CN110862959B - Method for inducing pluripotent adipose-derived stem cells - Google Patents
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Abstract
The invention discloses a method for inducing pluripotent adipose-derived stem cells, wherein sesamin F is applied in the culture process. The method comprises the following steps: (1) separating and culturing rat ADSCs; (2) osteogenic induced differentiation: the method comprises the steps of taking 3 rd generation ADSCs with good growth state, conventionally digesting, terminating digestion, centrifuging, resuspending cells, inoculating 100 cells/hole into a 6-hole plate with a cover glass, changing an experimental group into an osteogenesis induction culture medium 1 after the cells are completely attached to the wall, carrying out osteogenesis induction culture for 1 day, changing a liquid for 2 days, changing into an osteogenesis induction culture medium 2, and continuing osteogenesis induction for 7 days.
Description
Technical Field
The invention relates to a method for inducing pluripotent adipose-derived stem cells, belonging to the technical field of bioengineering.
Background
An adult stem cell refers to an undifferentiated cell that is present in an already differentiated tissue, which cell is capable of self-renewal and differentiation to form the cells of the tissue. Adult stem cells are present in various tissues and organs of the body. Adult stem cells in tissues of adult individuals are normally mostly dormant and may exhibit varying degrees of regenerative and renewal capacity in pathological states or under exogenous induction. Adult stem cells have a limited proliferative capacity compared to embryonic stem cells. Embryonic stem cells are pluripotent in terms of their differentiation potential and can form a variety of cells in vivo, whereas somatic stem cells can differentiate into only certain cell types of specific tissues. Adult stem cells are ubiquitous and the problem is how to find and isolate a variety of tissue-specific stem cells. Adult stem cells are often located in specific microenvironments. Mesenchymal cells in the microenvironment are capable of producing a range of growth factors or ligands that interact with stem cells to control their turnover and differentiation.
Among adult stem cells, bone marrow mesenchymal stem cells have been studied more frequently. However, the traditional bone marrow extraction method causes some pain to the patient, usually requires general anesthesia or intraspinal anesthesia, and has the disadvantages of small quantity, long time consumption for extracorporeal expansion and high price. There are two types of adipose tissue in an animal, one brown adipose tissue and one white adipose tissue. Mainly distributed on the shoulders and feet of the animals just born, and distributed in the body into two types, one is subcutaneous fat, and the other is visceral fat. Currently, scholars at home and abroad have obtained adipose stem cells from the two adipose tissues respectively. Because the adipose-derived stem cells have the characteristics of easy availability, safety, rapid amplification, multidirectional differentiation capability and the like, the adipose-derived stem cells become a class of adult stem cells with wide treatment application prospect.
The ability of adipose stem cells to differentiate into adipocytes, myocytes, chondrocytes, osteocytes, nerve cells, and the like, i.e., the mechanism of differentiation, is not known. Bone grafting is often required to treat common bone diseases in orthopedics, such as fracture, bone tumor, and bone tuberculosis, which can cause bone defects. The bone tissue engineering technology can be used for repairing human body defected bone tissue adipose-derived stem cells which can be used as seed cells of bone tissue engineering, so that the osteogenic differentiation efficiency of the bone tissue engineering is improved, and the problem to be solved is urgent.
The invention aims to provide a method for inducing pluripotent adipose-derived stem cells, in particular to a method for efficiently inducing differentiation from adipose-derived stem cells to osteoblasts, which has great application potential.
Disclosure of Invention
The invention aims to provide a method for inducing pluripotent adipose-derived stem cells, which particularly has great application potential for efficiently inducing differentiation from adipose-derived stem cells to osteoblasts.
Ganoderma lucidum (Ganoderma resinaceum Boud), also known as round-hole Ganoderma lucidum, Flat Ganoderma lucidum, Sanxiu Ganoderma lucidum, etc., as 1 species of Ganoderma genus, like Ganoderma lucidum and Ganoderma sinense, have been used for the prevention and treatment of various diseases for a long time. Podophenamine and the like isolate several compounds from sessile ganoderma, of which sessile ganoderma element F is 1 new heteroterpene. The sesamin F is yellow solid, and has a molecular formula as follows: c20H28O6. The spatial correlation structure is as follows:
the group of the invention unexpectedly discovers that the sesaminol F can be applied to the induction of pluripotent fat stem cells, particularly to the efficient induction of differentiation from fat stem cells to osteoblasts, and has great application potential in bone tissue engineering technology.
The research and development team applies the sesaminol F to the induction of the pluripotent adipose-derived stem cells into osteoblasts for the first time, and the osteoblasts are used as seed cells of bone tissue engineering to improve the osteogenic differentiation efficiency, so that the application has very important significance.
The technical problem to be solved by the invention can be realized by the following technical scheme.
A method of inducing pluripotent adipose stem cells, comprising:
during the culture process, sesamin F without stems is used.
Preferably, the steps are as follows:
(1) carrying out subculture on the primary ADSCs by separation culture;
(2) osteogenic induced differentiation:
taking 3 rd generation ADSCs with good growth state, digesting, terminating digestion, centrifuging, resuspending cells, inoculating 100 cells/well into a 6-well plate with a cover glass, changing the experimental group into an osteogenesis induction culture medium 1 after the cells are completely attached to the wall, carrying out osteogenesis induction culture for 1 day, changing the liquid for 2 days, changing into an osteogenesis induction culture medium 2, and continuing osteogenesis induction for 7d;
the osteogenesis inducer culture medium 1 and the osteogenesis inducer culture medium 2 are culture media containing sesaminol F.
More preferably, the osteogenesis inducer culture medium 1 and the osteogenesis inducer culture medium 2 are DMEM culture media containing sesaminol F.
Preferably, the osteogenesis inducer medium 1: DMEM medium containing 10% FBS, 0.1. mu. mol/L dexamethasone, 50mg/L vitamin C, and 20mg/L sessile ganoderin F.
Preferably, the osteogenesis inducer medium 2: DMEM medium containing 10% FBS, 50mg/L vitamin C, 100mg/L sesamin F.
Preferably, differentiation culture is followed by identification using alizarin red staining method.
Further preferably, the method comprises the following steps:
(1) separating and culturing rat ADSCs:
the methods of the references (Zuk P A, Zhu M, Mizuno H, et al, Multilineage cells from human subjects: experiments for cell-based therapeutics [ J ]. tissue Eng, 2001, 7: 211-28.): 1 healthy SD rat of 3-4 weeks is taken, soaked in 75% alcohol for 15min after anesthesia by 10% chloral hydrate, separated from subcutaneous fat of inguinal in an ultra-clean bench, and washed 3 times by PBS containing 1% double antibody. The adipose tissue was minced, and then a double volume of 0.1% collagenase i solution was added and mixed well. Placing the sealed centrifuge tube in a 37 deg.C constant temperature water bath kettle, slowly shaking for digestion for 60min, centrifuging at 1200r/min for 20 min, and removing upper layer oil, undigested adipose tissue and fibrous connective tissue. Phosphate Buffer Saline (PBS) resuspends the cells, filters the cells with a 200 μm filter screen, centrifuges the cells for 10 min at 1200r/min, removes the floating oil on the upper liquid surface, and repeats the above steps until the suspended oil is removed. The cells were resuspended in LG-DMEM containing 10% Fetal Bovine Serum (FBS), inoculated into a T25 flask, and cultured in a shaking incubator at 37 ℃ and 1 time per 3 days. After the primary cultured cells are fused into slices, subculture is carried out according to the proportion of 1: 3.
(2) Osteogenic induced differentiation:
taking 3 rd generation ADSCs with good growth state, conventionally digesting, terminating digestion, centrifuging, resuspending cells, inoculating 100 cells/well into a 6-well plate with a cover glass, changing an experimental group into an osteogenesis induction culture medium 1 after the cells are completely attached to the wall, carrying out osteogenesis induction culture for 1 day, changing a liquid for 2 days, changing into an osteogenesis induction culture medium 2, and continuing osteogenesis induction for 7 days;
osteogenic inducer medium 1: DMEM medium containing 10% FBS, 0.1. mu. mol/L dexamethasone, 50mg/L vitamin C, and 20mg/L sessile ganoderin F.
Osteogenic inducer medium 2: DMEM medium containing 10% FBS, 50mg/L vitamin C, 100mg/L sesamin F.
And (3) identification: when the alizarin red staining is observed under a mirror, the positive result is that the cell matrix is stained red by alizarin red. With specific reference to the prior art: washing with PBS for 2 times, fixing with 95% ethanol for 10 min, washing with PBS for 2 times, adding 1 ml of 0.2% alizarin red-Tris-HCl into each well, incubating for 30 min in dark, washing with PBS for 2 times, and observing under an inverted microscope.
Generally speaking, the induction of osteoblasts positive for alizarin red staining from adipose-derived stem cells takes 2-3 weeks, but the method of the present invention can significantly shorten the time to 6 days, greatly shorten the induction time, and improve the cell activity. Osteoporotic fracture (OPF), the most serious complication of osteoporosis, has become an intractable disease that seriously harms the quality of life of patients and occupies medical resources in today's society. After fracture of osteoporosis patients, the treatment period is long, the complications are many and the death rate is increased due to the weakening of osteoblast osteogenesis capacity and the enhancement of osteoclast bone resorption capacity. Therefore, how to promote fracture healing while treating primary diseases, shorten the treatment period and reduce the incidence of secondary fracture is a difficult point and hot point in the current osteoporosis fracture treatment. The adipose-derived stem cell induction method has great potential in the treatment of osteoporosis and fracture.
The invention has the advantages that:
adipose-derived mesenchymal stem cells (also called adipose-derived stem cells) have strong proliferation capacity and multidirectional differentiation potential, and can meet the requirements of tissue engineering on the number and types of cells, so that the adipose-derived mesenchymal stem cells are considered to be reliable sources of seed cells applied to bone tissue engineering. The invention innovatively applies the sessile ganoderin F to induce the pluripotent fat stem cells to quickly and efficiently (8 d) and differentiate the pluripotent fat stem cells into osteoblasts, and the sessile ganoderin F is derived from sessile ganodermalnoids, so that the long-term safety of the sessile ganoderin F is guaranteed, and the sessile ganoderin F has the potential of being directly applied to the bodies.
Drawings
FIG. 1 shows adipose-derived stem cells in primary culture for 3 d.
FIG. 2 is a graph showing the positive results of differentiation induction using the method of the present invention.
FIG. 3 shows a negative control group.
FIG. 4 shows that the bone induction culture of adipose-derived stromal stem cells in Oricell TMSD rat bone induction differentiation medium is performed for 8 days, and alizarin red staining is negative.
FIG. 5 shows that alizarin red stains positively in osteogenic differentiation induction medium for osteogenic differentiation induction culture of Oricell TMSD rat adipose-derived stromal cells for 3 weeks.
Detailed Description
The following examples of the present invention are described in detail, and are only for the purpose of illustrating the present invention and are not to be construed as limiting the present invention.
Specific examples of the present invention are described below.
Example 1
A method of inducing pluripotent adipose stem cells, comprising:
(1) separating and culturing rat ADSCs:
the methods of the references (Zuk P A, Zhu M, Mizuno H, et al, Multilineage cells from human subjects: experiments for cell-based therapeutics [ J ]. tissue Eng, 2001, 7: 211-28.): 1 healthy SD rat of 3-4 weeks is taken, soaked in 75% alcohol for 15min after anesthesia by 10% chloral hydrate, separated from subcutaneous fat of inguinal in an ultra-clean bench, and washed 3 times by PBS containing 1% double antibody. The adipose tissue was minced, and then a double volume of 0.1% collagenase i solution was added and mixed well. Placing the sealed centrifuge tube in a 37 deg.C constant temperature water bath kettle, slowly shaking for digestion for 60min, centrifuging at 1200r/min for 20 min, and removing upper layer oil, undigested adipose tissue and fibrous connective tissue. Phosphate Buffer Saline (PBS) resuspends the cells, filters the cells with a 200 μm filter screen, centrifuges the cells for 10 min at 1200r/min, removes the floating oil on the upper liquid surface, and repeats the above steps until the suspended oil is removed. The cells were resuspended in LG-DMEM containing 10% Fetal Bovine Serum (FBS), inoculated into a T25 flask, and cultured in a shaking incubator at 37 ℃ and 1 time per 3 days. After the primary cultured cells were fused into pieces, the ratio of 1: 3, subculturing. Primary culture 3d is shown in FIG. 1.
(2) Osteogenic induced differentiation:
taking 3 rd generation ADSCs with good growth state, conventionally digesting, terminating digestion, centrifuging, resuspending cells, inoculating 100 cells/well into a 6-well plate with a cover glass, changing an experimental group into an osteogenesis induction culture medium 1 after the cells are completely attached to the wall, carrying out osteogenesis induction culture for 1 day, changing a liquid for 2 days, changing into an osteogenesis induction culture medium 2, and continuing osteogenesis induction for 7 days;
osteogenic inducer medium 1: DMEM medium containing 10% FBS, 0.1. mu. mol/L dexamethasone, 50mg/L vitamin C, and 20mg/L sessile ganoderin F.
Osteogenic inducer medium 2: DMEM medium containing 10% FBS, 50mg/L vitamin C, 100mg/L sesamin F.
And (3) identification: when the alizarin red staining is observed under a mirror, the positive result is that the cell matrix is stained red by alizarin red. With specific reference to the prior art: washing with PBS for 2 times, fixing with 95% ethanol for 10 min, washing with PBS for 2 times, adding 1 ml of 0.2% alizarin red-Tris-HCl into each well, incubating for 30 min in dark, washing with PBS for 2 times, and observing under an inverted microscope. As shown in fig. 2, alizarin red stains red and the cell viability is high. (all experiments were repeated three times)
Example 2
OricellTMSD rat adipose-derived mesenchymal stem cell osteogenic induction differentiation medium kit (cyagen, China, cat # RAMD-90021) was used as a control.
A method of inducing pluripotent adipose stem cells, comprising:
(1) separating and culturing rat ADSCs:
the methods of the references (Zuk P A, Zhu M, Mizuno H, et al, Multilineage cells from human subjects: experiments for cell-based therapeutics [ J ]. tissue Eng, 2001, 7: 211-28.): 1 healthy SD rat of 3-4 weeks is taken, soaked in 75% alcohol for 15min after anesthesia by 10% chloral hydrate, separated from subcutaneous fat of inguinal in an ultra-clean bench, and washed 3 times by PBS containing 1% double antibody. The adipose tissue was minced, and then a double volume of 0.1% collagenase i solution was added and mixed well. Placing the sealed centrifuge tube in a 37 deg.C constant temperature water bath kettle, slowly shaking for digestion for 60min, centrifuging at 1200r/min for 20 min, and removing upper layer oil, undigested adipose tissue and fibrous connective tissue. Phosphate Buffer Saline (PBS) resuspends the cells, filters the cells with a 200 μm filter screen, centrifuges the cells for 10 min at 1200r/min, removes the floating oil on the upper liquid surface, and repeats the above steps until the suspended oil is removed. The cells were resuspended in LG-DMEM containing 10% Fetal Bovine Serum (FBS), inoculated into a T25 flask, and cultured in a shaking incubator at 37 ℃ and 1 time per 3 days. After the primary cultured cells are fused into slices, subculture is carried out according to the proportion of 1: 3.
(2) Osteogenic induced differentiation:
taking 3 rd generation ADSCs with good growth state, conventionally digesting, terminating digestion, centrifuging, resuspending cells, inoculating 100 cells/hole into a 6-hole plate with a cover glass, and changing the experimental group into an Oricell TMSD rat adipose-derived stromal stem cell osteogenic induction differentiation medium kit after the cells are completely attached to the wall to perform osteogenic induction culture for 8 days and 3 weeks respectively;
and (3) identification: when the alizarin red staining is observed under a mirror, the positive result is that the cell matrix is stained red by alizarin red. With specific reference to the prior art: washing with PBS for 2 times, fixing with 95% ethanol for 10 min, washing with PBS for 2 times, adding 1 ml of 0.2% alizarin red-Tris-HCl into each well, incubating for 30 min in dark, washing with PBS for 2 times, and observing under an inverted microscope.
The osteogenesis induction culture was carried out for 8d as shown in FIG. 4, and alizarin red staining was negative as shown in FIG. 5, respectively, and the culture was carried out for 3 weeks (all experiments were repeated three times).
Example 3
DMEM medium was used as a negative control.
A method of inducing pluripotent adipose stem cells, comprising:
(1) separating and culturing rat ADSCs:
the methods of the references (Zuk P A, Zhu M, Mizuno H, et al, Multilineage cells from human subjects: experiments for cell-based therapeutics [ J ]. tissue Eng, 2001, 7: 211-28.): 1 healthy SD rat of 3-4 weeks is taken, soaked in 75% alcohol for 15min after anesthesia by 10% chloral hydrate, separated from subcutaneous fat of inguinal in an ultra-clean bench, and washed 3 times by PBS containing 1% double antibody. The adipose tissue was minced, and then a double volume of 0.1% collagenase i solution was added and mixed well. Placing the sealed centrifuge tube in a 37 deg.C constant temperature water bath kettle, slowly shaking for digestion for 60min, centrifuging at 1200r/min for 20 min, and removing upper layer oil, undigested adipose tissue and fibrous connective tissue. Phosphate Buffer Saline (PBS) resuspends the cells, filters the cells with a 200 μm filter screen, centrifuges the cells for 10 min at 1200r/min, removes the floating oil on the upper liquid surface, and repeats the above steps until the suspended oil is removed. The cells were resuspended in LG-DMEM containing 10% Fetal Bovine Serum (FBS), inoculated into a T25 flask, and cultured in a shaking incubator at 37 ℃ and 1 time per 3 days. After the primary cultured cells are fused into slices, subculture is carried out according to the proportion of 1: 3.
(2) Osteogenic induced differentiation:
taking 3 rd generation ADSCs with good growth state, conventionally digesting, terminating digestion, centrifuging, resuspending cells, inoculating 100 cells/well into a 6-well plate with a cover glass in advance, and changing the experimental group into a DMEM culture medium for culture for 8 days after the cells are completely attached to the wall;
and (3) identification: when the alizarin red staining is observed under a mirror, the positive result is that the cell matrix is stained red by alizarin red. With specific reference to the prior art: washing with PBS for 2 times, fixing with 95% ethanol for 10 min, washing with PBS for 2 times, adding 1 ml of 0.2% alizarin red-Tris-HCl into each well, incubating for 30 min in dark, washing with PBS for 2 times, and observing under an inverted microscope. As shown in fig. 3, negative results (the experiments were repeated three times).
It can be seen that the induction of 8d significantly differentiated into osteoblasts using the induction method of the present invention. No significant change was observed in osteogenic induction differentiation medium 8d using commercially available Oricell TMSD rat adipose stromal stem cells until differentiation into osteoblasts was evident after 3 weeks. The invention innovatively applies the sessile ganoderin F to induce the pluripotent fat stem cells to quickly and efficiently differentiate into osteoblasts, and the sessile ganoderin F is derived from sessile ganoderma, so that the long-term safety of the sessile ganoderin F is guaranteed, and the sessile ganoderin F has the potential of being directly applied to the bodies.
It is to be understood that the foregoing is only a preferred embodiment of the invention and that modifications, variations and changes may be made in the invention without departing from the spirit or scope of the invention as defined in the appended claims.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (5)
1. A method of inducing pluripotent adipose stem cells, comprising:
during the induced differentiation culture process, sessile ganoderin F is applied, and the molecular formula of the sessile ganoderin F is as follows: c20H28O6。
2. The method for inducing pluripotent adipose stem cells according to claim 1, wherein the steps are as follows:
(1) carrying out subculture on the primary ADSCs by separation culture;
(2) osteogenic induced differentiation:
taking 3 rd generation ADSCs with good growth state, digesting, terminating digestion, centrifuging, resuspending cells, inoculating 100 cells/well into a 6-well plate with a cover glass, changing the experimental group into an osteogenesis induction culture medium 1 after the cells are completely attached to the wall, carrying out osteogenesis induction culture for 1 day, changing the liquid for 2 days, changing into an osteogenesis induction culture medium 2, and continuing osteogenesis induction for 7d;
the osteogenesis inducer culture medium 1 and the osteogenesis inducer culture medium 2 are culture media containing sesaminol F.
3. The method of inducing pluripotent adipose stem cells of claim 2, wherein the pluripotent adipose stem cells are:
the osteogenesis inducer culture medium 1 and the osteogenesis inducer culture medium 2 are DMEM culture media containing sesaminol F.
4. The method of inducing pluripotent adipose stem cells of claim 3, wherein the pluripotent adipose stem cells are:
the osteogenesis inducer culture medium 1: DMEM medium containing 10% FBS, 0.1 μmol/L dexamethasone, 50mg/L vitamin C, and 20mg/L sessile ganoderin F;
the osteogenesis inducer culture medium 2: DMEM medium containing 10% FBS, 50mg/L vitamin C, 100mg/L sesamin F.
5. The method of inducing pluripotent adipose stem cells according to any one of claims 1 to 4, wherein the method comprises:
differentiation culture and identification by alizarin red staining method.
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