CN112352047A - Preparation method and application of exosome derived from human umbilical cord mesenchymal stem cells - Google Patents
Preparation method and application of exosome derived from human umbilical cord mesenchymal stem cells Download PDFInfo
- Publication number
- CN112352047A CN112352047A CN202080002118.1A CN202080002118A CN112352047A CN 112352047 A CN112352047 A CN 112352047A CN 202080002118 A CN202080002118 A CN 202080002118A CN 112352047 A CN112352047 A CN 112352047A
- Authority
- CN
- China
- Prior art keywords
- supernatant
- mesenchymal stem
- umbilical cord
- human umbilical
- cord mesenchymal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Developmental Biology & Embryology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Immunology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Gastroenterology & Hepatology (AREA)
- Rheumatology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Epidemiology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention provides a preparation method and application of exosome derived from human umbilical cord mesenchymal stem cells, belonging to the technical field of biological preparations, wherein the preparation method comprises the following steps: culturing human umbilical cord mesenchymal stem cells to obtain an exosome-containing supernatant; and separating the supernatant containing the exosomes by a chemical precipitation method to obtain the MSC exosomes containing the protein kinase. The exosome derived from the human umbilical cord mesenchymal stem cells prepared by the preparation method contains protein kinase, and the exosome containing the protein kinase can enhance AMPK activity and/or inhibit liver cell steatosis, so that nonalcoholic fatty liver can be treated.
Description
Technical Field
The invention relates to the technical field of biological preparations, in particular to a preparation method and application of exosomes derived from human umbilical cord mesenchymal stem cells.
Background
The human umbilical cord mesenchymal stem cells are stem cells with the capability of multidirectional differentiation and the capability of self renewal. It enters Wharton's jelly on days 4-12 of the embryonic period and is active until the fetus is delivered. The umbilical cord has the advantages of easily-obtained materials, difficulty in pollution, no relation to moral, ethical and legal problems and the like, and is widely used for extracting the hUC-MSCs. Meanwhile, the research finds that the in vitro amplification and multidirectional differentiation capacity of the hUC-MSCs is stronger than that of MSCs from other tissues. Exosome is a membrane vesicle with a diameter of about 30-100 nm secreted from eukaryotic cells to the outside of cells, and contains components such as the cell membrane of the cell from which it is derived, proteins, and micro rna (mirna), and is fused with the adjacent cell membrane to transfer the intracellular substances of one cell to another cell, thereby performing information transfer between different cells. In the prior art, the use of exosome derived from human umbilical cord mesenchymal stem cells for immune regulation, skin injury repair, retinal neuronal cell repair and the like is disclosed, but the record and report of the use of exosome derived from human umbilical cord mesenchymal stem cells for treating non-alcoholic fatty liver disease are not available.
Disclosure of Invention
The invention aims to provide a preparation method and application of exosome derived from human umbilical cord mesenchymal stem cells.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of exosome derived from human umbilical cord mesenchymal stem cells, which comprises the following steps:
1) inoculating human umbilical cord mesenchymal stem cells into a low-sugar DMEM culture medium containing fetal calf serum for first amplification culture, transferring the cells to a serum-free culture medium for second amplification culture for 45-50 h when the cells are fused to 70% -80%, and collecting supernatant to obtain exosome-containing supernatant;
2) centrifuging the supernatant containing the exosome at 1800-2200 g for 25-35 min, and collecting the first supernatant; centrifuging the first supernatant at 8000-12000 g for 25-35 min, and collecting the second supernatant; centrifuging the second supernatant at 1400-1600 g for 25-30 min, and collecting the third supernatant;
3) mixing the third supernatant with an exosome extraction reagent, and extracting for 9-16 hours at 1-5 ℃ to obtain an extracting solution; and centrifuging the extracting solution for 25-30 min at 1400-1600 g, collecting the precipitate, and resuspending the precipitate to obtain the exosome derived from the human umbilical cord mesenchymal stem cells.
Preferably, the human umbilical cord mesenchymal stem cells in the step 1) comprise 3-5 generations of human umbilical cord mesenchymal stem cells.
Preferably, the volume percentage content of fetal calf serum in the low-sugar DMEM medium in the step 1) is 8% -12%.
Preferably, the temperature of the first amplification culture and the second amplification culture in the step 1) is 35-38 ℃ independently; the concentration of carbon dioxide in the first amplification culture and the concentration of carbon dioxide in the second amplification culture are independently 4-6%.
Preferably, the reagent used for resuspending the pellet in step 4) comprises phosphate buffered saline.
Preferably, after the heavy suspension precipitation, the heavy suspension is sequentially washed and filtered by a sterile filter membrane of 0.22 μm.
The invention also provides application of the exosome derived from the human umbilical cord mesenchymal stem cell prepared by the preparation method in the scheme in preparing a medicament for treating non-alcoholic fatty liver.
Preferably, the human umbilical cord mesenchymal stem cell-derived exosome is used for treating non-alcoholic fatty liver disease by enhancing AMPK activity and/or inhibiting liver cell steatosis.
The invention has the beneficial effects that: the invention provides a preparation method of exosome derived from human umbilical cord mesenchymal stem cells, which comprises the following steps: culturing human umbilical cord mesenchymal stem cells to obtain an exosome-containing supernatant; and separating the supernatant containing the exosomes by a chemical precipitation method to obtain the MSC exosomes containing the protein kinase. The exosome derived from the human umbilical cord mesenchymal stem cells prepared by the preparation method contains protein kinase, and the exosome containing the protein kinase can enhance AMPK activity and/or inhibit liver cell steatosis, so that nonalcoholic fatty liver can be treated.
Drawings
FIG. 1A is the morphology of MSC-Ex observed by electron microscopy in example 1;
FIG. 1B shows the results of the MSC-Ex diameter measurement in example 1;
FIG. 1C shows the Westernblot detection exosomes marker proteins CD9, CD63 of example 1;
FIG. 2A is the results of weight determination of the mouse model for MSC-Ex intervention NAFLD in example 2;
FIG. 2B shows the liver index measurement results of the mouse model with NAFLD intervened by MSC-Ex in example 2;
FIG. 2C shows the results of testing liver TG in mouse model with NAFLD intervened by MSC-Ex in example 2;
FIG. 2D shows the result of serum AST detection of the mouse model with NAFLD intervened by MSC-Ex in example 2;
FIG. 2E shows the results of the liver tissue HE and oil red "O" staining of the mouse model NAFLD with MSC-Ex intervention in example 2;
FIG. 3 is a schematic representation of MSC-Ex intervention in pAMPK protein expression in liver tissue of NAFLD mouse model in example 2;
FIG. 4A is a graph of the effect of Nile Red staining in example 3 to detect different concentrations of MSC-Ex in inhibiting LO2 steatosis;
fig. 4B is the imaging flow assay for LO2 lipid deposition level in example 3;
FIG. 4C shows the results of Westernblot detection of p-AMPK protein expression in example 3;
FIG. 5A shows the localization of CM-Dir membrane dye-labeled MSC-Ex in liver tissue of fatty liver mice; PBS, phosphate buffered saline; MSC-Ex, umbilical cord MSC exosomes;
FIG. 5B shows the location of PKH26 membrane dye-labeled MSC-Ex in hepatocytes LO 2; actin, Actin; DAPI, 4', 6-diamidino-2-phenylindole.
Detailed Description
The invention provides a preparation method of exosome derived from human umbilical cord mesenchymal stem cells, which comprises the following steps:
1) inoculating human umbilical cord mesenchymal stem cells into a low-sugar DMEM culture medium containing fetal calf serum for first amplification culture, transferring the cells to a serum-free culture medium for second amplification culture for 45-50 h when the cells are fused to 70% -80%, and collecting supernatant to obtain exosome-containing supernatant;
2) centrifuging the supernatant containing the exosome at 1800-2200 g for 25-35 min, and collecting the first supernatant; centrifuging the first supernatant at 8000-12000 g for 25-35 min, and collecting the second supernatant; centrifuging the second supernatant at 1400-1600 g for 25-30 min, and collecting the third supernatant;
3) mixing the third supernatant with an exosome extraction reagent, and extracting for 9-16 hours at 1-5 ℃ to obtain an extracting solution; and centrifuging the extracting solution for 25-30 min at 1400-1600 g, collecting the precipitate, and resuspending the precipitate to obtain the exosome derived from the human umbilical cord mesenchymal stem cells.
Firstly, inoculating human umbilical cord mesenchymal stem cells into a low-sugar DMEM culture medium containing fetal calf serum for first amplification culture, transferring the cells to a serum-free culture medium for second amplification culture for 45-50 h when the cells are fused to 70% -80%, and collecting supernatant to obtain exosome-containing supernatant; the preferable human umbilical cord mesenchymal stem cells comprise 3-5 generations of human umbilical cord mesenchymal stem cells; the human umbilical cord mesenchymal stem cells are from conventional markets; the preferred fusion degree of the human umbilical cord mesenchymal stem cells inoculated to the low-sugar DMEM medium containing fetal calf serum is 20-30%; the volume percentage content of fetal calf serum in the low-sugar DMEM medium is preferably 8% -12%, and more preferably 10%; the fusion degree of the transplanted cells to a serum-free culture medium is preferably 60-80%; the temperature of the first amplification culture and the second amplification culture is preferably 35-38 ℃ independently, and more preferably 37 ℃; the concentration of carbon dioxide in the first amplification culture and the concentration of carbon dioxide in the second amplification culture are independently preferably 4-6%, and more preferably 5%; after the collecting of the supernatant, it is preferable to further include centrifuging the supernatant at 300g for 10min to remove floating living cells.
After obtaining the exosome-containing supernatant, carrying out first centrifugation on the exosome-containing supernatant at 1800-2200 g for 25-35 min, and collecting the first supernatant; centrifuging the first supernatant at 8000-12000 g for 25-35 min, and collecting the second supernatant; centrifuging the second supernatant at 1400-1600 g for 25-30 min, and collecting the third supernatant; the centrifugal force of the first centrifugation is preferably 2000g, and the time is preferably 30min, and the first centrifugation is used for removing cell debris; the centrifugal force of the second centrifugation is preferably 10000g, and the time is preferably 30min, and the second centrifugation is used for removing organelles; the centrifugal force of the third centrifugation is preferably 1500g, and the time is preferably 30min, and the third centrifugation is used for concentrating the supernatant from which the organelles are removed.
After the third supernatant is obtained, mixing the third supernatant with an exosome extraction reagent, and extracting for 9-16 hours at the temperature of 1-5 ℃ to obtain an extracting solution; centrifuging the extracting solution at 1400-1600 g for 25-30 min, collecting the precipitate, and resuspending the precipitate to obtain exosomes derived from human umbilical cord mesenchymal stem cells; the exosome extraction reagent is preferably available from SBI corporation under the trade designation EXOTC 50A-1; the volume ratio of the third supernatant to the exosome extraction reagent is preferably (4-6): 1, more preferably 5: 1; the centrifugal force of the centrifugation is preferably 1500 g; the reagent used for resuspending the pellet preferably comprises phosphate buffered saline; after the heavy suspension precipitation, sequentially washing the heavy suspension and filtering by using a sterile filter membrane of 0.22 mu m; the washing mode comprises the steps of centrifuging the heavy suspension at the temperature of 4 ℃ for 30min at 1500g, collecting the precipitate, and re-suspending the precipitate; the number of washing is preferably not less than 3.
The storage temperature of the exosome derived from the human umbilical cord mesenchymal stem cells prepared by the preparation method is preferably-70 ℃.
The invention also provides application of the exosome derived from the human umbilical cord mesenchymal stem cell prepared by the preparation method in the scheme in preparing a medicament for treating non-alcoholic fatty liver; the human umbilical cord mesenchymal stem cell-derived exosome is preferably used for treating non-alcoholic fatty liver disease by enhancing AMPK activity and/or inhibiting liver cell steatosis.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
NAFLD (Non-alcoholic fatty liver disease); MSCs (Mesenchymal stem cells); exosomes (exosomes); AMPK (Adenosine-5' -monophosphoactivated protein kinase, AMP-dependent protein kinase).
Example 1 extraction and identification of exosomes derived from human umbilical cord mesenchymal Stem cells
The main materials and sources used in example 1 are as follows:
MSC culture reagent: a-MEM, fetal bovine serum (product of Bioind Co.), trypsin (product of Sigma Co.), carbon dioxide incubator (Forma Co.), serum-free medium (Shanghai Eikes Co.); inverted microscope, fluorescence microscope, biological microscope, electron microscope, superclean bench, bench top centrifuge, ultracentrifuge (beckman corporation, usa).
exosome extraction reagent: exosome extraction reagent (SBI corporation), rabbit anti-human CD9 antibody (biowork technology corporation, usa), rabbit anti-human CD63 antibody (Epitomics corporation, usa), horseradish peroxidase (HRP) -labeled goat anti-rabbit IgG secondary antibody (beijing kang century corporation), HRP chemiluminescent substrate, 100kda mwco centrifuge tube, 0.22um sterile filter membrane (Millipore corporation, usa); transmission electron microscopy (FEI Tecnai 12, Philips).
The specific implementation steps of embodiment 1 of the invention are described as follows:
(1) selecting 3-5 generation human umbilical cord mesenchymal stem cells with good growth state, firstly culturing with low-sugar DMEM culture medium of 10% fetal bovine serum, changing serum-free culture medium for culturing when the cells are fused to 80%, collecting culture supernatant after 48h, centrifuging for 10min at 300g to remove floating living cells, and separating MSC exosomes.
(2) Centrifuging the collected mesenchymal dry supernatant at 4 deg.C and 2000g for 30min to remove cell debris; centrifuging the collected cell-removed debris supernatant at 4 deg.C and 10000g for 30min to remove organelles; transferring the supernatant to a 100KDa MWCO ultrafiltration centrifugal tube, centrifuging at 4 ℃ and 1500g for 30min, and concentrating; mixing the concentrated solution with an exosome extraction reagent according to a ratio of 5:1(v/v), and precipitating at 4 ℃ overnight; centrifuging 1500g of the mixed solution for 30min the next day, collecting the lower layer of MSC exosome precipitate, and dissolving with PBS; adding the exosome after dissolving PBS into a 100KDa ultrafiltration centrifugal tube (Millipore corporation), centrifuging at 4 ℃ for 30min at 1500g, and washing with PBS for more than or equal to 3 times; and finally, filtering and sterilizing by using a 0.22um sterile filter membrane, subpackaging and storing at-70 ℃, and carrying out protein quantitative detection by using a BCA protein quantitative kit method.
(3) Exosomes morphology was observed by transmission electron microscopy (fig. 1A, morphological feature of exosomes under transmission electron microscopy): MSC-Ex 20uL, after mixing uniformly, dripping the mixture on a sample-carrying copper net with the diameter of 2mm, standing for 5min, absorbing residual liquid by using filter paper, reversing the copper net on 30g/L phosphotungstic acid (pH6.8) drops, carrying out negative dyeing for 5min at room temperature, drying under an incandescent lamp, and taking a picture under a transmission electron microscope, wherein the diameter of exosome shown in figure 1B is about 100nm of a vesicular structure;
westernblot detection of exosomes surface-tagged proteins: preparing 15% SDS-PAGE electrophoresis gel, fully cracking exosomes, adding 1/4 volumes of 5 XSDS loading buffer solution, boiling for 10min, loading electrophoresis according to 200ug of protein total amount, transferring the protein to a PVDF membrane by electrotransfer (300mA, 100min), sealing at room temperature for 1h with 50g/L skimmed milk, reacting with rabbit anti-human CD9 antibody and rabbit anti-human CD63 antibody (1: 500) at 4 ℃ overnight, washing the membrane 3 times with TBS/0.5% Tween20 on the next day, washing the membrane 5 times with HRP-labeled goat anti-rabbit IgG secondary antibody at 37 ℃ for 1h, and washing the membrane 5 times with TBS/0.5% Tween20, adding a premixed HRP incubation chemiluminescence substrate, and exposing and detecting by using a chemiluminescence gel imaging system, wherein the labeled CD9 and CD63 of exosomes derived from human umbilical cord mesenchymal stem cells are expressed positively as shown in figure 1C.
Example 2 mouse model construction of non-alcoholic fatty liver disease and therapeutic intervention Effect of MSC-Ex
The main materials and sources used in this example are as follows:
materials required for animal models: six-week-old ICR female mice, common feed (ShuxBeta), 60% high-fat feed (Jiangsu Medidson), oil red O (Sigma, USA), ELASA detection kit (AST, TG) (institute of national bioengineering, China).
The specific implementation steps of embodiment 2 of the invention are described as follows:
(1) constructing mouse model of MSC-Ex interfering NAFLD
Female ICR mice, 6 weeks old, were maintained under a 12h light/12 h dark cycle and provided free food and water. All mice were fed a High Fat Diet (HFD) (D12492i, 60% fat, 20% protein, 20% carbohydrate) for 10 weeks, tail vein injected with MSC-Ex for 4 weeks. Measuring weight change of the mouse every week, measuring the wet weight/weight ratio of the liver of the mouse, extracting liver lipid, collecting blood of the mouse, checking relevant indexes of liver function, detecting pathological changes of liver tissue structure by HE (high intensity intrinsic) staining, observing liver steatosis by oil red O staining, and successfully constructing an auxiliary judgment model. The model mice gradually increased in body weight, decreased liver index and increased serum AST with the progress of the disease, but the weight was reduced, the liver index was increased and the serum AST was decreased after the MSC-Ex treatment, and the MSC-Ex treatment reduced obesity and liver tissue damage caused by HFD diet (fig. 2A shows the results of the weight measurement, fig. 2B shows the results of the liver index measurement, and fig. 2D shows the results of the serum AST measurement).
The liver of the mouse in the model group is brownish yellow, the HE staining result shows that the liver inner focus flaky liver cells have steatosis, the liver lobule central area liver cells have serious fatty lesion, the MSC-Ex treatment group mouse has reduced steatosis of the liver cells, and the liver is dark red. After MSC-Ex treatment, the mouse liver tissue TG content is reduced, and oil red O staining shows that lipid droplets are reduced in deposition in the liver (FIG. 2C is a liver TG detection result, and FIG. 2E is a liver tissue HE and oil red O staining result); confocal immunofluorescence results showed enhancement of p-AMPK protein following MSC-Ex treatment (figure 3 is a schematic representation of pAMPK protein expression in liver tissue). It is suggested that MSC-Ex acts to reduce fatty liver by inhibiting hepatic cell steatosis by enhancing AMPK protein phosphorylation level.
Example 3 in vitro simulation of the effects of non-alcoholic steatosis and therapeutic intervention with MSC-Ex
The main materials and sources used in this example are as follows:
cell culture reagents: 1640DMEM (Gibco), fetal bovine serum (Gibco), trypsin (Gibco), carbon dioxide incubator (Forma); oleic acid (Sigma, usa), palmitic acid (Sigma, usa), Nile Red (Sigma, usa); the liver cell line LO2 was purchased from Shanghai cell institute of Chinese academy of sciences; inverted microscope, clean bench, bench top centrifuge, multifunction microplate reader (Biotek, usa), imaging flow cytometer (arnis).
The specific implementation steps of embodiment 3 of the invention are described as follows:
in vitro construction of MSC-Ex intervention hepatic cell steatosis model
(1) LO2 cells were plated at 8X 105The amount of (A) was inoculated into 6-well plates, and 2.0mM OPA/BSA and MSC-Ex and/or PBS were added for 24h after the cells were fully attached the next day. The results of Nile Red staining showed that LO2 cell steatosis was inhibited after MSC-Ex treatment (fig. 4A shows that different concentrations of MSC-Ex inhibit LO2 steatosis in Nile Red staining); imaging flow results showed that LO2 cells had reduced lipid deposition after MSC-Ex treatment (fig. 4B is imaging flow assay for the extent of LO2 lipid deposition).
The p-AMPK expression of LO2 cells is detected by Westernblot, and the result shows that the phosphorylation level of AMPK protein is enhanced after MSC-Ex treatment (FIG. 4C is the p-AMPK protein expression detection result of Westernblot). Therefore, MSC-Ex functions to reduce fatty liver by inhibiting hepatic cell steatosis by enhancing AMPK protein phosphorylation level.
Example 4 localization of CM-Dir-labeled MSC-Ex in non-alcoholic fatty liver mice and PKH 26-labeled MSC-Ex in LO2s
The main materials and sources used in this example are as follows:
100Kda MWCO ultrafiltration centrifuge tubes (MBI, china); vybrant CM-dill cell-labeling solution (Invitrogen, USA); phosphate buffer (PBS, self-prepared); PKH26 red fluorescent cell labeling kit (Sigma, germany); 0.22 μm filter (Millipore, Germany); hochest33342 dye (bodhands de biotechnology limited, china); 4% paraformaldehyde fixing solution: dissolving 20g of paraformaldehyde in 50mL of deionized water, stirring in a water bath at 65 ℃ until the paraformaldehyde is dissolved, and storing at normal temperature; maestro animal in vivo imager (CRI corporation, usa); confocal laser fluorescence microscopy (Nikon, japan).
The specific implementation steps of embodiment 4 of the present invention are described as follows:
(1) localization of CM-Dir-labeled MSC-Ex in non-alcoholic fatty liver mice
Sucking a certain volume of extracted MSC-Ex, mixing with red lipophilic membrane fluorescent dye CM-Dir (5 μ L/mL), and incubating at 37 deg.C in dark for 30 min. After the incubation is finished, sucking the mixed solution into a 100KDa MWCO ultrafiltration centrifugal tube, adding a finished product PBS with the volume of 10 times, gently blowing and beating the mixed solution uniformly, centrifuging the mixed solution at 4 ℃ for 30min at 1500g, removing the unbound CM-DiR dye, collecting the concentrated solution in the ultrafiltration centrifugal tube, sterilizing the concentrated solution by a filter with the diameter of 0.22 mu m, placing the sterilized concentrated solution into a new EP tube, and storing the sterilized concentrated solution in a dark place at 4 ℃.
Two non-alcoholic fatty liver model mice were injected in tail vein with 50. mu.L of CM-Dir-labeled MSC-Ex (experimental group) and 50. mu.L of LPBS (control group). After 12h, two mice were anesthetized, placed in a living body imager, fluorescent signals were collected, and the location of exosomes in the injury model was observed. The results showed that the MSC-Ex repair group accumulated in the mouse liver in a large amount, indicating that MSC-Ex could target the liver for repair (FIG. 5A).
(2) PKH26 marking the localization of MSC-Ex in LO2
Diluting the red fluorescent dye PKH26(1 μ L) with a diluent (100 μ L), and incubating for 5min at room temperature in the dark; mixing the extracted MSC-Ex (1 μ L) and diluent (100 μ L), and incubating at room temperature for 5 min; fully and uniformly mixing the cultured MSC-ex with a staining solution, and incubating for 5min at room temperature in a dark place; adding 5% BSA into the mixed solution, and standing for 5min in a dark place; and (3) sucking the mixed solution into a 100KDa MWCO ultrafiltration centrifugal tube, adding 10 times volume of finished product PBS, gently blowing and beating the mixture, uniformly mixing the mixture, centrifuging the mixture at the temperature of 4 ℃ for 30min at 1500g, removing unbound PKH-67 dye, collecting upper-layer concentrated solution, filtering and sterilizing the upper-layer concentrated solution through a filter of 0.22 mu m, and subpackaging the mixture in an EP tube to obtain the MSC-Ex (PKH26-MSC-Ex) marked by PKH 26.
LO2s at 3 × 104Is planted on the cell climbing sheetAfter the cells are completely attached to the wall, adding PKH26-MSC-Ex at different time points, incubating at 37 ℃, collecting cell slide after 48 hours, fixing for 30min at room temperature by 4% paraformaldehyde, and washing for 5min with PBS for 3 times. Hoechest stained nuclei for 10min, washed 3 times with PBS, 5min each time. And (3) dropwise adding a proper amount of an anti-fluorescence quencher sealing sheet, and observing and taking a picture by using a laser confocal microscope after the sheet is fixed. The results show that MSC-Ex is present in the cytoplasm of LO2s after 12h incubation (FIG. 5B).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A preparation method of exosomes derived from human umbilical cord mesenchymal stem cells comprises the following steps:
1) inoculating human umbilical cord mesenchymal stem cells into a low-sugar DMEM culture medium containing fetal calf serum for first amplification culture, transferring the cells to a serum-free culture medium for second amplification culture for 45-50 h when the cells are fused to 70% -80%, and collecting supernatant to obtain exosome-containing supernatant;
2) centrifuging the supernatant containing the exosome at 1800-2200 g for 25-35 min, and collecting the first supernatant; centrifuging the first supernatant at 8000-12000 g for 25-35 min, and collecting the second supernatant; centrifuging the second supernatant at 1400-1600 g for 25-30 min, and collecting the third supernatant;
3) mixing the third supernatant with an exosome extraction reagent, and extracting for 9-16 hours at 1-5 ℃ to obtain an extracting solution; and centrifuging the extracting solution for 25-30 min at 1400-1600 g, collecting the precipitate, and resuspending the precipitate to obtain the exosome derived from the human umbilical cord mesenchymal stem cells.
2. The preparation method according to claim 1, wherein the human umbilical cord mesenchymal stem cells in step 1) comprise 3-5 generation human umbilical cord mesenchymal stem cells.
3. The method according to claim 1, wherein the volume percentage of fetal bovine serum in the low-sugar DMEM medium in step 1) is 8-12%.
4. The preparation method according to claim 1, wherein the temperature of the first amplification culture and the second amplification culture in the step 1) is 35-38 ℃ independently; the concentration of carbon dioxide in the first amplification culture and the concentration of carbon dioxide in the second amplification culture are independently 4-6%.
5. The method of claim 1, wherein the reagents used in step 4) to resuspend the pellet comprise phosphate buffered saline.
6. The method of claim 1, further comprising washing the resuspended suspension and filtering through a sterile 0.22 μm filter in sequence after the resuspending precipitation.
7. The application of the exosome derived from the human umbilical cord mesenchymal stem cell prepared by the preparation method of any one of claims 1-6 in preparing a medicament for treating non-alcoholic fatty liver disease.
8. The use according to claim 7, wherein the human umbilical cord mesenchymal stem cell-derived exosomes are used for treating non-alcoholic fatty liver disease by enhancing AMPK activity and/or inhibiting liver cell steatosis.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2020/113910 WO2022051883A1 (en) | 2020-09-08 | 2020-09-08 | Preparation method for and application of exosome derived from human umbilical cord mesenchymal stem cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112352047A true CN112352047A (en) | 2021-02-09 |
Family
ID=74427565
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202080002118.1A Pending CN112352047A (en) | 2020-09-08 | 2020-09-08 | Preparation method and application of exosome derived from human umbilical cord mesenchymal stem cells |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN112352047A (en) |
| WO (1) | WO2022051883A1 (en) |
| ZA (1) | ZA202211314B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113171379A (en) * | 2021-04-28 | 2021-07-27 | 奥启(深圳)创投科技有限公司 | Application of mesenchymal stem cell exosome in preparation of drugs for treating fatty liver disease |
| CN113215094A (en) * | 2021-05-17 | 2021-08-06 | 山东大学齐鲁医院 | Mesenchymal stem cell exosome for reversing dedifferentiation of islet beta cells of type 2diabetes, and preparation method and application thereof |
| CN113388575A (en) * | 2021-06-11 | 2021-09-14 | 杭州露源生物科技有限公司 | Preparation method of mesenchymal stem cell exosome for skin injury repair |
| WO2023056923A1 (en) * | 2021-10-07 | 2023-04-13 | China Medical University | Use of exosomes derived from mesenchymal stem cells for treating non-alcoholic steatohepatitis |
| CN116286664A (en) * | 2023-03-10 | 2023-06-23 | 首都医科大学附属北京安贞医院 | Application of umbilical cord mesenchymal stem cell exosome |
| CN116891830A (en) * | 2023-08-03 | 2023-10-17 | 北京沃锶达细胞技术有限公司 | Liver cancer cell strain capable of dynamically monitoring AMPK activity in real time and application thereof |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114540296B (en) * | 2022-03-22 | 2023-11-24 | 和携科技有限公司 | Preparation method of composite exosome and application of composite exosome in directional enhancement of angiogenesis capacity |
| CN114574432A (en) * | 2022-04-01 | 2022-06-03 | 佛山热休生物技术有限公司 | Extraction method and application of placenta exosome |
| CN114984050A (en) * | 2022-06-10 | 2022-09-02 | 芙普瑞生物细胞科学(苏州)有限公司 | Preparation and use method of mesenchymal stem cell exosome freeze-dried powder |
| CN115025288B (en) * | 2022-06-17 | 2023-06-13 | 中南大学湘雅医院 | Exosome hydrogel mixed system and preparation method thereof |
| CN115125198B (en) * | 2022-07-11 | 2024-01-26 | 湖北沃德利派生物科技有限公司 | Human umbilical cord mesenchymal stem cell exosome and preparation method and application thereof |
| CN115521908A (en) * | 2022-10-24 | 2022-12-27 | 潍坊天创生物科技有限公司 | In-vitro culture and exosome separation and purification method of adipose-derived mesenchymal stem cells |
| CN116445401B (en) * | 2023-06-14 | 2023-08-22 | 成都康景生物科技有限公司 | Mesenchymal stem cell culture medium, stem cell exosome and preparation method |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101890050A (en) * | 2010-07-14 | 2010-11-24 | 江苏大学 | Human umbilical cordmesenchymal stem cell-derived exosome and application thereof |
| WO2014028494A1 (en) * | 2012-08-13 | 2014-02-20 | The Regents Of The University Of California | Detecting and treating liver damage |
| CN106974937A (en) * | 2017-03-30 | 2017-07-25 | 山东大学 | Application of the adipose-derived mescenchymal stem cell excretion body in treatment metabolic syndrome medicine is prepared |
| CN108103017A (en) * | 2018-02-28 | 2018-06-01 | 江苏大学 | The application of the isolation and purification method and people's umbilical cord mesenchymal stem cells excretion body of people's umbilical cord mesenchymal stem cells excretion body |
| CN108392494A (en) * | 2018-05-15 | 2018-08-14 | 江苏大学 | Applications of the exosome of people's umbilical cord mesenchymal stem cells secretion in preparing the drug for repairing skin ultraviolet injury |
| CN110038032A (en) * | 2019-05-10 | 2019-07-23 | 江苏大学 | The biological agent and preparation method of the novel anti-kidney fibrosis of people's umbilical cord MSC excretion body |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019055337A1 (en) * | 2017-09-13 | 2019-03-21 | Children's Hospital Medical Center | Role of exosomes, extracellular vesicles, in the regulation of metabolic homeostasis |
-
2020
- 2020-09-08 WO PCT/CN2020/113910 patent/WO2022051883A1/en active Application Filing
- 2020-09-08 CN CN202080002118.1A patent/CN112352047A/en active Pending
-
2022
- 2022-10-14 ZA ZA2022/11314A patent/ZA202211314B/en unknown
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101890050A (en) * | 2010-07-14 | 2010-11-24 | 江苏大学 | Human umbilical cordmesenchymal stem cell-derived exosome and application thereof |
| WO2014028494A1 (en) * | 2012-08-13 | 2014-02-20 | The Regents Of The University Of California | Detecting and treating liver damage |
| CN106974937A (en) * | 2017-03-30 | 2017-07-25 | 山东大学 | Application of the adipose-derived mescenchymal stem cell excretion body in treatment metabolic syndrome medicine is prepared |
| CN108103017A (en) * | 2018-02-28 | 2018-06-01 | 江苏大学 | The application of the isolation and purification method and people's umbilical cord mesenchymal stem cells excretion body of people's umbilical cord mesenchymal stem cells excretion body |
| CN108392494A (en) * | 2018-05-15 | 2018-08-14 | 江苏大学 | Applications of the exosome of people's umbilical cord mesenchymal stem cells secretion in preparing the drug for repairing skin ultraviolet injury |
| CN110038032A (en) * | 2019-05-10 | 2019-07-23 | 江苏大学 | The biological agent and preparation method of the novel anti-kidney fibrosis of people's umbilical cord MSC excretion body |
Non-Patent Citations (7)
| Title |
|---|
| ALEJANDRA HERNÁNDEZ 等: "Extracellular Vesicles in NAFLD/ALD: From Pathobiology to Therapy", 《CELLS》 * |
| SHUANGPENG CAI 等: "Emerging role of exosomes in liver physiology and pathology", 《HEPATOLOGY RESEARCH》 * |
| TINGFEN LI 等: "Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Alleviate Liver Fibrosis", 《STEM CELLS AND DEVELOPMENT》 * |
| 中国癌症基金会《中国肿瘤临床年鉴》编辑部: "《2015 中国肿瘤临床年鉴》", 30 September 2016, 北京：中国协和医科大学出版社 * |
| 于源华: "《生物工程与技术专业基础实验教程》", 30 June 2016, 北京：北京理工大学出版社 * |
| 周雪莲 等: "细胞外囊泡在非酒精性脂肪肝病中的研究进展", 《中国实用儿科杂志》 * |
| 梁馨苓 等: "《医院获得性急性肾损伤》", 30 June 2015, 北京：人民军医出版社 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113171379A (en) * | 2021-04-28 | 2021-07-27 | 奥启(深圳)创投科技有限公司 | Application of mesenchymal stem cell exosome in preparation of drugs for treating fatty liver disease |
| CN113215094A (en) * | 2021-05-17 | 2021-08-06 | 山东大学齐鲁医院 | Mesenchymal stem cell exosome for reversing dedifferentiation of islet beta cells of type 2diabetes, and preparation method and application thereof |
| CN113388575A (en) * | 2021-06-11 | 2021-09-14 | 杭州露源生物科技有限公司 | Preparation method of mesenchymal stem cell exosome for skin injury repair |
| WO2023056923A1 (en) * | 2021-10-07 | 2023-04-13 | China Medical University | Use of exosomes derived from mesenchymal stem cells for treating non-alcoholic steatohepatitis |
| TWI836656B (en) * | 2021-10-07 | 2024-03-21 | 中國醫藥大學 | Use of exosomes derived from mesenchymal stem cells for treating non-alcoholic steatohepatitis |
| CN116286664A (en) * | 2023-03-10 | 2023-06-23 | 首都医科大学附属北京安贞医院 | Application of umbilical cord mesenchymal stem cell exosome |
| CN116891830A (en) * | 2023-08-03 | 2023-10-17 | 北京沃锶达细胞技术有限公司 | Liver cancer cell strain capable of dynamically monitoring AMPK activity in real time and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| ZA202211314B (en) | 2023-02-22 |
| WO2022051883A1 (en) | 2022-03-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112352047A (en) | Preparation method and application of exosome derived from human umbilical cord mesenchymal stem cells | |
| CN105769916B (en) | Application of the source for mesenchymal stem cells excretion body in preparation treatment preeclampsia drug or preparation | |
| CN107937342B (en) | Method for amplifying neural stem cells through endothelial cell-derived exosomes | |
| CN111471650B (en) | Exosome derived from human umbilical cord mesenchymal stem cells, identification method and application | |
| CN109929799B (en) | Human umbilical cord mesenchymal stem cell exosome and preparation method and application thereof | |
| CN113717944B (en) | miRNA13896 over-expressed engineering human umbilical cord mesenchymal stem cell source exosome and preparation method and application | |
| CN114854676B (en) | Construction method and application of grass carp skeletal muscle myoblast cell line | |
| CN108148809A (en) | A kind of method that excretion body is detached in the supernatant from tumour cell | |
| WO2021147923A1 (en) | Vesicle and use thereof | |
| CN111759862A (en) | Application of stem cell exosome in preparation of anti-colitis-exacerbation medicine | |
| CN110038032A (en) | The biological agent and preparation method of the novel anti-kidney fibrosis of people's umbilical cord MSC excretion body | |
| CN110051694B (en) | Urine-derived stem cell preparation, preparation thereof and application thereof in preparation of acute immune rejection medicament after organ transplantation | |
| CN109266610A (en) | A method of promotion Derived from Mesenchymal Stem Cells is neuron | |
| CN112852717B (en) | Method for efficiently separating and culturing pig mammary gland epithelial cells | |
| CN112029717B (en) | Serum-free in vitro domestication culture of human mesenchymal stem cells | |
| CN112725266A (en) | Method for establishing simulation type valve interstitial cell calcification model | |
| CN102154203B (en) | Method for directionally inducing insulin-secreting cells by endometrial stem cells | |
| CN109512469A (en) | A method of uterine cavity liquid excretion body is extracted from pig living body | |
| CN111925985B (en) | Domestication culture method of mesenchymal stem cells | |
| CN106244539A (en) | For the test kit from Naive T cells induced amplification regulatory T cells | |
| CN113322226A (en) | Exosome derived from three-dimensional cultured embryonic stem cells and application of exosome in preparation of medicine for treating hepatic fibrosis | |
| CN106244525B (en) | A kind of Combined culture method of cardiac muscle cell and sustentacular cell of testis | |
| CN110917173A (en) | Active inflammation-tending anti-inflammatory engineered exosome and preparation method thereof | |
| CN114591890B (en) | In-vitro model for simulating interaction of resistance blood vessel smooth muscle cells and endothelial cells and construction method thereof | |
| CN112029718B (en) | Method for serum-free domestication culture of mesenchymal stem cells and culture medium used by same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |