CN108107221B - Purposes and kit of the specific agglutinin combination in terms of preparing the test carrier to identify ovarian epithelial carcinoma - Google Patents
Purposes and kit of the specific agglutinin combination in terms of preparing the test carrier to identify ovarian epithelial carcinoma Download PDFInfo
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Abstract
The invention proposes purposes and kit of a kind of specific agglutinin combination in terms of preparing the test carrier to identify ovarian epithelial carcinoma.For saliva sample, the specific agglutinin combination are as follows: (1) combination of PSA, SNA, MAL-II, ECA and HHL corresponds to healthy population；(2) combination of PHA-E, LTL and PNA corresponds to benign ovarian disease patient；(3) combination of RCA120, UEA-I, ConA, LCA, AAL, WFA, WGA, DBA, VVA, NPA and GNA corresponds to epithelial ovarian Disease.It is specifically bound by glycoprotein candy chain in agglutinin and saliva, the glycoprotein and sugar chain structure of differential expression in patient's saliva sample can be quickly detected, realize quick, the high-throughput detection to glycoprotein a variety of in sample and sugar chain structure.
Description
Technical field
The present invention relates to a kind of agglutinin purposes and kit for identifying ovarian epithelial carcinoma based on saliva glycoprotein.
Background technique
Oophoroma (Ovarian cancer, OC) is common one of feminine proses, in global common cancer
It is middle to occupy the 6th, the 5th is occupied in common female cancer, death toll arranges the in the major cancers for leading to global women die
Seven.In recent years, ovarian tumors rate is in rising trend in China, it is contemplated that China's ovarian tumors rate is up within 2016
6.43/10 ten thousand people；Since the early diagnosis for lacking apparent distinctive sign and screening method, oophoroma is very difficult.It is clinical
Studies have shown that 5 years survival rates of ovarian cancer patients that early stage (I/II) makes a definite diagnosis are up to 90% or more, however, about 70%-85%
Patient be in late stage (III/IV phase) making a definite diagnosis Shi Douyi, average 5 annual survival rates only have 20%-35%, cause ovary
Cancer becomes the highest gynecologic malignant tumor of case fatality rate.85%~90% histological type is ovarian epithelial carcinoma (EOC) in oophoroma.
Closely related in view of the survival rate and staging when making a definite diagnosis of patient, early detection becomes the key for improving oophoroma life cycle.
In human body, glycan molecule mainly exists in the form of monosaccharide or oligosaccharide, and oligonucleotide chain is both saccharide complex (sugared egg
White, glycolipid etc.) important component, while also have important biological function.As one kind, modification of protein glycosylation
It is one of most important posttranslational modification.Studies have shown that 70% human protein contains one or more sugar chains, these sugar chains
It is made of several monosaccharide of mannose, galactolipin and N-acetyl-glucosamine etc. ten, turns through different glycosyl transferases, glycosidase and sulfo group
Moving the catalysis of enzyme may make up huge sugar chain spectrum with different connection types.
During malignant change of cell and tumorigenesis, it is a universal phenomenon that glycosylation is abnormal, and this is changed
Become often earlier than the variation of P-glycoprotein expression amount.It is glycoprotein in view of the ovary carcinoma marker found at present, therefore to its sugar
Base mode and sugar chain structure do fine parsing, therefrom find or find new specific tumour marker, become in recent years
The new hot spot of oophoroma biomarker research.Compared to protein and lipid part, the change of sugar chain is often raw to body
Reason, the response of pathological state variation are sensitiveer, therefore study saccharide complex glycosylation pattern and Tumor marker can be improved
The specificity and sensitivity of detection, new method is provided for cancer early detection, diagnosis, prognosis and Monitoring of drug resistance.
Serum N-sugar chain structure of preliminary studies have shown that specificity, can effective identification of ovarian cancer and normal healthy controls,
It is possible that the detection for oophoroma provides the biomarker with more high accuracy, sensitivity and specificity.Leaving-one method intersects
Verifying, using specific N- sugar chain as marker, to the identification susceptibility of oophoroma up to 86%, specificity be 95.8%.
Saliva is the colorless and odorless formed by convergences such as salivary gland, salivary glands, weakly acidic Combination hypotonic fluid, is people
Important one of body fluid in vivo, though constituent is extremely complex, and it is relatively stable.There is data to show albumen in human saliva
Total amount is up to 0.5~3gL-1, therefrom identifies 1166 kinds of representational albumen, wherein 192 kinds of sialoproteins exist simultaneously
In plasma protein.The change that saliva glycoprotein group is constituted is able to reflect the variation of human body physiological state, by comparing sialic saccharide
The constituent of protein groups, can for know clearly body physiological status and body various change process provide one objectively see
Examine index.As previously mentioned, the Aberrant glycosylation of protein is a tumorigenic important symbol.Egg is glycosylated in sialoprotein
White ratio is higher, and mostly N- sugar chain glycoprotein.Therefore, human saliva becomes the ideal source of glycoprotein.In addition sialic saccharide
Albumen and seroglycoid have the similitude of height, and the composition and abundance of saliva glycoprotein can be very good in reflection blood
The variation of various glycoprotein levels.In view of saliva and human health and morbid state Close relation, as important human body fluid
One of personal information library, saliva sample are used for the early diagnosis of clinical disease, cannot be only used for effectively controlling for oral cavity partial disease
Treatment and disease prevention, it may also be used for the assessment of diagnosis, the course of disease monitoring and health of whole body system disease.
Agglutinin is a kind of carbohydrate-binding protein, can recognize different sugar chain structures and with multivalent forms and these sugar chains spy
The opposite sex combines.Sugared omics technology based on agglutinin is a kind of emerging, integrated technology in post-genomic science epoch, most generation
Table is lectin chip.Since the new century, agglutinin sugar omics technology has evolved into mutual with the methods of mass spectrum
Supplement, a kind of quick, sensitive, high-throughput glycobiology analysis tool, application field is also organized from sugar and learns basic research gradually
It is extended to the numerous areas such as biochemistry, medical test, immunology, molecular biology, bioinformatics.
Summary of the invention
The present invention relates to it is a kind of it is non-invasive, easy quickly, sample is micro, high-throughput passes through sugar in detection subjects saliva
Albumen and sugar chain variation carry out the agglutinin core of antidiastole ovarian epithelial carcinoma (epithelial ovarian cancer, EOC)
Chip technology and corresponding detection kit.
With this, to solve, the specificity present in ovarian epithelial carcinoma differential diagnostic method at present is not high, there are blood for sample
The harm of source property disease is unsuitable for the problems such as doing large-scale crowd generaI investigation.
Ovarian epithelial carcinoma saliva glycoprotein antidiastole chip provided by the present invention by a group agglutinin (ConA,
WGA, LCA, AAL, WFA, RCA120, UEA-I, VVA, SNA, PSA, ECA, MAL- II, HHL, PHA-E, LTL, PNA etc.) institute's structure
At detection kit includes above-mentioned agglutinin group chip and matched detection reagent.Testing result can be marked by apparatus measures
Object relative intensity of fluorescence realizes the quantitative analysis to detectable substance, can also estimate and do qualitative analysis；
The invention also includes the method for discrimination of testing result and differentiate mark.
When the agglutinin PSA sugar chain structure (α-D-Man, Fuc α -1,6GlcNAc, α-D-Glc) identified and SNA are known
Other sugar chain structure (Sia2-6Gal β 1-4Glc (NAc)) is significantly raised, the sugar chain structure that agglutinin MAL-II is identified
Sugar chain structure (the Gal β -1,4GlcNAc (type II), Gal β 1- that (Sia2-3Gal β 1-4Glc (NAc)), ECA are identified
3GlcNAc (type I)) and HHL identified sugar chain structure (High-Mannose, Man α 1-3Man, Man α 1-6Man,
Man5-GlcNAc2-Asn it) significantly lowers preliminary judgement sample and comes from healthy population.
As sugar chain structure (the Bisecting GlcNAc and biantennary N- that agglutinin PHA-E is identified
Glycans) significant up-regulation, sugar chain structure that agglutinin LTL is identified (Fucose α -1,3GlcNAc (core fucose),
Sia-Lex it) is significantly lowered with the sugar chain structure (Gal β 1-3GalNAc α-Ser/Thr (T)) that is identified of PNA, it can preliminary judgement sample
This comes from benign ovarian disease patient.
As sugar chain structure (β-gal, Gal β 1,4GlaNAc (type II) the Gal β 1- that agglutinin RCA120 is identified
3GlcNAc (type I) containing GlcNAc and MurNAc), sugar chain structure (the Fucose α 1- that is identified of UEA-I
2Gal β 1-4Glc (NAc)), ConA identified sugar chain structure (High Mannose, Mann α 1-6 (Mann α 1-3), Man,
Terminal GlcNac), the LCA sugar chain structure (α-D-Man, Fuc α -1,6GlcNAc, α-D-Glc), the AAL that are identified known
Other sugar chain structure (Fuc α -1,6GlcNAc (core fucose), Fuc α -1,3 (Gal β 1-4) GlcNac), WFA are identified
Sugar chain structure (the Multivalent that sugar chain structure (terminating in GalNAc α/β 1-3/6Gal) and WGA are identified
Sia and (GlcNAc) n) significantly up-regulation, sugar chain structure (α GalNAc, Tn antigen, the GalNAc α 1-3 that DBA is identified
((Fuc α 1-2)) Gal (blood group A antigen)), VVA identified sugar chain structure (terminal GalNAc,
GalNAc α-Ser/Thr (Tn), GalNAc α 1-3Gal), sugar chain structure (High-Mannose, Man the α 1- that is identified of NPA
6Man) sugar chain structure (High-Mannose, Man α 1-3Man) identified with GNA is significantly lowered, can preliminary judgement sample
From epithelial ovarian Disease.
The invention also includes the application of above-mentioned agglutinin group chip inspecting reagent unit, key step includes that saliva sample is adopted
Collection；Sialoprotein processing and fluorescent marker；Lectin chip detection and its data analysis.
The kit includes: protease inhibitors, 3 fluorescent dye of DMSO, Cy, Cy5 fluorescent dye, Sephadexg G-
25 columns, bovine serum albumin(BSA) (BSA), oxysilane reagent (GPTS), Bradford reagent, pvdf membrane, Tween-20, sheet glass
Base and more than specific agglutinin combination.
Five kinds of diagnostic models of subdivision can be quantified by further going back Primary Construction.
The present invention relatively has the advantage that compared with the existing detection method
The present invention is to be fixed on one group of specific agglutinin (19 kinds) on the substrate of surface modification as probe, by solidifying
Collection element is specifically bound with glycoprotein candy chain in saliva, can quickly detect the sugared egg of differential expression in patient's saliva sample
White and sugar chain structure realizes quick, the high-throughput detection to glycoprotein a variety of in sample and sugar chain structure.
For the present invention using saliva as object to be detected, acquisition is simple and easy to do, noninvasive, while can effectively avoid with blood mark
This is detectable substance and bring blood-borne pathogens (HIV, HBV, HCV, TP etc.) to the security threat in laboratory and reviewer.
Detailed description of the invention
Fig. 1 is lectin chip sample application array figure and the fluorescence detection figure applied to glycoprotein and sugar chain, and Fig. 1-A is agglutination
Plain chip sample application array figure, Fig. 1-B are respectively healthy women volunteer (HV), benign ovarian disease patient (OB), oophoroma I phase
Patient (OC-I), oophoroma II phase patient (OC-II) and oophoroma III phase patient's (OC-III) saliva glycoprotein and sugar chain structure
Fluoroscopic examination result figure.
Fig. 2-Fig. 7 is agglutinin scatter plot, in which:
Fig. 2 shows there is significant difference between HV and the reactivity and OB, OC of agglutinin PSA, SNA.
Fig. 3 shows the significant downward of reactivity of HV and agglutinin MAL- II, ECA, HHL.
Fig. 4 shows that OB and the reactivity of agglutinin PHA-E are significantly higher than HV, OC.
Fig. 5 shows the significant downward of the reactivity of OB and agglutinin LTL, PNA.
Fig. 6 shows the significant up-regulation of reactivity of OC and agglutinin RCA120, UEA- I, ConA, LCA, AAL, WFA and WGA.
Fig. 7 shows the significant downward of the reactivity of OC and agglutinin DBA, VVA, NPA, GNA.
Note: in the above scatter plot, P < 0.05 *；**P<0.01；* * P < 0.001.Rs:Spearman related coefficient.
Fig. 8 is the clustering figure of lectin chip result.Horizontal axis is an example sample, and the longitudinal axis is 37 kinds of agglutinins.Each
It is horizontal to the combination of sample that the intensity of square represents agglutinin.Right side grey scale indicates bond strength from high to low from top to bottom
(former clustering coloured picture is corresponding are as follows: red: height combines；It is green: low combination；It is black: moderate strength combination).
Fig. 9 is principal component analysis figure；Illustrated in figure the gray scale block of upper, middle and lower three (former coloured picture correspond to it is yellow, green,
It is red), as can be seen from the figure 63 healthy women volunteer (HV) saliva samples are all distributed in downside block, and 51 ovaries are good
Property disease (OB) patient's saliva sample almost all be distributed in middle part block, and ovarian epithelial carcinoma (OC) that 78 have been made a definite diagnosis is suffered from
Person's saliva sample is largely distributed in upside block, and oophoroma I phase patient (OC-I) saliva sample in part is located at middle part block, shows
There are cross reactions between OC-I and OB.But downside block absolutely not intersects with upside block, shows agglutinin core of the invention
Piece detection technique has very high specificity to the detection of HV and OC.
Specific embodiment
Below by the specific embodiment provided, the present invention will be further described, but not as a limitation of the invention.
Present invention screening from 37 kinds of agglutinins (as shown in table 1) is used for antidiastole ovarian epithelial carcinoma female patient
Agglutinin probe.
Table 1
Kit material used in the present invention includes: protease inhibitors, DMSO, Cy3, Cy5 fluorescent dye,
Sephadexg G-25 column, bovine serum albumin(BSA) (BSA), glass chip, oxysilane reagent (GPTS), Bradford reagent,
Pvdf membrane, Tween-20.
1. the collection of saliva sample
Acquisition saliva sample 192 altogether, healthy women volunteer (HV) saliva 63, ovary benign disease (OB) patient's saliva
Liquid 51, the ovarian epithelial carcinoma made a definite diagnosis (OC) patient saliva 78, wherein I phase patient 24, II phase patient 22, III
Phase patient 32.As shown in table 2.
2 sample information of table
2. sialoprotein processing and fluorescent marker
Saliva collection process is as follows, after subject's fasting 2 hours, is gargled with physiological saline, and keeping the tip of the tongue to withstand maxilla makes
Saliva naturally secret collects the saliva of nature outflow with sterile tube, protease inhibitors (1 μ L/mL) is added in the saliva of collection,
To prevent protein degradation, 4 DEG C, 12,000rpm × 10min be collected after centrifugation supernatant, 0.22 μm of filtering with microporous membrane degerming is collected
Saliva sample BCA method that treated measures protein concentration egg (100 μ g of Bai Hanliang), with Cy3 fluorochrome label, Sephadex
G-25 chromatographic purifying, the sialoprotein after the fluorescent marker being collected into are used for lectin chip test experience.
3. prepared by lectin chip
37 kinds of agglutinins and 2 BSA (negative control) and 1 BSA (positive control) for indicating Cy3 are dissolved in respectively
It is configured to the sampling liquid of 1mg/mL in buffer, uses rich brilliant 48 point sample instrument of core SmartArrayer (setting parameter: matrix difficult to understand
Number, point sample humidity, number of repetition, pre- point sample position and number, depth selection time etc.), by arrangement side shown in Fig. 1-A
Formula, by agglutinin and comparison liquid point in epoxidation chip base, put the chip made be placed in wet box be kept in dark place overnight, in 37 DEG C
Dry 2h is placed in chip cartridges in vacuum oven saves in 4 DEG C.
4. lectin chip experiment and its data analysis
Take the lectin chip a little made to be placed in 37 DEG C of vacuum ovens the 30min that rises again, respectively with 1 × PBST and 1 ×
PBS, each low speed shake washing and wash 1 time, and 5min/ times, centrifuge dripping chip is put into incubation box, and 650 μ L confining liquids are added, screw
It is incubated for box, is placed in chip constant temperature rotation hybridization instrument, room temperature low speed rotation closes 1h.After completing closing, chip is taken out, is ibid used
1 × PBST and 1 × PBS is respectively washed 2 times, 5min/ times.Centrifuge dripping be placed on be incubated for box in be added sample liquid (incubation buffer+
Labelled protein), it screws chip incubation box and is placed in chip constant temperature rotation hybridization instrument, room temperature low speed rotation is incubated for 3h.Completion is incubated
After educating, chip is taken out, ibid respectively washs 2 times with 1 × PBST and 1 × PBS, 5min/ times, centrifuge dripping, with Genepix 4000B
Chip scanner is scanned reading chip data.And the lectin chip normalized Analysis designed using laboratory self-programming
Software carries out automatic normalization analysis.
Fig. 8 and Fig. 9 is to show shape using same data source (192 saliva samples) with the difference that different disposal method obtains
Formula, it is shown that the glycoprotein candy chain of different sample groups has differences, and shows that glycoprotein candy chain can be used for distinguishing different sample groups.
5. the building of diagnostic model
Further, according to the lectin chip of 192 saliva samples as a result, utilizing stepwise logistic regression (forward
Stepwise logistic regression analysis) ovary benign disease (OB), oophoroma-I phase (OC- are constructed respectively
I), five kinds of oophoroma-II phase (OC-II), oophoroma-III phase (OC-III) and oophoroma (OC) diagnostic models.It is included in model
Agglutinin derive from the agglutinin that there were significant differences between each group.
1.Model OB model formation
2.Model OC-I model formation
3.Model OC-II model formation
4.Model OC-III model formation
5.Model OC model formation
Above-mentioned five kinds of diagnostic models why are further established, are desirable to quantify to segment OB, OC-I, OC-II, OC-
III and OC.The data of script have been carried out quantitative analysis by this five kinds of diagnostic models, obtain optimum sensitivity according to ROG curve
Cutoff value when specific.Judged according to cutoff value:
If cutoff value is 0.5, greater than cutoff value be for cancer, then sample substitute into model calculate resulting value closer to
A possibility that 0 health, is bigger, closer to 1, then bigger a possibility that cancer.And model itself is a sigmoid function,
90% data are extremely close to 0 or 1, close to the data of threshold value and few.The model gives specific threshold value, very convenient
It goes to be judged.
Above content is to the further description of the invention done in conjunction with specific embodiment, and it cannot be said that this hair
Bright specific implementation is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, it is not taking off
Under the premise of from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to protection of the invention
Range.
Claims (2)
1. purposes of the specific agglutinin combination in terms of preparing the test carrier to identify ovarian epithelial carcinoma, feature exist
In: it is directed to saliva sample, the specific agglutinin combination is one of following three kinds of combinations:
(1) combination of PSA, SNA, MAL-II, ECA and HHL corresponds to healthy population；
(2) combination of PHA-E, LTL and PNA corresponds to benign ovarian disease patient；
(3) combination of RCA120, UEA-I, ConA, LCA, AAL, WFA, WGA, DBA, VVA, NPA and GNA corresponds to epithelial
Disease of ovary patient；
Either to any recombinant of above (1) to (3)；
When the sugar chain structure that agglutinin PSA and SNA are identified significantly raises, the sugar that agglutinin MAL-II, ECA and HHL are identified
Chain structure is significantly lowered, then preliminary judgement sample comes from healthy population；
When the sugar chain structure that agglutinin PHA-E is identified significantly raises, the sugar chain structure that agglutinin LTL and PNA are identified is significant
It lowers, then preliminary judgement sample comes from benign ovarian disease patient；
When the sugar chain structure that agglutinin RCA120, UEA-I, ConA, LCA, AAL, WFA and WGA are identified significantly raises, DBA,
The sugar chain structure that VVA, NPA and GNA are identified significantly is lowered, then preliminary judgement sample comes from epithelial ovarian Disease.
2. it is a kind of based on detection saliva glycoprotein identify ovarian epithelial carcinoma kit, comprising: protease inhibitors, DMSO,
3 fluorescent dye of Cy, Cy5 fluorescent dye, Sephadexg G-25 column, bovine serum albumin(BSA) (BSA), oxysilane reagent
(GPTS), Bradford reagent, pvdf membrane, Tween-20, glass chip and agglutinin combination, it is characterised in that: described solidifying
Collection element combination is specific agglutinin combination described in claim 1.
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| CN111366632A (en) * | 2020-01-19 | 2020-07-03 | 深圳格道糖生物技术有限公司 | Lung small cell cancer screening and evaluating product based on saliva specific glycoprotein sugar chain structure and application |
| CN113702637B (en) * | 2021-08-09 | 2023-04-07 | 西北大学 | Lectin test carrier, kit and prediction model for predicting neoadjuvant chemotherapy curative effect of breast cancer |
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| 健康人和肝癌患者唾液糖蛋白糖链谱的研究;朱珉之;《中国优秀硕士学位论文全文数据库 医药卫生专辑》;20110815(第8期);第E072-186页 * |
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