Application of an oligosaccharide in promoting the proliferation of hUC-MSCs and preparing corresponding culture medium

Abstract

The present invention discloses an application of oligosaccharide in promoting the proliferation of hUC‑MSCs and preparing corresponding culture medium. The present invention proves through MTT experiment and clone formation experiment that neoagarotetrose can effectively promote the proliferation of hUC‑MSCs, and the higher the concentration of neoagarotetrose, the stronger the proliferation-promoting effect on hUC‑MSCs. The present invention further proves through Western blot experiment that while neoagarotetrose intervention culture promotes the proliferation of hUC‑MSCs, it does not significantly reduce the stem cell characteristics of hUC‑MSCs compared with conventional culture. There is no report on the effect of neoagarotetrose on the in vitro proliferation of hUC‑MSCs in the prior art.

Description

Technical Field

[0001] The invention belongs to the field of stem cells and relates to the application of oligosaccharides in promoting the proliferation of hUC-MSCs and preparing corresponding culture media. Background Art

[0002] Umbilical cord mesenchymal stem cells (UC-MSCs) are multifunctional stem cells found in the umbilical cord tissue of newborns, which can continue to proliferate and differentiate into one or more cell types under specific conditions. UC-MSCs have the advantages of being easy to obtain, isolate, culture and purify. After multiple passages and expansions, UC-MSCs can still maintain their original cell phenotype and differentiation characteristics. Human umbilical cord mesenchymal stem cells (hUC-MSCs) have the characteristics of non-invasive acquisition and low immunogenicity, which give them unique advantages in clinical applications. In recent years, hUC-MSCs have been widely used in the treatment of clinical diseases and have made some progress. For example, hUC-MSCs can target inflammatory tissues, regulate excessive immunity and inflammation, exert anti-inflammatory effects, and have functions such as promoting tissue repair. They have broad application prospects in the treatment of refractory diseases such as AID, NS, metabolic diseases, and CVDs.

[0003] As a new type of marine functional oligosaccharide, agar oligosaccharide has biological activities such as anti-tumor, anti-inflammatory, antioxidant, whitening and moisturizing, and has become a hot topic of research at home and abroad. According to the different reducing ends, agar oligosaccharides can be divided into agar oligosaccharides (AOs) and neo-agar oligosaccharides (NAOs). Neo-agarotetrose is a kind of NAOs.

[0004] There is no report in the prior art on the effect of neoagarotetrose on the proliferation of hUC-MSCs in vitro. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of an oligosaccharide in promoting the proliferation of hUC-MSCs and preparing a corresponding culture medium, so as to improve the proliferation efficiency of hUC-MSCs.

[0006] The object of the present invention is achieved through the following technical solutions:

[0007] An oligosaccharide is used to promote the proliferation of hUC-MSCs in vitro, wherein the oligosaccharide is neoagarotetrose. In a specific embodiment, it can be seen from the A value in Table 1 that 20μMA value>10μMA value>0μMA value, indicating that neoagarotetrose effectively promotes the proliferation of hUC-MSCs, and the higher the concentration of neoagarotetrose, the stronger the proliferation promoting effect on hUC-MSCs; Figure 1 As can be seen from Figure C, the number of clones at 20 μM is greater than the number of clones at 10 μM and greater than the number of clones at 0 μM, indicating that neoagarotetrose effectively promotes the proliferation of hUC-MSCs, and the higher the concentration of neoagarotetrose, the stronger the proliferation-promoting effect on hUC-MSCs.

[0008] The invention discloses an application of an oligosaccharide in preparing a culture medium for promoting the proliferation of hUC-MSCs, wherein the oligosaccharide is neoagarotetrose.

[0009] Preferably, the culture medium comprises a basal culture medium and an active ingredient, and the active ingredient is neoagarotetrose.

[0010] Preferably, the culture medium further comprises fetal bovine serum.

[0011] Preferably, the culture medium further comprises penicillin-streptomycin.

[0012] Preferably, the basal culture medium is DMEM / F12 culture medium.

[0013] A hUC-MSCs proliferation medium is prepared by adding neoagarotetrose to a basic medium DMEM / F12 medium. Furthermore, fetal bovine serum and penicillin-streptomycin can be pre-added to the proliferation medium, or the fetal bovine serum and penicillin-streptomycin can be added to the DMEM / F12 medium containing neoagarotetrose before use.

[0014] Beneficial effects achieved by the present invention:

[0015] The present invention proves through MTT experiment and clone formation experiment that neoagarotetrose can effectively promote the proliferation of hUC-MSCs, and the higher the concentration of neoagarotetrose, the stronger the promoting effect on the proliferation of hUC-MSCs. The present invention further proves through Western blot experiment that neoagarotetrose intervention culture promotes the proliferation of hUC-MSCs while not significantly reducing the stem cell characteristics of hUC-MSCs compared with conventional culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Middle: A is the expression of CD105, CD90, CD73, CD34, and CD45 determined by flow cytometry, with CD105, CD90, and CD73 being positive, and CD34 and CD45 being negative; B is the optical density (A) value at a wavelength of 490 nm determined by an ELISA reader, with 20μM A value > 10μM A value > 0μM A value; C is the result of clone formation, with the number of 20μM clones > the number of 10μM clones > the number of 0μM clones; D is the expression levels of stemness transcription factors Nanog, Oct4, and Sox2 determined by Western blot, with no significant differences in the expression levels of stemness transcription factors Nanog, Oct4, and Sox2 in hUC-MSCs cultured with 10μM, 20μM neoaganetetrose intervention and 0μM neoaganetetetrose intervention. DETAILED DESCRIPTION

[0017] In order to better introduce the present invention, the essential contents of the present invention are described in detail below through specific embodiments, but it should be noted that the protection scope of the present invention is not limited thereto.

[0018] Embodiment 1:

[0019] 1. Experimental Materials

[0020] DMEM / F12 culture medium was purchased from Gibco.

[0021] Fetal bovine serum (FBS) was purchased from Gibco.

[0022] Double antibody was purchased from Sigma-Aldrich.

[0023] Trypsin was purchased from Sigma-Aldrich.

[0024] Neoagarotetrose (HPLC≥98%) was purchased from Yuanye Biotechnology.

[0025] MTT was purchased from Sigma-Aldrich.

[0026] Crystal violet Sigma-Aldrich.

[0027] The primary and secondary antibodies were purchased from Wuhan Biotech Biotechnology Co., Ltd.

[0028] 2. Experimental Methods

[0029] 1. Isolation and culture of hUC-MSCs

[0030] Umbilical cord specimens were collected from normal parturients, washed with PBS to remove the umbilical artery and vein and amniotic membrane, and Wharton's jelly was obtained and cut into pieces of about 1 mm 3 Tissue blocks of different sizes were inoculated in a culture dish and cultured in DMEM / F12 medium containing 10% FBS and 1% double antibody at 37°C and 5% CO. 2 , cultured in a saturated humidity incubator, and inoculated in 25cm 2 In the culture flask, the medium was changed every 2 to 3 days, and the cells were digested with trypsin and passaged when the cell confluence reached 80%. The third generation cells were resuspended and the cell morphology was observed under an inverted microscope, and CD105, CD90, CD73, CD34, and CD45 were detected by flow cytometry.

[0031] 2. MTT assay for hUC-MSCs proliferation

[0032] The third generation hUC-MSCs with good growth status were taken, digested with trypsin, and resuspended in DMEM / F12 medium (complete medium) containing 10% FBS and 1% double antibody to adjust the cell density to 5×10 4After 100 μL / mL, the cells were plated in a 96-well plate, with 100 μL per well. After 12 hours, 100 μL of complete culture medium containing different concentrations of neoagarotetrose was added to each well, so that the final concentrations of neoagarotetrose were 0 μM, 10 μM, and 20 μM, respectively, with 5 replicate wells for each concentration. After 48 hours of continuous culture, 20 μL of freshly prepared 0.5% MTT solution was added to each well, and the cells were allowed to stand for 4 hours. The culture medium was discarded, and the cells were rinsed with PBS. 150 μL of DMSO solution was added to each well, and the cells were shaken for 15 minutes to fully dissolve the crystals. The cells were allowed to stand for 2 hours, and the supernatant was taken. The optical density (A) value at a wavelength of 490 nm was measured by an enzyme marker.

[0033] 3. Clone formation detection of hUC-MSCs proliferation

[0034] The third generation hUC-MSCs with good growth status were obtained, digested with trypsin, and resuspended in DMEM / F12 medium (complete medium) containing 10% FBS and 1% double antibody. 3 1 mL / well was inoculated into a 12-well plate. After 24 h, 1 mL of complete medium containing different concentrations of neoagarotetrose was added to each well, so that the final concentrations of neoagarotetrose were 0 μM, 10 μM, and 20 μM, respectively. Each concentration was replicated in triplicate and incubated at 37°C and 5% CO. 2 , and cultured in a saturated humidity incubator. After 9 days of continuous culture (complete medium containing corresponding concentrations of neotetraose was replaced every 2 to 3 days), the supernatant was discarded, the cells were washed 3 times with PBS, fixed with anhydrous ethanol for 30 minutes, the fixative was discarded, 10 mg / mL crystal violet stain was used for 30 minutes, the cells were washed 3 times with PBS, air-dried, and the number of clones in different neotetraose concentration groups was observed and photographed.

[0035] 4. Western blot detection of hUC-MSCs stemness

[0036] The third generation hUC-MSCs with good growth status were obtained, digested with trypsin, and resuspended in DMEM / F12 medium (complete medium) containing 10% FBS and 1% double antibody. 3 1 mL / well was inoculated into a 12-well plate. After 24 h, 1 mL of complete medium containing different concentrations of neoagarotetrose was added to each well, so that the final concentrations of neoagarotetrose were 0 μM, 10 μM, and 20 μM, respectively. Each concentration was replicated in triplicate and incubated at 37°C and 5% CO. 2, cultured in a saturated humidity incubator. After continuing to culture for 9 days (replace the complete medium containing the corresponding concentration of new agarose every 2 to 3 days), collect the cells, wash with PBS, lyse, carefully aspirate the supernatant, and quantify using a BCA kit. Take 35 μg of total protein samples, perform 10% SDS polyacrylamide gel electrophoresis, and transfer to a PVDF membrane. After the transfer, block with 50g / L skim milk powder at room temperature for 2 hours, add Nanog, Oct4, Sox2, GAPDH (internal reference) diluted primary antibodies, incubate at 4°C overnight, rinse with TBTS, add diluted secondary antibodies, incubate at room temperature for 2 hours, rinse with TBTS, add ECL luminescent solution for development, and take pictures.

[0037] 5. Statistical analysis

[0038] All data were expressed as mean ± standard deviation (x ± s). ANOVA analysis was used to compare multiple groups, and t-test was used to compare differences between two groups. P < 0.05 was considered significant. SPSS 20.0 was used for statistical analysis of relevant data.

[0039] 3. Experimental Results

[0040] 1. Isolation and culture of hUC-MSCs

[0041] The cells showed spindle-shaped vortex characteristics under an inverted microscope; flow cytometry showed positive expression of CD105, CD90, and CD73, and negative expression of CD34 and CD45 ( Figure 1 This indicates that hUC-MSCs have been successfully isolated and cultured.

[0042] 2. hUC-MSCs proliferation

[0043] The optical density (A) values ​​at 490 nm measured by the microplate reader are shown in Table 1 and Figure 1 In Figure B, the A value is directly proportional to the number of cells. From the A values ​​in Table 1, it can be seen that 20μMA value>10μMA value>0μMA value, indicating that neoagarotetrose effectively promotes the proliferation of hUC-MSCs, and the higher the concentration of neoagarotetrose, the stronger the proliferation-promoting effect on hUC-MSCs.

[0044] Table 1 Optical density (A) values ​​at 490nm wavelength

[0045] concentration A Value Significance analysis 0μM 0.683±0.022 / 10μM 0.949±0.027 VS0μM, P<0.05 20μM 1.371±0.024 VS0μM, P<0.05

[0046] The results of clone formation are compared in Figure 1 Figure C. Figure 1As can be seen from Figure C, the number of clones at 20 μM is greater than the number of clones at 10 μM and greater than the number of clones at 0 μM, indicating that neoagarotetrose effectively promotes the proliferation of hUC-MSCs, and the higher the concentration of neoagarotetrose, the stronger the proliferation-promoting effect on hUC-MSCs.

[0047] 3. Stemness of hUC-MSCs

[0048] Stemness transcription factors Nanog, Oct4, and Sox2 are important markers for hUC-MSCs stem cell characteristics. By tracking the changes in Nanog, Oct4, and Sox2 expression levels, we can semi-quantitatively analyze whether hUC-MSCs still maintain good stem cell characteristics. The results of Western blot determination of Nanog, Oct4, and Sox2 expression levels are shown in Figure 1 Figure D. Figure 1 As shown in Figure D, there is no significant difference in the expression levels of stemness transcription factors Nanog, Oct4, and Sox2 in hUC-MSCs cultured with 10μM, 20μM neoagaretose, and 0μM neoagaretose. This indicates that neoagaretose intervention culture promotes the proliferation of hUC-MSCs while not significantly reducing the stem cell characteristics of hUC-MSCs compared with conventional culture.

[0049] Embodiment 2:

[0050] A hUC-MSCs proliferation medium is prepared by adding neoagarotetrose to a basic medium DMEM / F12 medium. Furthermore, fetal bovine serum and penicillin-streptomycin can be pre-added to the proliferation medium, or the fetal bovine serum and penicillin-streptomycin can be added to the DMEM / F12 medium containing neoagarotetrose before use.

[0051] The purpose of the above embodiments is to elaborate on the essential content of the present invention, but it should be emphasized that those skilled in the art should not limit the protection scope of the present invention to the above specific embodiments.

Claims

1. Use of an oligosaccharide in promoting the proliferation of hUC-MSCs in vitro without reducing the stem cell properties of hUC-MSCs, wherein the oligosaccharide is neoagarotetrose.

2. Use of an oligosaccharide in preparing a culture medium that promotes the proliferation of hUC-MSCs without reducing the stem cell characteristics of hUC-MSCs, wherein the oligosaccharide is neoagarotetrose.

3. The use according to claim 2, characterized in that: The culture medium comprises a basal culture medium and an active component, and the active component is neoagarotetrose.

4. The use according to claim 3, characterized in that: The culture medium also includes fetal bovine serum.

5. The use according to claim 3, characterized in that: The culture medium also includes penicillin-streptomycin.

6. The use according to any one of claims 3 to 5, characterized in that: The basic culture medium was DMEM / F12 medium.

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