CN114381431A - Method for increasing content of exosomes released by cells - Google Patents
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Abstract
The invention relates to the technical field of engineered exosomes, and discloses a method for increasing the content of exosomes released by cells, which comprises the following steps: 1) treating a cancer cell line; 2) filtering the treated cancer cell supernatant by using a 0.45um filter membrane, and collecting the filtered liquid; 3) centrifuging at 120000g for 90min to extract exosome; 4) collecting exosomes; 5) performing each assay, wherein cancer cell line treatment comprises the steps of: 1) replacing the cancer cells with fresh culture medium; 2) irradiating the cancer cells for 1h by UVB, and paying attention to the state of the liver cancer cells at any time; 3) placing the cancer cells in an incubator for 2 h; 4) placing the cancer cells in an anoxic device for 1 h; 5) cancer cell supernatants were collected after 24 h. The invention can improve the release amount of exosome without changing cell genetic materials, is suitable for various cell lines, is simpler than methods such as an ultrasonic method, an LED lamp irradiation method and special culture medium treatment, and can better meet the requirement of improving the release amount of exosome of various cell lines.
Description
Technical Field
The invention relates to the technical field of engineered exosomes, in particular to a method for increasing the content of exosomes released by cells.
Background
The exosome is a micro vesicle with a lipid bilayer membrane structure, has the diameter of 30-150nm, can be secreted by almost all cells, can mediate substance exchange and information exchange among the cells, and researches show that the exosome carries various cancer promotion genes or cancer inhibition genes.
The biomedicine treatment technology based on exosomes is mature day by day, for example, the treatment drug is wrapped by utilizing the vesicle structure of the exosomes, the disease can be directly treated by targeting the focus, and compared with the prior method which widely applies liposome drug loading, the exosome drug loading has the advantages of good biocompatibility, strong stability, low immune rejection, easy absorption by cells and the like, but the extraction condition of the exosomes is harsh and the cost is high, so the method for stimulating the yield of the exosomes, which is simple, convenient, rapid and low in cost, has important social and economic significance and clinical value.
Exosomes are a new science hotspot in recent years, a plurality of mechanisms of the secretion process are not clear, the regulation and control of the secretion of exosomes in cells are a complex process, and the methods for improving the release amount of the exosomes in cells without changing gene expression comprise a low-intensity pulse ultrasound stimulation method, an LED different light irradiation stimulation method and a special culture medium treatment method, but the methods are complex to operate and poor in applicability to different cells, so that a method for improving the content of the exosomes released by the cells is provided to solve the problems.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a method for improving the content of exosomes released by cells, which is realized by exogenous stimulation, can obviously improve the release amount of exosomes under the conditions of not changing the genetic substances of the cells and not influencing the growth of the cells, does not influence the detection of downstream NTA, WB, electron microscope and the like, has simple and rapid operation, and can be applied to various cell lines.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme: a method of increasing the amount of exosomes released by a cell, comprising the steps of:
1) treating a cancer cell line;
2) filtering the treated cancer cell supernatant by using a 0.45um filter membrane, and collecting the filtered liquid;
3) centrifuging at 120000g for 90min to extract exosome;
4) collecting exosomes;
5) and (5) carrying out various detections.
Preferably, the cancer cell line treatment comprises the following steps:
1) replacing the cancer cells with fresh culture medium;
2) irradiating the cancer cells for 1h by UVB, and paying attention to the state of the liver cancer cells at any time;
3) placing the cancer cells in an incubator for 2 h;
4) placing the cancer cells in an anoxic device for 1 h;
5) cancer cell supernatants were collected after 24 h.
Preferably, the cancer cell line is any one of a liver cancer cell line, a colorectal cancer cell line, an oral cancer cell line and a stomach cancer cell line.
Preferably, the tests in the step 5) comprise NTA test, transmission electron microscope observation and Wb test.
Preferably, the transmission electron microscope observation of the exosome sample specifically comprises the following steps:
(1) taking out 10 mu L of exosome;
(2) sucking 10 μ L of sample, dripping on copper net, precipitating for 1min, and sucking away the supernatant with filter paper;
(3) dripping 10 μ L uranyl acetate on copper net for precipitation for 1min, and removing the floating liquid with filter paper;
(4) drying for several minutes at normal temperature;
(5)100kv was detected and imaged by electron microscopy (Hitachi, HT-7700).
Preferably, the Wb detection specifically includes the following steps:
(1) extracting protein;
(2) protein concentration quantification:
determining the sample protein concentration using a BCA protein concentration assay kit;
(3) SDS-PAGE electrophoresis:
1) sample treatment:
determining the sample loading amount according to the concentration of the sample, and ensuring that the total protein loading amount of each sample is 50 mu g;
adding proper amount of 5 times protein loading buffer solution into the protein sample, and boiling water bath at 95-100 ℃ for 5 min;
2) preparing glue and loading sample:
firstly, preparing separation glue, adding TEMED, immediately shaking up and pouring glue. Slowly adding 1ml of isopropanol on the glue surface to flatten the glue surface, pouring off the isopropanol on the upper layer of the glue surface after about 20min, and sucking the residual isopropanol with absorbent paper;
② preparing concentrated glue, adding TEMED and shaking up immediately for glue pouring. Filling the rest space with concentrated glue and then inserting a comb into the concentrated glue;
putting the electrophoresis frame into an electrophoresis tank, adding electrophoresis buffer solution, pulling out a comb, and adding a sample into the sample hole;
performing constant-pressure electrophoresis according to the concentration glue of 80V and the separation glue of 120V until the bromophenol blue reaches the lower edge of the gel plate;
(4) film transfer:
1) preparing membrane transfer filter paper and a PVDF membrane, wherein the PVDF membrane is activated by methanol before use, and the activation time is 3 min;
2) placing a 'sandwich' structure of a rotating film according to the directions of an anode and a cathode, sequentially arranging a rotating film sponge, 3 layers of filter paper, a PVDF film, glue, 3 layers of filter paper and a rotating film sponge from the anode to the cathode, and removing bubbles in each layer in the placing process;
3) the membrane is transferred constantly at 300mA, and the membrane transferring time is adjusted according to the molecular weight of the target protein;
(5) antibody incubation:
1) adding the transferred membrane into a closed liquid chamber, and sealing for 1h at a high temperature;
2) removing the blocking solution, adding primary antibody diluted with primary antibody diluent at 4 deg.C overnight (primary antibody concentration range of 0.5-2 ug/ml);
3) recovering primary antibody, washing with TBST for three times, each time for 5 min;
4) the diluted secondary antibody was added, incubated at room temperature for 30min, and washed four times with TBST on a shaker at room temperature, 5min each (secondary antibody dilution ratio 1: 10000) (ii) a
(6) And (3) chemiluminescence detection:
1) dripping a freshly prepared ECL mixed solution (A: B ═ 1:1) to the protein surface side of the membrane, and exposing in a dark room;
2) and adjusting exposure conditions according to different light intensities, developing and fixing.
(III) advantageous effects
Compared with the prior art, the invention provides a method for improving the content of exosomes released by cells, which has the following beneficial effects:
the invention can improve the release amount of exosome without changing cell genetic material, is suitable for various cell lines, can improve the yield of the exosome of HePG2 (liver cancer cell line) from 6.6E +11Particles/mL to 1.3E +12Particles/Ml, can improve the yield of the exosome of SW480 (colorectal cancer cell line) from 5.6E +11Particles/mL to 1.0E +12Particles/Ml, can improve the yield of the exosome of CAL-27 (oral cancer cell line) from 6.5E +11Particles/mL to 1.1E +12Particles/Ml, can improve the yield of the exosome of MGC-823 (gastric cancer cell line) from 5.3E +11Particles/mL to 9.1E +11Particles/Ml, and can more simply meet the release amount of various cell lines compared with the methods such as an ultrasonic method, an LED lamp irradiation method, culture medium treatment and the like.
Drawings
FIG. 1 is a schematic flow diagram of the present invention;
FIG. 2 is a schematic diagram of the results of NTA detection according to the present invention;
FIG. 3 is a schematic diagram of the results of transmission electron microscopy measurements according to the present invention;
FIG. 4 is a diagram showing the results of Wb detection according to the present invention.
Detailed Description
The main materials and reagents used in the invention are as follows:
0.45um filter (Jinteng), CD9\ TGS101 antibody (Abcam), cell culture medium, centrifuge (Hunan instrument).
The invention can meet the requirement of various cell lines on improving secretion of exosomes, so the experiment is synchronously completed by selecting HePG2 (liver cancer cell line), SW480 (colorectal cancer cell line), CAL-27 (oral cancer cell line) and MGC-823 (gastric cancer cell line).
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
referring to fig. 1, the method for increasing the content of exosomes released from cells in the present invention comprises the following steps:
1) replacing the fresh culture medium for the liver cancer cells;
2) irradiating the liver cancer cells for 1h by using UVB, and paying attention to the state of the liver cancer cells at any time;
3) placing the liver cancer cells in an incubator for 2 h;
4) placing the liver cancer cells in an anoxic device for 1 h;
5) collecting the liver cancer cell supernatant after 24 h;
6) filtering the treated liver cancer cell supernatant by using a 0.45um filter membrane, and collecting the filtered liquid;
7) centrifuging at 120000g for 90min to extract exosome;
8) collecting exosomes;
9) and (5) carrying out various detections.
Example 2:
referring to fig. 1, the method for increasing the content of exosomes released from cells in the present invention comprises the following steps:
1) replacing the colorectal cancer cells with fresh culture medium;
2) irradiating the colorectal cancer cells for 1h by UVB, and paying attention to the state of the colorectal cancer cells at any moment;
3) placing the colorectal cancer cells in an incubator for culturing for 2 h;
4) placing the colorectal cancer cells in an anoxic device for 1 h;
5) collecting the colorectal cancer cell supernatant after 24 hours;
6) filtering the treated colorectal cancer cell supernatant by using a 0.45um filter membrane, and collecting the filtered liquid;
7) centrifuging at 120000g for 90min to extract exosome;
8) collecting exosomes;
9) and (5) carrying out various detections.
Example 3:
referring to fig. 1, the method for increasing the content of exosomes released from cells in the present invention comprises the following steps:
1) replacing the oral cancer cells with fresh culture medium;
2) irradiating the oral cancer cells for 1h by UVB, and paying attention to the state of the oral cancer cells at any time;
3) placing the oral cancer cells in an incubator for culturing for 2 h;
4) placing the oral cancer cells in an anoxic device for 1 h;
5) collecting the oral cancer cell supernatant after 24 hours;
6) filtering the treated oral cancer cell supernatant by using a 0.45um filter membrane, and collecting the filtered liquid;
7) centrifuging at 120000g for 90min to extract exosome;
8) collecting exosomes;
9) and (5) carrying out various detections.
Example 4:
referring to fig. 1, the method for increasing the content of exosomes released from cells in the present invention comprises the following steps:
1) replacing the fresh culture medium for the gastric cancer cells;
2) irradiating the gastric cancer cells for 1h by UVB, and paying attention to the state of the gastric cancer cells at any time;
3) placing the gastric cancer cells in an incubator for 2 h;
4) placing gastric cancer cells in an anoxic device for 1 h;
5) collecting gastric cancer cell supernatant after 24 hours;
6) filtering the treated gastric cancer cell supernatant by using a 0.45um filter membrane, and collecting the filtered liquid;
7) centrifuging at 120000g for 90min to extract exosome;
8) collecting exosomes;
9) and (5) carrying out various detections.
The prepared exosomes of examples 1-4 were subjected to NTA (dachang magnifica zetaview) detection, the detection results are shown in fig. 2, and the NTA detection results show that:
after the treatment by the method, the yield of the HePG2 (liver cancer cell line) exosome can be increased from 6.6E +11Particles/mL to 1.3E +12Particles/Ml, the yield of the SW480 (colorectal cancer cell line) exosome can be increased from 5.6E +11Particles/mL to 1.0E +12Particles/mL, the yield of the CAL-27 (oral cancer cell line) exosome can be increased from 6.5E +11Particles/mL to 1.1E +12Particles/mL, and the yield of the MGC-823 (gastric cancer cell line) exosome can be increased from 5.3E +11Particles/mL to 9.1E +11 Particles/mL.
Carrying out transmission electron microscope observation on the exosome sample, and specifically comprising the following steps:
(1) taking out 10 mu L of exosome;
(2) sucking 10 μ L of sample, dripping on copper net, precipitating for 1min, and sucking away the supernatant with filter paper;
(3) dripping 10 μ L uranyl acetate on copper net for precipitation for 1min, and removing the floating liquid with filter paper;
(4) drying for several minutes at normal temperature;
(5)100kv was detected and imaged by electron microscopy (Hitachi, HT-7700).
And (3) the transmission electron microscope imaging result is shown in figure 3.
Performing Wb detection, specifically comprising the following steps:
(1) extracting protein;
(2) protein concentration quantification:
determining the sample protein concentration using a BCA protein concentration assay kit;
(3) SDS-PAGE electrophoresis:
1) sample treatment:
determining the sample loading amount according to the concentration of the sample, and ensuring that the total protein loading amount of each sample is 50 mu g;
adding proper amount of 5 times protein loading buffer solution into the protein sample, and boiling water bath at 95-100 ℃ for 5 min;
2) preparing glue and loading sample:
firstly, preparing separation glue, adding TEMED, immediately shaking up and pouring glue. Slowly adding 1ml of isopropanol on the glue surface to flatten the glue surface, pouring off the isopropanol on the upper layer of the glue surface after about 20min, and sucking the residual isopropanol with absorbent paper;
② preparing concentrated glue, adding TEMED and shaking up immediately for glue pouring. Filling the rest space with concentrated glue and then inserting a comb into the concentrated glue;
putting the electrophoresis frame into an electrophoresis tank, adding electrophoresis buffer solution, pulling out a comb, and adding a sample into the sample hole;
performing constant-pressure electrophoresis according to the concentration glue of 80V and the separation glue of 120V until the bromophenol blue reaches the lower edge of the gel plate;
(4) film transfer:
1) preparing membrane transfer filter paper and a PVDF membrane, wherein the PVDF membrane is activated by methanol before use, and the activation time is 3 min;
2) placing a 'sandwich' structure of a rotating film according to the directions of an anode and a cathode, sequentially arranging a rotating film sponge, 3 layers of filter paper, a PVDF film, glue, 3 layers of filter paper and a rotating film sponge from the anode to the cathode, and removing bubbles in each layer in the placing process;
3) the membrane is transferred constantly at 300mA, and the membrane transferring time is adjusted according to the molecular weight of the target protein;
(5) antibody incubation:
1) adding the transferred membrane into a closed liquid chamber, and sealing for 1h at a high temperature;
2) removing the blocking solution, adding primary antibody diluted with primary antibody diluent at 4 deg.C overnight (primary antibody concentration range of 0.5-2 ug/ml);
3) recovering primary antibody, washing with TBST for three times, each time for 5 min;
4) the diluted secondary antibody was added, incubated at room temperature for 30min, and washed four times with TBST on a shaker at room temperature, 5min each (secondary antibody dilution ratio 1: 10000) (ii) a
(6) And (3) chemiluminescence detection:
1) dripping a freshly prepared ECL mixed solution (A: B ═ 1:1) to the protein surface side of the membrane, and exposing in a dark room;
2) and adjusting exposure conditions according to different light intensities, developing and fixing.
The results were analyzed as shown in FIG. 4.
The invention has the beneficial effects that:
the invention can improve the release amount of exosome without changing cell genetic material, is suitable for various cell lines, can improve the yield of the exosome of HePG2 (liver cancer cell line) from 6.6E +11Particles/mL to 1.3E +12Particles/Ml, can improve the yield of the exosome of SW480 (colorectal cancer cell line) from 5.6E +11Particles/mL to 1.0E +12Particles/Ml, can improve the yield of the exosome of CAL-27 (oral cancer cell line) from 6.5E +11Particles/mL to 1.1E +12Particles/Ml, can improve the yield of the exosome of MGC-823 (gastric cancer cell line) from 5.3E +11Particles/mL to 9.1E +11Particles/Ml, and can more simply meet the release amount of various cell lines compared with the methods such as an ultrasonic method, an LED lamp irradiation method, culture medium treatment and the like.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. A method of increasing the amount of exosomes released from a cell, comprising the steps of:
1) treating a cancer cell line;
2) filtering the treated cancer cell supernatant by using a 0.45um filter membrane, and collecting the filtered liquid;
3) centrifuging at 120000g for 90min to extract exosome;
4) collecting exosomes;
5) and (5) carrying out various detections.
2. The method for increasing the content of exosomes released from cells according to claim 1, wherein the cancer cell line treatment comprises the following steps:
1) replacing the cancer cells with fresh culture medium;
2) irradiating the cancer cells for 1h by UVB, and paying attention to the state of the liver cancer cells at any time;
3) placing the cancer cells in an incubator for 2 h;
4) placing the cancer cells in an anoxic device for 1 h;
5) cancer cell supernatants were collected after 24 h.
3. The method for increasing the content of exosomes released from cells according to claim 2, wherein the cancer cell line is any one of a liver cancer cell line, a colorectal cancer cell line, an oral cancer cell line and a stomach cancer cell line.
4. The method for increasing the content of exosomes released from cells according to claim 1 or 3, wherein each test in the step 5) comprises NTA test, transmission electron microscopy and Wb test.
5. The method for increasing the content of exosomes released by cells according to claim 4, wherein the exosome sample comprises the following specific steps:
(1) taking out 10 mu L of exosome;
(2) sucking 10 μ L of sample, dripping on copper net, precipitating for 1min, and sucking away the supernatant with filter paper;
(3) dripping 10 μ L uranyl acetate on copper net for precipitation for 1min, and removing the floating liquid with filter paper;
(4) drying for several minutes at normal temperature;
(5)100kv was detected and imaged by electron microscopy (Hitachi, HT-7700).
6. The method according to claim 4, wherein the Wb detection specifically comprises the following steps:
(1) extracting protein;
(2) protein concentration quantification:
determining the sample protein concentration using a BCA protein concentration assay kit;
(3) SDS-PAGE electrophoresis:
1) sample treatment:
determining the sample loading amount according to the concentration of the sample, and ensuring that the total protein loading amount of each sample is 50 mu g;
adding proper amount of 5 times protein loading buffer solution into the protein sample, and boiling water bath at 95-100 ℃ for 5 min;
2) preparing glue and loading sample:
firstly, preparing separation glue, adding TEMED, immediately shaking up and pouring glue. Slowly adding 1ml of isopropanol on the glue surface to flatten the glue surface, pouring off the isopropanol on the upper layer of the glue surface after about 20min, and sucking the residual isopropanol with absorbent paper;
② preparing concentrated glue, adding TEMED and shaking up immediately for glue pouring. Filling the rest space with concentrated glue and then inserting a comb into the concentrated glue;
putting the electrophoresis frame into an electrophoresis tank, adding electrophoresis buffer solution, pulling out a comb, and adding a sample into the sample hole;
performing constant-pressure electrophoresis according to the concentration glue of 80V and the separation glue of 120V until the bromophenol blue reaches the lower edge of the gel plate;
(4) film transfer:
1) preparing membrane transfer filter paper and a PVDF membrane, wherein the PVDF membrane is activated by methanol before use, and the activation time is 3 min;
2) placing a 'sandwich' structure of a rotating film according to the directions of an anode and a cathode, sequentially arranging a rotating film sponge, 3 layers of filter paper, a PVDF film, glue, 3 layers of filter paper and a rotating film sponge from the anode to the cathode, and removing bubbles in each layer in the placing process;
3) the membrane is transferred constantly at 300mA, and the membrane transferring time is adjusted according to the molecular weight of the target protein;
(5) antibody incubation:
1) adding the transferred membrane into a closed liquid chamber, and sealing for 1h at a high temperature;
2) removing the blocking solution, adding primary antibody diluted with primary antibody diluent at 4 deg.C overnight (primary antibody concentration range of 0.5-2 ug/ml);
3) recovering primary antibody, washing with TBST for three times, each time for 5 min;
4) the diluted secondary antibody was added, incubated at room temperature for 30min, and washed four times with TBST on a shaker at room temperature, 5min each (secondary antibody dilution ratio 1: 10000) (ii) a
(6) And (3) chemiluminescence detection:
1) dripping a freshly prepared ECL mixed solution (A: B ═ 1:1) to the protein surface side of the membrane, and exposing in a dark room;
2) and adjusting exposure conditions according to different light intensities, developing and fixing.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113416695A (en) * | 2021-07-20 | 2021-09-21 | 泸州君益生物医学研究有限公司 | Method for improving exosome yield of mesenchymal stem cells |
| CN114540307A (en) * | 2022-02-16 | 2022-05-27 | 多莱泌生物科技(武汉)有限公司 | Preparation method of liver-targeting engineered exosome |
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