CN110951685A - Monocyte-derived exosome preparation applied to osteogenic differentiation of mesenchymal stem cells - Google Patents
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Abstract
The invention relates to a monocyte source exosome preparation applied to osteogenic differentiation of mesenchymal stem cells, which comprises exosomes secreted by monocytes in human peripheral blood and PBS (phosphate buffer solution), and the preparation method comprises the following steps: culturing monocytes selected from human peripheral blood at 37 deg.C to approximately 80%; replacing complete culture medium without exosomes fetal calf serum, culturing for 48h and collecting the culture medium; and (4) centrifuging the culture medium, and preserving the 1X PBS heavy suspension precipitate at-80 ℃ to obtain the monocyte source exosome preparation. In view of the pathophysiological process related to orthopedic diseases, the invention adopts the exosome prepared from the exosome secreted by the human peripheral blood mononuclear cells, has the characteristics of good biocompatibility, no toxicity and no immunoreaction, replaces the traditional osteogenesis inducer, avoids unknown risks caused by in vivo injection, and provides a new treatment application for clinical bone defects, osteonecrosis, osteoporosis, bone metabolic diseases and the like.
Description
Technical Field
The invention belongs to the field of biological medicine, relates to a technology for preparing an exosome preparation by using peripheral blood mononuclear cells, and particularly relates to an application of a mononuclear cell exosome in promoting osteogenic differentiation of mesenchymal stem cells.
Background
The bone tissue defect is formed by large gaps caused by the loss of some bones of the body due to trauma, infection, tumor, abnormal development of bones and other factors, and the problem is common and difficult to treat clinically. How to promote the repair of bone tissue defects has been the focus of research by researchers in this field. The traditional bone defect repairing method mainly comprises autologous bone transplantation, allogeneic bone transplantation, artificial biomaterial replacement and the like. But all have respective advantages and disadvantages. The source of autogenous bone is limited, allograft and xenogeneic bone transplantation has certain immunogenicity which can cause immunological rejection reaction, and also has the potential risk of spreading infectious diseases, and pure biomaterial filling usually only has bone conduction effect but lacks bone induction activity, and is not suitable for massive bone defect. Improvements on this basis are therefore needed to achieve treatment of bone defects.
At present, the research on bone tissues mainly focuses on the research on biological scaffold materials, seed cells and factors. The selection of seed cells is particularly important in bone tissue engineering, and as stem cell research is carried out, researchers lock the goal of seed cells to Mesenchymal Stem Cells (MSCs). Mesenchymal stem cells are a type of pluripotent cells with self-replicating ability, which can differentiate into various functional cells under certain conditions. The umbilical cord mesenchymal stem cell is one of the stem cells with obvious advantages, and has some special advantages: has stronger proliferation and differentiation capability, low tumor formation rate, lower risk of generating graft-versus-host disease after allograft and does not involve more disputes in the aspects of society, morality, ethics and the like.
Exosomes (exosomes) are membrane vesicles of about 30-150 nm in diameter. Exosomes are present in cell culture fluids and body fluids, including saliva, pleural fluid, cerebrospinal fluid, ascites, urine, plasma, serum, and all other body fluids. Exosomes contain but do not fixedly express a variety of biomolecules, such as mRNA, miRNA, mtDNA, proteins, lipids, and the like. The exosome has the characteristics of good biocompatibility, no toxicity and no immunoreaction. And, the cellular behavior of the target cell-mesenchymal stem cell, such as proliferation, differentiation, apoptosis, etc., is affected by carrying biomolecules. The invention provides an application of a cell-derived exosome in promoting differentiation of mesenchymal stem cells into osteogenesis, in particular to the osteogenesis differentiation of umbilical cord mesenchymal stem cells transplanted in vivo, which replaces a traditional osteogenesis inducer and avoids unknown risks caused by in vivo injection.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides the application of the monocyte exosome in promoting the osteogenic differentiation of the mesenchymal stem cells, replaces the traditional osteogenic inducer, and avoids unknown risks caused by in vivo injection.
The technical problem to be solved by the invention is realized by adopting the following technical scheme:
a monocyte-derived exosome preparation applied to osteogenic differentiation of mesenchymal stem cells comprises exosomes secreted by monocytes in human peripheral blood and PBS, and the exosomes secreted by the monocytes in the human peripheral blood are prepared by the following method:
(1) culturing monocytes selected from human peripheral blood at 37 deg.C to approximately 80%;
(2) replacing complete culture medium without exosomes fetal calf serum, culturing for 48h and collecting the culture medium;
(3) the culture medium is at 103Centrifuging at g/min for 10min, discarding precipitate, collecting supernatant, 2X103Centrifuging at g/min for 10min, removing the precipitate, and removing the cells 10min4Centrifuging at g/min for 30min, removing cell debris, and standing for 10min5Centrifuging at g/min for 70min, discarding supernatant, and resuspending in PBS (pH 7.2) for 105Centrifuging at g/min for 70min, discarding supernatant, and storing the 1 XPBS heavy suspension precipitate at-80 deg.C to obtain exosome secreted by monocyte.
Furthermore, the exosome-free fetal calf serum in the step (2) is normal fetal calf serum at 105Centrifuging at g/min for 70min, and collecting the upper light layerAnd (5) obtaining the product.
Further, the concentration of the exosome preparation stored at-80 ℃ in the step (3) was 100 ug/mL.
Moreover, the prepared exosome preparation secreted by the mononuclear cells is applied to promoting the osteogenic differentiation of the umbilical cord mesenchymal stem cells, and the specific method comprises the following steps:
(1) culturing Umbilical cord Mesenchymal Stem Cells (Umbilical code Mesenchyl Stem Cells, UCMSCs);
(2) taking 3 rd generation UCMSCs with good growth, and inducing the UCMSCs in osteogenesis by using a culture medium containing monocyte-derived exosomes;
(3) and (4) detecting the osteogenic differentiation of the UCMSCs.
Furthermore, the monocyte-derived exosome concentration in the step (2) was 10 mg/mL.
And the UCMSCs osteogenic differentiation detection method in the step (3) comprises an ALP staining detection method for UCMSCs osteogenic differentiation markers, an alizarin red staining detection method and an ALP, BMP-2 and COL-I gene detection method.
The invention has the advantages and positive effects that:
the invention provides a method for promoting osteogenic differentiation of umbilical cord mesenchymal stem cells by using exosomes secreted by monocytes, provides a new treatment method for clinical bone defects, osteonecrosis, osteoporosis, bone metabolic diseases and the like, and provides a material basis for developing and preparing medicaments for treating the diseases such as osteoporosis, bone fracture, bone defects, bone trauma and the like.
In view of the pathophysiological process related to orthopedic diseases, the invention adopts the exosome prepared from the exosome secreted by human peripheral blood mononuclear cells, has the characteristics of good biocompatibility, no toxicity and no immunoreaction, can adopt local injection administration or local filling after being mixed with absorbable gel to replace the traditional osteogenesis inducer, and avoids unknown risks caused by in vivo injection
Drawings
FIG. 1 is a diagram showing a preparation process of an exosome isolated by ultracentrifugation;
FIG. 2 is a graph showing the results of characterization of exosomes of monocytes, wherein A is a Transmission Electron Microscope (TEM) image of exosomes, and B is a particle size map of exosomes;
FIG. 3 is a graph showing the result of detection of alkaline phosphatase (ALP) staining for 7 days after induction of UCMSCs by monocyte-derived exosomes, wherein A is a control group (PBS) and B is an exosome-induced group;
FIG. 4 is a graph of alizarin red staining test results of monocyte-derived exosomes induced UCMSCs for 21 days, wherein A is a control group (PBS) and B is an exosome induced group;
FIG. 5 is a graph showing the results of expression levels of respective genes in stem cells after inducing UCMSCs for 21 days by monocyte-derived exosomes, wherein A is OCN group, B is BMP-2, and C is COL-I.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments, which are illustrative only and not limiting, and the scope of the present invention is not limited thereby.
Preparation of monocyte-derived exosome preparation
Culturing the mononuclear cells until 80% fusion is achieved, replacing the exosome-free serum low-sugar DMEM culture solution, starving for 48 hours, collecting the supernatant, extracting exosomes by adopting an ultracentrifugation step in figure 1, adding PBS (phosphate buffer solution) into the extracted exosomes for resuspension to prepare an exosome preparation of 100mg/mL, and freezing and storing at-80 ℃ for subsequent experiments.
Characterization of monocyte-derived exosome formulations
1. Electron microscopy to identify exosome morphology: taking 3 mu l of the separated and purified exosome preparation, adding an equal volume of PBS solution for dilution, dripping the diluted exosome preparation on a sample-carrying copper net with a 40-mesh carbon film, and slightly sucking redundant liquid by filter paper; counter-dyeing with 2% uranium oxoacetate solution 10ul at room temperature for 1min, slightly absorbing the redundant liquid with filter paper, and drying at room temperature for 15 min; observed under a transmission electron microscope and photographed. Observed under a transmission electron microscope, the monocyte-derived exosome is elliptical, has a diameter of about 50-100nm, has a complete envelope structure and contains low-density substances, and is shown in figure 2A.
2. Dynamic Light Scattering (DLS) determination of exosome size: and taking 10 mu l of the separated and purified exosome preparation, adding 1ml of LPBS solution for dilution, and collecting and analyzing reports after instrumental measurement is finished. The results are shown in FIG. 2B, with exosomes of about 50-100nm diameter.
UCMSCs osteogenic differentiation assay
UCMSCs cell culture: the 3 rd generation of well-grown UCMSCs were seeded in 24-well cell culture plates and divided into two groups when confluent to 50%. One group is as follows: DMEM/DF12 culture solution of 10% fetal bovine serum and PBS mixture treatment group; the two groups are as follows: group was treated with 10% fetal bovine serum in DMEM/DF12 culture medium and exosome preparation mixture. The culture conditions were: culturing at 37 deg.C in 5% CO2 incubator; medium was changed every 2 days.
ALP staining and alizarin red calcium nodule staining: the staining kit used in the two parts is commonly used in the market, has no special requirements, and the specific experimental steps are described in the specific kit. ALP staining showed that 10ug/ml of the exosome preparation had osteogenic effect on umbilical cord mesenchymal stem cells (FIGS. 3A, B). Alizarin red staining shows that calcified nodules which are stained positively in a dotted manner are visible in aggregation growth centers in the exosome preparation group, and calcium salt around cells is stained in an orange red color. (FIGS. 4A, B).
Detecting the expression level of each gene in the stem cells after inducing UCMSCs by the monocyte-derived exosomes: pouring out the culture medium in the pore plate, washing for 2 times by PBS, adding Trizol to extract RNA, carrying out reverse transcription to obtain cDNA, carrying out Q-PCR, and comparing the expression quantity of the genes of the umbilical cord mesenchymal stem cells osteogenesis related factors (OCN group, BMP-2 and COL-I) cultured by the two culture systems, wherein the result graph is shown in FIG. 5.
The experiment shows that the exosome secreted by the monocyte can promote the osteogenesis of the umbilical cord mesenchymal stem cells, and can provide a new treatment method for clinical bone defects, osteonecrosis, osteoporosis, fracture, bone trauma, bone metabolic diseases and the like.
Although the embodiments of the present invention and the accompanying drawings are disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.
Claims (6)
1. A monocyte-derived exosome preparation applied to osteogenic differentiation of mesenchymal stem cells is characterized in that: comprises exosome secreted by mononuclear cells in human peripheral blood and PBS, and the exosome secreted by the mononuclear cells in the human peripheral blood is prepared by the following method:
(1) culturing monocytes selected from human peripheral blood at 37 deg.C to approximately 80%;
(2) replacing complete culture medium without exosomes fetal calf serum, culturing for 48h and collecting the culture medium;
(3) the culture medium is at 103Centrifuging at g/min for 10min, discarding precipitate, collecting supernatant, 2X103Centrifuging at g/min for 10min, removing the precipitate, and removing the cells 10min4Centrifuging at g/min for 30min, removing cell debris, and standing for 10min5Centrifuging at g/min for 70min, discarding supernatant, and resuspending in PBS (pH 7.2) for 105Centrifuging at g/min for 70min, discarding supernatant, and storing the 1 XPBS heavy suspension precipitate at-80 deg.C to obtain exosome secreted by monocyte.
2. The monocyte-derived exosome preparation for osteogenic differentiation of mesenchymal stem cells according to claim 1, wherein: the exosome-free fetal calf serum in the step (2) is normal fetal calf serum in 105Centrifuging for 70min at g/min, and collecting the upper light layer.
3. The monocyte-derived exosome preparation for osteogenic differentiation of mesenchymal stem cells according to claim 1, wherein: the concentration of the exosome preparation stored at-80 ℃ in the step (3) is 100 ug/mL.
4. The monocyte-derived exosome preparation for osteogenic differentiation of mesenchymal stem cells according to claim 1, wherein: the prepared exosome preparation secreted by the mononuclear cells is applied to promoting the osteogenic differentiation of umbilical cord mesenchymal stem cells, and the specific method comprises the following steps:
(1) culturing Umbilical cord Mesenchymal Stem Cells (Umbilical code Mesenchyl Stem Cells, UCMSCs);
(2) taking 3 rd generation UCMSCs with good growth, and inducing the UCMSCs in osteogenesis by using a culture medium containing monocyte-derived exosomes;
(3) and (4) detecting the osteogenic differentiation of the UCMSCs.
5. The monocyte-derived exosome preparation for osteogenic differentiation of mesenchymal stem cells according to claim 4, wherein: the monocyte-derived exosome concentration in the step (2) is 10 mg/mL.
6. The monocyte-derived exosome preparation for osteogenic differentiation of mesenchymal stem cells according to claim 4, wherein: and (3) the UCMSCs osteogenic differentiation detection method comprises the steps of UCMSCs osteogenic differentiation marker ALP staining detection, alizarin red staining detection and ALP, BMP-2 and COL-I gene detection.
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| CN112716974A (en) * | 2021-01-11 | 2021-04-30 | 鱼丽娟 | Application of tumor-derived exosome in preparation of medicine for promoting osteoclast differentiation and osteolysis |
| CN114984047A (en) * | 2022-04-29 | 2022-09-02 | 山东克洛伊美生物医药科技有限公司 | Application of plasma exosome in preparation of medicine for treating osteoporosis |
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| US20150093363A1 (en) * | 2012-05-10 | 2015-04-02 | Biomatcell Ab | Osteogenic differentiation of mesenchymal stem cells |
| CN109745341A (en) * | 2019-01-25 | 2019-05-14 | 中国医学科学院北京协和医院 | Ferroso-ferric oxide superparamagnetic nano particle stimulates stem cell excretion body skeletonization |
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| CN114984047A (en) * | 2022-04-29 | 2022-09-02 | 山东克洛伊美生物医药科技有限公司 | Application of plasma exosome in preparation of medicine for treating osteoporosis |
| CN114984047B (en) * | 2022-04-29 | 2023-10-03 | 山东克洛伊美生物医药科技有限公司 | Application of plasma exosome in preparation of medicine for treating osteoporosis |
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