CN105675774B - The preparation method of saliva excretion body and its application in molecule diagnosis - Google Patents
The preparation method of saliva excretion body and its application in molecule diagnosis Download PDFInfo
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- CN105675774B CN105675774B CN201610035433.XA CN201610035433A CN105675774B CN 105675774 B CN105675774 B CN 105675774B CN 201610035433 A CN201610035433 A CN 201610035433A CN 105675774 B CN105675774 B CN 105675774B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
- G01N2030/146—Preparation by elimination of some components using membranes
Abstract
Application the invention discloses a kind of preparation method of saliva excretion body and its in molecule diagnosis；Include the following steps：High-abundance proteins are removed from saliva sample, filter off mucin, and separation and Extraction excretion body characterizes gained excretion body, extracts excretion body protein, identifies albumen, and the discovery of application and lung cancer biomarker.The saliva excretion Antibody Production Techniques not standardized still at present, there are high-abundance proteins interference and background impurities interference for existing technology of preparing, lead to the deficiencies of excretion body rate of recovery is low, identification albumen is few, it is difficult to reflect true excretion body protein group information.The present invention is using the saliva of be grown up healthy volunteer and patients with lung cancer as sample, using affinity chromatography chromatography and UF membrane combination technology, after the high-abundance proteins and mucin for eliminating saliva, the excretion body in saliva is extracted by centrifugation, and its protein groups is analyzed, identify more excretion body proteins.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of preparation method of saliva excretion body and its be diagnosed in molecule
In application.
Background technology
Excretion body (Extracellular Vesicles), which refers to the one kind for being secreted into extracellular microenvironment by cell, to be had
The vesica of membrane structure is small containing after birth ingredient that cell releases during fissioning to external budding and cell membrane
Grain, can carry the substances such as a variety of specific proteins, microRNA, lipid.The size of excretion body between 0.05~1.00 μm it
Between, cancer mechanism study in recent years confirms that excretion body is related to intercellular substance, energy, information transmission, especially cancer
Influencing each other between cell or between cancer cell and normal cell.Excretion body is widely present in tissue and body fluid, in disease
Clinical diagnosis, prognosis supposition, the health control etc. of disease have important directive function.Especially tumour cell source is outer
Secrete body, as the medium that tumour cell is exchanged with external environmental signals, by albumen, mRNA specific to tumour cell and
The bioactive substances such as miRNA pass to the environment and cell of surrounding, and microenvironment is transformed while invading other cells, makes it
Change { M.Baj-Krzyworzeka et al., Tumour-derived to the direction for being more advantageous to tumor development
microvesicles carry several surface determinants and mRNA of tumour cells and
transfer some of these determinants to monocytes.Cancer Immunology
Immunotherapy 55,808-818 (2006)；K.Al-Nedawi, B.Meehan, J.Rak, Microvesicles
Messengers and mediators of tumor progression.Cell Cycle 8,2014-2018 (2009)；
N.S.Barteneva, N.Maltsev, I.A.Vorobjev, Microvesicles and intercellular
communication in the context of parasitism.Frontiers in Cellular and
Infection Microbiology 3, (2013) }.Therefore cancer mechanism study, disease are had become for the research of excretion body
Important component { E.Colombo, B.Borgiani, C.Verderio, R.Furlan, the Microvesicles of disease diagnosis：
Novel biomarkers for neurological disorders.Frontiers in Physiology 3,63
(2012)；B.Frey, U.S.Gaipl, The immune functions ofphosphatidylserine in
Membranes of dying cells and microvesicles.Seminars in Immunopathology 33,
497-516(2011)；J.Conde-Vancells, E.Gonzalez, S.C.Lu, J.M.Mato, J.M.Falcon-Perez,
Overview of extracellular microvesicles in drug metabolism.Expert Opinion on
Drug Metabolism &Toxicology 6,543-554 (2010) }.
It is scarcely out of swaddling-clothes to the research of excretion body, due to being not fully understood to its constituent, it is given birth at present
Object functional study is in urgent need of strengthening.It is bright to be used for clinical diagnosis advantage as a kind of body fluid containing a large amount of biological informations for saliva
It is aobvious, because having the characteristics that be non-intruding, safe and simple, have broad application prospects in the molecular diagnosis field of disease.Together
When, be richly stored in saliva excretion body, and research is carried out to it and is expected to find the biological marker of early diagnosis for various diseases
Object.
Currently, that there are extracting methods is cumbersome, extraction efficiency is relatively low, background interference is serious etc. for the extracting method of saliva excretion body
It is insufficient.Such as the centrifugal force that ultracentrifugation method needs is 200000g or more, and need a large amount of biological sample ability of 30mL
Obtain albumen { M.Gonzalez-Begne et al., the Proteomic Analysis of Human Parotid of 10 μ g
Gland Exosomes by Multidimensional Protein Identification Technology(MudPIT)
.Journal of Proteome Research 8,1304-1314 (2009) }.It is although improved using the method for precipitation
Efficiency is taken, but the interference problem of background impurities can not be solved.
Invention content
It is an object of the invention to overcome above-mentioned the deficiencies in the prior art, a kind of preparation of saliva excretion body is provided
Method and its application in molecule diagnosis.Specifically, the present invention uses affinity chromatography chromatographic technique skill integrated with filter membrane
Art eliminates high-abundance proteins and mucin in saliva, then is prepared for saliva excretion body with the method for refrigerated centrifuge, and to it
Application in molecule diagnosis is studied, and the life of lung cancer is such as found according to the protein science difference of patients with lung cancer and Healthy People
Object marker.
The purpose of the present invention is what is be achieved through the following technical solutions：
The present invention relates to a kind of preparation methods of saliva excretion body, and described method includes following steps：
S1, the high-abundance proteins in desalivation are gone by affinity chromatography chromatography, mucin is removed in conjunction with filter membrane；
S2, refrigerated centrifuge is carried out to step S1 treated salivas, extracts saliva excretion body and saliva excretion body protein.
In the present invention, the repeatable operation of above-mentioned refrigerated centrifuge 3~5 times.The high-abundance proteins include ptyalin.
Preferably, the affinity chromatography chromatography is using starch as stationary phase, using phosphate buffer as mobile phase.Elution speed
Degree is 700~900 μ L/min.
Preferably, the filter membrane is 1.0~5.0 μm of polyvinylidene fluoride films.After the filter membrane is connected to affinity chromatography chromatographic column
To complete the integration of chromatography and membrane separation technique.In the present invention, using the filter membrane in the aperture, mucin can be effectively removed.
Preferably, the condition of the refrigerated centrifuge be 4 DEG C, 10000~20000g of centrifugal force, 30~90 minutes.More preferably
It is 60~80 minutes.
Preferably, extraction saliva excretion body protein includes cracking the albumen discharged in saliva excretion body, to lysate (supernatant
Liquid) carry out the step of precipitation obtains saliva excretion body protein.
Preferably, described be cracked into carries out the saliva excretion body with Triton X-100 (Triton-100)
It cracks 30~90 minutes on ice.More preferably every 150 μ L saliva excretion bodies addition, 1 μ L Triton-100 progress cracking 30 on ice~
60 minutes.When cracking, it is also added into 2.5 μ L protease inhibitors (every 150 μ L saliva excretions body).After cracking, it is added 10 times and splits
Solve the ice ethyl alcohol overnight precipitation of liquid product.
Preferably, further include that excretion body protein type mirror is carried out using Liquid chromatography coupled to mass spectrometry after step S2
Fixed step.
The invention further relates to a kind of purposes of the saliva excretion body of above method preparation in preparing biomarker.
Preferably, the biomarker is the biomarker for studying cancer purposes.The biomarker is
For studying cancer, while being not intended to the biomarker of diagnostic uses.
Preferably, the biomarker be lung cancer, carcinoma of mouth, gastric cancer or liver cancer biomarker.
Preferably, it extracts to obtain saliva excretion body protein by the preparation method, contrast difference obtains corresponding cancer
Saliva excretion body biomarker.
The present invention is directed saliva sample, since wherein 50~60% albumen is ptyalin and mucin, sternly
Ghost image rings the separation and Extraction of saliva excretion body.Specifically to remove ptyalin, the present invention chooses starch and is made of matrix
Affinity chromatography chromatographic column removes background interference by affinity interaction.Meanwhile filter membrane, structure are connected after affinity chromatography chromatographic column
Integralization column chromatography and membrane filtration system remove a large amount of mucins contained in saliva, reduce reunion and the saliva of GAP-associated protein GAP
The adherency of liquid excretion body, to greatly improve the extraction efficiency of excretion body.
Compared with prior art, the present invention has the advantages that：
1, the present invention provides a kind of preparation methods of saliva excretion body and its albumen easy to operate, the rate of recovery is high.
2, using the integrated saliva treatment technology of the present invention, the excretion body in extraction saliva can be efficiently separated, is avoided
The influence of high-abundance proteins and mucin is conducive to compare normal person and the excretion body in lung cancer patient saliva, in turn
The relevant biomarker of lung cancer is found from the excretion body protein of saliva.
3, the present invention, which takes, is combined by the affinity chromatography chromatographic technique of matrix of starch with filter centrifugation method, establishes
Affinity chromatography chromatography and film combination system eliminate the interference of peak background proteins while improving extraction efficiency, identification
A greater variety of excretion body proteins have been arrived, has helped to carry out excretion body protein group and further investigate.
Description of the drawings
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon：
Fig. 1 is the flow diagram of the method for the present invention；
Fig. 2 is to be utilized respectively excretion body protein after conventional centrifugal method and the method for the invention progress excretion body separation
Compare SDS-PAGE；Wherein, a is albumen marker, and b is 1.0 μ g sialoproteins, and c is the saliva that 1.0 μ g are obtained with the method for the present invention
Liquid excretion body protein, d are the saliva excretion body protein that 1.0 μ g conventional centrifugal methods obtain；
Fig. 3 is to be utilized respectively excretion body protein after conventional centrifugal method and the method for the invention progress excretion body separation
Particle size distribution figure；Wherein, A is saliva excretion body prepared by the method for the present invention, and B is saliva excretion body prepared by centrifugal method；
Fig. 4 is separation comparison diagrams of the HPLC to saliva excretion body protein peptide fragment, wherein A is saliva prepared by centrifugal method
Excretion body protein peptide fragment, B are saliva excretion body protein peptide fragments prepared by the method for the present invention.
Specific implementation mode
With reference to specific embodiment, the present invention is described in detail.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection domain.
Embodiment
1, saliva excretion body is extracted
The saliva sample of the preparation method healthy volunteer and patients with lung cancer as shown in Figure 1, acquisition is grown up of saliva excretion body,
The background interferences albumen such as albumen and the mucin of high abundance, specific side are removed by affinity chromatography chromatography and UF membrane combination technology
Method is that the stationary phase of affinity chromatography chromatographic column is prepared with 1: 1 starch and protein ratio, and the pvdf membrane of rear 5 μm of connection is to complete color
The integration of spectrum and membrane separation technique.The activation of the balance and film of chromatographic column is carried out with mobile phase PBS first.Afterwards in chromatography column feed materials
The saliva sample into corresponding proportion is held, and with the speed of 800 μ L/min, the mobile phase PBS that total volume is 2mL elutes saliva sample
This.The sample of elution is collected at 4 DEG C, 20000g is centrifuged 60 minutes, abandons supernatant, retains excretion body.It is slow with phosphate buffer
Excretion body is resuspended, at 4 DEG C, 20000g is centrifuged 60 minutes, is abandoned supernatant and is retained excretion body, repeats the above steps 2-3 times.Take 10 μ L salivas
Liquid excretion liquid suspension, Nanosight measure saliva excretion body granularity.The results are shown in Figure 2；The method of the present invention has obtained size
It is distributed broader saliva excretion body.
2, saliva excretion body protein is extracted
100 μ L phosphate buffers are added in the excretion body for taking 300 μ L salivas to extract.It is (poly- to add 2 μ L Triton-100
Ethylene glycol octyl phenyl ether), 5 μ L protease inhibitors cocktail crack 30 minutes on ice.At 4 DEG C, 20000g centrifuges 60 points
Clock, supernatant are excretion body protein lysate.The ice ethyl alcohol overnight precipitation for adding 10 times of volumes obtains saliva excretion body protein.
1.0 μ g saliva excretion body proteins are taken to carry out SDS-PAGE characterizations.The results are shown in Figure 3, and the composition of saliva excretion body and saliva is not
Together, and the method for the present invention obtains more saliva excretion body proteins.
3, excretion body protein is identified
It takes 10 μ g saliva excretion body proteins to carry out enzymolysis in solution, and uses q-TOF-MS/MS, to having obtained saliva excretion
Body peptide fragment is identified that the results are shown in Figure 4 for peptides separation, and the method for the present invention has obtained more peptide fragment components.
The comparison of 1 the method for the present invention of table and centrifugal method extraction excretion body protein
Table 2 is 63 differential proteins after being compared with patients with lung cancer saliva excretion body protein
As shown in Table 1, method of the invention compared to centrifuge method is more stable credible and obtained more excretion body eggs
In vain, illustrate that this method contributes to the extraction of saliva excretion body and excretion body protein.
As shown in Table 2, pass through the variance analysis to Healthy People and the saliva excretion body protein group of patients with lung cancer, it was found that 63
Species diversity albumen, wherein there have 12 kinds of albumen (Green Marker) to have document report to be related to lung cancer.Illustrate that this method helps to send out
Open up the lung cancer saliva molecular diagnostic techniques based on excretion body.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring the substantive content of the present invention.
Claims (3)
1. a kind of preparation method of saliva excretion body, which is characterized in that described method includes following steps：
S1, the high-abundance proteins in desalivation are gone by affinity chromatography chromatography, mucin is removed in conjunction with filter membrane；
The affinity chromatography chromatography is using starch as stationary phase, and using phosphate buffer as mobile phase, the filter membrane is connected to parent
After thin layer chromatography column；
S2, refrigerated centrifuge is carried out to step S1 treated salivas, extracts saliva excretion body and saliva excretion body protein；
The step S1 is specially：With 1:1 starch and protein ratio prepare the stationary phase of affinity chromatography chromatographic column, rear to connect 5 μ
The pvdf membrane of m is to complete the integration of chromatography and membrane separation technique；The balance and film of chromatographic column are carried out with mobile phase PBS first
Activation；Afterwards at chromatography column feed materials end into saliva sample, and with the speed of 800 μ L/min, the mobile phase PBS that total volume is 2mL is eluted
Saliva sample；
In the step S2, extraction saliva excretion body is specially：The saliva sample of collection step S1 elution at 4 DEG C, 20000g from
The heart 60 minutes abandons supernatant, retains excretion body；Excretion body is slowly resuspended with phosphate buffer, at 4 DEG C, 20000g centrifuges 60 points
Clock abandons supernatant and retains excretion body；The step of extraction saliva excretion body 2-3 times in repeating said steps S2；
In the step S2, extraction saliva excretion body protein is specially：100 μ L phosphorus are added in the excretion body for taking 300 μ L salivas to extract
Phthalate buffer；Add 2 μ L Triton X-100s, 5 μ L protease inhibitors cocktail are cracked 30 minutes on ice；
At 4 DEG C, 20000g is centrifuged 60 minutes, and supernatant is excretion body protein lysate；The ice ethyl alcohol for adding 10 times of volumes sinks overnight
Shallow lake obtains saliva excretion body protein.
2. the preparation method of saliva excretion body according to claim 1, which is characterized in that the filter membrane is 1.0~5.0 μm
Polyvinylidene fluoride film.
3. the preparation method of saliva excretion body according to claim 1, which is characterized in that further include using after step S2
Liquid chromatography coupled to mass spectrometry carries out the step of excretion body protein Identification of Species.
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Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108872578B (en) * | 2017-05-11 | 2021-07-02 | 首都医科大学附属北京口腔医院 | Novel method for diagnosing and monitoring oral cancer |
| CN107523536A (en) * | 2017-10-20 | 2017-12-29 | 上海市同济医院 | A kind of extracting method of tissue-derived excretion body and application |
| CN110308280B (en) * | 2019-05-30 | 2022-07-26 | 深圳大学 | Saliva exosome protein biomarker |
| CN110499287B (en) * | 2019-08-30 | 2021-07-23 | 博雅干细胞科技有限公司 | Method for simply preparing placenta mesenchymal stem cell exosome |
| CN110747158A (en) * | 2019-11-14 | 2020-02-04 | 赵凯 | Cell supernatant exosome extraction process based on precipitation reagent method |
| CN113801936B (en) * | 2020-03-30 | 2022-04-19 | 中国医学科学院肿瘤医院 | Kit, device and method for lung cancer diagnosis |
| CN111659158B (en) * | 2020-06-20 | 2022-02-08 | 大理大学 | Molecular marker method for extracting exosome peak position by exclusion chromatography |
| CN113774008A (en) * | 2021-08-14 | 2021-12-10 | 光武惠文生物科技(北京)有限公司 | Method for extracting exosome and application thereof |
| CN113533589B (en) * | 2021-09-16 | 2021-12-17 | 天九再生医学(天津)科技有限公司 | Method for analyzing exosomal charge heterogeneity |
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