CN112029705B - Method for promoting endothelial cells to produce exosomes, exosome preparation and application - Google Patents
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Abstract
The invention discloses a method for promoting endothelial cells to produce exosomes, an exosome preparation and application, wherein the method for promoting the endothelial cells to produce exosomes comprises the following operation steps: step 1: carrying out adherent culture and passage on human umbilical vein endothelial cell strains; step 2: adding panax notoginseng saponins into the culture solution of the adherent cells cultured in the step 1, and then continuously culturing in an incubator; and step 3: and (3) separating and purifying the exosome from the culture solution obtained in the step (2). The total arasaponin is added into the subculture liquid of human umbilical vein endothelial cells to induce the human umbilical vein endothelial cells to secrete exosomes, and the secretion amount is obviously improved.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a method for promoting endothelial cells to produce exosomes, an exosome preparation and application.
Background
At present, biological treatment becomes important research, development and application content in the field of biological medicine, and with the rapid development of biological cell technology and industry in the global scope, a plurality of support policies are issued successively by national governing departments and places in China to support the development of cell treatment.
Extracellular vesicles are important mediators of protein, mRNA, miRNA, and lipid transport to complete intercellular communication pathways, and are classified into three categories, including exosomes, microvesicles, and apoptotic bodies, according to their size and occurrence. Wherein the exosome is a packaging vesicle with a diameter of about 40-100nm, is secreted by various cells, and contains specific proteins, lipids, cytokines or genetic materials. Exosomes derived from different tissues not only have their specific proteins and nucleic acids, but also contain key molecules for their function.
Exosomes are membrane-enclosed vesicles of nanometer size, containing biologically active molecules, including proteins, RNA, miRNA, and the like. They act as messengers to regulate the function of both neighboring cells and remote target cells. Preclinical studies have shown that exosomes administered exogenously can, in some cases, redirect cellular activity and thus exert their therapeutic potential. Exosome-mediated delivery has several advantages: 1. are highly biocompatible and, if they are derived from appropriate cells [ e.g. Mesenchymal Stem Cells (MSCs) or immature Dendritic Cells (DC) ], are at the same time immunologically inert; 2. can cross the thick tissue barriers of human body, such as Blood Brain Barrier (BBB) and the like. Normally, exosomes can be used directly as potential therapeutic agents.
Exosomes have very broad treatment prospects and markets, but the yield is an important problem restricting the application of exosomes, and an effective technical method for improving the yield of exosomes is also searched internationally. At home and abroad, only the James Lee subject group of the chemical and biological Engineering college of State university of Ohio, USA invented a cell nano biochip, which can improve the production efficiency and nucleic acid encapsulation efficiency of exosomes in an order of magnitude, greatly surpasses the gene therapy drugs encapsulated by exosomes tested in clinical experiments at present in targeting and curative effect, and these results are published in "Nature biological Engineering" in 12 th and 16 th in 2019 (Zhaogang Yang, et al, Large-scale production of functional RNA-encapsulating exosomes via cellular nanoparticles).
Disclosure of Invention
In order to solve the above technical problems, the present invention aims to provide a method for preparing an active agent for improving the yield of an endothelial cell exosome.
In order to achieve the purpose, the technical scheme of the invention is as follows: a method for promoting endothelial cell production of exosomes, comprising the following steps:
step 1: carrying out adherent culture and passage on human umbilical vein endothelial cell strains;
step 2: adding panax notoginseng saponins into the culture solution of the adherent cells cultured in the step 1, and then continuously culturing in an incubator;
and step 3: and (3) separating and purifying the exosome from the culture solution obtained in the step (2).
The endothelial cells subcultured in the step 1 in the technical scheme are human umbilical vein endothelial cell strains.
In the technical scheme, the culture solution in the step 1 is an endothelial cell culture medium.
In the technical scheme, the concentration of the panax notoginseng saponins in the culture solution in the step 2 is 0.4mg/mL, and the culture solution is continuously cultured for 24 hours after the panax notoginseng saponins are added.
The specific steps of the exosome separation and purification in the step 3 in the above technical scheme are as follows: collecting the supernatant of the culture solution after completing the culture in the step 2, centrifuging for 10min at the temperature of 4 ℃ at 3000g, taking the supernatant after centrifugation, adding ECS reagent into the centrifuged supernatant, mixing, standing at 2-8 deg.C for 1-3 hr, wherein the volume ratio of the ECS reagent to the supernatant after centrifugation is 1:4, the supernatant is centrifuged for 60min at 10000g at 4 ℃ after standing, the supernatant is discarded after centrifugation, adding PBS solution which is 1/25 of ECS reagent dosage, uniformly blowing, beating and centrifuging the obtained precipitate, centrifuging at 12000g for 2min at 4 deg.C, collecting supernatant, transferring the supernatant into EPF column upper chamber, centrifuging at 4 deg.C for 10min at 3000g, and collecting liquid at the bottom of EPF column tube, which is purified exosome.
It is a further object of the present invention to provide an exosome-active agent prepared using the method as described above.
The invention also aims to apply the prepared exosome active preparation to the preparation of a medicament for improving the function of microvascular.
Compared with the prior art, the technical scheme of the invention has the beneficial effects that: the method has the advantages that the panax notoginseng saponins are added into the culture solution of the endothelial cells of the umbilical vein of the human to promote the endothelial cells of the umbilical vein of the human to secrete a large amount of exosomes, so that the exosomes can be prepared more conveniently, the yield is higher, the method has good specificity, the specificity is strong, and the accuracy is high.
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FIG. 1 is a standard curve prepared in the examples of the present invention.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
First, solution preparation
Preparing mother liquor with the concentration of TNF-alpha being 10 mug/mL;
because of low solubility of Panax notoginsenosides, it is necessary to slowly dissolve Panax notoginsenosides at 37 deg.C to obtain 40mg/mL total notoginsenosides solution (100mg total notoginsenosides powder added into 2.5mL endothelial cell culture medium), which is stored at 4 deg.C, and is preferably used in a short period (2-4 weeks), and no Panax notoginsenosides precipitate during each use.
Subculturing human umbilical vein endothelial cells
Taking human umbilical vein endothelial cells (purchased from cell center of basic medical research institute of Chinese medical science), performing primary culture by using an endothelial cell culture medium (ECM, produced by Sciencell, San Diego, CA), performing primary culture on the primary human umbilical vein endothelial cells for 2-3d, removing supernatant, adding PBS (phosphate buffer solution) for rinsing at least twice, adding a digestive juice (0.25% trypsin-0.02% EDTA mixed digestive juice) for digestion, wherein the addition amount of the digestive juice is preferably that of primary cells covering adherent cells, removing the digestive juice after complete digestion, supplementing the endothelial cell culture medium, and simultaneously blowing and beating the human umbilical vein endothelial cells until the human umbilical vein endothelial cells are uniformly distributed in the culture medium to obtain a subculture solution of the human umbilical vein endothelial cells.
Three, group experiment
And adding the same amount of subculture solution of the human umbilical vein endothelial cells into three culture dishes, wherein the three culture dishes correspond to a blank group, a control group and an experimental group respectively, the blank group is not treated, the control group is added with a dilution solution of a TNF-alpha solution to ensure that the concentration of TNF-alpha in the control group is 10ng/mL, the experimental group is added with a dilution solution of a panax notoginseng saponins solution to ensure that the concentration of panax notoginseng saponins in the experimental group is 0.4mg/mL, and then continuously culturing the three culture dishes for 24 hours.
Fourthly, collecting and purifying exosome
4.1 sample pretreatment
4.1.1 sampling: taking 20mL of cell culture solution supernatant from the blank group, the control group and the experimental group as samples (if the sample is frozen, the sample is unfrozen in a water bath at 25 ℃ after being taken out from a refrigerator, and the completely thawed sample is placed on ice, if the sample is fresh, the sample is collected and placed on ice);
4.1.2 centrifugation to remove cell debris: transferring each sample into a centrifuge tube respectively, centrifuging at 4 ℃ for 10min at 3000g to remove cell debris in the sample, and transferring the supernatant in each centrifuge tube into a round-bottom centrifuge tube respectively;
4.2 extraction of exosomes
4.2.1 pretreatment of the supernatant: adding 5mL of Exosome Concentration Solution (ECS reagent) into each round-bottom centrifuge tube, then covering a centrifuge tube cover tightly, uniformly mixing for 1min through a vortex oscillator, and standing for 2h at the temperature of 2-8 ℃;
4.2.2 precipitation of exosomes: centrifuging the treated round-bottom centrifuge tubes at 4 ℃ at 10000g for 60min, discarding the supernatant, and retaining the precipitate, wherein the precipitate is rich in exosome particles (note: the supernatant is completely aspirated as much as possible);
4.2.3 exosome weight suspensions: uniformly blowing 200 mu L of PBS on the precipitate obtained by centrifugation, and transferring the heavy suspension into a new 1.5mL centrifuge tube after the precipitate is uniformly suspended in the PBS;
4.2.4 harvest of exosome particles: centrifuging a 1.5mL centrifuge tube containing the resuspension at 4 ℃ at 12000g for 2min, and retaining a supernatant rich in exosome particles;
4.2.5 purification of exosomes: transferring the supernatant rich in the exosome particles into an upper chamber of an exosome purification Filter (EPF column), centrifuging at 4 ℃ for 10min at 3000g, and collecting liquid at the bottom of the EPF column after centrifugation, wherein the liquid is the purified exosome particles.
Fifthly, exosome quantification
First, a BCA protein quantification kit (manufactured by KANG SHIFT BIOLOGICAL TECHNOLOGY CO., LTD., operating according to the product instruction) was used to prepare a standard curve, and the obtained standard curve is shown in FIG. 1, wherein the regression equation is that y is 154.99x2+753.57x-11.06。
The exosome quantification is to reflect the amount of exosomes by the protein content in exosome lysate, crack the exosomes to be detected on ice by using protein lysate (RIPA lysate containing protease inhibitor and available from Shanghai Bin Yuntian biotechnology Co., Ltd. of model P0013C), detect the protein concentration in the lysate by using BCA method, calculate the concentration in the original stored exosome solution, and calculate the result as shown in Table 1.
Table 1 shows the results of quantifying exosome concentration in the blank group, control group and experimental group
From figure 1 and the table are the protein quantification standard curve and exosome quantification results, respectively. The exosome concentration extracted from the culture supernatant of different treated (blank, control, experimental) human umbilical vein endothelial cells was 327.83 μ g/mL,564.54 μ g/mL and 2515.03 μ g/mL (this is the concentration after concentration, not the concentration in the original supernatant). It can be seen that the total arasaponin induced human umbilical vein endothelial cells can significantly improve the yield of produced exosomes (7.69 times of that of the control).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (4)
1. A method for promoting the production of exosomes by endothelial cells, comprising the following operating steps:
step 1: carrying out adherent culture and passage on human umbilical vein endothelial cell strains;
step 2: adding panax notoginseng saponins into the culture solution of the adherent cells cultured in the step 1 until the concentration of the panax notoginseng saponins in the culture solution is 0.4mg/mL, and then continuously culturing in an incubator for 24 hours;
and step 3: and (3) separating and purifying the exosome from the culture solution obtained in the step (2).
2. The method for promoting endothelial cell production of exosomes according to claim 1, wherein the endothelial cells subcultured in step 1 are human umbilical vein endothelial cell lines.
3. The method for promoting endothelial cell production of exosomes according to claim 1, wherein the culture solution in step 1 is an endothelial cell culture medium.
4. The method for promoting endothelial cell to produce exosome according to claim 1, wherein the specific steps of the separation and purification of exosome in the step 3 are as follows: collecting the supernatant of the culture solution after completing the culture in the step 2, centrifuging for 10min at the temperature of 4 ℃ at 3000g, taking the supernatant after centrifugation, adding ECS reagent into the centrifuged supernatant, mixing, standing at 2-8 deg.C for 1-3 hr, wherein the volume ratio of the ECS reagent to the supernatant after centrifugation is 1:4, the supernatant is centrifuged for 60min at 10000g at 4 ℃ after standing, the supernatant is discarded after centrifugation, adding PBS solution which is 1/25 of ECS reagent dosage, uniformly blowing, beating and centrifuging the obtained precipitate, centrifuging at 12000g for 2min at 4 deg.C, collecting supernatant, transferring the supernatant into EPF column upper chamber, centrifuging at 4 deg.C for 10min at 3000g, and collecting liquid at the bottom of EPF column tube, which is purified exosome.
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| PCT/CN2021/115532 WO2022042739A1 (en) | 2020-08-31 | 2021-08-31 | Method for promoting endothelial cells to produce exosomes, and exosome preparation and application thereof |
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| CN117771348A (en) * | 2023-11-27 | 2024-03-29 | 山东省农业科学院畜牧兽医研究所 | Application of pig hip artery endothelial cell source exosome in preparation of bacterial vaccine |
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| CN107937342A (en) * | 2017-11-06 | 2018-04-20 | 中国人民解放军第四军医大学 | A kind of method that excretion body by source of endothelial cells expands neural stem cell |
| CN108823144B (en) * | 2018-05-03 | 2019-08-02 | 中国医学科学院微循环研究所 | Inhibit the excretion body active ingredient and its preparation method and application of endothelial cell migration |
| CN108753682B (en) * | 2018-05-03 | 2019-08-02 | 中国医学科学院微循环研究所 | Promote endothelial cell into the excretion body active ingredient and its preparation method and application of blood vessel |
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| CN108324735B (en) * | 2017-01-20 | 2024-02-09 | 李莉 | Exosome preparation for treating diseases and application thereof |
| CN107988153B (en) * | 2017-12-15 | 2021-05-18 | 英科博雅生命科技有限公司 | Method for separating exosome from human umbilical cord blood mesenchymal stem cell source and used reagent |
| JP7356672B2 (en) * | 2019-01-22 | 2023-10-05 | 東亞合成株式会社 | Exosome production method |
| CN112029705B (en) * | 2020-08-31 | 2021-10-08 | 中国医学科学院微循环研究所 | Method for promoting endothelial cells to produce exosomes, exosome preparation and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107937342A (en) * | 2017-11-06 | 2018-04-20 | 中国人民解放军第四军医大学 | A kind of method that excretion body by source of endothelial cells expands neural stem cell |
| CN108823144B (en) * | 2018-05-03 | 2019-08-02 | 中国医学科学院微循环研究所 | Inhibit the excretion body active ingredient and its preparation method and application of endothelial cell migration |
| CN108753682B (en) * | 2018-05-03 | 2019-08-02 | 中国医学科学院微循环研究所 | Promote endothelial cell into the excretion body active ingredient and its preparation method and application of blood vessel |
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| Antiangiogenesis Roles of Exosomes with Fei-Liu-Ping Ointment Treatment are Involved in the Lung Carcinoma with the Lewis Xenograft Mouse Model;Qi Zheng et al.;《Evidence-Based Complementary and Alternative Medicine》;20200316;全文 * |
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