CN112391341A - Application of SDF-1 protein activator in promoting human umbilical cord mesenchymal stem cell proliferation and differentiation in vitro - Google Patents

Application of SDF-1 protein activator in promoting human umbilical cord mesenchymal stem cell proliferation and differentiation in vitro Download PDF

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CN112391341A
CN112391341A CN202011370180.4A CN202011370180A CN112391341A CN 112391341 A CN112391341 A CN 112391341A CN 202011370180 A CN202011370180 A CN 202011370180A CN 112391341 A CN112391341 A CN 112391341A
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张川
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Henan Yuanchuang Life Stem Cell Bank Technology Co ltd
Zhengzhou Bain Biotechnology Co ltd
Zhengzhou Yuanchuang Gene Technology Co ltd
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Abstract

The invention discloses an application of an SDF-1 protein activator in promoting in-vitro proliferation and differentiation of human umbilical cord mesenchymal stem cells. The invention discovers that the SDF-1 protein activator has the activity of promoting the proliferation of the hUCMSCs and inducing the osteogenic differentiation of the hUCMSCs, and has concentration effect. Therefore, the SDF-1 protein activator can be used for preparing a culture medium for promoting the proliferation of the hUCMSCs and inducing the osteogenic differentiation of the hUCMSCs. The SDF-1 protein activator is N-trans-p-coumaroyl tyramine.

Description

Application of SDF-1 protein activator in promoting human umbilical cord mesenchymal stem cell proliferation and differentiation in vitro
Technical Field
The invention belongs to the field of stem cells, relates to culture and application of stem cells, and particularly relates to application of an SDF-1 protein activator for promoting in-vitro proliferation and differentiation of human umbilical cord mesenchymal stem cells.
Background
Mesenchymal Stem Cells (MSCs) are a class of adult stem cells derived from early-developing mesoderm and having multipotentiality and self-renewal potential. The sources of MSCs mainly include bone marrow, fat, umbilical cord, placenta, amnion, etc. Among them, human umbilical cord mesenchymal stem cells (hUC-MSCs) are the most potential seed cells due to the advantages of convenient material availability, wide source, low immunogenicity, no ethical dispute and the like.
Research shows that the hUC-MSCs have wide clinical application prospect and can be used for treating nervous system diseases, liver and kidney injuries, autoimmune diseases, heart diseases, orthopedic diseases, cardiovascular diseases, diabetes and the like.
The hUC-MSCs are induced to be differentiated into osteoblasts, and the hUC-MSCs can be used for treating bone related diseases by bone tissue engineering.
Disclosure of Invention
The invention aims to provide an application of an SDF-1 protein activator for promoting the in-vitro proliferation and differentiation of human umbilical cord mesenchymal stem cells, so as to induce the differentiation of hUC-MSCs into osteoblasts while amplifying the hUC-MSCs.
The technical scheme is as follows:
use of an SDF-1 protein activator for promoting human umbilical cord mesenchymal stem cell proliferation and differentiation in vitro, wherein the differentiation refers to osteogenic differentiation.
Further, the SDF-1 protein activator is N-trans-p-coumaroyl tyramine.
Use of an SDF-1 protein activator for preparing a medium for promoting the in vitro proliferation and differentiation of human umbilical cord mesenchymal stem cells, wherein the differentiation refers to osteogenic differentiation.
Further, the SDF-1 protein activator is N-trans-p-coumaroyl tyramine.
The technical effects are as follows:
the invention discovers that the N-trans-p-coumaroyl tyramine has the activity of promoting the proliferation of the hUCMSCs and inducing the osteogenic differentiation of the hUCMSCs, and has concentration effect. Therefore, the N-trans-p-coumaroyl tyramine can be used for preparing a culture medium for promoting the proliferation of the hUCMSCs and inducing the osteogenic differentiation of the hUCMSCs.
Drawings
FIG. 1 is an inverted microscope observation result of human umbilical cord mesenchymal stem cells; the visible cells are spindle-shaped and grow in a vortex shape, and the growth characteristics of the human umbilical cord mesenchymal stem cells are met.
FIG. 2 shows the results of alizarin red staining; the results show that the conventional induction group and the dosing induction group generate obvious calcium nodules, and the number of the calcium nodules of the high-concentration dosing induction group is obviously greater than that of the calcium nodules of the low-concentration dosing induction group, so that the obvious osteogenic differentiation is generated.
FIG. 3 shows the Western blot detection results; compared with a normal control group, the SDF-1 protein of the drug-added culture group containing 2 mu M or 5 mu M N-trans-p-coumaroyl tyramine is obviously increased, and the higher the concentration of the N-trans-p-coumaroyl tyramine is, the higher the SDF-1 protein content is. SDF-1 is known to be involved in cell proliferation, and the proliferation promoting effect of N-trans-p-coumaroyl tyramine on hUCMSCs is presumed to be involved in activating SDF-1 protein expression.
Detailed Description
First, experimental material
Human umbilical cord mesenchymal stem cells were purchased from Shanghai-passed autumn Biotechnology Ltd.
Fetal bovine serum, DMEM/F12(1:1) medium was purchased from Gibco, USA.
N-trans-p-coumaroyl tyramine is purchased from Dorper method, and the purity is 95-99%.
Second, Experimental methods
1. Culture, passage, morphological observation and phenotype detection of human umbilical cord mesenchymal stem cells
Recovering cryopreserved human umbilical cord mesenchymal stem cells by a conventional method, and culturing in a T25 culture flask with DMEM/F12 culture medium containing 10% fetal calf serum under the conditions of 37 ℃ and 5% CO2And saturated humidity. Changing the solution for 2-3 days, and carrying out passage when the cells grow to be full of 80-90% of the bottle bottom area. The passage ratio is 1:5, and the method comprises the following steps: placing 0.25% pancreatin-0.53 mM EDTA digestive juice and DPBS at 37 ℃ for preheating, pouring out the culture medium in a culture bottle, adding 3-5mLDPBS into the culture bottle, and removing after light shaking and washing. The pancreatin was diluted 4-fold with DPBS, 2mL of diluted pancreatin was added to the flask, incubated at room temperature for digestion, and after digestion was completed, 2mL of complete medium containing serum was added to stop digestion. Gently blowing the cells on the bottle wall by using a pipette gun to completely shed the cells, then collecting the cell suspension and blowing the cell suspension into single cell suspension, centrifuging at 1200rpm for 5min, removing the supernatant, adding the complete culture medium to suspend the cells, and carrying out passage.
And taking the human umbilical cord mesenchymal stem cells with good growth state after passage for subsequent experiments.
And (3) morphology observation: and (5) observing the form and growth condition of the human umbilical cord mesenchymal stem cells under an inverted microscope, and taking a picture.
And (3) phenotype detection: human umbilical cord mesenchymal stem cells are digested by 0.25% pancreatin and resuspended by PBS to prepare 1 × 106Cell suspension/mL. Then 100 mu L of cell suspension is taken and incubated with 20 mu L of labeled primary antibody (CD73-PE, CD90-FITC, CD105-APC, CD34-PE, CD45-APC, HLA-DR-FITC) for 1h at 4 ℃ in the dark, and the positive rate of the cell phenotype molecules is detected by an up-flow cytometer.
2. Cell proliferation Activity assay
Taking hUCMSCs with good growth state, preparing cell suspension with DMEM/F12 medium containing 10% fetal calf serum, and culturing at a rate of 1 × 10 per well4Inoculating each cell in 96-well culture plate, and dividing into normal control group and drug-adding culture group, each group having 5 multiple wells, 37 deg.C, 5% CO2After the culture for 24h at saturation humidity, the drug-adding culture group is replaced by DMEM/F12 culture medium containing 10% fetal calf serum and 2 mu M or 5 mu M N-trans-p-coumaroyl tyramine for continuous culture, and the normal control group is replaced by DMEM/F12 culture medium containing 10% fetal calf serum for continuous culture. After the culture is continued for 48h, 10 mu L of CCK8 reagent is added into each well, after incubation for 4h, the OD450 value of each well is measured at the wavelength of 450nm of an enzyme-linked immunosorbent assay, the average value of each well is taken, the cell proliferation activity of the drug-added culture group is calculated according to the following formula by taking the cell proliferation activity corresponding to the absorbance value of a normal control group as 100%.
Cell proliferation activity (%) ═ OD dosing culture group/OD normal control group × 100%.
4. Osteogenic differentiation assay
Taking hUCMSCs with good growth state, preparing cell suspension with DMEM/F12 medium containing 10% fetal calf serum, and culturing at a rate of 2 × 10 per well5The individual cells were seeded in 24-well plates at 37 ℃ with 5% CO2And after the culture is carried out for 24 hours under the saturated humidity, continuously culturing according to the following groups, and replacing the corresponding culture medium every 2-3 days.
Normal control group: the culture was continued by changing to DMEM/F12 medium containing 10% fetal bovine serum;
conventional induction group: changing to DMEM/F12 medium containing 10% fetal calf serum and conventional osteogenesis inducing factor (100nM dexamethasone +10mM sodium beta-glycerophosphate +50mg/L ascorbic acid) for continuous culture;
adding a medicine induction group: the culture was continued by changing to DMEM/F12 medium containing 10% fetal bovine serum, conventional osteogenic inducing factor (100nM dexamethasone +10mM sodium beta-glycerophosphate +50mg/L ascorbic acid) and 2. mu.M or 5. mu. M N-trans-p-coumaroyl tyramide.
And after continuously culturing for 9d, detecting the generation condition of each group of calcium nodules by adopting an alizarin red staining method, and photographing.
5. SDF-1 protein expression assay
Taking hUCMSCs with good growth state, preparing cell suspension with DMEM/F12 medium containing 10% fetal calf serum, and culturing at a rate of 2 × 10 per well5Inoculating each cell in 24-well culture plate, and dividing into normal control group and drug-adding culture group, each group having 5 multiple wells, 37 deg.C, 5% CO2After the culture for 24h at saturation humidity, the drug-adding culture group is replaced by DMEM/F12 culture medium containing 10% fetal calf serum and 2 mu M or 5 mu M N-trans-p-coumaroyl tyramine for continuous culture, and the normal control group is replaced by DMEM/F12 culture medium containing 10% fetal calf serum for continuous culture. After an additional 48h incubation, cells were collected, washed with PBS, lysed in lysates, and total protein was measured using BCA method. Separating the protein sample by SDS-PAGE gel electrophoresis to obtain the same amount of total protein, transferring to polyvinylidene fluoride membrane, sealing with 5% defatted milk-containing sealing solution, adding anti-SDF-1 and GAPDH primary antibody, shaking overnight at 4 deg.C, adding HRP-labeled secondary antibody, incubating at room temperature for 1h, developing by chemiluminescence method, and collecting the image by gel imager. The bands were scanned and quantified using Image J analysis.
6. Statistical treatment
Data were statistically processed using GraphPad Prism 5.0 software, expressed as x ± s, and comparisons between groups were by t-test, with P < 0.05 indicating significant differences.
Third, experimental results
1. Morphology observation and phenotype detection results of human umbilical cord mesenchymal stem cells
The observation result of the inverted microscope is shown in figure 1, the cells grow in a spindle shape and vortex shape, and the growth characteristics of the human umbilical cord mesenchymal stem cells are met. The results of the phenotypic detection are shown in table 1, the expression levels of CD34, CD45 and HLA-DR are very low, and the expression levels of CD73, CD90 and CD105 are very high, which are in line with the phenotypic characteristics of human umbilical cord mesenchymal stem cells.
TABLE 1 molecular Positive rates for cell phenotypes
Phenotype of cellsRate of positivity of seed Positive rate (%)
CD73 98.5
CD90 98.8
CD105 98.2
CD34 2.73
CD45 1.69
HLA-DR 0.87
2. Cell proliferation Activity assay results
As shown in Table 2, the cell growth activity of each group was higher in the drug-added culture group containing 2. mu.M or 5. mu. M N-trans-p-coumaroyl tyramine than in the normal control group.
TABLE 1 cell proliferation Activity of each group
Cell proliferation Activity
Normal control group (100.00±0.73)％
Medicine culture group (2 μ M) (175.28±1.15)％
Medicine culture group (5 mu M) (283.95±1.42)％
As can be seen from the above table, N-trans-p-coumaroyltyramine has a concentration-dependent effect on the proliferation-promoting activity of hUCMSCs.
3. Results of osteogenic differentiation assay
The results of osteogenic differentiation tests show that the conventional induction group and the drug-adding induction group both generate obvious calcium nodules, and the number of the calcium nodules in the high-concentration drug-adding induction group is obviously greater than that in the low-concentration drug-adding induction group (as shown in figure 2), so that the osteogenic differentiation is obvious. No obvious calcium nodules were detected in the normal control group.
The formation of mineralized nodules is the final stage of osteogenic differentiation and is an important marker of osteogenic differentiation in vitro. The results indicate that N-trans-p-coumaroyl tyramine has the effect of inducing osteogenic differentiation of hUCMSCs, and shows a dose-dependent effect.
4. SDF-1 protein expression test results
As shown in FIG. 3, the results of Western blot analysis show that SDF-1 protein in the drug-added culture group containing 2. mu.M or 5. mu. M N-trans-p-coumaroyl tyramine is significantly increased, and the higher the concentration of N-trans-p-coumaroyl tyramine is, the higher the SDF-1 protein content is. SDF-1 is known to be involved in cell proliferation, and the proliferation promoting effect of N-trans-p-coumaroyl tyramine on hUCMSCs is presumed to be involved in activating SDF-1 protein expression.

Claims (4)

1. Use of an SDF-1 protein activator for promoting human umbilical cord mesenchymal stem cell proliferation and differentiation in vitro, wherein the differentiation refers to osteogenic differentiation.
2. The use according to claim 1, wherein the SDF-1 protein activator is N-trans-p-coumaroyl tyramine.
3. Use of an SDF-1 protein activator for preparing a medium for promoting the in vitro proliferation and differentiation of human umbilical cord mesenchymal stem cells, wherein the differentiation refers to osteogenic differentiation.
4. The use according to claim 2, wherein the SDF-1 protein activator is N-trans-p-coumaroyl tyramine.
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