CN112592891B - Application of uralensin in-vitro promotion of human adipose-derived mesenchymal stem cell osteogenic differentiation - Google Patents
Application of uralensin in-vitro promotion of human adipose-derived mesenchymal stem cell osteogenic differentiation Download PDFInfo
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- CN112592891B CN112592891B CN202011504331.0A CN202011504331A CN112592891B CN 112592891 B CN112592891 B CN 112592891B CN 202011504331 A CN202011504331 A CN 202011504331A CN 112592891 B CN112592891 B CN 112592891B
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Abstract
The invention discloses an application of uralensin in promoting human adipose-derived mesenchymal stem cells to osteogenic differentiation in vitro. The invention discovers that uralensin has the effect of promoting the osteogenic differentiation of hASCs, can be used for promoting the osteogenic differentiation of human adipose-derived mesenchymal stem cells in vitro, and is used for preparing an in vitro culture medium for promoting the osteogenic differentiation of human adipose-derived mesenchymal stem cells.
Description
Technical Field
The invention belongs to the field of biology, and relates to stem cell in-vitro induced differentiation, in particular to application of uralense in-vitro promotion of human adipose-derived mesenchymal stem cell osteogenic differentiation.
Background
Tissue engineering has become one of the most promising treatments for bone defect repair. Human adipose-derived mesenchymal stem cells (hASCs) are receiving a great deal of attention in bone tissue engineering as an important source of mesenchymal stem cells. The use of inducers to culture hASCs in vitro to promote their directed differentiation is an important approach to obtaining bone tissue engineering cells. However, currently, such inducers are less.
Uralenol (CAS registry number: 139163-15-8) is a relatively common compound that is found in a variety of plants and has the following chemical structure:
disclosure of Invention
The invention aims at an application of uralensin in vitro promotion of human adipose-derived mesenchymal stem cell osteogenic differentiation.
The technical scheme for achieving the purposes of the invention is as follows:
an application of uralensin in promoting human adipose-derived mesenchymal stem cells osteogenic differentiation in vitro.
Application of uralensin in preparing culture medium for promoting human adipose-derived mesenchymal stem cells osteogenic differentiation in vitro.
The use of uralensin in vitro culture of hASCs to promote differentiation into osteoblasts to obtain bone tissue engineering cells for treating bone tissue diseases.
The invention has the outstanding advantages that:
the invention discovers that uralensin has the effect of promoting the osteogenic differentiation of hASCs, can be used for promoting the osteogenic differentiation of human adipose-derived mesenchymal stem cells in vitro, and is used for preparing an in vitro culture medium for promoting the osteogenic differentiation of human adipose-derived mesenchymal stem cells.
Drawings
FIG. 1 shows the morphology of hASCs cells observed by an inverted microscope;
FIG. 2A is a chart of alizarin red staining; b is agarose gel electrophoresis diagram of PCR products; c is SDS-polyacrylamide gel electrophoresis of hASCs total protein.
Detailed Description
The following describes the essential aspects of the invention in detail with reference to examples, and for reasons of brevity, the description of the experimental procedure will not be described in great detail, but will be routine to those skilled in the art.
1. Experimental materials
L-DMEM medium was purchased from Thermo and fetal bovine serum from GIBCO.
Diabodies, alizarin red, were purchased from Sigma, trizol reagent from Invitrogen, and M-MLV reverse transcriptase from TaKaRa.
Ural alcohol was purchased from Cinnamomum biosciences Inc. of Bao Ji City with a purity of 98%.
2. Experimental method
1. Isolation, culture and identification of hASCs
Placing the subcutaneous fat suction liquid into a centrifuge tube, adding a proper amount of type I collagenase, performing shaking table digestion at a constant temperature of 37 ℃ for 1h, adding an equal volume of L-DMEM medium, stopping digestion, centrifuging at 1500r/min for 10min, and discarding the supernatant. The precipitate is blown and evenly mixed with a proper amount of L-DMEM culture medium, filtered by a 200-mesh net, the filtrate is collected, centrifuged at 1000r/min for 5min, and the supernatant is discarded. The sediment is blown and mixed with a proper amount of L-DMEM medium containing 10 percent of fetal calf serum and 1 percent of double antibodyEvenly transferring into a cell culture flask, placing in a 37 ℃ CO with the volume fraction of 5 percent 2 Conventional culture and passage in a saturated humidity incubator. The cell morphology was observed with an inverted microscope.
2. hASCs grouping and induction culture
The 3 rd generation hASCs with good growth state are divided into a control group and an induction group, wherein the control group is cultured by using an L-DMEM medium containing 10% fetal bovine serum and 1% double antibody, and the induction group is cultured by using an L-DMEM medium containing 20 mug/mL uralense alcohol, 10% fetal bovine serum and 1% double antibody.
3. Identification of osteogenic differentiation by alizarin red staining
The 3 rd generation hASCs with good growth state are prepared into cell suspension by using L-DMEM medium containing 10% of fetal calf serum and 1% of diabody, and the cell suspension is prepared into a cell suspension with the concentration of 5 multiplied by 10 3 /cm 2 Is inoculated in 24-well plate at 37deg.C with a volume fraction of 5% CO 2 Culturing in a saturated humidity incubator, and continuously culturing by replacing the culture medium according to the groups when the culture medium grows to 80% and is fused, wherein the culture medium is replaced every 3 d. After 12d incubation, the medium was discarded, washed with PBS, fixed with 40g/L paraformaldehyde at room temperature for 10min, stained with 1g/L alizarin red staining working solution at room temperature for 30min, washed with PBS, observed for calcium deposition under an inverted microscope and photographed.
4. Identification of osteogenic differentiation by RT-PCR
The 3 rd generation hASCs with good growth state are prepared into cell suspension by using L-DMEM medium containing 10% of fetal calf serum and 1% of diabody, and the cell suspension is prepared into a cell suspension with the concentration of 5 multiplied by 10 3 /cm 2 Is inoculated in 24-well plate at 37deg.C with a volume fraction of 5% CO 2 Culturing in a saturated humidity incubator, and continuously culturing by replacing the culture medium according to the groups when the culture medium grows to 80% and is fused, wherein the culture medium is replaced every 3 d. After 12d of culture, the culture medium is discarded, PBS is used for washing, trizol reagent is used for extracting total RNA in hASCs, M-MLV reverse transcriptase kit is used for converting RNA into cDNA, then PCR reaction is carried out, PCR products are subjected to agarose gel electrophoresis, and gel imaging system is used for observing and comparing the expression condition of RUNX2 and OCN mRNA of each component bone marker gene, and the reference gene is beta-actin.
5. Western blotting method for identifying osteogenic differentiation
The 3 rd generation hASCs with good growth state are prepared into cell suspension by using L-DMEM medium containing 10% of fetal calf serum and 1% of diabody, and the cell suspension is prepared into a cell suspension with the concentration of 5 multiplied by 10 3 /cm 2 Is inoculated in 24-well plate at 37deg.C with a volume fraction of 5% CO 2 Culturing in a saturated humidity incubator, and continuously culturing by replacing the culture medium according to the groups when the culture medium grows to 80% and is fused, wherein the culture medium is replaced every 3 d. After 12d incubation, the medium was discarded and washed with PBS to extract total protein from the lysate. Protein concentration was measured with BCA protein assay kit. Equal amounts of total protein were loaded onto SDS-polyacrylamide gels for electrophoresis. And after electrophoresis, taking out the separation gel, and transferring the protein on the separation gel onto the PVDF membrane by using an electrotransfer instrument. Blocking with blocking solution containing 5% BSA at room temperature for 2 hr, adding primary antibody against RUNX2, OCN and GAPDH, shaking overnight at 4deg.C, adding secondary antibody labeled with HRP, incubating at room temperature for 1 hr, developing by chemiluminescence, and imaging gel.
3. Experimental results
1. Isolation, culture and identification of hASCs
The cell morphology observation result is shown in FIG. 1, and the cells are in long fusiform and are distributed in a vortex shape, so that the cell morphology observation result accords with the growth characteristics of hASCs.
2. Alizarin red staining
The alizarin red staining results are shown in fig. 2 a, and the induced group can see obvious calcareous deposition, which is unique to osteoblasts, compared with the control group, indicating that the induced group hASCs showed obvious osteogenic differentiation.
3. RT-PCR, western blotting results
The results of RT-PCR and Western blotting are shown in FIG. 2 as B, C, respectively, and the expression levels of RUNX2 mRNA/protein and OCN mRNA/protein in the induction group are significantly increased compared with the control group, and RUNX2 and OCN are important osteogenic marker genes/proteins, which indicates that the induction group hASCs have obvious osteogenic differentiation.
In conclusion, the uralensis alcohol has the effect of promoting the osteogenic differentiation of hASCs, can be used for promoting the osteogenic differentiation of human adipose-derived mesenchymal stem cells in vitro, and is used for preparing an in vitro culture medium for promoting the osteogenic differentiation of human adipose-derived mesenchymal stem cells.
The above-described embodiments are illustrative of the essential aspects of the present invention for better explaining the present invention, but those skilled in the art should understand that the scope of the present invention should not be limited to the above-described specific embodiments.
Claims (2)
1. Application of uralensin in promoting human adipose-derived mesenchymal stem cells osteogenic differentiation in vitro.
2. Application of uralensin in preparing culture medium for promoting human adipose-derived mesenchymal stem cells osteogenic differentiation in vitro.
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