CN114410577B - Method and device for repairing stem cell aging - Google Patents
Method and device for repairing stem cell aging Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M31/00—Means for providing, directing, scattering or concentrating light
- C12M31/12—Rotating light emitting elements
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2529/00—Culture process characterised by the use of electromagnetic stimulation
- C12N2529/10—Stimulation by light
Abstract
The invention belongs to the technical field of stem cell preparation, and particularly relates to a method and a device for repairing stem cell aging. The method for repairing stem cell aging is to irradiate mesenchymal stem cells with red light, green light and yellow light, wherein the wavelength of the red light is 609-657nm, the wavelength of the green light is 485-567nm, and the wavelength of the yellow light is 578-623nm. The invention further provides a device for realizing the method. The method and the device provided by the invention can reduce the expression of senescence genes of stem cells, reduce the number of senescence cells, and further remarkably increase the production capacity of mesenchymal stem cell exosomes, and have good clinical application prospect.
Description
Technical Field
The invention belongs to the technical field of stem cell preparation, and particularly relates to a method and a device for repairing stem cell aging.
Background
Mesenchymal Stem Cells (MSCs) are cells derived from adult tissues and having self-renewal and multi-directional differentiation ability, and widely exist in tissue organs such as bone marrow, skeletal muscle, umbilical cord, fat, skin, and the like. The mesenchymal stem cells have great application value in aspects of organ regeneration, repair and disease treatment.
Aging or biological aging is the gradual degradation of a functional characteristic of an organism. Aging refers to cellular aging, and also to aging of the whole body. At least later in the life cycle of an organism, with age, aging of the organism involves an increase in mortality and/or a decrease in fertility. Regenerative medicine is widely concerned with the development of cell therapies using Mesenchymal Stem Cells (MSCs) and their application to several diseases associated with aging. Successful treatment requires a large number of cells and thus extensive in vitro cell expansion is required. However, proliferation of bone marrow mesenchymal stem cells is limited, long-term culture is likely to cause continuous changes in bone marrow mesenchymal stem cells, and a significant portion of cells may undergo aging.
During disease progression, bone marrow mesenchymal stem cells undergo cellular senescence and mediate senescence-associated secretory phenotypes (SASPs), affecting the surrounding microenvironment. Thus, senescent bone marrow mesenchymal stem cells can accelerate tissue senescence by increasing the number of senescent cells and diffusing inflammation to neighboring cells. This results in that the in vitro culture passage of the mesenchymal stem cells can not maintain stable yield and quality at present, so that the mesenchymal stem cells lose normal functions.
Repairing aged mesenchymal stem cells, reducing inflammation penetration, improving surrounding microenvironment, and remodelling normal functions of cells and tissues. In the prior art, the repairing method of the aging mesenchymal stem cells is mainly implemented by drug intervention, for example, the literature' rule of aging induced bone marrow stem cell evolution and the intervention effect of traditional Chinese medicine, DOI: CNKI, SUN, zzyz.0.2009-01-044, intervenes in the senescence of stem cells by traditional Chinese medicine. However, since various drugs have adverse effects on organisms such as biotoxicity or side effects to some extent, it is still desirable to develop a physical method for repairing aged mesenchymal stem cells without using any drugs.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method and a device for repairing stem cell aging, and aims to provide a physical method for repairing the aging mesenchymal stem cells.
A method for repairing stem cell aging comprises irradiating mesenchymal stem cells with red light, green light and yellow light, wherein the red light has a wavelength of 609-657nm, the green light has a wavelength of 485-567nm, and the yellow light has a wavelength of 578-623nm.
Preferably, the light intensity of the red light is 100-130mW/sr, the light intensity of the green light is 20-30mW/sr, and the light intensity of the yellow light is 150-200mW/sr.
Preferably, the time for which the red light, green light and yellow light irradiate the mesenchymal stem cells is 30-60min.
Preferably, the irradiation directions of the red light, the green light and the yellow light are rotated relative to the mesenchymal stem cells, and the rotation speed is 30-50 revolutions per minute.
Preferably, the mesenchymal stem cells are adipose-derived mesenchymal stem cells.
The invention also provides mesenchymal stem cells obtained after stem cell aging is repaired by the method.
The invention also provides a device for repairing stem cell aging, which comprises:
the fixing component is used for fixing the container filled with the mesenchymal stem cells;
the light source assembly is provided with a red light source, a green light source and a yellow light source for irradiating the container.
Preferably, the light source device further comprises a rotating mechanism, wherein the rotating mechanism is used for driving the fixed assembly or the light source assembly so that the fixed assembly and the light source assembly relatively rotate.
Preferably, the portable lighting device further comprises an outer cover, the fixing assembly and the light source assembly are arranged inside the outer cover, the outer cover is provided with a container inlet, the rotating mechanism comprises a rolling bearing, an outer ring of the rolling bearing is fixedly connected with the inner wall of the container inlet, an inner ring of the rolling bearing is connected with the fixing assembly, and a driving device is connected with the inner ring of the rolling bearing.
Preferably, the light source assembly comprises a lamp barrel, a light source group is arranged on the lamp barrel and consists of a red light source, a green light source and a yellow light source, and a cavity for accommodating the container is arranged inside the lamp barrel.
According to the method and the device, the aged mesenchymal stem cells can be repaired by three light irradiation modes of red light, green light and yellow light, the expression of aged genes of the stem cells can be reduced, the number of the aged cells is reduced, and the yield and the quality of the cultured mesenchymal stem cells are obviously improved. The method is a pure physical method, only needs illumination, has no harm to mesenchymal stem cells and can not introduce medicines with toxic and side effects. Therefore, the invention has good application prospect.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 is a schematic diagram of the cell therapy transformation apparatus of example 2;
FIG. 2 is a schematic diagram showing the structure of a lamp tube in the cell therapy transformation apparatus of example 2;
FIG. 3 shows the primary cultured adipose-derived mesenchymal stem cells of Experimental example 1 cultured to 3 passages, and subcultured to 5 th, 7 th and 9 th passages after the cell therapy apparatus was subjected to light treatment for 30 minutes. RNA was extracted separately and quantitative PCR was performed to examine senescence genes p16, p21, p53 (< 0.05; p <0.05; #p < 0.05).
FIG. 4 shows the results of culturing adipose-derived stem cells to 3,9 passages in Experimental example 1, and culturing for 48 hours after 30 minutes of light treatment by a cytotherapeutic apparatus. The senescent cells were examined for beta-galactosidase. The blue color under the light microscope is senescent cells expressing β -galactosidase (< 0.05; #p < 0.05).
FIG. 5 is a diagram showing the detection of the expression of the exosome markers CD9, CD63, CD81 and TSG101 by Westernblotting.
The device comprises a 1-housing, a 2-lamp barrel, a 3-container inlet, a 4-liquid crystal display screen, a 5-fan, a 6-lamp holder, a 7-cavity, an 8-time reduction control key, a 9-time increase control key, a 10-instrument start key, an 11-on rotation control key and a 12-off rotation control key.
Detailed Description
It should be noted that, in the embodiments, algorithms of steps such as data acquisition, transmission, storage, and processing, which are not specifically described, and hardware structures, circuit connections, and the like, which are not specifically described may be implemented through the disclosure of the prior art.
Reagents and materials not specifically described in the examples were commercially available.
Example 1 methods for repairing Stem cell aging
Placing adipose-derived mesenchymal stem cells in a transparent needle cylinder, and irradiating the adipose-derived mesenchymal stem cells for 30min under an LED red light source, an LED green light source and an LED yellow light source with the rotation speed of 30 r/min, wherein the wavelength of the LED red light source is 620-625nm, and the light intensity is 120mW/sr; the wavelength of the LED green light source is 510-520nm, and the light intensity is 25mW/sr; the LED Huang Guangyuan has a wavelength of 585-590nm and a light intensity of 175mW/sr.
Example 2 device for repairing Stem cell aging
The embodiment provides a device (cell therapy transformation instrument) for repairing stem cell aging, the structure of which is shown in fig. 1 and 2, the device comprises a base, an outer cover 1 and a lamp tube 2 positioned in the outer cover, a container inlet 3 is arranged on one side of the outer cover 1, a liquid crystal display 4 and an instrument starting key 10 are arranged on the surface of the outer cover 1, a cavity 7 for accommodating a container (such as a transparent needle tube) filled with adipose-derived mesenchymal stem cells is arranged in the lamp tube 2, and one end of the cavity 7 is opened. The opening is directly opposite to the container inlet 3. The lamp tube 2 is provided with a light source group for illuminating the inside of the cavity 7, and the light source group is an array consisting of a red LED light source, a green LED light source and a yellow LED light source. The inside of the outer cover 1 is provided with a control board, the liquid crystal display 4, the light source group and the instrument start key 10 are electrically connected with the control board, and the outer cover 1 is fixedly connected with the base.
Further, the other side of the housing 1 opposite to the container inlet 3 has a fan 5, and the fan 5 is electrically coupled to the control board.
Further, a glass cover is arranged between the cavity 7 and the inner wall of the lamp tube 2.
Further, a rolling bearing is arranged at the container inlet 3, an outer ring of the rolling bearing is matched with the inner wall of the needle cylinder inlet 3, and inner rings of the rolling bearing extend out of two ends of the rolling bearing. The base is provided with a motor which is connected with one end of the inner ring through a belt. The other end of the inner ring is provided with three screw holes, screws are arranged in the screw holes, and one end with the screw holes is positioned at the outer side of the outer cover 1. The screw holes and the screws are used for installing a container filled with mesenchymal stem cells or a device for installing and fixing the container. The upper surface of the outer cover 1 is provided with an opening rotation control key 11 and a closing rotation control key 12, and the motor, the opening rotation control key 11 and the closing rotation control key 12 are electrically connected with a control panel.
Further, an increasing time control key 9 and a decreasing time control key 8 are arranged on the upper surface of the outer cover 1, and the increasing time control key 9 and the decreasing time control key 8 are electrically connected with the control panel.
Further, the top of the lamp tube 2 is provided with a lamp holder 6, the light source is arranged on the lamp holder 6 in a group, the lamp holders 6 are arranged in three rows in parallel, a red light LED light source, a green light LED light source and a yellow light LED light source are respectively installed, 10 lamp holders 6 are arranged in each row at equal intervals, and the row direction of the lamp holders 6 is parallel to the axis of the cavity 7.
Further, the outer cover 1 and the lamp tube 2 are made of medical lead-free stainless steel; the outer surface and the inner surface of the outer cover 1 and the outer surface of the lamp cylinder 2 are coated with baking finish, and the outer cover 1 is 17cm long, 14cm wide and 9.5cm high.
Methods of using the cytotherapeutic transformation apparatus of FIGS. 1 and 2:
the containers such as the needle cylinder enter the cavity 7 through the container inlet 3, screws in the screw holes are screwed, the needle cylinder and the inner ring of the rolling bearing are fixed together, the instrument start key 10 is started, the start rotation control key 11 is pressed down, and the red light LED light source, the green light LED light source and the yellow light LED light source are combined to stimulate cells in the needle cylinder. The built-in motor drives the inner ring of the rolling bearing to rotate through the belt, so that the needle cylinder is driven to rotate, the LED illumination is more uniform and sufficient, the aggregation phenomenon of cells can be prevented, and the cell growth activity is ensured. The irradiation time can be reduced or increased by reducing the time control key 8 or increasing the time control key 9, the fan 5 can radiate heat, and when the temperature reaches 25 ℃, the fan can be automatically started to ensure that the temperature of cells under the irradiation of the LED lamp is stably lower than 37 ℃; the liquid crystal display 4 can display time and temperature, the glass cover between the cavity 7 and the inner wall of the LED lamp tube 2 isolates the LED lamp from the detected sample and can balance the irradiation intensity of the LED lamp, and the motor and the fan 5 are controlled to be started and closed by controlling the automatic temperature and the illumination time of the panel.
The beneficial effects of the invention are further illustrated by experiments below.
Experimental example 1 repair of adipose-derived mesenchymal Stem cells
1. Instrument for measuring and controlling the intensity of light
The cell therapy transformation apparatus described in example 2.
2. Treatment method
1. Isolation of adipose-derived mesenchymal Stem cells
(1) Mice were sacrificed by dislocation and animals were sterilized with 70% ethanol for 5-10 minutes. The abdominal skin was dissected, the subcutaneous adipose tissue was exposed, and the subcutaneous adipose tissue was removed with sterile forceps and scissors.
(2) The extracted tissue was placed in a 6 cm pre-weighed petri dish. The tissue is weighed (N g) with an analytical balance.
(3) Add 1 XN ml (where N is the weight of adipose tissue in grams) of MSC isolation medium at room temperature to the dish containing adipose tissue.
(4) Adipose tissue was minced with sterile scissors until a fine cake was formed, and the minced tissue was accurately transferred to a 15 ml centrifuge tube using a 1ml filter with a cut end.
(5) 2.5 XN ml of MSC isolation medium was added to the petri dish, washed and transferred to place the remaining tissue pieces in the same 15 ml tube.
(6) The prepared collagenase solution 0.5xNml was added to a tube containing minced tissue and stirred at constant speed in a shaking flask at constant temperature of 37℃for 40 minutes at 200 revolutions per minute. The resulting cell suspension was blown up and down 20-30 times with a 10ml pipette until uniform.
(7) Centrifugation was performed at 22℃for 370g for 10 minutes, the supernatant was discarded, 1ml of mesenchymal stem cell isolation medium was added, and the cells were resuspended. The supernatant was discarded, 1ml was added, the cells were resuspended in 1ml of filtrate, 9ml of mesenchymal stem cell isolation medium was added, mixed, and centrifuged at 22℃for 10 minutes.
(8) Step 7 is repeated again. Finally, cells were resuspended in 1ml of mesenchymal stem cell growth medium.
(9) The total number of isolated cells was counted with a hemocytometer. (the average number of isolated cells at this stage is variable, but is usually around 5-15)
(10) 500-700 ten thousand cells are sown in each 10 cm culture dish, the growth medium of the mesenchymal stem cells is added in each culture dish, the final volume is 10ml, and CO is carried out at 37 ℃ under the condition of low oxygen 2 Culturing in an incubator.
(11) The cells were grown for approximately 5-7 days after culture passage to achieve 70-80% confluence, with mesenchymal stem cells replaced every 3 days the first and later. (the cells at this stage have a slightly elongated fibroblast-like shape).
(12) The dishes were tilted, carefully the medium was aspirated from the attached plastic, washed once with 1xPBS, 1ml trypsin solution was added to each 10 cm dish, and the cells were isolated at 37℃for 5-10 minutes.
(13) The cells were transferred to a 15 ml centrifuge tube, 5 ml of mesenchymal stem cell growth medium was added and gently mixed. Centrifuge at 370g for 7 min at room temperature. Cells were suspended in 1-2 ml of mesenchymal stem cell growth medium.
2. The experimental group is as follows: normal adipose-derived mesenchymal stem cells were subjected to no LED light irradiation (untreated group), and to an instrument LED light irradiation group (treated as in example 1, light-treated group). The stem cell culture passaging procedure, P3, P5, P7, P9, was followed for comparison as shown in fig. 3.
3. RNA extraction for quantitative real-time PCR analysis
Adipose-derived stem cells were collected and total RNA was extracted using TRIzol reagent (Invitrogen, carlsbad, calif., USA). RNA samples were pretreated with DNase I (Invitrogen Life Technologies, carlsbad, calif., USA) and cDNA was synthesized using the SuperScript kit (Invitrogen Life Technologies, carlsbad, calif., USA) according to manufacturer's recommendations. qRT-PCR was analyzed using miScript SYBR Green PCR Kits (Qiagen). Macrophage polarization and levels of oxidative stress marker mRNA were determined by ABI PRISM 7700 cycler (Applied Biosystems, foster City, calif.). Each sample was analyzed in duplicate with ribosomal 18S RNA as an internal control. All fold changes in gene expression were determined using the 2- ΔΔct method.
4. Senescence-associated-beta-galactosidase (SA-beta-gal) assays
SA- β -gal activity was measured using a senescence detection kit (ST 429, beyotidme) according to the manufacturer's instructions. Briefly, cells were incubated in ONPG for 12 hours at room temperature and then stained with the staining mixture overnight at 37 ℃ without CO2. Subsequently, the cells were observed and observed under an optical microscope (Zeiss HAL 100). These values were normalized to the total protein level assessed by the bicinchoninic acid (BCA) protein assay (Pierce).
3. Results:
as shown in fig. 3, the primary cultured adipose-derived mesenchymal stem cells were cultured to 3 passages, and after 30 minutes of light treatment by the cytotherapeutic apparatus, they were subcultured to 5 th, 7 th and 9 th passages. RNA was extracted and quantitative PCR was performed to examine senescence genes p16, p21, and p53, respectively. The results showed that the expression of senescence genes in the untreated group increased with the increase of passage, and that the senescence genes were significantly reduced by passage of culture to 5, 7, 9 passages after 30 minutes of light irradiation of the light-treated group, and maintained to 9 passages.
As shown in FIG. 4, adipose-derived stem cells were cultured to 3,9 passages, and the cell therapy apparatus was light-treated for 30 minutes and then cultured for 48 hours. The senescent cells were examined for beta-galactosidase. The blue color under the light microscope is senescent cells expressing beta-galactosidase. Showing a significant decrease in senescent cells in the light-treated group relative to the untreated group.
As shown in FIG. 5, westernblot identification of exosomes. After the cultured ASCs were subjected to light treatment for 30 minutes, the culture broth was collected to extract exosomes. The apocrine markers CD9, CD63, CD81, and TSG101 were examined. n=3/group.
4. Conclusion:
the adipose-derived mesenchymal stem cells are subjected to passaging, and after red, green and yellow light irradiation treatment, the anti-aging effect is enhanced, and the anti-aging efficiency is maintained.
The embodiment and experimental example show that the method and the device provided by the invention can reduce the expression of the senescence genes of the stem cells, reduce the number of the senescence cells, and further remarkably increase the production capacity of the exosomes of the mesenchymal stem cells, and have good clinical application prospect.
Claims (1)
1. A method of repairing stem cell senescence, comprising: the method comprises the steps of irradiating mesenchymal stem cells with red light, green light and yellow light, wherein the wavelength of the red light is 620-625nm, the wavelength of the green light is 510-520nm, and the wavelength of the yellow light is 585-590nm;
the light intensity of the red light is 120mW/sr, the light intensity of the green light is 25mW/sr, and the light intensity of the yellow light is 175mW/sr;
the time for irradiating the mesenchymal stem cells by the red light, the green light and the yellow light is 30 min;
the irradiation directions of the red light, the green light and the yellow light rotate relative to the mesenchymal stem cells, and the rotation speed is 30 revolutions per minute;
the mesenchymal stem cells are adipose-derived mesenchymal stem cells.
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