CN116023436A - High-density and high-activity proliferation method and culture medium for mesenchymal stem cells - Google Patents
High-density and high-activity proliferation method and culture medium for mesenchymal stem cells Download PDFInfo
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Abstract
The invention relates to a high-density and high-activity proliferation method and culture medium of mesenchymal stem cells. The invention obtains the active peptide M-428 which specifically promotes proliferation of human umbilical cord mesenchymal stem cells through high-throughput screening aiming at polypeptide libraries, and the polypeptide can promote the high expression of SDF-1 protein of human umbilical cord mesenchymal stem cells to promote proliferation of the stem cells through proteome comparison analysis. The polypeptide can inhibit apoptosis besides promoting cell proliferation after being used for culturing human umbilical cord mesenchymal stem cells.
Description
Technical Field
The application relates to the field of biology, in particular to a high-density and high-activity proliferation method and culture medium for mesenchymal stem cells.
Background
Stem cells are a population of primitive cells with self-proliferating and multipotent differentiation potential, and Mesenchymal Stem Cells (MSCs) are a member of the stem cell family, belonging to the category of adult stem cells. Stem cells are a population of primitive cells with self-proliferating and multipotent differentiation potential, and Mesenchymal Stem Cells (MSCs) are a member of the stem cell family, belonging to the category of adult stem cells. MSCs have the characteristic of paracrine or autocrine cytokines, and bioactive substances such as epidermal growth factors, chemotactic factors, immunoregulatory factors, nutritional factors and the like can be detected in the supernatant of stem cell culture, so that the MSCs are basic capability of acting in human bodies. After reaching the human body, the MSCs can secrete chemokines, and the chemokines and a plurality of signal paths (such as PI3K, p, ERK and the like) participate in the homing process of the MSCs and actively migrate to the target tissues through vascular endothelial cells. The tissue type of MSCs differentiation is very extensive, can differentiate into not only adipocyte, osteoblast, chondroblast, but also neuron, islet beta-like cell, liver-like cell, myocardial cell, epithelial cell, etc., participate in the tissue reconstruction effectively.
Umbilical Cord Mesenchymal Stem Cells (UCMSCs) refer to a multifunctional stem cell existing in neonatal umbilical cord tissues, can differentiate into a plurality of tissue cells, and has wide clinical application prospect. The hUCMSCs are adhered to the wall and then are in fibroblast-like cells, and the cells after passage are in a spindle shape with relatively uniform morphology. The surface markers are as high as those of MSCs from other sources, and express CD13, CD73, CD90, CD105, CD54, CD29 and CD44, HLA-ABC is expressed in a low level, CD34, CD45, CD14, CD33, CD38, CD133, HLA-DR, -DA, -DP, -DQ and the like, and meanwhile, the markers of partial human embryonic stem cells and some transcription factors (Oct-4, sox-2, nanog and the like) are expressed. It was found that huchcss can differentiate into cells with mature adipocyte structure and function, and into bone cells and chondrocytes. Many studies have found that MSCs facilitate in vitro expansion of hematopoietic stem cells, homing and engraftment of hematopoietic stem cells after transplantation, shorten the time for recovery of hematopoietic function of bone marrow after transplantation, by intercellular contact, secretion of various hematopoietic factors and production of bone marrow stroma, and have been confirmed in many animal experiments and clinical treatments; in addition, the mesenchymal stem cells can also have negative regulation and control effects on organism immunity through direct cell contact, cell factor secretion, chimeric body formation induction and immune tolerance induction, thereby opening up a new way for the generation of GVHD after the clinical treatment of the allogeneic hematopoietic stem cells. Human umbilical cord MSCs also secrete a variety of cytokines such as interleukin-6, metalloprotease-1/2 tissue inhibitor, monocyte chemotactic protein-1, growth-related oncogenes, hepatocyte growth factor, insulin-like growth factor binding protein 4, interleukin-8, etc., thereby maintaining the survival of islet-like cell mass and increasing its insulin expression level.
However, the research of huchtmscs still has a need to solve the problems, such as large differences of cell phenotypes, differentiation capacity, proliferation capacity, cell functions after long-term culture and the like in different laboratories caused by different separation methods, reagent use, culture conditions, serum use, added cell factor types, cell culture density and the like, so that the application of mesenchymal stem cells is directly affected. There is currently no unified protocol for the culture of MSCs. The following media are used: IMDM medium, LG-DMEM medium, DMEM+F12 medium, and MesencultTM medium; the serum concentrations used were also reported differently: the concentration used for LeeOK was 20%; cheng Fanjun and the like, the concentration of 2 percent and 10 percent of serum are compared to obtain a conclusion that a low-concentration serum culture system is adopted, so that multiplication and increment maintenance of MSCs are facilitated; there is no uniform knowledge of the selection of the planting density of MSCs. However, most experimental researches prove that the primary cultured umbilical cord blood MSCs are adhered to the umbilical cord blood MSCs after being cultured for 24-48 hours, form a fusiform shape about 1 week, and form clonal growth, and can be fused by more than 80% for 4-5 weeks. The stem cells cultured at first can be seen as multinucleated, flat-morphology osteoclast-like cells, and long spindle-shaped mesenchymal stem cells with higher purity and uniform morphology can be obtained after general passage to the 3 rd generation.
CN111235101A discloses a human umbilical mesenchymal stem cell culture medium and a culture method of human umbilical mesenchymal stem cells, which are mainly characterized in that sodium carboxymethylcellulose is added into the culture medium to promote the growth and adhesiveness of cells, silk fibroin is added into the culture medium to promote the proliferation and growth of cells, soybean oligopeptide, 3-picolinic acid, coenzyme Q10 and alpha-ketoglutaric acid are added for compounding, wherein the soybean oligopeptide and the coenzyme Q10 have good antioxidation, promote the metabolism of cells, improve the activity of intracellular enzymes, and the synergism of the alpha-ketoglutaric acid and 3-picolinic acid is used for regulating the metabolism of proteins, promoting the synthesis of proteins, improving the growth speed and proliferation quantity of cells and improving the performance of cell secretion growth factors. The microelements of magnesium, iron, zinc and selenium are added to provide better nutrition environment for the growth of cells and further promote the growth of cells. Increasing cellsActivity. CN108865989a provides a culture medium for umbilical mesenchymal stem cells, which comprises MEM/F12 culture medium and hedera helix extract, and the additional components comprise the following components: human serum albumin, transferrin, vascular endothelial growth factor, basic fibroblast growth factor, arginine, leukemia inhibitory factor, stem cell factor, beta-cytokine, parathyroid hormone, platelet-derived growth factor, tumor necrosis factor alpha, interleukin, erythropoietin, thrombopoietin, alpha-D-glucose, vitamin C, resveratrol, naHCO 3 Gangliosides; the Hedera helix extract is liquid, and is extracted from Hedera helix leaves by a conventional method, and the final concentration of the addition is 20-50 mg/L. However, the above patents are all complex in composition, high in use cost and unchanged in configuration. Therefore, the culture medium for umbilical cord mesenchymal stem cells is not an effective culture medium which can be quickly cultured and is convenient to prepare and effectively promote proliferation.
Disclosure of Invention
The invention obtains the active peptide M-428 which specifically promotes the proliferation of human umbilical cord mesenchymal stem cells through high-throughput screening aiming at polypeptide libraries.
Specifically, the amino acid sequence of the active peptide M-428 is CFNTIMCGALS.
Further, the polypeptide can be prepared by artificial synthesis.
Specifically, the invention provides a culture medium capable of promoting proliferation of human umbilical cord mesenchymal stem cells and reducing apoptosis, wherein the culture medium is added with the active peptide M-428 of the invention on the basis of a basic culture medium.
In particular, the invention also provides application of the active peptide M-428 in preparing a culture medium capable of promoting proliferation of human umbilical mesenchymal stem cells and reducing apoptosis.
Specifically, the culture medium is based on DMEM/F12 culture medium. Specifically, the volume ratio of DMEM basal medium to F12 basal medium is 1-3:1, preferably 1:1.
Further, the medium may be other media or reagents for stem cells as is common in the art.
Further, the stem cell culture medium comprises the following components: water-soluble non-polyelectrolyte polymers, inorganic salts, vitamins, amino acids, glucose, transferrin, insulin or insulin-like functional substitutes such as (IGF-1/2), fibroblast growth factor.
Specifically, the medium used in the present invention may contain additives known per se. The additive is not particularly limited as long as it does not inhibit proliferation of stem cells, and examples thereof include growth factors (e.g., insulin, etc.), iron sources (e.g., transferrin, etc.), polyamines (e.g., putrescine, etc.), minerals (e.g., sodium selenate, etc.), saccharides (e.g., glucose, etc.), organic acids (e.g., pyruvic acid, lactic acid, etc.), amino acids (e.g., L-glutamine, etc.), reducing agents (e.g., 2-mercaptoethanol, etc.), vitamins (e.g., ascorbic acid, d-biotin, etc.), steroids (e.g., β -estradiol, progesterone, etc.), antibiotics (e.g., streptomycin, penicillin, gentamicin, etc.), buffers (e.g., HEPES, etc.), and the like. In addition, additives conventionally used in stem cell culture may be appropriately contained. The additives are preferably contained in a concentration range known per se.
The medium used in the present invention may contain serum. The serum is not particularly limited as long as it does not inhibit proliferation of stem cells, and is preferably derived from a mammal (for example, fetal bovine serum, human serum, and the like). The concentration of serum may be within a concentration range known per se. However, it is known that the serum component also contains differentiation factors of human ES cells, etc., and that the difference between batches of serum may cause a variation in the culture result, so that the lower the serum content is, the more preferable, and the most preferable is no serum. Further, when cultured stem cells are used for medical purposes, components derived from different species may become infectious sources and heterologous antigens of blood-borne pathogens, and therefore, serum-free is preferable.
In addition, the culture medium of the present invention may be suitable for proliferation of stem cells from any animal. The stem cells that can be cultured using the medium of the present invention are, for example, rodents derived from mice, rats, hamsters, guinea pigs, etc.; rabbit, etc.; ungulates of pigs, cows, goats, horses, sheep, etc.; carnivorous of dogs, cats, etc.; stem cells of primates such as humans, monkeys, macaque, marmoset, gorilla, chimpanzee, etc., preferably stem cells derived from humans. Examples of the stem cells include mesenchymal stem cells, hematopoietic stem cells, neural stem cells, myeloid stem cells, germ stem cells, and the like. The pluripotent stem cells are preferably mesenchymal stem cells, and more preferably bone marrow mesenchymal stem cells. Mesenchymal stem cells broadly refer to a group of stem cells or their precursors that can differentiate into all or some of mesenchymal cells such as osteoblasts, chondroblasts, and adipoblasts.
Further, the polypeptide of the present invention is used at a concentration of 1. Mu.g/ml to 500. Mu.g/ml.
Further, the polypeptide of the present invention is used at a concentration of 1. Mu.g/ml to 200. Mu.g/ml.
Further, the polypeptide of the present invention is used at a concentration of 100. Mu.g/ml.
Further, the invention also provides application of the M-428 polypeptide in preparing a reagent for promoting high expression of SDF-1 protein in human umbilical cord mesenchymal stem cells, wherein the amino acid sequence of the M-428 polypeptide is shown in SEQ ID NO:1, wherein the concentration of the polypeptide is 25-200 mug/ml.
Advantageous effects
The invention obtains the active peptide M-428 which specifically promotes proliferation of human umbilical cord mesenchymal stem cells through high-throughput screening aiming at polypeptide libraries, and the polypeptide can promote the high expression of SDF-1 protein of human umbilical cord mesenchymal stem cells to promote proliferation of the stem cells through proteome comparison analysis. The polypeptide can inhibit apoptosis besides promoting cell proliferation after being used for culturing human umbilical cord mesenchymal stem cells.
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FIG. 1 shows the results of inverted microscope observation of human umbilical cord mesenchymal stem cells; the cells were seen to be fusiform and to be growing in a swirl.
FIG. 2M-428 validation of proliferation of stem cells by media prepared from peptides
FIG. 3 influence of M-428 peptide on SDF-1 protein expression
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The components, reagents, or apparatus used were conventional products commercially available without the manufacturer's knowledge.
EXAMPLE 1 preparation of human umbilical cord mesenchymal Stem cells
Washing the human umbilical cord with PBS containing the double antibodies of the green streptomycin for 4 times, and removing blood vessels in the umbilical cord to obtain the umbilical cord hua tong gum tissue. Repeatedly rinsing, and shearing umbilical cord into 1mm pieces 3 Is digested with 0.15% type I collagenase at 37deg.C for 15min, digested with 0.25% pancreatin for 15min, terminated with calf serum, filtered with 200 mesh sieve, centrifuged to obtain cell precipitate, and the cell precipitate is cultured in a T25 flask with DMEM/F12 complete culture medium containing 10% fetal calf serum in 5% CO 2 Culturing at 37 ℃. Cells grown as monolayers were digested with 0.025% pancreatin/EDTA and a cell suspension was prepared for passaging.
The cells were transferred to the fourth generation for culture morphology examination, and as can be seen in FIG. 1, the cells showed a uniform long spindle shape as a whole, formed a typical mediation-like arrangement growth, and exhibited the characteristics of typical umbilical cord mesenchymal stem cells.
And taking the 4 th generation of detection cell surface markers. Separating UC-MSCs into 2X 10 9 And (3) adding PE or FITC labeled mouse anti-human monoclonal antibodies CD13, CD14, CD44, CD45, CD71, CD90, CD34, CD105 and isotype control into a tube, incubating at 4 ℃ in the dark for 30min, fixing paraformaldehyde for 10min, detecting by a flow cytometry, wherein the immunophenotype of MSCs is expressed by the proportion of CD90+CD13+CD45-CD 14-cells/nucleated cells and CD44+CD34-CD105+CD71+ cells/nucleated cells. The results in Table 1 show that the cells highly expressed CD13, CD44, CD71, CD90 and CD105, but substantially more expressed the surface markers of hematopoietic cells such as CD45 and CD34, indicating that isolated pure umbilical cord mesenchymal stem cells.
TABLE 1UC-MSCs flow detection results
| Cell type | Proportion of |
| CD90+CD13+CD45-CD 14-cells/nucleated cells | (96.42±0.38)％ |
| CD44+CD34-CD105+CD71+ cells/nucleated cells | (96.23±0.24)％ |
Example 2 screening of Polypeptides promoting proliferation of umbilical mesenchymal Stem cells
The P4 generation human umbilical cord mesenchymal stem cell suspension of example 1 (1×10) was added to a 96-well culture plate 5 Per mL) of 100. Mu.L, 100. Mu.L of physiological saline was added to the control group, and the concentration of the polypeptide added to the polypeptide library by the experimental group was 10. Mu.g/mL. After gentle mixing, 5% CO was added 2 Culturing in incubator at 37deg.C for 24 hr, adding 40 μl of MTT solution into each well under aseptic condition, mixing gently, culturing for 4 hr, centrifuging for 10min, sucking supernatant with filter paper, standing for 15min at room temperature, and measuring A value of each well at reference wavelength of 630nm and test wavelength of 570 nm. The pro-cell proliferation rate was calculated as follows: cell proliferation rate = (a experiment-a control)/a experiment x 100%. The results are shown in Table 2.
| Group of | Cell proliferation Rate (%) |
| |
0 |
| Polypeptide 53 | 5.23 |
| Polypeptide 156 | 12.31 |
| Polypeptide 246 | 11.03 |
| Polypeptide 357 | 0.21 |
| Polypeptide 428 | 88.36 |
| Polypeptide 536 | 22.31 |
| Polypeptide 1186 | 10.1 |
| Polypeptide 1257 | 0.32 |
The inventor screens from the peptide library to obtain the polypeptide capable of promoting proliferation in table 2, wherein the polypeptide 428 has better proliferation promoting effect. The polypeptide is designated M-428 and its amino acid sequence is CFNTIMCGALS.
Example 3 validation of Medium for facilitating peptide preparation on Stem cell proliferation
Cell culture media containing 0. Mu.g/ml, 25. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml and 200. Mu.g/ml of the M-428 polypeptide were prepared using DMEM/F12 medium;
the P4 generation human umbilical cord mesenchymal stem cells prepared and cultured in example 1 are inoculated in a 96-well plate, 5000 cells per well and 100 μl of culture medium; after overnight incubation, the medium was removed and the cells were treated as follows:
group A: adding a cell culture medium containing 0 μg/ml of the M-428 polypeptide;
group B: adding a cell culture medium containing 25 μg/ml of the M-428 polypeptide;
group C: adding a cell culture medium containing 50 μg/ml of the M-428 polypeptide;
group D: adding a cell culture medium containing 100 μg/ml of the M-428 polypeptide;
group E: adding a cell culture medium containing 200 μg/ml of the M-428 polypeptide;
after the addition was completed, the cells were placed in a cell incubator and cultured for 72 hours, after the completion of the culture, 10. Mu.l of CCK-8 reagent was added to each well, and after incubation for 4 hours, the absorbance at 450nm was measured for each group of cells using a microplate reader. The results are shown in FIG. 2.
As can be seen from the results of fig. 2, the OD value of the blank group was 0.361±0.021, and the OD value of each group to which the polypeptide was added was significantly increased with the increase of the concentration. The OD value of the group E with the greatest concentration was 1.305±0.029, and the difference was extremely remarkable (P < 0.01) with respect to the blank group.
Example 4 detection of Effect of polypeptide-related protein expression Regulation and apoptosis Effect
Through proteomics comparison analysis, the SDF-1 protein of the polypeptide possibly acting on the umbilical cord mesenchymal stem cells is primarily determined, and the protein can be highly expressed so as to promote proliferation of the umbilical cord mesenchymal stem cells. Therefore, the experiment detects the influence of the polypeptide on the corresponding protein before and after the action, and adopts an immunoblotting method to determine the expression level of the cell SDF-1 protein. The method comprises the following specific steps:
cell culture media containing 0. Mu.g/ml, 25. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml and 200. Mu.g/ml of the M-428 polypeptide were prepared using DMEM/F12 medium;
the P4 generation human umbilical cord mesenchymal stem cells prepared and cultured in example 1 are inoculated in a 96-well plate, 5000 cells per well and 100 μl of culture medium; after overnight incubation, the medium was removed and the cells were treated as follows:
group A: adding a cell culture medium containing 0 μg/ml of the M-428 polypeptide;
group B: adding a cell culture medium containing 25 μg/ml of the M-428 polypeptide;
group C: adding a cell culture medium containing 50 μg/ml of the M-428 polypeptide;
group D: adding a cell culture medium containing 100 μg/ml of the M-428 polypeptide;
group E: adding a cell culture medium containing 200 μg/ml of the M-428 polypeptide;
after the addition is completed, placing the cells in a cell incubator for culturing for 72 hours, extracting total protein of the cells after the culturing is completed, measuring the protein concentration, performing electrophoresis treatment on the cells by using sodium dodecyl sulfate-polyacrylamide gel, transferring a film, washing, adding a primary antibody, adding a secondary antibody at 4 ℃ overnight in the next day, reacting for 60 minutes at room temperature, wherein the internal reference is betA-Actin, developing, and analyzing the gray value of a target protein band by using quality one software. The results are shown in FIG. 3.
The immunoblotting method shows that the SDF-1 protein expression level of the blank control group is significantly different from that of the polypeptide treatment group (P < 0.05). Compared with the blank control group, the expression level of the SDF-1 protein is obviously increased along with the increase of the concentration of the polypeptide, at the concentration of 200 mug/ml, the protein level of the SDF-1 reaches 3.14+/-0.04, and the difference is extremely obvious compared with the blank control group (1.27+/-0.09) (P < 0.01).
Detecting apoptosis by flow cytometry, and adjusting cell concentration by 2×10 5 Preparing a cell suspension by per ml, centrifuging at 1000r/min for 5min, washing with phosphate buffer, centrifuging, fixing with ethanol, centrifuging, re-suspending with phosphate buffer, adding 1ml of PI dye solution for dyeing, detecting by an up-flow cytometer, analyzing the fluorescence intensity according to Cellquest software, and calculating the apoptosis rate. The results are shown in Table 3 below.
| Group of | Apoptosis rate (%) |
| Group A | 5.04±0.23 |
| Group B | 3.66±0.18 |
| Group C | 3.14±0.24 |
| Group D | 2.56±0.13 |
| Group E | 2.13±0.17 |
From the results of Table 3, it can be seen that the polypeptide is capable of inhibiting apoptosis of stem cells while promoting high expression of SDF-1.
EXAMPLE 5 characterization of mesenchymal Stem cell differentiation characteristics before and after proliferation
Preparing a cell culture medium containing 200 μg/ml of M-428 polypeptide using DMEM/F12 medium; the control group was using DMEM/F12 medium; the cells prepared in example 1 of generation 3 were taken at a density of 5X 10 4 The cells were cultured in 24-well plates (sterile coverslips placed in wells) and the medium was used as described above, and the medium was changed for 2 days, wherein half of the wells were cultured in the induced cardiomyocyte medium (DMEM containing 15% FCS at a final concentration of 20. Mu. Mol/ml 5-Aza) and the medium was changed for 1 time for 3 days. Cells were taken before and 6 weeks after induction, and the expression of gene Connexin-43 was detected by RT-PCR. The PCR primer of the target gene Connecin-43 takes betA-Actin as an internal control:
5'-GACTTCAGCCTCCAAGGAGTTCCACC-3，
5'-AGTTGGAGATGGTGCTTCCGGCC-3' the PCR primers for betA-Actin are:
5'-GTGGGGCGCCCCAGGCACCA-3'，
5'-CTCCTTATTGTCACGCACGATTTC-3'. Semi-quantitative analysis was performed using betA-Actin as an internal control, the values being expressed as the ratio of the net areas of the two bands. The positive control was obtained with commercially available embryonic cardiomyocytes. The results show that the ratio of the stem cells induced by the polypeptide culture medium is (0.695+/-0.013), the ratio of the stem cells induced by the non-culture medium is (0.624+/-0.024), and the ratio of the positive control is (0.938+/-0.019), and the results show that the polypeptide can effectively promote the proliferation of the stem cells and improve the differentiation characteristics of the corresponding stem cells, so that the stem cells are easier to differentiate the myocardial cells.
Although embodiments of the invention have been disclosed above, they are not limited to the use listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art. Therefore, the invention is not to be limited to the specific details and figures shown and described herein, without departing from the general concepts defined in the claims and their equivalents.
Claims (7)
1. A polypeptide for promoting proliferation of umbilical cord mesenchymal stem cells, characterized in that: the amino acid sequence is shown in SEQ ID NO: 1.
2. A culture medium for promoting proliferation of human umbilical mesenchymal stem cells and reducing apoptosis, which is prepared by adding the polypeptide of claim 1 to DMEM/F12 culture medium, wherein the concentration of the polypeptide is 25 μg/ml-200 μg/ml.
3. Use of the polypeptide of claim 2 for preparing a medium for promoting proliferation of human umbilical mesenchymal stem cells and reducing apoptosis, wherein the polypeptide is added at a concentration of 25 μg/ml to 200 μg/ml.
4. The use according to claim 3, wherein the basal medium of the culture medium is DMEM/F12 medium.
5. The method according to claim 4, wherein the polypeptide is added to the medium at a concentration of 200. Mu.g/ml.
6. The method according to claim 4, wherein the polypeptide is added to the medium at a concentration of 25. Mu.g/ml.
7. Use of the polypeptide of claim 1 in the preparation of an agent for promoting high expression of SDF-1 protein in human umbilical cord mesenchymal stem cells, wherein the polypeptide is added at a concentration of 25 μg/ml to 200 μg/ml.
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| CN119161412A (en) * | 2024-10-12 | 2024-12-20 | 江苏启创细胞基因工程研究院有限公司 | A polypeptide for promoting the proliferation of human umbilical cord mesenchymal stem cells and its application in in vitro culture of human umbilical cord mesenchymal stem cells |
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| CN119161412A (en) * | 2024-10-12 | 2024-12-20 | 江苏启创细胞基因工程研究院有限公司 | A polypeptide for promoting the proliferation of human umbilical cord mesenchymal stem cells and its application in in vitro culture of human umbilical cord mesenchymal stem cells |
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