CN115404211A - Umbilical cord MSC-exos and beauty treatment application - Google Patents
Umbilical cord MSC-exos and beauty treatment application Download PDFInfo
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Abstract
The invention discloses an umbilical cord MSC-exos and a cosmetic application, wherein the MSC-exos is the MSC-exos secreted by human umbilical cord mesenchymal stem cells with high CCR5 expression. Experimental research shows that compared with the conventional MSC-exos, the MSC-exos secreted by the CCR5 high-expression umbilical cord mesenchymal stem cell provided by the invention can effectively inhibit the proliferation of human keloid fibroblasts, promote the apoptosis of the human keloid fibroblasts, and improve the effect of the exosome derived from the mesenchymal stem cell on treating keloid. Therefore, the MSC-exos has the prospect of being developed into anti-scar drugs or cosmetics.
Description
Technical Field
The invention belongs to the field of biology, relates to stem cell excretion, and particularly relates to umbilical cord MSC-exos and a beauty application.
Background
Keloid is an abnormal scar tissue caused by the hyperproliferation of fibroblasts, the massive deposition of collagen and extracellular matrix, and the deposition of core proteins. Since keloid scars seriously affect beauty, how to inhibit keloid scars is an important research direction for beauty researchers. At present, proliferation and migration of keloid fibroblasts are mainly inhibited.
The exosome is a microvesicle with the diameter of 30-120 nm which is actively secreted out of cells through a special endocytosis and efflux mechanism, and almost all living cells can secrete the exosome. In addition, exosomes are also present in various types of body fluids such as saliva, plasma, urine, and the like. The exosome mainly comprises lipid, protein and nucleic acid, wherein the lipid forms a membrane structure of the exosome, provides structural stability for the exosome and wraps active substances such as the protein, the nucleic acid and the like. Exosomes of normal cellular origin are able to efficiently eliminate cellular metabolic waste products and mediate intercellular information transduction, but exosomes secreted by some special cells have special biological functions due to carrying specific active substances of the secreting cells. The exosome derived from the stem cell contains various proteins and nucleic acids secreted by the stem cell, so that the exosome has the effects of promoting cell division and proliferation, regulating body immune response and the like.
The exosome derived from the mesenchymal stem cell is proved to be capable of inhibiting proliferation and migration of keloid fibroblasts. However, the suppression efficiency is not sufficiently high. Meanwhile, as the cost of the exosome derived from the mesenchymal stem cell is high, the cost is high and the efficiency of using the exosome derived from the mesenchymal stem cell to treat the keloid is low.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art, and provides umbilical cord MSC-exos and a beautifying application of inhibiting keloid so as to improve the effect of mesenchymal stem cell-derived exosome on treating keloid.
The above purpose of the invention is realized by the following technical scheme:
an umbilical cord MSC-exos is an MSC-exos secreted by human umbilical cord mesenchymal stem cells with high CCR5 expression.
The application of the umbilical cord MSC-exos in preparing an anti-scar medicine.
Use of the above umbilical cord MSC-exos for the preparation of an anti-scarring cosmetic product.
The technical effects are as follows:
compared with the conventional exosome, the exosome secreted by the CCR5 high-expression umbilical cord mesenchymal stem cell provided by the invention can effectively inhibit the proliferation of human keloid fibroblasts, promote the apoptosis of the human keloid fibroblasts, and improve the effect of the exosome derived from the mesenchymal stem cell on treating keloid. The exosome has the prospect of being developed into an anti-scar drug or a cosmetic product.
Drawings
FIG. 1 is a diagram of the morphology of human umbilical cord mesenchymal stem cells observed under an inverted microscope, which is in the shape of a long spindle and grows in a vortex, and meets the growth characteristics of the human umbilical cord mesenchymal stem cells;
FIG. 2 shows the immunofluorescence assay results of the stem cell surface markers, wherein the positive expression of CD44, CD90, CD73 and CD105 antigens and the negative expression of CD34 and CD45 antigens meet the phenotypic characteristics of human umbilical cord mesenchymal stem cells;
in FIG. 3, A is the expression level of CCR5 mRNA in each group of umbilical cord mesenchymal stem cells, and B is the expression level of CCR5 protein in each group of umbilical cord mesenchymal stem cells;
in FIG. 4, A is a representative diagram of exosome morphology observed under a transmission electron microscope, and B is the expression of exosome surface markers CD9, CD63 and CD81, all according with the characteristics of stem cell exosomes;
FIG. 5 is a graph of the relative clonal formation rates of various groups of keloid fibroblasts;
figure 6 shows the keloid fibroblast apoptosis in each group.
Detailed Description
1. Experimental materials
Fetal bovine serum, DMEM/F12 medium was purchased from Gibco. Penicillin was purchased from Sigma.
Exosome-free fetal bovine serum was purchased from SBI, usa.
pCDNA3.1-CCR5 recombinant plasmids, blank plasmids, were purchased from Shanghai \22331.
Lipofectamine TM 3000 was purchased from Thermo Fisher Scientific. The RT-PCR primer is designed and synthesized by a Kjeldahl gene.
CCR5, beta-actin primary and secondary antibodies, and CCK-8 reagents were purchased from Biyunnan Biotech.
Human keloid fibroblasts were purchased from Wuhan Proco Life technologies, inc., cat No. CP-H235. Complete medium for human keloid fibroblasts was purchased from Wuhan Proco Life technologies, inc., cat No. CM-H235.
2. Experimental methods
1. Umbilical cord mesenchymal stem cell isolated culture and identification
Collecting umbilical cord of newborn born in term of term, peeling artery and vein blood vessel, and cutting into 1mm 3 Tissue mass of size. Spreading the tissue block at 25cm 2 Adding DMEM/F12 medium containing 10% fetal bovine serum and 1% streptomycin to a culture flask, placing at 37 deg.C, 5% 2 Culturing under saturated humidity condition, discarding tissue block when cell climbs out of fusion about 60%, digesting with trypsin when cell fusion about 90%, adding DMEM/F12 culture medium to stop digestion when cell shrinkage and suspension are observed under the mirror, collecting cell suspension, centrifuging at 1000 Xg for 8min, collecting precipitate, and adding complete culture medium for resuspension. The primary cell 1 is subcultured into a new culture flask, namely the umbilical cord mesenchymal stem cell of the 1 st generation, liquid is changed for 1 time every 3d, when the cell is fused to be about 90%, the cell is digested by pancreatin, and the cells are subcultured according to the following steps of 1.
And taking the 4 th generation umbilical cord mesenchymal stem cells, observing the cell morphology under an inverted microscope and taking pictures.
Taking the 4 th generation umbilical cord mesenchymal stem cells, and measuring the phenotype of the surface marker of the stem cells by an immunofluorescence method.
2. Grouping and transfection
Culturing the 4 th generation umbilical cord mesenchymal stem cells in DMEM/F12 containing 10% fetal bovine serum, 1% streptomycin at 37 deg.C and 5% CO 2 Culturing under saturated humidity condition, taking cells with good growth state, and randomly dividing into: CCR5 transfection group (transfection pCDNA3.1-CCR5 recombinant plasmid), transfection control group (transfection blank plasmid), blank control group (no transfection operation). Transfection procedure was according to Lipofectamine TM 3000, performing the following experiment, replacing the fresh culture medium after 6h of transfection, and performing the subsequent experiment after 48h of transfection.
3. CCR5 mRNA expression level determination
Extracting total RNA of umbilical mesenchymal stem cells by a TRIzol method. cDNA is synthesized by reverse transcription using RNA as a template. Then using cDNA as template, PCR amplifying CCR5 gene, reaction condition is 94 deg.C for 4min, then 98 deg.C for 10s, 59 deg.C for 10s, 72 deg.C for 90s, amplifying for 35 cycles, finally 72 deg.C extending for 5min, the product is identified by 1% agarose gel electrophoresis. U6 is used as an internal reference. PCR primers were as follows.
CCR5 upstream primer: 5' ACAGAGACTTGCCACCATGGATTATCAAGGTGTC-3
CCR5 downstream primer: 5' GTGTCTCGAGTCACAAGCCCAGATATTTCC
U6 upstream primer: 5' CTCGCTTCGGCAGCACA-3
U6 downstream primer: 5' AACGCTTCACGAATTTGCGT-3
4. CCR5 protein expression level assay
Extracting total protein of umbilical cord mesenchymal stem cells of each group, determining protein concentration, loading the umbilical cord mesenchymal stem cells into SDS-PAGE gel for electrophoresis, transferring membranes, and sealing with 5% skimmed milk powder at room temperature for 1h. Antibody dilutions of CCR5 and β -actin primary antibody were added separately and incubated overnight at 4 ℃, secondary antibody dilutions were added and incubated for 1h at room temperature, followed by ECL assay.
5. Exosome extraction and identification
Extracting exosomes of all groups of umbilical cord mesenchymal stem cells by adopting a conventional ultracentrifugation method, and specifically comprising the following steps:
step S1, collecting umbilical cord mesenchymal stem cells of each group, culturing with DMEM/F12 containing 10% exosome-free fetal bovine serum and 1% streptomycin at 37 ℃ and 5% CO 2 Culturing for 48h under saturated humidity condition, and collecting culture supernatant;
s2, centrifuging at 300 Xg for 10min, discarding the precipitate, and collecting the supernatant;
s3, centrifuging at 2000 Xg for 10min, discarding the precipitated dead cells, and collecting the supernatant;
s4, performing 10000 Xg ultracentrifugation for 30min, removing cell debris, and collecting supernatant;
step S5, filtering the supernatant by using a 0.22-micron filter;
s6, performing ultracentrifugation at 100000 Xg for 70min, discarding supernatant, and collecting precipitate; resuspending the precipitate with PBS, ultracentrifuging at 100000 Xg for 70min, discarding the supernatant, and collecting the precipitate to obtain the exosome.
And taking a proper amount of exosomes, carrying out PBS (phosphate buffer solution) resuspension, and determining the concentration of the exosomes by using a BCA (burst cutting edge) method. And observing the form of the exosome by a transmission electron microscope. Western blot detection of the expression of exosome surface markers CD9, CD63 and CD 81.
6. Determination of proliferation Activity of keloid fibroblast (CCK-8 method)
Human keloid fibroblast culture: human keloid fibroblasts were treated with human keloid fibroblast complete medium at 37 ℃ and 5% CO according to the instructions of the manufacturer 2 Culturing under saturated humidity condition.
Inoculating human keloid fibroblast with good growth state in 96-well culture plate, and culturing at 5 × 10% 3 Per well, complete culture with human keloid fibroblasts at 37 ℃ and 5% CO 2 Culturing under the saturation humidity condition, and respectively adding exosomes of umbilical cord mesenchymal stem cells of a CCR5 transfection group, a transfection control group and a blank control group after 24 hours, wherein the final concentration of the exosomes is 1.5 mu g/mL. Meanwhile, a conventional culture control group without adding exosome is arranged. Each set was provided with 5 replicate wells.
After 48h, 10. Mu.L of CCK-8 solution was added to each well and the CO was 5% at 37 ℃ 2 Culturing for 2h under saturated humidity, and detecting absorbance value of each well at 450nm wavelength with enzyme-labeled detector.
7. Measurement of proliferation Activity of keloid fibroblast (clone formation method)
Inoculating human keloid fibroblast with good growth state into 6-well culture plate (1000 per well), and completely culturing human keloid fibroblast at 37 deg.C and 5% CO 2 Culturing under the saturated humidity condition, and respectively adding exosomes of the umbilical cord mesenchymal stem cells of a CCR5 transfection group, a transfection control group and a blank control group after 24 hours, wherein the final concentration of the exosomes is 1.5 mu g/mL. Meanwhile, a conventional culture control group without adding exosome is arranged. Each set was provided with 3 replicate wells.
After 7d, the medium was discarded, washed with PBS, fixed with 4% paraformaldehyde for 10 minutes at room temperature and stained with 0.1% crystal violet for 15 minutes. And (4) washing with PBS, drying, observing under a microscope to count the number of cell colonies of each group, and calculating the relative formation rate of the clones of other groups according to a formula by taking the clone formation rate of a conventional culture control group as 100%.
Relative clone formation ratio (%) = number of cell colonies of other groups/number of cell colonies of conventional culture control group × 100%.
8. Detection of apoptosis by flow cytometry
Inoculating human keloid fibroblast with good growth state into 96-well culture plate, and culturing at 5 × 10% 3 Per well, complete culture with human keloid fibroblasts at 37 ℃ and 5% CO 2 Culturing under the saturated humidity condition, and respectively adding exosomes of the umbilical cord mesenchymal stem cells of a CCR5 transfection group, a transfection control group and a blank control group after 24 hours, wherein the final concentration of the exosomes is 1.5 mu g/mL. Meanwhile, a conventional culture control group without adding exosome is arranged. Each set was provided with 5 replicate wells. And after 48h, collecting cells, and detecting the apoptosis rate of human keloid fibroblasts by an annexin V/PI double labeling method by taking equivalent cells from each group.
9. Statistical processing
Experimental data were analyzed using SPSS 20.0 software. The experimental data are expressed by mean +/-standard deviation, two groups of data are compared by adopting t test, and a plurality of groups of data are compared by adopting one-factor variance analysis. P < 0.05 is statistically significant.
3. Results of the experiment
1. Isolated culture result of human umbilical cord mesenchymal stem cells
The shape of the human umbilical cord mesenchymal stem cells observed under an inverted microscope is shown in figure 1, the human umbilical cord mesenchymal stem cells grow in a long fusiform shape and a vortex shape, and the growth characteristics of the human umbilical cord mesenchymal stem cells are met; the positive expression of CD44, CD90, CD73 and CD105 antigens and the negative expression of CD34 and CD45 antigens (figure 2) are consistent with the phenotypic characteristics of the human umbilical cord mesenchymal stem cells.
2. Results of transfection
The expression level of CCR5 mRNA in each group of umbilical cord mesenchymal stem cells is shown as A in figure 3. Compared with a blank control group, the expression level of CCR5 mRNA in the umbilical cord mesenchymal stem cells of the CCR5 transfection group is obviously increased.
The expression level of CCR5 protein in each group of umbilical cord mesenchymal stem cells is shown as B in figure 3. Compared with a blank control group, the expression level of the CCR5 protein in the umbilical cord mesenchymal stem cells of the CCR5 transfection group is obviously increased.
The results show that the group transfected by CCR5 successfully obtains umbilical cord mesenchymal stem cells with high CCR5 expression.
3. Exosome extraction and identification
The size and the shape of the exosomes in each group have no obvious difference, and are shown as A in figure 4, B is the expression of exosome surface markers CD9, CD63 and CD81, and all accord with the characteristics of stem cell exosomes.
4. Proliferative activity of keloid fibroblasts
Table 1 shows the absorbance values of each group, and FIG. 5 shows the relative formation rate of clones. As can be seen from table 1 and fig. 5, the proliferation of keloid fibroblasts was significantly inhibited in each exosome group compared to the conventional culture control group, of which the proliferation inhibition effect of umbilical cord mesenchymal stem cell exosomes on keloid fibroblasts was the strongest in CCR 5-transfected umbilical cord mesenchymal stem cell exosomes.
TABLE 1 Absorbance values for groups
5. Keloid fibroblast apoptosis
Figure 6 shows the keloid fibroblast apoptosis in each group. As can be seen from FIG. 6, compared with the conventional culture control group, the keloid fibroblast apoptosis rate of each exosome group is higher, wherein the apoptosis promoting effect of the umbilical cord mesenchymal stem cell exosomes of the CCR5 transfection group on the keloid fibroblasts is strongest.
The above examples are only intended to illustrate the essence of the present invention, but not to limit the scope of the present invention.
Claims (3)
1. An umbilical cord MSC-exos, comprising: the MSC-exos is secreted by human umbilical cord mesenchymal stem cells with high CCR5 expression.
2. Use of the umbilical cord MSC-exos of claim 1 for the preparation of a medicament for anti-scarring.
3. Use of the umbilical cord MSC-exos of claim 1 for the preparation of an anti-scarring cosmetic product.
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