CN112175899A - Mesenchymal stem cell osteogenic directional differentiation inducing liquid and application thereof - Google Patents
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Abstract
The invention provides a mesenchymal stem cell osteogenic directional differentiation inducing liquid and application thereof.
Description
Technical Field
The present invention belongs to the field of regenerative medicine. In particular to mesenchymal stem cell osteogenic directional differentiation inducing liquid and a preparation method and application thereof.
Background
The mesenchymal stem cells are ideal seed cells for repairing damaged tissues due to the self-renewal and multidirectional differentiation capacity. The directional induced differentiation of the mesenchymal stem cells can provide a new technical method for clinically treating tissue diseases and tissue defects. However, mesenchymal stem cells can be differentiated into other types of cells as well as the desired tissue cells and participate in tissue repair. Therefore, there is a need to regulate the differentiation of mesenchymal stem cells to allow directed differentiation into desired tissue cells or precursor cells. Improving the directional differentiation efficiency, reducing the differentiation capability of the mesenchymal stem cells to other types of cells in vivo, and improving the tissue treatment and repair effects. At present, the traditional method for inducing mesenchymal stem cells to differentiate generally uses chemical inducing molecules to induce mesenchymal stem cells to differentiate directionally. Such as dexamethasone or bone morphogenetic protein, can induce the mesenchymal stem cells to differentiate into osteoblasts.
Exosomes (exosomes) are micro-vesicles produced by the exocrine of cellular endosomes, with diameters between 40-100 nm. Microvesicles play an important role in signal communication between cells through interaction with target cell-associated receptors and transfer of proteins, mRNA, miRNA, DNAs, lipids, etc. It has been found that exosomes can be derived from various types of cells, such as B cells, T cells, dendritic cells, tumor cells, stem cells, endothelial cells, etc., and can produce different effects, such as clearance of nonfunctional proteins, antigen presentation, gene exchange, vascular proliferation, promotion of differentiation, inflammation, tumor metabolism, transmission of pathogenic or oncogenes, etc., and the effects of exosomes are generally consistent with the physiological properties of the cells from which they are derived.
As a bridge for transferring functional substances between adjacent cells, exosomes are increasingly gaining attention in the microenvironment of cell-cell interaction. In recent years, research on mesenchymal stem cells has been gradually expanded to research on the function of secreted exosomes, for example, exosomes secreted by mesenchymal stem cells contribute to the shift of the microenvironment of organ tissues from inflammatory to anti-inflammatory under injury stress conditions, and can promote fracture healing, regeneration of cardiomyocytes and chondrocytes, and the like. Based on the characteristics of the low-immunogenicity carrier (capable of transmitting genetic control substances such as miRNA, siRNA and the like), the mesenchymal stem cell has attractive prospect in the aspect of mesenchymal stem cell differentiation. Research has shown that dendritic cell exosome can induce the differentiation of mesenchymal stem cells into osteogenesis; the exosome derived from the human original mononuclear cell after lipopolysaccharide treatment has a certain effect of promoting the osteogenic differentiation of the mesenchymal stem cell. In the process of repairing bone defects, mesenchymal stem cells derived from bone marrow play an important role. Research shows that the bone marrow mesenchymal stem cells can promote bone regeneration, and exosomes derived from the bone marrow mesenchymal stem cells have the effect of promoting proliferation and differentiation of osteoblasts.
The traditional method for inducing mesenchymal stem cell differentiation generally uses chemical inducing molecules to induce mesenchymal stem cell differentiation directionally, but the inducing time is 3-4 weeks, the required time is too long, and the amount of calcium nodules is relatively small.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the mesenchymal stem cell osteogenic directional differentiation inducing liquid and the application thereof.
The purpose of the invention is realized by the following technical scheme:
the invention provides mesenchymal stem cell osteogenic directional differentiation inducing liquid, which comprises the following components: culture medium and exosomes.
Preferably, the culture substrate is an α -MEM medium.
Preferably, the exosome is prepared from a culture supernatant of mesenchymal stem cells, and the final concentration of the exosome is 5-10 mg/L. For example: 5mg/L, 6mg/L, 7mg/L, 8mg/L, 9mg/L, or 10 mg/L.
Preferably, the mesenchymal stem cell osteogenic directed differentiation inducing liquid further comprises fetal bovine serum, and the volume ratio of the fetal bovine serum is 5-10%. For example: 5%, 6%, 7%, 8%, 9% or 10%.
Preferably, the mesenchymal stem cell osteogenic directed differentiation inducing liquid further comprises sodium beta-glycerophosphate, wherein the concentration of the sodium beta-glycerophosphate is 5-10 mmol/L. For example: 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L, 9mmol/L or 10 mmol/L.
Preferably, the mesenchymal stem cell osteogenic directed differentiation inducing liquid further comprises ascorbic acid, and the concentration of the ascorbic acid is 40-50 mg/L. For example: 40mg/L, 41mg/L, 42mg/L, 43mg/L, 44mg/L, 45mg/L, 46mg/L, 47mg/L, 48mg/L, 49mg/L or 50mg/L.
Preferably, the mesenchymal stem cell osteogenic directed differentiation inducing solution further comprises dexamethasone, wherein the concentration of the dexamethasone is 0.1-0.5 [ mu ] mol/L. For example: 0.1. mu. mol/L, 0.2. mu. mol/L, 0.3. mu. mol/L, 0.4. mu. mol/L or 0.5. mu. mol/L.
Preferably, the mesenchymal stem cell osteogenic directed differentiation inducing liquid further comprises penicillin, wherein the concentration of the penicillin is 50-100U/mL. For example: 50U/mL, 60U/mL, 70U/mL, 80U/mL, 90U/mL, or 100U/mL.
Preferably, the mesenchymal stem cell osteogenic directed differentiation inducing liquid further comprises streptomycin, and the concentration of the streptomycin is 50-100 g/mL. For example: 50U/mL, 60U/mL, 70U/mL, 80U/mL, 90U/mL, or 100U/mL.
The invention further provides application of the mesenchymal stem cell osteogenic directed differentiation inducing liquid in promoting differentiation of mesenchymal stem cells into osteoblasts.
Compared with the prior art, the invention has the beneficial effects that: the inducing liquid for inducing the mesenchymal stem cells to differentiate into osteogenesis, which contains the exosomes, can obviously enhance the osteogenic differentiation efficiency of the mesenchymal stem cells, can be used for treating bone tissue regeneration and repair by the mesenchymal stem cells, enables the bone tissue regeneration and repair to differentiate directionally, and plays a role in regulating and controlling the differentiation of the mesenchymal stem cells. In addition, the mesenchymal stem cell osteogenic differentiation inducing liquid of the present invention has the following advantages:
1. the use is simple and convenient. In the process of inducing the mesenchymal stem cells to differentiate into the osteogenesis, the mesenchymal stem cells can be obviously improved by only using the mesenchymal stem cells to differentiate into the osteogenesis inducing liquid to culture the mesenchymal stem cells.
2. Is easy to prepare. The preparation method is realized by adding penicillin, streptomycin, fetal calf serum, beta-sodium glycerophosphate, ascorbic acid, dexamethasone and exosome into a commercially available alpha-MEM culture medium.
3. Exosome is easy to obtain and can be obtained by collecting cell supernatant of mesenchymal stem cells, the main extraction methods comprise an ultracentrifugation method, a gel chromatography method, an ultrafiltration method, an immunomagnetic bead method and a kit extraction method, and the prepared inducing liquid prepared by exosome can obviously improve the osteogenic differentiation efficiency of the mesenchymal stem cells.
Drawings
FIG. 1 shows the basic morphology of bone marrow mesenchymal stem cell exosomes observed under a transmission electron microscope;
FIG. 2 is a diagram showing the relationship between the activity of alkaline phosphatase in improving the differentiation efficiency of mesenchymal stem cells in a conventional osteogenesis inducing liquid and an exosome-containing mesenchymal stem cell osteogenic directed differentiation inducing liquid according to the present invention; wherein
Is a common osteogenesis inducing liquid and is characterized in that,
the invention relates to an exosome-containing mesenchymal stem cell osteogenic directional differentiation inducing liquid;
FIG. 3A is a calcium nodule staining pattern of a common osteogenesis inducing liquid for improving differentiation efficiency of mesenchymal stem cells osteoblasts;
fig. 3B is a calcium nodule staining diagram for improving mesenchymal stem cell osteoblast differentiation efficiency by the mesenchymal stem cell osteoblast directional differentiation inducing liquid according to the present invention.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The present invention will be described in further detail below. It should be noted that the following description is only an illustration of the claimed technical solutions, and does not limit these technical solutions in any way. The scope of the present invention is defined by the appended claims.
Example 1
The embodiment provides a mesenchymal stem cell osteogenic directional differentiation inducing liquid, which comprises the following components: culture medium and exosomes.
The culture medium described in this example was an α -MEM medium.
The exosome is prepared from a culture supernatant of mesenchymal stem cells, and the final concentration of the exosome is 5-10 mg/L.
In some embodiments, the final concentration of the exosomes is 6mg/L, 7mg/L, 8mg/L, 9mg/L, or 10 mg/L.
In some embodiments, the mesenchymal stem cell osteogenic directed differentiation inducing liquid further comprises fetal bovine serum, the volume ratio of which is 5%, 6%, 7%, 8%, 9% or 10%.
In some embodiments, the mesenchymal stem cell osteogenic directed differentiation inducing liquid further comprises sodium beta-glycerophosphate, the concentration of the sodium beta-glycerophosphate being 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L, 9mmol/L or 10 mmol/L.
In some embodiments, the mesenchymal stem cell osteogenic directed differentiation inducing liquid further comprises ascorbic acid at a concentration of 40mg/L, 41mg/L, 42mg/L, 43mg/L, 44mg/L, 45mg/L, 46mg/L, 47mg/L, 48mg/L, 49mg/L or 50mg/L.
In some embodiments, the mesenchymal stem cell osteogenic directed differentiation inducing solution further comprises dexamethasone at a concentration of 0.1. mu. mol/L, 0.2. mu. mol/L, 0.3. mu. mol/L, 0.4. mu. mol/L, or 0.5. mu. mol/L.
In some embodiments, the mesenchymal stem cell osteogenic committed differentiation-inducing liquid further comprises penicillin at a concentration of 50U/mL, 60U/mL, 70U/mL, 80U/mL, 90U/mL, or 100U/mL.
In some embodiments, the mesenchymal stem cell osteogenic directed differentiation inducing liquid further comprises streptomycin at a concentration of 50U/mL, 60U/mL, 70U/mL, 80U/mL, 90U/mL, or 100U/mL.
The preparation method of the mesenchymal stem cell osteogenic directed differentiation inducing liquid comprises the following steps:
1) preparing a bone marrow mesenchymal stem cell supernatant:
adopting 2-4 generation bone marrow mesenchymal stem cells, adding 10mL culture medium containing serum into the bone marrow mesenchymal stem cells, diluting to 1 × 106Cell suspension at a concentration of 25cm3The cell culture flask of (1) was placed at 37 ℃ with 5% CO2And collecting cell culture supernatant when the culture is carried out for 36-72 h in an incubator with saturated humidity, collecting the cell culture supernatant to more than 200mL, and extracting exosome.
2) Preparing bone marrow mesenchymal stem cell exosomes:
centrifuging the supernatant at 4 deg.C and 400 Xg for 10min, transferring the supernatant, and removing cell debris; centrifuging at 4 deg.C and 2000 Xg for 10min, transferring supernatant, and removing dead cells; filtering the supernatant with 0.22 μm sterile filter membrane to further remove impurities; precipitating exosome by ultracentrifugation for 150min at 120000 Xg at 4 ℃, adding 400 mu L of PBS after removing supernatant to dissolve and collect exosome precipitate, and finally filtering by using a sterile filter membrane of 0.22 mu m so as to ensure that the purity of the obtained exosome is higher. Subpackaging in a sterile centrifuge tube, storing at-80 deg.C for use, and identifying basic form of exosome, with the result shown in FIG. 1, and FIG. 1 is basic form of exosome of mesenchymal stem cell observed under transmission electron microscope.
3. Preparation of mesenchymal stem cell osteogenic directional differentiation inducing liquid
Adding 10% fetal calf serum, 10mmol/L beta-sodium glycerophosphate, 50mg/L ascorbic acid, 0.1 mu mol/L dexamethasone and exosome with the final concentration of 5-10mg/mL into a commercially available 1L alpha-MEM culture medium to obtain the mesenchymal stem cell osteogenic differentiation inducing liquid.
Example 2
The embodiment provides application of the mesenchymal stem cell osteogenic directed differentiation inducing solution in promoting differentiation of mesenchymal stem cells into osteoblasts. The application process specifically comprises the following steps:
the umbilical cord mesenchymal cells were inoculated into an alpha-MEM culture medium containing 10% fetal bovine serum, 100U/mL penicillin and 100g/mL streptomycin, and the medium was incubated at 37 ℃ with 5% CO2Culturing in the incubator.
After the umbilical cord mesenchymal cells grow to logarithmic phase and are fused by more than 80-85%, the mesenchymal stem cell osteogenic differentiation inducing liquid is replaced by the mesenchymal stem cell osteogenic differentiation inducing liquid, and the mesenchymal stem cell osteogenic differentiation inducing liquid is replaced every three days. And culturing for 14-21 days, and analyzing the marked protein alkaline phosphatase and the marked mineralized nodules in the osteogenic differentiation process by using an alkaline phosphatase kit and alizarin red staining respectively. The results are shown in fig. 2, fig. 3A and fig. 3B, wherein fig. 2 is a graph showing the relationship between the activity of alkaline phosphatase for improving the osteoblast differentiation efficiency of mesenchymal stem cells by using the ordinary osteogenic induction liquid and the osteoblast committed differentiation induction liquid for mesenchymal stem cells containing exosomes according to the present invention; wherein
Is a common osteogenesis inducing liquid and is characterized in that,
the invention relates to an exosome-containing mesenchymal stem cell osteogenic directional differentiation inducing liquid; FIG. 3A is a calcium nodule staining pattern of a common osteogenesis inducing liquid for improving differentiation efficiency of mesenchymal stem cells osteoblasts; FIG. 3B shows the osteogenic directed differentiation of mesenchymal stem cells according to the present inventionAnd the induction liquid is used for improving the calcium nodule staining pattern of the mesenchymal stem cell osteoblast differentiation efficiency.
As can be seen from fig. 2, fig. 3A and fig. 3B, the osteogenic differentiation inducing solution for mesenchymal stem cells according to the present invention can significantly improve the osteogenic differentiation efficiency of umbilical cord mesenchymal stem cells, indicating that it can be used for umbilical cord mesenchymal stem cells to treat bone tissue regeneration and repair, and enable them to differentiate directionally.
This summary merely illustrates some embodiments which are claimed, wherein one or more of the features recited in the claims can be combined with any one or more of the embodiments, and such combined embodiments are also within the scope of the present disclosure as if they were specifically recited in the disclosure.
Claims (10)
1. The mesenchymal stem cell osteogenic directional differentiation inducing liquid is characterized by comprising the following components: culture medium and exosomes.
2. The osteogenic directed differentiation inducing solution for mesenchymal stem cells according to claim 1, wherein the culture medium is α -MEM medium.
3. The osteogenic directed differentiation inducing solution of mesenchymal stem cells according to claim 1, wherein the exosomes are prepared from a culture supernatant of mesenchymal stem cells of bone marrow, and the final concentration of the exosomes is 5-10 mg/L.
4. The mesenchymal stem cell osteogenic directed differentiation inducing liquid according to claim 1, wherein the mesenchymal stem cell osteogenic directed differentiation inducing liquid further comprises fetal bovine serum, and the volume ratio of the fetal bovine serum is 5-10%.
5. The mesenchymal stem cell osteogenic directed differentiation inducing liquid according to claim 1, wherein the mesenchymal stem cell osteogenic directed differentiation inducing liquid further comprises sodium β -glycerophosphate, the concentration of the sodium β -glycerophosphate being 5 to 10 mmol/L.
6. The mesenchymal stem cell osteogenic directed differentiation inducing liquid according to claim 1, wherein the mesenchymal stem cell osteogenic directed differentiation inducing liquid further comprises ascorbic acid, and the concentration of the ascorbic acid is 40 to 50mg/L.
7. The mesenchymal stem cell osteogenic directed differentiation inducing liquid according to claim 1, wherein the mesenchymal stem cell osteogenic directed differentiation inducing liquid further comprises dexamethasone, the concentration of which is 0.1 to 0.5 μmol/L.
8. The mesenchymal stem cell osteogenic directed differentiation inducing liquid according to claim 1, wherein the mesenchymal stem cell osteogenic directed differentiation inducing liquid further comprises penicillin, and the concentration of the penicillin is 50-100U/mL.
9. The mesenchymal stem cell osteogenic directed differentiation inducing liquid according to claim 1, wherein the mesenchymal stem cell osteogenic directed differentiation inducing liquid further comprises streptomycin at a concentration of 50-100 g/mL.
10. Use of the mesenchymal stem cell osteogenic directed differentiation inducing liquid according to claims 1 to 10 for promoting differentiation of mesenchymal stem cells into osteoblasts.
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