CN104805051B - Method for inducing adipose-derived stem cells to differentiate into fibroblasts - Google Patents
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Abstract
The invention relates to the field of stem cell induction, in particular to a method for inducing adipose-derived stem cells to differentiate into fibroblasts. The method comprises the following steps: and (3) carrying out induction culture on the adipose-derived stem cells in the presence of vitamin C phosphate and bFGF to obtain fibroblasts. Experiments prove that the cells are still in a fusiform or vortex shape after being cultured for 7-10 days by adopting the method provided by the invention, and although the shape of the cells is very similar to that of the adipose-derived stem cells, the detection result of the expression condition of the type I collagen shows that the expression quantity of the type I collagen in the cultured cells is increased by 124 times and is obviously superior to that of a control group. The method provided by the invention successfully induces and differentiates the adipose-derived stem cells into fibroblasts, and the differentiation speed is better than that of a control group. During the continuous expansion culture and passage, the cells are further differentiated and expanded, so that a larger amount of fibroblasts can be obtained.
Description
Technical Field
The invention relates to the field of stem cell induction, in particular to a method for inducing adipose-derived stem cells to differentiate into fibroblasts.
Background
Stem cell therapy is considered to be a clinically safe and effective treatment for tissue damage repair. In the prior art, the technology of taking bone marrow or umbilical cord blood-filled mesenchymal stem cells as a wound repair material has been reported. However, bone marrow mesenchymal stem cells (BMSCs) have the hidden danger of virus pollution, and the cell number, the amplification and differentiation capacity of the BMSCs have obvious decline tendency along with the age of donors, so the BMSCs are not suitable for batch preparation. The mesenchymal stem cells derived from cord blood or placenta can overcome the defects, but can be preserved at birth and can not be provided by all patients. Adipose-derived mesenchymal stem cells (ADSCs) separated from human adipose tissue can control and regulate nearby damaged cells by secreting various growth factors, and play an important role, and the ADSCs are easily obtained from liposuction fat, have small wound on patients, high stem cell separation efficiency and multipotential differentiation capability, so the ADSCs have become a research hotspot of seed cells in recent years.
However, the first problem in using ADSCs for the preparation of wound repair materials is differentiation. 2008, it is reported that adipose-derived stem cells are compounded in a human acellular dermal matrix and transplanted to a skin defect of a nude mouse, and the implanted cells are found to be differentiated to vascular endothelial cells, fibroblasts and epithelial cells and can effectively promote wound healing. The skin is used as an organ with the largest human body area, large-area defect or difficult healing occurs, which is a great problem which troubles clinical work at present, and the ADSCs are undoubtedly the ideal choice for seed cells and scaffold materials. In the prior art, research on the differentiation of the fibroblasts of the ADSCs is less, and only research results show that the time for differentiating the ADSCs into the fibroblasts is longer.
Disclosure of Invention
In view of the above, the present invention provides a method for rapidly inducing adipose-derived stem cells to differentiate into fibroblasts.
The method for inducing the adipose-derived stem cells to differentiate into the fibroblasts provided by the invention comprises the following steps: and (3) carrying out induction culture on the adipose-derived stem cells in the presence of vitamin C phosphate and bFGF to obtain fibroblasts.
Vitamin C phosphate, also known as vitamin C phosphate, can promote collagen production.
bFGF is a fibroblast growth factor, has the function of promoting mitosis, and can promote wound tissue healing and tissue repair and tissue regeneration.
The invention adopts vitamin C phosphate and bFGF as inducers to induce adipose-derived stem cells to differentiate into fibroblasts. Experiments show that after 7-10 days of induction, the adipose-derived stem cells are differentiated into fibroblasts, and the differentiation speed is higher than that of other control groups.
In some embodiments, the medium of the induction culture is DMEM/F12 medium containing 5 wt% FBS.
In some embodiments, the concentration of bFGF is 5-15 μ g/mL; the concentration of the phosphoric acid vitamin C is 0.5-1.5 mmol/L.
In some embodiments, the concentration of bFGF is 10 μ g/mL; the concentration of the phosphoric acid vitamin C is 1 mmol/L.
Experiments show that the variety and concentration of the growth factors have obvious influence on the differentiation rate of the adipose-derived stem cell fibroblasts, and the differentiation rate is reduced by changing the variety or concentration of the growth factors.
In some embodiments, dexamethasone is also included in the medium of the induction culture.
In some embodiments, the concentration of dexamethasone is 0.01-0.2 μmol/L.
In some embodiments, the concentration of dexamethasone is 0.1 μmol/L.
Dexamethasone was used as an anti-inflammatory agent.
In some embodiments, the adipose stem cells are trypsinized and fused prior to induction culture.
Specifically, the trypsin digestion and fusion of the adipose-derived stem cells are as follows: taking adipose-derived stem cells of the third generation to the fifth generation, adding trypsin with the mass fraction of 0.25% and EDTA with the mass fraction of 0.02% for digestion, stopping digestion with serum and then centrifuging at 1200r/min for 5min, resuspending the cells with a DMEM/F12 culture medium, adjusting the density to 1 × 104cell/mL, and culturing until the fusion is 40-50%.
In some embodiments, the induction medium is replaced every 2 days during the induction culture.
In some embodiments, the induction culture is performed for 7d to 10d at 37 ℃ and CO2Volume fraction 5%, relative humidity 95%.
In some embodiments, the method for preparing the third-fifth generation adipose-derived stem cells comprises: the adipose tissues are washed by PBS buffer solution, digested by collagenase, centrifuged to take cell precipitates, cultured by an adipose stem cell complete culture medium until 80% of cells are fused, digested by trypsin and subcultured to the third generation to the fifth generation.
In some embodiments, the method further comprises the steps of expansion culture and passage after the induction culture.
In some embodiments, the expansion culture, passaging medium is fibroblast serum-free medium SFM.
In some embodiments, the medium is replaced every 2 days during the expansion culture, passage.
In some embodiments, the conditions for the amplification culture and the subculture are 37 ℃ CO2Volume fraction 5%, saturated humidity 95%.
The method for inducing the adipose-derived stem cells to differentiate into the fibroblasts provided by the invention comprises the following steps: and (3) carrying out induction culture on the adipose-derived stem cells in the presence of vitamin C phosphate and bFGF to obtain fibroblasts. Experiments prove that the cells are still in a fusiform or vortex shape after being cultured for 7-10 days by adopting the method provided by the invention, and although the shape of the cells is very similar to that of the adipose-derived stem cells, the detection result of the expression condition of the type I collagen shows that the expression quantity of the type I collagen in the cultured cells is increased by 124 times and is obviously superior to that of a control group. The method provided by the invention successfully induces and differentiates the adipose-derived stem cells into fibroblasts, and the differentiation speed is better than that of a control group. During the continuous expansion culture and passage, the cells are further differentiated and expanded, so that a larger amount of fibroblasts can be obtained.
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FIG. 1 shows the observation result of an inverted microscope (100X) of primary cultured adipose stem cells, wherein FIG. 1-a shows the observation result after 48h of primary culture; FIG. 1-b shows the observation after 5 days of primary culture;
FIG. 2 shows a flow cytometry for detecting the surface antigen expression of adipose-derived stem cells cultured by amplification to the third generation;
FIG. 3 shows the results of observing cell morphology at 40-fold magnification in an inverted microscope after induction culture of adipose-derived stem cell fibroblasts; wherein FIG. 3-a shows the morphology of the adipose-derived stem cells before induction culture, and FIG. 3-b shows the morphology of the cells after induction culture in the experimental group; FIG. 3-c shows the morphology of control 1 cells after induction culture; FIG. 3-d shows the morphology of control 2 cells after induction culture; FIG. 3-e shows the morphology of control 3 cells after induction culture;
FIG. 4 shows immunofluorescence staining assays for expression of collagen type I in cells following induction of differentiation; wherein, FIG. 4-a shows the expression of type I collagen in the cells of the experimental group; FIG. 4-b shows collagen type I expression in control 1 cells; FIG. 4-c shows collagen type I expression in control group 2 cells; FIG. 4-d shows collagen type I expression in control 3 cells.
Detailed Description
The invention provides a method for inducing adipose-derived stem cells to differentiate into fibroblasts, and a person skilled in the art can use the content to appropriately improve the process parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The instruments adopted by the invention are all common commercial products and can be purchased in the market.
Wherein the complete culture medium of the adipose-derived stem cells is purchased from Guangzhou Tao scientific instruments Co
Fibroblast serum-free medium was purchased from Guangzhou Tao scientific instruments, Inc
The invention is further illustrated by the following examples:
example 1 isolation and culture of human adipose-derived stem cells (ADSCs)
The extract of subcutaneous adipose tissue from abdomen of human body obtained by liposuction (endocrine diseases and infectious diseases excluded before operation) is centrifuged at 800g, washed with PBS buffer, soaked, and washed 3 times at 800gx4 min.
Removing macroscopic blood vessels and crusted hoof tissues, sufficiently shearing the tissues by using an ophthalmological scissors, putting the sheared tissues into a 50ml centrifuge tube, adding 2 times of 0.5% collagenase by volume, 1 time of 1% BSA by volume and double antibiotics by volume, uniformly mixing, sealing, and putting the mixture into a shaking table to digest for 50min at 37 ℃ until the mixture is pasty.
Adding isovolumetric low-sugar DMEM containing 10% fetal calf serum by volume fraction to terminate digestion, centrifuging at 1800r/min for 10min, dividing into 3 layers after centrifugation, wherein the upper layer is grease and undigested complete adipose tissue, the middle layer is supernatant, and the lower layer is sediment of mixed cells of adipose stem cells, red blood cells and the like.
Discarding the upper and middle layers, adding complete culture medium into the lower layer to resuspend cell precipitate, filtering with 70um cell screen,
adjusting the cell concentration of the filtered filtrate to 5x105monocytes/mL inoculated in T-25cm2In a culture flask, 5% CO at 37 deg.C2And culturing in saturated humidity.
After 48h, the solution is changed, and the adipose-derived stem cells are added into the complete culture medium for continuous culture.
When the cells are cultured until 80% of fusion, the cells are digested and passaged to the third generation to the fifth generation by adopting 0.25% trypsin/0.02% EDTA.
The results of observation with an inverted microscope are shown in FIG. 1, and show: after the primary cells are cultured for 24 hours, a small amount of cells can adhere to the wall; after 48h (FIG. 1-a), round, short spindle, irregular cells were seen under an inverted microscope; after the liquid is changed, the cells grow rapidly, spindle cells are increased obviously, a part of the cells can see a plurality of bulges, monolayer fusion is started after 4-5 days (figure 1-b), the cells are in a fibroblast-like shape, the cells grow rapidly, passage is carried out once after 3 days, and the cell shape is gradually uniform along with the increase of the generation number.
Example 2 identification of surface markers for human adipose-derived stem cells (ADSCs)
Taking the 3 rd generation cells in the logarithmic phase, sucking out the culture medium, adding a digestive juice of 0.25% trypsin and 0.02% EDTA for digestion, stopping digestion with a proper amount of serum, and gently blowing and beating to form a single cell suspension.
Centrifuging at 1000rpm for 10min, discarding supernatant, washing cells with 4 deg.C cold PBS for 3 times, uniformly suspending, and adjusting cell concentration to about 105-106One per ml.
Taking 2 sample loading tubes, adding 500ul of single cell suspension into each tube, centrifuging, marking tube No. 1 as a standard control, and adding 2 μ l FITC or PE labeled mouse anti-human cell surface molecules CD59, CD45, CD34 and CD105 antibody working solution into tube No. 2.
Incubate for 20min at room temperature in the dark.
PBS was washed twice to remove unbound antibody, 500ul of 1640 medium was resuspended, and surface markers were identified on a flow cytometer.
The identification result is shown in fig. 2, and the flow cytometry detection of the surface antigen of the ADSCs by taking the experiment without adding the antibody as a control shows that: the ADSCs CD59 and CD90 of the 3 rd generation are positively expressed, and HLA-DR and CD45 are negatively expressed, which accords with the characteristics of stem cells.
Example 3 induced differentiation and characterization of human adipose-derived stem cells into fibroblasts
The ADSCs of the third to fifth generations prepared in example 1 were aspirated from the culture medium, digested with a digestion solution of 0.25% trypsin + 0.02% EDTA, and then digested with a suitable amount of serum.
Centrifuging at 1200rpm for 5min, resuspending adipose-derived stem cells in complete culture medium, and adjusting the density to 1x104Perml, seeded in 6-well plates, 2ml per well.
When the cells reach 40% -50% fusion, the culture medium shown in the table 1 is applied, the induction culture is carried out under the same environment, the induction culture medium is changed every 2 days, after the cells are cultured for 7-10 days, part of the cells are taken to detect related indexes, the rest of the cells are continuously cultured and passaged by using a fibroblast complete culture medium, and the complete culture medium is changed every 2 days.
TABLE 1 Experimental design for induced differentiation of adipose-derived stem cells into fibroblasts
Example 4 Effect of inducing differentiation of human adipose-derived Stem cells into fibroblasts
The results of observing cell morphology by an inverted microscope after culture are shown in fig. 3, after the ADSCs are induced and differentiated for 10 days, the cell morphology has no obvious change, and the cells still grow in a fusiform or vortex shape. This is because the fibroblast morphology is very similar to that of adipose stem cells.
Example 5 Real-time PCR detection of CK10 and CK19 expression
The expression conditions of CK10 and CK19 are detected by Real-time PCR,
1. total RNA extraction from cells
Digesting the cells of the control group and the experimental group in example 2 by using pancreatin, centrifuging, collecting cell precipitates, adding 500 μ L of the Trizol reagent, and standing at room temperature for 5min for full lysis; adding 100 μ L chloroform into the lysate, shaking with a vortex oscillator to mix the solution thoroughly, and standing at room temperature for 3 min; centrifuging at 4 deg.C and 10000rpm for 15 min; taking out the centrifugal tube after centrifugation, dividing the sample into three layers, and carefully absorbing the upper clear water into a new EP tube; adding isopropanol with the same volume into the upper clear water phase, shaking and mixing uniformly by a vortex oscillator, standing at room temperature for 5min, and centrifuging at 4 ℃ at 10000rpm for 15 min; pouring off the supernatant, slowly adding 1mL of 75% precooled ethanol, and gently mixing uniformly to wash the precipitate; carefully absorbing the liquid by using a gun head, adding 50 mu L of DEPC water, uniformly mixing by using a vortex oscillator, and fully dissolving RNA precipitate; 2. mu.L of the RNA sample was sampled and the concentration of the sample was measured by a nucleic acid protein analyzer.
2. Cellular cDNA harvesting
By using Prime Script of TAKARATMThe RT reagent Kit reverse transcription Kit can efficiently synthesize cDNA for Real Time PCR in a short Time, and comprises the following specific steps:
RT reactions were prepared according to the composition of Table 2 (the reaction preparation had to be carried out on ice).
TABLE 2 reaction solution
| Reagent | Amount of the composition used | Final concentration |
| 5*Prime Script Buffer(for Real Time) | |
1* |
| Prime Script RT Enzyme | 0.5μL | |
| Oligo dT Primer(50μM) | 0.5μL | 25pmol |
| Random 6mers(100μM) | 0.5μL | 50pmol |
| Total RNA | ||
| RNase Free dH2O | up to 10μL |
The reaction solution was dispensed into PCR tubes, and placed in a PCR apparatus to perform reverse transcription reaction under the conditions shown in Table 3
TABLE 3 reverse transcription conditions
| The step of circulation | Temperature of | Time of day | |
| Reverse transcription reaction | 37℃ | 15min | |
| Inactivation of reverse transcriptase | 85 | 5sec | |
| 4℃ | Holding |
The cDNA obtained after reverse transcription was diluted and added with 65. mu.L of water containing LDEPC per 10. mu.L of the system and stored at 4 ℃ for further use.
3. Real-time PCR detection factor expression
Detecting the type I collagen expression condition by adopting Real-time PCR and an immunofluorescence method, wherein: the factor Real-timePCR specific primer sequence is provided by the website NCBI and synthesized by Shanghai bioengineering, Inc. GAPDH, type I collagen Real-time PCR specific primers are shown in Table 4:
TABLE 4 Real-time PCR specific primers
| Gene | Primer F (upstream Primer) | Primer R (downstream Primer) |
| GAPDH | 5’-GTCATTGAGAGCAATGCCAG-3’ | 5’-GTGTTCCTACCCCCAATGTG-3’ |
| Type I collagen | 5’-CTGGTACGGCGAGAGCAT-3’ | 5’-CAGGCTCCGGTGTGACTC-3’ |
After obtaining the primers, carrying out short-time centrifugation, adding a certain amount of DEPC water to dissolve the primers according to the requirements on a centrifuge tube, diluting the primers into 10 mu M, and storing the diluted primers at-20 ℃ for later use.
Using the reverse-transcribed cDNA as a template, SsoFast from BIO-RADTMThe EvaGreen Supermix kit is used for carrying out real-time fluorescent quantitative PCR amplification on inflammatory factors, and comprises the following specific steps:
(1) Real-timePCR reaction solution was prepared according to the following composition
TABLE 5 Real-timePCR reaction solution
| Reagent | Amount of the composition used | Final concentration | |
| SsoFast | 5μL | 1* | |
| Forward primer | 0.5μL | 300-500nM | |
| Reverse primer | 0.5μL | 300-500nM | |
| DNA template | 4μL | ||
| Total volume | 10μL |
The reaction solution was mixed well and added to a 96-well Real-time PCR plate, 10. mu.L per well, and set to 4 replicate wells. The reaction plate was placed in BIO-RAD CFX96TMIn a Real-time system instrument, Real-time PCR was carried out under the following reaction conditions, and finally based on the obtained C(t)The expression level of the target gene in the cell after induction relative to the expression level of the gene before induction was calculated by referring to the expression level of the gene in the cell before induction:
TABLE 6 Real-time PCR program
The results are shown in Table 7:
the results of the detection of Real-timePCR are shown in Table 7:
TABLE 7 test results
| Relative expression of type I collagen | |
| Before |
1 |
| Experimental group | 124** |
| |
1.1 |
| Control group 2 | 78** |
| Control group 3 | 57** |
Note that there is a significant difference (p <0.01)
After the ADSCs are induced and differentiated to the fibroblasts for 10 days, extracting cell RNA and carrying out reverse transcription to form cDNA, and detecting the expression condition of the type I collagen by using a fluorescence quantitative PCR instrument, wherein the expression quantity of the type I collagen of the control group 1 has no obvious change compared with that before induction as shown in Table 7; the expression level of type I collagen in the control group 2 was increased 78-fold; the expression level of type I collagen in the control group 3 was increased 57-fold; the expression level of the experimental group is increased by 124 times, which indicates that the adipose-derived stem cells have differentiated into the fibroblasts, and the induced differentiation speed of the experimental group is obviously faster than that of the control group 1, the control group 2 and the control group 3.
Example 6 immunofluorescence staining assay for CK19 expression on cell surface after induced differentiation
CK19 expression was detected by immunofluorescence to identify the effect of cell differentiation. The specific method comprises the following steps:
the cell suspensions of the control group and the experimental group in example 2 were inoculated into 6-well plates and cultured until 50-60% confluency, and washed 3 times with PBS for 5min each.
4% paraformaldehyde was fixed at room temperature for 10min and washed 3 times with PBS for 5min each.
Cells were permeabilized with 0.2% TritonX-100 for 10min and washed 3 times with PBS for 5min each.
Adding 10% serum, incubating at 37 deg.C for 15min, decanting the serum, washing twice with PBS, adding 50ul mouse anti-human CK19 antibody working solution, incubating at room temperature for 1h, and replacing the antibody with PBS as negative control.
Washing with PBS for 3 times (5 min each time), adding fluorescently-labeled secondary antibody working solution dropwise, incubating at 37 deg.C for 30min, and washing with PBS for 3 times (5 min each time).
0.5ug/ml DAPI staining was added for 10min and washed 3 times with PBS for 5min each.
50% glycerol mounting, observing the experimental results under a fluorescence microscope and taking pictures. The results are shown in FIG. 4: the expression of collagen type I in the cells after differentiation induction by immunofluorescence staining was shown in fig. 4: after the ADSCs are induced and differentiated to the fibroblasts for 10 days, the expression condition of the type I collagen is detected by an immunofluorescence staining method, and the result shows that the immunofluorescence staining of the control group 1 is negative, the immunofluorescence staining of the control group 2, the control group 3 and the experimental group is positive, but the number of the immunofluorescence staining cells of the experimental group is more than that of the control group 2 and the control group 3.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (6)
1. A method for inducing adipose-derived stem cells to differentiate into fibroblasts is characterized in that human adipose-derived stem cells are induced and cultured in the presence of vitamin C phosphate and bFGF to obtain fibroblasts;
the culture medium for induction culture comprises: DMEM/F12 containing 5% FBS, and the inducers were 1mmol/L vitamin C phosphate, 10. mu.g/L bFGF and 0.1. mu. mol/L dexamethasone.
2. The method as claimed in claim 1, wherein the induction culture is carried out for 7d to 10d at 37 ℃ and CO2Volume fraction 5%, relative humidity 95%.
3. The method provided by claim 1, wherein the adipose-derived stem cells are trypsinized and fused before induction culture, and specifically: taking adipose-derived stem cells of the third generation to the fifth generation, adding trypsin with the mass fraction of 0.25% and EDTA with the mass fraction of 0.02% for digestion, stopping digestion with serum, centrifuging at 1200r/min for 5min, resuspending the cells with a DMEM/F12 culture medium, and adjusting the density to1×104cell/mL, and culturing until the fusion is 40-50%.
4. The method according to any one of claims 1 to 3, further comprising the steps of performing amplification culture and subculture after the induction culture.
5. The method as claimed in claim 4, wherein the medium for the expanded culture and subculture is the epidermal cell serum-free medium SFM.
6. The method according to claim 4, wherein the conditions for the amplification culture and the subculture are 37 ℃ and CO2Volume fraction 5%, saturated humidity.
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