CN109212227A - Hepatopathy based on the special glycoprotein candy chain structure of saliva/cirrhosis correlation screening, the product of assessment and application - Google Patents
Hepatopathy based on the special glycoprotein candy chain structure of saliva/cirrhosis correlation screening, the product of assessment and application Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis
Abstract
The invention discloses the hepatopathy based on the special glycoprotein candy chain structure of saliva/cirrhosis correlation screenings, the product of assessment and application, for saliva sample, two kinds are provided for hepatopathy/cirrhosis screening, early diagnosis, risk assessment, the product of drug screening and/or curative effect evaluation, three kinds of specific sugar albumen sugar chain structure β-D-GlcNAc in one of product testing saliva sample, the expression (whether high expression) of Gal β 1-4GlcNAc and (GlcNAc β 1-4) n, whether any or any combination of specific 11 kind N- sugar chain is contained in another product testing saliva sample.Correspondingly, play specific binding recognition reaction agglutinin be DSA, can separately as identification the special glycoprotein candy chain structure of saliva reagent to prepare related medical product.
Description
Technical field
The determination of hepatopathy (especially cirrhosis) marker that the present invention relates to a kind of based on the special glycoprotein of saliva and its
Related product, application.
Background technique
Cirrhosis (Hepatic Cirrhosis, HC) is a kind of common chronic liver disease in China, is more common in viral liver
Scorching, fatty liver disease prognosis.Currently, the various detection methods of cirrhosis are limited by its technical characterstic respectively, there is spy
The defects of anisotropic low, sampling is inconvenient and cannot early diagnose.In recent years studies have shown that: with hepatopathy occurrence and development, patient
Hepatic tissue and the body fluid such as serum and saliva in have occurred glycoprotein glycosylation abnormal change.
Patent document CN105675893A disclose it is a kind of based on serum/be common based on Protein Detection sugar in serum and saliva
Chain logo object is to identify the scheme of hepatitis.Wherein:
Based on serum detection when, when PTL-I, PNA, AAL, LTL, STL, BS-I, PTL-II, SBA, UEA-I, MAL-I,
PHA-E+L is raised, while timing can distinguish it with Healthy People under ACA, that is, suffer from hepatopathy；On this basis, when WFA,
GSL-II, MAL-II, SJA, LEL, PWM, BPL are raised, while timing under EEL, can determine that the corresponding crowd of sample suffers from
Heavy hepatitis B.
When being detected jointly based on serum and saliva, when STL, SBA, WFA, GSL-II are raised, while under ACA, timing can be with
It is distinguished with Healthy People, that is, suffers from hepatopathy.
Above scheme required agglutinin when detecting sample is more, and need to make combination statistics, therefore for cirrhosis (HC)
Screening, diagnosis and assessment it is still not easy enough, effective.
It is important to point out that some factors in blood can cycle to reach salivary gland with body fluid, or with salivary secretion
Into oral cavity or the gene expression profiling in saliva is caused to change, the abundance of certain protein or type is caused to change, thus
Reflect the variation of part protein level in blood.But it has now been found that having 1939 kinds of protein, human plasma in people's saliva
In have 3020 kinds of protein, only 27% sialoprotein matter group is overlapped with plasma proteins group.Plasma Proteomics discovery
177 potential source biomolecule markers relevant to cardiovascular disease protein in, also only 40% protein appears in saliva
Liquid Proteomics；In the protein of listed 1058 potential cancer associated biomarkers, only 34% albumen
Matter appears in sialoprotein matter group.This shows that the biomarker in many blood circulations can not be reflected in saliva
It is fixed.Even, compared to serum, the content in saliva is usually significant lower in addition, same albumen.So practicing
In, the two not inevitable connection generally requires researcher by a large amount of experiment and analysis, can just judge whether there is albumen
A possibility that matter group is overlapped.
Also therefore, above scheme have to be based respectively on serum detection, detected jointly based on serum and saliva provide it is different
Scheme.
Summary of the invention
Screening, diagnosis and assessment etc. about cirrhosis (HC), inventor pass through many experiments and analysis, it is determined that with
Draw a conclusion:
One, compared to other glycoprotein candy chain structures in saliva, β-D-GlcNAc, the Gal β 1- that agglutinin DSA is identified
4GlcNAc and (GlcNAc β 1-4) n sugar chain structure are in hepatopath (hepatitis B (HB), cirrhosis (HC), liver cancer (HCC)) saliva
High expression (P≤0.0343) is rendered as in liquid；And compared to healthy volunteer (HV), hepatitis B and liver cancer, β-D-GlcNAc, Gal β 1-
4GlcNAc and (GlcNAc β 1-4) n sugar chain structure expression highest (p≤0.0034) in HC patient's saliva.
Two, following 11 kinds of N- sugar chains exist only in the saliva of HC patient, and are not found in the saliva of HV, HB and HCC.
m/z.1996.7238(Fuc)1(GalNAc)1(Gal)1(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2006.7316(Gal)3(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2037.7503(Fuc)1(GalNAc)2(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2281.8321(Sia)1(Fuc)1(GalNAc)1(Gal)2(GlcNAc)2+(Man)3(GlcNAc)2；
m/z.2573.9480(Sia)1(Fuc)2(Gal)2(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2772.9572(Sia)2(Gal)4(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2794.0427(Fuc)3(Gal)3(GlcNAc)4+(Man)3(GlcNAc)2(Fuc)1；
m/z.2806.9879(Fuc)1(Gal)6(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2924.0805(Gal)4(GlcNAc)6+(Man)3(GlcNAc)2(Fuc)1；
m/z.3232.1912(Fuc)2(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2；
m/z.3813.3617(Sia)3(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2)。
According to above first conclusion, following application scheme can be obtained:
A kind of product for the screening of hepatopathy, early diagnosis, risk assessment, drug screening and/or curative effect evaluation, for
Saliva sample, the product include:
A, for obtaining the device of the expression of three kinds of specific sugar albumen sugar chain structures, three kinds of specific sugar protein sugars
Chain structure is respectively β-D-GlcNAc, Gal β 1-4GlcNAc and (GlcNAc β 1-4) n；
B, for differentiating the whether highly expressed mark of three of the above specific sugar albumen sugar chain structure, module or processor.This
In " mark " tend to similar test strips form make qualitative conclusions display (whether there is or not, compare), " module ", " place
Reason device " tends to numerical quantization the results show that can also show and/or export certainly qualitative conclusions；Concrete methods of realizing category
In conventional technical means.
By application, the product carries out drug screening, for curative effect evaluation, can be before and after measure object administration (course for the treatment of)
The expression of the above two specific sugar albumen sugar chain structure of different time points shows the medicine if expression has no reduction
The unsatisfactory curative effect of object.
Above-mentioned apparatus includes lectin chip, is incubated for box and biochip scanning system, wherein on lectin chip at least
Being provided with agglutinin DSA, (agglutinin being capable of β-D-GlcNAc, Gal β 1-4GlcNAc and (GlcNAc on specific recognition glycoprotein
β 1-4) n sugar chain structure).
Above-mentioned mark, module or the corresponding reference value of the pre-recorded unsoundness of processor (HV) are used for and sample results pair
According to determining whether high expression.For example, fluorescence signal inatheadearomatizationazone can be set greater than 1.35, determine that it is high expression.
A kind of agglutinin prepares the use of Related product separately as the reagent of the special glycoprotein candy chain structure recognition of saliva
On the way, the agglutinin is DSA, the Related product be kit, equipment, can operating system and/or their combination, purposes is
Screening, early diagnosis, risk assessment, drug screening and/or the curative effect evaluation of hepatopathy.
According to above second conclusion, following application scheme can be obtained:
The recognition unit of specific sugar chain is in building for making cirrhosis (HC) screening, early diagnosis, wind based on saliva sample
Danger assessment, drug screening and/or curative effect evaluation product in terms of purposes, the following 11 kinds of N- sugar chains of recognition unit identification it
Any or any combination (passes through judgement " having ", "None" you can get it conclusion):
m/z.1996.7238(Fuc)1(GalNAc)1(Gal)1(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2006.7316(Gal)3(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2037.7503(Fuc)1(GalNAc)2(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2281.8321(Sia)1(Fuc)1(GalNAc)1(Gal)2(GlcNAc)2+(Man)3(GlcNAc)2；
m/z.2573.9480(Sia)1(Fuc)2(Gal)2(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2772.9572(Sia)2(Gal)4(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2794.0427(Fuc)3(Gal)3(GlcNAc)4+(Man)3(GlcNAc)2(Fuc)1；
m/z.2806.9879(Fuc)1(Gal)6(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2924.0805(Gal)4(GlcNAc)6+(Man)3(GlcNAc)2(Fuc)1；
m/z.3232.1912(Fuc)2(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2；
m/z.3813.3617(Sia)3(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2。
Product of the one kind for cirrhosis (HC) screening, early diagnosis, risk assessment, drug screening and/or curative effect evaluation,
For saliva sample, which includes:
A, agglutinin DSA；
B, for being coupled the affinity chromatography solid phase carrier of agglutinin DSA；
C, for from containing β-D-GlcNAc, in the glycoprotein of Gal β 1-4GlcNAc and (GlcNAc β 1-4) n sugar chain structure
Isolate the reagent and device of N- sugar chain；
D, mass spectrograph, for showing the m/z value for corresponding to various N- sugar chains；
E, for identifying mark, module or the processor of aforementioned 11 kinds of rocks N- sugar chain according to mass spectrogram, to obtain whether contain
There is any or any combination judgement result of aforementioned 11 kinds of N- sugar chains.
The above-mentioned affinity chromatography solid phase carrier for being coupled agglutinin DSA can choose Fe3O4Magnetic particle, agarose are solidifying
Glue, sephadex, glass microsphere etc..
Detailed description of the invention
Fig. 1 is lectin chip to 215 saliva samples (HV, n=51；HB, n=51；HC, n=68；HCC, n=45)
Detect by example, the wherein case map analysis of agglutinin DSA result.In figure, ordinate indicates the agglutinin on lectin chip
Corresponding normalization fluorescence intensity NFI, in each case figure top-down each horizontal line respectively represent this group of data maximum value,
Quartile, median, preceding quartile and minimum value afterwards.Horizontal line represents the comparison shown in both ends between two groups in figure, according to p
One-way analysis of variance P-Value obtained, wherein p* < 0.05 indicates that significant difference, p** < 0.01 indicate that difference is very aobvious
It writes, p*** < 0.001 indicates that difference is extremely significant.HV: healthy volunteer's saliva, HB: hepatitis B patient saliva, HC: liver cirrhosis patient saliva
Liquid, HCC: Saliva of Primary Hepatocellular Carcinoma.
Fig. 2 is each probe combination DSA intensity scatter plot of saliva chip.Horizontal line represents the ratio shown in both ends between two groups in figure
Compared with P is according to one-way analysis of variance P-Value obtained, and wherein P* < 0.05 indicates that significant difference, P** < 0.01 indicate
Difference highly significant, P*** < 0.001 indicate that difference is extremely significant.HV: healthy volunteer's saliva, HB: hepatitis B patient saliva, HC: liver
Sclerosis patients' saliva, HCC: Saliva of Primary Hepatocellular Carcinoma
Fig. 3 is four groups of mixing saliva sample SDS-PAGE silver staining colour developings and DSA Blot experiment result, in which: A is aobvious for silver staining
The result figure of color；B is that the result figure of acquisition is scanned under 635nm wavelength after DSA agglutinin trace.Each band sample information are as follows:
1:Protein Ladder, 2: healthy volunteer's saliva, 3: hepatitis B patient saliva, 4: liver cirrhosis patient saliva, 5: liver cancer patient
Saliva.
Specific embodiment
The associated verification of the application described in detail below is tested and analysis, the specific R&D process of inventor are without being limited thereto.
One, the screening of chronic hepatitis B, cirrhosis and Saliva of Primary Hepatocellular Carcinoma protein difference sugar chain structure
Research method:
1.1, the collection and pretreatment of saliva sample
The saliva sample of healthy volunteer used by this experiment and chronic hepatitis B, cirrhosis and liver cancer patient strictly passes through
Cross Northwest University, Tang Dou hospital of The Fourth Military Medical University, The People's Hospital and Xi'an Communications University the second affiliated hospital
Ethics examines (Human Research Ethics Committees (HRECs)).The volunteer of all donations saliva samples connects
With assist sampling guidance clinician to this research work know, agree to and height cooperate, uniform sampling request it
The lower acquisition for completing saliva sample.Its specific requirement are as follows: sample donor needs non-diabetic, and other organs should nothing in addition to liver
The chronic diseases such as inflammation and tumour, in sampling, donor need to be determined does not feed in 3 hours and 24 is small before acquiring saliva
When it is interior do not take drugs, then gargled three times with the physiological saline of cleaning sterile (0.9%NaCl), it is ensured that donor's oral hygiene
Under the premise of no swill, donor's the tip of the tongue resist maxilla and in it is sublingual collect naturally secret saliva sample collect to
2mL centrifuge tube, be added immediately 10 μ L protease inhibitors (Protease Inhibitor Cocktail, Sigma-Aldrich,
U.S.A) temporary in ice bath.Saliva sample 215 are collected altogether under clinician's guidance: wherein 51 (HV, n of healthy volunteer
=51), chronic hepatitis patient 51 (HB, n=51) caused by hepatitis B, chronic cirrhosis patient 68 (HC, n=68), liver cancer
Patient 45 (HCC, n=45).Specific sample information is as shown in table 1.
Saliva acquired in 12 hours, and saliva is dispensed by 1mL into centrifuge tube, if the amount of having it is few can be added 1 less than 1mL person ×
PBS complements to 1mL, 10,000g × 15min centrifugation, careful Aspirate supernatant, through trace dna protein assay (Nano-
Drop press 1mg sialoprotein after) measuring concentration: protease inhibitors is added in the amount of 10 μ L protease inhibitors, dispenses after mixing
It is stored in -80 DEG C.
Table 1 is used for the saliva sample information that the small-scale lectin chip of diagnosing hepatism is detected by example
1.2, the fluorescent marker of a sialoprotein and quantitative
The Nano-drop that learns from else's experience quantitative 100 μ g of saliva sample, is added 0.1mol/L Na in equal volume2CO3/
NaHCO3It is molten that 5 μ L fluorescence are added according to 1mg:120 μ L using DMSO dissolution Cy3 fluorescence dry powder and into sample for pH9.3 buffer
Liquid was in incubation at room temperature 3 hours, and during which sample is strictly protected from light and keeps concussion.After reaction, 10 μ L 4M hydroxyls are added into sample
Amine aqueous solution reacts 5 minutes on ice, after the excessive free fluorescence of adequate closure, utilizes Sephadex G-25 molecular sieve gel
Column separates albumen sample.Fluorescent sample is collected with new 1.5mL centrifuge tube and is quantified, and the sample after fluorescent marker is kept away
Light prevents fluorophor from quenching, and stores in -20 DEG C.
1.3, lectin chip detects sialoprotein matter sugar chain differential expression by example
This experiment utilizes the homemade small-scale lectin chip of 14 probe in a laboratory, specifically includes agglutinin MAL-
II,UEA-I,PTL-I,LTL,RCA120,DSA,GNA,PHA-E+L,EEL,AAL,STL,ACA,WGA,MAL-I.High standard is super
Clean, surface microscopic is smooth 75 × 25mm glass slide utilizes (3- after decontamination lotion, strong acid cleaning
Glycidyloxypropyl) trithoxysilane reagent carries out epoxy silane modification to surface of glass slide, has surface of glass slide
Agglutinin-NH can be coupled2Epoxy group.Using 48 point sample instrument of Smart Arrayer quantify point sample feminine gender Quality Control (BSA),
Internal reference probe PTL-II, 14 kinds of detection probes and positive quality control (BSA of Cy3 fluorescent marker), each Quality Control or probe parallel point
The agglutinin micro-array chip of a 9 × 7 (850 μm of spacing of point), 4 DEG C of preservations after point sample are made in 3 points of sample.Every epoxidation
4 arrays of slide point system, array covering surface ensure to be matched with the adhesive tape enclosed area of chip reaction box, and every chip can be same accordingly
When detect 4 different albumen samples.
Before carrying out lectin chip detection sample, from 4 DEG C of taking-up chips, rise again in negative pressure in 37 DEG C of vacuum desiccators
30min cleans 5min × 2 time in level concussion shaking table 70rpm followed by 1 × PBST, then cleans 5min × 2 with 1 × PBS
It is secondary, sufficiently to clean the free agglutinin not being coupled on slide.It is dried and is remained with small-sized glass slide centrifuge machine after the completion of cleaning
PBS.Before loading, 120 μ L lectin chip confining liquids are first added to slide blank table into each array region for being incubated for box
The signal value of slide back end when the epoxidation group in face is closed to reduce fluorescent scanning.It is incubated after being incubated for box sealing in constant temperature
37 DEG C of reaction 1h in case are educated, after the completion of closing, 1 × PBST cleans 5min × 2 time, and 1 × PBS cleans 5min × 2 time, and drying is backward
It is incubated in each array region of box and albumen sample incubation system (80 μ L lectin chip Incubating Solutions, 8 μ L 4M azanols, 2 μ L is added
The sialoprotein sample of 0.1%Tween-20,4 μ g Cy3 label, ultrapure water supply final volume to 120 μ L), it is incubated in 37 DEG C of constant temperature
3h is educated, cleans 5min × 2 time to 1 × PBST after reaction, 1 × PBS cleans 5min × 2 time, dries and scan.Agglutinin core
The final reading data of piece realizes that sweep parameter is set using the produced Genepix 4000B chip scanner of Axon company are as follows:
Excitation wavelength 532nm, PMT power 70%, excitation intensity 100%.
1.4, lectin chip data are analyzed
The numeralization process of lectin chip fluorescence signal is completed by GenePix Pro (4000B) software, by every
The data of a array are extracted, and the data of acquisition include: that probe signals subtract background signal net difference value FI obtained
The data such as the standard variance SD (Standard Deviation) of (Fluorescent Intensity), background.In analytic process
In, the validity of the point data is judged according to the FI/SD of each point first, takes the point of FI/SD >=1.5 as valid data, meter
Each probe standard normalization fluorescence signal value NFI (Normalized Fluorescent Intensity) is calculated, i.e., with each
The Median FI value of probe is indicated are as follows: NFI divided by the sum of 14 detection probe FI values with formulax=Median FIx/
(Median FI1+Median FI2+Median FI3+…+Median FI14), it can get 14 kinds of agglutinations on each array accordingly
NFI corresponding to plain probe is simultaneously used as statistical analysis.
Here statistical analysis mainly utilize GraphPad Prism 6.0 to tetra- groups of data of HV, HB, HC and HCC into
One-way analysis of variance (One-Way ANOVA Analysis) between row group, specific method are by data by group typing " Column "
Formula is analyzed, case figure " Box&Shiskers " or scatter plot " Scatter&SD " etc. are selected according to mapping demand.It subsequently enters
" Analyze " analyzes formula by comparing simultaneously report discrepancy conspicuousness P-Value between group two-by-two.
Result of study:
2.1 lectin chips analyze hepatopath's saliva glycoprotein sugar chain differential expression
215 saliva samples detect by example using lectin chip array shown in FIG. 1, at chip data
Reason obtains each NFI value, and by the calculated average NFI of each group of data, the results are shown in Table 2.Each average NFI result can be intuitive
Ground shows the combination situation of each group saliva sample and each probe, and can assess each agglutinin to a according to its standard deviation SD
The dispersion degree of example pattern detection.The sugar chain of each agglutinin probe institute specific recognition is also enumerated in addition to experimental data, in table
Sugar-type, the Sia α 2-3Gal identified such as agglutinin MAL-II, the sialylated sugar chain structure of Sia α 2-3GalNAc, GNA identification
Man α 1-3Man mannan sugar chain structure etc..
2 example saliva lectin chip testing results of table
aLectin Probe, 14 kinds of agglutinin probes contained by the chip array；bSpecificity to
Glycopatterns, sugar chain sugar-type (Glc, the glucose of each agglutinin specific recognition；GlcNAc, acetylglucosamine；Gal, half
Lactose；GalNAc, acetylgalactosamine；Man, mannose；Sia, sialic acid；Fuc, fucose)；cNFI, chip data normalizing
Each probe normalization fluorescence intensity is corresponded to after change processing, data show sample mean NFI and its standard deviation SD in table.
The screening of 2.2 Saliva of Primary Hepatocellular Carcinoma difference glycoprotein candy chains
Carry out between group that comparison acquires the Ratio respectively compared between group two-by-two using each average NFI value enumerated in table 2
Value, and define Ratio>1.500 represent the sugar chain that the agglutinin is identified compare between group in embody it is high express, Ratio<
0.667 represent the sugar chain that the agglutinin is identified compared between group in embody low expression.After comparison among groups, agglutinin is found
MAL-II, AAL, STL, ACA, WGA, MAL-I identification sugar chain compared between group in without differential expression.Exist between other 8 kinds of groups
The agglutinin (UEA-I, PTL-I, LTL, RCA120, DSA, GNA, PHA-E+L, EEL) of difference and detailed data such as 3 institute of table
Show.Analysis is found, compared to HV and HB, identifies GalNAc α -1,3Gal and GalNAc α -1,3Gal β -1,3/4Glc structure is coagulated
Collect its average NFI of element PTL-I high expression in HCC group, and compares indifference between HCC and HC；Compared to HB, Gal α 1- is identified
Its average NFI of the agglutinin EEL of 3 (Fuc α 1-2) Gal structures high expression in HCC group, and HCC is compared to comparing nothing between HV, HC
Difference；Compared to HV, HB and HC, the solidifying of Fuc α 1-2Gal β 1-4GlcNAc and Fuc α 1-3 (Gal β 1-4) GlcNAc structure is identified
Its average NFI of collection element LTL embodies high expression in HCC group.The variance analysis of average NFI shows this 3 kinds of agglutinin identifications
Sugar chain have relatively highly expressed trend in Saliva of Primary Hepatocellular Carcinoma.
3 four groups of salivas of table detect the variance analysis of average NFI
Tetra- groups of HV, HB, the HC and HCC average NFI Ratio values that comparing calculation obtains two-by-two are listed in table, and are defined
The expression of Ratio A/B > 1.500 is when the expression of the specific sugar chain structure of a certain agglutinin identification of two comparison among groups of A, B, A
Group is marked with upward arrow compared with the high expression of B group in table；Ratio A/B < 0.667 indicates to work as a certain agglutinin of two comparison among groups of A, B
When the expression of the specific sugar chain structure of identification, A group is marked with down arrow compared with B group low expression in table.
Using a number of cases evidence corresponding to statistical analysis agglutinin DSA probe, judged by one-way analysis of variance solidifying
Collect the significance of difference of the sugar chain structure of element DSA identification in liver cirrhosis patient saliva, result is as shown in Figure 1, a series of systems
Meter learns data can intuitively reflect the significance of difference and dispersion degree of each comparison among groups on the diagram.According to single factor test between each group
From the point of view of the result of variance analysis: compared to other glycoprotein candy chain structures in saliva, the β-D-GlcNAc that agglutinin DSA is identified,
Gal β 1-4GlcNAc and (GlcNAc β 1-4) n sugar chain structure are in hepatopath (hepatitis B, cirrhosis, liver cancer) saliva
It is now high expression (P*≤0.0343)；And compared to healthy volunteer (HV), hepatitis B (HB) and liver cancer (HCC), β-D-GlcNAc,
Gal β 1-4GlcNAc and (GlcNAc β 1-4) n sugar chain structure expression highest (p**≤0.0034) in HC patient's saliva.
And this result and average NFI comparison result are almost the same.
So last it follows that compare other glycoprotein, β-D-GlcNAc, the Gal β 1- of agglutinin DSA identification
4GlcNAc and (GlcNAc β 1-4) n sugar chain structure are high expression in HB, HC, HCC patient's saliva, and compared to HV, HB and
β-D-GlcNAc, Gal the β 1-4GlcNAc and (GlcNAc β 1-4) n sugar chain structure that HCC, agglutinin DSA are identified are in HC patient
Expression highest (p**≤0.0034) in saliva.It can be considered in this, as cirrhosis (HC) screening, early diagnosis,
The foundation of risk assessment, drug screening and/or curative effect evaluation.
Two, healthy volunteer and hepatitis B, cirrhosis and the specific glycoprotein of Saliva of Primary Hepatocellular Carcinoma are divided using agglutinin DSA
From and sugar chain identification
Research method:
Utilize 90 patients with chronic liver saliva sample (HB, the n=30 in strict conformity with each stage characteristic feature；HC, n=
30；HCC, n=30) and the verifying DSA identification of 30 healthy volunteer's saliva samples β-D-GlcNAc, Gal β 1-4GlcNAc and
(the GlcNAc β 1-4) up-regulated expression of n sugar chain structure in HC group.By the way that saliva chip is made in 120 saliva samples, utilize
Agglutinin DSA detects on the chip β-D-GlcNAc, Gal β 1-4GlcNAc and (GlcNAc β 1-4) n sugar in each example patient saliva
The abundance and comparative analysis difference of chain structure.The mixing saliva sample of each group, benefit are obtained additionally by equal protein qualities mixing
Each group mixing saliva sample is verified with SDS-PAGE albuminous degeneration electrophoresis and agglutinin Blot experiment (Lectin Blotting)
The abundance difference of β-D-GlcNAc, Gal β 1-4GlcNAc and (GlcNAc β 1-4) n sugar chain structure in this.It a series of is tested through above-mentioned
Upper agglutinin DSA is coupled using a kind of ferriferrous oxide nano magnetic grain surface after card, and agglutinin magnetic particle compound is made
(DSA-Magnetic Particle Compounds, LMPCs) come the specific glycoprotein that is enriched in each group saliva mixing sample,
N- sugar chain and O- sugar chain on specific glycoprotein are separated using PNGase F enzyme cutting method, NaClO oxidizing process, and is swashed by Matrix-assisted
Photo-ionisation flight time mass spectrum (Matrix-Assisted Laser Desorption Ionization-Time of
Flight-Mass Spectrum, MALDI-TOF-MS) Mass Spectrometric Identification is carried out to sugar chain and sugar chain structure is speculated, it obtains
The healthy volunteer of agglutinin DSA identification and the specific glycoprotein candy chain spectrum of hepatitis B, cirrhosis, Saliva of Primary Hepatocellular Carcinoma and comparative analysis
Difference, it is specific as follows.
The selection of 3.1 saliva samples is mixed and is quantified
This experiment sample standard deviation collected comes from The Fourth Military Medical University Tang Dou hospital, and the volunteer for providing sample all has second
The chemical examination of liver five indices and liver function test data are grouped sample according to the clinician of guidance sampling.It unites in data
On meter, the statistics and clinical threshold reference of following analysis data: hepatitis B surface antigen HBsAg are taken；Hepatitis B virus e antigen HBeAg；
Hepatitis B core antibody HBcAb；Glutamic-pyruvic transaminase ALT (normal reference value: 0~40U/L)；Glutamic-oxalacetic transaminease AST (nominal reference
Value: 0~40U/L)；Glutamyl transpeptidase GGT (normal reference value: 0~50U/L)；Albumin A lbumin (normal reference value: 35
~55g/L)；AFP AFP (normal reference value: 0~200ng/mL).In addition, making age and gender to count, statistics letter
Breath such as table 4.
Table 4 is used to verify and the saliva sample Information Statistics of Mass Spectrometric Identification
The acquisition of saliva sample and processing method are with described previously, using trace dna protein assay to listed in table
120 saliva samples are quantified, and then take 500 μ g of every saliva sample to be mixed by quality.Secure good health volunteer
(HV), four chronic hepatitis B (HB), chronic cirrhosis (HC) and liver cancer (HCC) saliva mixing samples.It is fixed using BCA albumen
It measures kit (green skies biotechnology, Chinese Shanghai) and measures mixing sample concentration.
The design of 3.2 saliva chips and the verifying of sugar chain sugar-type difference
This experimental design is a kind of with healthy volunteer and chronic hepatitis, cirrhosis, liver cancer patient each 30 saliva samples
The saliva chip of probe is reacted in this conduct, amounts to 120 saliva probes, in addition 4 negative Quality Controls and 4 positive quality controls, each
Sample makes continuous 3 repetitions in point on chip, and the saliva micro-array chip of a 12 × 32 (point spacing 1mm) is made.It utilizes
Agglutinin DSA relative quantification detect on saliva chip in each example saliva sample β-D-GlcNAc, Gal β 1-4GlcNAc and
When the gene expression abundance of (GlcNAc β 1-4) n sugar chain structure, using Cy5 fluorochrome label agglutinin DSA and it is made into reaction system
(400 μ L lectin chip Incubating Solutions, 40 μ L 4M azanols, the agglutinin of 10 μ L 0.1%Tween-20,20 μ g Cy5 labels
DSA, ultrapure water supply final volume to 600 μ L) it is splined on saliva chip.Saliva chip and Cy5-DSA association reaction are completed and clear
After washing, most data acquisition (parameter setting are as follows: excitation wavelength 635nm, PMT on Genepix 4000B chip scanner finally
Power 70%, excitation intensity 100%).
After the NFI numerical value for obtaining each probe, by the standard deviation (SD) of each data provided by checkout scanning system,
Then the data for rejecting SD < 1.5 take the Median value of 3 repetition points of each probe, by the Median Value Data of each saliva sample
By production scatter plot in tetra- groups of 6.0 softwares of typing GraphPad Prism of HV, HB, HC and HCC and pass through Ordinary One-
Way ANOVA Analysis carries out statistical analysis.
3.3 poly- propionamide gel electrophoresises (SDS-PAGE) and band colour developing
SDA-PAGE gel is transferred in electrophoresis tank, checks gel slide direction and electrode direction to ensure electric current logical
Under line state.In our current research, the albumen sample separated through SDS-PAGE needs to identify mesh by two kinds of coloration methods on gel
Band, i.e., using silver oxide silver staining colour developing (Silver Staining) comparison total protein difference and utilize agglutinin trace
It tests to verify the specific sugar protein sugar chain differential expression of DSA identification.Sialoprotein sample applied sample amount for silver staining is 8 μ g,
Sialoprotein sample applied sample amount for Lectin Blotting is 30 μ g, and each group sample of quality is taken etc. according to experiment demand
And 4 μ L 5 × Loading Buffer are added, 20 μ L are complemented to ultrapure water, sample system is placed in boiling water by concussion after mixing
Water-bath 5min, rapid centrifugation and the loading after cooled on ice after water-bath.According to Protein Ladder, HV, HB, HC, HCC it is suitable
Sequence is successively to each gel pore loading.Electrophoretic parameters setting are as follows: under 80V constant voltage mode, to Loading Buffer in concentration glue
When forming an elongated and horizontal straight line in layer, adjustment power supply to 110V constant voltage mode to electrophoresis terminates.
Silver staining colour developing: the PAGE gel for silver staining should take out from offset plate in time after reaction in electrophoresis, in
2h or more is impregnated in fixer (50% ethyl alcohol, 10% glacial acetic acid, water).Gel volume is fixed to narrow down to after half in soak
30min is reacted in (30% ethyl alcohol, 6.8% 5 water sodium acetate, 0.2% sodium thiosulfate, 0.15% glutaraldehyde, water).It is soaking
It impregnates in ultrapure water after liquid reaction and is swollen again, change water and soak 20min × 3 time.Using dyeing liquor (0.5% silver nitrate,
0.1% formaldehyde, water) impregnate gel the purpose of be to make silver ion in conjunction with glue internal protein.All reaction process are in low on shaking table
Speed concussion keeps each step reaction solution to contact with the uniform of gel.Ultrapure water ringing gel and reactor are used after staining reaction 20min
Ware 1min is subsequently poured into developing solution (2.5% sodium carbonate, 0.1% formaldehyde, water).Band develops the color to appropriate degree in gel
It is terminated in time with terminate liquid (1% glacial acetic acid, water) and acquires image, while strictly using ultrapure water in overall process, avoid each step
Cl is introduced in solution-、CO3 2-、SO4 2-Plasma impacts chromogenic reaction.
Agglutinin Blot experiment: the PAGE gel for Blot experiment takes out of offset plate after reaction in electrophoresis
Out, it cuts and is immersed in 10min in 1 × transferring film buffer after removing redundance, impregnate should also have with gel etc. together with it
The filter paper 4 of area is opened, the PVDF blotting membrane 1 through methanol immersion activation 30s is opened.From the anode of electrophoresis tank access to cathode, trace
System should are as follows: filter paper 2 is opened, PVDF blotting membrane, gel, filter paper 2 are opened, and has been discharged layers of material in order and has been guaranteed layers of material
Between fit closely bubble-free and be placed in electrophoresis tank, setup parameter 300mA constant current electrophoresis 1h 45min completes trace reaction.To
The pvdf membrane after trace is taken out after the reaction was completed, pollution and physical damage should be avoided in the process, and film is placed in to be diluted to work dense
1h is in the Carbo-Free Blocking confining liquid of degree to slough absorption of the non-specific free polysaccharide on film.It will after closing
Pvdf membrane is transferred in 1 × TBS, and the Ca of the Cy5-DSA and final concentration of 0.1mM of final concentration of 5 μ g/mL is added2+, 4 DEG C are protected from light
Shake 12h.15min × 3 time are cleaned with 1 × TBST after reaction, entire cleaning process is it must also be noted that be strictly protected from light to prevent
Fluorescent quenching.Fluorescence signal image is scanned using 840 multifunctional image analyzer of Storm, parameter setting PMT is 800, differentiates
Rate 100DPI, excitation wavelength 635nm.
The Magnetic Isolation of the preparation of 3.4 agglutinins-magnetic particle complex and corresponding glycoprotein
The epoxidation Fe that this experiment is prepared using a kind of laboratory3O4Magnetic particle, agglutination is made in agglutinin DSA in coupling
Biscuit porcelain composite material of particles (DSA-Magnetic Particle Compounds, LMPCs) then passes through LMPCs and sample
Mixing, Magnetic Isolation, cleaning and elution obtain the special glycoprotein of agglutinin DSA institute affine combination.Epoxidation is taken first
Fe3O4(material is obtained by a kind of co-immunoprecipitation method and is modified through epoxidation magnetic particle 3mg, and specific method is shown in text
It offers[46]), the coupling buffer that 1mL is added repeats after overturning until mixing well, and is placed in complete to magnetic particle on magnetic separator
Liquid is discarded supernatant when being adsorbed on magnet entirely realizes cleaning, cleaning step × 3 time.Buffer solution is coupled followed by 600 μ L
The DSA agglutinin for dissolving 300 μ g is added to be resuspended in the magnetic particle system and mix, and is placed in 180rpm, 25 on horizontal concussion shaking table
DEG C reaction 6h complete agglutinin DSA and magnetic particle coupling.It in Magneto separate on magnetic separator while being abandoned after the completion of being coupled
Supernatant is removed, 1mL magnetic grain confining liquid is added by resuspension, concussion, Magneto separate and abandons the LMPCs of supernatant step cleaning at this time, repeats 3
600 μ L magnetic grain confining liquids are added after secondary cleaning and close its extra epoxy group in shaking bed reaction 1h.After completing closing, benefit
It is cleaned LMPCs totally 3 times in conjunction with buffer solution, simultaneously, dissolves saliva sample using the combination buffer solution of 600 μ L volumes
2mg (if saliva original concentration is too small, is containing correspondence using 10KDa ultra-filtration centrifuge tube (Millipore Corp, U.S.A)
In 14 after quality sample, 000 × g centrifugal ultrafiltration be concentrated after sample, be then settled to 600 μ using in conjunction with buffer solution
L it) is added in LMPCs system and is resuspended afterwards, 180rpm concussion, 25 DEG C of reaction 3h.Before elution, using cleaning buffer solution, repeat
Foregoing cleaning step at least 5 times.It is to be checked to measure clearly using protein concentration in Nano-Drop the real time measure cleaning solution
Illustrate that the protein of non-specific binding is separated thoroughly when protein concentration is 0 in washing lotion.400 μ L elution buffers are added
Liquid shakes 20min in 180rpm on shaking table, collects supernatant after Magnetic Isolation, and specific glycoprotein is due to agglutinin change at this time
Property and be dissolved in supernatant, then add 200 μ L elution buffers and be repeated once elution step and collect supernatant.
3.5 enzyme process separate glycoprotein N- sugar chain (N-Glycans)
The LMPCs that learns from else's experience separation obtain 0 μ g of specific sugar protein 20 be added 10KDa super filter tube (Millipore Corp,
U.S.A it in), is firstly added in 8M urea to super filter tube at maximum carrier fluid amount scale, then through 14,000 × g is centrifuged 10min will
Inorganic ion, non-protein micromolecular are extremely collected in casing with solvent through ultrafiltration membrance filter；The mixing of 400 μ L 8M urea is added
It is centrifuged afterwards and repeats this step 3 time so that protein sample is sufficiently denaturalized；1 × DTT working solution, the 400 μ L diluted is added and fills
Divide pressure-vaccum to mix, is reacted at 56 DEG C 14 after 45min, 000 × g is centrifuged 10min；400 μ 1 × IAM of L working solutions are added into pipe simultaneously
It is protected from light 30min closing disulfide bond at room temperature；The 40mmol/L NH of 400 μ L is added4HCO3Centrifugation changes liquid to remove remnants
Each step reaction solution and repeat this step 3 time；The 20 preactivated Trypsin protease of μ L hydrochloric acid, 37 DEG C of overnight digestions are added；
After reaction then boiling water bath 1min changes new collection casing, to ultrafiltration membrane to inactivate Trypsin protease for ultrafiltration membrane
2 μ L PNGase-F glycosidases of interior addition react 14 after 10h, and 000 × g is centrifuged 10min, and 400 μ L 40mmol/L are added
NH4HCO3It is repeated once centrifugation, N- sugar chain has been filtered into new collection casing after being centrifuged twice, and N- sugar chain solution is dense in being centrifuged
- 20 DEG C of traditional vacuum freeze-dryings on contracting instrument.
3.6NaClO oxidizing process separates O- sugar chain (O-Glycans)
The LMPCs that learns from else's experience separates 200 μ g of glycoprotein obtained and is added in 10KDa super filter tube, and 14,000 × g is centrifuged 10min
Former solvent phase is removed, is added after 400 μ L ultrapure waters mix and is centrifuged, repeat the cleaning at least 7 times, NaClO is in subsequent reaction
There is strict demand to pH, therefore pays special attention to the quality of ultrapure water and the thorough degree of cleaning in experimentation；For the last time
200 μ L ultrapure waters, 100 μ L 6%NaClO are sequentially added after cleaning, are placed on horizontal concussion shaking table and are reacted 30min at room temperature；Instead
Ice bath is pre-chilled after answering, and the oxidation that 10% formic acid (in advance in pre-cooling preparation on ice) termination sodium hypochlorite of 15 μ L is added is anti-
It answers, to avoid reaction excessively violent, reaction system is always held at 10min in ice bath；The repetition of 400 μ L ultrapure waters is added after centrifugation
Cleaning at least 7 times to walk the remaining formic acid of reaction in abundant removal；After completing last time cleaning, it is centrifuged in filter membrane at this time and is answered
The solution of remaining about 30 μ L volumes, 170 μ L ultrapure waters is added into filter membrane and with formic acid tune pH to 7.6 or so, is then added
6.64 μ L 6%NaClO change new collection casing and are sealed system with sealed membrane and react at room temperature for 24 hours.Reaction
After be added 8 μ L pre-cooling 10% formic acid terminate reaction, 200 μ L ultrapure waters are added after centrifugation and mixes and is centrifuged, twice from
The sufficiently filter of O- sugar chain is into new collecting pipe after the heart, by O- sugar chain solution in -20 DEG C of traditional vacuum freeze-dryings on centrifuge concentrator.
The gel desalination of 3.7 sugar chains
The cleaning of Sepharose CL-4B and nonpolarity balance: take 1.5mL without enzyme centrifuge tube, be added 100 μ L's
The water methanol eluent of 800 μ L is added in Sepharose CL-4B gel, is resuspended 9 after mixing, 000 × g is centrifuged 5min, from centrifugation
30s is stood vertically in centrifuge tube orifice plate after taking out in machine, is carefully inhaled after gel planar horizontal with pipettor and abandons supernatant, is used
Water methanol eluent repeats the above steps 5 times；Acquisition is to upper after n-butanol cleaning solution repeated centrifugation is added, abandons supernatant step 2 time
The gel of sample.
Sugar chain loading desalination: the N/O- glycosylation sugar chain sample after taking 500 μ L n-butanol cleaning solutions to re-dissolve freeze-drying, so
Loading is into the good Sepharose CL-4B gel of pre-balance afterwards, in 80rpm concussion reaction 1h, root on level concussion shaking table
According to the polarity difference of solid-liquid two-phase, polar sugar chain can be negligent of liquid phase and be bound in gel particles；After sugar chain is in conjunction with gel,
9,000 × g, which is centrifuged 5min and is inhaled with pipettor, abandons supernatant, then adds after 500 μ L n-butanol cleaning solutions mix to be centrifuged and abandon
Supernatant repeats this cleaning step 5 times to remove nonpolar polypeptide and the salt ion in sample；500 μ L first are added after the completion of cleaning
Alcohol water elution is redissolved in sugar chain because of the polarity of methanol solution at this time and washes in 180rpm concussion reaction 20min on shaking table
In de- liquid, after reaction 9,000 × g be centrifuged and use newly without enzyme pipe collection supernatant, then with 500 μ L water methanol eluent weights
After backwashing is 1 time de-.By N/O- sugar chain solution after purification in -20 DEG C of traditional vacuum freeze-dryings on centrifuge concentrator.
The MALDI-TOF-MS interpretation of mass spectra of 3.8N-/O- sugar chain
Utilize the 1:1 methanol of 5 μ L: aqueous solution (v:v) re-dissolves the sugar chain crystallization after freeze-drying, and pressure-vaccum, which mixes, guarantees tube bottom
Micro sugar chain sample after completely dissolution, takes the target plate sample well of 2 μ L loadings to 384 points of Bruker MTP Anchorchip
On, it is crystallized to sample natural air drying, observes the shape of sample crystallization on stainless steel target plate, sugar chain sample is formed under suitable concentration
Crystal form should star-shaped in the range of circular drop come radially outward.Then add again in the range of original sample crystallizes
Upper 2 μ L DHB matrix waits natural air drying to recrystallize, 384 target plates of MTP Anchorchip for having sugar chain to crystallize loading
It is loaded into MALDI-TOF/TOF-MS (Bruker Daltonic, Germany), passes through first mass spectrometric solution under TOF-MS mode
Original sample is analysed, parameter is selected are as follows: under the polysaccharide determination spectral coverage of " RP_700-4000_Da.Par ", according to standards calibration
Parsing precision within the scope of this successively parses N- glycosylation sugar chain (1200-4000Da) and O- sugar according to the target position of record
Base sugar chain (500-1800Da).
3.9 MASS SPECTRAL DATA ANALYSIS
The mass spectrometric data with " .Ref " for suffix is opened using analysis software flexAnalysis, spectral peak signal-to-noise ratio is set
(S/N) >=3 be automatic screening condition, read the data at each peak, if because believe it is hot-tempered influence so that individual visible peak is unread comes out, can
Individual higher peaks are added in data list manually by "+(Find MASS Spectrum) " in software interface.By upper
Sequence of operations is stated, available " m/z. ", " S/N ", " Quality Fac. ", " Res. ", " Intens. " and " Area " totally 6
Quantified data." m/z. " and " Intens. " that (S/N >=3) choose each peak under the conditions of signal-to-noise ratio is effective enumerates generation text
This document imports Glycoworkbench software and automatically analyzes sugar chain structure, analyzes parameter setting are as follows: selection Glycome DB
Database selects ion channel for " [M+Na]+" and " [M+H]+", charge at most+1, precursor ion tolerance is ± 1Da, fragment
Ion tolerance is ± 0.5Da.
Result of study:
Verifying of the 4.1 saliva chips to DSA identification saliva sugar chain sugar-type difference
120 quantitative saliva samples, with agglutinin DSA competitive binding, pass through each saliva probe under same incubation environment
The NFI of point reflects each sample β-D-GlcNAc, the gene expression abundance of Gal β 1-4GlcNAc and (GlcNAc β 1-4) n sugar chain structure.
Blood Group-H Antigen and the T-Antigen structure that agglutinin PTL-II is identified is steady in HV, HB, HC and HCC
Fixed expression and indifference.It is reacted using Cy5-PTL-II with saliva chip, chip scans are shown: each saliva probe on chip
Point sample is uniform, and negative Quality Control and positive quality control have good indicative function, and show after internal reference agglutinin PTL-II loading
It is good to quantify homogeneity for sample probe out, without quantitative inaccuracy caused by experimental error.It is anti-using Cy5-DSA and saliva chip
It answers, is based on chip scans, be grouped using the Median value of each saliva probe FI according to HV, HB, HC and HCC and draw scatterplot
Figure, as shown in Figure 2.
Pass through Ordinary One-Way ANOVA Analysis statistical analysis between tetra- groups of HV, HB, HC and HCC of groups
(such as table 5) is found compared to HV group, β-D-GlcNAc, Gal the β 1-4GlcNAc and (GlcNAc β 1- that agglutinin DSA is identified
4) n sugar chain structure has significantly in HB (p**=0.0021), HC (p**** < 0.0001) and HCC (p*=0.00320) group
Express increasing trend.It is worth noting that, the sugar chain structure significantly high expression in HC group of agglutinin DSA institute specific recognition,
With HV group p**** < 0.0001), significantly increase compared with HB group (p*=0.0216) and HCC group (p**=0.0013)
Krustal-Wallis test and One-Way ANOVA are analyzed between the group of 5 four groups of samples of table
aMultiple comparison (1vs.2), compares the grouping of formation two-by-two between four groups of samples；bMean rank
Diff., mean value is graded difference between the group obtained by Krustal-Wallis test, Mean rank diff.=(Mean
rank 2–Mean rank 1)；cSignificant, the significance of difference, if diff>20 Mean rank between group and p<0.05
For Yes, on the contrary is No.
4.2 agglutinin traces verify mixing sample result
Three groups of patients with chronic liver and healthy volunteer's saliva sample pass through respectively in mixing elimination group after individual difference, mixed
Sample is closed by SDS-PAGE electrophoresis and through silver staining experimental evaluation HV, HB, HC and HCC sialoprotein difference (such as Fig. 3 A).Silver staining
The result shows that the band of four groups of sialoproteins is almost the same, the quantity and coloring of the high kurtosis protein band between 10~100KDa
Depth is almost the same (wider lane near such as 70KDa, 60KDa).
By agglutinin Blot experiment (Lectin Blotting) result (such as Fig. 3 B) of DSA it can be observed that agglutinin
The glycoprotein of DSA and tetra- groups of saliva samples of HV, HB, HC and HCC are specifically bound, and are shown about 7 apparent bands, are removed
3 combine outside bands similar in level, clearly illustrate 1 band (about 60KDa for having obvious combination difference in figure
Place), it is shown that β-D-GlcNAc, Gal β 1-4GlcNAc and (GlcNAc β 1-4) n sugar chain structure are specific in HC patient's saliva
Expression increases, and the band that 1 molecular weight is about 35KDa shows β-D-GlcNAc, Gal β 1-4GlcNAc and (GlcNAc β 1-4)
N sugar chain structure is specific expressed in HV saliva to be increased, and the combination of DSA and HV group is significantly stronger than tri- groups of HB, HC and HCC.This
As a result it is consistent substantially with the result of lectin chip, saliva chip, verifying illustrates in the mixing sample for eliminating individual difference,
The expression of sialoprotein matter is limited by silver staining developing sensitivity does not embody apparent difference, and in protein level without change
β-D-GlcNAc, Gal the β 1-4GlcNAc and (GlcNAc β 1-4) n sugar chain structure that DSA is identified on the basis of change are in HC patient
There is the phenomenon that increasing in saliva glycoprotein.
The 4.3 glycoprotein N- sugar chain MALDI-TOF-MS through DSA-LMPCs separation are parsed
Using the specific glycoprotein of DSA identification in LMPCs separation HV, HB, HC and HCC patient saliva, then utilize
PNGase F restriction endonuclease exclusively separates the N- sugar chain on specific glycoprotein, passes through each sugar chain matter of MALDI-TOF-MS Mass Spectrometric Identification
Sugar chain database Glycome is simultaneously searched in lotus ratio (m/z.), each N- sugar chain peak for signal-to-noise ratio S/N > 3 for taking Mass Spectrometric Identification to obtain
DB is analyzed, thus it is speculated that is obtained N- sugar chain structure information, and is obtained the corresponding mass spectrometric data in each N- sugar chain peak, including m/
Z., N- sugar chain structure, relative peak intensities etc..
48 kinds of N- sugar chains are identified altogether by MALDI-TOF-MS, wherein reflecting respectively in HV, HB, HC and HCC each group
Determine to 20,13,26 and 25 kind of N sugar chain.It is worth noting that, the saliva sample for thering are following 11 kinds of N- sugar chains to exist only in HC group
In.
m/z.1996.7238(Fuc)1(GalNAc)1(Gal)1(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2006.7316(Gal)3(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2037.7503(Fuc)1(GalNAc)2(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2281.8321(Sia)1(Fuc)1(GalNAc)1(Gal)2(GlcNAc)2+(Man)3(GlcNAc)2；
m/z.2573.9480(Sia)1(Fuc)2(Gal)2(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2772.9572(Sia)2(Gal)4(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2794.0427(Fuc)3(Gal)3(GlcNAc)4+(Man)3(GlcNAc)2(Fuc)1；
m/z.2806.9879(Fuc)1(Gal)6(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2924.0805(Gal)4(GlcNAc)6+(Man)3(GlcNAc)2(Fuc)1；
m/z.3232.1912(Fuc)2(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2；
m/z.3813.3617(Sia)3(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2。
So last detected it follows that being directed to saliva sample, it need to only measure whether it contains this 11 kinds of rock N- sugar
Any or any combination of chain can make cirrhosis (HC) screening, early diagnosis, risk assessment, drug screening and/or curative effect
The result of assessment.
Claims (7)
1. a kind of product for the screening of hepatopathy, early diagnosis, risk assessment, drug screening and/or curative effect evaluation, for saliva
Liquid sample characterized by comprising
A, for obtaining the device of the expression of three kinds of specific sugar albumen sugar chain structures, three kinds of specific sugar protein sugars link
Structure is respectively β-D-GlcNAc, Gal β 1-4GlcNAc and (GlcNAc β 1-4) n；
B, for differentiating the whether highly expressed mark of three of the above specific sugar albumen sugar chain structure, module or processor.
2. product according to claim 1, it is characterised in that: described device includes lectin chip, is incubated for box and biology
Chip scanning system, wherein at least provided with agglutinin DSA on lectin chip.
3. product according to claim 1, it is characterised in that: the mark, module or the pre-recorded unsoundness of processor
(HV) corresponding reference value determines whether high expression for compareing with sample results.
4. a kind of agglutinin prepares the purposes of Related product separately as the reagent of the special glycoprotein candy chain structure recognition of saliva,
It is characterized by: the agglutinin be DSA, the Related product be kit, equipment, can operating system and/or their group
It closes, purposes is screening, early diagnosis, risk assessment, drug screening and/or the curative effect evaluation of hepatopathy.
5. the recognition unit of specific sugar chain is in building for making cirrhosis (HC) screening, early diagnosis, risk based on saliva sample
Purposes in terms of the product of assessment, drug screening and/or curative effect evaluation, it is characterised in that: the recognition unit identification following 11
Any or any combination of kind N- sugar chain:
m/z.1996.7238(Fuc)1(GalNAc)1(Gal)1(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2006.7316(Gal)3(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2037.7503(Fuc)1(GalNAc)2(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2281.8321(Sia)1(Fuc)1(GalNAc)1(Gal)2(GlcNAc)2+(Man)3(GlcNAc)2；
m/z.2573.9480(Sia)1(Fuc)2(Gal)2(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2772.9572(Sia)2(Gal)4(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2794.0427(Fuc)3(Gal)3(GlcNAc)4+(Man)3(GlcNAc)2(Fuc)1；
m/z.2806.9879(Fuc)1(Gal)6(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2924.0805(Gal)4(GlcNAc)6+(Man)3(GlcNAc)2(Fuc)1；
m/z.3232.1912(Fuc)2(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2；
m/z.3813.3617(Sia)3(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2。
6. product of the one kind for cirrhosis (HC) screening, early diagnosis, risk assessment, drug screening and/or curative effect evaluation, needle
To saliva sample, which is characterized in that the product includes:
A, agglutinin DSA；
B, for being coupled the affinity chromatography solid phase carrier of agglutinin DSA；
C, for being separated in Gal β 1-4GlcNAc and the glycoprotein of (GlcNAc β 1-4) n sugar chain structure from β-D-GlcNAc is contained
The reagent and device of N- sugar chain out；
D, mass spectrograph, for showing the m/z value for corresponding to various N- sugar chains；
E, for identifying mark, module or the processor of following 11 kinds of rock N- sugar chains according to mass spectrogram, with obtain whether containing with
Any or any combination judgement result of lower 11 kinds of N- sugar chains:
m/z.1996.7238(Fuc)1(GalNAc)1(Gal)1(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2006.7316(Gal)3(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2037.7503(Fuc)1(GalNAc)2(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2281.8321(Sia)1(Fuc)1(GalNAc)1(Gal)2(GlcNAc)2+(Man)3(GlcNAc)2；
m/z.2573.9480(Sia)1(Fuc)2(Gal)2(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2772.9572(Sia)2(Gal)4(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2794.0427(Fuc)3(Gal)3(GlcNAc)4+(Man)3(GlcNAc)2(Fuc)1；
m/z.2806.9879(Fuc)1(Gal)6(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2924.0805(Gal)4(GlcNAc)6+(Man)3(GlcNAc)2(Fuc)1；
m/z.3232.1912(Fuc)2(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2；
m/z.3813.3617(Sia)3(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2。
7. product according to claim 6, it is characterised in that: described for being coupled the affinity chromatography solid phase of agglutinin DSA
Carrier is Fe3O4Magnetic particle, Ago-Gel, sephadex or glass microsphere.
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