CN111849878A - Method for improving osteogenesis capacity of mesenchymal stem cells - Google Patents
Method for improving osteogenesis capacity of mesenchymal stem cells Download PDFInfo
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- CN111849878A CN111849878A CN202010749546.2A CN202010749546A CN111849878A CN 111849878 A CN111849878 A CN 111849878A CN 202010749546 A CN202010749546 A CN 202010749546A CN 111849878 A CN111849878 A CN 111849878A
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Abstract
The invention provides a method for improving osteogenesis capacity of mesenchymal stem cells, which comprises the steps of inoculating the mesenchymal stem cells into an alpha-MEM culture medium containing 10% fetal bovine serum for culture, and digesting and passaging by adopting 0.2% of pancreatin when the fusion degree of the mesenchymal stem cells is 80-85%; inoculating the obtained mesenchymal stem cells into an alpha-MEM culture medium containing 10% fetal calf serum for culture, and adding osteogenic inducing liquid and metformin aqueous solution for induction when the density of the mesenchymal stem cells is more than 90%, wherein the induction time is 14-21 days. Metformin acts on mesenchymal stem cells, and the alizarin red staining result shows that the metformin can obviously improve the calcium mineralization nodule degree of the cells, which indicates that the metformin has obvious induction effect on the osteogenic differentiation of the mesenchymal stem cells; metformin can remarkably improve the expression of mesenchymal stem cell osteogenic differentiation related genes SIRT1, ALP, COL1A1, RUNX2 and BGLAP; the improvement of the osteogenic differentiation capacity of the mesenchymal stem cells has very important significance for stem cell transplantation and bone biological research.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to a method for improving osteogenesis capacity of mesenchymal stem cells.
Background
The bone tissue engineering means that the separated autologous high-concentration osteoblasts, bone marrow stromal stem cells or chondrocytes are cultured and amplified in vitro and then planted on a natural or artificially synthesized cell scaffold or extracellular matrix which has good biocompatibility and can be gradually degraded and absorbed by a human body, the biomaterial scaffold can provide a living three-dimensional space for the cells, is beneficial to the cells to obtain enough nutrient substances, performs gas exchange, removes waste materials, enables the cells to grow on the prefabricated three-dimensional scaffold, then implants the cell hybrid material into a bone defect part, and continuously proliferates the planted bone cells while the biomaterial is gradually degraded, so that the aim of repairing the bone tissue defect is fulfilled, and therefore, the enough osteoblasts become a key factor in the bone tissue engineering. How to obtain a sufficient amount of osteoblast seed cells by induction and maintain the phenotypic characteristics of the seed cells during induction has become a hot spot in bone tissue engineering research.
Mesenchymal Stem Cells (MSCs) are important members of the stem cell family, are derived from early-developing mesoderm, belong to pluripotent stem cells, and are originally found in bone marrow, so that MSCs are increasingly concerned by people due to the characteristics of multidirectional differentiation potential, hematopoietic support, stem cell implantation promotion, immune regulation, self-replication and the like. For example, under in vivo or in vitro specific induction conditions, mesenchymal stem cells can be differentiated into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium and the like, still have multidirectional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed cells for repairing tissue and organ injuries caused by aging and pathological changes. Due to the self-renewal, high proliferation and mesodermal differentiation potential of the MSC, the MSC can repair damaged tissues and promote the repair of tissue functions in clinical practical application. However, as research on mesenchymal stem cells proceeds, more and more unknown fields are revealed and await further research. At present, the effects of promoting the differentiation of mesenchymal stem cells into osteoblasts and improving bone regeneration and repair are important problems in scientific research and clinical application in related fields, and the efficiency of the existing method for differentiating the mesenchymal stem cells into osteoblasts is low, so that the research and discovery of a new osteogenic differentiation method become more important.
Disclosure of Invention
The invention aims to provide a method for improving osteogenic differentiation capacity of mesenchymal stem cells, aiming at the problems in the prior art.
The purpose of the invention is realized by the following technical scheme:
a method for improving osteogenic differentiation capacity of mesenchymal stem cells comprises the following steps:
(1) the mesenchymal stem cells are inoculated in an alpha-MEM culture medium containing 10% fetal bovine serum and placed in CO2Culturing in an incubator, and digesting and passing by adopting 0.2 percent of pancreatin when the fusion degree of the mesenchymal stem cells is 80 to 85 percent;
(2) inoculating the mesenchymal stem cells obtained in the step (1) into an alpha-MEM culture medium containing 10% fetal bovine serum, and placing the culture medium in CO2Culturing in an incubator, adding osteogenic inducing liquid and metformin aqueous solution for induction when the density of the mesenchymal stem cells is more than 90%, wherein the induction time is 14-21 days, the induction time is too short, the osteogenic differentiation induction effect of the mesenchymal stem cells is influenced, and the induction time is too long, which can cause mass death of the mesenchymal stem cells.
Metformin is a commonly used drug for resisting type 2 diabetes mellitus, glucose utilization is realized by reducing glucose production of the liver and increasing the sensitivity of peripheral tissues to insulin, and the clinical drug effect of metformin is well established. In addition, metformin promotes osteoblast activity, increases mineral precipitation, and simultaneously inhibits osteoclast activity and prevents bone mass reduction. Metformin induces the growth and differentiation of osteoblast-like cell lines (UMR 106, MC3T 3) with the ability to increase mineralization of the extracellular matrix. The metformin is adopted to improve the osteogenic differentiation capacity of the mesenchymal stem cells, and has very important significance for stem cell transplantation and bone biological research.
Preferably, alizarin red staining, calcium nodule quantification and intracellular osteogenic differentiation related gene expression measurement are carried out on the mesenchymal stem cells obtained in the step (2).
Preferably, the genes associated with the determination of intracellular osteogenic differentiation include SIRT1, ALP, COL1A1, RUNX2 and BGLAP.
Preferably, the mesenchymal stem cells of step (1) comprise bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, synovial mesenchymal stem cells or umbilical cord mesenchymal stem cells. The mesenchymal stem cells are primary cultured mesenchymal stem cells or subcultured mesenchymal stem cells.
Preferably, the osteogenesis inducing solution in step (2) is an α -MEM medium containing 50ug/mL ascorbic acid, 100nM dexamethasone, and 10mM β sodium glycerophosphate.
Preferably, the concentration of the metformin in the step (2) is 50-400 ummol/L; preferably, the concentration of the metformin in the step (2) is 100-200 ummol/L. The induction effect cannot be well achieved when the concentration of the metformin is lower than 50 ummol/L, and the cell activity is influenced when the concentration of the metformin is higher than 400 ummol/L.
Preferably, CO is present in step (1) and step (2)2The temperature of the incubator is 37 ℃ and CO2The concentration was 5%.
Preferably, CO is present in step (1) and step (2)2The replacement frequency of the culture solution in the incubator is 2-3 days/time.
The invention provides a product prepared by a method for improving osteogenic differentiation capacity of mesenchymal stem cells, and the product comprises metformin for improving osteogenic differentiation capacity of the mesenchymal stem cells.
The invention provides application of the product in improving the osteogenic differentiation capacity of mesenchymal stem cells.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a method for improving the osteogenesis capacity of mesenchymal stem cells, which is characterized in that metformin acts on the mesenchymal stem cells, can promote the osteoblast activity, increase the mineral precipitation, inhibit the osteoclast activity, prevent the bone mass from reducing, induce the growth and differentiation of osteogenesis-like cell lines (UMR 106, MC3T 3) and simultaneously have the capacity of increasing the mineralization of extracellular matrixes. The alizarin red staining result shows that metformin can remarkably improve the degree of calcium mineralization nodules of cells, and the metformin has remarkable induction effect on osteogenic differentiation of mesenchymal stem cells; the metformin can obviously improve the expression of mesenchymal stem cell osteogenic differentiation related genes SIRT1, ALP, COL1A1, RUNX2 and BGLAP. The improvement of the osteogenic differentiation capacity of the mesenchymal stem cells has very important significance for stem cell transplantation and bone biological research.
Drawings
Fig. 1A is a flow detection result of a positive surface marker of umbilical cord mesenchymal stem cells CD90 in an embodiment of the present invention;
fig. 1B is a flow detection result of an umbilical cord mesenchymal stem cell CD105 positive surface marker in an embodiment of the present invention;
fig. 1C is a flow detection result of a positive surface marker of umbilical cord mesenchymal stem cells CD73 in an embodiment of the present invention;
fig. 1D is a flow detection result of an umbilical cord mesenchymal stem cell CD34 negative surface marker in an embodiment of the present invention;
fig. 1E is a flow detection result of an umbilical cord mesenchymal stem cell CD45 negative surface marker in an embodiment of the present invention;
fig. 1F is a flow detection result of an umbilical cord mesenchymal stem cell CD14 negative surface marker in an embodiment of the present invention;
fig. 1G is a flow detection result of an umbilical cord mesenchymal stem cell CD19 negative surface marker in an embodiment of the present invention;
FIG. 1H shows the result of flow detection of HLA-DR negative surface marker of umbilical cord mesenchymal stem cells according to an embodiment of the present invention;
FIG. 2A shows alizarin red staining results of osteogenic differentiation of umbilical cord mesenchymal stem cells of a control group in the example of the present invention;
FIG. 2B shows alizarin red staining results of osteogenic differentiation of umbilical cord mesenchymal stem cells in metformin group in the example of the present invention;
FIG. 3 is a graph comparing quantitative values of calcium nodules of osteoblastic differentiation of umbilical cord mesenchymal stem cells according to an embodiment of the present invention;
FIG. 4 is a relative expression diagram of a SIRT1 gene in an example of the invention;
FIG. 5 is a graph showing relative expression of ALP gene in the examples of the present invention;
FIG. 6 is a diagram showing the relative expression of COL1A1 gene in examples of the present invention;
FIG. 7 is a relative expression diagram of RUNX2 gene in the example of the present invention;
FIG. 8 is the BGLAP gene relative expression map in the example of the present invention.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Description of the reagents:
alpha-MEM medium was purchased from Sigma;
metformin was purchased from Sigma;
the human umbilical cord mesenchymal stem cell osteogenesis inducing liquid can be prepared by the following method:
to alpha-MEM medium were added 50ug/mL ascorbic acid, 100nM dexamethasone, 10mM sodium beta glycerophosphate, 5% fetal bovine serum.
Example 1
Isolated culture of human umbilical cord mesenchymal stem cells
1. The surface of the umbilical cord is thoroughly washed with Phosphate Buffered Saline (PBS) in a biosafety cabinet, the superficial membrane of the umbilical cord tissue is stripped off, the umbilical cord is dissected open, the residual blood in the two umbilical arteries and one umbilical vein is removed and the arteriovenous vessels are removed. The treated umbilical cord tissue is cut into the size of about 1-2 mm3Then, the tissue blocks were placed in an α -MEM medium containing 10% FBS, the tissue blocks were shaken and spread on a culture plane, and then 5% CO was added2And culturing in a saturated humidity incubator at 37 ℃, wherein the culture solution is replaced once every two days, the growth state of the cells is closely concerned, and the solution replacement time can be adjusted according to the growth condition of the cells.
2. Cells were observed to approximately fill 80% to 85% of the bottom of the flask and passaged at a ratio of 1: 3. After washing the cells twice with PBS, 2ml of 0.20% trypsin was added to the flask and digested for 1 min. The morphology of the cells during the enzymatic digestion was observed by an inverted microscope, and if it was observed that the gaps between the cells became large and the cytoplasm retracted, the digestion solution was immediately aspirated, and α -MEM medium containing 10% FBS was added to terminate the digestion. And repeatedly blowing and beating the cells until the cells are blown down. And (3) collecting the liquid, pouring the liquid into a centrifuge tube, placing the centrifuge tube in a 1000rprn centrifuge at a rotating speed, centrifuging for 8min, pouring out the supernatant, adding an alpha-MEM culture medium containing 10% FBS into the centrifuge tube for resuspension, and carrying out inoculation culture to obtain the P4 generation human umbilical cord mesenchymal stem cells.
Identification of human umbilical cord mesenchymal stem cells
The P4 generation human umbilical cord mesenchymal stem cells obtained by the subculture are digested by 0.20% trypsin, and the stably proliferating cells are collected. Will 106The collected human umbilical cord mesenchymal stem cells are suspended in 100ml of PBS buffer solution containing 1% bovine serum albumin, referring to FIGS. 1A-H, FIGS. 1A-H are the loss detection results of mesenchymal stem cells CD90, CD73, CD34, CD45, CD14, CD19 and HLA-DR surface markers, and the results show that the positive surface markers CD90, CD105 and CD73 are all greater than or equal to 95%, the negative surface markers CD45, CD34, CD11b, CD19 and HLA-DR are all less than 2%, so the human umbilical cord mesenchymal stem cells cultured in the embodiment meet the stem cell standard.
Osteogenic differentiation induction of human umbilical cord mesenchymal stem cells
Digesting the P4 generation human umbilical cord mesenchymal stem cells obtained by subculture with 0.20% trypsin to obtain the product with the concentration of 1 × 106The cell suspension is inoculated on a 12-well plate, liquid is changed every three days, the density change of the cells is observed, the cells are randomly divided into a control group and a metformin group after the cell density is more than 90 percent, osteogenesis inducing liquid (10 percent FBS, 50ug/mL ascorbic acid, 100nM dexamethasone and alpha-MEM of 10mM beta sodium glycerophosphate) is added into the control group, 100ummol/L metformin is added into the metformin group on the basis of the osteogenesis inducing liquid, alizarin red staining and calcium nodule quantification are carried out after the induction day 14, and the expression of genes related to osteogenesis differentiation in the cells is measured.
1. Alizarin red staining and calcium nodule quantification
The culture solution in the well plate was removed and the cells to be tested were washed 3 times with PBS. 4% paraformaldehyde is added into a 12-well plate, fixed at room temperature for 15min and then completely aspirated, and washed with PBS 3 times. Then, 300uL alizarin red dye solution is added into each hole in the hole plate, the mixture is completely sucked after being dyed for 15min at room temperature, and the mixture is washed for 3 times by PBS. And (3) collecting images by using a microscope, adding 200uL of 5% perchloric acid into each hole of a 12-hole plate after the images are collected, and standing for 10min at room temperature. And transferring the dissolved solution into a 96-well plate, grouping, detecting the light absorption value at the wavelength of 420nm by using an enzyme-labeling instrument, and averaging 2 multiple detection wells in each group.
RT-PCR detection of osteogenic marker Gene expression
The culture medium in the culture plate was aspirated, and the cells to be tested were washed 3 times with PBS. mu.L of TRIzol reagent (total RNA extraction reagent, Invitrogen) was added to each well of a 6-well plate, and the mixture was pipetted and mixed at room temperature and then placed in a 1.5 mL EP tube. Each sample was added with 100. mu.L of chloroform, shaken well, left to stand at room temperature for 15min, and centrifuged at 12000g at 4 ℃ for 12 min. And (3) sucking the colorless and transparent supernatant to another EP tube, adding 200 mu L of isopropanol into each sample, fully shaking, standing at room temperature for 10min, centrifuging at 12000g at 4 ℃ for 8min, and removing the supernatant to obtain the trace feather-shaped RNA precipitate at the tube bottom. Adding appropriate amount of 75% ethanol solution, shaking EP tube, mixing the above RNA precipitate, centrifuging at 8000g and 4 deg.C for 5min, removing supernatant, and air drying at room temperature for 30 min. The RNA precipitate was mixed with RNase-free water and left at 60 ℃ for 10 min. A small amount of sample is taken, and the RNA concentration is measured by a microplate reader.
Using PrimeScriptTMRT Master Mix (TaKaRa) kit reverse transcription of RNA. Reaction system: (calculated as 500. mu.g RNA described below) test RNA, 5. mu.L of 5 XPrimeScript RT Master Mix, followed by addition of RNase-free water to a 10. mu.L system. The reaction conditions of the PCR instrument are set to 42 ℃ for 60min, 70 ℃ for 5min and 4 ℃ for 60 min. RT-PCR detection was performed using SYBR Green PCR master mix (ThermoFisher) kit using primers corresponding to SIRT1, ALP, COL1A1, RUNX2, and BGLAP genes, respectively. Reaction system 10 ul: after diluting the cDNA 4 times, the cDNA to be tested, SYBR Green, the upstream primer, the downstream primer and the RNase-free water were mixed in volumes of 1. mu.L, 5. mu.L, 0.4. mu.L and 3.2. mu.L. Reaction conditions are as follows: the reaction conditions of the PCR instrument were set to 95 ℃ for 3min, 95 ℃ for 10s, 56 ℃ for 30s, and 60 ℃ for 5s, and 40 cycles were performed. The calculation formula of the relative expression quantity of the gene is chi = 2−ΔΔCtΔ Δ Ct = Δ E- Δ C, Δ E = Ctexp-Ct internal reference, Δ C = ctect 1-Ct internal reference (Ctexp is the cycle value of the target gene, and ctet 1 is the cycle value of each test sample control group).
Referring to fig. 2A-2B, fig. 2A is a result of alizarin red staining for osteogenic differentiation of umbilical cord mesenchymal stem cells in a control group, and fig. 2B is a result of alizarin red staining for osteogenic differentiation of umbilical cord mesenchymal stem cells in a metformin group, it can be seen that, after metformin is added for 14 days during osteogenic induction using UCMSC, alizarin red staining of the metformin group is deepened compared with that of the control group, fig. 3 is a comparison graph of quantitative values of calcium nodules for osteogenic differentiation of umbilical cord mesenchymal stem cells, and quantitative improvement of calcium nodules of the metformin group compared with that of the control group, that is, metformin can significantly improve the degree of calcium mineralization nodules of cells, indicating that metformin has a significant inducing effect on osteogenic differentiation of mesenchymal stem cells. Referring to fig. 4-8, which are relative expression graphs of five genes SIRT1, ALP, COL1a1, RUNX2 and BGLAP, respectively, RT-PCR results show that the addition of metformin during osteogenesis induction can increase the expression of genes SIRT1, ALP, COL1a1, RUNX2 and BGLAP, which indicates that metformin induction can increase the osteogenesis capacity of umbilical cord mesenchymal stem cells.
The above description is only an embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions within the technical scope of the present disclosure should be covered by the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.
Claims (6)
1. A method for improving osteogenic differentiation capacity of mesenchymal stem cells is characterized by comprising the following steps:
(1) the mesenchymal stem cells are inoculated in an alpha-MEM culture medium containing 10% fetal bovine serum and placed in CO2Culturing in an incubator, and digesting and passing by adopting 0.2 percent of pancreatin when the fusion degree of the mesenchymal stem cells is 80 to 85 percent;
(2) inoculating the mesenchymal stem cells obtained in the step (1) into an alpha-MEM culture medium containing 10% fetal bovine serum, and placing the culture medium in CO2Culturing in an incubator, and adding osteogenic inducing liquid and metformin aqueous solution for induction when the density of the mesenchymal stem cells is more than 90%, wherein the induction time is 14-21 days.
2. A method of improving osteogenic differentiation capacity of mesenchymal stem cells according to claim 1, wherein the mesenchymal stem cells of step (1) comprise bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, synovial mesenchymal stem cells or umbilical cord mesenchymal stem cells.
3. The method for improving osteogenic differentiation ability of mesenchymal stem cells according to claim 1, wherein the osteogenic induction solution in step (2) is α -MEM culture medium comprising 50ug/mL ascorbic acid, 100nM dexamethasone, 10mM β sodium glycerophosphate.
4. A method for improving osteogenic differentiation ability of mesenchymal stem cells according to claim 1, wherein the concentration of metformin in step (2) is 50 to 400 ummol/L.
5. A product for increasing the osteogenic capacity of mesenchymal stem cells, wherein the product comprises metformin.
6. Use of the product according to claim 5 for improving osteogenic differentiation capacity of mesenchymal stem cells.
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