CN114574429B

CN114574429B - Composite reagent for extracting lung-derived exosome component substances

Info

Publication number: CN114574429B
Application number: CN202210257946.0A
Authority: CN
Inventors: 张庆华; 宋凯; 匡鑫
Original assignee: Shanghai Wayen Biomedical Technology Co ltd
Current assignee: Shanghai Wayen Biomedical Technology Co ltd
Priority date: 2022-03-16
Filing date: 2022-03-16
Publication date: 2024-04-19
Anticipated expiration: 2042-03-16
Also published as: CN114574429A

Abstract

The invention provides a compound reagent for extracting a lung-derived exosome component substance, which comprises the following steps: centrifuging the serum sample to obtain a supernatant; adding an enrichment reagent group to obtain a compound; adding an extraction reagent group to obtain corresponding composition substances, wherein the composition substances comprise proteins and RNA, the proteins comprise NSE and CEA, and the RNA comprises miR-21-5p, miR-141 and miR-23a. The lung cancer can be detected in advance by extracting the lung-derived protein and RNA, and the lung cancer treatment method is beneficial to early treatment.

Description

Composite reagent for extracting lung-derived exosome component substances

Technical Field

The invention relates to the field of molecular biology, in particular to a compound reagent for extracting exosome composition substances from lung sources.

Background

Lung cancer is a malignant tumor with the first incidence and mortality rate in China. Early lung cancer is hidden and diversified in expression, clinical characteristics and signs are not obvious, and most lung cancer patients are found to be in late stage. The survival rate of lung cancer in the late 5 years is extremely low, and the survival rate of lung cancer in the early (stage I) five years can reach more than 50 percent. Thus, early detection and diagnosis of lung cancer is a key element for reducing the incidence rate and death rate of lung cancer.

At present, a plurality of detection means for lung cancer, such as CT, X-ray chest radiography, abscission cytology examination, CT positioning puncture biopsy and the like, exist clinically. Although the screening research of the LDCT in some areas has made a certain research progress, patients meeting the screening standard of the high-risk group of the LDCT are few, the screening accuracy is not high enough, and the like.

The exosome is a microvesicle with a lipid bilayer membrane having a diameter of about 30-150nm which can be secreted by most cells in the body, and is widely present in various body fluids such as blood, tears, urine, saliva, milk, ascites, etc., and is also derived from various cells such as erythrocytes, leukocytes, macrophages and epithelial cells in the body. The exosomes contain proteins, rRNA, miRNA, DNA and the like derived from blast cells, mediate signal transduction between cells under physiological and disease states, and have the effects of disease diagnosis, immune regulation and the like. The lung-derived exosome-mediated cell interaction may play an important role in the occurrence and transfer process of lung cancer, and lung cancer specific proteins and RNA of the lung-derived exosome in circulating blood can become ideal biomarkers for lung cancer diagnosis, which provides objective basis for early diagnosis, early treatment and prognosis judgment of lung cancer.

Liquid diagnosis taking serum lung derived exosomes as an example has the following advantages: 1. the wound is small, and the compliance of patients is good; 2. the collection is convenient, and the storage is facilitated; 3. simple, convenient and easy to operate. However, it is difficult to extract lung-derived proteins and RNA directly from serum.

In view of this, the present invention has been made.

Disclosure of Invention

The first object of the invention is to provide a compound reagent for extracting lung-derived exosome components, wherein the reagent components provided by the reagent group are matched with each other, and proteins and RNA in exosome can be completely extracted after the compound reagent is used.

The second purpose of the invention is to provide a using method of the compound reagent, which has simple steps and can completely extract protein and RNA of lung source exosomes, thereby solving the problem that the protein and RNA of lung source are difficult to extract in the prior art.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

The invention provides a compound reagent for extracting a lung source exosome component substance, which comprises an enrichment reagent group and an extraction reagent group;

The enrichment reagent group comprises an enrichment reagent 1, an enrichment reagent 2 and an enrichment reagent 3, wherein the enrichment reagent 1 comprises, by weight, 10-20 parts of PEG20000 with concentration of 25%, 5-15 parts of PEG10000 with concentration of 20%, 1-4 parts of Tween 20 with concentration of 0.05%, 1-4 parts of potassium dihydrogen phosphate solution, 1-4 parts of disodium hydrogen phosphate solution, 1-4 parts of sodium chloride solution and 1-4 parts of potassium chloride solution, the enrichment reagent 2 comprises, by weight, 5-15 parts of PEG20000 with concentration of 20%, 5-15 parts of PEG8000 with concentration of 20%, 5-15 parts of glucose solution and 2-8 parts of sodium acetate solution, and the enrichment reagent 3 comprises 0.1-5 parts of Protein A/G magnetic beads coated with SFTPC antibodies;

the extraction reagent group comprises a protein extraction reagent group and an RNA extraction reagent group;

The protein extraction reagent group comprises RIPA strong lysate;

The RNA extraction reagent set comprises an RNA extraction reagent 1, an RNA extraction reagent 2, an RNA extraction reagent 3, an RNA cleaning buffer solution 1 and an RNA cleaning buffer solution 2, wherein the RNA extraction reagent 1 comprises Trizol lysate, the RNA extraction reagent 2 comprises 100% trichloromethane, the RNA extraction reagent 3 comprises 100% ethanol, the RNA cleaning buffer solution 1 is 3 parts of RWP buffer solution, and the RNA cleaning buffer solution 2 is 2 parts of RPE buffer solution.

Wherein the purposes of each reagent are as follows:

25% PEG20000: for enriching exosomes;

20% PEG10000: for enriching exosomes;

0.05% tween: the buffer solution system is used for stabilizing the buffer solution system, so that the subsequent washing is convenient;

potassium dihydrogen phosphate solution, disodium hydrogen phosphate solution, sodium chloride solution and potassium chloride solution: a buffer component;

20% PEG20000: for enriching exosomes;

20% PEG8000: for enriching exosomes;

Glucose solution: is used for assisting in enriching exosomes;

Sodium acetate solution: negative anions used for neutralizing the surface of exosomes promote exosomes to gather and settle;

Protein A/G magnetic beads coated with SFTPC antibody: is used for enriching lung source exosomes.

The PEG can be used for exosome enrichment experiments, and PEG solutions with different molecular weights and different concentrations can be used in combination with glucose and the like to increase exosome enrichment effect, so that more exosomes are collected, and the separation efficiency is far more than that of single PEG. In addition, the combined application of the reagents such as sodium acetate solution, tween and the like also increases the dispersibility and stability of the exosomes, has better impurity removing effect, and greatly improves the extraction efficiency and purity of the exosomes.

Meanwhile, the enrichment reagent group is adopted to perform the optimization selection of exosomes step by step, impurities distributed from large to small are removed step by step through the enrichment reagent 1, the enrichment reagent 2 and the enrichment reagent 3, the purity of the enriched exosomes is ensured, the proportion among the reagents disclosed by the invention is extremely important, the amount of the used buffer reagent is also required to be controlled, a large amount of experiments prove that the exosomes are damaged when the use amount of the buffer reagent is too large, the phenomenon of aggregation among the exosomes is caused when the use amount of the buffer reagent is too small, the enriched exosomes have obvious impurities, the purity of the collected exosomes cannot be ensured, and the use proportion of the reagents is also particularly important, and the optimal proportion is obtained through a plurality of experiments.

For the enrichment reagent group, three enrichment reagents are needed, because practice shows how to use only any one of the enrichment reagent 1, the enrichment reagent 2 and the enrichment reagent 3, the purity of the enriched exosomes cannot be ensured without combination, and the single enrichment reagent has weaker matching action, the quality in the exosome enrichment process cannot be ensured, the phenomenon of exosome aggregation can be easily caused by breakage, however, when the three enrichment reagents are matched, the quality of the exosomes is ensured, and the purity of the obtained exosomes is also ensured, so the combination is an optimal scheme.

Likewise, for the composition of the enrichment reagent 1, the enrichment reagent 2 and the enrichment reagent 3, the selected components or the dosage of each component is also used for ensuring that the enriched exosomes can reach the required purity, and substances such as tween, glucose and the like which are added are not easy to be aggregated on the one hand for improving the dispersibility of the exosomes and are not easy to be aggregated on the other hand, but the adding amount of the substances is not excessive, otherwise, the purity of the exosomes enriched cannot be ensured, and the normal operation of enrichment can be influenced by introducing excessive reagents.

It can be seen that, for the scheme of the present invention, the types, numbers, and amounts of the enrichment reagents, as well as the components and amounts of the enrichment reagents themselves, are all determined by a series of inventive practices, and are not obtained by simple operational practices, and a great deal of inventive labor is required.

The unique composite reagent provided by the invention optimizes the purification steps of exosomes, has poor enrichment effect and low exosome purity of a single enrichment reagent, can increase the enrichment effect of the exosomes through the combined use of the enrichment reagent, can collect more exosomes, has better impurity removal effect, greatly improves the extraction efficiency and purity of the exosomes, and has far more separation efficiency than single PEG. In addition, specific lung-derived exosomes are obtained, which also achieve results that are difficult to achieve with conventional exosome-enriching agents.

After the exosome is obtained through enrichment, the protein extraction reagent group and the RNA extraction reagent group are adopted to collect protein and RNA, the internal RNA extraction reagent group is used as a composite reagent, RNA in the exosome can be completely extracted, and the RNA in the exosome is removed and buffered to obtain pure RNA through the mutual matching of the RNA extraction reagent 1, the RNA extraction reagent 2, the RNA extraction reagent 3, the RNA cleaning buffer 1 and the RNA cleaning buffer 2.

Preferably, the enriching reagent 1 comprises 13-18 parts by weight of PEG20000 with the concentration of 25%, 8-12 parts by weight of PEG10000 with the concentration of 20%, 1.5-3 parts by weight of Tween 20 with the concentration of 0.05%, 1.5-3 parts by weight of potassium dihydrogen phosphate solution, 1.5-3 parts by weight of disodium hydrogen phosphate solution, 1.5-3 parts by weight of sodium chloride solution and 1.5-3 parts by weight of potassium chloride solution, the enriching reagent 2 comprises 8-12 parts by weight of PEG20000 with the concentration of 20%, 8-12 parts by weight of PEG8000 with the concentration of 20%, 8-12 parts by weight of glucose solution and 3-7 parts by weight of sodium acetate solution, and the enriching reagent 3 comprises 0.5-4 parts by weight of Protein A/G magnetic beads coated with SFTPC antibody.

Preferably, the enriching reagent 1 comprises 15 parts of PEG20000 with the concentration of 25%, 10 parts of PEG10000 with the concentration of 20%, 2 parts of Tween 20 with the concentration of 0.05%, 2 parts of potassium dihydrogen phosphate solution, 2 parts of disodium hydrogen phosphate solution, 2 parts of sodium chloride solution and 2 parts of potassium chloride solution in parts by weight, the enriching reagent 2 comprises 10 parts of PEG20000 with the concentration of 20%, 10 parts of PEG8000 with the concentration of 20%, 10 parts of glucose solution and 5 parts of sodium acetate solution in parts by weight, and the enriching reagent 3 comprises 1 part of Protein A/G magnetic beads coated with SFTPC antibody.

The invention also provides a using method of the reagent set, which comprises the following steps:

Centrifuging the serum sample to obtain a supernatant;

Adding an enrichment reagent group to obtain a compound;

Adding an extraction reagent group to obtain corresponding composition substances, wherein the composition substances comprise proteins and RNA, the proteins comprise NSE and CEA, and the RNA comprises miR-21-5p, miR-141 and miR-23a.

Preferably, the adding of the enriched reagent set comprises the steps of:

adding enrichment reagent 1, standing at 3-5deg.C, centrifuging, and removing clear liquid to obtain heavy suspension 1;

Adding enrichment reagent 2, standing at 3-5deg.C, centrifuging, and removing clear liquid to obtain heavy suspension 2;

adding enrichment reagent 3, and incubating overnight at 3-5 ℃ to remove the clear liquid to obtain heavy suspension 3.

Preferably, the volume ratio of the enrichment reagent 1 to the supernatant is 1: (3-5) the volume ratio of the enriching reagent 2 to the resuspension 1 is 1: (4-6) the volume ratio of the enriching reagent 3 to the resuspension 2 is 1: (4-6).

Preferably, when the constituent material is a protein, a protein extraction reagent is selected, and the method of using the protein extraction reagent comprises the steps of: adding a protein extraction reagent into the heavy suspension 3, placing in a refrigerator, swirling, repeating for three times, centrifuging, and taking supernatant to obtain the protein.

Preferably, the volume ratio of the protein extraction reagent to the resuspension 3 is 1: (0.5-2).

Preferably, when the constituent substance is RNA, an RNA extraction reagent set is selected, and the method of using the RNA extraction reagent set includes the steps of:

Adding an RNA extraction reagent 1 into the heavy suspension 3, and standing at room temperature after vortex oscillation;

adding an RNA extraction reagent 2, standing at room temperature, and centrifuging to obtain colorless supernatant;

And adding an RNA extraction reagent 3 for adsorption.

Preferably, the method further comprises the step of washing the adsorbed RNA:

And (3) adding an RNA cleaning buffer solution 1 after centrifugation, adding an RNA cleaning buffer solution 2 after centrifugation at room temperature, adding deionized water after centrifugation, and obtaining RNA after centrifugation at room temperature.

Preferably, the volume ratio of the RNA extraction reagent 1 to the resuspension 3 is (4-6): 1, the volume ratio of the RNA extraction reagent 1 to the RNA extraction reagent 2 is (3-5): 1, a step of; the volume ratio of the RNA extraction reagent 3 to the colorless supernatant is (1-2): 1.

The concentration of all the reagents mentioned in the invention is volume percent, and the reagents comprise an enrichment reagent 1, an enrichment reagent 2, an enrichment reagent 3, a protein extraction reagent, an RNA extraction reagent 1, an RNA extraction reagent 2, an RNA extraction reagent 3, an RNA cleaning buffer solution 1 and an RNA cleaning buffer solution 2.

Compared with the prior art, the invention has at least the following advantages:

(1) The compound reagent provided by the invention can conveniently and accurately extract lung-derived protein and RNA from a serum sample, and the lung cancer can be diagnosed by the specific protein and RNA obtained by the invention. The extracted protein can be used in protein detection schemes such as protein chips, protein mass spectrum and the like, RNA can be used for gene second generation sequencing, the gene state of a detected person can be comprehensively known in real time, accurate detection and accurate diagnosis can be achieved, the possibility of suffering from diseases can be predicted or clinical diagnosis can be guided, and the patient can be treated in an individual targeting mode.

(2) Through the technical scheme, the method is simple to operate and high in practicability, can reduce coprecipitation pollution of non-exosome proteins and other impurities in the exosome extraction process, is used for separating exosome in a sample from the sample, and greatly improves the purity and yield of the exosome. Based on this, one sample can be subjected to 2-level extraction of proteins and nucleic acids and subsequent detection.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to designate like parts throughout the figures. In the drawings:

FIG. 1 is a transmission electron microscope of an exosome according to an embodiment of the present invention;

FIG. 2 is a graph showing the particle size concentration of exosomes according to an embodiment of the present invention, wherein the abscissa represents the particle size and the ordinate represents the vesicle concentration;

FIG. 3 shows the exosome marker band provided by the examples of the present invention, wherein the positive marker CD9 has a molecular weight of 25kDa;

FIG. 4 shows the results of ELISA analysis of NSE and CEA in lung derived exosomes as provided in experimental examples of the present invention;

FIG. 5 shows the results of PCR analysis of miR-21-5p, miR-141 and miR-23a in lung derived exosomes provided by experimental examples of the invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1

The embodiment provides a method for extracting lung-derived exosome constituent substances, which can conveniently and accurately extract lung-derived proteins and RNA directly from a serum sample, the extracted proteins and RNA can be greatly applied to early diagnosis, screening and diagnosis of lung cancer, the wound is small, the collection and the storage are convenient, and the discovery rate of lung cancer is greatly improved.

The extraction and detection of the lung source exosome protein and RNA mainly comprises the following steps:

1. Enriching the blood lung-heat source exosome;

(1) Taking out serum sample, thawing in water bath at 25deg.C, and placing on ice;

(2) Centrifuging at 2000 Xg for 10min at 4deg.C, collecting supernatant;

(3) Centrifuging at 10000 Xg for 30min at 4deg.C, collecting supernatant;

(4) Adding an enrichment reagent 1, and standing at 3-5 ℃ for 30min, wherein the volume ratio of the enrichment reagent 1 to the supernatant is 1: (3-5) in order to achieve better effect, the volume ratio of the enrichment reagent 1 to the supernatant was chosen to be 1:4, selecting the temperature to be 4 ℃;

(5) Centrifuging at 3000 Xg for 10min, and removing supernatant to obtain heavy suspension 1;

(6) Adding an enrichment reagent 2, and standing at 3-5 ℃ for 30min, wherein the volume ratio of the enrichment reagent 2 to the heavy suspension 1 is 1: (4-6) in order to ensure the enrichment effect, the volume ratio of the enrichment reagent 2 to the resuspension 1 is selected as 1:5, selecting the temperature to be 4 ℃;

(7) Centrifuging at 3000 Xg for 10min, removing supernatant to obtain heavy suspension 2, wherein the obtained heavy suspension 2 can be used for exosome characterization;

(8) Adding an enrichment reagent 3, incubating overnight at 3-5 ℃, and placing the mixture on a magnetic rack to remove supernatant to obtain a heavy suspension 3 containing lung source exosome magnetic bead complexes, wherein the volume ratio of the enrichment reagent 3 to the heavy suspension 2 is 1: (4-6) in order to achieve a better complex, the volume ratio of the enriching reagent 3 to the resuspension 2 is chosen to be 1:5, the temperature was chosen to be 4 ℃.

The resuspension 3 was split into two parts, and protein and RNA were extracted separately.

2. Extracting serum lung-derived proteins;

(1) Adding a protein extraction reagent into the heavy suspension 3, standing for 10min in a refrigerator, and then swirling for 30s, wherein the repeated process is carried out for 3 times, and the volume ratio of the protein extraction reagent to the heavy suspension 3 is 1: (0.5-2), in order to achieve better effect, the volume ratio of protein extraction reagent to resuspension 3 was chosen to be 1:1, a step of;

(2) After centrifugation at 13200 Xg for 5min, the supernatant was taken, which was the serum lung-derived protein solution extracted.

3. Extracting serum lung-derived RNA;

(1) Adding an RNA extraction reagent 1 into the heavy suspension 3, and then carrying out vortex oscillation for 1min and then standing for 10min at room temperature, wherein the volume ratio of the RNA extraction reagent 1 to the heavy suspension 3 is (4-6): 1, in order to achieve better effect, the volume ratio of the RNA extraction reagent 1 to the resuspension 3 is selected to be 5:1, a step of;

(2) Then adding an RNA extraction reagent 2, standing for 5min at room temperature, and centrifuging for 15min at a speed of 12000g/min to obtain colorless supernatant, wherein the volume ratio of the RNA extraction reagent 1 to the RNA extraction reagent 2 is (3-5): 1, in order to obtain a higher supernatant, the volume ratio of the RNA extraction reagent 1 to the RNA extraction reagent 2 was selected to be 4:1, a step of;

(3) Adding an RNA extraction reagent 3 into the colorless supernatant, transferring the supernatant to an adsorption column, placing the adsorption column into a collecting pipe, centrifuging for 15s at a speed of 12000g/min, removing filtrate, and repeating until all the colorless supernatant is treated, wherein the volume ratio of the RNA extraction reagent 3 to the colorless supernatant is (1-2): 1, in order to obtain a better effect, the volume ratio of the RNA extraction reagent 3 to the colorless supernatant was selected to be 1.5:1, a step of;

(4) Adding 500 μl of RNA washing buffer 1, centrifuging at 12000 Xg for 15s at room temperature, and washing the adsorption column;

(5) Adding 500 μl of RNA washing buffer 2, centrifuging at 12000 Xg for 2min at room temperature, and washing the adsorption column;

(6) 15 μl of deionized water was added to the forgetting adsorption column, and the mixture was centrifuged at 12000 Xg for 2min at room temperature to obtain RNA.

4. Detecting exosome by using an electron microscope;

And (3) observing the size, the shape and the like of the exosomes by adopting an exosome electron microscope detection technology. The specific steps are as follows:

(1) Dripping 5 μl of exosome sample onto copper mesh, and incubating at room temperature for 5min;

(2) After the incubation is finished, the water absorbing paper is used for absorbing the redundant liquid on one side;

(3) Dropwise adding 2% uranyl acetate into a copper wire, and incubating for 1min at room temperature;

(4) After the incubation is finished, the water absorbing paper is used for absorbing the redundant liquid on one side;

(5) Drying at room temperature for about 20min, and observing on a machine;

(6) Instrument information used for the test:

instrument name: nano transmission electron microscope detector

Production company: FEI;

Instrument model: tecnai G2 Spirit BioTwin;

acceleration voltage setting: 80Kv.

The detection results are shown in FIG. 1.

5. Detecting the particle size of exosomes;

And determining the dispersivity, the particle size and the distribution of the exosomes by adopting a nanoparticle tracking analysis technology. The specific steps are as follows:

(1) Taking a frozen sample, thawing in a water bath at 25 ℃, and placing on ice;

(2) Mounting nanopore: nanopore is arranged on qNano, and stretch is adjusted to be about 47mm by rotating a handle;

(3) Wetting nanopore: mu.L of filtered Wetting solution was carefully added to the lower fluid cell to ensure no bubbles were generated. Upper fluid cells were installed and capped. The mixture was brought to-20 mPa by VPM and was left to stand for 5min.

(4) Adjusting the baseline current: wetting solution in lower fluid cell was replaced with 75 μl of electrolyte and 35 μl of electrolyte was added to nanopore through upper fluid cell. VPM was adjusted to 20 mPa and the voltage to 0.1V for 5min. Then the voltage is regulated to be about 140nA, and the current is waited for 5-10min;

(5) Coating nanopore: replacing the electrolyte in the lower fluid cell with 75 mu LCoating solution, adding 35 mu L Coating solution to nanopore via upper fluid cell, covering with a protective cap, adjusting VPM to 20mPa, and waiting for 5min;

(6) Repeating the step 3;

(7) Determining calibration particles and samples: and 35 μl of calibration particle solution was added to nanopore via upper fluid cell, the VPM was set to 2mPa, and after the current stabilized and particles passed, the data recording was started. The pressure was adjusted to 5mPa and recorded again. The samples are the same procedure;

(8) Clicking ANALYSE DATE to open a file to be analyzed, clicking unprocesswd files on the right button, and processing the file;

(9) Selecting a sample and calibration particles with corresponding pressure, analyzing and generating an image, previewing a report and storing the report;

(10) Information of the instrument used for the test:

instrument name: qNano nanoparticle analyzer;

production company: iZON Science;

Instrument model: qNano Gold;

analysis software version: izon Control Suite V3.3.2.2001.

The detection results are shown in FIG. 2.

6. Western Blot detection of exosomes;

And (3) taking the exosome heavy suspension 3, adding an equal volume of RIPA (strong) lysate for cracking, extracting protein, and detecting exosome markers by Western blot. The specific steps are as follows:

(1) And (3) glue preparation: according to the molecular weight of the target protein, a 1.5mm glass plate and a 15-hole sample comb are used for preparing a piece of 10% separating gel and 5% concentrating gel;

(2) Sample loading;

(3) Electrophoresis: performing electrophoresis with voltage stabilization of 80v until Loading Buffer (indicator) enters into the separation gel, changing into voltage stabilization of 120v, and continuing electrophoresis until Loading Buffer (indicator) reaches the bottom of the gel, and stopping electrophoresis;

(4) Transferring: PVDF film with aperture of 0.22 μm is selected, constant current is 200mA, and film transferring time is 90min;

(5) Closing: diluting 5% skimmed milk powder with PBST, sealing for 1 hr, and washing with PBST for 3 times each for 10min;

(6) Incubating primary antibodies: placing the membrane into a hybridization box, adding corresponding antibody, and placing the hybridization box at a decolorizing shaker for overnight at 4 ℃;

(7) Incubating a secondary antibody: the hybridization cassette was slowly shaken on a shaker to restore room temperature. Removing the primary antibody, washing the membrane 3 times by using PBST (poly (butylene glycol) for 10min each time, putting the secondary antibody and the membrane into a hybridization box again, putting the hybridization box on a shaking table, slowly shaking, and incubating for 1h at room temperature;

(8) Removing the secondary antibody, and washing the membrane 3 times by using PBST for 10min each time;

(9) Exposure: an appropriate amount of ECL luminescence was added and the film was photographed using a digital imager.

The detection results are shown in FIG. 3.

7. Quantitative detection of exosome protein BCA;

(1) Diluting the sample;

(2) Configuring a standard curve with BSA;

(3) Preparing BCA working solution according to BCA kit requirements;

(4) Adding the diluted sample into a 96-well ELISA plate, and adding BCA working solution (sample: working solution=1:8);

(5) Oven at 37 ℃ for 12min;

(6) The concentration of the protein was measured by an enzyme-labeled instrument, and the measurement results are shown in Table 1.

TABLE 1 protein BCA quantitative results

8. Quantitatively detecting lung-derived RNA;

1. Mu.L of RNA was quantitatively detected by denovix ultra-micro spectrophotometer, and the detection results are shown in Table 2.

Table 2 RNA quantitative results

	Sample name	Denovix concentration (ng/. Mu.L)	Volume (mu L)	Total amount (ng)	A260/A280
						1	Exosome	25.23	15	378.5	1.831
2	Magnetic bead enrichment	7.12	10	71.2	1.449

In the above scheme, the enriching reagent 1 comprises 15 parts of PEG20000 with the concentration of 25%, 10 parts of PEG10000 with the concentration of 20%, 2 parts of Tween 20 with the concentration of 0.05%, 2 parts of potassium dihydrogen phosphate solution, 2 parts of disodium hydrogen phosphate solution, 2 parts of sodium chloride solution and 2 parts of potassium chloride solution, the enriching reagent 2 comprises 10 parts of PEG20000 with the concentration of 20%, 10 parts of PEG8000 with the concentration of 20%, 10 parts of glucose solution and 5 parts of sodium acetate solution, and the enriching reagent 3 comprises 1 part of Protein A/G magnetic beads coated with SFTPC antibody; the protein extraction reagent group comprises RIPA strong lysate; the RNA extraction reagent 1 comprises Trizol lysate, the RNA extraction reagent 2 comprises chloroform with the concentration of 100%, the RNA extraction reagent 3 comprises ethanol with the concentration of 100%, the RNA cleaning buffer solution 1 is 3 parts of RWP buffer solution, and the RNA cleaning buffer solution 2 is 2 parts of RPE buffer solution.

Examples 2 to 5

The specific embodiment corresponds to example 1, except that the following tables 3 and 4 are shown.

Comparative example 1

The specific embodiment corresponds to example 1, except that the amount of 25% peg20000 was changed as shown in tables 3 and 4 below.

Comparative example 2

The specific embodiment was identical to example 1, except that the amount of disodium hydrogen phosphate solution was varied as shown in tables 3 and 4 below.

Comparative example 3

The specific embodiment corresponds to example 1, except that the amount of 20% peg20000 was changed as shown in tables 3 and 4 below.

Comparative example 4

The specific embodiment corresponds to example 1, except that the amount of the glucose solution was changed as shown in tables 3 and 4 below.

Comparative example 5

The specific embodiment corresponds to example 1, except that no enrichment reagent 1 was used, as shown in tables 3 and 4 below.

Comparative example 6

The specific embodiment corresponds to example 1, except that no enriching reagent 3 was used, as shown in tables 3 and 4 below.

TABLE 3 enrichment of the Components of reagent 1

TABLE 4 enrichment of the Components of reagents 2, 3

	20％PEG20000	20％PEG8000	Glucose solution	Sodium acetate solution	Protein A/G magnetic beads
						Example 2	5	15	5	8	0.1
Example 3	15	5	15	2	5
						Example 4	8	12	8	7	0.5
Example 5	12	8	12	3	4
						Comparative example 1	10	10	10	5	1
Comparative example 2	10	10	10	5	1
						Comparative example 3	20	10	10	5	1
Comparative example 4	10	10	0	5	1
						Comparative example 5	10	10	10	5	1
Comparative example 6	10	10	10	5	0

Comparative example 7

The specific embodiment corresponds to example 1, except that the RNA extraction reagent set is RNA extraction reagent 1 and RNA extraction reagent 2.

Comparative example 8

Experiments were performed using commercially available exosome extraction reagents.

Kit cargo number: cat# EXORG50 0A-1, mainly comprising exosome extraction reagent (Exosome Extraction Reagent), sample dilution (sample solution), purification column/1.5 ml collection tube (Collection Tubes 1.5.5 ml).

Exosome extraction is carried out according to the instruction of the kit, and the specific operation steps are as follows:

1. sample pretreatment

Thawing the frozen sample in a water bath at room temperature or 25 ℃, and placing the completely thawed sample on ice; for fresh samples, samples were collected and placed on ice, centrifuged at 3,000Xg for 15min at 4℃to remove cells or cell debris, and the supernatant was aspirated into a new tube after centrifugation.

2. Precipitation exosomes

Mu.l (at least 100. Mu.l) of the treated serum was aspirated, placed in a 1.5mL centrifuge tube (kit not provided), an equal volume (250. Mu.l) of sample diluent was added, mixed well, then 125. Mu.l of exosome extraction reagent was added, inverted and mixed well, and then allowed to stand at 4℃for at least 30min. After standing, the sample was centrifuged at 1,500×g at 4℃for 30min, white precipitate was seen at the bottom of the tube, the supernatant was aspirated off, and the residual liquid was completely removed by centrifugation at 1,500×g at 4℃for 5min, taking care not to destroy the exosome precipitate.

3. Exocrine weight suspension

Adding 100ul of sample diluent or corresponding buffer solution required by downstream application, repeatedly blowing or vortex mixing with a pipette, thoroughly dissolving precipitate, and collecting the whole exosome.

4. Exosome purification

The resuspended exosomes were transferred to a purification column, placed in 1.5ml collection tubes (provided by the kit, previously placed), centrifuged at 2,000Xg for 5min at 4 ℃, the purification column discarded, and the exosomes extracted in the collection tubes. The extracted exosomes can be used directly in downstream experiments (e.g. particle size analysis, nucleic acid extraction, etc.), or stored at 2-8 ℃ for one week, or at-80 ℃ for about three months.

Experimental example

The methods provided in example 1, example 3 and comparative examples 1-8 are used for extracting lung-derived proteins and RNA from serum samples of 20 lung cancer patients and 20 healthy people, and the detection results of example 1 are shown in FIG. 4 in combination with ELISA experiments for detecting lung cancer disease markers NSE and CEA. miR-21-5p, miR-141 and miR-23a are detected by combining an RT-PCR experiment, and the detection result of example 1 is shown in FIG. 5.

The specific steps are as follows:

1. Enriching the blood lung-heat source exosome;

the experimental methods of examples and comparative examples are substantially identical to those described above, except that the composition tables of the enrichment reagents 1, 2,3 are shown.

2. Extracting blood lung-heat-clearing source protein and RNA;

The experimental procedure was the same as in the examples.

3. ELISA is used for detecting the content of specific proteins;

the experimental method is specifically referred to the instruction book of the kit.

4. QPCR (quantitative polymerase chain reaction) detection of miRNA expression quantity;

(1) First strand cDNA synthesis;

RNA was removed from the-80℃refrigerator, thawed at 4℃and then the following reaction solution was prepared in a 0.2ml PCR tube. The PCR tube was placed in a PCR apparatus, and the procedure was run: incubation was carried out at 37℃for 15min, denaturation at 98℃for 5min and incubation at 4 ℃.

TABLE 5 reverse transcription reaction system

Total RNA	1μg
		5xRTBuffer	2μL
RTEnzymeMix	0.5μL
		PrimerMix	0.5μL
H2O	XμL
		RNAtmplate	XμL
Total volume	20μL

(2)SYBRGreenqPCR；

The following reaction solution was prepared in a 0.2ml PCR tube. The reaction solution was added to a 96-well plate, and the 96-well plate was placed in an ABI7500 real-time fluorescent quantitative PCR instrument. Running a program: incubating at 50 ℃ for 2min;95 ℃ for 10min;40 cycles: 95 ℃,15 seconds, 60 ℃ and 1min; dissolution profile.

TABLE 6 PCR reaction system

2×SYBRGreenPCRbuffer	10μL
		Forwardprimer(10μM)	0.5μL
Reverseprimer(10μM)	0.5μL
		Template	10ng
ddH2O	XμL
		Total volume	10μL

(3) Reagents and apparatus;

Reverse transcription reagent: TOYOBOReverTraAceqPCRRTKit;

Quantitative PCR reagents: ABIPowerSYBRGreenPCRMasterMix;

primer synthesis: shanghai Bioengineering Co., ltd;

quantitative PCR instrument: ABI7500 real-time fluorescence quantitative PCR system.

The results of the tests of example 3 and comparative examples 1 to 8 are shown in tables 7 and 8 below:

TABLE 7 results of healthy person sample experiments

TABLE 8 results of lung cancer patient sample experiments

From the experimental results, it can be seen that:

The ratio of the reagents used in example 3 was not an optimal combination, and thus had an influence on the quantitative results of protein and nucleic acid, and the yield was only about 50% of the results in example 1. In ELISA detection results of lung cancer samples, the protein detection level in example 1 was 8-12ng/ml, while the concentration in example 3 was only half that in example 1, with a protein concentration of about 5 ng/ml. The comparison between the PCR quantitative detection result and the protein detection result can obtain the same conclusion.

The changes in experimental conditions for the single factors of comparative examples 1-4 all had different degrees of influence on the results of marker detection. Although in the detection results of healthy samples, comparative examples 1 to 4 were very close to the detection results of the markers in example 1, this was due to the low level of the markers in the healthy samples themselves. In the lung cancer sample detection results, the concentration of the protein markers (NSE and CEA) in comparative examples 1-4 is in the range of 2.5-5.5ng/ml, which is far lower than the protein detection level of 8-12ng/ml in example 1. The relative expression quantity of miRNA markers (miR-21-5 p, miR-141 and miR-23 a) is 0.8-4.4, which is far lower than the relative expression quantity of miRNA in example 1 by 5.9-8.2, so that the change of a single factor in the invention has great influence on the scheme of the invention, and the requirement of the scope of the invention is ensured to be met when the method is used, so that the excellent detection effect of the invention can be achieved.

In comparative example 5, because no enrichment reagent 1 was used, the non-ideal state of too little initial exosome enrichment total amount occurred, the protein content and RNA were low, the total protein amount did not exceed 1ug, the nucleic acid was only about 5ng, the detection of the marker was indirectly affected, the protein detection level of the lung cancer sample was only 1ng/ml, the relative expression level of miRNA was also only about 1, and the detection result was not as ideal as the detection data of example 1.

The use of the enrichment reagent 3 was absent in comparative example 6, and lung-origin specific exosomes were not obtained, thus giving a negative result.

The RNA extraction reagent described in comparative example 7 showed normal protein quantification results, whereas the nucleic acid quantification results were significantly lower, only about 8ng, whereas the total amount of nucleic acid in example 1 could reach 60ng. Too low total nucleic acid directly influences the detection level of miRNA markers, the relative expression level of miRNA is only about 1, and the detection result is not ideal as that of the detection data of the embodiment 1.

A commercially available kit was prepared using the exosome in comparative example 8, and it was found that a negative result was found through experiments. The detection level of the lung cancer sample protein is not more than 2ng/ml, the relative expression level of miRNA is not more than 2, and the detection result is not ideal as that of the detection data of the embodiment 1.

According to the experiment, the method has the characteristics of simple operation and strong practicability, and in the embodiment 1, the total exosomes can be rapidly extracted from the serum sample by the composite reagent with a specific proportion, and the coprecipitation pollution of non-exosome proteins and other impurities in the exosome extraction process is reduced, so that the purity and the yield of the exosomes are greatly improved. In addition, the application of the specific antibody and the matched use of the extraction reagent can conveniently and accurately extract the lung source protein and RNA, and the lung cancer can be diagnosed by the specific protein and RNA obtained by the invention.

In the comparative example, the ratio of the compound reagent is greatly changed, so that the extraction effect of exosomes is obviously influenced, the enrichment purity of exosomes from a subsequent lung source and the extraction detection results of protein and nucleic acid are indirectly influenced, and a negative result is presented. In addition, commercial kits cannot be directly accepted with subsequent lung-derived exosome enrichment in the present invention due to uncertainty in reagent composition, and a negative result appears. Since the comparative example is not within the scope of the present invention, the intended detection effect cannot be achieved.

Meanwhile, it is known through experiments that: for exosome yield, the total exosome concentration of the combined use of the reagents can reach 11 orders of magnitude per milliliter, whereas the conventional method only has about 10 orders of magnitude. Compared with the understanding range of exosomes, the exosomes prepared by the method have the advantages that the grain size range is concentrated, the report of the literature is met, the grain size uniformity is good, and the effect is far superior to that of the exosomes prepared by the traditional method: the exosome range span of the preparation is large and as small as 20nm and as large as 300nm, and the exosome range span exceeds the cognition reported in the traditional literature. In addition, compared with the detection result of an electron microscope, the compound reagent provided by the invention has the advantages that the prepared exosome has clean background and no obvious impurities, the quantity and the structure are good, and the exosome is in a vesicle shape. The exosomes prepared by the traditional method are easy to aggregate and damage. The exosome purity is estimated by calculating the ratio of the particle number to the protein amount, the ratio of the number to the total protein amount is high and can reach 200, and the ratio is far higher than that of the traditional method by 50-100, so that the exosome purity enriched by the enrichment reagent is higher. Finally, the exosomes prepared by the method are detected by combining a Western Blot technology to obtain specific high-abundance proteins Albumin and IgG (L), so that the method has a more remarkable high-abundance removal effect, and the combined use effect of reagents is better.

Finally, it is to be understood that the above embodiments are merely exemplary embodiments employed for the purpose of illustrating the principles of the present invention, however, the present invention is not limited thereto. Various modifications and improvements may be made by those skilled in the art without departing from the principles and spirit of the invention, and such modifications and improvements are also considered within the scope of the invention.

Claims

1. The compound reagent for extracting the lung source exosome component substances is characterized by comprising an enrichment reagent group and an extraction reagent group;

The protein extraction reagent group comprises RIPA strong lysate;

The RNA extraction reagent group comprises an RNA extraction reagent 1, an RNA extraction reagent 2 and an RNA extraction reagent 3, wherein the RNA extraction reagent 1 comprises Trizol lysate, the RNA extraction reagent 2 comprises chloroform with the concentration of 100%, and the RNA extraction reagent 3 comprises ethanol with the concentration of 100%.

2. The composite reagent according to claim 1, wherein the enriching reagent 1 comprises 13-18 parts by weight of PEG20000 having a concentration of 25%, 8-12 parts by weight of PEG10000 having a concentration of 20%, 1.5-3 parts by weight of tween 20 having a concentration of 0.05%, 1.5-3 parts by weight of potassium dihydrogen phosphate solution, 1.5-3 parts by weight of disodium hydrogen phosphate solution, 1.5-3 parts by weight of sodium chloride solution and 1.5-3 parts by weight of potassium chloride solution, the enriching reagent 2 comprises 8-12 parts by weight of PEG20000 having a concentration of 20%, 8-12 parts by weight of PEG8000 having a concentration of 20%, 8-12 parts by weight of glucose solution and 3-7 parts by weight of sodium acetate solution, and the enriching reagent 3 comprises 0.5-4 parts by weight of Protein a/G magnetic beads coated with SFTPC antibody.

3. The composite reagent according to claim 1, wherein the enriching reagent 1 comprises 15 parts by weight of PEG20000 with a concentration of 25%, 10 parts by weight of PEG10000 with a concentration of 20%, 2 parts by weight of tween 20 with a concentration of 0.05%, 2 parts by weight of potassium dihydrogen phosphate solution, 2 parts by weight of disodium hydrogen phosphate solution, 2 parts by weight of sodium chloride solution and 2 parts by weight of potassium chloride solution, the enriching reagent 2 comprises 10 parts by weight of PEG20000 with a concentration of 20%, 10 parts by weight of PEG8000 with a concentration of 20%, 10 parts by weight of glucose solution and 5 parts by weight of sodium acetate solution, and the enriching reagent 3 comprises 1 part by weight of Protein a/G magnetic beads coated with SFTPC antibody.

4. A method of using the composite reagent of any one of claims 1-3, comprising the steps of:

Centrifuging the serum sample to obtain a supernatant;

adding the enrichment reagent group to obtain a compound;

Adding the extraction reagent group to obtain corresponding composition substances, wherein the composition substances comprise proteins or RNA, the proteins comprise NSE and CEA, and the RNA comprises miR-21-5p, miR-141 and miR-23a.

5. The method of claim 4, wherein the sequence of operations for adding the collection reagent set comprises the steps of:

6. The method of claim 5, wherein the volume ratio of the enriching reagent 1 to the supernatant is 1: (3-5) the volume ratio of the enriching reagent 2 to the resuspension 1 is 1: (4-6) the volume ratio of the enriching reagent 3 to the resuspension 2 is 1: (4-6).

7. The method of claim 5, wherein when the constituent material is a protein, a protein extraction reagent is selected, and the method of using the protein extraction reagent comprises the steps of: adding a protein extraction reagent into the heavy suspension 3, placing in a refrigerator, swirling, repeating for three times, centrifuging, and taking supernatant to obtain the protein.

8. The method of claim 5, wherein the volume ratio of protein extraction reagent to resuspension 3 is 1: (0.5-2).

9. The method according to claim 5, wherein when the constituent substance is RNA, a RNA extraction reagent set is selected, and the method of using the RNA extraction reagent set comprises the steps of:

and adding an RNA extraction reagent 3 for adsorption to obtain RNA.

10. The method according to claim 9, wherein the volume ratio of RNA extraction reagent 1 to the resuspension 3 is (4-6): 1, the volume ratio of the RNA extraction reagent 1 to the RNA extraction reagent 2 is (3-5): 1, a step of; the volume ratio of the RNA extraction reagent 3 to the colorless supernatant is (1-2): 1.