CN111690686A - Application of miRNA high expression in promoting in-vitro proliferation and osteogenic differentiation of umbilical cord mesenchymal stem cells - Google Patents
Application of miRNA high expression in promoting in-vitro proliferation and osteogenic differentiation of umbilical cord mesenchymal stem cells Download PDFInfo
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Abstract
The invention discloses application of miRNA high expression in promoting in-vitro proliferation and osteogenic differentiation of umbilical cord mesenchymal stem cells. miRNAs play an important role in cells. The miR-575 plays an important role in the proliferation and osteogenic differentiation of hUCMSCs, and the improvement of the expression level of miR-575 can effectively promote the in vitro proliferation and osteogenic differentiation of hUCMSCs, so that the in vitro proliferation and osteogenic differentiation of hUCMSCs can be promoted by the method for improving the expression of miR-575in hUCMSCs through intervention, and the miR-575 can be used as a target to screen components for promoting the in vitro proliferation and osteogenic differentiation of hUCMSCs.
Description
Technical Field
The invention belongs to the field of biology, relates to stem cells, and particularly relates to application of miRNA high expression in promoting in-vitro proliferation and osteogenic differentiation of umbilical cord mesenchymal stem cells.
Background
Mesenchymal stem cells are widely applied to the field of tissue engineering and regenerative medicine at present due to self-renewal capacity and multidirectional differentiation potential, have wide sources and are easy to operate in vitro, so that the mesenchymal stem cell therapy has a wide prospect, wherein human umbilical cord-derived mesenchymal stem cells (hucml mesenchymal stem cells, hUCMSCs) are considered to be an original mesenchymal stem cell group with higher plasticity and lower immunogenicity, and are convenient and easy to obtain. A large number of researches prove that the hUCMSCs can be differentiated into various adult cells through induction culture, can be differentiated into osteocytes, chondrocytes, hepatocytes, cardiac muscle cells and the like in vitro, and can be differentiated into dopaminergic neurons, skeletal muscle cells, endothelial cells, islet cells and the like in vivo. The hUCMSCs have wide application prospect in emerging transformation medicine.
Based on the multidirectional differentiation capacity of the hUCMSCs, the hUCMSCs provide a new feasible approach for the problem of the source of seed cells of the artificial bone in bone tissue engineering.
microRNA (miRNA) is an endogenous non-coding RNA that is evolutionarily highly conserved among viruses and higher organisms. Each miRNA can regulate and control a plurality of target genes, and a plurality of miRNAs can cooperatively regulate and control the same target gene, the miRNA realizes macroscopic and fine regulation and control, and a plurality of physiological and pathological characteristics are related to miRNA-mediated regulation and control. Research shows that miRNA also has important function in the differentiation regulation of hUCMSCs. Zhang Yan et al explores the action and the action mechanism of miR-92b-3P in the osteogenic differentiation process of human umbilical cord mesenchymal stem cells (hUCMSCs), and finds that the expression quantity of miR-92b-3P in the osteogenic process of hUCMSCs is obviously increased (P is less than 0.05) compared with that in a control group; the overexpression of miR-92b-3P can promote in-vitro osteogenic differentiation and in-vivo ectopic osteogenesis capacity of hUCMSCs (P is less than 0.05); and the inhibition of miR-92b-3P can reduce in-vitro osteogenic differentiation (P is less than 0.05) of hUCMSCs, and finally, the conclusion is drawn: miR-92b-3p can promote osteogenic differentiation of hUCMSCs by inhibiting DKK1 expression (miR-92b-3p promotes osteogenic differentiation of human umbilical cord mesenchymal stem cells, basic medicine and clinic, Vol. 36, No.5, 2016 (5 months)).
miRNAs are in a wide variety of types, and more miRNAs playing an important role in osteogenic differentiation of hUCMSCs are continuously discovered.
Disclosure of Invention
The invention aims to provide application of miRNA high expression in promoting in-vitro proliferation and osteogenic differentiation of umbilical cord mesenchymal stem cells, wherein the miRNA is miR-575.
The technical scheme is as follows:
an application of a method for improving miR-575 expression level in promoting in-vitro proliferation and osteogenic differentiation of umbilical cord mesenchymal stem cells. In a specific embodiment, compared with a Control group, the in vitro proliferation activity of the hUCMSCs in the miR-575mimic group is remarkably improved, the in vitro proliferation activity of the hUCMSCs in the miR-575inhibitor group is remarkably reduced, and the in vitro proliferation activity of the hUCMSCs in the miR-575NCmimic group and the miR-575NC inhibitor group is not remarkably different; compared with a Control group, the expression levels of Runx2 and OCN protein after bone induction are obviously increased after the miR-575mimic group forms the bone induction, the expression levels of Runx2 and OCN protein after the miR-575inhibitor forms the bone induction are obviously reduced, and the expression levels of Runx2 and OCN protein after the bone induction are not obviously different after the miR-575NC mimic group and the miR-575NCinhibitor form the bone induction.
A method for promoting in-vitro proliferation and osteogenic differentiation of umbilical cord mesenchymal stem cells is characterized in that a component for improving miR-575 expression level is added in the culture process of the umbilical cord mesenchymal stem cells. In a specific embodiment, compared with a Control group, the in vitro proliferation activity of the hUCMSCs in the miR-575 mic group is remarkably improved, the in vitro proliferation activity of the hUCMSCs in the miR-575inhibitor group is remarkably reduced, and the in vitro proliferation activity of the hUCMSCs in the miR-575NC mic group and the miR-575NC inhibitor group is not remarkably different; compared with a Control group, the expression levels of Runx2 and OCN protein after osteoinduction are obviously increased by the miR-575mimic group, the expression levels of Runx2 and OCN protein after osteoinduction are obviously reduced by the miR-575inhibitor group, and the expression levels of Runx2 and OCN protein after osteoinduction are not obviously different by the miR-575NCmimic group and the miR-575NC inhibitor group.
The miR-575 is used for screening components for promoting in-vitro proliferation and osteogenic differentiation of hUCMSCs as a target.
The technical effects are as follows:
miRNAs play an important role in cells. The invention discovers that miR-575 plays an important role in the proliferation and osteogenic differentiation of hUCMSCs, and the improvement of the expression level of miR-575 can effectively promote the in vitro proliferation and osteogenic differentiation of hUCMSCs, so that the in vitro proliferation and osteogenic differentiation of hUCMSCs can be promoted by the method for improving the expression of miR-575in hUCMSCs through intervention, and the miR-575 can be used as a target to screen a culture medium additive for promoting the in vitro proliferation and osteogenic differentiation of hUCMSCs.
Drawings
FIG. 1 shows that the OD value and the proliferation rate of each group are respectively, compared with the Control group, the in vitro proliferation activity of the miR-575 mic group hUCMSCs is remarkably improved, the in vitro proliferation activity of the miR-575inhibitor group hUCMSCs is remarkably reduced, and the in vitro proliferation activity of the miR-575NC mic group and the miR-575NC inhibitor group hUCMSCs has no remarkable difference.
FIG. 2 shows relative expression levels of genes related to bone differentiation after transfection, compared with Control group, relative expression levels of Runx2 mRNA and OCN mRNA after bone induction by miR-575mimic composition, relative expression levels of Runx2 mRNA and OCN mRNA after bone induction by miR-575inhibitor composition, and relative expression levels of Runx2 mRNA and OCN mRNA after bone induction by miR-575NCmimic group and miR-575NC inhibitor composition have no significant difference.
FIG. 3 shows the expression levels of Runx2 and OCN proteins in each group after osteogenesis induction, compared with the Control group, the expression levels of Runx2 and OCN proteins after osteogenesis are obviously increased after miR-575 mic composition, the expression levels of Runx2 and OCN proteins after osteoinduction are obviously decreased after miR-575inhibitor composition, and the expression levels of Runx2 and OCN proteins after osteoinduction are not obviously different after miR-575NC mic group and miR-575NC inhibitor composition.
Detailed Description
The following description and drawings are included to describe the general nature of the invention, and are not intended to limit the scope of the invention.
First, test materials
Human umbilical cord tissue was taken from healthy, full-term, birth foetuses and placed in sterile PBS solution containing 1% double antibody for use within 8 h.
alpha-MEM medium, fetal bovine serum FBS and 0.25% trypsin were purchased from Gibco, USA.
FITC or PE labeled mouse anti-human CD90, CD105, CD73, CD14, CD19, CD34, CD45, and HLA-DR antibodies and respective related isotype controls were purchased from eBioscince, USA.
The miR-575 mici, miR-575NC mici, miR-575inhibitor and miR-575NC inhibitor are synthesized by Shanghai Jima biotechnology Limited.
MTT assay reagents were purchased from petunia.
The human umbilical cord mesenchymal stem cell osteogenesis inducing differentiation medium is purchased from Guangzhou Seisakul Biotech limited.
Second, test method
1. Isolation, culture and identification of umbilical cord mesenchymal stem cells
The contents of the test are the conventional operation in the field, and the method is the same as the invention patent application named as "a CXCR4 agonist and the application in the aspect of in vitro culture of the umbilical cord mesenchymal stem cells" and the invention patent application named as "a method for promoting the in vitro proliferation of the umbilical cord mesenchymal stem cells". The method comprises the following specific steps:
separating and culturing hUCMSCs: placing a 10-15 cm term newborn umbilical cord in a sterile PBS solution containing 1% double antibody, transferring to a superclean bench, repeatedly washing with the sterile PBS solution containing 1% double antibody, cleaning blood in blood vessels, transferring to a Hank's solution, removing two umbilical veins and one umbilical artery, peeling off the outer skin, exposing the middle jelly-like Huatong glue, cutting into small segments, drying in the air, and cutting into 1mm pieces3Size, spread to the bottom of T75 flask, inverted flask, and 10mL of α -MEM medium containing 10% FBS and 1% diabody was added contralaterally to the tissue mass at 37 deg.C and 5% CO2And culturing under saturated humidity conditions. And after 12h, reversing the culture flask to enable the culture medium to overflow the tissue block, continuously culturing for about 14d until cells climb out of the periphery of the tissue block, removing the tissue block, digesting for about 30-60 s by using 0.25% trypsin, centrifuging for 5min at 1000r/min after digestion is stopped, inoculating the tissue block into a culture dish after heavy suspension, changing the solution once every 3d, and observing the 5 th generation cells under an inverted microscope for subsequent experiments.
Identification of hUCMSCs by taking 5 th generation hUCMSCs, digesting with 0.25% pancreatin, and adjusting cell density to 1 × 106Adding 1mL of cell suspension into each centrifuge tube, washing with sterile PBS for 2 times, centrifuging at 1000 × g for 5min, discarding supernatant, resuspending cells at 100 mu LPBS, adding appropriate amount of CD90, CD105, CD73, CD14, CD19, CD34, CD45 and HLA-DR antibody and isotype control, incubating at room temperature in dark for 30min, centrifuging at 1000 × g for 5min, washing with PBS for 2 minThen, 500. mu.L of LPBS was added for resuspension to prepare a single cell suspension, which was then detected by flow cytometry.
2. Grouping and transfection
The 5 th generation hUCMSCs were cultured in α -MEM containing 10% FBS and 1% diabody at 37 deg.C under 5% CO2And cultured under saturated humidity conditions, inoculated into 6-well plates, and grouped as follows:
miR-575mimic group: transfecting miR-575 mici and miR-575 high expression;
miR-575NC mimic group: transfecting miR-575NC mimic and miR-575 to normally express;
miR-575inhibitor group: transfecting miR-575inhibitor, wherein miR-575 is expressed at low level;
miR-575NC inhibitor group: transfecting miR-575NC inhibitor, wherein the miR-575 is normally expressed;
control group: not transfected.
After 24h, cell transfection is carried out according to the Lipofectamine 2000 instruction and the grouping, a mimic (mimic) of miR-575, a negative control mimic (NC mimic), an inhibitor (inhibitor) and a negative control inhibitor (NCinhibitor) are transfected into the cells respectively, and after 48h, each group of cells are washed and collected for subsequent experiments.
3. RT-PCR method for detecting miR-575 gene expression after transfection
Collecting transfected cells, extracting total RNA of each group of cells by using a TRIzol kit, measuring the concentration and purity of the RNA, synthesizing cDNA by using a reverse transcription kit, carrying out PCR amplification, taking U6 as an internal reference, calculating the relative expression level of miR-575 of each group according to a formula miR-575/U6 according to the operation method of the kit, and calculating the relative expression level of miR-575 of a Control group as 1.00.
Primers for miR-575 and U6 are as follows:
miR-575 upstream primer (Sequence NO. 1): 5'-CGTAAAGCACCACATCTAGGT-3', respectively;
miR-575 downstream primer (Sequence NO. 2): 5'-GGTAAGGAGCTGTCCAGCC-3', respectively;
u6 upstream primer (Sequence No. 3): 5'-GCTTCGGCAGCACATATACT-3', respectively;
u6 downstream primer (Sequence No. 4): 5'-GCAGGGTCCGAGGTATTC-3' are provided.
4. Determination of in vitro proliferation activity of umbilical cord mesenchymal stem cells by MTT method
The transfected cells were collected and plated in 96-well plates, 1 × 10 per well4Cells/200. mu.L, at 37 ℃ with 5% CO2After culturing for 48h under the saturation humidity condition, 20 μ L of MTT solution with the concentration of 5mg/mL is added into each well, culturing is carried out for 4h, supernatant is removed, 150 μ L of LDMSO is added, the mixture is gently shaken, the absorbance (OD value) at 490nm is detected by a microplate reader, the proliferation rate of the hbucmscs in the transfection group is calculated according to the OD value as the following formula, the proliferation rate of the hbucmscs in the Control group is 100%, and the proliferation rate of the transfection group is × 100% of the OD value of the transfection group/OD value of the Control group.
5. Osteogenic induced differentiation culture
Collecting transfected cells, inoculating the cells in a 24-well plate, and completely replacing the transfected cells with umbilical cord mesenchymal stem cells in osteogenic induced differentiation culture based on 5% CO at 37 DEG C2Culturing under saturated humidity condition, and collecting cells after 72h for detection.
6. RT-PCR method for detecting mRNA expression level of osteogenic related genes Runx2 and OCN
Collecting cells cultured by osteogenesis induction differentiation, washing with PBS, extracting total RNA of each group of cells by using a TRIzol kit, measuring the concentration and purity of the RNA, synthesizing cDNA by using a reverse transcription kit, carrying out PCR amplification, calculating relative expression levels of Runx2 mRNA and OCN mRNA according to the formula Runx2 mRNA/U6 and OCN mRNA/U6 by using U6 as an internal reference according to the operation method of the kit, and calculating the relative expression levels of Runx2 mRNA and OCN mRNA of a Control group to be 1.00. The primers are as follows:
runx2 upstream primer (Sequence NO. 5): 5'-GAATGAAGTCGCTGTCCTCG-3', respectively;
runx2 downstream primer (Sequence NO. 6): 5'-CCTTCCCTGCTCTTGTTGG-3', respectively;
OCN upstream primer (Sequence NO. 7): 5'-GTGCAGCCTTTGTGTCCAAG-3', respectively;
OCN downstream primer (Sequence NO. 8): 5'-TCAGCCAACTCGTCACAGTC-3', respectively;
u6 upstream primer (Sequence No. 3): 5'-GCTTCGGCAGCACATATACT-3', respectively;
u6 downstream primer (Sequence No. 4): 5'-GCAGGGTCCGAGGTATTC-3' are provided.
7. Western Blot method for measuring expression level of osteogenesis related proteins Runx2 and OCN
Collecting cells cultured by osteogenesis-inducing differentiation, washing with PBS, adding a proper amount of RIPA lysate (100mmol/LPMSF), performing ice lysis for 30min, determining the protein concentration by a BCA method, adjusting the protein concentration to be consistent, adding 5 Xloading buffer solution, boiling for 10min at 100 ℃, transferring to a PVDF membrane after 10% SDS-polyacrylamide gel electrophoresis, sealing with 5% skimmed milk powder at room temperature for 2h, adding Runx2, OCN and GAPDH primary antibody, and incubating overnight at 4 ℃. After TBST repeatedly washing the membrane, incubating the secondary antibody marked by HRP for 1h, developing the color of the chemiluminescent reagent after washing the membrane, photographing by using a gel imaging system, and analyzing the gray value by using Image J software.
8. Statistical analysis
All data were analyzed using SPSS 17.0 statistical software and expressed as mean ± standard deviation (mean ± SD). The comparison between the two groups adopts one-factor analysis of variance, and P is less than 0.05 to indicate that the difference has statistical significance.
Third, test results
1. Separation, culture and identification results of umbilical cord mesenchymal stem cells
Observation under an inverted microscope shows that the hUCMSCs grow adherent to the wall, are fusiform, have relatively uniform sizes, grow in parallel or vortex, and accord with the cell biology characteristics of the hUCMSCs. Flow cytometry detection results show that positive expression (more than 95%) of CD90, CD105 and CD73 and negative expression (less than 2%) of CD14, CD19, CD34, CD45 and HLA-DR are consistent with the surface marker characteristics of hUCMSCs.
2. miR-575 gene expression level in transfected hUCMSCs
The expression levels of miR-575 genes in each group after transfection are shown in Table 1, compared with the Control group, the expression level of miR-575in the miR-575 mic group hUCMSCs is obviously increased, the expression level of miR-575in the miR-575inhibitor group hUCMSCs is obviously reduced, and the expression levels of miR-575in the miR-575NC mic group and the miR-575NC inhibitor group hUCMSCs have no obvious difference. The result shows that the transfection operation is successful, hUCMSCs with different expression levels of miR-575 have been successfully constructed, and subsequent experiments can be carried out.
TABLE 1 relative expression levels of miR-575 genes in each group after transfection
3. In vitro proliferation Activity of transfected hUCMSCs
The OD value and the proliferation rate of each group are shown in a table 2 and a figure 1, compared with the Control group, the in vitro proliferation activity of the miR-575 mic group hUCMSCs is remarkably improved, the in vitro proliferation activity of the miR-575inhibitor group hUCMSCs is remarkably reduced, and the in vitro proliferation activity of the miR-575NC mic group and the miR-575NC inhibitor group hUCMSCs has no remarkable difference. The result shows that the hUCMSCs with high miR-575 expression have higher in vitro proliferation activity.
TABLE 2 OD values and proliferation rates of the respective groups
4. Osteogenic differentiation Activity (mRNA level) of transfected hUCMSCs
Relative expression levels of Runx2 mRNA and OCN mRNA in each group after osteogenic induction are shown in table 3 and figure 2, compared with a Control group, the relative expression levels of Runx2 mRNA and OCN mRNA after osteoinduction formed by miR-575mimic are obviously increased, the relative expression levels of Runx2 mRNA and OCN mRNA after osteoinduction formed by miR-575inhibitor are obviously reduced, and the relative expression levels of Runx2 mRNA and OCN mRNA after osteoinduction formed by miR-575NCmimic group and miR-575NC inhibitor are not obviously different. This shows that the hUCMSCs with high miR-575 expression have higher osteogenic differentiation activity.
TABLE 3 relative expression levels of the constitutive osteodifferentiation-associated genes after transfection
5. Osteogenic differentiation Activity (protein level) of transfected hUCMSCs
As shown in figure 3, the expression levels of Runx2 and OCN proteins in each group after osteogenesis induction are obviously increased, compared with a Control group, the expression levels of Runx2 and OCN proteins after osteogenesis are formed by miR-575 mic are obviously increased, the expression levels of Runx2 and OCN proteins after osteoinduction are obviously reduced after miR-575inhibitor is formed, and the expression levels of Runx2 and OCN proteins after osteoinduction are formed by miR-575NC mic group and miR-575NC inhibitor are not obviously different. This shows that the hUCMSCs with high miR-575 expression have higher osteogenic differentiation activity.
In conclusion, miR-575 plays an important role in the proliferation and osteogenic differentiation of hUCMSCs, and the improvement of the expression level of miR-575 can effectively promote the in vitro proliferation and osteogenic differentiation of hUCMSCs, so that the in vitro proliferation and osteogenic differentiation of hUCMSCs can be promoted by the method for improving the expression of miR-575in hUCMSCs through intervention, and the miR-575 can be used as a target to screen a culture medium additive for promoting the in vitro proliferation and osteogenic differentiation of hUCMSCs.
The above-mentioned embodiments and the accompanying drawings are used to specifically describe the material of the present invention, but should not be construed as limiting the scope of the present invention.
Sequence listing
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Claims (3)
1. An application of a method for improving miR-575 expression level in promoting in-vitro proliferation and osteogenic differentiation of umbilical cord mesenchymal stem cells.
2. A method for promoting in vitro proliferation and osteogenic differentiation of umbilical cord mesenchymal stem cells is characterized in that: adding a component for improving the expression level of miR-575in the culture process of the umbilical cord mesenchymal stem cells.
The application of the miR-575 as a target for screening components for promoting the in-vitro proliferation and osteogenic differentiation of the hUCMSCs.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112391341A (en) * | 2020-11-30 | 2021-02-23 | 张川 | Application of SDF-1 protein activator in promoting human umbilical cord mesenchymal stem cell proliferation and differentiation in vitro |
| CN112481219A (en) * | 2020-11-30 | 2021-03-12 | 张川 | Application of high gene expression in promoting proliferation of human umbilical cord mesenchymal stem cells in-vitro culture |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102014934A (en) * | 2008-02-22 | 2011-04-13 | 新加坡科技研究局 | Mesenchymal stem cell particles |
| CN102858997A (en) * | 2009-11-02 | 2013-01-02 | 新加坡科技研究局 | Methods for monitoring cellular states and for immortalizing mesenchymal stem cells |
| KR20180132785A (en) * | 2016-03-31 | 2018-12-12 | 샌바이오, 인크. | Medium, method, cell and secretory factor for stem cell culture and treatment |
-
2020
- 2020-07-19 CN CN202010695023.4A patent/CN111690686B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102014934A (en) * | 2008-02-22 | 2011-04-13 | 新加坡科技研究局 | Mesenchymal stem cell particles |
| CN102858997A (en) * | 2009-11-02 | 2013-01-02 | 新加坡科技研究局 | Methods for monitoring cellular states and for immortalizing mesenchymal stem cells |
| KR20180132785A (en) * | 2016-03-31 | 2018-12-12 | 샌바이오, 인크. | Medium, method, cell and secretory factor for stem cell culture and treatment |
| CN109219658A (en) * | 2016-03-31 | 2019-01-15 | 赛百奥公司 | Culture medium, method, cell and the factor of secretion cultivated and treated for stem cell |
Non-Patent Citations (2)
| Title |
|---|
| HONG ZHUANG 等: "Altered expression of microRNAs in the neuronal differentiation of human Wharton’s Jelly mesenchymal stem cells", 《NEUROSCIENCE LETTERS》 * |
| 吴家昌 等: "miRNA与细胞分化的研究进展", 《现代生物医学进展》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112391341A (en) * | 2020-11-30 | 2021-02-23 | 张川 | Application of SDF-1 protein activator in promoting human umbilical cord mesenchymal stem cell proliferation and differentiation in vitro |
| CN112481219A (en) * | 2020-11-30 | 2021-03-12 | 张川 | Application of high gene expression in promoting proliferation of human umbilical cord mesenchymal stem cells in-vitro culture |
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