CN105675774A - Preparation method of saliva extracellular vesicles and application of saliva extracellular vesicles to molecular diagnosis - Google Patents
Preparation method of saliva extracellular vesicles and application of saliva extracellular vesicles to molecular diagnosis Download PDFInfo
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- CN105675774A CN105675774A CN201610035433.XA CN201610035433A CN105675774A CN 105675774 A CN105675774 A CN 105675774A CN 201610035433 A CN201610035433 A CN 201610035433A CN 105675774 A CN105675774 A CN 105675774A
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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
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Abstract
The invention discloses a preparation method of saliva extracellular vesicles and application of the saliva extracellular vesicles to molecular diagnosis.The preparation method includes the steps of removing high-abundance proteins from saliva samples, filtering out mucoproteins, separating and extracting the extracellular vesicles, characterizing the obtained extracellular vesicles, extracting extracellular vesicle proteins, identifying the extracellular vesicle proteins and applying the extracellular vesicle proteins to lung cancer biomarker discovery.Currently, a normalized saliva extracellular vesicle preparation technology is absent, and high-abundance protein interference and background impurity interference, existing in existing preparation technologies, result in shortcomings of low extracellular vesicle recovery rate, fewer identified proteins and the like, so that true extracellular vesicle proteomics information can be reflected difficultly.The preparation method of the saliva extracellular vesicles and application of the saliva extracellular vesicles to molecular diagnosis have the advantages that saliva of healthy adult volunteers and lung cancer patients is taken as the samples, and an affinity chromatography and membrane separation combined technology is adopted, so that after the high-abundance proteins and the mucoproteins of the saliva are removed, the extracellular vesicles in the saliva are extracted centrifugally, proteomes of the extracellular vesicles are analyzed and more extracellular vesicle proteins are identified.
Description
Technical field
The invention belongs to biological technical field, outside being specifically related to a kind of saliva, secrete the preparation method of body and the application in molecular diagnosis thereof.
Background technology
Secreting body (ExtracellularVesicles) outward refers to by a kind of vesica with membrane structure of emiocytosis to extracellular microenvironment, it is the molecule containing born of the same parents' film component that cell discharges in the process to external budding and cytolemma fission, the materials such as multiple specific protein, microRNA, lipid can be carried. the size secreting body outward is between 0.05～1.00 μm, and cancer study mechanism in recent years confirms that outer to secrete the material between body to cell, energy, information transmission relevant, particularly influencing each other between cancer cell or between cancer cell and normal cell. secreting body outward is extensively present in tissue and body fluid, has important guidance effect in the clinical diagnosis of disease, prognosis supposition, health control etc. particularly the outer of tumour cell source secretes body, it is as the medium of tumour cell and external environment signal communication, by albumen specific to tumour cell, the biologically active substances such as mRNA and miRNA pass to environment and the cell of surrounding, transformation microenvironment while other cells of invasion, it is made to change { M.Baj-Krzyworzekaetal. in direction of tumor development to being more conducive to, Tumour-derivedmicrovesiclescarryseveralsurfacedeterminan tsandmRNAoftumourcellsandtransfersomeofthesedeterminants tomonocytes.CancerImmunologyImmunotherapy55, 808-818 (2006), K.Al-Nedawi, B.Meehan, J.Rak, MicrovesiclesMessengersandmediatorsoftumorprogression.Ce llCycle8,2014-2018 (2009), N.S.Barteneva, N.Maltsev, I.A.Vorobjev, Microvesiclesandintercellularcommunicationinthecontextof parasitism.FrontiersinCellularandInfectionMicrobiology3, (2013) .}.Therefore the research secreting body outward has been become to the important component part { E.Colombo of cancer study mechanism, medical diagnosis on disease, B.Borgiani, C.Verderio, R.Furlan, Microvesicles:novelbiomarkersforneurologicaldisorders.Fr ontiersinPhysiology3,63 (2012); B.Frey, U.S.Gaipl, Theimmunefunctionsofphosphatidylserineinmembranesofdying cellsandmicrovesicles.SeminarsinImmunopathology33,497-51 6 (2011); J.Conde-Vancells, E.Gonzalez, S.C.Lu, J.M.Mato, J.M.Falcon-Perez, Overviewofextracellularmicrovesiclesindrugmetabolism.Exp ertOpiniononDrugMetabolism&Toxicology6,543-554 (2010) .}.
The research externally secreting body at present is also in the primary stage, owing to its moiety is still not exclusively clear, the research of its biological function is in urgent need of strengthening. Saliva is as a kind of body fluid containing large number of biological information, and it is with the obvious advantage for clinical diagnosis, and because having features such as being non-intruding, safety, simplicity, the molecular diagnosis field in disease has broad application prospects. Meanwhile, saliva is contained and abundant outer secretes body, it is studied and is expected to as various disease finds the biomarker of early diagnosis.
At present, there is the deficiencies such as extracting method is loaded down with trivial details, extraction efficiency is lower, background interference is serious in the extracting method secreting body outside saliva. The centrifugal force that such as ultra-high speed centrifugal method needs is more than 200000g, and the large number of biological sample needing 30mL just can obtain the albumen { M.Gonzalez-Begneetal. of 10 μ g, ProteomicAnalysisofHumanParotidGlandExosomesbyMultidimen sionalProteinIdentificationTechnology (MudPIT) .JournalofProteomeResearch8,1304-1314 (2009) .}. Although the method adopting precipitation improves extraction efficiency, but cannot solve the interference problem of background impurities.
Summary of the invention
It is an object of the invention to overcome above-mentioned the deficiencies in the prior art, it is provided that outside a kind of saliva, secrete the preparation method of body and the application in molecular diagnosis thereof. Specifically, the present invention uses affinity chromatography chromatographic technique and filter membrane integrated technique, eliminate the high-abundance proteins in saliva and mucoprotein, body is secreted outside having prepared saliva by the method for frozen centrifugation again, and its application in molecular diagnosis has been studied, as found the biomarker of lung cancer according to the protein science difference of patients with lung cancer and healthy people.
It is an object of the invention to be achieved through the following technical solutions:
Secreting the preparation method of body outside the present invention relates to a kind of saliva, described method comprises the steps:
S1, removed the high-abundance proteins in saliva by affinity chromatography chromatography, remove mucoprotein in conjunction with filter membrane;
S2, step S1 is processed after saliva carry out frozen centrifugation, extract and secrete body protein outside secreting body and saliva outside saliva.
In the present invention, above-mentioned frozen centrifugation can repetitive operation 3～5 times. Described high-abundance proteins comprises salivin.
Preferably, described affinity chromatography chromatography is stationary phase taking starch, take phosphate buffered saline buffer as moving phase. Elution speed is 700～900 μ L/min.
Preferably, described filter membrane is 1.0～5.0 μm of polyvinylidene fluoride films. This filter membrane be connected to affinity chromatography chromatographic column after to complete the integration of chromatogram and membrane separation technique. In the present invention, adopt the filter membrane in this aperture, can effectively remove mucoprotein.
Preferably, the condition of described frozen centrifugation be 4 DEG C, centrifugal force 10000～20000g, 30～90 minutes.It is more preferably 60～80 minutes.
Preferably, extract secrete outside saliva secrete in body outside body protein comprises cracking release saliva albumen, lysate (supernatant liquor) carries out precipitating the step secreting body protein outside obtaining saliva.
Preferably, it is cracked into described in Triton X-100 (Triton-100) that body carries out cracking on ice 30～90 minutes to secreting outside described saliva. More preferably secrete body outside every 150 μ L salivas to add 1 μ LTriton-100 and carry out cracking on ice 30～60 minutes. During cracking, also add 2.5 μ L proteinase inhibitor (secreting body outside every 150 μ L salivas). After cracking, the ice ethanol adding 10 times of lysate volumes spends the night precipitation.
Preferably, after step S2, also comprise and adopt Liquid chromatography coupled to mass spectrometry to carry out the outer step secreting the qualification of body protein kind.
The present invention secretes body in the purposes preparing in biomarker outside also relating to saliva prepared by a kind of aforesaid method.
Preferably, described biomarker is the biomarker for studying cancer purposes. Described biomarker is for studying cancer, is not the biomarker for diagnostic uses simultaneously.
Preferably, described biomarker is the biomarker of lung cancer, oral carcinoma, cancer of the stomach or liver cancer.
Preferably, being extracted by described preparation method and secrete body protein outside obtaining saliva, difference contrasts, and secretes body biomarker outside obtaining the saliva of corresponding cancer.
The present invention is directed saliva sample, due to wherein 50～60% albumen be salivin and mucoprotein, secrete the separation and Extraction of body outside seriously affecting saliva. For removing salivin specifically, it is that matrix makes affinity chromatography chromatographic column that the present invention chooses starch, removes background interference by affinity interaction. Meanwhile, after affinity chromatography chromatographic column, connect filter membrane, being integrally formed column chromatography and membrane filtration system, remove in saliva contain mucoprotein in a large number, reduce reuniting and secreting the adhesion of body outside saliva of associated protein, thus greatly improve the outer extraction efficiency secreting body.
Compared with prior art, the present invention has following useful effect:
1, the present invention secretes the preparation method of body and albumen thereof outside providing a kind of simple to operate, saliva that the rate of recovery is high.
2, the integrated saliva process technology of the present invention is utilized, effectively outer in separation and Extraction saliva can secrete body, avoid high-abundance proteins and mucoprotein impact, be conducive to contrasting the outer body of secreting in normal people and lung cancer patient saliva, and then secrete, from the outer of saliva, the biomarker finding body protein that lung cancer is relevant.
3, the present invention take taking starch be matrix affinity chromatography chromatographic technique with filtration centrifugal method combine, establish affinity chromatography chromatogram and film coupling system, while improve extraction efficiency, eliminate the interference of peak background albumen, identify and a greater variety of outer secreted body protein, contributed to externally secreting body protein group and carry out further investigation.
Accompanying drawing explanation
By reading with reference to the detailed description that non-limiting example is done by the following drawings, the other features, objects and advantages of the present invention will become more obvious:
Fig. 1 is the schematic flow sheet of the inventive method;
Fig. 2 utilizes conventional centrifugal method to carry out outer body of secreting with the method for the invention to be separated the rear outer comparison SDS-PAGE secreting body protein respectively; Wherein, a is albumen marker, b is 1.0 μ g sialoproteins, and c secretes body protein outside the saliva that obtains of 1.0 μ g the inventive method, and d secretes body protein outside the saliva that obtains of the conventional centrifugal methods of 1.0 μ g;
Fig. 3 utilizes conventional centrifugal method to carry out outer body of secreting with the method for the invention to be separated the rear outer particle size distribution figure secreting body protein respectively;Wherein, A secretes body outside the saliva prepared of the inventive method, and B secretes body outside the saliva prepared of centrifugal method;
Fig. 4 be HPLC to the separation comparison diagram secreting body protein peptide section outside saliva, wherein, A secretes body protein peptide section outside the saliva prepared of centrifugal method, and B secretes body protein peptide section outside the saliva prepared of the inventive method.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail. The technician contributing to this area is understood the present invention by following examples further, but does not limit the present invention in any form. It should be appreciated that to those skilled in the art, without departing from the inventive concept of the premise, it is also possible to make some distortion and improvement. These all belong to protection scope of the present invention.
Embodiment
1, body is secreted outside extracting saliva
The preparation method secreting body outside saliva is as shown in Figure 1, gather the saliva sample of adult healthy volunteer and patients with lung cancer, the albumen of high abundance and the background interference albumen such as mucoprotein is removed by affinity chromatography chromatogram and membrane sepn combination technology, concrete grammar is the stationary phase that the starch with 1: 1 and albumen ratio prepare affinity chromatography chromatographic column, and the pvdf membrane of rear connection 5 μm is to complete the integration of chromatogram and membrane separation technique. First the balance of chromatographic column and the activation of film is carried out with moving phase PBS. After enter the saliva sample of corresponding proportion at chromatography column feed materials end, and the speed with 800 μ L/min, cumulative volume is the moving phase PBS wash-out saliva sample of 2mL. The sample collecting wash-out is at 4 DEG C, and centrifugal 60 minutes of 20000g, abandons supernatant, secretes body outside retaining. Resuspended outer secreting body with phosphate buffered saline buffer is slow, at 4 DEG C, centrifugal 60 minutes of 20000g, abandons supernatant and retains and outer secrete body, repeat above-mentioned steps 2-3 time. Secreting body suspension outside getting 10 μ L salivas, Nanosight secretes body granularity outside measuring saliva. Result is as shown in Figure 2; The inventive method secretes body outside obtaining the wider saliva of distribution of sizes.
2, body protein is secreted outside extracting saliva
Get 300 the outer of μ L salivas extraction and secrete body, add 100 μ L phosphate buffered saline buffers. Add 2 μ LTriton-100 (Triton X-100), 5 μ L proteinase inhibitor cocktail cracking on ice 30 minutes again. At 4 DEG C, centrifugal 60 minutes of 20000g, supernatant secretes body protein lysate outside being. The ice ethanol adding 10 times of volumes again spends the night outside precipitation obtains saliva and secretes body protein. Secrete body protein outside getting 1.0 μ g salivas and carry out SDS-PAGE sign. Result as shown in Figure 3, secretes body different with the composition of saliva outside saliva, and the inventive method obtain more saliva outside secrete body protein.
3, body protein qualification is secreted outward
Secreting body protein outside getting 10 μ g salivas and carry out enzymolysis in solution, and adopt q-TOF-MS/MS, identify secreting body peptide section outside obtaining saliva, as shown in Figure 4, the inventive method obtains more peptide section component to peptides separation result.
Table 1 the inventive method and centrifugal method extract the outer contrast secreting body protein
Table 2 be with patients with lung cancer saliva outside secrete body protein contrast after 63 differential proteins
As shown in Table 1, the method for the present invention is compared centrifugal method and is more stablized credible and obtain and more outer secrete body protein, illustrates and secretes body outside present method contributes to saliva and secrete the extraction of body protein outward.
As shown in Table 2, by the variance analysis secreting body protein group outside healthy people and the saliva of patients with lung cancer, it has been found that 63 species diversity albumen, the existing document of 12 kinds of albumen (Green Marker) is wherein had to report relevant to lung cancer. Illustrate that present method contributes to development based on the lung cancer saliva molecular diagnostic techniques secreting body outward.
Above specific embodiments of the invention are described.It is understood that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect the flesh and blood of the present invention.
Claims (10)
1. outside a saliva, secrete the preparation method of body, it is characterised in that, described method comprises the steps:
S1, removed the high-abundance proteins in saliva by affinity chromatography chromatography, remove mucoprotein in conjunction with filter membrane;
S2, step S1 is processed after saliva carry out frozen centrifugation, extract and secrete body protein outside secreting body and saliva outside saliva.
2. outside saliva according to claim 1, secrete the preparation method of body, it is characterised in that, described affinity chromatography chromatography is stationary phase taking starch, take phosphate buffered saline buffer as moving phase.
3. outside saliva according to claim 1, secrete the preparation method of body, it is characterised in that, described filter membrane is 1.0～5.0 μm of polyvinylidene fluoride films.
4. outside saliva according to claim 1, secrete the preparation method of body, it is characterised in that, the condition of described frozen centrifugation is 4 DEG C, centrifugal force 10000～20000g, 60～80 minutes.
5. outside saliva according to claim 1, secrete the preparation method of body, it is characterised in that, extract secrete outside saliva secrete in body outside body protein comprises cracking release saliva albumen, lysate carries out precipitating the step secreting body protein outside obtaining saliva.
6. outside saliva according to claim 5, secrete the preparation method of body, it is characterised in that, described in be cracked into and with Triton X-100, described outer saliva secreted body and carry out cracking on ice 30～90 minutes.
7. outside saliva according to claim 1, secrete the preparation method of body, it is characterised in that, after step S2, also comprise and adopt Liquid chromatography coupled to mass spectrometry to carry out the outer step secreting the qualification of body protein kind.
8. outside the saliva that prepared by the method for claim 1, secrete body in the purposes preparing in biomarker.
9. purposes according to claim 8, is characterized in that, described biomarker is the biomarker for studying cancer purposes.
10. purposes according to claim 8, is characterized in that, described biomarker is the biomarker of lung cancer, oral carcinoma, cancer of the stomach or liver cancer.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108872578A (en) * | 2017-05-11 | 2018-11-23 | 首都医科大学附属北京口腔医院 | A kind of new method of diagnosis and monitoring carcinoma of mouth |
| CN110308280A (en) * | 2019-05-30 | 2019-10-08 | 深圳大学 | A kind of saliva excretion body protein biomarker |
| CN110499287A (en) * | 2019-08-30 | 2019-11-26 | 博雅干细胞科技有限公司 | The method for simply preparing placenta mesenchyma stem cell excretion body |
| CN110747158A (en) * | 2019-11-14 | 2020-02-04 | 赵凯 | Cell supernatant exosome extraction process based on precipitation reagent method |
| CN111659158A (en) * | 2020-06-20 | 2020-09-15 | 大理大学 | Molecular marker method for extracting exosome peak position by exclusion chromatography |
| CN113533589A (en) * | 2021-09-16 | 2021-10-22 | 天九再生医学(天津)科技有限公司 | Method for analyzing exosomal charge heterogeneity |
| 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 |
| CN114231638A (en) * | 2020-03-30 | 2022-03-25 | 中国医学科学院肿瘤医院 | Kit, device and method for lung cancer diagnosis |
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Non-Patent Citations (7)
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108872578A (en) * | 2017-05-11 | 2018-11-23 | 首都医科大学附属北京口腔医院 | A kind of new method of diagnosis and monitoring carcinoma of mouth |
| 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 |
| CN110308280A (en) * | 2019-05-30 | 2019-10-08 | 深圳大学 | A kind of saliva excretion body protein biomarker |
| CN110499287A (en) * | 2019-08-30 | 2019-11-26 | 博雅干细胞科技有限公司 | The method for simply preparing placenta mesenchyma stem cell excretion body |
| 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 |
| CN114231638A (en) * | 2020-03-30 | 2022-03-25 | 中国医学科学院肿瘤医院 | Kit, device and method for lung cancer diagnosis |
| CN114231638B (en) * | 2020-03-30 | 2023-06-27 | 中国医学科学院肿瘤医院 | Application of exosome-7 f-2-3p, ARPC5 and the like in lung cancer diagnosis |
| CN111659158A (en) * | 2020-06-20 | 2020-09-15 | 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 |
| CN113533589A (en) * | 2021-09-16 | 2021-10-22 | 天九再生医学(天津)科技有限公司 | Method for analyzing exosomal charge heterogeneity |
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