CN113174368A - Application of bone morphogenetic protein in promoting differentiation of human skin fibroblasts into adipocytes - Google Patents
Application of bone morphogenetic protein in promoting differentiation of human skin fibroblasts into adipocytes Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to application of bone morphogenetic protein in promoting differentiation of human skin fibroblasts into adipocytes. The application specifically comprises the steps that before the human skin fibroblasts are induced to be differentiated into fat cells, the human skin fibroblasts are pretreated by adopting bone morphogenetic proteins; the concentration of the bone morphogenetic protein is 50-400 ng/mL, and the treatment time is 12-48 h. The method adopts bone morphogenetic protein for pretreatment before inducing the human skin fibroblasts to differentiate into the adipocytes, effectively improves the efficiency of the human skin fibroblasts to differentiate into the adipocytes, and provides a new thought and a new method for clinical wound repair, scar repair and treatment of lipodystrophy.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of bone morphogenetic protein in promoting differentiation of human skin fibroblasts into adipocytes.
Background
Bone Morphogenetic Proteins (BMPs), also known as bone morphogenetic proteins, are a group of highly conserved functional proteins with similar structures and belong to the TGF- β family. The bone morphogenetic protein can stimulate mesenchymal cells to directionally differentiate into osteoblasts, is a main factor for inducing the formation of bones and cartilages in vivo, and plays an important role in the development, regeneration and repair of bones. Bone morphogenic proteins are widely present in tissues such as embryos, blood cells, kidneys and spleens of pigs, cows, sheep, rabbits, mice and humans, and not only participate in regulation of bones, but also play a role in development of kidneys, livers, bones and nervous systems.
In the prior art, various reagent combinations are used for promoting the human skin fibroblasts to be differentiated into the adipocytes, but the efficiency of differentiating the human skin fibroblasts into the adipocytes is relatively low in the methods, so that the methods are difficult to be widely used for wound repair, scar repair, lipodystrophy treatment and the like in clinic.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides application of bone morphogenetic protein in promoting differentiation of human skin fibroblasts into adipocytes.
In a first aspect, the present invention provides the use of a bone morphogenetic protein for promoting the differentiation of human skin fibroblasts into adipocytes.
The invention discovers that the efficiency of the human skin fibroblasts for differentiating into the adipocytes can be effectively improved by adopting the bone morphogenetic protein for pretreatment before inducing the human skin fibroblasts to differentiate into the adipocytes.
In a second aspect, the present invention provides a method for promoting differentiation of human skin fibroblasts into adipocytes, comprising:
pretreating human skin fibroblasts with bone morphogenetic protein before inducing the human skin fibroblasts to differentiate into adipocytes;
the concentration of the bone morphogenetic protein is 50-400 ng/mL, and the treatment time is 12-48 h.
Further, the concentration of the bone morphogenetic protein is 100-200 ng/mL, and the treatment time is 12-24 h.
Further, the bone morphogenetic protein is bone morphogenetic protein 2 or bone morphogenetic protein 4.
Further, the pretreatment process is carried out when the confluence of the human skin fibroblasts reaches 50-70%.
Further, the inducing the differentiation of human skin fibroblasts into adipocytes is:
placing the pretreated human skin fibroblasts into a first differentiation culture medium for culturing for 6-10 days; then placing the mixture in a second differentiation culture medium to culture for 2-4 days; then placing the mixture in a basic culture medium for culturing for 8-12 days;
the first differentiation culture medium is a basic culture medium additionally added with at least two of dexamethasone, insulin, rosiglitazone or 1-methyl-3-isobutyl xanthine;
and the second differentiation culture medium is a basal culture medium additionally added with insulin.
Further, the basic culture medium comprises: 10 to 20 percent of fetal calf serum, 50 to 150U/mL of penicillin and 50 to 150U/mL of streptomycin.
Further, the first differentiation medium is a basic medium additionally added with 0.5-1 mu M dexamethasone, 10-20 mu g/mL insulin, 1-2 mu M rosiglitazone and 0.5-1 mM 1-methyl-3-isobutyl xanthine;
and the second differentiation culture medium is a basal culture medium additionally added with 10-20 mu g/mL of insulin.
Furthermore, the generation number of the human skin fibroblasts is less than or equal to 10.
The invention further provides the fat cell prepared by the method.
The invention has the following beneficial effects:
before the induction differentiation of human skin fibroblasts, the invention firstly carries out bone morphogenetic protein pretreatment on the cells, and then carries out the induction differentiation according to a differentiation mode of '6 +2+ 8'. The mode of 'firstly pretreating bone morphogenetic protein and then inducing differentiation' greatly improves the efficiency of differentiating human skin fibroblasts into fat cells and improves the efficiency by about 100 percent.
The induction method provided by the invention can efficiently induce and differentiate human skin fibroblasts into adipocytes, thereby providing a new thought and a new method for clinical wound repair, scar repair and treatment of lipodystrophy.
Drawings
FIG. 1 shows the results of oil red staining provided in example 1 of the present invention.
FIG. 2 is a statistical result of oil red staining provided in example 1 of the present invention; p <0.01, P <0.001, P <0.0001, and the differences between the different treatment groups were statistically significant.
FIG. 3 shows the detection results of the marker genes of mature adipocytes provided in example 1 of the present invention; p <0.01, P <0.001, P <0.0001, and the differences between the different treatment groups were statistically significant.
FIG. 4 shows the results of oil red staining provided in example 2 of the present invention.
FIG. 5 shows the statistical results of oil red staining provided in example 2 of the present invention; p <0.01, P <0.001, P <0.0001, and the differences between the different treatment groups were statistically significant.
FIG. 6 shows the detection results of the marker genes of mature adipocytes provided in example 2 of the present invention; p <0.01, P <0.001, P <0.0001, and the differences between the different treatment groups were statistically significant.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
This example provides a method of promoting differentiation of human skin fibroblasts into adipocytes:
1. acquisition of Primary human skin fibroblasts (HDF)
(1) Removing skin tissue with a diameter of 1mm from volunteers, and rinsing with Phosphate Buffered Saline (PBS) for 3 times;
(2) removing excessive adipose tissue with ophthalmologic scissors in an intercellular superclean bench, and cutting the remaining tissue, washing with PBS 3 times, and placing into 10cm cell culture dish;
(3) the PBS was aspirated off, 1-2mL of 0.25% pancreatin was added, all tissue pieces were submerged and placed in a container containing 5% CO2Digesting for 5-10 minutes at 37 ℃ in the cell culture box;
(4) after digestion, adding 1mL of common culture medium to terminate digestion, and then gently and repeatedly blowing and sucking by using a pipette gun to blow and disperse cell clusters;
(5) transferring the cell suspension to 35mm petri dishes, adding 4mL of common culture medium, placing in a cell incubator under 5% CO2Culturing at 37 deg.C for 4-6 days;
(6) as the culture proceeds, the culture medium may be appropriately supplemented depending on the amount of the culture medium in the culture dish. Finally, the separated cells are primary human skin fibroblasts;
after the cells grow full, subculturing is carried out;
in the whole cell separation process, the aseptic operation is ensured, and the pollution is avoided. And (4) carrying out mycoplasma detection after the cells are separated out, and carrying out subsequent experiments after confirming that no mycoplasma is polluted.
In this embodiment, multiple induced differentiation experiments find that the HDF cell induced differentiation efficiency is lower when the number of generations of HDF cells used for induced differentiation is higher, and the higher the number of generations of cells is, the harder the induced differentiation is, and the lower the differentiation efficiency is. Therefore, in this example, the subsequent induced differentiation experiment was performed using HDF cells within 10 passages.
2. Induced differentiation of cells
2.1 Induction of differentiation
(1) Taking out the cover glass soaked in 75% alcohol, burning and sterilizing on an alcohol lamp, and then paving in 35mm culture dishes, wherein 4 cover glass are paved on each culture dish;
(2) add 4mL of normal media to the dish and spread the overlapping coverslips with forceps for later cell passage.
(3) After the primary HDF cells in the 10cm culture dish were overgrown, the culture medium in the culture dish was aspirated off, and washed 2 times with 2mL of Phosphate Buffered Saline (PBS) each time; then sucking off PBS, adding 1mL of pancreatin with the concentration of 0.25%, and repeatedly shaking the culture dish by two hands to enable the pancreatin to uniformly infiltrate the bottom of the culture dish; finally placing in a container containing 5% CO2Digesting for 1-2min at 37 ℃ in the cell culture box;
(4) the digested cells were removed and 0.5mL basal medium was added to the culture dish to stop the digestion; then repeatedly blowing and sucking by using a pipettor, uniformly blowing and transferring the cells into a 1.5mL centrifuge tube, and centrifuging for 4 minutes at 1000 rpm/min;
(5) sucking off the supernatant, adding 1mL of a basal medium, slightly and repeatedly sucking, and uniformly mixing the cells; then, according to the following steps of 1: 4, evenly dividing the cell suspension into 4 35mm culture dishes paved with cover glass, repeatedly shaking the culture dishes by two hands to uniformly mix the cells, and then putting the cells in a cell culture box for culture;
(6) after the confluence degree of the cells reaches 50-70%, adding bone morphogenetic protein 2 with the final concentration of 200ng/mL, and pretreating the cells for 24 hours;
(7) sucking off the basic culture medium, adding 3mL of differentiation medium I, putting the cell culture box back for continuous induction culture for 6 days, and replacing the fresh culture medium every two days; when the culture medium is replaced, the culture medium is gently added along the wall of the culture dish;
(8) sucking off the first differentiation medium, adding 3mL of a second differentiation medium, and performing induced culture for 2 days; as the induced differentiation proceeded, a shiny circular fat drop was found in the cells under a 10-fold microscope;
(9) absorbing the differentiation medium II, adding 3mL of basic medium, continuing culturing for 8 days, and replacing the fresh medium every two days; under a microscope, cells containing fat drops are more and more, and fat drops in the cells are larger and larger;
(10) after the induction differentiation is finished, taking out the cover glass in the culture dish for oil red staining, and detecting the induction differentiation effect; in addition, RNA of the residual cells of the culture dish is extracted, fluorescent quantitative PCR is carried out, the expression of marker genes Adiponectin, Fabp4 and Leptin of the mature adipocytes is detected, and the induced differentiation result is further verified.
Basic culture medium: high-sugar Dulbecco's Modified Eagle Medium (DMEM) + 20% fetal bovine serum +100U/mL penicillin + 100. mu.g/mL streptomycin;
differentiation medium one: dexamethasone (Dexamethasone), insulin (insulin), Rosiglitazone (Rosiglitazone), 3-isobutyl-1-methyl xanthine (1-methyl-3-isobutylxanthine) were added to the basal medium to give final concentrations of 1. mu.M, 20. mu.g/mL, 2. mu.M, 0.5mM, respectively;
and (3) differentiation medium II: to the basal medium, insulin was added at a final concentration of 20. mu.g/mL.
3. Analysis of induced differentiation results
3.1 oil Red dyeing
Oil red O is a very strong fat solvent and dye agent, capable of binding triglycerides and is therefore frequently used for fat dyeing. In this example, fat droplets in mature adipocytes were detected by oil red staining of cells after induced differentiation. The specific operation is as follows:
(1) the coverslip was washed 3 times with PBS and then fixed with 4% Paraformaldehyde (PFA) for 20 minutes;
(2) after 3 washes with PBS, equilibrate with 60% isopropanol for 5 minutes;
(3) hermetically dyeing with oil red O (dissolved in 60% isopropanol) at a concentration of 3g/L for 10 minutes;
(4) washing with 60% isopropanol for 3 times;
(5) staining with hematoxylin for 2 minutes;
(6) washing with PBS for 3 times, air drying, and sealing to form image;
(7) counting the number of cells which are positive in oil red staining;
3.2 detection of marker Gene of mature adipocytes
Adipoectin, Fabp4 and Leptin are marker genes for mature adipocytes, and are often used to measure the effect of adipocyte differentiation induction. In this example, Trizol was used to extract total RNA from induced cells, reverse transcription was performed, and then fluorescent quantitative PCR was performed to detect the expression levels of three genes, i.e., Adiponectin, Fabp4, and Leptin.
The PCR primer sequences used were as follows:
Adiponectin:5’-GATGGCAGAGATGGCAC-3’
5’-GCTGAGCGGTATACATAGG-3’
Fabp4:5’-ACGAGAGGATGATAAACTGGTGG-3’
5’-GCGAACTTCAGTCCAGGTCAAC-3’
Leptin:5’-CACCAAAACCCTCATCAAGACA-3’
5’-CTTTCTGTTTGGAGGAGACTGACT-3’
4. analysis of detection results
The results of oil red staining are shown in FIG. 1, where the dark parts are stained fat, and the uninduced HDF cells have no fat accumulation, while the HDF cells induced to differentiate for 16 days have significant fat accumulation. Compared with a control group, after being pretreated for 24 hours by 200ng/mL BMP2, the induction can obviously promote the differentiation of fat cells;
the statistical results of oil red staining are shown in fig. 2, where the number of cells positive for oil red staining in the non-induced HDF cells is 0, and the number of cells positive for oil red staining in the HDF cells induced to differentiate for 16 days are: 475 pieces/cm of control group2200ng/mL BMP2 pretreatment group 950/cm2Compared with a control group, the pretreatment group is improved by about 100 percent;
mature adipocyte marker gene detection As shown in FIG. 3, in this example, by quantitative PCR (quantitative PCR), marker genes of three mature adipocytes, i.e., Adiponectin, Fabp4, and Leptin, were detected. The expression of Fabp4 and Leptin of 200ng/mL BMP2 pretreatment group is obviously higher than that of a control group.
Example 2
The culture medium and the specific experimental procedures involved in this example were the same as in example 1, and the "6 +2+ 8" induced differentiation mode was also used. Except that the pretreatment was changed to 200ng/mL BMP4 pretreatment for 24 hours.
After induction differentiation is finished, the result of induction differentiation is detected through oil red staining and mature fat cell marker gene detection. The results are as follows:
the results of oil red staining are shown in FIG. 4, where the dark parts are stained fat, and the uninduced HDF cells have no fat accumulation, while the HDF cells induced to differentiate for 16 days have significant fat accumulation. Compared with a control group, after being pretreated for 24 hours by 200ng/mL BMP4, the induction can obviously promote the differentiation of fat cells;
the statistical results of oil red staining are shown in fig. 5, where the number of cells positive for oil red staining in the non-induced HDF cells is 0, and the number of cells positive for oil red staining in the HDF cells induced to differentiate for 16 days are: control group 260 pieces/cm2200ng/mL BMP4 pretreatment446 pieces/cm in group2；
The detection results of the marker genes of mature adipocytes are shown in FIG. 6, and in this example, by quantitative PCR (quantitative PCR), the marker genes of Adiponectin, Fabp4, and Leptin were detected. The expression of adipoectin, Fabp4 and Leptin in the BMP4 pretreatment group at 200ng/mL is obviously higher than that in the control group.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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Claims (10)
1. Application of bone morphogenetic protein in promoting differentiation of human skin fibroblasts into adipocytes.
2. A method of promoting differentiation of human skin fibroblasts into adipocytes, comprising:
pretreating human skin fibroblasts with bone morphogenetic protein before inducing the human skin fibroblasts to differentiate into adipocytes;
the concentration of the bone morphogenetic protein is 50-400 ng/mL, and the treatment time is 12-48 h.
3. The method according to claim 2, wherein the concentration of the bone morphogenetic protein is 100-200 ng/mL and the treatment time is 12-24 h.
4. The method according to claim 2 or 3, wherein said bone morphogenetic protein is bone morphogenetic protein 2 and/or bone morphogenetic protein 4.
5. The method of claim 2, wherein the pretreatment procedure is performed when the human skin fibroblasts reach a confluency of 50-70%.
6. The method according to any one of claims 2 to 5, wherein the inducing differentiation of human skin fibroblasts into adipocytes is:
placing the pretreated human skin fibroblasts into a first differentiation culture medium for culturing for 6-10 days; then placing the mixture in a second differentiation culture medium to culture for 2-4 days; then placing the mixture in a basic culture medium for culturing for 8-12 days;
the first differentiation culture medium is a basic culture medium additionally added with at least two of dexamethasone, insulin, rosiglitazone or 1-methyl-3-isobutyl xanthine;
and the second differentiation culture medium is a basal culture medium additionally added with insulin.
7. The method of claim 6, wherein the basal medium is a high-glucose DMEM medium comprising 10-20% fetal bovine serum, 50-150U/mL penicillin and 50-150U/mL streptomycin.
8. The method according to claim 6 or 7, wherein the first differentiation medium is a basal medium supplemented with 0.5-1 μ M dexamethasone, 10-20 μ g/mL insulin, 1-2 μ M rosiglitazone and 0.5-1 mM 1-methyl-3-isobutylxanthine;
and/or the presence of a gas in the gas,
and the second differentiation culture medium is a basal culture medium additionally added with 10-20 mu g/mL of insulin.
9. The method of any one of claims 2-8, wherein the number of passages of the human skin fibroblasts is less than or equal to 10.
10. Adipocytes produced by a method according to any one of claims 2 to 9.
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| CN110093309A (en) * | 2018-01-29 | 2019-08-06 | 中国科学院动物研究所 | A kind of induced fibroblast transdifferentiation is the method for fat cell |
| CN110592003A (en) * | 2019-09-17 | 2019-12-20 | 北京大学 | Method for inducing human skin fibroblast to differentiate into fat cell in vitro |
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| CN110093309A (en) * | 2018-01-29 | 2019-08-06 | 中国科学院动物研究所 | A kind of induced fibroblast transdifferentiation is the method for fat cell |
| CN110592003A (en) * | 2019-09-17 | 2019-12-20 | 北京大学 | Method for inducing human skin fibroblast to differentiate into fat cell in vitro |
| CN111518753A (en) * | 2020-04-07 | 2020-08-11 | 北京大学 | Application of insulin-like growth factor 2 in promoting differentiation of human skin fibroblasts into adipocytes |
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