CN106501225A - The sugar chain of agglutinin group identification is distinguishing pancreatic mucinous cystic tumors and the application in pancreas serosity cystic tumor - Google Patents
The sugar chain of agglutinin group identification is distinguishing pancreatic mucinous cystic tumors and the application in pancreas serosity cystic tumor Download PDFInfo
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
The invention discloses the sugar chain of agglutinin group identification is distinguishing pancreatic mucinous cystic tumors and the application in pancreas serosity cystic tumor.The sugar chain of agglutinin group identification disclosed by the invention is by WGA, BPL, STL, DBA, sugar chain in the glycosylation albumen of the agglutinin group identification of PTL I and MAL I compositions, these sugar chains are different in the pancreas cyst fluid of mucinous cystic tumors and serosity cystic tumor patient, STL, WGA, content of the sugar chain of BPL and DBA identifications in MCN patient's pancreas cyst fluid is significantly higher than SCN patient, and content of the sugar chain of PTL I and MAL I identifications in MCN patient's pancreas cyst fluid is substantially less than SCN patient, it is 0.714 sensitivity when distinguishing SCN patient with MCN patient is combined using WGA with BPL, specificity is 1.Show that the agglutinin group of the present invention can be used to distinguish SCN patient and MCN patient.
Description
Technical field
The present invention relates in biological technical field, the sugar chain of agglutinin group identification distinguish pancreatic mucinous cystic tumors and
Application in pancreas serosity cystic tumor.
Background technology
With Imaging Technology progress and extensively apply, cystic pancreatic disease (pancreatic cyst lesion,
PCL) recall rate is improved year by year.The nearest Research statistics PCL total prevalence rates in the U.S. are 2.5%, and cystic pancreatic disease is by one group
Have the heterogenous lesion of the pathological changes composition of the different pathological types of common symptoms, mainly include the false capsule of simple pancreas
Swollen (PPs) and cystic Tumor of Pancreas (pancreatic cystic neoplasms, PCNs).PCNs category invisible morbidity early stage
Without obvious sign, which forms cyst as principal character with glandular tube or acinar epithelia hypertrophy, ischesis, in all constitutional pancreases
1%～5% is accounted in adenoncus tumor.Cystic Tumor of Pancreas is subdivided into mucinous neoplasms by 2010 editions WHO stagings：Mucuss capsule
Property tumor (Mucinous cystic neoplasm, MCN), intraductal papilloma (Intraductal papillary
Mucinous neoplasm, IPMN) and non-mucinous neoplasms：Serosity cystic tumor (Serous cystic neoplasm,
SCN), solid-pseudopapillary tumors (Solid pseudopaillary neoplasm, SPN.Wherein, malignant pancreatic cystic lesion bag
Include mucinous cystic tumors (MCN) and intraductal papillary-mucinous tumor (IPMN) and mucuss cystadenocarcinoma (Mucinous
Cystic adenocarcinoma, MCA)；And serosity cystic tumor (SCN) mostly then is benign lesion.Different pathological types its
Therapeutic Method is different, but single be difficult to distinguish which is good pernicious from clinical symptoms and Radiologic imaging, cystic lesion is generally sent out by CT
Existing, but CT is less than 70% for the sensitivity of diagnosing malignant tumor, and specificity is then between 87%-98%, the T2WI of MRI
More preferable soft tissue contrast can be provided mutually, thus is more often available to the good pernicious Differential Diagnosiss of cyst, but its accuracy is still
Stay in the relatively low interval interior of 20%-80%.Single accuracy from the good evil of iconography discriminating cystic lesion is still relatively low, while conventional
Serology tumor markerses：CEA；CA19-9；CA72-4 and CA153 etc., specificity, sensitivity in terms of precancerous lesion is differentiated
Property is relatively low.Histopathologic examination is still the goldstandard of current clinical diagnosises, but in view of the intrinsic defect of histological examination is as damaged
The inspection of wound property, dynamic detection etc. is unable to, the method for early diagnosis for seeking invasive is the key of prevention and control disease canceration. ultrasonic endoscope
Lower fine needle aspiration biopsy (endoscopic ultrasonography-guided fine needle aspiration, EUS-
FNA) the intuitively firsthand information can be provided for the diagnosis of cystic Tumor of Pancreas.
Protein glycosylation is modification after a kind of modal protein translation, is under glycosyl transferase effect to turn saccharide
Move to the process that special amino acid residue on protein, and protein forms glycosidic bond.Research shows, 70% human protein bag
Contain one or more sugar chains, 1% human genome take part in the synthesis and modification of sugar chain.The glycosyl of protein in mammal
Change type and can be divided into three kinds：N- glycosylations, O- glycosylations and GPI glycosyl-phosphatidyl inositol anchors.Most sugars protein is comprised only
A kind of type of glycosylation, but some protein polypeptides are connected with N- sugar chains, O- sugar chains or ammonia polyose of candy simultaneously.
Agglutinin due to its can specific recognition difference sugar chain structure can be combined with sugar chain high-affinity with multivalent forms
Characteristic be widely used in the research that sugar group is learned, lectin chip is by the agglutinin probe that is fixed on chip and sugar in sample
Albumen sugar chain specifically binds, and can detect the change of glycoprotein candy chain structure and connected mode in sample with high throughput, be
One of maximally effective analytical tool of research glycoprotein candy chain structure change.
Content of the invention
The technical problem to be solved is how to distinguish pancreatic mucinous cystic tumors and pancreas serosity capsule
Tumor.
For solving above-mentioned technical problem, present invention firstly provides the material of the sugar chain of detection agglutinin group identification is following
A1 the application in) or A2)：
A1) prepare and distinguish or supplementary globe pancreatic mucinous cystic tumors (Mucinous cystic neoplasm, MCN)
Patient and pancreas serosity cystic tumor (Serous cystic neoplasm, SCN) patient product；
A2) distinguish or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystic tumor；
The agglutinin group includes b1) or b2)：
B1) WGA and/or BPL；
B2) by b1) with STL, DBA, PTL-I and MAL-I in the compositionss that constitute of all or part.
In above-mentioned application, the agglutinin group may also include at least one in a kind of 30 agglutinins；Described 31
Kind of agglutinin be Jacalin, ECA, HHL, WFA, GSL- II, MAL- II, PHA-E, SJA, PNA, EEL, AAL, LTL, MPL,
LEL、GSL-Ⅰ、LCA、RCA120、BS-Ⅰ、ConA、PTL-Ⅱ、DSA、SBA、VVA、NPL、PSA、ACA、UEA-Ⅰ、PWM、GNA、
PHA-E+L and SNA.
Wherein, in the agglutinin group in the sugar chain such as embodiment of each agglutinin identification shown in table 2 or/and table 4.
In above-mentioned application, the agglutinin group can be only b1) or b2), can also be by b1) or b2) a kind of with described 30
The compositionss of at least one composition in agglutinin.
In above-mentioned application, the sugar chain content of agglutinin group identification can be the sugar chain of agglutinin group identification in pancreas
Content in cyst fluid.
In above-mentioned application, the material of the sugar chain content for detecting the agglutinin group identification may include also to can be only detection
Reagent and/or instrument needed for the sugar chain content of the agglutinin group identification.The sugar chain for detecting the agglutinin group identification
The material of content specifically may include or can be the agglutinin group or the chip containing the agglutinin group.
The reagent of the sugar chain content of detection agglutinin group identification may include the agglutinin group, fluorescent dye,
Block buffer, incubation buffer, PBST and/or PBS.The reagent of the sugar chain content for detecting the agglutinin group identification
Can only by the agglutinin group, the fluorescent dye, the Block buffer, the incubation buffer, PBST and/or PBS groups
Into.The fluorescent material can be Cy3 (Amerhsma companies of the U.S.).The Block buffer can be to PBS in add BSA, sweet
The solution that propylhomoserin and Tween-20 are obtained, in the Block buffer, the concentration of BSA, glycine and Tween-20 is respectively 2%
(mass percent concentration), 500mmol/L and 0.1% (mass percent concentration).The incubation buffer can be to PBS in plus
Enter the solution that BSA, glycine and Tween-20 are obtained, the concentration of BSA, glycine and Tween-20 point in the incubation buffer
Wei not 2% (mass percent concentration), 500mmol/L and 0.1% (mass percent concentration).PBST is to add to PBS
The Tween-20 mass percent concentrations that Tween-20 is obtained are 0.1% solution.Glycine can be U.S. Sigma-Aldrich
Products.Tween-20 can be Sigma-Aldrich's product.The pH of PBS above can be 7.4.
The instrument of the sugar chain content of detection agglutinin group identification may include the chip and/or
GenPix4000B chip scanners.The sugar chain content of detection agglutinin group identification also can be only the chip and/
Or GenPix 4000B chip scanners.GenPix 4000B chip scanners are U.S.'s Axon Products.
The material of the sugar chain content for detecting the agglutinin group identification can also be by the detection agglutinin group identification
Reagent and/or instrument needed for sugar chain content is constituted with data processing equipment, and the data processing equipment is used for will be to be measured
The content of the sugar chain of the agglutinin group identification of object is converted to the diagnostic value of the object to be measured, according to the object to be measured
Diagnostic value determine the object to be measured for MCN patient or SCN patient.The data processing equipment can be software and/or module.
The software can be 3.0 softwares of GenePix.
In one embodiment of the invention, elaborate with the WGA in the agglutinin group and BPL distinguish MCN patient and
The method of SCN patient, methods described include：Fluorescence by the content of the sugar chain of the reaction WGA and BPL identification from object to be measured
Normalization numerical value substitutes into the diagnostic value that formula 1 obtains the object to be measured；
In formula 1, WGA represents fluorescence signal normalization numerical value when detecting the sample to be tested using WGA；BPL is represented
Detect that using BPL the fluorescence signal normalization numerical value during sample to be tested, Y represent the diagnostic value of the sample to be tested.Formula 1
In WGA be specially using WGA detect the sample to be tested when fluorescence signal with using WGA, BPL, STL, DBA, PTL-I,
MAL-I、Jacalin、ECA、HHL、WFA、GSL-Ⅱ、MAL-Ⅱ、PHA-E、SJA、PNA、EEL、AAL、LTL、MPL、LEL、
GSL-Ⅰ、LCA、RCA120、BS-Ⅰ、ConA、PTL-Ⅱ、DSA、SBA、VVA、NPL、PSA、ACA、UEA-Ⅰ、PWM、GNA、PHA-E
The ratio of fluorescence signal sum during+the L and SNA detection samples to be tested, BPL are specially and treat test sample using described in BPL detections
The fluorescence signal of this when with using WGA, BPL, STL, DBA, PTL-I, MAL-I, Jacalin, ECA, HHL, WFA, GSL- II,
MAL-Ⅱ、PHA-E、SJA、PNA、EEL、AAL、LTL、MPL、LEL、GSL-Ⅰ、LCA、RCA120、BS-Ⅰ、ConA、PTL-Ⅱ、
DSA, SBA, VVA, NPL, PSA, ACA, UEA- I, PWM, GNA, PHA-E+L and SNA detects the fluorescence letter during sample to be tested
The ratio of number sum.
It is object to be measured to MCN patient using formula 1 with the cystic Tumor of Pancreas patient of unknown MCN patient and SCN patient
When making a distinction with SCN patient, such as Y is more than 0.70, and the object to be measured is or candidate is MCN patient, and such as Y is less than or equal to
0.70, the object to be measured is or candidate is SCN patient.
In actual applications, when the content of the sugar chain for being recognized using the agglutinin group distinguishes MCN patient and SCN patient,
From the agglutinin group, suitable agglutinin and corresponding model can be selected to distinguish MCN patient and SCN trouble according to practical situation
Person, as long as the content for being available with the sugar chain of the agglutinin group identification of the present invention is made a distinction to MCN patient and SCN patient,
Belong to protection scope of the present invention.
The lectin chip is that each agglutinin in the agglutinin group is fixed on the chip obtained on holder.Described
Holder can be epoxide chip base.The epoxide chip base is the holder prepared using epoxide.Described
Lectin chip can contain negative Quality Control point and/or position mark.The negative Quality Control point of the lectin chip can be BSA.Institute
The position mark for stating agglutinin core can be the BSA of the fluorochrome label.Agglutinin is fixed on holder using rich
Crystalline substance core SmartArrayer48 point sample instruments (Beijing Capitalbio Corporation Co., Ltd.) difficult to understand is carried out.
For solve above-mentioned technical problem, present invention also offers the agglutinin group identification sugar chain content in following A 1)
Or A2) in application：
A1) prepare and distinguish or supplementary globe pancreatic mucinous cystic tumors (Mucinous cystic neoplasm, MCN)
Patient and pancreas serosity cystic tumor (Serous cystic neoplasm, SCN) patient product；
A2) distinguish or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystic tumor.
For solving above-mentioned technical problem, present invention also offers the sugar chain of agglutinin group identification is sticked as pancreas is distinguished
Fluidity cystic tumor and pancreas serosity cystic tumor mark are in differentiation or supplementary globe pancreatic mucinous cystic tumors and pancreas
Application in gland serosity cystic tumor.
For solving above-mentioned technical problem, present invention also offers using the sugar chain of agglutinin group identification as differentiation pancreas
The differentiation pancreatic mucinous cystic tumors and pancreas serosity of mucinous cystic tumors and pancreas serosity cystic tumor mark
The material of cystic tumor following A 1) or A2) in application；
A1) prepare and distinguish or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystic tumor product；
A2) distinguish or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystic tumor.
For solving above-mentioned technical problem, present invention also offers distinguishing or supplementary globe pancreatic mucinous cystic tumors and pancreas
Gland serosity cystic tumor product, is the material of the sugar chain content of the detection agglutinin group identification.
In the said goods, the product may include the agglutinin group or the chip containing the agglutinin group.
Chip described in the said goods can be chip described in above-mentioned application.
In the said goods, each agglutinin in the agglutinin group can be by fluorescent material labelling.The fluorescent material can
For Cy3.
The material of the sugar chain content for detecting the agglutinin group identification can be the detection mentioned above agglutinin group
The material of the sugar chain content of identification.
For solve above-mentioned technical problem, present invention also offers the product following A 1) or A2) in application；
A1) prepare and distinguish or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystic tumor product；
A2) distinguish or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystic tumor.
For solving above-mentioned technical problem, present invention also offers build distinguishing pancreatic mucinous cystic tumors and pancreas serosity
The method of property cystic tumor.
The method for building differentiation pancreatic mucinous cystic tumors and pancreas serosity cystic tumor provided by the present invention, bag
Include：Respectively the sugar chain in pancreatic mucinous cystic tumors group and pancreas serosity cystic tumor group pancreas cyst fluid is quantitatively divided
Analysis, obtains cystic Tumor of Pancreas sugar chain characteristic spectrum according to analysis result；The cystic Tumor of Pancreas sugar chain characteristic spectrum is by described solidifying
The sugar chain composition of collection element group identification.
In said method, the pancreatic mucinous cystic tumors group may include that at least one pancreatic mucinous cystic tumors are suffered from
Person.The pancreas serosity cystic tumor group may include at least one pancreas serosity cystic tumor patient.
In said method, quantitative analyses are carried out to the sugar chain in pancreas cyst fluid, carried out using the agglutinin group.Described
Each agglutinin in agglutinin group can be by fluorescent material labelling.The fluorescent material can be Cy3.
In said method, carrying out quantitative analyses to the sugar chain in pancreas cyst fluid may include to utilize containing the agglutinin group
Chip is carried out quantitatively to the sugar chain in pancreatic mucinous cystic tumors group and pancreas serosity cystic tumor group pancreas cyst fluid respectively
Analysis.Specifically may include：Protein in testing sample is incubated with the chip with after the fluorescent material labelling, institute is determined
State the content of the sugar chain of the identification of agglutinin group described in testing sample.
The cystic Tumor of Pancreas sugar chain characteristic spectrum can be formula 1.
Pancreatic mucinous cystic tumors can be distinguished using cystic Tumor of Pancreas sugar chain characteristic spectrum and pancreas serosity capsule is swollen
Tumor.
In another embodiment of the present invention, carrying out quantitative analyses to the sugar chain in pancreas cyst fluid is included using described solidifying
It is fixed that collection element group is carried out to the sugar chain in pancreatic mucinous cystic tumors group and pancreas serosity cystic tumor group pancreas cyst fluid respectively
Amount analysis.
In the present invention, the sugar chain is sugar chain in glycosylation albumen.
Difference by comparing the glycoprotein candy chain content of SCN patient's agglutinin identification different from MCN patient of the invention,
And by correlation analysiss, it is found that for distinguishing the sugar chain that six kinds of agglutinins of SCN patient and MCN patient are recognized, this six kinds are coagulated
Collection element is respectively STL, WGA, BPL, DBA, PTL-I and MAL-I, the every kind of agglutinin of these four agglutinins of STL, WGA, BPL and DBA
Identification content of the sugar chain in MCN patient's pancreas cyst fluid is significantly higher than SCN patient, and both agglutinins of PTL-I and MAL-I are every
Plant agglutinin content of the identification sugar chain in MCN patient's pancreas cyst fluid and be substantially less than SCN patient, using WGA and BPL association regions
It is 0.714 with sensitivity during MCN patient to divide SCN patient, and specificity is 1.Show the sugar chain of the agglutinin group identification of the present invention
Can identify as biomolecule, for distinguishing SCN patient and MCN patient, it is possible to use the agglutinin group of the present invention and its knowledge
Other sugar chain distinguishes SCN patient and MCN patient.
Description of the drawings
Fig. 1 is lectin chip sample application array figure and the fluoroscopic examination figure for being applied to glycoprotein candy chain, and in figure, A is agglutinin
Chip sample application array figure, B are pancreas Mucinous cystoadenoma (MCN), pancreatic serous cystadenoma (SCN) patient's cystic Tumor of Pancreas
Cyst fluid glycoprotein candy chain fluoroscopic examination result figure；
Fig. 2 is GenePix3.0 software analysis chip scanning figures, and wherein abscissa represents fluorescence intensity.
Fig. 3 is ROC curve analysis result.
Fig. 4 is the proof diagram by taking STL and BPL as an example.
Specific embodiment
The present invention is further described in detail with reference to specific embodiment, the embodiment for being given is only for explaining
The bright present invention, rather than in order to limit the scope of the present invention.
Experimental technique in following embodiments, if no special instructions, is conventional method.
In following embodiments, material used, reagent etc., if no special instructions, commercially obtain.
Block buffer in following embodiments be to PBS in add the solution that BSA, glycine and Tween-20 obtain,
In Block buffer the concentration of BSA, glycine and Tween-20 be respectively 2% (mass percent concentration), 500mmol/L and
0.1% (mass percent concentration).Incubation buffer be to PBS in add the solution that BSA, glycine and Tween-20 obtain,
In incubation buffer the concentration of BSA, glycine and Tween-20 be respectively 2% (mass percent concentration), 500mmol/L and
0.1% (mass percent concentration).PBST is that the Tween-20 mass percent concentrations obtained to PBS addition Tween-20 are
0.1% solution.Glycine can be Sigma-Aldrich's product.Tween-20 can be U.S. Sigma-Aldrich
Products.The pH of PBS above is 7.4.
Embodiment 1, for distinguishing the glycosylation albumen sugar chain of MCN patient and SCN patient
(1) object of study and sample collection
Object of study：Pass through (ethics number through Hospital Ethical Committee：S2014-108-01), exclude malignant tumor medical history；
Serious pulmonary insufficiency；Bleeding and clotting obstacle；Through imaging diagnosises clearly, Chinese People's Liberation Army General Hospital digests early stage
Internal medicine combination tumor surgery lesion accepts PCNs patient for medical treatment, the MCN patient 17 (entering group MCN groups) for wherein making a definite diagnosis, SCN patient 18
(entering group SCN groups).
Cyst fluid collection at least 0.2ml is simultaneously immediately placed on ice, adds protease inhibitor (to add 1 μ per 0.1 milliliter of cyst fluid
L) protein degradation is prevented.
(2) cystic Tumor of Pancreas cyst fluid albumen is processed and fluorescent labeling
35 cyst fluid samples respectively take respective volume (including 100 μ g total proteins) and 100 μ L 0.1M Na2CO3(pH 9.3) delays
Liquid mixing, mixed liquor and Cy3 fluorescent dye incubated at room 3h is rushed, with the albumen of Sephadex G-25 column separating purification Cy3 labellings
Matter, and the concentration with labeled protein in spectrophotometric determination collection liquid.
(3) lectin chip and data analysiss
The agglutinin group of 37 kinds of agglutinin compositions, A institutes in the sample application array such as Fig. 1 of agglutinin is fixed with lectin chip
Show, the details of each agglutinin are as shown in table 1.
The point sample that the operation instruction that 37 kinds of agglutinins in table 1 are provided according to each agglutinin producer respectively is made into 1mg/mL
Liquid.Wherein Cy3 labellings and cold BSA be respectively position mark and negative Quality Control point.By rich crystalline substance core difficult to understand
The every kind of agglutinin of 48 spotting system arrange parameter of SmartArrayer48 point sample instruments (Beijing Capitalbio Corporation Co., Ltd.)
Repeat point sample three times, its point sample matrix repeats 4 areas of system for 4 × 10 array, and point is formed in epoxide chip base.Point sample
Finish and chip is placed in overnight incubation in the environment that humidity is 50%, then in 37 DEG C of vacuum drying ovens, dry 3h, be easy to coagulate
Collection element is effectively combined with chip base, standby.Before closing, put the chip for making and core is respectively cleaned with the 1 × PBST and 1 × PBS of pH7.4
Piece 2 times, each 5min are dried, obtain lectin chip.
Table 1, lectin chip coagulation prime information
In chip hybridization box, in Block buffer, 4rpm at the uniform velocity rotates 25 DEG C of closing 1h, with PBST, the PBS of pH7.4
Respectively wash 2 times, each 5min, dry；MCN groups and SCN group example samples respectively take protein after 5 μ g labellings, respectively with incubation buffering
After liquid mixing, with chip incubated at room 3h, respectively washed 2 times with 10mmol/L PBST and 10mmol/L PBS, each 5min, dried；
GenPix 4000B chip scanners (Axon companies of the U.S.) scan chip.Then with 3.0 softwares of GenePix from scanning result
Obtain the information such as fluorescence signal intensity value and background value to be analyzed in figure, can obtain every on MCN groups and SCN group agglutinin chips
The fluorescence signal value of individual point, in this chip, corresponding 3 of every kind of agglutinin repeats a little, i.e., actual every kind of agglutinin duplicate detection 3
Secondary, 3 signal datas are obtained, the corresponding intermediate value of intermediate value, i.e. each agglutinin is extracted.Calculate the intermediate value institute of each agglutinin
The ratio for accounting for 37 kinds of agglutinin intermediate value sums completes data normalization.Each sample does three repetitions.Agglutinin normalization fluorescence
Intensity is expressed as the mean value ± standard deviation value of three repetitions.Each agglutinin fluorescence intensity is calculated in MCN group normalization numerical value
Compare the relative change of glycosylated protein with the ratio (i.e. ratio values) of SCN group normalization numerical value.Data are utilized simultaneously
SPSS19 carries out t check analyses.
The result of the GenPix 4000B chip scanners scanning chip of one MCN groups sample and a SCN group sample is such as
In Fig. 1 shown in B.GenePix3.0 software analysis chip scanning figures are as shown in Figure 2.As a result show, in (1) MCN groups and SCN groups,
In glycosylation albumen, the signal intensity average of Jacalin, LEL, LCA, ConA and ACA is all higher than 0.05, shows its specificity
Based on high mannose and core fucose sugar chain structure, it is high sweet that LEL and ConA is recognized the sugar chain of the glycosylation albumen of identification
Dew sugar-type N- sugar chain, Gal β 1-3GalNAc α-Ser/Thr (T) and GalNAc α-Ser/Thr (Tn), ACA of Jacalin identifications
Gal β 1-3GalNAc α-Ser/Thr (T antigen) of identification, in two class cystadenoma cyst fluids, content is higher；(2) coagulation
The terminating in GalNAc α/β 1-3/6Gal structures of plain WFA identifications, the trimers and of STL identifications
The N- sugar chain structures of tetramers of GlcNAc and core (GlcNAc) only have high level in MCN group samples. and (3)
And the GalNAc, GalNAc α -1,3Gal, GalNAc α -1,3Gal β -1,3/4Glc, agglutinin MAL-I of agglutinin PTL-I identifications
Only in SCN group samples, content is raised the Gal β -1,4GlcNAc structures of identification.
The change of MCN, SCN cyst fluid glycoprotein candy chain spectrum：
Respectively MCN groups are detected with SCN group cyst fluid samples using lectin chip, chip data is obtained by software
And after normalized, ratio values (table 2, table 3 and table 4) being calculated, normalization data is as shown in Figure 2.
The coagulation of the sugar chain structure of the glycoprotein candy chain structure and specific recognition of differential expression in table 2, MCN and SCN cyst fluids
Element
Table 3, each sample fluorescence signal normalization numerical value
The Ratio values of table 4, remaining agglutinin
According to statistical theory, Ratio values between 0.5 and 2, then in sample identical glycoprotein candy chain content in MCN
Zero difference between two groups of samples of group and SCN groups；Ratio value >=2, then the glycoprotein candy chain content in MCN groups sample be significantly higher than SCN
Identical glycoprotein candy chain content in group；Ratio value≤0.5, then the glycoprotein candy chain content in MCN groups sample be substantially less than SCN
The content of identical glycoprotein candy chain in group.
As a result find, the glycosylation albumen sugar chain content of 6 kinds of agglutinin identifications has in MCN groups with SCN group cyst fluids significantly
Difference.In glycoprotein candy chain, the N- of trimers and tetramers of GlcNAc and core (GlcNAc) of STL identifications
Sugar chain structure, the multivalence sialic acid structure of WGA identifications, Gal β 1-3GalNAc and Terminal GalNAc, DBA of BPL identifications
The α GalNAc of identification, Tn antigen, GalNAc α 1-3 ((Fuc α 1-2)) Gal (blood group A antigen) are in MCN
In group, content substantially increases.The sugar chain structure of PTL-I and MAL-I both agglutinins identifications content in SCN groups is dramatically increased
(R<0.5, p<0.05).Show the glycoprotein candy chain that can be recognized using this 6 kinds of agglutinins and containing which distinguish MCN patient with
SCN patient.
For distinguishing MCN patient and SCN patient, construct by taking WGA and BPL as an example and built using Influencing factors
The mathematical model of MCN patient and SCN patient is distinguished, formula 1 is seen.
In formula 1, WGA represents that sample fluorescence signal normalization numerical value when being detected using WGA, BPL are represented and utilizes BPL
Sample fluorescence signal normalization numerical value when being detected, Y represent the diagnostic value of sample.
ROC is carried out during using formula 1 with the cystic Tumor of Pancreas patient of unknown MCN patient and SCN patient as object to be measured
Tracing analysiss (Fig. 3), area under curve AUC=0.853, sensitivity are 0.714, and specificity is 1, and the method is judged to cancer of pancreas
The threshold value of patient is 0.70, i.e., when Y is more than 0.70, object to be measured is doubtful for MCN patient, when Y is less than or equal to 0.70, treats
Survey object is SCN patient.
(4) agglutinin trace
For further verifying the result of lectin chip, by taking agglutinin STL and BPL as an example, by fluorescently-labeled for Cy3 coagulation
Element is incubated with the cystic Tumor of Pancreas cyst fluid albumen after transferring film, and scanned instrument is scanned and 3.0 software analysis of Genepix, and determination contains
Content situation of the glycosylation albumen of the sugar chain for having STL and BPL to recognize in MCN groups with SCN group cyst fluids.Concrete operation step is such as
Under：
By 30 μ g albumen samples first through PAGE gel electrophoresis, after electrophoresis terminates, gel is taken out, in transferring film buffer
The rinsing several seconds.Pvdf membrane using front first with go to after absolute methanol pretreatment 5min balance in transferring film buffer good after use.Take
Go out gel using the half-dried transferring film instrument of TE 70PWR according to pvdf membrane electric current 0.8mA every square centimeter, constant current transferring film 1.5h.Transferring film is complete
Bi Hou, is placed into pvdf membrane in preprepared TBST immediately and cleans twice, and each 10min washes away the transferring film remained on film
Buffer. and then proceed in Carbo-free confining liquids, jog 1h closings on shaking table.After closing terminates, directly by Cy3 fluorescence
The agglutinin of labelling is pressed 2 μ g/mL of final concentration and is added in Carbo-free confining liquids, and on shaking table, 4 DEG C of lucifuges of jog are overnight.TBST
Cleaning film 3 times, each 10min, it is 800 then to arrange PMT in Storm840 gel imaging systems, red fluorescence channel
Scanogram under (635nm excitation wavelengths/650LP launch wavelengths), reads protein band gray value with Image J softwares.
As a result (Fig. 4) shows, fluorescence intensities of the STL and BPL in the cyst fluid sample of 18 MCN patients is apparently higher than 17
Fluorescence intensity in example SCN patient's cyst fluid sample, shows, in glycosylation albumen, the sugar chain that STL and BPL is combined is in MCN patient's capsule
Content in liquid is higher than the content in SCN patient's cyst fluid.At two protein band (white edge is sketched the contours) places of 60kDa and 50kDa,
Fluorescence intensities of the STL as probe in MCN patient is significantly higher than the fluorescence intensity (P in SCN patient<0.05)；?
Protein band (white edge is sketched the contours) place of 120kDa, fluorescence intensities of the BPL as probe in MCN patient is significantly higher than to be suffered from SCN
Fluorescence intensity (P in person<0.05).As a result show consistent with lectin chip result.Prove that these agglutinins can be used as spy
Pin distinguishes SCN patient and MCN patient by the detection to the glycoprotein candy chain in cystic Tumor of Pancreas cyst fluid.
Claims (10)
1. the material of the sugar chain of detection agglutinin group identification following A 1) or A2) in application：
A1) prepare and distinguish or supplementary globe pancreatic mucinous cystic tumors patient and pancreas serosity cystic tumor patient product；
A2) distinguish or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystic tumor；
The agglutinin group includes b1) or b2)：
B1) WGA and/or BPL；
B2) by b1) with STL, DBA, PTL-I and MAL-I in the compositionss that constitute of all or part.
2. application according to claim 1, it is characterised in that：The agglutinin group also include Jacalin, ECA, HHL,
WFA、GSL-Ⅱ、MAL-Ⅱ、PHA-E、SJA、PNA、EEL、AAL、LTL、MPL、LEL、GSL-Ⅰ、LCA、RCA120、BS-Ⅰ、
All or part in ConA, PTL- II, DSA, SBA, VVA, NPL, PSA, ACA, UEA- I, PWM, GNA, PHA-E+L and SNA.
3. application according to claim 1 and 2, it is characterised in that：The sugar chain content of the agglutinin group identification is described
Content of the sugar chain of agglutinin group identification in pancreas cyst fluid.
4. agglutinin group described in claim 1 or 2 identification sugar chain following A 1) or A2) in application：
A1) prepare and distinguish or supplementary globe pancreatic mucinous cystic tumors patient and pancreas serosity cystic tumor patient product；
A2) distinguish or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystic tumor.
5. the sugar chain of the identification of agglutinin group described in claim 1 or 2 is used as differentiation pancreatic mucinous cystic tumors and pancreas slurry
Fluidity cystic tumor mark is in differentiation or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystic tumor
Application.
6. the sugar chain using the identification of agglutinin group described in claim 1 or 2 is used as differentiation pancreatic mucinous cystic tumors and pancreas
The material of the differentiation pancreatic mucinous cystic tumors of serosity cystic tumor mark and pancreas serosity cystic tumor is following
A1 the application in) or A2)：
A1) prepare and distinguish or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystic tumor product；
A2) distinguish or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystic tumor.
7. distinguish or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystic tumor product, be that test right is required
The material of the sugar chain content of the identification of agglutinin group described in 1 or 2.
8. product according to claim 7, it is characterised in that：The product includes agglutinin described in claim 1 or 2
Group or the chip containing agglutinin group described in claim 1 or 2.
9. product described in claim 7 or 8 following A 1) or A2) in application：
A1) prepare and distinguish or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystic tumor product；
A2) distinguish or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystic tumor.
10. the method for distinguishing pancreatic mucinous cystic tumors and pancreas serosity cystic tumor is built, including：Glutinous to pancreas respectively
Sugar chain in fluidity cystic tumor group and pancreas serosity cystic tumor group pancreas cyst fluid carries out quantitative analyses, according to analysis result
Obtain cystic Tumor of Pancreas sugar chain characteristic spectrum；The cystic Tumor of Pancreas sugar chain characteristic spectrum is by coagulation described in claim 1 or 2
The sugar chain composition of element group identification.
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