CN108225875A - The preparation of nasal cavity washing liquid excretion body, identification method - Google Patents

The preparation of nasal cavity washing liquid excretion body, identification method Download PDF

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Publication number
CN108225875A
CN108225875A CN201810033509.4A CN201810033509A CN108225875A CN 108225875 A CN108225875 A CN 108225875A CN 201810033509 A CN201810033509 A CN 201810033509A CN 108225875 A CN108225875 A CN 108225875A
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Prior art keywords
excretion body
liquid
nasal cavity
washing liquid
cavity washing
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陈静
张薇
尤易文
游波
单颖
施思
包丽丽
乐慧君
张洁
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Affiliated Hospital of Nantong University
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Affiliated Hospital of Nantong University
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34Purifying; Cleaning
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Pathology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • General Physics & Mathematics (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Urology & Nephrology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The nasal cavity washing preparation of liquid excretion body, identification method the invention discloses a kind of non-diagnostic purposes, the presence including the nasal cavity washing acquisition of liquid, the extraction and purification of excretion body, Electronic Speculum identification excretion body, immune-blotting method PROTEIN C D9, CD63.The method of the present invention simple possible, easily and effectively realizes the extraction of excretion body in nasal cavity washing liquid;Accurately and reliably, overcoming existing technology of preparing, there are background impurities interference, and the excretion body rate of recovery is low, identifies the deficiencies of albumen is few for the method for the present invention.Moreover, the method for the present invention can be directly used in the advantage of subsequent detection, there are the application values such as higher scientific research.

Description

The preparation of nasal cavity washing liquid excretion body, identification method
Technical field
Preparation, identification method the present invention relates to a kind of nasal cavity washing liquid excretion body.
Background technology
Important tool of the excretion body as cell and intercellular communication, be invaginated by intracellular lysosome particle formed it is more Foam, under specific stimulation, and cell membrane fusion, to the vesicles of a diameter of 50-100nm of exocytosis.Excretion Body is widely present in tissue and body fluid, by transporting the bioactive substances such as protein, mRNA, miRNA that it is rich in, ginseng Face cytoproximal signal path etc. with immunological regulation, the reconstruct of regulating cell epimatrix, activation, there is weight in scientific research etc. The directive function wanted.
Nasal cavity washes one's hands and face liquid(nasal lavage fluid, NLF)A kind of body containing biological information as nasal cavity part Liquid because its access approaches has the characteristics that non-intruding, safe and simple, has broad application prospects.
At present, there are extracting method is cumbersome, extraction efficiency is relatively low, background interference for the extracting method of nasal cavity washing liquid excretion body The deficiencies of serious, and there is no unified standard step.
Invention content
The purpose of the present invention is to provide preparation, the identifications of nasal cavity washing liquid excretion body that a kind of method is easy, effect is good Method.
The present invention technical solution be:
A kind of preparation of the nasal cavity washing liquid excretion body of non-diagnostic purposes, identification method, it is characterized in that:Include the following steps:
(One)Nasal cavity washes one's hands and face the acquisition of liquid
Healthy volunteer and patients with chronic sinusitis nasal cavity washing liquid are acquired respectively.Specific operation process is as follows:By 5 milliliters of physiology Brine instils in the left nose hole of subject, and head is advised to be tilted backwards in 30 °, and soft palate is closed, and then head turns forward, and collects nose Chamber washes one's hands and face liquid;Right nostril repeats the above steps;
(Two)The extraction and purification of excretion body
1. the nasal cavity washing liquid of collection is filtered by strainer, preliminary impurity screening and bacterium;
2. filtered nasal cavity washing liquid centrifuge 10 minutes under 4 DEG C, the centrifugal condition of 3000g, separation removal nasal cavity is washed one's hands and face Cell and other impurities in liquid, then obtain supernatant liquid;
3. 2. supernatant liquid that step is obtained is moved into the second centrifuge tube, 30 points are centrifuged under 4 DEG C, the centrifugal condition of 6000g Clock, further separating and removing impurities, obtains supernatant liquid;
4. 3. supernatant liquid that step is obtained is moved into third centrifuge tube, centrifugation 1 is small under 4 DEG C, the centrifugal condition of 10000g When, obtain supernatant liquid;
5. surpassing the immigration of 4. supernatant liquid that step obtains from pipe, centrifuged 1 hour under 4 DEG C, the centrifugal condition of 10,0000g, After removing supernatant liquid, precipitated;
6. after 5. precipitation that step obtains is resuspended washing with PBS, it is further purified by molecular exclusion chromatography;
7. surpassing the washing liquid immigration of step 6. treated nasal cavity from pipe, centrifuged again under 4 DEG C, the centrifugal condition of 10,0000g 1 hour, obtain being deposited in total excretion body of the nasal cavity washing liquid of the centrifugation bottom of the tube;
(Three)Electronic Speculum identifies excretion body
1. above-mentioned obtained excretion body is resuspended with PBS solution;
2. 20mL/L, pH6.8 Salkowski's solution is added dropwise, 1:1 mixing;
3. liquid takes above-mentioned mixing liquid 5ul drops on load sample copper mesh, at room temperature negative staining 10 minutes, dried under incandescent lamp;
4. transmission electron microscope observing is found:Products therefrom is the bilayer membrane structure foam of 50-100nm sizes;
(Four)The presence of immune-blotting method PROTEIN C D9, CD63
1. 1 × PBS for the excretion body 1ml that extraction purification is obtained is resuspended, excretion weight suspension is obtained, adds in 1ml lysates, Extract total protein;
2. BCA methods measure excretion body protein content;
3. making 10% and 4% gradient glue, enough buffer solutions are added in, the excretion body total protein per hole loading 50ug;
4. installing electrode, connect with the mains, constant pressure 80V makes protein go on same starting line, and protein is run to separation gel Shi Gaiwei 120V;
5. cutting glue, it is put into transferring film buffer solution;Pvdf membrane placement is polarized in methyl alcohol, is put into transferring film liquid in distilled water after aquation, With 15 min are balanced together with cotton pad and filter paper in transferring film buffer solution;
6. pvdf membrane, glue, cotton pad and filter paper is fabricated to " sandwich " structure, first layer is cotton pad, and the second layer is filter paper, third Layer is glue, and the 4th layer is pvdf membrane, and layer 5 is filter paper, and layer 6 is cotton pad, is clamped;It is put into transferring film slot, adds in transferring film liquid And ice chest, transferring film slot are placed in the basin for filling ice water;Installation electrode connects power supply, and transferring film condition is set as 200 mA of constant current, when Between 90 min;
7. after transferring film, pvdf membrane is taken out, is marked, is placed on shaking table, TBST washes 5 min × 3 time, 5 % degreasing oxen Milk closes 1-2h;TBST washes 5 min × 3 time of film;Primary antibody CD9, CD63 is added to be incubated, is stayed overnight for 4 DEG C after 1 h of room temperature;
8. TBST washes 10 min × 3 time of film;Secondary antibody is incubated, 2 h of room temperature;TBST washes 20 min × 3 time;
9. developing in darkroom, X photographic films are rinsed;
Through the expression that distinctive PROTEIN C D9, CD63 in excretion body surface face is detected in above method products therefrom.
Excretion body is further identified using nano particle tracer technique, analysis has detected the excretion body that extraction purification obtains Concentration, size are consistent with the characterization of excretion body.
The method of the present invention simple possible, easily and effectively realizes the extraction of excretion body in nasal cavity washing liquid;The method of the present invention Accurately and reliably, overcoming existing technology of preparing, there are background impurities interference, and the excretion body rate of recovery is low, identifies the deficiencies of albumen is few. Moreover, the method for the present invention can be directly used in the advantage of subsequent detection, there are the application values such as higher scientific research.
Description of the drawings
The invention will be further described with reference to the accompanying drawings and examples.
Fig. 1 is the electron micrograph that the present invention implements products therefrom.
Fig. 2 is to implement the immune-blotting method result that PROTEIN C D9, CD63 in products therefrom is expressed to the present invention.
Fig. 3 is that nano particle tracer technique analysis testing result is carried out to products therefrom of the present invention.
Specific embodiment
Not specified various instruments and reagent are commercial product well known in the art in the following example, can be passed through Commercial sources obtain.
A kind of preparation of the nasal cavity washing liquid excretion body of non-diagnostic purposes, identification method, include the following steps:
First, the acquisition of nasal cavity washing liquid
Obtain participant know and the agreement of ethics under, respectively acquire healthy volunteer and patients with chronic sinusitis nasal cavity washing Liquid.Specific operation process is as follows:5 milliliters of physiological saline are instiled in the left nose hole of subject, head is advised to be tilted backwards in 30 °, Soft palate is closed, and then head turns forward, and collects nasal cavity washing liquid.Right nostril repeats the above steps.
2nd, the extraction and purification of excretion body
1. the nasal cavity washing liquid of collection is filtered by strainer, preliminary impurity screening and bacterium.
2. filtered nasal cavity washing liquid centrifuge 10 minutes under 4 DEG C, the centrifugal condition of 3000g, separation removes nasal cavity The cell and other impurities in liquid are washed one's hands and face, then obtains supernatant liquid.
3. the supernatant liquid 2. obtained is moved into the second centrifuge tube, 30 points are centrifuged under 4 DEG C, the centrifugal condition of 6000g Clock, further separating and removing impurities, obtains supernatant liquid.
4. the supernatant liquid 3. obtained is moved into third centrifuge tube, centrifugation 1 is small under 4 DEG C, the centrifugal condition of 10000g When, obtain supernatant liquid.
5. surpassing the supernatant liquid 4. obtained immigration from pipe, centrifuged 1 hour under 4 DEG C, the centrifugal condition of 10,0000g, After removing supernatant liquid, precipitated.
6. after the precipitation 5. obtained is resuspended washing with PBS, it is further purified by molecular exclusion chromatography.
7. surpassing the washing liquid immigration of 6. treated nasal cavity from pipe, centrifuged again under 4 DEG C, the centrifugal condition of 10,0000g 1 hour, obtain being deposited in total excretion body of the nasal cavity washing liquid of the centrifugation bottom of the tube.
3rd, Electronic Speculum identification excretion body
1. above-mentioned obtained excretion body is resuspended with PBS solution.
2. 20mL/L Salkowski's solutions (pH6.8) 1 are added dropwise:1 mixing.
3. liquid takes above-mentioned mixing liquid 5ul drops on load sample copper mesh, at room temperature negative staining 10 minutes, dried under incandescent lamp.
4. the photo that transmission electron microscope observing arrives as shown in Figure 1, it can be found that:Products therefrom divides for the double of 50-100nm sizes The excretion body of sublayer membrane structure foam, morphology and other document reports is consistent.
4th, the presence of immune-blotting method PROTEIN C D9, CD63
1. the product (the excretion body extracted in nasal cavity washing liquid) of gained is resuspended with 1 × PBS of 1ml, excretion weight is obtained Suspension adds in 1ml lysates, extracts total protein.
2. BCA methods measure excretion body protein content.
3. making 10% and 4% gradient glue, enough buffer solutions are added in, the excretion body total protein per hole loading 50ug.
4. installing electrode, connect with the mains, 80 V of constant pressure makes protein go on same starting line, protein run to point 120 V are changed to during from glue.
5. cutting glue, it is put into transferring film buffer solution.Pvdf membrane placement is polarized in methyl alcohol, is put into transferring film in distilled water after aquation Liquid balances 15 min together with cotton pad and filter paper in transferring film buffer solution.
6. pvdf membrane, glue, cotton pad and filter paper are fabricated to " sandwich " structure, first layer is cotton pad, and the second layer is filter paper, Third layer is glue, and the 4th layer is pvdf membrane, and layer 5 is filter paper, and layer 6 is cotton pad, is clamped.It is put into transferring film slot, adds in and turn Film liquid and ice chest, transferring film slot are placed in the basin for filling ice water.Installation electrode connects power supply, and transferring film condition is set as constant current 200 MA, 90 min of time.
7. after transferring film, pvdf membrane is taken out, is marked, is placed on shaking table, TBST washes 5 min × 3 time, and 5 % take off Fat milk closing 1-2 h.TBST washes 5 min × 3 time of film.Add primary antibody(CD9、CD63)It is incubated, is stayed overnight for 4 DEG C after 1 h of room temperature.
8. TBST washes 10 min × 3 time of film.Secondary antibody is incubated, 2 h of room temperature.TBST washes 20 min × 3 time.
9. developing in darkroom, X photographic films are rinsed.
Testing result is as shown in Fig. 2, through detecting the distinctive albumen in excretion body surface face in above method products therefrom The expression of CD9, CD63, therefore, it is excretion body that products therefrom is illustrated from molecular characterization.
Nano particle tracer technique(NTA)Further identify excretion body
NTA is a kind of method of relatively new characterization nano particle, it can directly and in real time observe nano particle.NTA passes through Light microscope collects the scattered light signal of nano particle, and one section of nano particle of shooting makees the image of Brownian movement in the solution, The Brownian movement of each particle is tracked and analyzed, so as to calculate the hydrodynamic radius of nano particle and concentration.
In clinical diagnosis, quickly and easily under complicated biological context(Such as nasal cavity washing liquid, blood plasma, urine)It measures Concentration, size and the characterize data of excretion body are indispensable requirements, presently, there are conventional method all can not perfectly solve this One problem.NTA as a kind of completely new measuring technique, have real-time observation, higher resolution ratio, accurate measurement of concetration and Fluorescence measurement function provides the new approaches to excretion body size and concentration studies.
Testing result is as shown in figure 3, NTA analyses have detected the concentration through above method products therefrom, size etc., with excretion The characterization of body is consistent.

Claims (2)

1. a kind of preparation of the nasal cavity washing liquid excretion body of non-diagnostic purposes, identification method, it is characterized in that:Include the following steps:
(One)Nasal cavity washes one's hands and face the acquisition of liquid
Healthy volunteer and patients with chronic sinusitis nasal cavity washing liquid are acquired respectively;Specific operation process is as follows:By 5 milliliters of physiology Brine instils in the left nose hole of subject, and head is advised to be tilted backwards in 30 °, and soft palate is closed, and then head turns forward, and collects nose Chamber washes one's hands and face liquid;Right nostril repeats the above steps;
(Two)The extraction and purification of excretion body
1. the nasal cavity washing liquid of collection is filtered by strainer, preliminary impurity screening and bacterium;
2. filtered nasal cavity washing liquid centrifuge 10 minutes under 4 DEG C, the centrifugal condition of 3000g, separation removal nasal cavity is washed one's hands and face Cell and other impurities in liquid, then obtain supernatant liquid;
3. 2. supernatant liquid that step is obtained is moved into the second centrifuge tube, 30 points are centrifuged under 4 DEG C, the centrifugal condition of 6000g Clock, further separating and removing impurities, obtains supernatant liquid;
4. 3. supernatant liquid that step is obtained is moved into third centrifuge tube, centrifugation 1 is small under 4 DEG C, the centrifugal condition of 10000g When, obtain supernatant liquid;
5. surpassing the immigration of 4. supernatant liquid that step obtains from pipe, centrifuged 1 hour under 4 DEG C, the centrifugal condition of 10,0000g, After removing supernatant liquid, precipitated;
6. after 5. precipitation that step obtains is resuspended washing with PBS, it is further purified by molecular exclusion chromatography;
7. surpassing the washing liquid immigration of step 6. treated nasal cavity from pipe, centrifuged again under 4 DEG C, the centrifugal condition of 10,0000g 1 hour, obtain being deposited in total excretion body of the nasal cavity washing liquid of the centrifugation bottom of the tube;
(Three)Electronic Speculum identifies excretion body
1. above-mentioned obtained excretion body is resuspended with PBS solution;
2. 20mL/L, pH6.8 Salkowski's solution is added dropwise, 1:1 mixing;
3. liquid takes above-mentioned mixing liquid 5ul drops on load sample copper mesh, at room temperature negative staining 10 minutes, dried under incandescent lamp;
4. transmission electron microscope observing is found:Products therefrom is the bilayer membrane structure foam of 50-100nm sizes;
(Four)The presence of immune-blotting method PROTEIN C D9, CD63
1. 1 × PBS for the excretion body 1ml that extraction purification is obtained is resuspended, excretion weight suspension is obtained, adds in 1ml lysates, Extract total protein;
2. BCA methods measure excretion body protein content;
3. making 10% and 4% gradient glue, enough buffer solutions are added in, the excretion body total protein per hole loading 50ug;
4. installing electrode, connect with the mains, constant pressure 80V makes protein go on same starting line, and protein is run to separation gel Shi Gaiwei 120V;
5. cutting glue, it is put into transferring film buffer solution;Pvdf membrane placement is polarized in methyl alcohol, is put into transferring film liquid in distilled water after aquation, With 15 min are balanced together with cotton pad and filter paper in transferring film buffer solution;
6. pvdf membrane, glue, cotton pad and filter paper is fabricated to " sandwich " structure, first layer is cotton pad, and the second layer is filter paper, third Layer is glue, and the 4th layer is pvdf membrane, and layer 5 is filter paper, and layer 6 is cotton pad, is clamped;It is put into transferring film slot, adds in transferring film liquid And ice chest, transferring film slot are placed in the basin for filling ice water;Installation electrode connects power supply, and transferring film condition is set as 200 mA of constant current, when Between 90 min;
7. after transferring film, pvdf membrane is taken out, is marked, is placed on shaking table, TBST washes 5 min × 3 time, 5 % degreasing oxen Milk closes 1-2h;TBST washes 5 min × 3 time of film;Primary antibody CD9, CD63 is added to be incubated, is stayed overnight for 4 DEG C after 1 h of room temperature;
8. TBST washes 10 min × 3 time of film;Secondary antibody is incubated, 2 h of room temperature;TBST washes 20 min × 3 time;
9. developing in darkroom, X photographic films are rinsed;
Through the expression that distinctive PROTEIN C D9, CD63 in excretion body surface face is detected in above method products therefrom.
2. the preparation of the nasal cavity washing liquid excretion body of non-diagnostic purposes according to claim 1, identification method, feature It is:Excretion body is further identified using nano particle tracer technique, analysis have detected the concentration of excretion body that extraction purification obtains, Size is consistent with the characterization of excretion body.
CN201810033509.4A 2018-01-15 2018-01-15 The preparation of nasal cavity washing liquid excretion body, identification method Pending CN108225875A (en)

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CN101498734A (en) * 2009-03-09 2009-08-05 昆明理工大学 Method for simultaneously turning two films and marking protein TRX and procaspase12
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