CN115386547A - Application of oligosaccharide in promoting proliferation of hUC-MSCs and preparing corresponding culture medium - Google Patents
Application of oligosaccharide in promoting proliferation of hUC-MSCs and preparing corresponding culture medium Download PDFInfo
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Abstract
The invention discloses application of oligosaccharide in promoting proliferation of hUC-MSCs and preparation of corresponding culture media. MTT experiments and clone formation experiments prove that the new qiong tetrasaccharide can effectively promote the proliferation of the hUC-MSCs, and the higher the concentration of the new qiong tetrasaccharide is, the stronger the proliferation promoting effect on the hUC-MSCs is. The invention further proves that the stem cell characteristics of the hUC-MSCs are not obviously reduced compared with the conventional culture by the intervention culture of the neoagarotetraose through a Westernblot experiment. In the prior art, no report is provided on the influence of neoagarotetraose on the in vitro proliferation of hUC-MSCs.
Description
Technical Field
The invention belongs to the field of stem cells, and relates to application of oligosaccharide in promoting proliferation of hUC-MSCs and preparing a corresponding culture medium.
Background
Umbilical cord mesenchymal stem cells (UC-MSCs) are a pluripotent stem cell that is present in umbilical cord tissue of a newborn and can continue to proliferate and differentiate into one or more cell types under specific conditions. The UC-MSCs have the advantages of easy acquisition, separation, culture, purification and the like. After multiple passages and amplification, the UC-MSCs can still maintain the original cell phenotype and differentiation characteristics. The human umbilical cord mesenchymal stem cells (hUC-MSCs) have the characteristics of non-invasive acquisition property and low immunogenicity, so that the human umbilical cord mesenchymal stem cells have unique advantages in clinical application. In recent years, the hUC-MSCs have been widely used in clinical disease treatment and some progress has been made. For example, the hUC-MSCs can target inflammatory tissues, regulate excessive immunity and inflammation, play an anti-inflammatory role, have the functions of promoting tissue repair and the like, and have wide application prospects in treatment of refractory diseases such as AID, NS, metabolic diseases, CVDs and the like.
Agar oligosaccharide as a novel marine functional oligosaccharide has biological activities of resisting tumor, inflammation and oxidation, whitening, moisturizing and the like, and becomes a hot point of domestic and foreign researches. Agar oligosaccharides can be classified into Agar Oligosaccharides (AOs) and neo-agar oligosaccharides (NAOs) according to the difference in reducing ends. Neoagarotetraose is one of NAOs.
In the prior art, no report is reported on the influence of neoagarotetraose on the in-vitro proliferation of hUC-MSCs.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides the application of oligosaccharide in promoting the proliferation of hUC-MSCs and preparing corresponding culture media so as to improve the proliferation efficiency of the hUC-MSCs.
The purpose of the invention is realized by the following technical scheme:
an oligosaccharide is used for promoting in-vitro proliferation of hUC-MSCs, and the oligosaccharide is neoqiron tetrasaccharide. In a specific embodiment, as can be seen from the a values in table 1, the 20 μ MA value > 10 μ MA value > 0 μ MA value, which indicates that the neoqiong tetrasaccharide effectively promotes the proliferation of the hUC-MSCs, and the higher the concentration of the neoqiong tetrasaccharide is, the stronger the proliferation promoting effect on the hUC-MSCs is; as can be seen from the graph C in FIG. 1, the clone number of 20. Mu.M > 10. Mu.M > 0. Mu.M indicates that the neoagarotetraose effectively promotes the proliferation of the hUC-MSCs, and the higher the concentration of the neoagarotetraose, the stronger the proliferation promoting effect on the hUC-MSCs.
An application of oligosaccharide in preparing a culture medium for promoting the proliferation of hUC-MSCs, wherein the oligosaccharide is neoagarotetraose.
Preferably, the culture medium comprises a basic culture medium and an active ingredient, wherein the active ingredient is neoagarotetraose.
Preferably, the medium further comprises fetal bovine serum.
Preferably, the medium further comprises streptomycin.
Preferably, the basal medium is DMEM/F12 medium.
A hUC-MSCs proliferation culture medium is prepared by adding neoagarotetraose into a basal culture medium DMEM/F12 culture medium. Furthermore, the proliferation medium may be supplemented with fetal calf serum and streptomycin, or may be supplemented with fetal calf serum and streptomycin into DMEM/F12 medium containing neoagarotetraose at the time of use.
The invention has the following beneficial effects:
MTT experiments and clone formation experiments prove that the neoagarotetraose can effectively promote the proliferation of the hUC-MSCs, and the higher the concentration of the neoagarotetraose is, the stronger the proliferation promoting effect on the hUC-MSCs is. The invention further proves that the stem cell characteristics of the hUC-MSCs are not obviously reduced compared with the conventional culture by the intervention culture of the neoagarotetraose through a Westernblot experiment.
Drawings
In fig. 1: a is CD105, CD90, CD73, CD34 and CD45 expression determined by flow cytometry, CD105, CD90 and CD73 expression is positive, and CD34 and CD45 expression is negative; b is the optical density (A) value at 490nm wavelength measured by the microplate reader, 20 mu MA value is more than 10 mu MA value is more than 0 mu MA value; c is the clone formation result, the 20 mu M clone number is more than 10 mu M clone number is more than 0 mu M clone number; d is expression levels of the dry transcription factors Nanog, oct4 and Sox2 measured by Westernblot, and the expression levels of the dry transcription factors Nanog, oct4 and Sox2 in hUC-MSCs cultured by intervention of 10 mu M and 20 mu M neoqiong tetrasaccharide and cultured by intervention of 0 mu M neoqiong tetrasaccharide are not obviously different.
Detailed Description
In order to better illustrate the invention, the following detailed description of the invention with specific examples, but it should be noted that the scope of the invention is not limited thereby.
Example 1:
1. experimental materials
DMEM/F12 medium was purchased from Gibco.
Fetal Bovine Serum (FBS) was purchased from Gibco.
The diabody was purchased from Sigma-Aldrich.
Pancreatin was purchased from Sigma-Aldrich.
Neoagarotetraose (HPLC. Gtoreq.98%) was purchased from a source leafy organism.
MTT was purchased from Sigma-Aldrich.
Crystal Violet Sigma-Aldrich.
The primary antibody and the secondary antibody are purchased from Wuhan Boohot Biotech limited.
2. Experimental methods
1. hUC-MSCs separation and culture
Collecting umbilical cord specimen of parturient with normal childbirth, washing with PBS to remove umbilical artery, vein and amnion to obtain Wharton's jelly, shearing into pieces of about 1mm 3 The size of the tissue mass, inoculated into a culture dish, cultured with DMEM/F12 containing 10% FBS, 1% double antibody at 37 deg.C and 5% CO 2 Culturing in a saturated humidity incubator, and inoculating to 25cm when the cell fusion degree reaches about 85% 2 And (4) changing the culture solution for 1 time every 2-3 days in a culture bottle, and carrying out trypsinization passage when the cell fusion reaches 80%. Taking 3 rd generation cells, resuspending, observing cell morphology under an inverted microscope, and detecting CD105, CD90, CD73, CD34 and CD45 by flow cytometry.
2. MTT detection of hUC-MSCs proliferation
Collecting the 3 rd generation hUC-MSCs with good growth state, trypsinizing, re-suspending with 10% FBS-containing 1% double antibody DMEM/F12 medium (complete medium), and adjusting cell density to 5 × 10 4 After each mL, the cells were plated in 96-well plates at 100. Mu.L/well. After 12h, 100 μ L of complete medium containing neoagarotetraose with different concentrations was added to each well, so that the final concentration of neoagarotetraose was 0 μ M, 10 μ M, 20 μ M, and 5 wells per concentration. After further culturing for 48 hours, 20. Mu.L of a new preparation 0.5% MTT solution was added to each well, the mixture was allowed to stand for 4 hours, the medium was discarded, PBS was washed, 150. Mu.L of MSO solution was added to each well, the mixture was shaken for 15 minutes to dissolve the crystals sufficiently, the mixture was allowed to stand for 2 hours, the supernatant was taken, and the optical density (A) value at a wavelength of 490nm was measured by an enzyme-linked microplate reader.
3. Cloning formation detection of hUC-MSCs proliferation
Collecting 3 rd generation hUC-MSCs with good growth state, trypsinizing, resuspending with DMEM/F12 medium (complete medium) containing 10% FBS and 1% double antibody, and adding into the medium at 2 × 10% 3 One/well was inoculated into 12-well plates, 1mL per well. After 24h, 1mL of complete medium containing neoagarotetraose at different concentrations was added to each well to give final neoagarotetraose concentrations of 0. Mu.M, 10. Mu.M, and 20. Mu.M, 3 more wells per concentration, and the concentration was 5% CO at 37 ℃ in each well 2 And culturing in a saturated humidity incubator. After continuing to culture for 9 days (replacing the complete culture containing the neoagarotetraose with the corresponding concentration every 2 to 3 days)And (3) removing supernatant, washing with PBS (phosphate buffer solution) for 3 times, fixing with absolute ethyl alcohol for 30min, removing fixing liquid, dyeing with 10mg/mL crystal violet dye for 30min, washing with PBS for 3 times, air-drying, and observing and photographing to compare the clone number of different concentration groups of neoagarotetraose.
4. Westernblot detection of dryness of hUC-MSCs
Collecting 3 rd generation hUC-MSCs with good growth state, trypsinizing, resuspending with DMEM/F12 medium (complete medium) containing 10% FBS and 1% double antibody, and adding into the medium at 2 × 10% 3 One/well was inoculated to 12-well plates, 1mL per well. After 24h, 1mL of complete medium containing neoagarotetraose at different concentrations was added to each well to give final neoagarotetraose concentrations of 0. Mu.M, 10. Mu.M, and 20. Mu.M, 3 more wells per concentration, and the concentration was 5% CO at 37 ℃ in each well 2 And culturing in a saturated humidity incubator. After continuing culturing for 9d (changing the complete medium containing neoagarotetraose with the corresponding concentration every 2-3 d), collecting cells, washing with PBS, lysing, carefully aspirating the supernatant, and quantifying with a BCA kit. Samples of 35. Mu.g of total protein were each subjected to 10-% (w/w) SDS polyacrylamide gel electrophoresis, and transferred onto a PVDF membrane. After the membrane transfer is finished, 50g/L of skimmed milk powder is sealed for 2h at room temperature, nanog, oct4, sox2 and GAPDH (internal reference) are added to dilute the primary antibody, incubated overnight at 4 ℃, rinsed by TBTS, added with diluted secondary antibody, incubated for 2h at normal temperature, rinsed by TBTS, added with ECL luminescent solution for development and photographed.
5. Statistical analysis
All data are expressed by means of the mean +/-standard deviation (x +/-s), the ANOVA analysis method is adopted for comparison among multiple groups, the t test method is adopted for comparison of differences between the two groups, the difference is obvious when P is less than 0.05, and statistical analysis of related data is carried out by using SPSS 20.0.
3. Results of the experiment
1. hUC-MSCs separation and culture
Observing the fusiform vortex characteristic of the cells under an inverted microscope; flow cytometry showed positive expression of CD105, CD90, CD73, and negative expression of CD34, CD45 (panel a in fig. 1). This indicates that the hUC-MSCs have been successfully isolated and cultured.
2. Proliferation of hUC-MSCs
The optical density (A) at 490nm, measured by the microplate reader, is shown in Table 1 and B in FIG. 1, and the A value is proportional to the number of cells. As can be seen from the A values in Table 1, the 20 mu MA value is greater than the 10 mu MA value and is greater than the 0 mu MA value, which indicates that the neoqiong tetrasaccharide can effectively promote the proliferation of the hUC-MSCs, and the higher the concentration of the neoqiong tetrasaccharide is, the stronger the proliferation promoting effect on the hUC-MSCs is.
TABLE 1 optical density (A) at 490nm wavelength
| Concentration of | Value of A | Significance analysis |
| 0μM | 0.683±0.022 | / |
| 10μM | 0.949±0.027 | V.S.0μM，P＜0.05 |
| 20μM | 1.371±0.024 | V.S.0μM，P＜0.05 |
The results of clone formation are compared in panel C of FIG. 1. As can be seen from the graph C in FIG. 1, the clone number of 20. Mu.M is greater than that of 10. Mu.M and that of 0. Mu.M, which indicates that the neoagarotetraose effectively promotes the proliferation of the hUC-MSCs, and the higher the concentration of the neoagarotetraose, the stronger the proliferation promoting effect on the hUC-MSCs.
3. Dry hUC-MSCs
The dry transcription factors Nanog, oct4 and Sox2 are important markers for characterizing the characteristics of the hUC-MSCs stem cells, and whether the hUC-MSCs still maintain good stem cell characteristics can be semi-quantitatively analyzed by tracking the changes of the expression levels of Nanog, oct4 and Sox 2. The results of the expression levels of Nanog, oct4, and Sox2 in the Westernblot assay are shown in FIG. 1, panel D. As can be seen from the graph D in FIG. 1, the expression levels of the dry transcription factors Nanog, oct4 and Sox2 in hUC-MSCs cultured with 10. Mu.M and 20. Mu.M of neoagarotetraose intervention culture and 0. Mu.M of neoagarotetraose intervention culture are not obviously different. This indicates that the stem cell characteristics of the hUC-MSCs are not significantly reduced compared with conventional culture while the proliferation of the hUC-MSCs is promoted by the intervention culture of neoagarotetraose.
Example 2:
a hUC-MSCs proliferation culture medium is prepared by adding neoagarotetraose into a basal culture medium DMEM/F12 culture medium. Furthermore, the proliferation medium may be supplemented with fetal calf serum and streptomycin, or may be supplemented with fetal calf serum and streptomycin into DMEM/F12 medium containing neoagarotetraose at the time of use.
The above-described embodiments are intended to illustrate the material of the invention in detail, but it should be emphasized that those skilled in the art should not limit the scope of the invention to the above-described specific embodiments.
Claims (6)
1. An application of oligosaccharide in promoting in-vitro proliferation of hUC-MSCs, wherein the oligosaccharide is neoagarotetraose.
2. An oligosaccharide is an application of a culture medium for promoting the proliferation of hUC-MSCs, wherein the oligosaccharide is neoagarotetraose.
3. Use according to claim 2, characterized in that: the culture medium comprises a basic culture medium and an active ingredient, wherein the active ingredient is neoagarotetraose.
4. Use according to claim 3, characterized in that: the culture medium also includes fetal bovine serum.
5. Use according to claim 3, characterized in that: the culture medium further comprises streptomycin.
6. Use according to any one of claims 3 to 5, characterized in that: the basic culture medium is DMEM/F12 culture medium.
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