CN115584342A - Method for delaying replicative senescence of mesenchymal stem cells - Google Patents
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Abstract
The invention provides a method for delaying replicative senescence of mesenchymal stem cells. The acetate-containing culture solution is used for culturing the mesenchymal stem cells, so that the activity of the cells can be effectively improved, the proliferation and migration of the cells can be promoted, the metabolic stability of the cells can be maintained, the proportion of aging cells can be reduced, the replicative aging of the mesenchymal stem cells can be effectively delayed, and the amplification efficiency and the cell activity of the mesenchymal stem cells cultured in vitro can be obviously improved.
Description
Technical Field
The invention belongs to the technical field of cell culture. More particularly, it relates to a method for delaying the replicative senescence of mesenchymal stem cells.
Background
Aging is a common phenomenon in the biological world, and aging of organisms is caused by cellular aging, which is mainly divided into two types: one is that the cell gradually stops division and proliferation after limited division times along with the increase of the passage times in the in vitro culture process, and the phenomena of proliferation slowing, growth retardation, dryness decline, differentiation loss, replication capacity weakening and the like appear, and the cell enters an aging state, namely replicative aging; the other is that various harmful stimuli to the cells from external local factors are gradually accumulated, so that the cells gradually lose the self-proliferation, differentiation or repair functions, namely 'premature aging'. The aging of stem cells is a complex process of multi-factor interaction, and the aging stem cells can cause the stability of human tissues and organs to be reduced and can also cause aging-related diseases when the stability is severe. In the stem cell transplantation process, the change of the cell activity and the aging degree of the aged cells severely limits the treatment effect and clinical application of the aged cells.
Mesenchymal Stem Cells (MSCs) are derived from adult stem cells of mesodermal stromal tissue or ectodermal neuroepithelium and nerve ridges in early development, are a heterogeneous population of cells, with multi-lineage differentiation potential. The main sources of mesenchymal stem cells are bone marrow, fat, umbilical cord blood, muscle, epithelium, placenta and the like, however, the number of mesenchymal stem cells isolated in these tissues is small, in order to obtain sufficient mesenchymal stem cells for clinical treatment, in vitro amplification is necessary, but continuous in vitro amplification causes senescence of mesenchymal stem cells, i.e., replicative senescence, and the main phenotypes include: (1) the shape of the cell is changed from a long fusiform shape to a polygonal shape or a star shape; the cell volume is increased and is in a wide and flat shape; fine particles are visible in the cytoplasm; (2) After a certain number of passages, the growth rate of the cells is obviously slowed down, and the cell proliferation capacity is reduced; (3) The activity of the galactosidase relevant to aging is increased, and the blue-staining positive cells of the beta galactosidase are increased.
Although mesenchymal stem cells have better development in clinical treatment, aged stem cells not only influence the result of experimental research, but also limit the treatment effect of stem cell transplantation, for example, aged stem cells are locally injected into non-diabetic and obese diabetic mice, the volume and density of blood vessels of the skin wound of the mice are reduced, and the wound healing is inhibited; in addition, as in the research of improving the heart function by bone marrow mesenchymal stem cell transplantation, the recovery effect of the mesenchymal stem cells of the young rat on the heart function is obviously better than that of the mesenchymal stem cells of the old rat. Therefore, the method for effectively delaying the replicative senescence of the mesenchymal stem cells has considerable necessity for improving the amplification efficiency and cell activity of the mesenchymal stem cells cultured in vitro and increasing the number of clinically available mesenchymal stem cells.
The acetate is a product obtained by replacing carboxyl hydrogen of acetic acid with metal, and mainly comprises sodium acetate, potassium acetate, magnesium acetate, ammonium acetate, zinc acetate and the like, and has wide application fields, for example, the sodium acetate and the ammonium acetate can be used as meat preservatives, the potassium acetate can be used as diuretics and softeners, the zinc acetate can be used as a wood preservative, and for example, the sodium acetate is used as an additional carbon source of a sewage treatment plant and the potassium acetate is used for preparing sludge activated carbon and the like in the prior art, but no related research report on the replicative aging effect of the acetate on mesenchymal stem cells is found at present.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for delaying the replicative senescence of mesenchymal stem cells, so as to obviously improve the amplification efficiency and cell activity of the mesenchymal stem cells cultured in vitro and increase the number of the mesenchymal stem cells available for clinic.
The first purpose of the invention is to provide application of acetate in delaying replicative senescence of mesenchymal stem cells.
The second purpose of the invention is to provide a method for delaying the replicative senescence of the mesenchymal stem cells.
The third purpose of the invention is to provide a culture solution for delaying the replicative senescence of the mesenchymal stem cells.
The above purpose of the invention is realized by the following technical scheme:
in the process of in vitro culture of the mesenchymal stem cells, along with the increase of passage times, the cell activity and the proliferation capacity are obviously reduced, the cell senescence phenomenon is obviously enhanced, the yield of the mesenchymal stem cells in vitro amplification is reduced, and the application of the mesenchymal stem cells in the transplantation treatment of clinical diseases is restricted.
Preferably, the acetate is one or more of sodium acetate, potassium acetate or magnesium acetate.
Preferably, the mesenchymal stem cells are one or more of umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, placenta mesenchymal stem cells, dental pulp mesenchymal stem cells or menstrual blood mesenchymal stem cells.
Most preferably, the mesenchymal stem cell is an umbilical cord mesenchymal stem cell.
Preferably, the method for delaying the replicative senescence of the mesenchymal stem cells is one or more of improving cell activity, promoting cell proliferation, promoting cell migration, keeping cell metabolism stable or reducing the proportion of senescent cells.
The invention also provides a method for delaying the replicative senescence of the mesenchymal stem cells, which uses culture solution containing acetate to culture the mesenchymal stem cells.
Preferably, the preparation method of the culture solution comprises the following steps: fetal bovine serum and acetate were added to DMEM/F12 medium.
Preferably, the acetate is present in the culture broth at a final concentration of 2.5 to 15mM.
Further preferably, the final concentration of the acetate in the culture solution is 2.5 to 10mM.
Most preferably, the acetate is present in the culture broth to a final concentration of 5mM.
Preferably, the mass ratio of the DMEM/F12 culture medium to the fetal calf serum is 85-95: 5 to 15.
Most preferably, the mass ratio of the DMEM/F12 medium to the fetal bovine serum is 90:10.
preferably, the culturing is subculture.
The mesenchymal stem cells are cultured by adopting the culture solution, so that the activity of the cells can be effectively improved, the proliferation and migration of the cells can be promoted, the metabolic stability of the cells can be maintained, the proportion of aging cells can be reduced, the cell aging (namely replicative aging) of the mesenchymal stem cells in the in-vitro continuous subculture process can be effectively delayed, and the amplification efficiency and the cell activity of the mesenchymal stem cells cultured in vitro can be obviously improved, therefore, the culture solution containing a DMEM/F12 culture medium, fetal bovine serum and acetate and capable of delaying the replicative aging of the mesenchymal stem cells is also within the protection range of the invention.
The invention has the following beneficial effects:
according to the research of the invention, the acetate-containing culture solution is used for culturing the mesenchymal stem cells, so that the activity of the cells can be effectively improved, the proliferation and migration of the cells can be promoted, the metabolic stability of the cells can be maintained, the proportion of aging cells can be reduced, the replicative aging of the mesenchymal stem cells can be effectively delayed, and the amplification efficiency and the cell activity of the mesenchymal stem cells cultured in vitro can be obviously improved.
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FIG. 1A shows the cell morphology of WJMSCs, FIG. 1B shows the detection result of CCK-8 in sodium acetate group, FIG. 1C shows the detection result of CCK-8 in potassium acetate group, and FIG. 1D shows the detection result of CCK-8 in magnesium acetate group.
FIG. 2A shows the result of EdU detection, and FIG. 2B shows the statistics of the proportion of proliferating cells in total cells.
FIG. 3A shows the survival assay results of WJMSCs, and FIG. 3B shows the percentage of dead cells in total cells.
Fig. 4A is the result of detecting apoptosis of WJMSCs, and fig. 4B is the statistical result of the fraction of apoptotic cells in total cells.
Fig. 5A is a result of measuring ROS levels in WJMSCs cells, and fig. 5B is a result of measuring mitochondrial distribution and damage in WJMSCs cells.
FIG. 6 shows the results of the migration ability test of WJMSCs.
Fig. 7A is a result of measuring senescence in WJMSCs in the sodium acetate group, fig. 7B is a result of measuring senescence in WJMSCs in the potassium acetate group, and fig. 7C is a result of measuring senescence in WJMSCs in the magnesium acetate group.
Wherein, control and WJMSCs represent blank Control groups, WJMSCs + SA represents a sodium acetate group, WJMSCs + PA represents a potassium acetate group, and WJMSCs + MA represents a magnesium acetate group.
Detailed Description
The invention is further described with reference to the drawings and specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Complete medium of WJMSCs: 90% DMEM/F12 medium +10% Fetal Bovine Serum (FBS).
Umbilical cord mesenchymal stem cells WJMSCs: obtained by tissue block adherence method.
Example 1 Effect of acetate on mesenchymal Stem cell Activity
1. Experimental method
Sodium acetate (Sodium acetate, SA) was added to the complete medium of WJMSCs at final concentrations of 2.5mM, 5mM, 10mM, 15mM, respectively, for 2X 10 culture 4 WJMSCs (Potassium acetate, PA) were added to the complete medium of WJMSCs at final concentrations of 2.5mM, 5mM, 10mM, 15mM, respectively, for 3X 10 culture 4 Magnesium Acetate (MA) was added to the complete medium of WJMSCs at final concentrations of 2.5mM, 5mM, 10mM, and 15mM, respectively, for 3X 10 culture 4 WJMSCs/mL.
And taking a complete culture medium (added with corresponding amount of WJMSCs) of WJMSCs without acetate as a blank control, respectively culturing for 24h, observing the cell morphology of the WJMSCs, and detecting the cell activity of each group of WJMSCs after 48h treatment by using a CCK-8 method.
2. Results of the experiment
The morphology of the cells in the 5mM group is shown in FIG. 1A, and it can be seen that the morphology of the cells is not substantially changed after 5mM of acetate is added, and the cells grow adherently like fibroblasts.
The detection result of the CCK-8 in the sodium acetate group is shown in figure 1B, the detection result of the CCK-8 in the potassium acetate group is shown in figure 1C, and the detection result of the CCK-8 in the magnesium acetate group is shown in figure 1D, so that the acetate (sodium acetate, potassium acetate and magnesium acetate) can obviously improve the activity of the WJMSCs and further effectively delay the replicative senescence of the WJMSCs, wherein the activity is improved most obviously by adding 5mM of the acetate.
Example 2 Effect of acetate on proliferation of mesenchymal Stem cells
1. Experimental methods
Adding sodium acetate, potassium acetate and magnesium acetate into a complete culture medium of the WJMSCs at a final concentration of 5mM for culturing the WJMSCs, taking the complete culture medium of the WJMSCs without the acetate as a blank control, respectively culturing for 48h, detecting the proliferation capacity of each group of WJMSCs by using an EdU kit, counting the number of EdU positive cells, and calculating the proportion of the proliferation cells in total cells.
2. Results of the experiment
The EdU measurement results are shown in fig. 2A, and the proportion statistics of proliferating cells in total cells are shown in fig. 2B. Therefore, when the complete culture medium of the WJMSCs added with acetate (sodium acetate, potassium acetate and magnesium acetate) is used for culturing the WJMSCs, the proliferation capacity of the WJMSCs is remarkably improved, and the acetate can remarkably promote the proliferation of the WJMSCs, so that the replicative senescence of the WJMSCs is effectively delayed.
Example 3 Effect of acetate on survival of mesenchymal Stem cells
1. Experimental methods
Adding sodium acetate, potassium acetate and magnesium acetate into the complete culture medium of WJMSCs at a final concentration of 5mM for culturing WJMSCs, and culturing for 48h with the complete culture medium of WJMSCs without acetate as blank control, and using Live/Dead TM And the Cell Imaging Kit detects the survival condition of each group of WJMSCs, counts the number of dead cells and calculates the proportion of the dead cells in the total cells.
2. Results of the experiment
The survival test results of WJMSCs are shown in FIG. 3A, and the proportion of dead cells in the total cells is shown in FIG. 3B. As can be seen, when the complete culture medium of WJMSCs supplemented with acetate (sodium acetate, potassium acetate, magnesium acetate) was used to culture WJMSCs, the survival of WJMSCs was not significantly changed (although the proportion of dead cells in the potassium acetate group was slightly increased in fig. 3B, there was no statistical difference), indicating that acetate did not significantly affect the survival rate of WJMSCs.
Example 4 Effect of acetate on apoptosis of mesenchymal Stem cells
1. Experimental method
Adding sodium acetate, potassium acetate and magnesium acetate into a complete culture medium of WJMSCs at a final concentration of 5mM for culturing the WJMSCs, taking the complete culture medium of the WJMSCs without the acetate as a blank control, respectively culturing for 48h, detecting the apoptosis condition of each group of WJMSCs by TUENL immunofluorescence, counting the number of apoptotic cells, and calculating the proportion of the apoptotic cells in total cells.
2. Results of the experiment
The detection result of apoptosis of WJMSCs is shown in FIG. 4A, and the statistical result of the proportion of apoptotic cells in total cells is shown in FIG. 4B. Therefore, when the complete culture medium of the WJMSCs added with acetate (sodium acetate, potassium acetate and magnesium acetate) is used for culturing the WJMSCs, the apoptosis of the WJMSCs is not obviously changed, and the condition that the acetate does not obviously influence the apoptosis rate of the WJMSCs is shown.
Example 5 Effect of acetate on the oxidative metabolism of mesenchymal Stem cells and mitochondria
1. Experimental method
Adding sodium acetate, potassium acetate and magnesium acetate into a complete culture medium of the WJMSCs at a final concentration of 5mM for culturing the WJMSCs, taking the complete culture medium of the WJMSCs without the acetate as a blank control, respectively culturing for 48H, detecting the intracellular ROS level of each group of WJMSCs by using a CM-H2DCFDA probe, and detecting the mitochondrial distribution and damage condition of each group of WJMSCs by using MitoTracker immunofluorescence.
2. Results of the experiment
The detection result of ROS level in WJMSCs cells is shown in figure 5A, and the detection result of mitochondrial distribution and damage condition in WJMSCs cells is shown in figure 5B. Therefore, the complete culture medium of the WJMSCs added with the acetate (sodium acetate, potassium acetate and magnesium acetate) is used for culturing the WJMSCs, the oxidation free radical level of the WJMSCs is not obviously changed, the number, the form and the distribution of mitochondria are not obviously different and are not damaged, namely the acetate has no obvious influence on the oxidative metabolism of the umbilical cord mesenchymal stem cells, and the acetate can keep the cell metabolism stability of the WJMSCs, so that the replicative senescence of the WJMSCs is effectively delayed.
Example 6 Effect of acetate on migration Capacity of mesenchymal Stem cells
1. Experimental method
Adding sodium acetate, potassium acetate and magnesium acetate into a complete culture medium of the WJMSCs respectively at a final concentration of 5mM for culturing the WJMSCs, taking the complete culture medium of the WJMSCs without the acetate as a blank control, culturing respectively until the cell confluence rate reaches 100%, detecting the migration capacity of each group of WJMSCs by using a cell scratch experiment, and counting the migration rate of the WJMSCs.
2. Results of the experiment
The results of the migration ability test of WJMSCs are shown in FIG. 6. Therefore, when the complete culture medium of the WJMSCs added with acetate (sodium acetate, potassium acetate and magnesium acetate) is used for culturing the WJMSCs, the migration capability of the WJMSCs is remarkably improved, wherein the improvement effect of the potassium acetate is most remarkable, which shows that the acetate can remarkably promote the migration of the WJMSCs, and further, the replicative senescence of the WJMSCs is effectively delayed.
Example 7 Effect of acetate on aging of mesenchymal Stem cells
1. Experimental methods
Adding sodium acetate, potassium acetate and magnesium acetate into complete culture media of WJMSCs at a final concentration of 5mM respectively for culturing WJMSCs of P10, P15 and P20, and detecting the aging condition of each group of WJMSCs by a beta-galactosidase detection kit after culturing for 24 h.
2. Results of the experiment
The results of aging tests on WJMSCs in the sodium acetate group are shown in FIG. 7A, the results of aging tests on WJMSCs in the potassium acetate group are shown in FIG. 7B, and the results of aging tests on WJMSCs in the magnesium acetate group are shown in FIG. 7C. Therefore, when the complete culture medium added with acetate (sodium acetate, potassium acetate and magnesium acetate) is used for culturing the WJMSCs, the senescence of the WJMSCs is effectively delayed, and the acetate can effectively relieve the cell senescence of the WJMSCs in the subculture process, namely delay the replicative senescence of the WJMSCs.
In conclusion, the research of the invention finds that the culture solution containing acetate is used for culturing the mesenchymal stem cells, so that the activity of the cells can be effectively improved, the proliferation and the migration of the cells can be promoted, the metabolic stability of the cells can be maintained, the proportion of aging cells can be reduced, the replicative aging of the mesenchymal stem cells can be effectively delayed, and the amplification efficiency and the cell activity of the mesenchymal stem cells cultured in vitro can be obviously improved.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. Use of acetate in delaying replicative senescence of mesenchymal stem cells.
2. The use according to claim 1, wherein the acetate is one or more of sodium acetate, potassium acetate or magnesium acetate.
3. The use of claim 1, wherein the mesenchymal stem cell is one or more of umbilical cord mesenchymal stem cell, adipose mesenchymal stem cell, bone marrow mesenchymal stem cell, placenta mesenchymal stem cell, dental pulp mesenchymal stem cell or menstrual mesenchymal stem cell.
4. The use of claim 1, wherein the delaying of the replicative senescence of mesenchymal stem cells is one or more of increasing cell activity, promoting cell proliferation, promoting cell migration, maintaining stable cell metabolism, or decreasing the proportion of senescent cells.
5. A method for delaying replicative senescence of mesenchymal stem cells is characterized in that the mesenchymal stem cells are cultured by using a culture solution containing acetate.
6. The method of claim 5, wherein the culture medium is prepared by: fetal bovine serum and acetate were added to DMEM/F12 medium.
7. The method according to claim 6, wherein the acetate is present in the culture medium at a final concentration of 2.5 to 15mM.
8. The method according to claim 7, wherein the acetate is present in the culture medium at a final concentration of 2.5 to 10mM.
9. The method according to claim 6, wherein the mass ratio of the DMEM/F12 medium to the fetal bovine serum is 85-95: 5 to 15.
10. A culture solution for delaying replicative senescence of mesenchymal stem cells is characterized by comprising a DMEM/F12 culture medium, fetal bovine serum and acetate.
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