CN108982856A - Based on liver cancer correlation screening/assessment product of the special glycoprotein candy chain structure of saliva and application - Google Patents
Based on liver cancer correlation screening/assessment product of the special glycoprotein candy chain structure of saliva 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
- G01N33/53—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
Abstract
The invention discloses based on liver cancer correlation screening/assessment product of the special glycoprotein candy chain structure of saliva and application, for saliva sample, two kinds are provided for liver cancer (HCC) screening, early diagnosis, risk assessment, the product of drug screening and/or curative effect evaluation, the expression (whether high expression) of two kinds of specific sugar albumen sugar chain structure Fuc α 1-2Gal β 1-4GlcNAc and Fuc α 1-3 (Gal β 1-4) GlcNAc in one of product testing saliva sample, whether any or any combination of specific eight kind fucosylation O- sugar chain is contained in another product testing saliva sample.Correspondingly, the agglutinin that can play specific binding recognition reaction is LTL, the purposes of Related product can be prepared separately as the reagent of the special glycoprotein candy chain structure recognition of saliva.
Description
Technical field
The present invention relates to a kind of determination of liver tumour marker based on the special glycoprotein of saliva and its Related product, answer
With.
Background technique
Liver cancer (Hepatocellular Carcinoma, HCC) belongs to the cancer of high incidence and high mortality in China.
Currently, the various detection methods of HCC are limited by its technical characterstic respectively, presence specificity is low, sampling is inconvenient and cannot be early
The defects of phase diagnoses.Have in recent years a large number of studies show that: with the occurrence and development of tumour, tumor tissues, serum and the saliva of patient
Glycoprotein glycosylation abnormal change has occurred in the body fluid such as liquid.
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 liver cancer (HCC)
Screening, diagnosis and assessment are 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 is based respectively on serum detection, is detected jointly based on serum and saliva and give different sides
Case.
Summary of the invention
Screening, diagnosis and assessment etc. about liver cancer (HCC), 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, the Fuc α 1-2Gal β 1-4GlcNAc that agglutinin LTL is identified
It is high expression in cirrhosis (HC), HCC patient's saliva with Fuc α 1-3 (Gal β 1-4) GlcNAc sugar chain structure, and in stabilization
Increasing trend；And compared to healthy volunteer (HV), hepatitis B (HB) and HC, Fuc α 1-2Gal β 1-4GlcNAc and Fuc α 1-3 (Gal β
1-4) GlcNAc sugar chain structure expression highest (p≤0.0005) in HCC patient's saliva.
Two, further, 8 kinds of fucosylation O- sugar chain (m/z 652.216 (Fuc)1(GlcNAc)1(GalNAc)1,
828.269(Fuc)1(Gal)1(GlcNAc)1(GalNAc)1,833.314(Fuc)1(GlcNAc)1(GalNAc)2,995.367
(Fuc)1(Gal)1(GlcNAc)2(GalNAc)1,1144.295(Fuc)1(Sia)1(Sulf-Gal)1(Gal)1(GalNAc)1,
1157.420(Fuc)1(Gal)2(GlcNAc)2(GalNAc)1,1163.417(Fuc)2(GlcNAc)2(Gal)1(GalNAc)1,
1193.402(Fuc)1(Gal)2(GlcNAc)2(GalNAc)1) exist only in the saliva of HCC patient, and be not found in HV, HB and
In the saliva of HC.
According to above first conclusion, following application scheme can be obtained:
Product of the one kind for liver cancer (HCC) screening, early diagnosis, risk assessment, drug screening and/or curative effect evaluation,
For saliva sample, comprising:
A, for obtaining the device of the expression of two kinds of specific sugar albumen sugar chain structures, described two specific sugar protein sugars
Chain structure is respectively Fuc α 1-2Gal β 1-4GlcNAc and Fuc α 1-3 (Gal β 1-4) GlcNAc；
B, for differentiating the whether highly expressed mark of both 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 LTL, (this agglutinin being capable of Fuc α 1-2Gal β 1-4GlcNAc and Fuc α 1-3 on specific recognition glycoprotein
(Gal β 1-4) GlcNAc sugar chain structure).
Above-mentioned mark, module or the pre-recorded unsoundness of processor (HV), hepatitis B (HB), cirrhosis (HC), liver cancer (HCC)
Corresponding reference value determines whether high expression for compareing with sample results.It is greater than for example, fluorescence signal inatheadearomatizationazone can be set
1.5, determine that it is high expression；Fluorescence signal inatheadearomatizationazone determines that it is low expression less than 0.67；Ratio 0.67~1.5 it
Between express and do not change.
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 LTL, the Related product be kit, equipment, can operating system and/or their combination, purposes is
Liver cancer (HCC) screening, early diagnosis, risk assessment, drug screening and/or curative effect evaluation.
According to above second conclusion, following application scheme can be obtained:
The recognition unit of specific sugar chain is in building for making liver cancer (HCC) screening, early diagnosis, risk based on saliva sample
Purposes in terms of the product of assessment, drug screening and/or curative effect evaluation, the recognition unit identify following eight kinds of fucosylations
Any or any combination (passing through judgement " having ", "None" you can get it conclusion) of O- sugar chain:
m/z 652.216(Fuc)1(GlcNAc)1(GalNAc)1,
m/z 828.269(Fuc)1(Gal)1(GlcNAc)1(GalNAc)1,
m/z 833.314(Fuc)1(GlcNAc)1(GalNAc)2,
m/z 995.367(Fuc)1(Gal)1(GlcNAc)2(GalNAc)1,
m/z 1144.295(Fuc)1(Sia)1(Sulf-Gal)1(Gal)1(GalNAc)1,
m/z 1157.420(Fuc)1(Gal)2(GlcNAc)2(GalNAc)1,
m/z 1163.417(Fuc)2(GlcNAc)2(Gal)1(GalNAc)1,
m/z 1193.402(Fuc)1(Gal)2(GlcNAc)2(GalNAc)1。
Product of the one kind for liver cancer (HCC) screening, early diagnosis, risk assessment, drug screening and/or curative effect evaluation,
For saliva sample, which includes:
A, agglutinin LTL；
B, for coupling the affinity chromatography solid phase carrier of agglutinin LTL；
C, it is used for from the sugar for containing Fuc α 1-2Gal β 1-4GlcNAc and Fuc α 1-3 (Gal β 1-4) GlcNAc sugar chain structure
The reagent and device of O- sugar chain are isolated in albumen；
D, for parsing the device of the chemical constitution formula of various O- sugar chains；
E, whether any containing aforementioned eight kinds of fucosylations O- sugar chain in the various O- sugar chains parsed for differentiating
Or the mark, module or processor of any combination.
The above-mentioned affinity chromatography solid phase carrier for coupling agglutinin LTL can choose Fe3O4Magnetic particle, agarose are solidifying
Glue, sephadex, glass microsphere etc..
For the above-mentioned device for parsing the chemical constitution formula of various O- sugar chains there are many optional type, working principle can be with
It is MALDI TOF MS method MALDI-MS, fast atom bombardment mass spectroscopy FAB-MS, electrospray ionization mass spectrum ES-
MS, liquid chromatography, Liquid Chromatography-Mass Spectrometry, nuclear magnetic resonance nmr etc., by m/z the obtaining of information for obtaining O- sugar chain
Learn composition formula.
Detailed description of the invention
Fig. 1 is the NFI case figure check analysis of agglutinin LTL, PTL-I, EEL.In figure, ordinate is indicated on lectin chip
Normalization fluorescence intensity NFI corresponding to the agglutinin, top-down each horizontal line respectively represents this group of data in each case figure
Maximum value, rear quartile, median, preceding quartile and minimum value.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: cirrhosis is suffered from
Person's saliva, HCC: Saliva of Primary Hepatocellular Carcinoma.
Fig. 2 is each probe combination LTL 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: cirrhosis is suffered from
Person's saliva, HCC: Saliva of Primary Hepatocellular Carcinoma.
Fig. 3 is four groups of mixing saliva sample SDS-PAGE silver staining colour developings and LTL 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 LTL agglutinin trace；B1~b4 is respectively to mark
The apparent band of differential expression in agglutinin trace.Each band sample information are as follows: 1:Protein Ladder, 2: healthy will
Hope person's saliva, 3: hepatitis B patient saliva, 4: liver cirrhosis patient saliva, 5: Saliva of Primary Hepatocellular Carcinoma.
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, according to mapping demand selection case figure " Box&Shiskers " or scatter plot " Scatter&SD "
Deng." Analyze " analysis formula is subsequently entered 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 LTL, PTL-I and EEL, judged by one-way analysis of variance
The significance of difference of the sugar chain structure of this 3 kinds of agglutinins identification in Saliva of Primary Hepatocellular Carcinoma, result is as shown in Figure 1, a series of
Statistical data can intuitively reflect the significance of difference and dispersion degree of each comparison among groups on the diagram.According to Dan Yin between each group
From the point of view of the result of plain variance analysis, the Fuc α 1-2Gal β 1-4GlcNAc and Fuc α 1-3 (Gal β 1-4) of agglutinin LTL identification
GlcNAc structure significantly increases (p≤0.0005) compared with other three groups in HCC, and equal indifference between other three groups, illustrates this
High expression specifically occurs in Saliva of Primary Hepatocellular Carcinoma glycoprotein for one sugar chain structure.GalNAc α -1,3Gal that PTL-I is identified,
Though GalNAc α -1,3Gal β -1,3/4Glc structure representation intensity relatively show that it is expressed in liver cancer saliva and has through NFI average value
Certain up-regulation, but apparently through one-way analysis of variance, the expression intensity of the sugar chain structure occurs larger in HB group, HCC group
Floating range, a kind of trend of unstable expression is presented in this two groups, so be not present the significance of difference.Agglutinin
Gal α 1-3 (Fuc α 1-2) Gal structure that EEL is identified significantly increases (p=0.0015) compared with HB group in HCC group, this result
It is almost the same with average NFI comparison result, but HCC group compared to HV group, HC group without significant difference.
So last it follows that compare other glycoprotein, the Fuc α 1-2Gal β 1-4GlcNAc of agglutinin LTL identification
It is high expression in HB, HC, HCC patient's saliva with Fuc α 1-3 (Gal β 1-4) GlcNAc sugar chain structure, and is incremented by stablizing
Trend；And HV, HB and HC are compared, Fuc α 1-2Gal β 1-4GlcNAc and Fuc α 1-3 (Gal β 1-4) GlcNAc structure is suffered from HCC
Expression highest (p≤0.0005) in person's saliva.Therefore, it may be considered that in this, as liver cancer (HCC) screening, early diagnosis, wind
The foundation of danger 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 LTL
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 LTL identification of 30 healthy volunteer's saliva samples Fuc α 1-2Gal β 1-4GlcNAc and Fuc α
1-3 (the Gal β 1-4) up-regulated expression of GlcNAc structure in HCC group.By the way that saliva chip is made in 120 saliva samples, benefit
Fuc α 1-2Gal β 1-4GlcNAc and Fuc α 1-3 (Gal β 1-4) in each example patient saliva is detected on the chip with agglutinin LTL
The abundance and comparative analysis difference of GlcNAc structure.The mixing saliva sample of each group is obtained additionally by equal protein qualities mixing
This, each group mixing is verified using SDS-PAGE albuminous degeneration electrophoresis and agglutinin Blot experiment (Lectin Blotting)
The abundance difference of Fuc α 1-2Gal β 1-4GlcNAc and Fuc α 1-3 (Gal β 1-4) GlcNAc structure in saliva sample.Through above-mentioned one
Using a kind of ferriferrous oxide nano magnetic bead surfaces being coupled upper agglutinin LTL after series of validation, that agglutinin magnetic particle is made is compound
Object (LTL-Magnetic Particle Compounds, LMPCs) is come the specific sugar egg that is enriched in each group saliva mixing sample
It is white, N- sugar chain and O- sugar chain on specific glycoprotein are separated using PNGase F enzyme cutting method, NaClO oxidizing process, and auxiliary by matrix
Help laser ionization 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 LTL 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 LTL relative quantification detects on saliva chip Fuc α 1-2Gal β 1-4GlcNAc, Fuc α 1-3 (Gal in each example saliva sample
β 1-4) GlcNAc structure gene expression abundance when, using Cy5 fluorochrome label agglutinin LTL and be made into reaction system (400 μ L
Lectin chip Incubating Solution, 40 μ L 4M azanols, the agglutinin LTL of 10 μ L 0.1%Tween-20,20 μ g Cy5 label are ultrapure
Water supplies final volume to 600 μ L) it is splined on saliva chip.After saliva chip and Cy5-LTL association reaction are completed and cleaned, finally
In data acquisition on Genepix 4000B chip scanner (parameter setting are as follows: excitation wavelength 635nm, PMT power 70%,
100%) excitation intensity is.
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 Krustal-Wallis
Test and one-way ANOVAanalysis 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 LTL 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 1h45min 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-LTL 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 LTL in coupling
Biscuit porcelain composite material of particles (LTL-Magnetic Particle Compounds, LMPCs) then passes through LMPCs and sample
Mixing, Magnetic Isolation, cleaning and elution obtain the special glycoprotein of agglutinin LTL 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 LTL 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 LTL 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 (1000-3750Da) 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 LTL identification saliva sugar chain sugar-type difference
120 quantitative saliva samples, with agglutinin LTL competitive binding, pass through each saliva probe under same incubation environment
The NFI of point reflects that the expression of each sample Fuc α 1-2Gal β 1-4GlcNAc and Fuc α 1-3 (Gal β 1-4) GlcNAc structure is rich
Degree.Blood Group-H Antigen and the T-Antigen structure that agglutinin PTL-II is identified is in HV, HB, HC and HCC
Stable expression and indifference.It is reacted using Cy5-PTL-II with saliva chip, chip scans are shown: each saliva on chip
Probe spotting is uniform, and negative Quality Control and positive quality control have good indicative function, and after internal reference agglutinin PTL-II loading
It is good to show that sample probe quantifies homogeneity, without quantitative inaccuracy caused by experimental error.Utilize Cy5-LTL and saliva chip
Reaction is based on chip scans, is grouped using the Median value of each saliva probe FI according to HV, HB, HC and HCC and draws scatterplot
Figure, as shown in Figure 2.
Pass through Krustal-Wallis test and One-Way ANOVA between tetra- groups of HV, HB, HC and HCC of groups
The Fuc α 1-2Gal β 1-4GlcNAc and Fuc α 1-3 that Analysis statistical analysis (such as table 5) discovery agglutinin LTL is identified
(Gal β 1-4) GlcNAc structure has significant expression increasing trend (p < 0.0001) compared to HV group and HB group in HCC group,
There is significant expression increasing trend compared to HV group (p=0.0002) and HB (p=0.0019) group in HC group, and in HV and HB
Between group, then there was no significant difference between HC and HCC group.
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 high molecular weight protein band greater than 100KDa dye in this experiment compared with
Shallowly and make band unobvious because of " hangover " phenomenon.The quantity of high kurtosis protein band between 10~100KDa and color depth
Spend almost the same (wider lane near such as 70KDa, 60KDa, 15KDa, 10KDa).And between 25~15KDa about
There is one to be transparent to show that out at this that protein band is darker compared with HV, HB group in HC, HCC group in the band of yellow at 20KDa
Some, this illustrates that band corresponding part albumen hepatopathy caused by HBV development later period has the tendency that expression up-regulation.
By agglutinin Blot experiment (Lectin Blotting) result (such as Fig. 3 B) of LTL it can be observed that agglutinin
The glycoprotein of LTL 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, and figure acceptance of the bid shows 4 bands (b1~b4) for having obvious combination difference, such as molecule
Fuc α 1-2Gal β 1- during amount is about the b1 of 90KDa, the b4 of the b2 of about 60KDa, about 25KDa are shown from health to liver cancer
4GlcNAc, Fuc α 1-3 (Gal β 1-4) GlcNAc sugar-type express the trend increased, and wherein b2 band reflects LTL and HCC group
In conjunction with being significantly stronger than tri- groups of HV, HB and HC；Have and band b3 that only 1 molecular weight is about 34KDa is shown in this course
The sugar-type expresses reduced trend out, and the combination of LTL and HV group is significantly stronger than tri- groups of HB, HC and HCC.This result substantially with it is solidifying
Collect plain chip, the result of saliva chip is consistent, verifying illustrates in the mixing sample for eliminating individual difference, sialoprotein matter
Expression is limited by silver staining developing sensitivity does not embody apparent difference, and on the basis of protein level is unconverted
Fuc α 1-2Gal β 1-4GlcNAc and Fuc α 1-3 (Gal β 1-4) GlcNAc sugar chain structure that LTL is identified is in HCC patient's saliva
There is the phenomenon that increasing in glycoprotein.
The 4.3 glycoprotein N- sugar chain MALDI-TOF-MS through LTL-LMPCs separation are parsed
Using the specific glycoprotein of LTL 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..
29 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 21,25,29 and 28 kind of N- sugar chain.In these sugar chains, 21 kinds of N- sugar chains (such as m/z 1419.476 (Man) is shared3
+(Man)3(GlcNAc)2, 1444.507 (Gal)1(GlcNAc)1(Fuc)1+(Man)3(GlcNAc)2Deng) reflected in four groups
Surely it arrives, 4 kinds of N- sugar chain (m/z 1743.581 (Man)5+(Man)3(GlcNAc)2, 1905.634 (Man)6+(Man)3
(GlcNAc)2, 2158.777 (Fuc)2(Gal)2(GalNAc)3+(Man)3(GlcNAc)2And 2905.036 (Fuc)1(Gal)5
(GlcNAc)5+(Man)3(GlcNAc)2) be accredited in the saliva of tri- groups of patients with chronic liver of HB, HC, HCC.It is worth noting
, there is a kind of N- sugar chain (m/z 2205.766 (NeuAc)1(Fuc)2(Gal)1(GlcNAc)1(Man)2+(Man)3
(GlcNAc)2) exist only in the saliva sample of HC group, there is 3 kinds of N- sugar chains (m/z 1257.423 (Man)2+(Man)3
(GlcNAc)2, 1501.529 (Gal)1(GlcNAc)2+(Man)3(GlcNAc)2And 1704.608 (Gal)1(GlcNAc)3+
(Man)3(GlcNAc)2) exist only in HC and two groups of HCC of saliva sample.
The 4.4 glycoprotein O- sugar chain MALDI-TOF-MS through LTL-LMPCs separation are parsed
The O- sugar chain on specific glycoprotein is separated using NaClO oxidizing process, passes through each sugar of MALDI-TOF-MS Mass Spectrometric Identification
Each N- sugar chain peak of chain mass-to-charge ratio (m/z.), signal-to-noise ratio S/N > 3 for taking Mass Spectrometric Identification to obtain passes through search sugar chain database
Glycome DB is analyzed (according to the modification feature of O- sugar chain acid, to cope with when carrying out sugar chain information comparison by mass-to-charge ratio
Analyse and compare plus sugar chain modified group A:58Da and B:72Da with answering), thus it is speculated that O- sugar chain structure information is obtained, and is obtained
Each O- sugar chain peak corresponding mass spectrometric data, including m/z., N- sugar chain structure, relative peak intensities etc. out.
35 kinds of O- sugar chains are identified altogether by MALDI-TOF-MS, wherein reflecting respectively in HV, HB, HC and HCC each group
Determine to 20,18,19 and 24 kind.Wherein, 9 kinds of O- sugar chain (such as m/z 572.256 (NeuAc)1(GalNAc)1, 1097.383
(Fuc)1(NeuAc)1(Gal)1(GalNAc)1Deng) identify in tetra- groups of saliva samples of HV, HB, HC and HCC, there are also 5 kinds
O- sugar chain (such as m/z 781.256 (Gal)3(GalNAc)1,938.345(Fuc)2(Gal)1(GlcNAc)1(GalNAc)1Deng) only
Being present in HV group and 9 kinds of O- sugar chains (wherein has 8 kinds of sugar chains containing Fuc, such as m/z 828.269 (Fuc)1(Gal)1
(GlcNAc)1(GalNAc)1, 995.367 (Fuc)1(Gal)1(GlcNAc)2(GalNAc)1Deng) exist only in HCC group.In addition,
Early stage during this hepatopathy occurrence and development, that is, 2 kinds of O- sugar chains are identified in HV and HB group saliva sample jointly
(m/z 600.115(Sulf-GlcNAc)1(GalNAc)1With 935.296 (Fuc)1(Gal)3(GalNAc)1), accordingly in the disease
3 kinds of O- sugar chains (m/z 1322.444 (Fuc) has been identified in the advanced stage of sick development process, that is, HC and HCC group jointly1
(NeuAc)1(Gal)1(GlcNAc)2(GalNAc)1, 1339.392 (Fuc)1(NeuAc)1(Sulf-Gal)1(Gal)1
(GlcNAc)1(GalNAc)1With 1355.454 (Fuc)1(Gal)3(GlcNAc)2(GalNAc)1).In addition, 4 kinds of O- sugar chain (such as m/
z 762.168(Gal)1(Sulf-GlcNAc)1(GalNAc)1, 1259.436 (Fuc)1(NeuAc)1(Gal)2(GlcNAc)1
(GalNAc)1Deng) be present in HV, HB and HC group saliva, and have 3 kinds of O- sugar chains (m/z 617.224 (Fuc)1(Met-Gal)1
(GalNAc)1, 1177.407 (Fuc)2(GlcNAc)2(Gal)1(GalNAc)1With 1308.444 (Fuc)1(NeuAc)1(Gal)1
(GlcNAc)2(GalNAc)1) be present in the saliva of HB, HC and HCC group.
And the characteristic of glycoprotein candy chain terminal fucose is identified according to agglutinin LTL, above-mentioned 35 kinds of sugar chains are carried out into one
The analysis of step, discovery have identified 8,8,10 and 16 kind of fucosylation O- sugar chain respectively in HV, HB, HC and HCC each group.Its
In, 2 kinds of fucosylation O- sugar chain ((m/z 751.261/773.243 (Fuc)1(Gal)2(GalNAc)1,1097.383
(Fuc)1(NeuAc)1(Gal)1(GalNAc)1) identified in tetra- groups of saliva samples of HV, HB, HC and HCC, a kind of fucose
Base O- sugar chain (m/z 935.296 (Fuc)1(Gal)3(GalNAc)1) be present in HV and HB group, 3 kinds of fucosylation O- sugar
Chain (m/z 1322.444 (Fuc)1(NeuAc)1(Gal)1(GlcNAc)2(GalNAc)1,1339.392(Fuc)1(NeuAc)1
(Sulf-Gal)1(Gal)1(GlcNAc)1(GalNAc)1,and 1355.454(Fuc)1(Gal)3(GlcNAc)2(GalNAc)1)
It is present in HC and HCC group.In addition, there is 2 kinds of fucosylation O- sugar chain (m/z 896.289 (Fuc)1(NeuGc)1(Gal)1
(GalNAc)1,and 1259.436(Fuc)1(NeuAc)1(Gal)2(GlcNAc)1(GalNAc)1) it is present in HV, HB and HC group
In, 3 kinds of fucosylation O- sugar chain (m/z 617.224 (Fuc)1(Met-Gal)1(GalNAc)1, 1177.407 (Fuc)2
(GlcNAc)2(Gal)1(GalNAc)1,and 1308.444(Fuc)1(NeuAc)1(Gal)1(GlcNAc)2(GalNAc)1) exist
In a series of hepatopathys caused by HBV (i.e. in HB, HC and HCC group).Importantly, there is 3 kinds of fucosylation O- sugar chain (m/z
938.345(Fuc)2(Gal)1(GlcNAc)1(GalNAc)1,1083.383(Fuc)1(NeuAc)1(Gal)1(GalNAc)1,and
1402.520(Fuc)1(NeuAc)1(Gal)1(GlcNAc)1(Sulf-GlcNAc)1(GalNAc)1) exist only in HV group, with
And 8 kinds of fucosylation O- sugar chain (m/z 652.216 (Fuc)1(GlcNAc)1(GalNAc)1,828.269(Fuc)1(Gal)1
(GlcNAc)1(GalNAc)1,833.314(Fuc)1(GlcNAc)1(GalNAc)2,995.367(Fuc)1(Gal)1(GlcNAc)2
(GalNAc)1,1144.295(Fuc)1(Sia)1(Sulf-Gal)1(Gal)1(GalNAc)1,1157.420(Fuc)1(Gal)2
(GlcNAc)2(GalNAc)1,1163.417(Fuc)2(GlcNAc)2(Gal)1(GalNAc)1),1193.402(Fuc)1(Gal)2
(GlcNAc)2(GalNAc)1) exist only in HCC group.
So last it follows that 8 kinds of fucosylation O- sugar chain (m/z 652.216 only identified in HCC group
(Fuc)1(GlcNAc)1(GalNAc)1, 828.269 (Fuc)1(Gal)1(GlcNAc)1(GalNAc)1, 833.314 (Fuc)1
(GlcNAc)1(GalNAc)2, 995.367 (Fuc)1(Gal)1(GlcNAc)2(GalNAc)1, 1144.295 (Fuc)1(Sia)1
(Sulf-Gal)1(Gal)1(GalNAc)1, 1157.420 (Fuc)1(Gal)2(GlcNAc)2(GalNAc)1, 1163.417 (Fuc)2
(GlcNAc)2(Gal)1(GalNAc)1) and 1193.402 (Fuc)1(Gal)2(GlcNAc)2(GalNAc)1)；For saliva sample
It is detected, only need to measure its any or any combination for whether containing this 8 kinds of fucosylation O- sugar chains, liver cancer can be made
(HCC) result of screening, early diagnosis, risk assessment, drug screening and/or curative effect evaluation.
Claims (8)
1. product of the one kind for liver cancer (HCC) screening, early diagnosis, risk assessment, drug screening and/or curative effect evaluation, needle
To saliva sample characterized by comprising
A, for obtaining the device of the expression of two kinds of specific sugar albumen sugar chain structures, described two specific sugar protein sugar links
Structure is respectively Fuc α 1-2Gal β 1-4GlcNAc and Fuc α 1-3 (Gal β 1-4) GlcNAc；
B, for differentiating the whether highly expressed mark of both 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 LTL 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), hepatitis B (HB), cirrhosis (HC), the corresponding reference value of liver cancer (HCC), determine whether high table for compareing with sample results
It reaches.
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 LTL, the Related product be kit, equipment, can operating system and/or their group
It closes, purposes is liver cancer (HCC) screening, early diagnosis, risk assessment, drug screening and/or curative effect evaluation.
5. the recognition unit of specific sugar chain building for making liver cancer (HCC) screening based on saliva sample, early diagnosis, risk are commented
Estimate, the purposes in terms of the product of drug screening and/or curative effect evaluation, it is characterised in that: the recognition unit identification is eight kinds following
Any or any combination of fucosylation O- sugar chain:
m/z 652.216(Fuc)1(GlcNAc)1(GalNAc)1,
m/z 828.269(Fuc)1(Gal)1(GlcNAc)1(GalNAc)1,
m/z 833.314(Fuc)1(GlcNAc)1(GalNAc)2,
m/z 995.367(Fuc)1(Gal)1(GlcNAc)2(GalNAc)1,
m/z 1144.295(Fuc)1(Sia)1(Sulf-Gal)1(Gal)1(GalNAc)1,
m/z 1157.420(Fuc)1(Gal)2(GlcNAc)2(GalNAc)1,
m/z 1163.417(Fuc)2(GlcNAc)2(Gal)1(GalNAc)1,
m/z 1193.402(Fuc)1(Gal)2(GlcNAc)2(GalNAc)1。
6. product of the one kind for liver cancer (HCC) 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 LTL；
B, for coupling the affinity chromatography solid phase carrier of agglutinin LTL；
C, it is used for from the glycoprotein for containing Fuc α 1-2Gal β 1-4GlcNAc and Fuc α 1-3 (Gal β 1-4) GlcNAc sugar chain structure
In isolate the reagent and device of O- sugar chain；
D, for parsing the device of the chemical constitution formula of various O- sugar chains；
E, for differentiating in the various O- sugar chains parsed whether any containing following eight kinds of fucosylations O- sugar chain or to appoint
Meaning combined mark, module or processor；
m/z 652.216(Fuc)1(GlcNAc)1(GalNAc)1,
m/z 828.269(Fuc)1(Gal)1(GlcNAc)1(GalNAc)1,
m/z 833.314(Fuc)1(GlcNAc)1(GalNAc)2,
m/z 995.367(Fuc)1(Gal)1(GlcNAc)2(GalNAc)1,
m/z 1144.295(Fuc)1(Sia)1(Sulf-Gal)1(Gal)1(GalNAc)1,
m/z 1157.420(Fuc)1(Gal)2(GlcNAc)2(GalNAc)1,
m/z 1163.417(Fuc)2(GlcNAc)2(Gal)1(GalNAc)1,
m/z 1193.402(Fuc)1(Gal)2(GlcNAc)2(GalNAc)1。
7. product according to claim 6, it is characterised in that: described for coupling the affinity chromatography solid phase of agglutinin LTL
Carrier is Fe3O4Magnetic particle, Ago-Gel, sephadex or glass microsphere.
8. product according to claim 6, it is characterised in that: described for parsing the chemical constitution formula of various O- sugar chains
The working principle of device be MALDI TOF MS method MALDI-MS, fast atom bombardment mass spectroscopy FAB-MS,
Electrospray ionization mass spectrum ES-MS, liquid chromatography, Liquid Chromatography-Mass Spectrometry or nuclear magnetic resonance nmr, by obtaining O- sugar chain
M/z information obtains chemical constitution formula.
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