CN115558638A

CN115558638A - Exosome prepared from placenta mesenchymal stem cells and application thereof

Info

Publication number: CN115558638A
Application number: CN202211289694.6A
Authority: CN
Inventors: 魏卿; 肖海蓉; 刘庆喜; 裴丽环
Original assignee: BOYALIFE Inc
Current assignee: BOYALIFE Inc
Priority date: 2022-10-20
Filing date: 2022-10-20
Publication date: 2023-01-03

Abstract

The invention relates to an exosome prepared from placenta mesenchymal stem cells and application thereof. In one aspect, the invention relates to exosome separated and extracted by using placenta mesenchymal stem cells, which has an average particle size of 50-200 nm and expresses membrane protein CD9 and membrane protein CD81, wherein the positive expression rate of the exosome membrane protein CD9 is more than 65%, and the positive expression rate of the membrane protein CD81 is more than 75%. The exosome method comprises the following steps: inoculating the mesenchymal stem cells into a culture bottle, culturing to make the cells adhere to the wall, and adding IL-1 beta into a culture solution for culturing; after changing the complete medium, 2%O at 37 deg.C ₂ 、5％CO ₂ Culturing in an incubator; the cells were subjected to differential centrifugation to obtain exosomes in the form of suspensions. The invention also relates to the use of the prepared exosomes in the preparation of a medicament for treating inflammatory diseases. The method of the invention has the advantages ofHigh secretion yield, high activity and the like.

Description

Exosome prepared from placenta mesenchymal stem cells and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a method for obtaining exosomes by culturing mesenchymal stem cells. In particular to a method for inducing and culturing mesenchymal stem cell secretion exosomes under inflammatory factors and a hypoxia environment and extracting the exosomes.

The mesenchymal stem cells of the invention are obtained from placenta isolation and subculture.

Background

Mesenchymal Stem Cells (MSCs) are a type of non-hematopoietic pluripotent stem cells derived from mesoderm, and it has been confirmed that MSCs have strong anti-inflammatory and multiple immune cell-suppressing abilities in addition to self-renewing self-replication and multi-directional differentiation potential, and are capable of inducing peripheral immune tolerance. Research has shown that MSCs are produced by secreting various immunomodulatory factors, such as IFN- γ, PGE2, etc. (see: polchert D, sobinsky J, douglas G, kidd M, moadsiri A, reina E, et al., IFN-gamma activation of sensory stem cells for treatment and preservation of graft summary host disease. European journal of immunology.2008; 1745-55, spaggiari GM, abdelrazik H, becchetti F, moretta L, MSCs in bit monocyte-derived DC mapping and function by selective interaction with the generation of image DCs, central role of MSC-derived pro-stable N2. Blood.2009;113 (26): 6576-83.), and thereby inhibiting the function of immune cells (e.g., T cells, B cells, NK cells, antigen presenting cells, etc.) (see: corcinone A, benvenuto F, ferretti E, giunti D, cappiello V, cazzani F, et al., human sensory stem cells modulation. Blood.2006;107 (1): 367-72. And Di Nicola M, carlo stella C, magni M, milanesi M, longoni PD, matteucci P, et al., human bone row cells suspension samples T-simulation induced by cell or non-specific immune tissue blood.2002; 99-38): 10-38).

The immunogenicity of mesenchymal stem cells is low. Cell therapy based on MSCs has been successfully applied to the treatment of cardiovascular diseases, bone and cartilage defects, diabetes and other diseases. MSCs release various cytokines and growth factors through paracrine and autocrine, and the secreted bioactive factors can inhibit fibrosis and apoptosis, enhance angiogenesis and participate in tissue repair and regeneration.

The mesenchymal stem cells have rich sources and can be taken from tissues such as umbilical cord, placenta, bone marrow, umbilical blood, fat and the like.

Exosomes (exosomes) are membrane microvesicles secreted by cells with a diameter of about 30-200 nm and a density ranging between 1.13-1.19 g/ml. Exosomes can carry a variety of proteins, mrnas, mirnas similar to the source cell, and are involved in immune regulation, cellular communication, cell migration, angiogenesis, etc. (see: yanez-Mo M, siljander PR, andreu Z, zavec AB, borras FE, buzas EI, et al. Biological properties of extracellular fluids and the same physiological functions. Journal of extracellular fluids. 2015; 4. Lai et al found that MSCs-derived exosomes reduced myocardial ischemia reperfusion injury and demonstrated that miRNA of exosomes could promote angiogenesis, and thus exosomes could be a new approach to the treatment of cardiovascular disease (see: lai RC, arslan F, lee MM, sze NS, choo A, chen TS, et al. Xin et al found that MSCs-derived exosomes can promote growth of neural axons by transferring miR-133B to neural cells (see: xin H, li Y, buller B, katakowski M, zhang Y, wang X, et al. Exosome medium transfer of miR-133b from multi-potential cells sensing bacterial cells to neural cells connected to neural cells output from stem cells.2012 (7): 1556-64.). Filipazzi et al found that tumor cell-derived exosomes could inhibit cytotoxicity of T-cells and NK-cells through the NK cell activating receptor NKG2D (natural killer group 2, cell D), thereby affecting the host's immune system (see: filipazzi P, burdek M, villa A, rivolltini L, huber V.Recent Advances on the roll of tumor exosomes in immune suppression and disease progression. Secondary in cancer biology.2012;22 (4): 342-9).

Exosomes (exosomes), membrane vesicles secreted by living cells, were first discovered in 1983. With the progress of research, it is continuously paid attention to find that it has the functions of carrying out protein and nucleic acid transport, specifically targeting receptor cells, exchanging proteins and lipids or triggering downstream signaling events, and participating in intercellular communication. The exosome-carrying protein includes two types of protein molecules, source cell non-specific and source cell specific. The former may be associated with biogenesis and biological actions of exosomes, mainly including: cytosolic proteins, proteins involved in intracellular signal transduction, various metabolic enzymes, heat shock proteins, and tetraspanin proteins; the other is a special protein, which exists only in an exosome secreted by a special cell, and the exosome of the special cell source is closely related to the biological function, such as: the exosomes of molecular origin contain MHCII class molecules. Therefore, exosomes from different cell sources carry different signaling molecules and exert different functions. For example: exosomes secreted by tumor cells can mediate angiogenesis tumor cell proliferation and immune escape, while dendritic cell-derived exosomes can cause an effective anti-tumor immune response of an organism. The existing research finds that the exosome contains protein rRNA and microRNA related to cell sources, and the exosome can transmit functional nucleic acid molecules among cells through a biological barrier, so that various biological functions are exerted, and the exosome is expected to become a novel administration way and a gene therapy vector.

The exosome is used as a nano-scale lipid inclusion structure, and substances such as protein, mRNA (messenger ribonucleic acid), microRNA (microRNA) and the like are encapsulated inside the exosome. Exosomes are naturally present in body fluids, including blood, saliva, urine, breast milk, and the like. Exosomes are membrane vesicles secreted by living cells derived from late endosomes (also known as multivesicular bodies). Almost all cells, including tumors, can produce and release exosomes. The exosome is secreted and released by cells, spreads in body fluids such as blood and can be phagocytized by other cells finally, and is an important medium for intercellular communication. The host cell or the exosome secreted by the tumor cell participates in the growth, proliferation, metabolism, regulation and the like of the cell. The information exchange between immune cells and tumor cells can also be carried out through exosomes, and the communication mode plays a dual role in regulating tumor immunity: exosomes can either elicit anti-tumor responses by suppressing immune cells (DCs, NK cells, CD4+, CD8+ T cells, etc.), or induce immunosuppression or the immunosuppression of regulatory cell populations (MDSCs, tregs, bregs).

At present, researches show that exosomes of mesenchymal stem cells carry various effective cytokines, proteins and small molecular nucleic acid substances, and can effectively mediate cell proliferation, apoptosis and function regulation. Researches show that the exosome contains vascular endothelial growth factor, fibroblast growth factor, platelet proliferation factor, tumor necrosis factor, tumor growth factor and the like, and has the functions of inhibiting apoptosis and the fibrosis degree of cells, promoting angiogenesis mitosis, mediating immunoreaction and the like. Experiments prove that insulin-like growth factors and vascular endothelial cell growth factors carried by exosomes secreted by using mesenchymal stem cells are key leading factors for treating acute kidney injury. In immunology, the surface of the exosome lipid membrane secreted by the mesenchymal stem cell expresses various membrane proteins, such as: coagulation factors, tumor necrosis factors, MHC I/II molecules, and CCR5 chemokine receptors, among others, which are important for their role in resisting inflammation.

At present, methods and ways for extracting exosomes are various, an ultracentrifugation method or an expensive kit column-passing method is mainly adopted in the existing exosome extraction method, however, the obtained exosomes by the ultracentrifugation method are uneven in quality, the obtained exosomes cannot be guaranteed, the step-by-step centrifugation time is long, sometimes even the exosomes cannot be obtained by separation due to the centrifugation time or the centrifugation speed, and time and cost are wasted. The cartridge-over-column method is generally only suitable for obtaining relatively small amounts of exosomes and is expensive.

The prior art has reported some methods for the isolation and extraction of exosomes. For example, CN106282107a (chinese patent application No. 201610779165.2) discloses a method for separating exosome from human placenta mesenchymal stem cell source, CN105708861a (chinese patent application No. 201610149852.6) discloses application of exosome derived from bone marrow mesenchymal stem cell in preparation of a medicament for treating ankylosing spondylitis, CN105267240a (chinese patent application No. 201410781765.3) discloses application of exosome derived from mesenchymal stem cell, CN104382827a (chinese patent application No. 201410705462.3) discloses application of exosome derived from human amniotic mesenchymal stem cell, CN103767985a (chinese patent application No. 201210402915.6) discloses preparation and application of human blood or mesenchymal stem cell secretory exosome. However, in the prior art, the exosomes are extracted by adopting mesenchymal stem cell culture supernatants from various sources, and the methods have the defects of low yield, complex process and the like.

Therefore, there is still a need in the art for a new method for isolating and extracting exosomes, particularly for extracting exosomes secreted by mesenchymal stem cells in a simple, easily available and high-yield manner. Furthermore, it would be desirable in the art to provide a method of treating inflammatory diseases, for example by using exosomes.

Disclosure of Invention

The invention aims to provide a novel method for separating and extracting exosome, and particularly the method can extract exosome through mesenchymal stem cell secretion in a simple and easily-obtained and high-yield manner. It has surprisingly been found that the above object can be achieved advantageously by the process according to the invention. The present invention has been completed based on this finding.

The mesenchymal stem cells of the present invention are of placental origin.

To this end, the present invention provides, in a first aspect, an isolated and extracted exosome having an average particle size of 50 to 200nm, for example, having an average particle size of 75 to 150nm, using placental mesenchymal stem cells.

The exosome according to the first aspect of the invention, which expresses the membrane protein CD9 and the membrane protein CD81. In one embodiment, the positive expression rate of the exosome membrane protein CD9 is greater than 65%, such as greater than 70%, for example greater than 75%. In one embodiment, the positive expression rate of the exosome membrane protein CD81 is greater than 75%, such as greater than 80%, for example greater than 85%.

The exosome according to the first aspect of the present invention is prepared by a method comprising the following steps using placental mesenchymal stem cells:

(1) Inoculating mesenchymal stem cells into culture bottle, adding MSC complete culture medium, standing at 37 deg.C and 5% CO ₂ Culturing in an incubator to make cells adhere to the wall, adding IL-1 beta into the culture solution to the concentration of 8-12 ng/mL, and continuing culturing;

(2) The medium was aspirated off and replaced with fresh MSC complete medium to allow the cells to continue at 37 ℃ with 5% CO ₂ Culturing in an incubator until the cell fusion degree is more than or equal to 80 percent;

(3) The medium was aspirated off, washed with PBS, then MSC complete medium was added, and the mixture was placed at 37 ℃ under 2% O ₂ 、5％ CO ₂ Culturing in an incubator for 42-56 hours;

(4) The cell supernatant was aspirated into a centrifuge tube and centrifuged as follows:

centrifuging for 8-12 minutes at 4 ℃ at 250-350 g, and sucking supernatant into another centrifugal tube;

centrifuging at 1500-2500 g and 4 ℃ for 18-25 minutes, and sucking supernatant into another centrifugal tube;

centrifuging at 8000-12000 g and 4 deg.c for 25-35 min, filtering the supernatant with 0.22 micron filter membrane and setting in another centrifuge tube;

centrifuging at 80000-120000 g and 4 deg.C for 75-120 min, and discarding the supernatant;

(5) Adding sterile PBS into a centrifuge tube to enable the exosome sediment to be resuspended, centrifuging for 75-120 minutes at 80000-120000 g and 4 ℃, removing supernate, adding sterile PBS to enable the exosome to be suspended, and obtaining exosome in the form of suspension.

According to the exosome of the first aspect of the present invention, in step (1), the mesenchymal stem cell is a cell of P2 to P8 generations, for example, a cell of P3 to P6 generations.

The exosomes according to the first aspect of the invention, in step (1), the cells are added to the culture flask at a density of (0.5-5). Times.10 ^4 cells/cm ^2, for example at a density of (0.5-2). Times.10 ^4 cells/cm ^ 2. In one embodiment, step (1) uses T75 flasks, each flask inoculated with (2-10). Times.10 ^5 cells, e.g., 7.5X 10^5 cells, with 10-20 ml of medium added.

The exosome according to the first aspect of the present invention, in step (1), the cells are allowed to adhere by culturing for 20 to 30 hours, for example, for 24 hours.

The exosome according to the first aspect of the present invention is cultured for 20 to 30 hours, for example, 24 hours, in step (1) after addition of IL-1. Beta.

The exosome according to the first aspect of the present invention, in step (3), 2% O at 37 ℃ ₂ 、5％ CO ₂ Culturing in an incubator for 48 hours;

the exosome according to the first aspect of the present invention is, in step (4), first centrifuged at 250g at 4 ℃ for 12 minutes, then at 2500g at 4 ℃ for 18 minutes, then at 8000g at 4 ℃ for 35 minutes, and the supernatant is filtered through a 0.22 μm filter and then centrifuged at 120000g at 4 ℃ for 75 minutes.

The exosome according to the first aspect of the present invention is, in step (4), first centrifuged at 350g at 4 ℃ for 8 minutes, then at 1500g at 4 ℃ for 25 minutes, then at 12000g at 4 ℃ for 25 minutes, and the supernatant is filtered through a 0.22 μm filter and then centrifuged at 80000g at 4 ℃ for 120 minutes.

The exosome according to the first aspect of the present invention, in step (4), is first centrifuged at 300g at 4 ℃ for 10 minutes, then at 2000g at 4 ℃ for 20 minutes, then at 10000g at 4 ℃ for 30 minutes, and the supernatant is filtered through a 0.22 μm filter and then centrifuged at 100000g at 4 ℃ for 90 minutes.

The exosome according to the first aspect of the present invention is centrifuged at 100000g at 4 ℃ for 90 minutes, or 80000g at 4 ℃ for 120 minutes, or 120000g at 4 ℃ for 75 minutes in step (5).

The exosome according to the first aspect of the present invention, in step (5), the resulting exosome suspension is stored at-80 ℃.

The exosome according to the first aspect of the present invention, in step (5), the exosome obtained as 1.5 × 10^6 cells of step (1) is resuspended in 0.5-5 ml of sterile PBS, such as 0.5-2 ml of sterile PBS, such as 1ml of sterile PBS.

According to the exosome of the first aspect of the present invention, in step (1), sodium tartrate and lysine are added to the culture solution at concentrations of 0.15 to 0.2mg/mL and 2.0 to 2.5mg/mL, respectively, for example, at concentrations of 0.15mg/mL and 2.2mg/mL, respectively, together with IL-1 β.

The exosome according to the first aspect of the present invention, wherein the placental mesenchymal stem cells are prepared by a method comprising the steps of:

(a) Treatment of placental tissue

Removing amnion from human placenta, cutting placenta lobular epidermis membrane sample tissue, and cleaning with normal saline;

mixing placenta hominis She BiaocengCutting the membrane-like tissue to a volume of 0.2cm ³ The tissue fragment of (a);

placing the tissue fragments in a centrifuge tube, adding a proper amount of 0.9% physiological saline, filtering by using a 300-mesh filter screen, and cleaning twice by using a proper amount of 0.9% physiological saline until filtrate is clear;

adding the cleaned tissue into HBSS digestive juice containing 0.005% Liberase MNP-S enzyme and 0.05% DNA I type enzyme, mixing well, and digesting in shaking table at 37 deg.C and 100rpm for 30min;

(b) Obtaining placental progenitor cells

After digestion is finished, adding fetal calf serum into a centrifuge tube to stop digestion, uniformly mixing, diluting with 50ml of normal saline containing 5% dextran, 2.5% human serum albumin and 200U of DNase I, filtering by using a 300-mesh filter screen, washing tissues for multiple times by using 100ml of normal saline, and collecting filtrate;

centrifuging the obtained filtrate at 1400rpm for 5min, removing supernatant, collecting precipitate, resuspending with physiological saline, centrifuging again, and collecting precipitate;

suspending the precipitated cells by DMEM/F12, sampling and counting to obtain P0 generation cells;

(c) Purification culture

The culture conditions are as follows: DMEM/F12 medium containing 10% fetal bovine serum and 2mM L-glutamine at 37 ℃ with 5% CO ₂ Culturing in a constant-temperature wet incubator;

digestion conditions are as follows: 0.25% pancreatin, digested at 37 ℃ for 2 minutes;

harvesting conditions are as follows: terminating digestion with complete medium, centrifuging at 1400rpm for 5min, and collecting precipitate;

at a rate of 5000 to 15000 pieces/cm ² The placenta P0 generation cells are inoculated in a T75 culture bottle at the inoculation density, and the liquid is changed all at the 3 rd to 4 th days; cloning cells on days 6-7, growing fusiform cells in vortex form on days 10-11, namely P1 generation placental mesenchymal stem cells, collecting and then carrying out passage;

(d) Purifying and culturing the P1 generation cells in the mode of the step (c) and carrying out passage to obtain P2 generation cells; and by analogy, sequentially obtaining the P3-P8 generation mesenchymal stem cells.

Further, the second aspect of the present invention provides a method for isolating and extracting exosomes using placental mesenchymal stem cells, comprising the steps of:

(2) The medium was aspirated off and replaced with fresh MSC complete medium to allow the cells to continue to incubate at 37 ℃ with 5% CO ₂ Culturing in an incubator until the cell fusion degree is more than or equal to 80 percent;

(3) The medium was aspirated off, washed with PBS, then MSC complete medium was added, and the cells were incubated at 37 ℃ with 2% O ₂ 、5％ CO ₂ Culturing in an incubator for 42-56 hours;

According to the method of the second aspect of the present invention, in step (1), the mesenchymal stem cells are cells of P2 to P8 generations, for example, cells of P3 to P6 generations.

According to the method of the second aspect of the present invention, in step (1), the flask is charged with cells at a density of (0.5-5). Times.10 ^4 cells/cm ^2, for example, (0.5-2). Times.10 ^4 cells/cm ^ 2. In one embodiment, step (1) uses T75 flasks, each flask inoculated with (2-10). Times.10 ^5 cells, e.g., 7.5X 10^5 cells, with 10-20 ml of medium added.

According to the method of the second aspect of the present invention, in step (1), the cells are allowed to adhere by culturing for 20 to 30 hours, for example, for 24 hours.

According to the method of the second aspect of the present invention, in step (1), the culture is continued for 20 to 30 hours, for example, for 24 hours after the addition of IL-1. Beta. In the above step.

The process according to the second aspect of the present invention, step (3), is carried out at 37 ℃ with 2% O ₂ 、5％ CO ₂ Culturing in an incubator for 48 hours;

according to the method of the second aspect of the present invention, in the step (4), the supernatant is first centrifuged at 250g and 4 ℃ for 12 minutes, then at 2500g and 4 ℃ for 18 minutes, then at 8000g and 4 ℃ for 35 minutes, and then filtered through a 0.22 μm filter and then at 120000g and 4 ℃ for 75 minutes.

According to the method of the second aspect of the present invention, in the step (4), the supernatant is first centrifuged at 350g and 4 ℃ for 8 minutes, then at 1500g and 4 ℃ for 25 minutes, then at 12000g and 4 ℃ for 25 minutes, and then filtered through a 0.22 μm filter and centrifuged at 80000g and 4 ℃ for 120 minutes.

According to the method of the second aspect of the present invention, in the step (4), the supernatant is first centrifuged at 300g at 4 ℃ for 10 minutes, then at 2000g at 4 ℃ for 20 minutes, then at 10000g at 4 ℃ for 30 minutes, and then the supernatant is filtered through a 0.22 μm filter and then centrifuged at 100000g at 4 ℃ for 90 minutes.

According to the method of the second aspect of the present invention, in the step (5), the sample is centrifuged at 100000g at 4 ℃ for 90 minutes, or at 80000g at 4 ℃ for 120 minutes, or at 120000g at 4 ℃ for 75 minutes.

According to the method of the second aspect of the present invention, in step (5), the resulting exosome suspension is stored at-80 ℃.

According to the method of the second aspect of the present invention, in step (5), the exosomes obtained from 1.5X 10^6 cells of step (1) are resuspended in 0.5-5 ml of sterile PBS, such as 0.5-2 ml of sterile PBS, such as 1ml of sterile PBS.

According to the method of the second aspect of the present invention, in step (1), sodium tartrate and lysine are added to the culture solution at concentrations of 0.15 to 0.2mg/mL and 2.0 to 2.5mg/mL, respectively, for example, at concentrations of 0.15mg/mL and 2.2mg/mL, respectively, together with IL-1. Beta. Being added.

According to the method of the second aspect of the invention, the resulting exosomes have an average particle size of 50 to 200nm, for example an average particle size of 75 to 150nm.

According to the method of the second aspect of the invention, the resulting exosomes express the membrane protein CD9 and the membrane protein CD81. In one embodiment, the positive expression rate of the exosome membrane protein CD9 is greater than 65%, such as greater than 70%, for example greater than 75%. In one embodiment, the positive expression rate of the exosome membrane protein CD81 is greater than 75%, such as greater than 80%, for example greater than 85%.

According to the method of the second aspect of the present invention, the placental mesenchymal stem cells are prepared by a method comprising the steps of:

(a) Treatment of placental tissue

cutting the membrane sample tissue of placenta lobule surface to 0.2cm ³ The tissue fragment of (a);

placing the tissue fragments in a centrifuge tube, adding a proper amount of 0.9% normal saline, filtering by using a 300-mesh filter screen, and cleaning twice by using a proper amount of 0.9% normal saline until filtrate is clear;

(b) Obtaining placental progenitor cells

(c) Purification culture

Further, the third aspect of the present invention provides a use of exosomes extracted from isolated placental mesenchymal stem cells, which has an average particle size of 50-200 nm, for example, which has an average particle size of 75-150 nm, in the preparation of a medicament for treating inflammatory diseases.

According to the use of the third aspect of the invention, the exosomes express the membrane protein CD9 and the membrane protein CD81. In one embodiment, the positive expression rate of the exosome membrane protein CD9 is greater than 65%, such as greater than 70%, for example greater than 75%. In one embodiment, the positive expression rate of the exosome membrane protein CD81 is greater than 75%, such as greater than 80%, for example greater than 85%.

According to the use of the third aspect of the present invention, the exosome is prepared by a method comprising the following steps of:

(1) Inoculating mesenchymal stem cells into a culture bottle, adding MSC complete culture medium, placing at 37 deg.C and 5% CO ₂ Culturing in incubator to make cells adhere to wall, then moving toAdding IL-1 beta into the culture solution to the concentration of 8-12 ng/mL, and continuing culturing;

centrifuging at 1500-2500 g and 4 ℃ for 18-25 minutes, and sucking supernatant into another centrifuge tube;

According to the use of the third aspect of the present invention, in the step (1), the mesenchymal stem cells are cells of P2 to P8 generations, for example, cells of P3 to P6 generations.

According to the use of the third aspect of the invention, in step (1), the flask is filled with cells at a density of (0.5-5). Times.10 ^4 cells/cm ^2, for example at a density of (0.5-2). Times.10 ^4 cells/cm ^ 2. In one embodiment, step (1) uses T75 flasks, each flask inoculated with (2-10). Times.10 ^5 cells, e.g., 7.5X 10^5 cells, with 10-20 ml of medium added.

According to the use of the third aspect of the present invention, in the step (1), the cells are allowed to adhere by culturing for 20 to 30 hours, for example, for 24 hours.

According to the use of the third aspect of the present invention, in the step (1), the culture is continued for 20 to 30 hours, for example, for 24 hours after the addition of IL-1. Beta. In the above step.

According to a third aspect of the invention, in step (3), 2% O at 37 ℃ ₂ 、5％ CO ₂ Culturing in an incubator for 48 hours;

according to the use of the third aspect of the present invention, in the step (4), the supernatant is first centrifuged at 250g and 4 ℃ for 12 minutes, then at 2500g and 4 ℃ for 18 minutes, then at 8000g and 4 ℃ for 35 minutes, and then filtered through a 0.22 μm filter and then at 120000g and 4 ℃ for 75 minutes.

According to the use of the third aspect of the present invention, in the step (4), the supernatant is first centrifuged at 350g and 4 ℃ for 8 minutes, then at 1500g and 4 ℃ for 25 minutes, then at 12000g and 4 ℃ for 25 minutes, and then filtered with a 0.22 μm filter and centrifuged at 80000g and 4 ℃ for 120 minutes.

According to the use of the third aspect of the present invention, in the step (4), the supernatant is first centrifuged at 300g at 4 ℃ for 10 minutes, then at 2000g at 4 ℃ for 20 minutes, then at 10000g at 4 ℃ for 30 minutes, and then filtered with a 0.22 μm filter and then centrifuged at 100000g at 4 ℃ for 90 minutes.

According to the use of the third aspect of the present invention, in step (5), the sample is centrifuged at 100000g at 4 ℃ for 90 minutes, or at 80000g at 4 ℃ for 120 minutes, or at 120000g at 4 ℃ for 75 minutes.

According to the use of the third aspect of the present invention, in step (5), the resulting exosome suspension is stored at-80 ℃.

According to the use of the third aspect of the present invention, in step (5), the exosomes obtained from 1.5X 10^6 cells of step (1) are resuspended in 0.5-5 ml of sterile PBS, such as 0.5-2 ml of sterile PBS, such as 1ml of sterile PBS.

According to the use of the third aspect of the present invention, in the step (1), sodium tartrate and lysine are added to the culture solution together with IL-1. Beta. At concentrations of 0.15 to 0.2mg/mL and 2.0 to 2.5mg/mL, for example, at concentrations of 0.15mg/mL and 2.2mg/mL, respectively.

According to the use of the third aspect of the present invention, the placental mesenchymal stem cells are prepared by a method comprising the steps of:

(a) Treatment of placental tissue

(b) Obtaining placental progenitor cells

(c) Purification culture

at a rate of 5000 to 15000 pieces/cm ² Inoculating the placenta P0 generation cells into a T75 culture bottle at the inoculation density, and completely changing the liquid on the 3 rd to 4 th days; cloning cells on 6-7 days, growing fusiform cells in vortex form on 10-11 days, collecting P1 generation mesenchymal stem cellsPassage can be carried out;

Of the various process steps described above, although specific steps are described in some detail or in language specific to the process steps described in the examples of the following detailed description, those skilled in the art will be able to fully appreciate the above-described process steps from the detailed disclosure of the invention as a whole.

Any embodiment of any aspect of the invention may be combined with other embodiments, as long as they do not contradict. Furthermore, in any embodiment of any aspect of the invention, any feature may be applicable to that feature in other embodiments, so long as they do not contradict. The invention is further described below.

All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.

Although the MSC-derived exosomes have many advantages such as promoting angiogenesis, promoting cell proliferation, growth and migration, the role of most of the MSC-derived exosomes cultured by the conventional method in mediating inflammation regulation is not obvious. Based on the design, the invention designs the exosome which is secreted by the MSC and is induced by the low oxygen partial pressure and inflammatory factor stimulation mode, shows good inflammation regulation and control effects in vitro and provides a new scheme for the exosome to treat inflammatory diseases.

Drawings

FIG. 1: electron microscope image of placenta mesenchymal stem cell exosome.

FIG. 2: expression level of exosome membrane protein CD 9.

FIG. 3: expression level of exosome membrane protein CD81.

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.

Unless otherwise indicated, some of the reagents used in the present invention are conventional in the art or readily available from commercial sources. For example, serum-free medium (Gibco) was purchased from ThermoFisher Scientific; human platelet lysates were purchased from Preciion Biomedical, inc.; the phosphate buffer (pH6.8, PBS) was prepared by: taking 250ml of 0.2mol/L potassium dihydrogen phosphate solution, adding 118ml of 0.2mol/L sodium hydroxide solution, diluting with water to 1000ml, shaking up, and sterilizing at 121 ℃ for 15min to obtain the final product; tissue washes (PRS-TCR-1) were purchased from Purey. The MSC complete medium described herein is a serum-free medium containing 2% human platelet lysate.

Example 1: primary culture and subculture of placenta mesenchymal stem cells

Methods for obtaining primary and passaged mesenchymal stem cells from placenta have been reported in many documents, and the mesenchymal stem cells used in the preparation of exosomes of the present invention can be obtained using these literature methods, and although the key core of the present technology is not, exemplarily, the present invention is willing to describe herein a method for preparing mesenchymal stem cells.

In this example, the method described in chinese patent No. ZL 2019108190702 is referred to for preparing placental mesenchymal stem cells, and the materials and sources used in the method can also be referred to in this patent document.

(1) Treating the placenta tissue: removing amnion from a fresh human placenta, shearing placental lobule surface membrane sample tissue, and cleaning with normal saline; cutting the placental lobule superficial membrane sample tissue into pieces having a volume of about 0.2cm ^ 3; placing the tissue fragments in a centrifuge tube, adding a proper amount (100 ml) of 0.9% physiological saline, filtering by using a 300-mesh filter screen, and cleaning twice by using a proper amount (100 ml) of 0.9% physiological saline until filtrate is clear; adding the cleaned tissue into HBSS digestive juice (100 ml) containing 0.005% Liberase MNP-S enzyme and 0.05% DNA I enzyme, mixing well, and shaking and digesting in shaker for 30min (37 deg.C, 100 rpm);

(2) Obtaining placenta original cells: after digestion, adding fetal bovine serum (2 ml) into a centrifuge tube to terminate digestion, uniformly mixing, diluting with 50ml of normal saline containing 5% dextran, 2.5% human serum albumin and 200U of DNase I, filtering with a 300-mesh filter screen, washing tissues for multiple times with 100ml of normal saline, and collecting filtrate; centrifuging the obtained filtrate at 1400rpm for 5min (acceleration 9, deceleration 7), removing supernatant, collecting precipitate, resuspending with physiological saline (100 ml), centrifuging again, and collecting precipitate; suspending the precipitated cells by DMEM/F12 (100 ml), sampling and counting to obtain P0 generation cells;

(3) Purification culture and passage:

(4) Purifying and culturing the P1 generation cells in the mode of the step (3) and carrying out passage to obtain P2 generation cells; by analogy, the P3-P8 generation mesenchymal stem cells are obtained in sequence.

The cells obtained by the method have excellent performances in aspects of cell morphology, cell phenotype, differentiation potential and the like as described in ZL 2019108190702.

Example 2: treatment of mesenchymal stem cells with IL-1 beta and isolation of purified exosomes by differential centrifugation

(1) The mesenchymal stem cells (generation P4) obtained in example 1 were seeded into T75 culture flasks at 7.5X 10^5 cells, 15mL of MSC complete medium was added to each flask, and the flasks were kept at 37 ℃ with 5% CO ₂ Culturing in an incubator (24 hours) to allow the cells to adhere to the wall, adding IL-1 beta into the culture solution to reach the concentration of 10ng/mL, and continuing culturing for 24 hours; [ those skilled in the art understand that other sizes of culture bottles such as T25 and T75 bottles can be used instead, and the number of cell inoculations and/or the amount of culture medium added can be varied according to the culture area, for example, it is routine practice in the art to add about 15ml of culture medium at the above concentrations in MSC culture]

(3) The medium was aspirated, washed 3 times with PBS, and then 15mL of MSC complete medium was added to each flask, which was then placed at 37 ℃ and 2%O ₂ 、5％ CO ₂ Culturing in an incubator for 48 hours;

[ the above 2% of O ₂ 、5％ CO ₂ The atmosphere in the incubator is nitrogen-balanced atmosphere, and the 5% CO mentioned above ₂ The atmosphere in the incubator is an atmosphere in which 21% oxygen and the balance nitrogen are balanced, and the meaning of such expressions is generally recognized by those skilled in the art]

(4) Cell supernatants (30 ml, amount of supernatant from 2 flasks) were pipetted into a (50 ml) centrifuge tube and centrifuged as follows:

centrifuge at 300g for 10min at 4 ℃ (to remove dead cells and larger cell debris) and aspirate the supernatant into another (50 ml) centrifuge tube;

centrifuging at 2000g and 4 deg.C for 20min (further removing impurities such as cell debris), and sucking the supernatant into another (high-speed) centrifuge tube;

centrifuging at 10000g and 4 deg.C for 30min (to further remove smaller cell debris and impurities), filtering the supernatant with 0.22 μm filter membrane (PES filter membrane, millipore), and placing in another (ultra-speed) centrifuge tube;

centrifuging at 100000g and 4 deg.C for 90 min, and discarding the supernatant;

(5) Adding 20mL of sterile PBS into a centrifuge tube to resuspend the exosome precipitate, centrifuging at 100000g and 4 ℃ for 90 minutes, removing supernatant, adding 1mL of sterile PBS to resuspend the exosome to obtain 1mL of exosome suspension, and storing the exosome suspension at-80 ℃ and/or subpackaging the exosome suspension for performance determination.

Centrifuge tubes and centrifuges used in the above steps with different centrifugal force requirements were purchased from Beckman Coulter.

Example 2a: treatment of mesenchymal stem cells with IL-1 beta and isolation of purified exosomes by differential centrifugation

(1) The mesenchymal stem cells (generation P3) obtained in example 1 were seeded into T75 culture flasks at 7.5X 10^5 cells, 17.5mL of MSC complete medium was added to each flask, and the flasks were incubated at 37 ℃ with 5% CO ₂ Culturing in an incubator (20 hours) to allow the cells to adhere to the wall, adding IL-1 beta into the culture solution to the concentration of 8ng/mL, and continuing culturing for 24 hours;

(3) The medium was aspirated, washed 3 times with PBS, and 15mL of MSC complete medium was added to each flask, which was then placed at 37 ℃ and 2%O ₂ 、5％ CO ₂ Culturing in an incubator for 48 hours;

centrifuge at 250g for 12 min at 4 ℃ (to remove dead cells and larger cell debris) and aspirate the supernatant into another (50 ml) centrifuge tube;

centrifuging at 2500g and 4 deg.C for 18 min (to further remove impurities such as cell debris), and sucking the supernatant into another (high-speed) centrifuge tube;

centrifuging at 8000g and 4 deg.C for 35 min (to further remove small cell debris and impurities), filtering the supernatant with 0.22 μm filter membrane (PES filter membrane, millipore), and placing in another (ultracentrifuge) centrifuge tube;

centrifuging at 120000g and 4 deg.C for 75 min, and discarding the supernatant;

(5) And adding 15mL of sterile PBS into the centrifuge tube to resuspend the exosome precipitate, centrifuging at 80000g and 4 ℃ for 120 minutes, discarding the supernatant, adding 1mL of sterile PBS to resuspend the exosome to obtain 1mL of exosome suspension, and storing the exosome suspension at-80 ℃ and/or subpackaging the exosome suspension for performance measurement.

Example 2b: treatment of mesenchymal stem cells with IL-1 beta and isolation of purified exosomes by differential centrifugation

(1) The mesenchymal stem cells (P6 generation) obtained in example 1 were inoculated into T75 culture flasks at 7.5X 10^5 cells, 12.5mL of MSC complete medium was added to each flask, and the flasks were incubated at 37 ℃ with 5% CO ₂ Culturing in an incubator (30 hours) to allow the cells to adhere to the wall, adding IL-1 beta into the culture solution to reach the concentration of 12ng/mL, and continuing culturing for 24 hours;

centrifuge at 350g for 8 min at 4 ℃ (remove dead cells and larger cell debris) and aspirate supernatant into another (50 ml) centrifuge tube;

centrifuging at 1500g and 4 deg.C for 25 min (further removing impurities such as cell debris), and sucking the supernatant into another (high-speed) centrifuge tube;

centrifuging at 12000g and 4 deg.C for 25 min (to further remove smaller cell debris and impurities), filtering the supernatant with a 0.22 μm filter (PES filter, millipore) and placing in another (ultracentrifuge) centrifuge tube;

centrifuging at 80000g at 4 ℃ for 120 minutes, and discarding the supernatant;

(5) The exosome pellet was resuspended by adding 25mL sterile PBS to the centrifuge tube, centrifuged at 120000g for 75 minutes at 4 ℃, the supernatant was discarded, and 1mL sterile PBS was added to resuspend the exosomes, resulting in 1mL exosome suspension, which could be stored at-80 ℃ and/or split for performance assay.

Example 3: treatment of mesenchymal stem cells with IL-1 beta and isolation of purified exosomes by differential centrifugation

(1) The mesenchymal stem cells (P4 generation) obtained in example 1 were inoculated into T75 culture flasks at 7.5X 10^5 cells, 15mL of MSC complete medium was added to each flask, and the mixture was set at 37 ℃ and 5% CO ₂ Culturing in incubator (24 hr) to allow cell to adhere to wall, adding IL-1 β, sodium tartrate and lysine to the culture solution to reach concentration of 10ng/mL, 0.175mg/mL and 2.2mg/mL respectively, and culturing for 24 hr;

[ 2% of O above ₂ 、5％ CO ₂ The atmosphere in the incubator is nitrogen-balanced atmosphere, and the 5% CO mentioned above ₂ The atmosphere in the incubator is an atmosphere in which 21% oxygen and the balance nitrogen are balanced, and the meaning of such expressions is generally recognized by those skilled in the art]

centrifuging at 100000g and 4 deg.C for 90 min, and discarding the supernatant;

Example 3a: treatment of mesenchymal stem cells with IL-1 beta and isolation of purified exosomes by differential centrifugation

(1) The mesenchymal stem cells (P3 generation) obtained in example 1 were inoculated into T75 culture flasks at 7.5X 10^5 cells, 17.5mL of MSC complete medium was added to each flask, and the mixture was incubated at 37 ℃ with 5% CO ₂ Culturing in an incubator (20 hr) to allow the cells to adhere to the wall, adding IL-1 β, sodium tartrate and lysine hydrochloride into the culture solution to reach concentrations of 8ng/mL, 0.2mg/mL and 2mg/mL respectively, and continuing culturing for 24 hr;

centrifuging at 120000g and 4 deg.C for 75 min, and discarding the supernatant;

Example 3b: treatment of mesenchymal stem cells with IL-1 beta and isolation of purified exosomes by differential centrifugation

(1) The mesenchymal stem cells (P6 generation) obtained in example 1 were inoculated into T75 culture flasks at 7.5X 10^5 cells, 12.5mL of MSC complete medium was added to each flask, and the flasks were incubated at 37 ℃ with 5% CO ₂ Culturing in an incubator (30 hours) to allow cells to adhere to the wall, adding IL-1 beta, sodium tartrate and lysine hydrochloride into the culture solution to reach the concentrations of 12ng/mL, 0.15mg/mL and 2.5mg/mL respectively, and continuing culturing for 24 hours;

Example 3c: reference is made to example 3, example 3a and example 3b, respectively, except that no sodium tartrate is added in step (1) to give three batches of exosomes, which can be designated as example 3c1, example 3c2, example 3c3, respectively.

Example 3d: reference is made to example 3, example 3a and example 3b, respectively, except that no lysine hydrochloride is added in step (1) to give three exosomes, which can be designated as example 3d1, example 3d2 and example 3d3, respectively.

Example 4: transmission electron microscope observation of exosome particles

1. Collecting the separated and purified exosome precipitate 50 μ l, adding 2.5% glutaraldehyde with the same volume, and fixing in a refrigerator at 4 deg.C for 1hr;

2. dripping 20 μ l of the fixed exosome suspension to the front surface of a copper mesh, and standing for 20min;

3. carefully blotting excess solution with absorbent filter paper; then, the copper mesh is cleaned by ultrapure water for 5 times, and is sucked and dried by filter paper in 30 seconds each time;

4. dripping 1 drop of 2% uranyl acetate staining solution on the front surface of the copper mesh, staining for 1min, and then using filter paper to extend the edge of the copper mesh and suck the redundant staining solution;

5. and (3) placing the copper mesh in air at room temperature for natural drying, airing the copper mesh, then putting the copper mesh on a machine for observation, and calculating and counting the particle size and distribution of the exosomes.

The exosomes (suspensions) obtained from each example of the present invention were tested using the above method. The number of exosomes from each example was calculated from 1.5X 10^6 cells (2 flasks) with the results: the numbers of exosome particles for example 2, example 2a and example 2b are 7.6 x 10^9, 6.8 x 10^9 and 8.2 x 10^9, respectively, the numbers of exosome particles for example 3, example 3a and example 3b are 93.3 x 10^9, 98.7 x 10^9 and 90.4 x 10^9, the numbers of exosome particles for example 3c1, example 3c2 and example 3c3 are 8.3 x 10^9, 7.7 x 10^9 and 8.9 x 10^9, respectively, and the numbers of exosome particles for example 3d1, example 3d2 and example 3d3 are 5.4 x 10^9, 4.8 x 10^9 and 6.2 x 10^9, respectively. Using the exosome of example 2 as an example, the initial amount of MSC cells was 1.5 x 10^6 cells, yielding an exosome volume of 1mL, where the exosome concentration was measured to be 7.6 x 10^9 grains/mL, corresponding to 7.6 x 10^9 grains of exosome from 1.5 x 10^6 cells. The above "7.6 x 10^9 exosomes" means 7.6 x 10 power-of-9 exosomes, and other similar expressions have similar meanings.

Fig. 1 shows an electron microscope image of the placental mesenchymal stem cell exosomes obtained in example 3. All exosomes obtained in example 2, example 2a and example 2b, example 3a and example 3b, example 3c and example 3d above were determined to have average particle sizes in the range of 98-131 nm, for example, the peak particle size of the exosome of example 3 was 113.7 ± 4.2nm and the average particle size was 107.7 ± 0.8nm.

Example 5: flow cytometry for detecting exosome particle surface protein expression

1. Inverting the components of the Thermofisher CD63 magnetic beads (cat No. 10606D) and mixing for 10min; pipetting 20. Mu.l of the magnetic bead suspension into a 1.5mL round-bottom EP tube;

2. adding 200 mul of magnetic bead cleaning solution into an EP tube, and fully and uniformly mixing by using a gun head;

3. placing EP on magnetic frame for 1min; then the supernatant was aspirated off;

4. taking 50 mul of the extracted plasma exosome suspension, adding 50 mul of cleaning solution to a final volume of 100 mul, and fully and uniformly mixing;

5. placing the exosome-cleaning fluid mixed solution on a rotary mixer, setting the rotating speed to be 10rpm, and placing the mixer at the temperature of 2-8 ℃ for rotary incubation overnight;

6. centrifuging the sample for 3-5 seconds at intervals, and collecting the precipitate;

7. adding 300 mul of cleaning fluid into the sample, and fully and uniformly mixing the solution for 30 seconds by using a gun head;

8. placing the sample on a magnetic frame for about 1min, and absorbing and removing supernatant;

9. adding 400 mul of cleaning solution into the sample, and fully and uniformly mixing the solution for 30 seconds by using a gun head;

10. placing the sample on a magnetic frame for about 1min, sucking and removing the supernatant, and resuspending the sample with 300 mu l of cleaning solution;

11. adding CD9-PE and CD81-FITC flow antibodies into 100 μ l samples, incubating at 4 deg.C in dark for 30min,

12. placing the incubated sample on a magnetic frame for about 1min, sucking and removing supernatant, and adding 300 mu l of cleaning solution for washing;

13. after repeating step 12 once, the samples were resuspended with 300. Mu.l PB and tested on the machine.

FIG. 2 shows the expression level of the exosome membrane protein CD9 obtained in example 3, and FIG. 3 shows the expression level of the exosome membrane protein CD81 obtained in example 3, wherein the positive expression rate of CD9 is 82.7%, and the positive expression rate of CD81 is 94.4%. Other examples the expression levels of the membrane proteins CD9 and CD81 of the resulting exosomes were not significantly different from those of FIGS. 2 and 3.

Example 6: exosome protein content detection

The embodiment uses Pierce ^TM Protein quantification kits (cat # 23225, thermo Scientific) were tested.

1. Taking 10 mu L of a BCA (2,2-biquinoline-4,4-disodium phthalate) standard substance with the concentration of 5mg/mL, diluting the BCA standard substance with PBS to the final concentration of 0.5mg/mL to be used as a BCA standard substance solution, adding the standard substance solution into a protein standard substance well of a 96-well plate according to the proportion of 0, 2, 4, 6, 8, 12, 16 and 20 mu L, and adding PBS to make up to 20 mu L;

2. exosome samples were diluted appropriately and 20 μ Ι _ were added to the sample wells of a 96-well plate. Because the pipettor has a large error when taking a small amount of sample, the point in front of the standard line may not be very accurate, so that the sample concentration point falls behind 1/2 of the standard line as much as possible.

3. 200. Mu.L of BCA working solution was added to each well, and left at 37 ℃ for 15 to 30 minutes. And (4) measuring the OD value of A562nm by using a microplate reader, and calculating the protein concentration according to the standard curve.

The standard curve is y =0.9999x-0.0488, where x is the OD value and y is the exosome protein concentration in mg/ml.

The exosomes (suspensions) obtained from each example of the present invention were tested using the above method. The amount of secretion protein obtained from 1.5X 10^6 cells was calculated for each example and the results are: the protein contents of example 2, example 2a and example 2b were 59.7. Mu.g, 56.3. Mu.g and 64.2. Mu.g, respectively, the protein contents of example 3, example 3a and example 3b were 325.3. Mu.g, 346.2. Mu.g and 337.4. Mu.g, respectively, and the protein contents of the 6 exosome samples of example 3c and example 3d were in the range of 58-74. Mu.g.

Example 7: elisa immune regulation experiment for detecting TNF-alpha level secreted by exosome to PBMC (peripheral blood mononuclear cell)

Tumor Necrosis Factor-alpha (TNF-alpha) is a proinflammatory cytokine, participates in normal inflammatory reaction and immune reaction, is mainly produced by activated monocytes and macrophages, and acts in vivo in two forms of transmembrane type (tmTNF) and secretory type (sTNF), wherein tmTNF is distributed on cells secreting TNF-alpha in the form of membrane protein, and is cleaved by TACE (TNF-alpha-converting enzyme) enzyme activation to produce sTNF. TNF α receptors are classified into 2 types (TNFR i and TNFR ii) and exist on various cell surfaces, and the binding of TNF α to TNFR often causes apoptosis, inflammation, tumor occurrence, and the like.

In the experiment, the exosome extracted and purified from the mesenchymal stem cells and PBMC are co-cultured according to a certain proportion, then the TNF-alpha expression level in cell supernatant is detected by adopting an ELISA method, and the inhibition capability of the exosome on the TNF-alpha release of the lymphocytes is detected by analyzing the change of the TNF-alpha expression level.

1. Separating mononuclear cells (PBMC) from healthy adult peripheral blood by Ficoll method, resuspending PBMC in immune cell serum-free medium (Miltenyi, containing 2.5% serum replacement), adjusting the density of PBMC to 4X 10^5/ml, and then adding 50. Mu.l of CD3/CD28 magnetic beads (Thermofisiher) to each ml of PBMC suspension; after mixing the PBMCs and magnetic beads, 0.5ml of PBMCs were added to the wells of a 24-well plate and 50. Mu.l of a suspension containing 1X 10^7 exosomes was added to the wells. Setting a test group hole (PBMC activated by magnetic beads and added with exosome), a control group hole (PBMC activated by magnetic beads and not added with exosome), a negative group hole (PBMC not activated by magnetic beads), and a blank group hole (only containing immune cell serum-free culture medium);

2. after 5 days of co-culture, the supernatant of the co-culture medium was collected; then centrifuged at 2000rpm for 5 minutes; collecting the supernatant;

3. taking out the ELISA kit (R & D Systems) for detecting TNF-alpha and reagents from a refrigerator, placing at room temperature (balancing to room temperature before use), taking down the microporous plate strips, placing the microporous plate strips in the microporous plate strip support, placing the rest microporous plate strips back into the foil paper bag, sealing, and placing back into the refrigerator;

4. taking out a TNF-alpha standard substance, adding 0.95ml of deionized water, and lightly blowing and beating for several times by using a suction head to dissolve PGE2 so as to prepare a standard substance stock solution with the concentration of 10000 pg/ml; placing the dissolved PGE2 standard product at room temperature for minutes, and shaking the TNF-alpha standard product by hand for 2-3 times every 4-5 minutes; then, 7 1.5ml EP tubes were taken and TNF-. Alpha.standards were diluted to different concentration gradients with Calibrator dilution RD 6-12: 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.3pg/ml, and 15.6pg/ml;

5. sample release: for each detection sample, taking a proper amount of the sample to be detected, and diluting the sample to be detected to the exosome protein concentration of 5 mu g/ml by using a Calibrator dilution RD 6-12;

6. sample loading and incubation: separately adding 50. Mu.l of Assay Diluent RD1F to each well of the microplate; adding 50 μ l of culture medium into the microplate, repeating 3 wells to obtain a control group; adding 50 mul of standard solution with different concentrations into the micro-porous plate, and repeating the steps for 3 holes respectively; adding 50 mul of diluted sample into a micro-porous plate, and repeating 3 holes respectively; carefully covering a microporous plate with a film, and oscillating for 2 hours at room temperature at the rotating speed of 450 rpm;

7. carefully tearing off the adhesive film, pouring out the liquid in the microporous plate, and inversely buckling the microporous plate on absorbent paper for beating for several times; adding 300 mu l of 1 × Wash Buffer into each hole, then pouring out the 1 × Wash Buffer, and inversely buckling the microporous plate on absorbent paper for beating for several times; repeating for 3 times;

8. add 200. Mu.l TNF-. Alpha.conjugate to each well, carefully cover a new patch, and incubate at room temperature for 2 hours;

9. carefully tearing off the adhesive film, pouring out the liquid in the microporous plate, and inversely buckling the microporous plate on absorbent paper for beating for several times; adding 300 mu l of 1 × Wash Buffer into each hole, then pouring off the 1 × Wash Buffer, and inversely buckling the microporous plate on absorbent paper for beating for several times; repeating for 3 times;

10. adding 200 μ l of Substrate Solution into each well, and standing for 30 minutes at room temperature in the absence of light; then adding 50 mu l of Stop Solution into each hole;

11. reading OD value: placing the microplate into an enzyme-labeling instrument (Thermofisor, multiscan), setting an enzyme-labeling instrument program, detecting absorbance at a wavelength of 450nm, and performing data analysis;

12. and subtracting the average value of the OD values of the culture medium blank group from the result, then utilizing Origin software, taking the theoretical concentration of each point of a standard curve as an X axis, taking the light absorption value as a Y axis, simultaneously carrying out logarithmic conversion on two sides, fitting by a four-parameter method, drawing a standard curve, inputting the light absorption value of the sample, and calculating the concentration value of TNF-alpha of the sample.

The level of TNF- α secretion by exosomes-inhibiting PBMCs was determined using the above method, with the results:

the TNF-alpha level of the negative group was 802.2pg/ml,

the level of TNF-alpha in the control group was 5203.7pg/ml,

the test groups of example 2, example 2a and example 2b had TNF- α levels of 1227 to 1413pg/ml for example the test group of example 2 had TNF- α levels of 1316.2pg/ml,

the TNF-alpha levels in the test groups of examples 3, 3a and 3b were 1087-1348 pg/ml, e.g.the TNF-alpha level in the test group of example 3 was 1284.7pg/ml,

the TNF- α levels in the experimental groups of examples 3c and 3d ranged from 1232 to 1446 pg/ml.

Example 8: RT-PCR analysis of inhibitory effect of exosome on TNF-alpha gene expression level of PBMC

1. Extraction of total RNA of PBMC: 1ml of PBMC cells of each group (experimental group, control group, negative group) obtained after co-culturing for 5 days in example 7, step 2 were aspirated from a 24-well plate, placed in a 1.5ml EP tube at 300g, centrifuged for 5 minutes, and then the supernatant was aspirated off, and 1ml of Trizol reagent (Life) was added to the pellet, and the pellet was left at room temperature for 5 minutes and sufficiently lysed;

2. placing the EP tube into a high-speed centrifuge, centrifuging at 4 ℃ and 12000rpm for 5 minutes, and then removing the precipitate;

3. adding 200 mul of chloroform, shaking and mixing evenly, and then placing for 15 minutes at room temperature;

4. placing the EP tube into a high-speed centrifuge at 12000rpm, and centrifuging for 15 minutes;

5. absorbing the upper water phase into another centrifuge tube; adding 0.5ml of isopropanol solution, uniformly mixing, and standing for 10 minutes at room temperature;

6. placing the EP tube into a high-speed centrifuge, centrifuging at 4 ℃ and 12000rpm for 10 minutes, then removing supernatant and precipitating RNA at the bottom of the tube;

7. adding 1mL of 75% ethanol solution, and gently shaking the centrifugal tube to perform suspension precipitation;

8. placing the EP tube into a high-speed centrifuge, centrifuging at 4 ℃ and 8000rpm for 5 minutes, and then removing supernatant; air-drying at room temperature for 5-10 min, and resuspending RNA with 20 μ l of sterile deionized water;

9. detecting the RNA concentration by using a nanodrop instrument;

10. reverse transcription of cDNA: performing reverse transcription of RNA into cDNA according to the operation instruction of a Bestar qPCR kit (cat # DBI-2220);

11. qPCR detection of TNF-alpha Gene expression levels: the TNF-alpha gene expression level was detected by real-time fluorescent quantitative PCR according to the Bestar qPCR (SyberGreen) kit (cat # DBI-2043) kit instructions.

The expression level of TNF-alpha gene detected by real-time fluorescent quantitative PCR is characterized by relative mRNA level (times), the relative mRNA level of the negative group is 1, the relative mRNA level of other groups is calculated, and the result is that:

the relative mRNA level of the control group was 114.6-fold,

the relative mRNA levels in the test groups of example 2, example 2a and example 2b were 64 to 73 times, for example, the relative mRNA level in the test group of example 2 was 70.6 times,

the relative mRNA levels in the test groups of example 3, example 3a and example 3b were 66 to 74 times, for example, 68.2 times in the test group of example 3,

the relative mRNA levels in the experimental groups of example 3c and example 3d were 67-79 fold.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

1. The exosome separated and extracted by using the placenta mesenchymal stem cells has the average particle size of 50-200 nm, expresses membrane protein CD9 and membrane protein CD81, and has the positive expression rate of the membrane protein CD9 of more than 70 percent and the positive expression rate of the membrane protein CD81 of more than 80 percent.

2. Exosome according to claim 1 having an average particle size of 75-150 nm and a positive expression rate for membrane protein CD9 of greater than 75% and for membrane protein CD81 of greater than 85%.

3. The exosome according to claim 1, which is prepared using placental mesenchymal stem cells by a method comprising the steps of:

(1) Inoculating mesenchymal stem cells into culture flask, adding MSC complete culture medium, setting at 37 deg.C, and 5% CO ₂ Cultivation in incubatorCulturing to make the cells adhere to the wall, adding IL-1 beta into the culture solution to the concentration of 8-12 ng/mL, and continuing culturing;

(2) Removing the medium by aspiration, replacing with fresh MSC complete medium, and continuing the cell culture at 37 deg.C, 5% ₂ Culturing in an incubator until the cell fusion degree is more than or equal to 80 percent;

(3) The medium was aspirated off, washed with PBS, then MSC complete medium was added, and the mixture was placed at 37 ℃ and 2%O ₂ 、5％CO ₂ Culturing in an incubator for 42-56 hours;

4. Exosome according to claim 3, in step (1),

mesenchymal stem cells are cells of generations P2 to P8, for example, cells of generations P3 to P6;

adding cells at a density of (0.5-5). Times.10 ^4 cells/cm ^2, for example, adding cells at a density of (0.5-2). Times.10 ^4 cells/cm ^ 2;

using T75 culture bottles, inoculating (2-10). Times.10 ^5 cells such as 7.5.10 ^5 cells in each bottle, adding 10-20 ml of culture medium;

culturing for 20-30 hours, for example, 24 hours to allow the cells to adhere to the wall;

continuing the culture for 20 to 30 hours, for example, for 24 hours after the IL-1 beta is added;

sodium tartrate and lysine were added to the culture solution at concentrations of 0.15 to 0.2mg/mL and 2.0 to 2.5mg/mL, respectively, for example, at concentrations of 0.15mg/mL and 2.2mg/mL, respectively, in addition to IL-1. Beta.

5. Exosome according to claim 3, step (3), at 37 ℃, 2%O ₂ 、5％CO ₂ The culture was carried out in an incubator for 48 hours.

6. Exosome according to claim 3, in step (4),

first, the supernatant was centrifuged at 250g at 4 ℃ for 12 minutes, then at 2500g at 4 ℃ for 18 minutes, then at 8000g at 4 ℃ for 35 minutes, and the supernatant was filtered through a 0.22 μm filter and then at 120000g at 4 ℃ for 75 minutes;

centrifuging at 350g and 4 deg.C for 8 min, then at 1500g and 4 deg.C for 25 min, then at 12000g and 4 deg.C for 25 min, filtering the supernatant with 0.22 μm filter membrane, and centrifuging at 80000g and 4 deg.C for 120 min;

first, the supernatant was centrifuged at 300g and 4 ℃ for 10 minutes, then at 2000g and 4 ℃ for 20 minutes, then at 10000g and 4 ℃ for 30 minutes, and the supernatant was filtered through a 0.22 μm filter and then centrifuged at 100000g and 4 ℃ for 90 minutes.

7. Exosome according to claim 3, in step (5),

centrifuging at 100000g and 4 deg.C for 90 min, or at 80000g and 4 deg.C for 120 min, or at 120000g and 4 deg.C for 75 min;

storing the obtained exosome suspension at-80 ℃;

exosomes obtained from 1.5 x 10^6 cells of step (1) are resuspended in 0.5-5 ml sterile PBS, e.g. 0.5-2 ml sterile PBS, e.g. 1ml sterile PBS.

8. An exosome according to claim 3, said placental mesenchymal stem cells being prepared by a method comprising the steps of:

(a) Treatment of placental tissue

adding the cleaned tissue into HBSS digestive juice containing 0.005% Liberase MNP-S enzyme and 0.05% DNAI type enzyme, mixing well, and performing shake digestion in a shaker at 37 deg.C and 100rpm for 30min;

(b) Obtaining placental progenitor cells

(c) Purification culture

at a rate of 5000 to 15000 pieces/cm ² Inoculating the placenta P0 generation cells into a T75 culture bottle at the inoculation density, and completely changing the liquid on the 3 rd to 4 th days; cloning cells on days 6-7, growing fusiform cells in vortex form on days 10-11, namely P1 generation placental mesenchymal stem cells, collecting and then carrying out passage;

9. A method for separating and extracting exosome by using placenta mesenchymal stem cells comprises the following steps:

(1) Inoculating mesenchymal stem cells into culture flask, adding MSC complete culture medium, setting at 37 deg.C, and 5% CO ₂ Culturing in an incubator to make cells adhere to the wall, adding IL-1 beta into the culture solution to the concentration of 8-12 ng/mL, and continuing culturing;

(2) Discard the medium and replace with fresh MSC complete medium, continue the cells at 37 ℃ and 5% CO ₂ Culturing in an incubator until the cell fusion degree is more than or equal to 80 percent;

centrifuging at 8000-12000 g and 4 deg.c for 25-35 min, filtering the supernatant with 0.22 micron filter film and setting in another centrifuge tube;

(5) Adding sterile PBS into a centrifuge tube to enable the exosome precipitate to be resuspended, centrifuging for 75-120 minutes at 80000-120000 g and 4 ℃, removing supernatant, adding sterile PBS to enable the exosome to be suspended, and obtaining exosome in a suspension form;

further, it is as described in any one of the second aspect of the present invention.

10. Use of exosome isolated from placental mesenchymal stem cells according to any one of claims 1 to 8 in the preparation of a medicament for the treatment of inflammatory disease.