 87.74%, specificity 0%.
Embodiment 10 is with 5,6- dihydrouracil and behenic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil and behenic acid, which are combined, examines
When disconnected BDTT, AUC (area under ROC curve) is 0.9587；Diagnostic sensitivity is 91.51%, specificity 5.405%.
Embodiment 11 is with 5,6- dihydrouracil and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil and behenic acid, which are combined, examines
When disconnected BDTT, AUC (area under ROC curve) is 0.9737；Diagnostic sensitivity is 92.45%, specificity 0%.
Embodiment 12 is with D-Asp and threonic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), D-Asp and when threonic acid Combining diagnosis BDTT,
AUC (area under ROC curve) is 0.9669；Diagnostic sensitivity is 95.28%, specificity 10.81%.
Embodiment 13 is with D-Asp and stearic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), D-Asp and when threonic acid Combining diagnosis BDTT,
AUC (area under ROC curve) is 0.9878；Diagnostic sensitivity is 96.23%, specificity 5.405%.
Embodiment 14 is with D-Asp and behenic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), D-Asp and behenic acid Combining diagnosis
When BDTT, AUC (area under ROC curve) is 0.9878；Diagnostic sensitivity is 97.76%, specificity 5.405%.
Embodiment 15 is with D-Asp and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), D-Asp and tetracosanoic acid Combining diagnosis BDTT
When, AUC (area under ROC curve) is 0.9913；Diagnostic sensitivity is 96.23%, specificity 2.703%.
Embodiment 16 is with threonic acid and stearic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), threonic acid and when stearic acid Combining diagnosis BDTT, AUC
(area under ROC curve) is 0.8534；Diagnostic sensitivity is 76.42%, specificity 16.22%.
Embodiment 17 is with threonic acid and behenic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), threonic acid and when behenic acid Combining diagnosis BDTT,
AUC (area under ROC curve) is 0.8457；Diagnostic sensitivity is 78.30%, specificity 18.92%.
Embodiment 18 is with threonic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), threonic acid and when tetracosanoic acid Combining diagnosis BDTT,
AUC (area under ROC curve) is 0.8386；Diagnostic sensitivity is 64.15%, specificity 0%.
Embodiment 19 is with stearic acid and behenic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), stearic acid and when behenic acid Combining diagnosis BDTT,
AUC (area under ROC curve) is 0.8962；Diagnostic sensitivity is 80.19%, specificity 8.108%.
Embodiment 20 is with stearic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), stearic acid and when tetracosanoic acid Combining diagnosis BDTT,
AUC (area under ROC curve) is 0.8677；Diagnostic sensitivity is 72.64%, specificity 5.405%.
Embodiment 21 is with behenic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), behenic acid and tetracosanoic acid Combining diagnosis BDTT
When, AUC (area under ROC curve) is 0.8651；Diagnostic sensitivity is 77.36%, specificity 5.405%.
Four, BDTT is diagnosed with three kinds of markers
Embodiment 22 is with 5,6- dihydrouracil, D-Asp and threonic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, D-Asp and threonic acid
When Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9827；Diagnostic sensitivity is 97.17%, and specificity is
10.81%.
Embodiment 23 is with 5,6- dihydrouracil, D-Asp and stearic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, D-Asp and stearic acid
When Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9929；Diagnostic sensitivity is 94.34%, and specificity is
2.703%.
Embodiment 24 is with 5,6- dihydrouracil, D-Asp and behenic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, D-Asp and 22
When alkanoic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9893；Diagnostic sensitivity is 98.11%, and specificity is
5.405%.
Embodiment 25 is with 5,6- dihydrouracil, D-Asp and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, D-Asp and 24
When sour Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9982；Diagnostic sensitivity is 98.11%, and specificity is
2.703%.
Embodiment 26 is with 5,6- dihydrouracil, threonic acid and stearic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, threonic acid and stearic acid joint
When diagnosing BDTT, AUC (area under ROC curve) is 0.9605；Diagnostic sensitivity is 87.74%, specificity 0%.
Embodiment 27 is with 5,6- dihydrouracil, threonic acid and behenic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, threonic acid and behenic acid
When Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9587；Diagnostic sensitivity is 91.51%, and specificity is
5.405%.
Embodiment 28 is with 5,6- dihydrouracil, threonic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, threonic acid and tetracosanoic acid connection
When closing diagnosis BDTT, AUC (area under ROC curve) is 0.9755；Diagnostic sensitivity is 92.45%, specificity 0%.
Embodiment 29 is with 5,6- dihydrouracil, stearic acid and behenic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, stearic acid and behenic acid
When Combining diagnosis BDTT, AUC (area under ROC curve) is 0.987；Diagnostic sensitivity is 97.17%, and specificity is
5.405%.
Embodiment 30 is with 5,6- dihydrouracil, stearic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, stearic acid and tetracosanoic acid connection
When closing diagnosis BDTT, AUC (area under ROC curve) is 0.9855；Diagnostic sensitivity is 93.4%, specificity 0%.
Embodiment 31 is with 5,6- dihydrouracil, behenic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, behenic acid and 24
When sour Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9842；Diagnostic sensitivity is 96.23%, and specificity is
2.703%.
Embodiment 32 is with D-Asp, threonic acid and stearic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), D-Asp, threonic acid and stearic acid Combining diagnosis
When BDTT, AUC (area under ROC curve) is 0.9895；Diagnostic sensitivity is 97.17%, specificity 5.405%.
Embodiment 33 is with D-Asp, threonic acid and behenic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), D-Asp, threonic acid and behenic acid joint
When diagnosing BDTT, AUC (area under ROC curve) is 0.9776；Diagnostic sensitivity is 96.23%, specificity 5.405%.
Embodiment 34 is with D-Asp, threonic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), D-Asp, threonic acid and tetracosanoic acid, which are combined, examines
When disconnected BDTT, AUC (area under ROC curve) is 0.9913；Diagnostic sensitivity is 96.23%, specificity 2.703%.
Embodiment 35 is with D-Asp, stearic acid and behenic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), D-Asp, stearic acid and behenic acid joint
When diagnosing BDTT, AUC (area under ROC curve) is 0.9921；Diagnostic sensitivity is 94.34%, specificity 2.703%.
Embodiment 36 is with D-Asp, stearic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), D-Asp, stearic acid and tetracosanoic acid, which are combined, examines
When disconnected BDTT, AUC (area under ROC curve) is 0.9944；Diagnostic sensitivity is 99.06%, specificity 2.703%.
Embodiment 37 is with D-Asp, behenic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), D-Asp, behenic acid and tetracosanoic acid connection
When closing diagnosis BDTT, AUC (area under ROC curve) is 0.9926；Diagnostic sensitivity is 97.17%, and specificity is
2.703%.
Embodiment 38 is with threonic acid, stearic acid and behenic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), threonic acid, stearic acid and behenic acid Combining diagnosis
When BDTT, AUC (area under ROC curve) is 0.898；Diagnostic sensitivity is 80.19%, specificity 8.108%.
Embodiment 39 is with threonic acid, stearic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), threonic acid, stearic acid and tetracosanoic acid Combining diagnosis
When BDTT, AUC (area under ROC curve) is 0.8756；Diagnostic sensitivity is 69.81%, specificity 5.405%.
Embodiment 40 is with threonic acid, behenic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), threonic acid, behenic acid and tetracosanoic acid, which are combined, examines
When disconnected BDTT, AUC (area under ROC curve) is 0.8929；Diagnostic sensitivity is 79.25%, specificity 10.81%.
Embodiment 41 is with stearic acid, behenic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), stearic acid, behenic acid and tetracosanoic acid, which are combined, examines
When disconnected BDTT, AUC (area under ROC curve) is 0.9044；Diagnostic sensitivity is 83.96%, specificity 8.108%.
Five, BDTT is diagnosed with four kinds of markers
Embodiment 42 is with 5,6- dihydrouracil, D-Asp, threonic acid and stearic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, D-Asp, threonic acid
When with stearic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9946；Diagnostic sensitivity is 96.23%, specifically
Degree is 2.703%.
Embodiment 43 is with 5,6- dihydrouracil, D-Asp, threonic acid and behenic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, D-Asp, threonic acid
When with behenic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9898；Diagnostic sensitivity is 98.11%,
Specificity is 5.405%.
Embodiment 44 is with 5,6- dihydrouracil, D-Asp, threonic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, D-Asp, threonic acid
When with tetracosanoic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9985；Diagnostic sensitivity is 99.06%, special
Different degree is 2.703%.
Embodiment 45 is with 5,6- dihydrouracil, D-Asp, stearic acid and behenic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, D-Asp, stearic acid
When with behenic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9977；Diagnostic sensitivity is 97.17%,
Specificity is 0%.
Embodiment 46 is with 5,6- dihydrouracil, D-Asp, stearic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, D-Asp, stearic acid
When with tetracosanoic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9985；Diagnostic sensitivity is 100%, specifically
Degree is 2.703%.
Embodiment 47 is with 5,6- dihydrouracil, D-Asp, behenic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, D-Asp, 22
Alkanoic acid and when tetracosanoic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9982；Diagnostic sensitivity is
98.11%, specificity 2.703%.
Embodiment 48 is with 5,6- dihydrouracil, threonic acid, stearic acid and behenic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, threonic acid, stearic acid and two
When dodecanoic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9867；Diagnostic sensitivity is 96.23%, specifically
Degree is 2.703%.
Embodiment 49 is with 5,6- dihydrouracil, threonic acid, stearic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, threonic acid, stearic acid and two
When tetradecylic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9847；Diagnostic sensitivity is 93.40%, specificity
It is 0%.
Embodiment 50 is with 5,6- dihydrouracil, threonic acid, behenic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, threonic acid, behenic acid
When with tetracosanoic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9852；Diagnostic sensitivity is 96.23%, special
Different degree is 2.703%.
Embodiment 51 is with 5,6- dihydrouracil, stearic acid, behenic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, stearic acid, behenic acid
When with tetracosanoic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9916；Diagnostic sensitivity is 97.17%, special
Different degree is 2.703%.
Embodiment 52 is with D-Asp, threonic acid, stearic acid and behenic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), D-Asp, threonic acid, stearic acid and 22
When alkanoic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9916；Diagnostic sensitivity is 95.28%, and specificity is
2.703%.
Embodiment 53 is with D-Asp, threonic acid, stearic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), D-Asp, threonic acid, stearic acid and 24
When sour Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9944；Diagnostic sensitivity is 99.06%, and specificity is
2.703%.
Embodiment 54 is with D-Asp, threonic acid, behenic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), D-Asp, threonic acid, behenic acid and two
When tetradecylic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9926；Diagnostic sensitivity is 97.17%, specificity
It is 2.703%.
Embodiment 55 is with D-Asp, stearic acid, behenic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), D-Asp, stearic acid, behenic acid and two
When tetradecylic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9944；Diagnostic sensitivity is 99.06%, specificity
It is 2.703%.
Embodiment 56 is with threonic acid, stearic acid, behenic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), threonic acid, stearic acid, behenic acid and 24
When sour Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9097；Diagnostic sensitivity is 82.08%, and specificity is
8.108%.
Six, BDTT is diagnosed with five kinds of markers
Embodiment 57 is combined with 5,6- dihydrouracil, D-Asp, threonic acid, stearic acid and behenic acid examines
Disconnected BDTT
By ROC curve (Receiver operating curve) test, 5,6- dihydrouracil, D-Asp, threonic acid,
Stearic acid and when behenic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9521；Diagnostic sensitivity is
93.4%, specificity 0%.
Embodiment 58 is with 5,6- dihydrouracil, D-Asp, threonic acid, stearic acid and tetracosanoic acid Combining diagnosis
BDTT
By ROC curve (Receiver operating curve) test, 5,6- dihydrouracil, D-Asp, threonic acid,
Stearic acid and when tetracosanoic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.999；Diagnostic sensitivity is 100%,
Specificity is 2.703%.
Embodiment 59 is combined with 5,6- dihydrouracil, D-Asp, threonic acid, behenic acid and tetracosanoic acid
Diagnose BDTT
By ROC curve (Receiver operating curve) test, 5,6- dihydrouracil, D-Asp, threonic acid,
Behenic acid and when tetracosanoic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9516；Diagnostic sensitivity is
95.28%, specificity 2.703%.
Embodiment 60 is combined with 5,6- dihydrouracil, D-Asp, stearic acid, behenic acid and tetracosanoic acid
Diagnose BDTT
By ROC curve (Receiver operating curve) test 5,6- dihydrouracil, D-Asp, stearic acid,
Behenic acid and when tetracosanoic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9995；Diagnostic sensitivity is
99.06%, specificity 0%.
Embodiment 61 is with 5,6- dihydrouracil, threonic acid, stearic acid, behenic acid and tetracosanoic acid Combining diagnosis
BDTT
It is tested by ROC curve (Receiver operating curve), 5,6- dihydrouracil, threonic acid, stearic acid, 20
Two alkanoic acids and when tetracosanoic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9913；Diagnostic sensitivity is
97.17%, specificity 2.703%.
Embodiment 62 is with D-Asp, threonic acid, stearic acid, behenic acid and tetracosanoic acid Combining diagnosis BDTT
It is tested by ROC curve (Receiver operating curve), D-Asp, threonic acid, stearic acid, docosane
When acid and tetracosanoic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.9944；Diagnostic sensitivity is 99.06%,
Specificity is 2.703%.
Seven, BDTT is diagnosed with six kinds of markers
Embodiment 63 is with 5,6- dihydrouracil, D-Asp, threonic acid, stearic acid, behenic acid and 24
Sour Combining diagnosis BDTT
By ROC curve (Receiver operating curve) test 5,6- dihydrouracil, D-Asp, threonic acid,
When stearic acid, behenic acid and tetracosanoic acid Combining diagnosis BDTT, AUC (area under ROC curve) is 0.04118；Diagnosis
Sensitivity is 0%, specificity 0%.General status see the table below 2
Table 2
Comparative example 1：
When using alpha-fetoprotein as diagnosis index, sensitivity 100%, but its specificity is 54.3%.
Comparative example 2：
When with CA19-9 (tumor markers) being diagnosis index, it can obviously increase, BDTT and EHCC can not be carried out
Good discriminating, therefore do not calculate it and diagnose specificity and susceptibility.
The clinical value of tumor markers is the diagnosis and monitoring of tumour, and aspect, tumour mark are failed to pinpoint a disease in diagnosis in reduction tumour
The high sensitivity of will object is particularly important.To improve the recall rate of tumour, therefore clinic advocates Diagnostic Value of Several Serum Tumor Markers joint-detection
This research to tumor markers joint-detection BDTT, primary cholelithiasis, congenital biliary duct cyst, cholangiocarcinoma patient into
Evaluation of methodology is gone, and overall merit sensitivity, specificity.It is obtained in conjunction with ROC curve, when diagnosing BDTT, with 5,6- bis-
Sensitivity when five hydrogen uracil, D-Asp, stearic acid, behenic acid and tetracosanoic acid index joints and specificity
It is significantly improved when being diagnosed compared with single index, has respectively reached 99.06% and 0%, it is sensitive can effectively to make up alpha-fetoprotein diagnosis
Spend insufficient defect.In conclusion when differentiating BDTT with primary cholelithiasis, congenital biliary duct cyst, cholangiocarcinoma,
Stearic acid and threonic acid two indices joint-detection have important clinical value.
Claims (1)
1. a kind of marker answering in preparing the kit for distinguishing hepatocellular carcinoma complicated cholangiocarcinoma acid and biliary tract disease
With, which is characterized in that the marker is by 5,6- dihydrouracil, D-Asp, threonic acid, stearic acid and tetracosanoic acid group
At.
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