CN110777142A - Method for promoting secretion of extracellular fluid by low-intensity pulse ultrasonic stimulation - Google Patents
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Abstract
The invention discloses a method for promoting secretion of a cell exosome by low-intensity pulse ultrasonic stimulation, which comprises the following steps: culturing cells in a 37 ℃ constant-temperature incubator, removing culture solution in a culture dish when the cells grow to 80%, adding PBS solution along the dish wall to clean the cells, replacing fetal calf serum culture solution without exosomes, wrapping the culture dish with a sealing film, performing low-intensity pulse ultrasonic stimulation for 20 minutes, removing the sealing film in a super clean bench, putting the culture dish into the 37 ℃ constant-temperature incubator to culture for 24 hours, collecting culture solution in the culture dish, separating exosomes and performing morphological identification, and performing exosome concentration detection after enriching exosomes. The method promotes the secretion of the cell exosomes through low-intensity pulse ultrasonic stimulation, solves the problem that the traditional method depends on the low yield of the cell exosomes, has low cost and easy popularization, ensures that the highest amount of the cell-secreted exosomes can reach 150-200%, has the structure and the function of normal exosomes, and is simple, convenient, safe and efficient.
Description
Technical Field
The invention relates to the field of biomedical application, relates to a method for promoting secretion of a cell exosome, and particularly relates to a method for promoting secretion of a cell exosome by low-intensity pulse ultrasonic stimulation.
Background
The exosome is a lipid bimolecular vesicle structure which is almost secreted by all cells and wraps intracellular proteins and RNA, has the diameter of 30-150 nm, is mainly derived from a multivesicular body formed by invagination of intracellular lysosome particles, is fused with a cell membrane through an outer membrane of the multivesicular body and then is released into an extracellular matrix, and plays an important information transmission mode in normal physiological cells and diseased cells. At present, the biomedical 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 current liposome drug loading widely applied, the exosome drug loading has the advantages of good biocompatibility, strong stability, low immune rejection, easy absorption by cells and the like, however, the extraction condition of the exosomes is harsh and the cost is high, so the method for stimulating the exosome yield, 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, and the regulation of the secretion of exosomes in cells is a complex process:
(1) from the viewpoint of the method of exosome extraction, the mainstream separation techniques include ultracentrifugation and polyethylene glycol precipitation. The ultracentrifugation technology separates the exosome from the liquid containing the exosome under the ultracentrifugation of 10,000 g, which needs an ultracentrifuge to be carried out, and has strict requirements on equipment and complicated procedures. The polyethylene glycol precipitation method generates precipitation by combining polyethylene glycol and exosome, and centrifugally separates exosome, but the further application of exosome is influenced due to excessive impurities and overlong extraction period.
(2) From the viewpoint of extracting raw materials of exosomes, the exosomes are cheap and convenient materials such as milk, but animal exosomes have species difference with human, whether good compatibility can be achieved needs to be verified in a large quantity, and exosomes directly extracted from mammalian cells face low production efficiency due to different cell secretion capacities, and cannot meet the requirement of mass production.
(3) From the perspective of the method for regulating exosomes, the biological method for regulating exosome secretion by genetically modifying key proteins of cell-regulated exosomes faces many technical problems which are not overcome yet, and the biological function of secreted exosomes can be influenced by the stimulation of cells to produce exosomes by chemical drugs. Although ultrasound therapy is widely used clinically, there is no research and report on the secretion stimulation of extracellular bodies and secretion mechanisms thereof by physical ultrasound.
Disclosure of Invention
The invention solves the technical problems that the existing method for extracting exosomes has strict requirements on equipment, complicated procedures, excessive impurities and overlong extraction period, and the existence of the exosomes regulated and controlled by biological and chemical methods is not yet clear, and provides the method for promoting the secretion of the exosomes by low-intensity pulse ultrasonic stimulation.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the method for promoting the secretion of the extracellular fluid by low-intensity pulse ultrasonic stimulation specifically comprises the following steps:
step 1: culturing cells in a constant-temperature incubator at 37 ℃, removing culture solution in a culture dish when the cells grow to 80%, adding PBS (phosphate buffer solution) along the dish wall to clean the cells, and replacing with fetal calf serum culture solution without exosomes;
step 2: wrapping the culture dish in the step 1 with a sealing film, performing low-intensity pulse ultrasonic stimulation for 20 minutes, removing the sealing film in a super clean bench, and putting the culture dish into a constant-temperature incubator at 37 ℃ for culturing for 24 hours; wherein the ultrasonic intensity of the low-intensity pulse ultrasonic is less than 5W/cm
2；
And step 3: collecting the culture solution in the culture dish in the step 2, ultracentrifuging to separate the exosome and carrying out morphological identification;
and 4, step 4: enriching the exosomes obtained in the step 3; wherein the ultrasonic intensity of the low-intensity pulse ultrasonic is less than 5W/cm
2。
Further, the ultrasonic intensity of the low-intensity pulse ultrasonic is 0.6-3.4W/cm
2。
Furthermore, the ultrasonic intensity of the low-intensity pulse ultrasonic is 0.6-2.1W/cm
2。
Further, the cell is derived from a mammal.
Still further, the cells include, but are not limited to, mammalian-derived cells of the lung cancer cell line SPC-A1, lung cancer cell line SPC-A1-BM, lung cancer cell line A549, or human embryonic kidney cell 293T.
Further, the exosome concentration detection employs a nanoparticle tracking analysis system (NTA).
In the invention, the low-intensity pulse ultrasound can affect the fluidity of cell membranes so as to affect cell membrane related signal transduction such as mTOR signal pathway, and the mTOR signal pathway can inhibit autophagy so as to promote secretion of exosomes.
Compared with the prior art, the invention has the beneficial effects that:
the method promotes the secretion of the exosome of the cell through low-intensity pulse ultrasonic stimulation, solves the problem of low yield of the exosome depending on the secretion of the cell in the prior art, and has low cost and easy popularization. Under the low-intensity pulse ultrasonic stimulation, the highest amount of the exosome secreted by the cells can reach 150-200%, and the exosome has the structure and the function of a normal exosome, and the method is simple, convenient, safe and efficient.
Drawings
FIG. 1 is a schematic diagram of a device for promoting secretion of extracellular bodies by low-intensity pulsed ultrasonic stimulation according to the present invention;
FIG. 2 shows the morphological examination of the extracellular secretion of SPC-A1 and SPC-A1-BM cells extracted in the examples;
FIG. 3 shows the correlation between the concentrations of extracellular secretion of SPC-A1 and SPC-A1-BM cells and the intensity of ultrasound in the examples.
FIG. 4 shows the relationship between the exosome concentration extracted from human embryonic kidney cell 293T and lung cancer cell line A549 cells and the ultrasound intensity in the examples.
Detailed Description
The invention is further illustrated with reference to the following figures and examples.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The conditions of the cell culture chamber in the following examples were 37 ℃ and 5% CO
2。
The ultrasound stimulation parameter in the following examples is center frequency 0.5MH
ZDuty Cycle (DC) of 0.5, pulse duration of 5ms, Pulse Repetition Frequency (PRF) of 100H
ZEnergy intensity (I) of sound field of ultrasonic probe
SPPA) 0.6 to 5W/cm
2The ultrasonic stimulation mode is pulse ultrasonic stimulation.
In the embodiment, low-intensity pulse ultrasound is adopted to stimulate lung cancer cell lines SPC-A1, SPC-A1-BM cells, lung cancer cell lines A549 and human embryo kidney cells 293T respectively, and the culture solution is enriched and detected for exosomes, so that the secretion amount of the cell exosomes can be increased through the low-intensity pulse ultrasound. The specific experimental steps are as follows:
(1) cells were cultured normally, plated 24 hours before stimulation, and controlled at 10 cells per 35mm dish
6Individual cells, cells attached over 24 hours.
(2) Before ultrasonic stimulation, the culture solution in the culture dishes of the experimental group and the control group is removed, 1mL of PBS solution is added along the dish wall to clean cells, the culture solution of fetal calf serum without exosome is replaced, and then the 35mm culture dish is wrapped by a sealing film to avoid cell contamination.
(3) The experimental group was sonicated for 20 minutes and the control group was in the same apparatus except that it was left without sonication for 20 minutes. After the stimulation is finished, the sealing film is removed in a super clean bench and the culture medium is placed in a constant temperature incubator at 37 ℃ for 24 hours. After 24 hours, the culture solution in the culture dish is collected to separate the exosomes.
As shown in figure 2, surface marker molecules, particle size and Transmission Electron Microscope (TEM) morphological analysis are carried out on the separated exosomes, CD9, Alix and TSG101 exosome markers are enriched in the exosomes, β -actin serving as an internal reference only exists in cells, the peak value of the particle size is between 100 and 150nm, the characteristic of the exosomes is met, and the classical exosome cup-shaped structure is observed under the TEM, so that the exosomes are successfully extracted.
(4) After the exosomes are enriched, a nanoparticle tracking analysis system (NTA) is used for detecting the exosome concentration, and the comparison of the exosome concentration level change under different conditions is shown in figures 3 and 4, so that the ultrasonic intensity is 0.6-2.1W/cm
2Or 0.6 to 3.4W/cm
2When the secretion amount of the exosome is obviously higher than that of the control group, the maximum energy intensity is 0.6W/cm
2About 200, gradually exceeds 3.4W/cm with the ultrasonic intensity
2Later, the exosome concentration was lower than the control group. All data in the statistical analysis are given as mean + standard deviation, the data are evaluated using one-way analysis of variance, and P values less than 0.05 are considered statistically significant.
In conclusion, under the low-intensity pulse ultrasonic stimulation, the highest secretion amount of the cell can reach 150-200%, and the cell has the structure and the function of a normal exosome, and the method is simple, convenient, safe and efficient.
Claims (6)
1. A method for promoting secretion of extracellular bodies by low-intensity pulse ultrasonic stimulation is characterized by comprising the following steps:
the method comprises the following steps: culturing cells in a constant-temperature incubator at 37 ℃, removing culture solution in a culture dish when the cells grow to 80%, adding PBS (phosphate buffer solution) along the dish wall to clean the cells, and replacing with fetal calf serum culture solution without exosomes;
step two: wrapping the culture dish in the first step with a sealing film, performing low-intensity pulse ultrasonic stimulation for 20 minutes, removing the sealing film in a super clean bench, and putting the culture dish into a constant-temperature incubator at 37 ℃ for culturing for 24 hours;
step three: collecting the culture solution in the culture dish in the step two, ultracentrifuging to separate exosomes, and performing morphological identification;
step four: enriching the exosomes obtained in the third step; wherein the ultrasonic intensity of the low-intensity pulse ultrasonic is less than 5W/cm
2。
2. The method for stimulating and promoting secretion of extracellular bodies according to claim 1, wherein the ultrasonic intensity of the low-intensity pulsed ultrasound is 0.6-3.4W/cm
2。
3. The method for stimulating and promoting secretion of extracellular bodies according to claim 2, wherein the ultrasonic intensity of the low-intensity pulsed ultrasound is 0.6-2.1W/cm
2。
4. A method for stimulating the secretion of exosomes according to any one of claims 1 to 3, wherein the cell is derived from a mammal.
5. The method for stimulating and promoting secretion of extracellular secretion according to claim 4, wherein the cell includes one or a combination of two or more of lung cancer cell line SPC-A1, lung cancer cell line SPC-A1-BM, lung cancer cell line A549 and human embryonic kidney cell 293T.
6. The method for stimulating the secretion of exosomes from cells according to claim 1, wherein said exosome concentration detection is performed by a nanoparticle tracking analysis system.
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Cited By (7)
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|---|---|---|---|---|
| CN112980791A (en) * | 2019-12-12 | 2021-06-18 | 中国科学院深圳先进技术研究院 | Production method of microvesicle, microvesicle obtained based on production method of microvesicle and application of microvesicle |
| CN113684130A (en) * | 2021-08-17 | 2021-11-23 | 深圳高性能医疗器械国家研究院有限公司 | Equipment and method for stimulating secretion of cell exosome, obtained exosome and application thereof |
| WO2022032337A1 (en) * | 2020-08-11 | 2022-02-17 | Royal Melbourne Institute Of Technology | Stimulating cellular production of exosomes |
| CN114107205A (en) * | 2021-11-29 | 2022-03-01 | 厦门大学附属翔安医院 | Method for stimulating cells to quickly secrete exosomes and application thereof |
| CN114672456A (en) * | 2022-03-01 | 2022-06-28 | 上海交通大学医学院附属第九人民医院 | Method for improving extracellular vesicle secretion efficiency of adipose-derived stem cells by utilizing ultrasonic stimulation and application |
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| CN116716288A (en) * | 2023-04-28 | 2023-09-08 | 四川大学 | Method for improving exosome yield by acoustic wave vibration |
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| CN112980791A (en) * | 2019-12-12 | 2021-06-18 | 中国科学院深圳先进技术研究院 | Production method of microvesicle, microvesicle obtained based on production method of microvesicle and application of microvesicle |
| WO2022032337A1 (en) * | 2020-08-11 | 2022-02-17 | Royal Melbourne Institute Of Technology | Stimulating cellular production of exosomes |
| WO2022193430A1 (en) * | 2021-03-15 | 2022-09-22 | 广州赛莱拉生物基因工程有限公司 | Preparation method for bovine placenta exosome |
| CN113684130A (en) * | 2021-08-17 | 2021-11-23 | 深圳高性能医疗器械国家研究院有限公司 | Equipment and method for stimulating secretion of cell exosome, obtained exosome and application thereof |
| CN113684130B (en) * | 2021-08-17 | 2024-04-26 | 深圳高性能医疗器械国家研究院有限公司 | Device and method for stimulating secretion of cell exosomes, exosomes obtained by method and application of exosomes |
| CN114107205A (en) * | 2021-11-29 | 2022-03-01 | 厦门大学附属翔安医院 | Method for stimulating cells to quickly secrete exosomes and application thereof |
| CN114107205B (en) * | 2021-11-29 | 2023-08-22 | 哈尔滨医科大学 | Method for stimulating cells to secrete exosomes rapidly and application of method |
| CN114672456A (en) * | 2022-03-01 | 2022-06-28 | 上海交通大学医学院附属第九人民医院 | Method for improving extracellular vesicle secretion efficiency of adipose-derived stem cells by utilizing ultrasonic stimulation and application |
| CN116716288A (en) * | 2023-04-28 | 2023-09-08 | 四川大学 | Method for improving exosome yield by acoustic wave vibration |
| CN116716288B (en) * | 2023-04-28 | 2024-04-26 | 四川大学 | Method for improving exosome yield by acoustic wave vibration |
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