CN111218420A

CN111218420A - Extraction method of bitter gourd exosomes and application of bitter gourd exosomes in preparation of antitumor drugs

Info

Publication number: CN111218420A
Application number: CN202010090930.6A
Authority: CN
Inventors: 齐素华; 马萍; 王斌; 顾兵; 王婉
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2020-02-13
Filing date: 2020-02-13
Publication date: 2020-06-02
Anticipated expiration: 2040-02-13
Also published as: CN111218420B

Abstract

The invention discloses an extraction method of bitter gourd exosomes and application of the bitter gourd exosomes in preparation of antitumor drugs, wherein the extraction steps are as follows: taking a proper amount of fresh Yunnan wild balsam pear, removing seeds, cleaning and airing; squeezing to obtain juice, centrifuging, collecting supernatant, and removing precipitate; ultracentrifuging the obtained supernatant, removing the supernatant, taking the precipitate, and suspending the precipitate in 1-2mL of phosphate buffer saline; the suspension was filtered through a 0.22 μm filter, the filtrate was centrifuged again in an ultracentrifuge, the supernatant was discarded, and the pellet was suspended in 1-2mL of phosphate buffered saline. The obtained exocrine can be used for preparing antitumor drugs. The extraction method is convenient and effective, the extraction purity is high, and the extracted bitter gourd exosomes can inhibit proliferation and migration infiltration of tumor cells, can promote apoptosis of the tumor cells, and achieve the effect of effectively treating tumor diseases.

Description

Extraction method of bitter gourd exosomes and application of bitter gourd exosomes in preparation of antitumor drugs

Technical Field

The invention relates to the field of medicines, and in particular relates to an extraction method of a bitter gourd exosome and application of the bitter gourd exosome in preparation of an anti-tumor medicine.

Background

The clinically applied antitumor drugs include chemotherapeutic drugs, biological agents and the like, but the drugs have strong killing effect on normal cells while killing tumor cells. In recent years, with the development of molecular oncology and molecular pharmacology, the research and development of antitumor drugs are shifting from traditional non-selective single cytotoxic drugs to novel drugs with multi-link effects, and the research and development of antitumor drugs have entered a new era.

Exosomes are composed of specific proteins, lipids and nucleic acid substances, carry multiple mirnas, and can bind to specific receptors on the surface of target cell membranes to regulate multiple signal pathways and regulate various physiological and pathological processes. Whether exosome can be extracted from bitter gourd and used in anti-tumor treatment is not reported.

Disclosure of Invention

One of the purposes of the invention is to provide an extraction method of bitter gourd exosomes, which can extract exosomes from bitter gourd plants.

The second purpose of the invention is to provide the application of the balsam pear exosome extracted by the method in preparing anti-tumor drugs.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: an extraction method of bitter gourd exosomes comprises the following steps:

(1) taking a proper amount of fresh Yunnan wild balsam pear, removing seeds, cleaning and airing;

(2) air drying, squeezing to obtain juice, and continuously centrifuging at 4 deg.C for 10min at 1000 g; 3000g, 20 min; 10000g, 40 min; centrifuging, taking the supernatant, and discarding the precipitate;

(3) ultracentrifuging the obtained supernatant, centrifuging for 90min at 4 deg.C and 150000g in an ultrarefrigerated centrifuge, discarding the supernatant after centrifugation, collecting the precipitate, and suspending the precipitate in 1-2mL PBS buffer solution;

(4) the suspension was filtered through a 0.22 μm filter, the filtrate was again centrifuged at 150000g for 90min at 4 ℃ in an ultrafreeze centrifuge, the supernatant was discarded after centrifugation, and the pellet was suspended in 1-2mL of PBS buffer.

The invention also provides application of the balsam pear exosome extracted by the method in preparing an anti-tumor medicament.

The invention also provides application of the momordica charantia exosome extracted by the method in preparation of a tumor cell proliferation inhibitor. Wherein the tumor cell is T98G glioma cell or HepG2 liver cancer cell.

The bitter gourd exosome is co-cultured with a human glioma cell (T98G), a normal nerve cell (HT22), a human hepatoma cell (HepG2) and a normal hepatocyte (QSG7701), and a cell counting kit (CCK-8) experiment shows that the bitter gourd exosome can obviously inhibit the proliferation of tumor cells T98G and HepG2 within the concentration range of 0.3-3 mu g/mL, and has no inhibition effect on normal cells HT22 and QSG 7701.

Under the condition that the concentration of the bitter gourd exosomes is 3 mug/mL, a cell clone formation experiment and a cell migration experiment (Transwell chamber) prove that the bitter gourd exosomes can inhibit the proliferation and migration infiltration of glioma cells of a T98G line and HepG2 human liver cancer cells; the reduction of the ratio of Bcl-2/Bax through Western blotting experiments proves that the exosome can promote the apoptosis of tumor cells.

Compared with the prior art, the invention has the following beneficial effects:

1. the method for extracting the balsam pear exosomes can extract exosomes from plant balsam pears, is convenient and effective, and has high extraction purity;

2. the bitter gourd exosome extracted by the invention can inhibit proliferation and migration infiltration of tumor cells, can promote apoptosis of the tumor cells, and achieves the effect of effectively treating tumor diseases, thereby realizing the application in preparing anti-tumor medicaments.

Drawings

FIG. 1 is a flow chart of the extraction of Momordica Charantia exosomes of example 1 of the present invention;

FIG. 2 is a transmission electron micrograph of an exosome of Momordica charantia extracted in example 1 of the present invention;

FIG. 3 is a graph showing the particle size distribution of the isolated exosomes of Momordica charantia of example 1 of the present invention;

FIG. 4 is a band diagram of the marker protein of the exosomes of Momordica charantia extracted in example 1 of the present invention;

FIG. 5 shows the activity detection CCK8 assay of tumor cells T98G, HepG2 and normal cells HT22 and QSG7701 in example 2 of the present invention;

FIG. 6 shows the T98G glioma cell clonogenic assay of example 2 of the invention: in the A picture, ctrl represents a blank control group, Exosome represents a bitter gourd Exosome group, and the B picture is a statistical chart of the number of formed cell clones;

FIG. 7 shows the HepG2 liver cancer cell clone formation experiment of example 2 of the present invention: in the A picture, ctrl represents a blank control group, Exosome represents a bitter gourd Exosome group, and the B picture is a statistical chart of the number of formed cell clones;

FIG. 8 shows the cell migration and infiltration experiment (Transwell chamber) of T98G glioma cells of example 2 of the present invention: in the A picture, ctrl represents a blank control group, Exosome represents a bitter gourd Exosome group, and the B picture is a statistical graph of the number of migration infiltrating cells;

FIG. 9 shows the cell migration and infiltration experiment (Transwell chamber) of HepG2 liver cancer cells of example 2 of the present invention: in the A picture, ctrl represents a blank control group, Exosome represents a bitter gourd Exosome group, and the B picture is a statistical graph of the number of migration infiltrating cells;

FIG. 10 shows Western blot analysis of T98G glioma cells in example 2 of the present invention: a is a Western blot detection result graph, B is a statistical graph of Bcl-2/Bax ratio;

FIG. 11 shows the Western blot analysis of HepG2 liver cancer cells in example 2 of the present invention: the C picture is a Western blot detection result picture, and the D picture is a statistical picture of the Bcl-2/Bax ratio.

Detailed Description

The invention is described in further detail below with reference to the figures and specific examples.

Example 1: extraction method of bitter gourd exosomes

Referring to the extraction process shown in figure 1, removing seeds of fresh Yunnan wild fructus Momordicae Charantiae 500g, washing for three times, each time for 1-3min, and air drying;

air drying, squeezing juice with fresh juice extractor to obtain juice (about 200 mL), centrifuging juice continuously at 4 deg.C for 10min at 1000 g; 3000g, 20 min; 10000g, 40 min; centrifuging, taking the supernatant, and discarding the precipitate;

ultracentrifuging the obtained supernatant, centrifuging for 90min at 4 deg.C and 150000g in an ultrarefrigerated centrifuge, discarding the supernatant after centrifugation, collecting the precipitate, and suspending the precipitate in 1-2mL PBS buffer solution;

the suspension was filtered through a 0.22um filter, the filtrate was again centrifuged in an ultracentrifuge at 4 ℃ and 150000g for 90min, and after centrifugation, the pellet was removed, the supernatant was discarded, and the pellet was suspended in 1-2mL of PBS buffer.

Placing the extracted bitter gourd exosomes under a transmission electron microscope to observe the form, and the steps are as follows: dripping the balsam pear exosome solution obtained by fresh low-temperature ultracentrifugation on the clean surface of the sealing film; placing the copper mesh membrane surface on the droplets of the balsam pear exosomes, suspending for 10 minutes, and slowly sucking dry by using filter paper; transferring a copper net to a 3% glutaraldehyde solution drop, suspending for 5 minutes, and sucking dry by using filter paper; transferring the copper net to the DW liquid drop for 10 times, each time for 2 minutes, and drying by using filter paper each time; transferring the copper mesh onto 4% uranyl acetate droplets, carrying out suction drying by using filter paper for 10 minutes; after natural drying (30 min), the record was observed by TEM. As a result, as shown in fig. 2, most of the exosomes extracted in this example were in a saucer-like membrane bubble structure with a clear membrane of about 120nm in an electron microscope field.

The particle size of the extracted bitter gourd exosomes is measured, the exosomes and phosphate buffer solution are diluted by 1:10 and mixed uniformly, 500 microliters of the exosomes are added into a special cuvette for detection, and the result is shown in fig. 3, wherein the sizes of the exosomes are mainly concentrated at about 120nm and belong to the range of the sizes (30-200nm) of the exosomes.

Three marker proteins of exosome, CD54, CD63 and TSG101, were determined by immunoblotting, and the exosome marker protein bands were clearly visible (as shown in FIG. 4).

Example 2: application of bitter gourd exosome in preparation of antitumor drugs

(1) Experimental Material and drug

1 experimental drug: DMEM culture medium, calf serum, 0.25% trypsin, phosphate buffer, bitter gourd exosomes, crystal violet, 4% paraformaldehyde, a western blot kit, an electron microscope observation exosome kit and a CCK-8 kit.

2 laboratory instruments and materials: the kit comprises a carbon dioxide incubator, an inverted microscope, an ultra-clean bench, an electron microscope, a culture dish, a Pasteur pipette, a centrifuge tube, a Haliotis diversicolor counting plate, a sample adding gun, a disposable gun head, a 6-hole plate, an alcohol lamp and cultured cells (HT22 hippocampal neuron cells, T98G human glioma cells, 7701 human liver cells and HepG2 human liver cancer cells which are all purchased from Shanghai cell banks of Chinese academy of sciences).

(2) Experimental procedure

1. Cellular pancreatin digestion

1.1 Wash hands with soap and wipe hands with 75% alcohol before entering sterile room.

1.2 observing the cell morphology under an inverted microscope to determine whether the cell state is good. The medium required for the experiment was preheated to 37 ℃.

1.3 disinfecting the hand before entering the super clean bench; the clean bench surface of the super clean bench is wiped with 75% alcohol solution.

1.4 the ultraviolet lamp of the super clean bench is turned on to irradiate the table-board for about 30 minutes, the exhaust fan is turned on, the ultraviolet lamp of the super clean bench is turned off, the air is cleaned, and the ozone is removed.

1.5 igniting the alcohol lamp; everything was sterilized with 75% alcohol before entering the clean bench.

1.6 the mouth of the culture medium bottle is sterilized by 75 percent alcohol, and the bottle is obliquely arranged on a frame beside an alcohol lamp after passing through the flame of the alcohol lamp.

1.7 pour off the old medium of the cultured cells. The dishes were washed with an appropriate amount of phosphate buffer solution to remove the residual old medium.

1.8 Add 1 ml pancreatin per large dish, reduce the amount of the dish, immerse the bottom cells in the enzyme solution. Visually, digestion was stopped when the bottom of the dish was seen to have cells sliding off and the enzyme solution became turbid. Typical room temperature digestion times are about 1-3 minutes.

1.9 Add a small amount of fresh medium containing serum to stop the reaction, repeatedly blow the digested cells to detach and disperse them, transfer them to a centrifuge tube with a Pasteur pipette, centrifuge for 5 minutes at 1000 rpm and discard the supernatant. 2ml of fresh medium was added, and after being blown up uniformly, the portions were transferred with a pasteur pipette into a 0.5 ml plastic centrifuge tube.

2. Counting on a Bowden counting plate

2.1 Wash the surface of the counting plates with 70% ethanol.

2.2 Wash coverslip, slightly water on the edge, then press the coverslip onto the counting plate groove and half silver counting area.

2.3 mix the cell sample well, blow vigorously to disperse all cell clumps, and then aspirate 20 microliters of sample with a pipette tip.

2.4 immediately move the cell suspension to the edge of the chamber of the blood cell counting plate, squeeze the cell suspension out, and fill the gap between the counting plate and the cover slip by capillary action. The liquid in the small chamber can be neither more nor less; the volume of the cells would otherwise change due to changes in surface tension, subject to the liquid just flowing to the edge of the counting plate recess.

2.5 according to the above operation, the other chamber of the counting plate is filled with the cell suspension.

2.6 feathering excess liquid, the counter plate was placed on the microscope stage.

2.7 select a 10X objective, focus with gridlines. If focusing is difficult due to weak contrast, the iris can be adjusted to reduce the incoming light, or the light can be slightly tilted by biasing the condenser.

2.8 move the counting plate so that the view under the mirror is exactly one large square in the center of the whole network area and is the maximum range you can see bounded by 3 parallel lines. The four corners of the large square may be slightly out of view due to the eyepiece.

2.9 the number of cells in this 1 mm square range was calculated using a finer division (again 3 parallel line divisions) and a single line grid. To avoid double counting, cells of the line were counted for left and top lines only, and right and bottom lines were not counted.

2.10 the second chamber into the field of view for the second counting.

3. Cell count CCK8 cell Activity assay

3.1 cell inoculation and treatment

3.1.1 preparation of cell suspension with cell concentration of 10X 10⁴/mL；

3.1.2 adding the prepared cell suspension into the balsam pear exosomes to ensure that the concentration of the balsam pear exosomes is 3 ng/mu L;

3.1.3 Add 100. mu.L of cell suspension per well in 96-well plates (5 replicate wells per concentration), fill the other wells without cell suspension with DI water, and incubate the plates in an incubator for 48 hours (37 ℃, 5% CO)₂)。

3.2 CCK8 Activity

3.2.1 Add 10. mu.L of CCK8 solution to each well.

3.2.2 incubate the plates in the incubator for 1-4 hours.

3.2.3 Absorbance at 450nm was measured using a microplate reader.

3.3 result processing: OD value curves were prepared using graphpad prism7 software.

3.4 repeat the experiment: the experiment was repeated 3 times independently.

4. Tumor cell clonogenic assay

4.1 conversion to a volume of 500 cells per dish based on the counted cell concentration, the volume of cell suspension was added to a 6-well plate (2 wells per cell) and the medium was added to 1 ml per dish.

4.2 the first well of each cell served as a control well, and the remaining wells received Momordica Charantia exosomes at a ratio of exosomes to culture medium of 3. mu.g/mL.

4.3 cover the dish lid, put the dish back into the carbon dioxide incubator, and culture is continued for 7 to 14 days until the cells proliferate about 50 times.

4.4 pour off the medium, aspirate off the residual medium with a gun and wash 3 times with phosphate buffer.

4.5 Per well 4% paraformaldehyde was added for 10 minutes fixation. The methanol was decanted, the residual methanol was removed by gun and washed 3 times with phosphate buffer.

4.6 adding crystal violet into each hole (only when the bottom of each hole is fully paved), and dyeing for 10 to 15 minutes.

4.7 discard the dye liquor.

5. Cell migration (Transwell chamber) assay

5.1 seeding of cells

5.1.1 cell suspension 200. mu.L was added to the Transwell chamber.

The 5.1.224 well plate lower chamber was typically filled with 500. mu.L of medium containing 15% FBS, and it was noted that bubbles were often formed between the lower layer of medium and the chamber, and once they were formed, the chemotactic effect of the lower layer of medium was reduced or even eliminated. Care is taken in the case of plating, and once the air bubbles are present, the chamber is lifted, the air bubbles removed, and the chamber is placed in the plate.

5.2 culturing cells: culturing for 12-48h (mainly according to the invasion capacity of cancer cells). 24h is common, and the time point is selected to take the invasiveness of the cells into consideration, so that the influence of the treatment factors on the cell number cannot be ignored.

5.3, fixed dyeing: the Transwell chamber was removed, the medium from the wells was discarded, washed 2 times with calcium-free PBS, the upper non-migrated cells were gently wiped off with a cotton swab, fixed with methanol or formaldehyde for 30 minutes, and the chamber was appropriately air-dried. Stained with 0.1% crystal violet for 30-60min and washed 3 times with PBS. The upper chamber was gently wiped with a cotton swab.

6. Western blot

6.1 electrophoresis

6.1.1 SDS-PAGE gel formulation: SDS-PAGE gels (split and concentrated gels) were prepared according to the instructions for the reagents.

6.2 sample treatment: an appropriate amount of concentrated SDS-PAGE protein loading buffer was added to the collected protein samples and heated in a water bath at 100 ℃ for 5 minutes to fully denature the proteins.

6.3 Loading and electrophoresis

6.3.1 after cooling to room temperature, the protein samples were loaded directly into SDS-PAGE gel wells.

6.3.2 electrophoresis is generally recommended using low voltage, constant voltage electrophoresis on the top gel and high voltage, constant voltage electrophoresis on the bromophenol blue into the bottom gel. The low voltage can be set at 80-100V and the high voltage can be set at around 120V.

6.3.3 based on the electrophoresis of the prestained protein molecular weight standards, it is expected that the electrophoresis will be stopped after the desired protein has been properly isolated.

6.4 film transfer: in the experiment, a PVDF membrane is selected, the membrane rotating current is set to be 300mA, and the membrane rotating time is set to be 30-60 minutes. The larger the molecular weight of the target protein, the longer the required transmembrane time, and the smaller the molecular weight of the target protein, the shorter the required transmembrane time.

6.5 sealing

6.5.1 after the membrane is transferred, immediately placing the protein membrane into a washing solution prepared in advance, and rinsing for 1-2 minutes to wash off the membrane transfer solution on the membrane. In all steps after the film is transferred, the moisture retention of the film must be paid attention to avoid the drying of the film, otherwise, a higher background is easily generated.

6.5.2 Add blocking solution, shake slowly on a shaker, block for 120 minutes at room temperature.

6.6 Primary antibody incubation

6.6.1 reference to the description of the Primary antibody (three exosome marker proteins CD54, CD63, TSG 101), the Primary antibody was diluted with Primary antibody diluent at the appropriate ratio

6.6.2 immediately add diluted primary antibody, incubate overnight at 4 ℃.

6.6.3 recovering the primary antibody. Adding the washing solution, and slowly shaking and washing on a side shaking table for 5-10 minutes. After the washing liquid is absorbed completely, the washing liquid is added again for washing for 5-10 minutes. The total number of washes was 3. If the result background is high, the washing time can be prolonged and the number of washing can be increased appropriately.

6.7 incubation with Secondary antibody

6.7.1 horseradish peroxidase (HRP) -labelled secondary antibodies were diluted with secondary antibody dilutions at appropriate ratios, with reference to the secondary antibody specifications.

6.7.2 diluted secondary antibody was immediately added and incubated on a side shaker for one hour at room temperature or 4 ℃ with slow shaking.

6.7.3 recovering the secondary antibody. Adding the washing solution, and slowly shaking and washing on a side shaking table for 5-10 minutes. After the washing liquid is absorbed completely, the washing liquid is added again for washing for 5-10 minutes. The total number of washes was 3. If the result background is high, the washing time can be prolonged and the number of washing can be increased appropriately.

6.8 protein detection. Bio-rad berle ChemiDoc MP chemiluminescent gel imaging system/totipotent imager western blot imaging.

(3) Results of the experiment

The results of the CCK8 experiment are shown in FIG. 5. The activity of normal cells (HT22 and QSG7701) is not reduced under the condition that the concentration of the balsam pear exosomes reaches 3 mu g/ml, while the activity of tumor cells (T98G and HepG2) is obviously reduced, which shows that the concentration of the balsam pear exosomes can reduce the activity of the tumor cells under the condition of 3 mu g/ml.

The results of the colony formation experiments are shown in fig. 6 and 7. Under the condition that the concentration of the bitter gourd exosomes is 3 mu g/ml, the clone formation numbers of the T98G glioma cells in the figure 6 and the HepG2 liver cancer cells in the figure 7 are obviously reduced, and the bitter gourd exosomes can inhibit the proliferation of tumor cells.

The results of the cell migration and infiltration experiments are shown in fig. 8 and 9. The cell migration and infiltration numbers of the T98G glioma cells in the figure 8 and the HepG2 liver cancer cells in the figure 9 are obviously reduced under the condition that the concentration of the bitter gourd exosomes is 3 mu g/ml, and the bitter gourd exosomes can inhibit the migration and infiltration of tumor cells.

The results of the western blot are shown in fig. 10 and 11. The ratio of Bcl-2/Bax of T98G glioma cells and HepG2 liver cancer cells is reduced under the condition that the concentration of bitter gourd exosomes is 3 mu g/ml, the Bcl-2/Bax ratio determines the trend of apoptosis, the Bcl-2/Bax ratio is up-regulated, and the apoptosis is inhibited; the Bcl-2/Bax ratio is reduced to promote apoptosis, and the reduction of the Bcl-2/Bax ratio indicates that the balsam pear exosome can induce tumor apoptosis.

Claims

1. A method for extracting bitter gourd exosomes is characterized by comprising the following steps:

(3) ultracentrifuging the obtained supernatant, centrifuging for 90min at 4 deg.C and 150000g in an ultrarefrigerated centrifuge, discarding the supernatant after centrifugation, taking the precipitate, and suspending the precipitate in 1-2mL phosphate buffer solution;

(4) the suspension was filtered through a 0.22 μm filter, the filtrate was again centrifuged at 150000g for 90min at 4 ℃ in an ultrafreeze centrifuge, the supernatant was discarded after centrifugation, and the precipitate was taken and suspended in 1-2mL phosphate buffered saline.

2. The application of the momordica charantia exosome extracted by the method of claim 1 in preparing an anti-tumor drug.

3. The use of an exosome extracted from Momordica charantia of claim 1 in the preparation of an inhibitor of tumor cell proliferation.

4. The use of claim 3, wherein said tumor cell is a T98G glioma cell or a HepG2 hepatoma cell.