CN114517178A - Application of Trolox in delaying mesenchymal stem cell aging - Google Patents
Application of Trolox in delaying mesenchymal stem cell aging Download PDFInfo
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Abstract
The invention provides an application of Trolox in delaying mesenchymal stem cell aging, belonging to the technical field of stem cell culture. In the invention, Trolox can increase the proliferation rate of cells, resist the aging of the cells and improve the production of an immunoregulatory factor PGE-2 and the expression of microRNA17-92 under the condition of not changing a mesenchymal stem cell surface marker. Therefore, Trolox can improve the anti-aging of the mesenchymal stem cells, simultaneously keep the cells from differentiating, has higher cell quality and provides a new material for clinical application.
Description
Technical Field
The invention belongs to the technical field of stem cell culture, and particularly relates to application of Trolox in delaying mesenchymal stem cell senescence.
Background
Mesenchymal Stem Cells (MSCs) have multipotent differentiation and self-renewal capacity and low immunogenicity, and have become important research targets in the fields of cell therapy and regenerative medicine. Human umbilical cord mesenchymal stem cells (hUC-MSC) are convenient to obtain, low in ethical limit and rich in source, and become an important sample source. MSCs have better proliferative activity in vitro, but after limited passage, they show signs of senescence. This senescence-generating property during in vitro culture limits the number of MSCs that can be expanded, thereby affecting their clinical utility. One of the research hotspots of regenerative medicine is how to delay the aging of mesenchymal stem cells or make aged MSCs reappear the state of rejuvenation to prolong the utility value.
In the prior art, cell anti-aging is usually realized by adding an antioxidant in a culture process. The antioxidant has antioxidant activity, and can prevent membrane oxidative damage by scavenging lipid peroxy radicals in cells and inhibiting peroxidation of polyunsaturated fatty acids. However, antioxidants are selective for cell type and not any antioxidant of any kind can exert anti-aging effects on cells. At present, no anti-aging drugs of mesenchymal stem cells are reported.
Disclosure of Invention
In view of the above, the present invention aims to provide an application of Trolox in delaying mesenchymal stem cell senescence.
The invention provides an application of Trolox in delaying mesenchymal stem cell aging.
Preferably, the mesenchymal stem cell anti-aging is manifested in that the proliferation rate of the mesenchymal stem cell is improved and the positive staining proportion of beta-galactosidase in the mesenchymal stem cell is reduced.
Preferably, when the proliferation rate of the mesenchymal stem cells is taken as a detection index, the number of passages of the mesenchymal stem cells is less than 10.
Preferably, when the positive staining proportion of beta-galactosidase in the mesenchymal stem cells is used as a detection index, the number of passages of the mesenchymal stem cells comprises 0-15 passages.
Preferably, the working concentration of Trolox is not less than 2 mu mol/l.
The invention provides application of Trolox in preparation of an anti-aging culture medium for mesenchymal stem cell culture or passage.
Preferably, the concentration of Trolox in the medium is not less than 2. mu. mol/l.
Preferably, the mesenchymal stem cells are cultured or passaged in a medium containing Trolox.
The invention provides application of Trolox in improving the biological function of mesenchymal stem cells.
Preferably, the improvement of the biological functions of the mesenchymal stem cells comprises immune regulation function and micro RNA expression;
preferably, the number of passages of the mesenchymal stem cells is not more than 18.
The invention provides an application of Trolox in delaying mesenchymal stem cell senescence. According to the invention, Trolox is obtained by screening from a plurality of compounds with antioxidant function, experiments prove that when the Trolox is applied to the culture of the mesenchymal stem cells, the cell proliferation characteristics of the mesenchymal stem cells with different generations can be improved, and by detecting the activity of an aging related index beta-galactosidase, the Trolox has an anti-aging effect on the mesenchymal stem cells, so that a new thought is provided for the subsequent preparation of a culture medium or a medicament for culture of the mesenchymal stem cells.
Meanwhile, when the Trolox is applied to the culture of the mesenchymal stem cells, the expression of a mesenchymal stem cell surface marker is not influenced, the original biological characteristics of the mesenchymal stem cells are kept, and no adverse influence is caused on the subsequent cell application. Therefore, Trolox can increase the proliferation rate of cells and resist the aging of the cells under the condition of not changing the surface marker of the mesenchymal stem cells, and provides higher utilization value for the clinical application of the Trolox.
Drawings
FIG. 1 illustrates an embodiment of the present invention in an MSC under-the-mirror configuration;
FIG. 2 is a graph showing the effect of Trolox on the proliferation of MSC at the P4 generation in an example of the present invention;
FIG. 3 is a graph of the effect of Trolox on the proliferation of P12 generation MSCs in an example of the present invention;
FIG. 4 shows the staining results of beta-galactosidase in the examples of the present invention;
FIG. 5 shows the statistical analysis of the beta-gal staining according to the present invention;
FIG. 6 is a result of detecting hUC-MSC surface markers by flow cytometry in the present embodiment, wherein FIG. 6a is Blank set, FIG. 6b is control set, and FIG. 6c is Trolox set;
FIG. 7 shows the expression of PGE2 in MSC according to the embodiment of the present invention, FIG. 7a shows the generation P4 MSC, FIG. 7b shows the generation P12 MSC, FIG. 7c shows the statistical result of the group without adding Trolox, and FIG. 7d shows the statistical result of the group with adding Trolox;
FIG. 8 shows the effect of Trolox on the expression of microRNA17-92 in MSC in the embodiment of the present invention, FIG. 8a shows the result of microRNA17-92 expression after MSC is cultured by adding Trolox once, and FIG. 8b shows the result of microRNA17-92 expression after MSC is cultured by continuously (totally 8 times) adding Trolox.
Detailed Description
The invention provides an application of Trolox in delaying mesenchymal stem cell aging.
In the invention, in order to screen compounds capable of improving the anti-aging effect of the mesenchymal stem cells, dozens of compounds with anti-oxidation effect are selected from an anti-oxidation compound library for culturing the mesenchymal stem cells, and the anti-aging experiment verification of the mesenchymal stem cells is carried out by selecting the compound Trolox with the highest PEG2 expression level in the cells by taking the PEG2 expression level in the cells as a detection index. In the embodiment of the invention, the anti-aging effect of the mesenchymal stem cells is shown in that the proliferation rate of the mesenchymal stem cells is improved, and the positive staining proportion of beta-galactosidase in the mesenchymal stem cells is reduced. When the proliferation rate of the mesenchymal stem cells is taken as a detection index, the number of passages of the mesenchymal stem cells is preferably less than 10 passages, more preferably 0-8 passages, further preferably 0-6 passages, and most preferably 0-4 passages. The number of passages of the mesenchymal stem cells is a key factor influencing the Trolox to promote the proliferation of the mesenchymal stem cells, and when the number of passages is large, for example, over 12 passages, the ability of the Trolox to promote the proliferation of the mesenchymal stem cells is reduced. When the positive staining proportion of beta-galactosidase in the mesenchymal stem cells is used as a detection index, the number of passages of the mesenchymal stem cells preferably comprises 0-15 passages, more preferably 2-12 passages, and further preferably 5-10 passages. In the embodiment of the invention, beta galactosidase staining is respectively carried out on the mesenchymal stem cells of passage 5 and passage 12, and the result shows that Trolox has an anti-aging effect on the mesenchymal stem cells.
In the invention, the concentration of Trolox is also a key factor influencing the anti-aging effect of the mesenchymal stem cells. The working concentration of Trolox is preferably not less than 2 mu mol/l, and more preferably 3-100 mu mol/l. In the embodiment of the invention, the working concentration of Trolox is set to be 5,10,20,50,100 mu mol/l, and the Trolox has the effect of improving the cell proliferation rate. When the concentration is less than 2. mu. mol/l, the working concentration of Trolox is 0.25, 0.5, 1. mu. mol/l, the cell proliferation rate cannot be significantly increased.
The source of the mesenchymal stem cells in the invention is not limited, and the mesenchymal stem cells known in the art can be used, for example, umbilical cord-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, and the like. In the embodiment of the invention, umbilical cord-derived mesenchymal stem cells are taken as an example to illustrate the role of Trolox in the aspect of mesenchymal stem cell anti-aging.
Meanwhile, the Trolox does not change cell surface markers while improving the anti-aging performance of the mesenchymal stem cells, which indicates that the cell types cannot be changed and differentiation cannot occur.
The invention provides an application of Trolox in preparation of an anti-aging culture medium for mesenchymal stem cell culture or passage, and aims to improve the anti-aging performance of mesenchymal stem cells based on Trolox.
In the present invention, the medium is cultured based on a conventional medium in the art for culturing mesenchymal stem cells, and a Trolox component is added. The concentration of Trolox in the culture medium is preferably not less than 2 mu mol/l, and more preferably 3-100 mu mol/l.
The invention provides application of Trolox in improving the biological function of mesenchymal stem cells.
In the present invention, the improvement of the biological function of the mesenchymal stem cell preferably includes an immune regulatory function and the expression of microrna. The immune regulation function and the expression of the micro RNA are the quality standard of mesenchymal stem cell culture, wherein the immune regulation function takes the capability of expressing PGE2 by the mesenchymal stem cell as an evaluation index, the immune regulation function has anti-inflammatory effect, and the higher the expression level of PGE2 is, the stronger the immune microenvironment regulation capability of the mesenchymal stem cell is, thus being beneficial to the mesenchymal stem cell to play a cell treatment role in clinic. The low expression of the microRNA is the function of characterizing the secretion of nucleic acid of the mesenchymal stem cell. The function of Trolox is limited by the number of passages of mesenchymal stem cells, and the number of passages of the mesenchymal stem cells is preferably not more than 18, more preferably 0-15, and further preferably 5-10. In the embodiment of the invention, in the case of not adding Trolox, the generation of PGE-2 is gradually reduced along with the increase of the generation number. After the experimental group is cultured by adding Trolox, the generation of PGE-2 is gradually increased from P6 to P18 generation, but the generation of Trolox is obviously reduced after P18, which shows that Trolox takes cells from P0 generation to P18 generation as objects in improving the biological function of the mesenchymal stem cells.
In the invention, the culture medium containing Trolox is preferably adopted for culturing or subculturing the mesenchymal stem cells each time, which is favorable for maintaining the anti-aging performance of the mesenchymal stem cells. When the cells are subjected to passage or culture, the biological function of the mesenchymal stem cells cannot be obviously improved as expected only by using a medium containing Trolox once to culture the cells, and when the Trolox is added during the passage and every passage culture, the biological function of the mesenchymal stem cells is favorably maintained, and particularly the expression of microRNA17-3p, microRNA17-5p, microRNA18a-3p, microRNA19b-3p, microRNA20a-5p and microRNA92a-3p in the cells is improved.
Therefore, the culture or passage of the mesenchymal stem cells by using the medium containing Trolox is beneficial to improving the anti-aging performance of the cells, maintaining higher proliferation rate, generating a large amount of cells, and simultaneously keeping higher cell activity, thereby providing a material for clinically applying the mesenchymal stem cells to cell therapy of various diseases.
The following examples are provided to illustrate the application of Trolox in delaying the aging of mesenchymal stem cells, but they should not be construed as limiting the scope of the present invention.
Example 1
Screening for antioxidant Compounds
Selecting 28 conventional antioxidant compounds including Trolox and BHT from antioxidant compound library (see Table 1), adding 28 compounds into culture medium of mesenchymal stem cell respectively to final concentration of 2 μmol/l, and culturing at 37 deg.C with 5% CO2After culturing in an incubator for 48h, the expression of PGE-2 protein in the cells was measured using ELISA kits.
The results are shown in Table 1.
Example 2
Culture of hUC-MSCs
Inoculating umbilical cord-derived mesenchymal stem cells to a serum-free medium under aseptic conditions,standing at 37 deg.C and 5% CO2Culturing in an incubator. The liquid is replenished at the 2d of inoculation, and the liquid is changed 1 time every 3 d. The morphology and growth of the cells were observed under an inverted microscope. When the adherent cells are fused to 80% -90%, digesting and passaging by pancreatin, and collecting the cells for later use.
Observing under an inverted microscope, finding that cells climb out from the edges of the tissue blocks 6-8 days after the umbilical cord tissue blocks are planted, wherein the cells are small in size, short in fusiform and vortex-shaped in growth. After passage, the growth rate of the cells is increased, and the cells can be transferred to the next generation after 2-3 days (figure 1).
Example 3
CCK8 detection of hUC-MSCs cell proliferation
Respectively taking P4 and P12 generation MSCs at 1 × 104The density per well was seeded in 48 well plates with Trolox concentration gradients set at 0, 0.25. mu. mol/l, 0.5. mu. mol/l, 1. mu. mol/l, 2. mu. mol/l, 5. mu. mol/l, 10. mu. mol/l, 20. mu. mol/l, 50. mu. mol/l, 100. mu. mol/l, 3 multiple wells per concentration, and each concentration Trolox was added to 48 well plates. Adherence was routinely done for 24 hours and 48 hours. The supernatants were collected at each concentration, centrifuged and the supernatants were taken in a new EP tube and stored at-20 ℃ until use. After 10% CCK-8 reagent was added at the corresponding time point, the culture was continued for 2h and the absorbance at 450nm was measured.
As can be seen from fig. 2 and 3, both P4 and P12 showed no significant proliferation-promoting effect of low concentration Trolox on MSCs. At 24 hours of culture, both P4 and P12 exhibited significant proliferation-promoting effects starting at a concentration of 2. mu. mol/l. At 48 hours of culture, P4 generation MSCs showed significant proliferation promoting effect starting at 2. mu. mol/l, but in P12 MSCs, Trolox did not show significant proliferation promoting effect on MSCs.
Example 4
Cell beta-galactosidase staining for detecting senescence
The MSCs of the P6 generation are planted in a six-hole plate and divided into an experimental group and a control group, the MSCs are transmitted to P12 from the P6 generation, 5 mu L DMSO is added into the control group, and Trolox with the concentration of 5 mu mol/L is added into the experimental group. The number of P12 generation cells was 1X 105Inoculating the cells in a six-hole plate at a density, adding hydrogen peroxide with a concentration of 200 mu M/L after 24 hours of adherence to the wall to induce cell agingAfter 2 hours of induction, the medium was changed to a medium containing 10% serum for 24 hours. Staining MSCs with beta-galactosidase kit at 37 ℃ without CO2The incubator is kept overnight, then the staining condition is observed under a mirror, 6 fields are randomly taken for counting, and the positive rate is calculated according to the formula I.
Positive rate (%). staining positive cell count/total number of cells in field x 100% formula I
The results are shown in FIGS. 4 and 5. After culturing the MSC from P6 by 7-generation Trolox, the appearance of MSC morphological change under the mirror can be observed, the volume is large, and a plurality of antennae are flat. After staining, six fields were randomly selected for observation and calculation of positive cell rate in each of the control group and the experimental group, which indicated that the average positive rates of the control group and the experimental group were 0.55 and 0.43(p equals to 0.015) respectively after the mesenchymal stem cells were cultured by Trolox (fig. 5). The result shows that the mesenchymal stem cells can resist aging after being cultured by Trolox.
Example 5
Detecting surface markers by flow cytometry:
the MSCs of P22 obtained in the other experimental and control groups of step 3 were trypsinized, centrifuged, and a portion of each cell was re-mixed in an EP tube. Staining the experimental group and the control group of the cell fraction 3, performing isotype control on the group 1, and adding IgG; and 2 groups of negative color matching, respectively adding CD34, CD45, and 3 groups of positive color matching, respectively adding CD73, CD90 and CD105, incubating for 15 minutes in a dark place, adding 500 mu ml PBS for washing after incubation is finished, centrifuging to remove supernatant, adding 0.5ml PBS for cell resuspension, filtering, and analyzing by an up-flow cytometer.
The results are shown in FIG. 5. Detecting the expression of hUC-MSC surface markers CD34, CD45, CD73, CD90 and CD105 by a flow cytometer. The results show that Trolox has no significant effect on the expression of MSC surface markers compared to the control group. Both control and experimental groups showed CD34 (0.09%, 0.03%, respectively), CD45 (0.05%, 0.03%, respectively) negative, CD73 (99.98%, 100%, respectively), CD90 (100%, respectively), and CD105 (99.97%, 99.98%, respectively) positive (fig. 6a, fig. 6b, and fig. 6 c).
Example 6
ELISA for detecting PGE-2 protein expression
Culture supernatants collected in steps 2 and 3 were removed and analyzed for PGE-2 content in the supernatants by ELISA. The content of PGE-2 in the culture supernatant is determined according to the kit specification, the optical density value of each sample is read at 450nm by using an enzyme-labeling instrument, a standard curve is established according to the concentration of the standard substance and the corresponding optical density value thereof, and the content of PGE-2 is respectively determined according to the optical density value of each sample within the range of the standard curve. The detection range of the ELISA kit is PGE-20.200-400 mu g/L respectively, and the detection range lower than the lower limit value of the kit is not listed.
MSCs can secrete various cytokines to participate in regulation of immune microenvironment, wherein PGE-2 is an important immunoregulator which is produced, and the influence of Trolox on the expression condition of PGE-2 in culture supernatant is detected by using an ELISA method. As a result, the P4 and P12 generation MSCs show that the low concentration of Trolox has no obvious influence on the generation of PGE-2 in 24 hours and 48 hours, and when the concentration of Trolox reaches more than 2 mu mol/l, the generation of PGE-2 can be obviously promoted. In addition, ELISA results of culture supernatants collected at intervals from P6 to P22 showed that the production of PGE-2 was gradually reduced with the passage number of generation in the control group without addition of Trolox. The experimental group showed a gradual increase in PGE-2 production from P6 to P18 in the case of Trolox-supplemented cultures, but a marked decrease in Trolox production after P18 (FIGS. 7a, 7b, 7c and 7 d).
Example 7
RT-PCR analysis of expression of microRNA17-92
Planting P5 generation MSC in two six-hole plates with density of 5 × 105And each ml, adding 5 mu mol/l of Trolox into one plate of experiment group, adding the same volume of DMSO into a control group, transferring one generation, culturing for 48 hours until the cells are completely attached, removing culture supernatant, adding 500 mu ml of trizol to extract RNA, transferring the other plate to P12, adding 5 mu mol/l of Trolox into the experiment group to culture, adding the same volume of DMSO into the control group, adding trizol to extract RNA after 48 hours of complete attachment, and freezing and storing at-80 ℃ for later use. The total RNA extraction, reverse transcription and PCR reaction operation of the cells are carried out according to the Kit instruction of an RNA rapid extraction Kit, PrimeScript TMRTreagent Kit and RNAPCR Kit (AMV) Ver.3.0; PCR reactionThe conditions are as follows: pre-denaturation at 95 ℃ for 2min, denaturation at 94 ℃ for 30s, annealing at 30s, extension at 72 ℃ for 1min, 30 cycles, and final extension at 72 ℃ for 10 min; calculated using the following formula:
ΔCT(test)＝CT(target,test)-CT(ref,test)
ΔCT(calibrator)＝CT(target,calibrator)-CT(ref,calibrator)L
ΔΔCT()＝ΔCT(test)-ΔCT(calibrator)L
by use of 2-ΔΔCTThe expression level of each microRNA was calculated.
TABLE 1 sequences of microRNAs and RT-PCR amplimers corresponding thereto
| Micro RNA | Forward primer |
| hsa-miR-92a-3p | TATTGCACITGTCCCGGCCT(SEQ ID NO:1) |
| hsa-miR-20a-5p | CGCGTAAAGTGCTTATAGTGCAGGTAG(SEQ ID NO:2) |
| hsa-miR-17-3p | CACTGCAGTGAAGGCACTTGTAG(SEQ ID NO:3) |
| hsa-miR-17-5p | GCAAAGTGCTTACAGTGCAGGTAG(SEQ ID NO:4) |
| hsa-miR-18a-3p | ACTGCCCTAAGTGCTCCTTCTG(SEQ ID NO:5) |
| hsa-miR-19b-3p | TCGTGTGCAAATCCATGCAAAACTGA(SEQ ID NO:6) |
The reverse primer is a universal primer carried in the detection kit during PCR.
When the change of microRNA17-92 of the P5 generation MSC is measured by qPCR after only one Trolox culture, the result shows that: compared with the control group, single Trolox has no obvious effect on MSC microRNA 17-92. Addition of Trolox from P5 cultured MSCs to P12, with qPCR results showing: compared with a control group, Trolox has obvious improvement effect on the expression of microRNA17-3p, microRNA17-5p, microRNA18a-3p, microRNA19b-3p, microRNA20a-5p and microRNA92a-3p (figure 8a and figure 8 b).
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.
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Application of Trolox in delaying mesenchymal stem cell aging
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Claims (10)
- Application of Trolox in delaying mesenchymal stem cell aging.
- 2. The use of claim 1, wherein the mesenchymal stem cell anti-aging is manifested by increasing the proliferation rate of mesenchymal stem cells and decreasing the positive proportion of β -galactosidase staining in mesenchymal stem cells.
- 3. The use of claim 2, wherein the number of passages of the mesenchymal stem cells is less than 10 passages when the proliferation rate of the mesenchymal stem cells is used as a detection index.
- 4. The use of claim 2, wherein when the positive staining ratio of beta-galactosidase in the mesenchymal stem cell is used as a detection index, the number of passages of the mesenchymal stem cell comprises 0-15 passages.
- 5. The use according to any one of claims 1 to 4, wherein the working concentration of Trolox is not less than 2 μmol/l.
- Application of Trolox in preparation of an anti-aging culture medium for mesenchymal stem cell culture or passage.
- 7. The use as claimed in claim 6, wherein the concentration of Trolox in the medium is not less than 2 μmol/l.
- Application of Trolox in improving biological functions of mesenchymal stem cells.
- 9. The use of claim 8, wherein the enhancement of the biological function of the mesenchymal stem cell comprises an immunoregulatory function and the expression of microRNAs;preferably, the number of passages of the mesenchymal stem cells is not more than 18.
- 10. Use according to claim 8 or 9, wherein the mesenchymal stem cells are cultured or passaged each time in a medium comprising Trolox.
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