CN116496983A - Hypoxia-induced human umbilical mesenchymal stem cell exosome and preparation method and application thereof - Google Patents
Hypoxia-induced human umbilical mesenchymal stem cell exosome and preparation method and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a hypoxia-induced human umbilical cord mesenchymal stem cell exosome and a preparation method and application thereof, and belongs to the technical field of biomedicine. The invention provides a preparation method of hypoxia-induced human umbilical cord mesenchymal stem cell exosomes, comprising the following steps: culturing human umbilical cord mesenchymal stem cells in a low-oxygen environment to obtain a cell culture solution; the oxygen volume percentage content of the low-oxygen environment is 0.5-1.5%; and (3) performing differential ultracentrifugation on the cultured human umbilical cord mesenchymal stem cells to obtain hypoxia-induced human umbilical cord mesenchymal stem cell exosomes. The exosome obtained by the method for preparing the exosome by hypoxia induction is used for the diabetic wound surface, the treatment effect of the exosome on the diabetic wound surface is obviously better than that of the exosome prepared under the normoxic condition, the exosome has more advantages in healing speed, and has greater application potential in the aspects of stimulating the regeneration of skin appendages and improving healing quality.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a hypoxia-induced human umbilical mesenchymal stem cell exosome, and a preparation method and application thereof.
Background
The wound surface difficult to heal is one of serious complications of diabetes, and seriously affects the life quality of patients and even life health. Under this high sugar environment of the organism, the skin acts as a protective barrier for the human body and is in a state of serious metabolic disturbance. The development of a safe diabetes wound healing treatment method with potential application value has important value.
Exosomes are extracellular microvesicles having a lipid bilayer membrane structure and containing various small molecule active ingredients such as proteins, DNA, RNA, and the like. Exosomes act as one of the paracrine pathways, and can act as carriers to transport their contents into the target cells through fusion cell membranes or endocytosis, thereby exerting an effective biological effect. Compared with medicines, small molecular materials, stem cell treatment and the like, the stem cell-derived exosome has better biosafety and histocompatibility. With the increasing demands of disease treatment, the exosomes are stable in quality, suitable for large-scale extraction and safer in clinical application. The effect of exosomes applied to diabetic wound repair at present is often poor.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a preparation method of a hypoxia-induced human umbilical mesenchymal stem cell exosome, which can be effectively applied to repairing a diabetic wound surface.
The invention aims at realizing the following technical scheme:
the invention provides a preparation method of hypoxia-induced human umbilical cord mesenchymal stem cell exosomes, which comprises the following steps:
culturing human umbilical cord mesenchymal stem cells in a low-oxygen environment to obtain a cell culture solution; the oxygen volume percentage content of the low-oxygen environment is 0.5-1.5%;
extracting exosomes from the cell culture solution to obtain hypoxia-induced human umbilical mesenchymal stem cell exosomes.
Preferably, the culture medium for the culture comprises a high sugar DMEM medium; the high-sugar DMEM culture medium contains 5% -10% of exosome-free serum.
Preferably, the time of the culture is 36-48 hours; the temperature of the culture was 37 ℃.
Preferably, the step of extracting includes:
centrifuging the cell culture solution at a low speed, and taking a supernatant to obtain a first suspension;
centrifuging the first suspension at medium speed, and taking the supernatant to obtain a second suspension;
and filtering the second suspension, performing high-speed centrifugation, taking the precipitate, re-suspending, and re-suspending after high-speed centrifugation again to obtain the hypoxia-induced human umbilical mesenchymal stem cell exosome suspension.
Preferably, the rotating speed of the low-speed centrifugation is 1000-2000 g; the low-speed centrifugation time is 5-10 min;
the rotation speed of the medium-speed centrifugation is 10,000-20,000 g; the middle speed centrifugation time is 20-30 min;
the rotating speed of the high-speed centrifugation is 100,000-200,000 g; the high-speed centrifugation time is 75-90 min.
Preferably, the pore size of the filter membrane for filtration is 0.22 μm; the resuspended reagent comprises sterile PBS buffer; the pH of the sterile PBS buffer is 7.2-7.4.
Preferably, the human umbilical cord mesenchymal stem cells comprise human umbilical cord mesenchymal stem cells obtained by passage for 2-4 times.
The invention provides the hypoxia-induced human umbilical mesenchymal stem cell exosome prepared by the preparation method.
The invention provides application of the hypoxia-induced human umbilical cord mesenchymal stem cell exosome in preparation of a reagent with high miR-17-5p expression.
The invention also provides application of the hypoxia-induced human umbilical cord mesenchymal stem cell exosome in preparing a medicament for promoting diabetic wound healing.
The invention has the beneficial effects that:
the invention provides a preparation method of hypoxia-induced human umbilical cord mesenchymal stem cell exosomes, which comprises the following steps: culturing human umbilical cord mesenchymal stem cells in a low-oxygen environment to obtain a cell culture solution; the oxygen volume percentage content of the low-oxygen environment is 0.5-1.5%; extracting exosomes from the cell culture solution to obtain hypoxia-induced human umbilical mesenchymal stem cell exosomes. The invention prepares the human umbilical cord mesenchymal stem cell exosome by utilizing the hypoxia condition, and the human umbilical cord mesenchymal stem cell exosome can realize the high expression of miR-17-5p. The results of the examples show that: the method for preparing the exosome by utilizing hypoxia induction provided by the invention has the advantages that the obtained exosome is used for the diabetic wound surface, the treatment effect on the diabetic wound surface is obviously better than that of the exosome prepared under the normoxic condition, the healing speed is higher, and the application potential is higher in the aspects of stimulating the regeneration of skin appendages and improving the healing quality.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the results of measurement of the particle size and concentration of exosomes prepared in example 1;
FIG. 2 is a graph showing the results of measurement of the particle size and concentration of exosomes prepared in comparative example 1;
FIG. 3 is a transmission electron microscope image of the exosomes prepared in example 1;
FIG. 4 is a transmission electron microscope image of the exosomes prepared in comparative example 1;
FIG. 5 is a graph showing the expression levels of surface-specific marker proteins of exosomes and cellular proteins prepared in example 1 and comparative example 1;
FIG. 6 is a graph of the abundance of miR-17-5p in the exosomes prepared in example 1 and miR-17-5p in the exosomes prepared in comparative example 1;
FIG. 7 is a graph of the wound on day 0, day 3, day 7 and day 14 for the control, normoxic and hypoxic exosomes;
FIG. 8 is a graph showing the relationship between the ratio of wound surface and the number of days of wound healing in the control group, normoxic exosome group and hypoxic exosome group;
FIG. 9 is a chart of HE staining of wound tissue from the control, normoxic and hypoxic exosomes, day 7 and day 14;
fig. 10 is a graph of wound tissue wound margin spacing ratio for the control, normoxic and hypoxic exosomes, day 7 and day 14.
Detailed Description
The invention provides a preparation method of hypoxia-induced human umbilical cord mesenchymal stem cell exosomes, which comprises the following steps:
culturing human umbilical cord mesenchymal stem cells in a low-oxygen environment to obtain a cell culture solution; the oxygen volume percentage content of the low-oxygen environment is 0.5-1.5%;
extracting exosomes from the cell culture solution to obtain hypoxia-induced human umbilical mesenchymal stem cell exosomes.
The invention cultures human umbilical cord mesenchymal stem cells in a low-oxygen environment to obtain a cell culture solution; the oxygen volume percentage of the low-oxygen environment is 0.5-1%, preferably 1%.
In the present invention, the human umbilical cord mesenchymal stem cells preferably include human umbilical cord mesenchymal stem cells obtained by passaging 2 to 4 times. In the present invention, the method for obtaining human umbilical cord mesenchymal stem cells preferably comprises the steps of:
cutting umbilical cord tissue, adding type II collagenase for digestion to obtain umbilical cord cell digestive juice;
stopping digestion, filtering, centrifuging, and discarding supernatant to obtain human umbilical cord mesenchymal stem cells.
In the invention, umbilical cord tissues are preferably sheared, and type II collagenase is added for digestion to obtain umbilical cord cell digestive juice.
In the present invention, the umbilical cord tissue is preferably fresh umbilical cord tissue. In the present invention, the preservation mode of the umbilical cord tissue preferably comprises placing the umbilical cord tissue in physiological saline containing 0.1% green chain diabody. In the invention, the 0.1% physiological saline of the green chain double antibody is specifically that 1 mu L of the green chain double antibody is added into 1ml of physiological saline. In the present invention, the green chain diabody is preferably a mixture of green chain and streptomycin (100×, P1400, soribao) available from soribao biotechnology limited. The preservation mode is preferably suitable for short-term preservation of umbilical cord tissues; the short storage time is preferably less than or equal to 2 hours. The present invention preferably includes a pretreatment of the umbilical cord tissue prior to shearing the umbilical cord tissue. The pretreatment according to the invention preferably comprises: soaking umbilical cord tissue in alcohol to obtain soaked umbilical cord tissue; the soaked umbilical tissue is removed from the adventitia of the umbilical cord, umbilical arteries and umbilical veins are stripped, and the umbilical cord tissue is washed clean with physiological saline. In the pretreatment according to the present invention, the soaking time is preferably 10 to 120 minutes, more preferably 10 minutes. The invention soaks umbilical cord tissue to maintain the activity of tissue cells, reduce the cell death rate and prevent infection. After obtaining the pretreated umbilical cord tissue, the present invention preferably shears the umbilical cord tissue. The method of the present invention is not particularly limited, and any shearing method conventional in the art may be used. In the present invention, the umbilical cord tissue is preferably sheared into pieces of umbilical cord tissue. In the present invention, the volume of the umbilical cord tissue mass is preferably 1-2 mm 3 More preferably 1mm 3 . In the present invention, the umbilical cord tissue blocks are preferably spread uniformly in a cell culture dish and then digested by adding type II collagenase. The time of digestion according to the present invention is preferably 0.5 to 1 hour, more preferably 1 hour. After the digestion is completed, umbilical cord cell digestive juice is obtained.
After obtaining the umbilical cord cell digestive juice, the invention preferably stops digestion, filters, centrifugates, and discards the supernatant to obtain the human umbilical cord mesenchymal stem cells. In the present invention, the termination of digestion is preferably performed using an equal volume of medium; the medium is preferably a serum-containing DMEM complete medium. In the present invention, the volume percentage of serum in the DMEM complete medium containing serum is preferably 15 to 20%, more preferably 20%. After termination of digestion, the present invention preferably filters the termination digest. The method of filtration according to the invention is preferably cell-screen filtration. After filtration is completed, the filtrate is preferably centrifuged. The rotation speed of the centrifugation is preferably 1000r/min; the time of the centrifugation is preferably 5min. After the centrifugation is completed, the supernatant is discarded, and the sediment is the digested human umbilical cord mesenchymal stem cells.
After the digested human umbilical cord mesenchymal stem cells are obtained, primary culture is carried out in the invention, and the primary cultured human umbilical cord mesenchymal stem cells are obtained. In the present invention, the medium used for the primary culture is preferably DMEM complete medium. The time of the primary culture according to the present invention is preferably 3d.
After obtaining the primary cultured human umbilical cord mesenchymal stem cells, the invention preferably carries out subculture. The method of the present invention is not particularly limited, and conventional methods for subculturing human umbilical cord mesenchymal stem cells known to those skilled in the art may be used. In the present invention, the temperature of the subculture is preferably 37 ℃; the subculture is preferably carried out at a volume percentage of 5% CO 2 Is performed in a cell culture tank. In the present invention, the number of times of subculture is preferably 2 to 4 times, more preferably 3 times.
After the passaged human umbilical cord mesenchymal stem cells are obtained, the passaged human umbilical cord mesenchymal stem cells are cultured in a low-oxygen environment to obtain a cell culture solution.
In the present invention, the volume percentage of oxygen in the low-oxygen environment is 0.5% to 1.5%, preferably 0.5% to 1%, and more preferably 1%. In the present invention, the culture medium for the culture preferably includes a high sugar DMEM medium or a medium dedicated to stem cells. When the culture medium for the culture of the invention is a high-sugar DMEM medium, the high-sugar DMEM medium preferably includes an exosome-free serum, and the content of the exosome-free serum in the high-sugar DMEM medium is preferably 5% -10%, more preferably 10%. The purpose of adding serum into the culture medium is mainly to provide cell nutrition support and ensure the output of exosomes. In the present invention, the time of the cultivation is preferably 36 hours to 48 hours, more preferably 48 hours; the temperature of the culture is preferably 37 ℃. In the present invention, the carbon dioxide content in the low-oxygen environment is preferably 5% by volume. After the culture in the low-oxygen environment is completed, the cell culture solution is obtained.
After obtaining a cell culture solution, extracting exosomes from the cell culture solution to obtain hypoxia-induced human umbilical mesenchymal stem cell exosomes.
The extraction step of the present invention preferably comprises:
centrifuging the cell culture solution at a low speed, and taking a supernatant to obtain a first suspension;
centrifuging the first suspension at medium speed, and taking the supernatant to obtain a second suspension;
and filtering the second suspension, performing high-speed centrifugation, taking the precipitate, re-suspending, and re-suspending after high-speed centrifugation again to obtain the hypoxia-induced human umbilical mesenchymal stem cell exosome suspension.
In the present invention, the temperature is preferably controlled to 4 ℃ during the differential ultracentrifugation. In the present invention, the rotational speed of the low-speed centrifugation is preferably 1000 to 2000g, more preferably 2000g; the low-speed centrifugation time is preferably 5 to 10 minutes, more preferably 10 minutes. After the low speed centrifugation is completed, the invention preferably uses the supernatant to obtain a first suspension. The first suspension is obtained, and the first suspension is preferably subjected to moderate speed centrifugation. In the present invention, the rotational speed of the medium speed centrifugation is preferably 10,000 to 20,000g, more preferably 10,000g; the time of the medium speed centrifugation is preferably 20 to 30 minutes, more preferably 30 minutes. After medium speed centrifugation is complete, the invention preferably uses the supernatant to obtain a second suspension. The present invention preferably removes large particulate matter, including cells, dead cells, debris, and the like, by low-speed centrifugation and medium-speed centrifugation. After the second suspension is obtained, the present invention preferably filters the second suspension to obtain a filtrate. The pore size of the filter membrane for filtration of the present invention is preferably 0.22. Mu.m. After the filtrate is obtained, the filtrate is preferably subjected to high-speed centrifugation in the present invention. In the present invention, the rotational speed of the high-speed centrifugation is preferably 100,000 to 200,000g, more preferably 100,000g; the time for the high-speed centrifugation is preferably 60 to 90 minutes, more preferably 75 minutes. After the first high speed centrifugation is completed, the present invention preferably takes the pellet for resuspension. In the present invention, the resuspension is preferably performed using a PBS buffer to obtain a resuspension. After obtaining the heavy suspension, the invention preferably re-centrifiigates the heavy suspension at high speed. In the present invention, the rotational speed of the high-speed centrifugation again is preferably 100,000 to 200,000g, more preferably 100,000g; the time for the high-speed centrifugation is preferably 60 to 90 minutes, more preferably 75 minutes. After the high-speed centrifugation is finished again, sediment is preferably selected for re-suspension, and the hypoxia-induced human umbilical mesenchymal stem cell exosome suspension is obtained. The re-suspension according to the invention is preferably carried out using PBS buffer. In the present invention, the pH of the PBS buffer is preferably 7.2 to 7.4. After the hypoxia-induced human umbilical cord mesenchymal stem cell exosome suspension is obtained, the hypoxia-induced human umbilical cord mesenchymal stem cell exosome suspension can be directly used or frozen for later use.
The preparation method of the hypoxia-induced human umbilical cord mesenchymal stem cell exosome provided by the invention utilizes hypoxia conditions to induce the human umbilical cord mesenchymal stem cell and then extracts the exosome. The preparation method increases the content of miR-17-5p in the exosome while maintaining the natural structure of the exosome, and can effectively improve the healing of diabetic wounds.
The invention provides the hypoxia-induced human umbilical mesenchymal stem cell exosome prepared by the technical scheme. In the invention, the hypoxia-induced human umbilical cord mesenchymal stem cell exosome maintains the natural structure of exosomes, and the hypoxia-induced human umbilical cord mesenchymal stem cell exosomes can be verified to be capable of expressing miR-17-5p in a high degree.
The invention also provides a reagent for preparing miR-17-5p high expression by using the hypoxia-induced human umbilical cord mesenchymal stem cell exosome.
The invention also provides application of the hypoxia-induced human umbilical cord mesenchymal stem cell exosome in preparing a medicament for promoting diabetic wound healing. The method for preparing the exosome by utilizing hypoxia induction provided by the invention has the advantages that the obtained exosome is used for the diabetic wound surface, the treatment effect on the diabetic wound surface is obviously better than that of the exosome prepared under the normoxic condition, the healing speed is higher, and the application potential is higher in the aspects of stimulating the regeneration of skin appendages and improving the healing quality.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
A hypoxia-induced human umbilical cord mesenchymal stem cell exosome is prepared by the following steps:
1) The method for obtaining the human umbilical cord mesenchymal stem cells comprises the following steps:
(1) Fresh umbilical cord tissue is obtained from an operating room of a clinical hospital (all operations are approved by the clinical committee of the hospital), placed into physiological saline containing 0.1% green chain double antibody and transported to a laboratory (the physiological saline containing 0.1% green chain double antibody is used for preserving umbilical cord tissue for no more than 2 hours and can only be used for short preservation of umbilical cord tissue); soaking umbilical cord in alcohol for several minutes, removing umbilical cord adventitia, removing umbilical artery and umbilical vein, and washing with physiological saline;
(2) Cutting the treated umbilical cord tissue into pieces of about 1mm in an ultra clean bench 3 Uniformly spreading the tissue blocks in a cell culture dish, adding type II collagenase, and placing the tissue blocks in a constant temperature cell culture box for digestion;
(3) Adding an equal volume of complete culture medium DMEM containing 20% by volume of serum after 1h to terminate digestion, filtering with a cell sieve, centrifuging the filtered cell suspension at a speed of 1000r/min for 5min;
(4) The supernatant was discarded and the cells were resuspended in 5% CO with DMEM complete medium 2 Primary culture is carried out in a cell culture box at 37 ℃ for 3 daysChanging the culture medium, changing the primary culture solution 2-3 days later, performing subculture after the primary culture is completed, and taking the human umbilical cord mesenchymal stem cells obtained by the third-generation subculture for subsequent experiments.
2) The low oxygen environment was set to 1% oxygen concentration and 5% carbon dioxide concentration. Culturing the human umbilical cord mesenchymal stem cells obtained in step 1) in Gibco high-sugar DMEM medium containing 10% exosome-free serum in a low-oxygen environment. The temperature of the culture was 37℃and the time of the culture was 48 hours (the cell fusion degree reached about 80%).
3) Centrifuging culture supernatant of human umbilical cord mesenchymal stem cells cultured under hypoxia condition in a centrifuge at a low speed of 2,000g for 10min, and collecting supernatant to obtain a first suspension; the first suspension was centrifuged at 10,000g for 30min to give a second suspension.
4) The second suspension was filtered using a 0.22 μm ultrafiltration membrane, and the resulting filtrate was centrifuged at 100,000g for 75min. The supernatant was removed, the pellet resuspended in sterile PBS buffer and centrifuged again at 100,000g for 75min. The temperature is controlled at 4 ℃ in the centrifugation process of low-speed centrifugation, medium-speed centrifugation and high-speed centrifugation. After centrifugation again, the supernatant was removed and the pellet was resuspended in sterile PBS buffer to give the hypoxic exosome suspension reagents, respectively. Can be used at present or frozen for later use.
Comparative example 1
A human umbilical cord mesenchymal stem cell exosome is prepared by the same steps as in example 1, except that an normoxic environment is set to have an oxygen concentration of 21% and a carbon dioxide concentration of 5%.
Application example 1
The hypoxia-induced human umbilical cord mesenchymal stem cell exosomes prepared in example 1 and the human umbilical cord mesenchymal stem cell exosomes prepared in comparative example 1 were subjected to correlation identification.
1. Nanoparticle analysis detects the concentration and particle size of exosomes.
The detection method comprises the following steps: the exosome suspension was separated into 20 μl, diluted with 5-10 mL PBS buffer, placed into a nanosystem sample chamber (particle metric) for detection, and then the results were processed and analyzed using software ZetaView 8.04.02 to record particle size and particle concentration for each example and comparative exosome. The particle size and particle concentration of the exosomes prepared in example 1 are shown in fig. 1; the particle size and particle concentration of the exosomes prepared in comparative example 1 are shown in fig. 2.
The results in FIGS. 1 and 2 show that the particle sizes and concentrations of normoxic and hypoxic exosomes are similar, and the particle sizes of normoxic and hypoxic exosomes are 123.4+ -36.7 nm and 121.8+ -28.3 nm, respectively; the concentrations of the normoxic and hypoxic exosomes are both about 1×10 9 particles/mL. Indicating that the exosomes obtained from normoxic and hypoxic groups have no obvious differences in particle size and particle concentration.
2. Transmission electron microscope for detecting and observing morphology of exosomes
The external secretion form is photographed and recorded by using a transmission electron microscope, and the specific detection method is operated as follows:
(1) Osmium acid fixation: 20. Mu.L of the sEVs-containing suspension was added to 500. Mu.L of a 2% osmium acid solution, and the mixture was allowed to stand in a refrigerator at 4℃for 2 hours for fixation;
(2) Dehydrating: after the fixation, the sample was washed with PBS buffer for 3 times, each washing was allowed to stand for about 15min, and then dehydrated in ethanol solutions of 50%, 70%, 80% and 90% concentration gradients in order for 15min. And after dehydration is finished, standing for 15min, and finally soaking in absolute ethyl alcohol for dehydration for 15min. Dewatering again according to the above process;
(3) Replacement: immersing the dehydrated sample in acetone for 15min, displacing the dehydrated solution, and repeating the displacement process once;
(4) Dipping: the impregnating solution I is prepared from acetone and embedding agent according to the volume ratio of 2:1, the impregnating solution II is prepared from acetone and embedding agent according to the volume ratio of 1:1, the impregnating solution III is prepared from acetone and embedding agent according to the volume ratio of 1:2, and the impregnating solution IV is prepared from 100% embedding agent. Sequentially soaking in the soaking solution I, the soaking solution II and the soaking solution III for 2 hours, finally soaking in the soaking solution IV for 24 hours, and repeating the soaking in the soaking solution IV once;
(5) Embedding: after the impregnation is completed, placing the sample into an embedding plate filled with a pure embedding agent for embedding treatment;
(6) Polymerized slices: polymerizing the sample at 65 ℃ after embedding, cutting the sample into slices after 48 hours, wherein the thickness is 60-80 nm;
(7) Dyeing: dyeing a slice sample by using uranium diacetate, soaking for 10min, cleaning, then dyeing by using lead acetate, cleaning again after 10min, and observing by using an electron microscope;
(8) Photographing: electron microscope (Hitachi) detection imaging was performed at 100kV with an acceleration voltage set at 40-120kV (100V increment).
The exosome suspensions prepared in example 1 and comparative example 1 were photographed using a transmission electron microscope using the above method, the photographed pictures of the exosomes prepared in example 1 are shown in fig. 3, and the photographed pictures of the exosomes prepared in comparative example 1 are shown in fig. 4. Wherein the white scale in fig. 3 and 4 is 200nm.
As can be seen from fig. 3 and 4, the normoxic exosomes and the hypoxic exosomes have classical bilayer membrane-like structures of exosomes, which overall take on cup-like or spherical morphology, and the diameters of the normoxic exosomes and the hypoxic exosomes are not significantly different.
3. Extracting total protein of exosome for detection
Protein concentration was detected by BCA protein detection kit, and surface specific markers of exosomes were detected by electrophoresis, transfer, blocking, secondary antibody incubation and chemiluminescent imaging analysis using western blot experiments: CD63, CD9, TSG101 and Calnexin. The specific operation is as follows:
(1) BCA assay detects protein concentration: about 50. Mu.L of each of the exosome suspensions prepared in example 1 and comparative example 1 was added to 50. Mu.L of RIPA lysate (R0010, soy Co.) and the mixture was thoroughly lysed at 4℃for 15min, centrifuged (12000 g,4 ℃) for 20min, and the supernatant was slowly aspirated by a pipette and placed in a 1.5mL EP tube. The cell lysate was prepared by culturing human umbilical cord mesenchymal stem cells in a cell culture dish having a diameter of 6cm, sucking off the culture supernatant, lysing the cells sufficiently with 100. Mu.L of RIPA lysate at 4℃for 15min, scraping the cells down and transferring them into a 1.5mL EP tube, centrifuging (12000 g,4 ℃) for 20min, and then slowly sucking out the supernatant with a pipette, and placing it in a 1.5mL EP tube. After measuring the protein concentration of the cell lysate and the two sets of exosomes using BCA protein concentration detection kit (PC 0020, soribao), three sets of samples were diluted to the same concentration by calculation with PBS buffer, following protein samples: the loading buffer (5X) was mixed well at a volume ratio of 4:1 and boiled in an iron bath at 105℃for 10min.
(2) Electrophoresis: according to the description operation of the glue preparation kit, the lower glue is separation glue, and the upper glue is concentrated glue. After the glue is added and removed (glue separation), absolute ethyl alcohol is used for flattening, after the glue is added and removed (glue concentration), a comb is inserted as soon as possible, and bubbles in the groove (the existence of the bubbles affects the electrophoresis of a sample) are avoided. The boiled protein sample in step (1) was thawed after being taken out of the refrigerator, and vortexed and flash-off (because a small amount of water vapor would condense at the tube cap, affecting the sample concentration). The sample loading amount is selected according to the protein concentration, the concentration is 10 mu L higher, and the concentration is 30-40 mu L lower. Rainbow markers are added into sample grooves on two sides of the sample, and are used as target protein position references, and the sample loading sequence sequentially comprises cellular proteins, normoxic exosome proteins and hypoxic exosome proteins. Electrophoresis was performed at room temperature, 70mV was concentrated at constant pressure, 130mV was constant pressure after entering the separation gel, and electrophoresis was performed to the position where the experiment was required.
(3) Electric conversion: an electric transfer apparatus (electric transfer tank, ice-making, etc.) and a reagent (electric transfer liquid preparation: 100mL of 10 Xelectric transfer liquid, 700mL of pure water, and 200mL of methanol were contained in each 1000mL of electric transfer liquid) were prepared. When transferring the membrane, the PVDF membrane is soaked in methanol for more than 10 seconds, no bubble can appear between the glue and the membrane, and the electric transfer liquid can be put into ice for precooling in advance. In the ice tank, 300mA is constant current for 1h (the time can be adjusted according to the size of the target protein);
(4) Closing: PVDF film was taken out from the electrotransfer cell, immersed in skimmed milk (5%, 100mL, and 5 g milk powder), and sealed for 1h or more. The sealed milk is recycled and put into a refrigerator at the temperature of 4 ℃, and the next day is used for diluting the secondary antibody;
(5) Incubation resistance: diluting primary antibodies (CD 63, CD9, TSG101 and Calnexin) by using primary anti-dilution liquid (1:1000, namely 1mL of primary anti-dilution liquid is added with 1 mu L of primary antigen liquid), cutting a film according to the size of a target protein strip, putting the cut strip into the primary antibodies, and shaking the film slowly by a shaking table at 4 ℃ for overnight;
(6) Secondary antibody incubation: primary antibodies were recovered, and membranes were rinsed with PBST (pbs+0.1% tween) or TBST (tbs+0.1% tween) and swished 3 times for 10min on a shaker. Then soaking the membrane in a secondary antibody (the secondary antibody is diluted by using 5% skimmed milk, and the ratio is 1:10000) and incubating for 1h at room temperature;
(7) Exposure: and (3) preparing luminous liquid, putting the strip after incubation of the secondary antibody into an exposure machine (GE company), dripping the luminous liquid for exposure, and collecting pictures. The protein bands can be analyzed for gray values using Image J software if necessary.
The total protein in the exosome suspensions and cell lysates prepared in example 1 and comparative example 1 was detected by the above method.
The results of measuring the expression levels of the surface-specific marker proteins of the exosomes and the cellular proteins prepared in example 1 and comparative example 1 are shown in fig. 5.
As can be seen from fig. 5, both normoxic and hypoxic exosomes were able to express exosome specific markers CD63, CD9 and TSG101, but not Calnexin (Calnexin is expressed only in cellular proteins). The extracted normoxic and hypoxic exosomes were confirmed to be exosomes.
4. After extraction of total RNA of exosomes, qRT-PCR was used to detect the expression level of miR-17-5p in hypoxia-derived exosomes.
(1) Sample preparation: taking 500 mu L of exosome suspension respectively, adding 200 mu L of LTrilzol lysate respectively, vortex shaking, and standing at room temperature for 30min;
(2) RNA precipitation: 200. Mu.L of chloroform was added to each EP tube, vigorously vortexed for 10s, allowed to stand still at room temperature for 5min, and centrifuged at 13000g at 4℃for 15min. After centrifugation, the visible liquid was layered, the upper colorless clear liquid layer was aspirated (avoiding aspiration to the lower pink liquid layer), and added to an enzyme-free EP tube containing 400. Mu.L of isopropanol, gently inverted and mixed well, and allowed to stand at room temperature for 10min. Centrifugation was continued at 13000g for 20min at 4 ℃. White RNA precipitation was visible after centrifugation (invisible to naked eyes if RNA amount is small);
(3) RNA washing: the supernatant was carefully aspirated, 1mL of 75% ethanol (using DEPC water with absolute ethanol) was added and centrifuged at 10000g for 5min at 4 ℃. The supernatant was removed by aspiration and repeated washes with absolute ethanol were continued. Centrifuging, removing supernatant, opening the EP tube cover, and air drying to volatilize ethanol;
(4) RNA lysis and concentration determination: RNA was solubilized using an appropriate amount of DEPC and RNA concentration, OD, was detected using Nanodrop 260 And OD (optical density) 280 The RNA concentration was recorded on the tube wall and stored in a-80℃refrigerator.
(5) Reverse transcription of miRNA: cDNA was obtained according to the TakaraPrimeScript II 1st Strand cDNA Synthesis Kit (D6210A) kit instructions;
the following solutions and reverse transcription systems were prepared according to Table 1
TABLE 1 reverse transcription system for miRNAs
| Reagent(s) | Dosage of |
| 5×primerscript buffer | 1μL |
| RT MixI | 2μL |
| RT primer for U6(U6-R) | 0.25μL |
| RT primer for miRNA(miR-RT) | 0.25μL |
| RNA | 400ng |
| DEPC water | 6.5μL |
| Total volume | 10μL |
And (3) vibrating and centrifuging the PCR tube filled with the mixed solution, and then placing the PCR tube into a PCR instrument at 16 ℃ for 30min, at 42 ℃ for 30min and at 85 ℃ for 1min. The cDNA can be stored in a refrigerator at-20 ℃;
the primer sequences are shown in Table 2.
TABLE 2 reverse transcription primer sequences
(6) PCR of cDNA
The PCR reaction liquid system is shown in Table 3 (the whole process was performed on ice).
TABLE 3 PCR reaction liquid System
| Reagent(s) | Usage amount |
| cDNA | 2μL |
| TB green Premix Ex TaqII | 12.5μL |
| Upstream/downstream primer | 1 mu L each |
| ddH 2 O | 8.5μL |
| Total volume | 25μL |
The PCR reaction conditions are shown in Table 4.
TABLE 4 PCR reaction conditions
The primers for the PCR reaction are shown in Table 5.
TABLE 5 primers for PCR reactions
After 35 cycles, the results were analyzed relatively quantitatively.
After extracting total RNA of exosomes prepared in example 1 and comparative example 1 by the above method, the expression level of miR-17-5p in hypoxia source exosomes was detected using qRT-PCR.
The abundance of miR-17-5p in the exosomes prepared in example 1 and miR-17-5p in the exosomes prepared in comparative example 1 is shown in FIG. 6.
As can be seen from FIG. 6, the relative expression level of miR-17-5p in the hypoxic exome reached 12.5, whereas the relative expression level of miR-17-5p in the normoxic exome was only 1.5. The significantly high abundance of miR-17-5p in the hypoxia exosome group, yu Changyang exosome group, demonstrates that hypoxia induction can increase the content of miR-17-5p in exosome.
Application example 2
Control test of exosome preparation for treating diabetes mice
1. Diabetes wound model establishment
12 diabetic gene mice (db/db mice) similar in sex, age, weight and growth state were randomly divided into two groups of 6. After anesthesia, back dehairing and disinfection towel spreading treatment are carried out, a wound surface with the diameter of 20mm is manufactured on the back of a mouse in a sterile environment, and contracture prevention fixation is carried out by using a rubber ring.
2. Treatment of diabetic wound healing by stem cell exosomes
The experiments were divided into PBS control group, normoxic exosome group and hypoxic exosome group. After the wound models of each group are established, PBS control group, normoxic exosome group and hypoxia exosome group are injected at intervals of 3, 6, 9 and 12 points around the wound margin, and 12.5 mu L of exosome suspension is injected at each point, which is 50 mu L. Wherein, the exosomes prepared in comparative example 1 were injected into normoxic exosomes group; hypoxia exosomes prepared in example 1 were injected; the PBS control group was injected with sterile PBS buffer. The injection method and injection volume of PBS control group, normoxic exosome group and hypoxic exosome group are the same.
3. Evaluation of wound healing and evaluation of exosome efficacy
(1) Wound healing assessment
Photographing the wound surfaces of 3 groups of mice, recording the healing condition of the daily wound surfaces, and replacing and updating the damaged rubber ring. The wound area healing rate is as follows: (A) 0 -A n )/A 0 X 100%, where A 0 Is the wound area of the same day of molding, A n Is the area of the wound surface on the nth day after molding. The area is obtained through image J software calculation, and the wound healing rate is statistically analyzed and plotted after the experiment is finished. On days 3, 7 and 14 after constructing the wound model, 6 mice were randomly picked from group 3, euthanized, and wound skin was cut completely (up to the muscular layer) along the periphery of the wound (about 1cm from the wound), cut off with tissue scissors, and fixed in 4% paraformaldehyde fixing solution for subsequent HE staining analysis.
Wherein, the control group, normoxic exosome group and hypoxic exosome group were photographed on the wounds of day 0, day 3, day 7 and day 14, and the obtained pictures are shown in fig. 7. The relationship between the wound surface ratio and the number of days of wound healing in the control group, normoxic exosome group and hypoxic exosome group is shown in fig. 8 and table 6.
TABLE 6 relationship between wound ratio and days of wound healing in control group, normoxic exosome group and hypoxic exosome group
| Days (days) | Day 0 | Day 3 | Day 7 | Day 10 | Day 14 | Day 17 | Day 21 |
| Control group 1 | 100 | 88.36 | 78.76 | 46.74 | 17.14 | 6.95 | 1.53 |
| Control group 2 | 100 | 87.04 | 76.39 | 49.52 | 20.25 | 7.53 | 0 |
| Control group 3 | 100 | 88.19 | 79.35 | 44.79 | 24.26 | 9.78 | 1.04 |
| Control group 4 | 100 | 87.02 | 80.72 | 52.42 | 17.49 | 5.64 | 0 |
| Control group 5 | 100 | 85.94 | 75.38 | 44.74 | 27.59 | 7.64 | 0 |
| Control group 6 | 100 | 87.35 | 80.99 | 42.45 | 23.72 | 9.75 | 0 |
| Normoxic exosome 1 | 100 | 86.87 | 67.04 | 31.85 | 6.09 | 1.75 | 0 |
| Normoxic exosome 2 | 100 | 81.34 | 60.73 | 28.14 | 10.16 | 2.92 | 0 |
| Normoxic exosome 3 | 100 | 85.38 | 66.31 | 37.86 | 9.19 | 1.96 | 0 |
| Normoxic exosome 4 | 100 | 84.29 | 68.16 | 33.76 | 8.25 | 1.64 | 0 |
| Normoxic exosome 5 | 100 | 87.43 | 61.33 | 31.53 | 5.48 | 0 | 0 |
| Normoxic exosome 6 | 100 | 89.57 | 70.69 | 36.84 | 9.68 | 2.17 | 0 |
| Hypoxia exosome 1 | 100 | 89.55 | 48.34 | 19.52 | 0.26 | 0 | 0 |
| Hypoxia exosome 2 | 100 | 90.42 | 46.83 | 26.74 | 2.3 | 0 | 0 |
| Hypoxia exosome 3 | 100 | 87.86 | 43.16 | 21.75 | 1.03 | 0 | 0 |
| Hypoxia exosome 4 | 100 | 85.63 | 42.08 | 23.75 | 2.11 | 0 | 0 |
| Hypoxia exosome 5 | 100 | 88.43 | 40.83 | 21.67 | 0.84 | 0 | 0 |
| Hypoxia exosome 6 | 100 | 90.42 | 48.48 | 24.42 | 0 | 0 | 0 |
From fig. 7 and 8, table 6, it is clear that the control group, normoxic exosome group and hypoxic exosome group start from day 4 to day 5, and the wound healing rates between the three groups start to be different. On day 7, the wound areas of the normoxic exosome group and the hypoxia exosome group are smaller than those of the control group, wherein the wound proportion of the control group is 78.6+/-2.3%, the wound proportion of the normoxic exosome group is 65.7+/-3.9%, and the wound proportion of the hypoxia exosome group is 44.9+/-3.3%. By day 14, the control group still had a small wound, and the normoxic exosome treated group and the hypoxic exosome treated group had nearly complete healing, with the control group having a wound ratio of 21.7±4.1%, the normoxic exosome group having a wound ratio of 8.1±1.9%, and the hypoxic exosome group having a wound ratio of 1.1±0.9%. These results demonstrate that exosomes under both normoxic and hypoxia induction promote wound healing, but hypoxia exosomes can reduce wound area faster than normoxic exosomes.
(2) HE staining evaluation
Tissue dehydration: taking out wound tissue blocks on the 7 th day and the 14 th day from the fixing solution, washing in flowing water for 30min, sequentially dewatering the tissue blocks in 75% ethanol, 85% ethanol, 95% ethanol and absolute ethanol, and finally putting the tissue blocks in xylene solution to remove alcohol in the tissue.
Paraffin embedded sections: placing the tissue blocks in melted paraffin according to the sequence of wound surface upwards for embedding, and slicing the tissue blocks by using a slicing machine after the paraffin is cooled and solidified, wherein the thickness of the tissue blocks is 5 mu m. Slightly spreading the cut slice on warm water, inserting the slice under the water surface of the slice by using a slide glass, slightly lifting the slide glass, flatly attaching the slice on the slide glass, and marking the slide glass by using a pencil in a grouping way;
dewaxing: the paraffin sections were placed in a flaker for 2 hours to soften the paraffin. Paraffin sections were then immersed in xylene (I) followed by xylene (II) and allowed to stand at room temperature for 10 minutes. Sequentially adding into absolute ethanol, 95% ethanol, 85% ethanol and 75% ethanol, standing for 2 min each time, and soaking in clear water for 2 min;
dyeing: the dewaxed slice is soaked in hematoxylin solution, washed 3 times with pure water after 20 minutes, differentiated in 1% ethanol hydrochloride for 10 seconds, dipped in 5% ammonia solution for 3 seconds after differentiation, and then washed 3 times with pure water. Finally, soaking in eosin staining solution, standing for 5 minutes at room temperature, and rinsing with pure water for 3 times;
dewatering, transparentizing and sealing: the sections were dehydrated using low to high concentration gradient ethanol, then soaked in xylene for 2 minutes to transparentize the sections, finally a suitable amount of neutral gum was dropped onto the tissue sections of the slides, the coverslips were mounted for sealing, and the slides were photographed and analyzed using a microscope (Olympus).
The results of HE staining of wound tissue from the control group, normoxic exosome group and hypoxic exosome group on days 7 and 14 are shown in FIG. 9; the wound tissue wound edge spacing ratios of the control, normoxic and hypoxic exosomes at day 7 and day 14 are shown, for example, in fig. 10 and table 7.
TABLE 7 control, normoxic and hypoxic exosomes, wound tissue wound margin spacing ratio on days 7 and 14
After the collected wound tissues are dyed by HE, experimental results show that when the wound is treated by normoxic exosomes or hypoxic exosomes, compared with a control group, the epithelialization of the diabetic wound can be obviously promoted, the newly-born skin tissues at the wound edges can be covered and gathered towards the center of the wound more quickly, the gap between the wound edges is reduced, and more skin appendages such as sebaceous glands, hair follicles and the like can be regenerated (figure 9). And the epithelialization promoting effect of the hypoxia exosome is superior to that of the normoxic exosome, so that the migration of the newly-born skin tissue at the wound margin to the center can be better accelerated, the wound margin interval is shortened, the healing speed of the wound surface is increased, and the tissue regeneration quality is improved (figure 10).
In conclusion, the human umbilical cord mesenchymal stem cell exosome prepared by the preparation method of the hypoxia-induced human umbilical cord mesenchymal stem cell exosome provided by the invention is applied to the wound surface of a diabetic mouse, has advantages in healing speed, and has greater application potential in the aspects of stimulating regeneration of skin appendages and improving healing quality.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. A preparation method of hypoxia-induced human umbilical mesenchymal stem cell exosomes, which is characterized by comprising the following steps:
culturing human umbilical cord mesenchymal stem cells in a low-oxygen environment to obtain a cell culture solution; the oxygen volume percentage content of the low-oxygen environment is 0.5-1.5%;
extracting exosomes from the cell culture solution to obtain hypoxia-induced human umbilical mesenchymal stem cell exosomes.
2. The method according to claim 1, wherein the culture medium for cultivation comprises a high-sugar DMEM medium; the high-sugar DMEM culture medium contains 5% -10% of exosome-free serum.
3. The method according to claim 1 or 2, wherein the time of the cultivation is 36 to 48 hours; the temperature of the culture was 37 ℃.
4. The method of claim 1, wherein the step of extracting comprises:
centrifuging the cell culture solution at a low speed, and taking a supernatant to obtain a first suspension;
centrifuging the first suspension at medium speed, and taking the supernatant to obtain a second suspension;
and filtering the second suspension, performing high-speed centrifugation, taking the precipitate, re-suspending, and re-suspending after high-speed centrifugation again to obtain the hypoxia-induced human umbilical mesenchymal stem cell exosome suspension.
5. The method according to claim 4, wherein the low-speed centrifugation is performed at a rotational speed of 1000 to 2000g; the low-speed centrifugation time is 5-10 min;
the rotation speed of the medium-speed centrifugation is 10,000-20,000 g; the middle speed centrifugation time is 20-30 min;
the rotating speed of the high-speed centrifugation is 100,000-200,000 g; the high-speed centrifugation time is 60-90 min.
6. The method according to claim 4, wherein the pore size of the filter membrane is 0.22. Mu.m; the resuspended reagent comprises sterile PBS buffer; the pH of the sterile PBS buffer is 7.2-7.4.
7. The method of claim 1, wherein the human umbilical cord mesenchymal stem cells comprise human umbilical cord mesenchymal stem cells obtained by passaging 2 to 4 times.
8. The hypoxia-induced human umbilical mesenchymal stem cell exosome prepared by the preparation method of any one of claims 1 to 7.
9. The hypoxia-induced human umbilical cord mesenchymal stem cell exosome of claim 8 for preparing a reagent for high expression of miR-17-5p.
10. The use of hypoxia-induced human umbilical cord mesenchymal stem cell exosomes of claim 8 in the preparation of a medicament for promoting diabetic wound healing.
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