CN115466722A - Method for promoting stem cell proliferation and collecting stem cell exosomes - Google Patents
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Abstract
The invention discloses a method for promoting stem cell proliferation and collecting a stem cell exosome, and particularly discloses application of an FGL1 inhibitor in promoting stem cell in-vitro proliferation, wherein the stem cell is an umbilical cord mesenchymal stem cell. Experiments prove that the umbilical cord mesenchymal stem cells with the inhibited FGL1 have stronger proliferation activity in vitro, still maintain good multidirectional differentiation capacity, and the secreted exosomes conform to the characteristics of stem cell exosomes. The FGL1 inhibitor can be used for promoting the in-vitro proliferation of umbilical cord mesenchymal stem cells, thereby improving the efficiency of obtaining mesenchymal stem cell exosomes.
Description
Technical Field
The invention belongs to the field of stem cells, relates to stem cell culture and exosome preparation, and particularly relates to a method for promoting stem cell proliferation and collecting stem cell exosomes.
Background
Exosomes (Exo) are vesicle-like substances that can be released by most cells, approximately 40-160 nm in diameter. It originates from the endocytic pathway: the cytoplasmic membrane is endocytosed to form an early endosome, which can develop into a late endosome, which buds inwards to form luminal vesicles and become a multivesicular body, part of which is fused with the cytoplasmic membrane, and the released luminal vesicles are exosomes. The process of multivesicular bodies occurs such that the exosomes eventually contain nucleic acids and proteins associated with the source cell, which is a necessary condition for the exosomes to exert similar physiological functions as the source cell.
Mesenchymal Stem Cells (MSCs), which are adult stem cells having the ability to self-renew and differentiate in multiple directions, are first isolated from bone marrow, and then it is found that MSCs can be isolated and prepared from periosteum, fat, muscle, placenta, umbilical cord, and umbilical cord blood. In the early days, it was thought that the repair of the injury by MSCs was based on differentiation and replacement, and further studies revealed that MSCs have a short retention time in vivo, and that MSCs are retained in many organs such as lung and spleen by intravenous injection, and therefore the paracrine action of MSCs has been attracting attention. Researches find that the MSCs exosome (MSCs-Exo) can play a similar physiological role as the MSCs, can improve the cell function and phenotype, regulate the immune system and the like, and achieves the aim of treating diseases.
At present, the collection and preparation efficiency of the MSCs-Exo is low. Improving the efficiency of MSCs-Exo production is generally done from two perspectives: 1. the proliferation efficiency of the MSCs is improved, and the cell base number is improved; 2. improve the secretion efficiency of the MSCs and improve the MSCs-Exo secretion capability of a unit amount of cells. In comparison, the first angle implementation is rich.
The invention solves the problem of low efficiency of MSCs-Exo collection and preparation from the first angle.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art, provides a method for promoting stem cell proliferation and collecting stem cell exosomes, and improves the efficiency of stem cell culture and collection of the stem cell exosomes.
The above purpose of the invention is realized by the following technical scheme:
the application of the FGL1 inhibitor in promoting the in-vitro proliferation of stem cells, wherein the stem cells are umbilical cord mesenchymal stem cells.
Preferably, the FGL1 inhibitor is a siRNA that inhibits FGL1 expression.
A method of collecting stem cell exosomes, comprising the steps of:
step S1, collecting umbilical cord mesenchymal stem cells treated with FGL1 inhibitor, 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 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, discarding 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.
Preferably, the FGL1 inhibitor is a siRNA that inhibits FGL1 expression.
The technical effects are as follows:
experiments prove that the umbilical cord mesenchymal stem cells with the FGL1 inhibited have stronger proliferation activity in vitro and still maintain good multidirectional differentiation capability, and the secreted exosomes conform to the characteristics of stem cell exosomes. Therefore, the FGL1 inhibitor can be used for promoting the in-vitro proliferation of umbilical cord mesenchymal stem cells, and further improving the efficiency of obtaining mesenchymal stem cell exosomes.
Drawings
FIG. 1 is a morphological diagram of umbilical cord mesenchymal stem cells observed under an inverted microscope;
FIG. 2 shows the expression level of FGL1 protein in each group of umbilical cord mesenchymal stem cells;
FIG. 3 is a diagram showing the results of osteogenic differentiation and adipogenic differentiation of the siRNA group umbilical cord mesenchymal stem cells under the induction culture of osteogenic differentiation medium and adipogenic differentiation medium;
FIG. 4 shows the identification result of exosome secreted by umbilical cord mesenchymal stem cells in siRNA group; wherein A is the form of the exosome observed under a transmission electron microscope, B is the expression of exosome surface markers CD9, CD63 and CD81, and C is the particle size distribution of the exosome, which all accord with the characteristics of the stem cell exosome.
Detailed Description
1. Experimental Material
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.
Markers for use in the flow assay, CD44, CD90, CD73, CD105, CD34, CD45, were purchased from BD.
FGL1siRNA, siRNActive control and RT-PCR primers were designed and synthesized from the Shanghai Jikai gene.
Lipofectamine TM 3000 was purchased from Thermo Fisher Scientific.
TRIzol reagent, oneStep RT-PCRKit kit was purchased from Thermo Fisher Scientific.
FGL1, GAPDH primary antibody, secondary antibody, and CCK-8 reagents were purchased from bi yun tian biotechnology.
The culture medium for osteogenic and adipogenic induction and differentiation of human umbilical cord mesenchymal stem cells is purchased from Wuhan Punuo Sai Life technologies, inc.
2. Experimental method
1. Umbilical cord mesenchymal stem cell isolated culture
Collecting umbilical cord of newborn born in term of month, peeling artery and vein, 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, setting the culture at 37 deg.C, 5% CO 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. And (3) passaging the primary cells 1. Taking the 4 th generation of human umbilical cord mesenchymal stem cells, observing the cell morphology under an inverted microscope and taking a picture; and (3) identifying cell phenotypes by using a flow cytometer, and carrying out subsequent experiments by positively expressing CD44, CD90, CD73 and CD105 antigens and negatively expressing CD34 and CD45 antigens.
2. Grouping and transfection
Culturing the 4 th generation umbilical cord mesenchymal stem cells in DMEM/F12 containing 10% fetal bovine serum and 1% streptomycin at 37 ℃ in 5% CO 2 Culturing under saturated humidity conditions, taking cells with good growth state, and randomly dividing the cells into: siRNA group (transfection FGL1 siRNA), siRNA-NC group (transfection siRNAactive control), blank group (no transfection). 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. FGL1 mRNA expression level determination
Extracting total RNA of umbilical cord mesenchymal stem cells of each group by using a TRIzol method. RNA reversal and PCR amplification were performed according to the OneStep RT-PCR Kit instructions, and the PCR amplification reaction system was as follows: 5 XQIAGEN One Step RT-PCR Buffer 10. Mu.L, dNTP Mix 2. Mu.L, upstream and downstream primers 1. Mu.L each, RNA 1. Mu.g, QIAGEN One Step RT-PCR Enzyme Mix 2. Mu.L, RNase-free water make-up 50. Mu.L. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5min, followed by annealing at 95 ℃ for 10s and 60 ℃ for 40min for 40 cycles. GAPDH as internal reference, according to 2 —△△Ct The method calculates the relative expression level of FGL1 mRNA in each group of human umbilical cord mesenchymal stem cells.
The primer sequences are as follows:
FGL1 upstream primer: 5' GCTGGTGGTTTAACAGGTGTGC
FGL1 downstream primer: 5' AGAATACCACCCACCATGCC-3
GAPDH upstream primer: 5' AGAAGACTGTGGATGG-3
GAPDH downstream primer: 5' AGCTCAGGGATGACCTTG-3
4. FGL1 protein expression level determination
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 FGL1, GAPDH primary antibodies were added separately and incubated overnight at 4 ℃, and secondary antibody dilutions were added and incubated for 1h at room temperature, followed by ECL assay.
5. CCK-8 method for measuring cell proliferation activity
Inoculating each group of umbilical cord mesenchymal stem cells into a 96-well culture plate, and culturing at 5 × 10 3 Cell/well, incubation with DMEM/F12 containing 10% fetal bovine serum, 1% streptomycin at 37 ℃ with 5% CO 2 Under saturated humidity conditionCulture, each group of 5 repeat 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 condition, and detecting absorbance value of each well at 450nm wavelength by using enzyme-labeled detector.
6. Determination of multidirectional differentiation Capacity
Taking the umbilical cord mesenchymal stem cells of the siRNA group, digesting the umbilical cord mesenchymal stem cells by 5 multiplied by 10 4 The cell density of each well is inoculated in a 6-well plate, 2mL osteogenic inducing liquid (commercial human umbilical cord mesenchymal stem cell osteogenic induced differentiation culture medium provided by Wuhan Poncirus Life technologies, ltd.) is added into each well, 1 inducing liquid is replaced every 3d in the period, alizarin red staining is carried out on the 14 th day, and the formation condition of mineralized nodules is observed under a microscope. Taking the umbilical cord mesenchymal stem cells of the siRNA group, digesting the umbilical cord mesenchymal stem cells by 5 multiplied by 10 4 The cell density per well was inoculated in a 6-well plate, 2mL of the adipogenic induction liquid (a commercial adipogenic induction differentiation medium for human umbilical cord mesenchymal stem cells provided by GmbH) was added to each well, 1 induction liquid was changed every 3d during the period, oil red O staining was performed on day 14, and lipid droplets were observed under a microscope.
7. Exosome extraction and identification
The exosome of the siRNA group umbilical cord mesenchymal stem cells is extracted by adopting a conventional ultracentrifugation method, and the method comprises the following specific steps:
step S1, collecting the siRNA-group umbilical cord mesenchymal stem cells, culturing the cells in DMEM/F12 containing 10% of exosome-free fetal bovine serum and 1% of streptomycin at 37 ℃ and 5% of 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 dead cells of the precipitate, and collecting the supernatant;
s4, performing 10000 Xg ultracentrifugation for 30min, discarding 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; and (4) resuspending the precipitate with PBS, ultracentrifuging at 100000 Xg for 70min again, discarding the supernatant, and collecting the precipitate to obtain the exosome.
And taking a proper amount of exosome, carrying out PBS (phosphate buffer solution) resuspension, and determining the concentration of the exosome 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. The particle size analyzer measures the particle size distribution.
8. Statistical treatment
Experimental data were analyzed using SPSS 20.0 software. The experimental data are expressed by mean plus or minus 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. Result of isolated culture 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 conform to the phenotypic characteristics of human umbilical cord mesenchymal stem cells.
2. FGL1 mRNA, FGL1 protein expression level
The relative expression level of FGL1 mRNA in each group of umbilical cord mesenchymal stem cells is shown in Table 1. Compared with blank group, the expression level of FGL1 mRNA in the umbilical cord mesenchymal stem cells of the siRNA group is obviously reduced.
TABLE 1FGL1 mRNA relative expression levels
The expression level of FGL1 protein in each group of umbilical cord mesenchymal stem cells is shown in FIG. 2. Compared with blank group, FGL1 protein expression level in umbilical cord mesenchymal stem cells of siRNA group is obviously reduced.
The results indicate that inhibition of FGL1 is successfully achieved after siRNA transfection into umbilical cord mesenchymal stem cells.
3. Cell proliferation Activity
The absorbance values of all groups are shown in table 2, and compared with blank group, the absorbance value of siRNA group is obviously increased, which shows that the umbilical cord mesenchymal stem cells with FGL1 inhibited have obviously stronger proliferation activity.
TABLE 2 Absorbance values for each set
4. Capability of multidirectional differentiation
The results are shown in fig. 3, the siRNA group umbilical cord mesenchymal stem cells directionally generate osteogenic differentiation and adipogenic differentiation under the induction culture of osteogenic differentiation medium and adipogenic differentiation medium, respectively. This result demonstrates that FGL 1-inhibited umbilical mesenchymal stem cells maintain good multipotential differentiation capacity.
5. Exosome extraction and identification
In fig. 4, a is the form of the exosome observed under a transmission electron microscope, B is the expression of the exosome surface markers CD9, CD63 and CD81, and C is the particle size distribution of the exosome, all of which conform to the characteristics of the stem cell exosome.
The experimental results show that the umbilical cord mesenchymal stem cells with the FGL1 inhibited have stronger proliferation activity in vitro, still maintain good multidirectional differentiation capability, and the secreted exosomes accord with the characteristics of stem cell exosomes. Therefore, the FGL1 inhibitor can be used for promoting the in-vitro proliferation of umbilical cord mesenchymal stem cells, and further improving the efficiency of obtaining mesenchymal stem cell exosomes.
The above examples are only intended to illustrate the essence of the present invention in detail, but are not intended to limit the scope of the present invention.
Claims (4)
- Use of an FGL1 inhibitor for promoting the in vitro proliferation of stem cells, which are umbilical cord mesenchymal stem cells.
- 2. The use according to claim 1, wherein the FGL1 inhibitor is an siRNA that inhibits FGL1 expression.
- 3. A method of collecting stem cell exosomes, comprising the steps of:step S1, collecting umbilical cord mesenchymal stem cells treated with FGL1 inhibitor, 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 supernatant;s2, centrifuging at 300 Xg for 10min, discarding the precipitate, and collecting the supernatant;s3, centrifuging at 2000 Xg for 10min, discarding the dead cells of the precipitate, and collecting the supernatant;s4, performing 10000 Xg ultracentrifugation for 30min, discarding 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; and (4) resuspending the precipitate with PBS, ultracentrifuging at 100000 Xg for 70min again, discarding the supernatant, and collecting the precipitate to obtain the exosome.
- 4. The method of claim 3, wherein the FGL1 inhibitor is an siRNA that inhibits FGL1 expression.
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| CN116478997B (en) * | 2023-06-06 | 2023-12-29 | 苏州吉玛基因股份有限公司 | siRNA for inhibiting FGL1, and modification and application thereof |
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