CN116850193B - Method for slowing down skin aging by regulating hair follicle stem cells - Google Patents
Method for slowing down skin aging by regulating hair follicle stem cells Download PDFInfo
- Publication number
- CN116850193B CN116850193B CN202310627929.6A CN202310627929A CN116850193B CN 116850193 B CN116850193 B CN 116850193B CN 202310627929 A CN202310627929 A CN 202310627929A CN 116850193 B CN116850193 B CN 116850193B
- Authority
- CN
- China
- Prior art keywords
- stem cells
- hair follicle
- follicle stem
- taurolidine
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 57
- 210000003780 hair follicle Anatomy 0.000 title claims abstract description 53
- 230000009759 skin aging Effects 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title abstract description 8
- 230000001105 regulatory effect Effects 0.000 title abstract description 5
- AJKIRUJIDFJUKJ-UHFFFAOYSA-N taurolidine Chemical compound C1NS(=O)(=O)CCN1CN1CNS(=O)(=O)CC1 AJKIRUJIDFJUKJ-UHFFFAOYSA-N 0.000 claims abstract description 50
- 229960004267 taurolidine Drugs 0.000 claims abstract description 50
- 210000003491 skin Anatomy 0.000 claims abstract description 17
- 230000008439 repair process Effects 0.000 claims abstract description 13
- 230000032683 aging Effects 0.000 claims abstract description 10
- 239000000203 mixture Substances 0.000 claims description 14
- 230000000694 effects Effects 0.000 claims description 13
- 238000009472 formulation Methods 0.000 claims description 11
- 230000009758 senescence Effects 0.000 claims description 10
- 230000002708 enhancing effect Effects 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 abstract description 8
- 108090000623 proteins and genes Proteins 0.000 abstract description 8
- 239000006143 cell culture medium Substances 0.000 abstract description 4
- 230000001934 delay Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 41
- 239000002609 medium Substances 0.000 description 15
- 239000012528 membrane Substances 0.000 description 13
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 12
- 239000006285 cell suspension Substances 0.000 description 8
- 238000001514 detection method Methods 0.000 description 7
- 239000002033 PVDF binder Substances 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 6
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 102000006311 Cyclin D1 Human genes 0.000 description 5
- 108010058546 Cyclin D1 Proteins 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 101710142276 Protein P21 Proteins 0.000 description 4
- 239000006180 TBST buffer Substances 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000002791 soaking Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 102000005936 beta-Galactosidase Human genes 0.000 description 3
- 108010005774 beta-Galactosidase Proteins 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 210000004209 hair Anatomy 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 208000032840 Catheter-Related Infections Diseases 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 102100033270 Cyclin-dependent kinase inhibitor 1 Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 101000596404 Homo sapiens Neuronal vesicle trafficking-associated protein 1 Proteins 0.000 description 1
- 101000891649 Homo sapiens Transcription elongation factor A protein-like 1 Proteins 0.000 description 1
- 208000019693 Lung disease Diseases 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 229940121363 anti-inflammatory agent Drugs 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 230000032677 cell aging Effects 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 1
- 229960002327 chloral hydrate Drugs 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000019612 pigmentation Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 208000037922 refractory disease Diseases 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000007665 sagging Methods 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 210000004927 skin cell Anatomy 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229910052979 sodium sulfide Inorganic materials 0.000 description 1
- GRVFOGOEDUUMBP-UHFFFAOYSA-N sodium sulfide (anhydrous) Chemical compound [Na+].[Na+].[S-2] GRVFOGOEDUUMBP-UHFFFAOYSA-N 0.000 description 1
- 238000009168 stem cell therapy Methods 0.000 description 1
- 238000009580 stem-cell therapy Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
- 230000037314 wound repair Effects 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
- A61K31/549—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame having two or more nitrogen atoms in the same ring, e.g. hydrochlorothiazide
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/18—Antioxidants, e.g. antiradicals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0627—Hair cells
- C12N5/0628—Hair stem cells; Hair progenitors
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Dermatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Birds (AREA)
- Developmental Biology & Embryology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Cell Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Engineering & Computer Science (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Cosmetics (AREA)
Abstract
A method for slowing down skin aging by regulating hair follicle stem cells belongs to the technical field of skin aging. The invention cultures the hair follicle stem cells by using the cell culture medium containing Taurolidine (Taurolidine), effectively delays the aging of the hair follicle stem cells and regulates and controls the proteins related to the aging of the stem cells. Meanwhile, the present invention has found that hair follicle stem cells treated with Taurolidine can repair damaged skin faster than untreated hair follicle stem cells. Therefore, the invention expands the new application of Taurolidine, effectively solves the limitation of the prior application, and improves the practicability.
Description
Technical Field
The invention belongs to the technical field of skin aging, and particularly relates to a method for slowing down skin aging by regulating and controlling hair follicle stem cells.
Background
Stem cells are a class of cells that sustain the development of vital tissue and organs and repair and regeneration of lesions. With the rapid development of regenerative medicine in recent years, stem cell therapy is increasingly widely applied in clinic and has remarkable curative effect. It has become the ultimate therapeutic approach for many refractory diseases, and stem cell research has become a research hotspot worldwide.
Hair follicle stem cells are a population of stem cells that have the ability to self-renew and differentiate, and can be derived into various types of skin cells. Skin aging is a complex process in which a variety of cellular factors and molecular mechanisms play an important role, and changes in hair follicle stem cells are also considered one of them.
Recent studies have shown that the number and function of hair follicle stem cells gradually decrease with age. This results in a series of aging manifestations of the skin, such as loss of elasticity, skin sagging, increased wrinkles, pigmentation, dryness, and susceptibility to injury. In addition, either intrinsic or extrinsic stimuli may affect the function of hair follicle stem cells, further accelerating skin aging.
Thus, the relationship between hair follicle stem cells and skin aging is very close, and changes in hair follicle stem cells may be an early sign of skin aging. Protecting and promoting the health of hair follicle stem cells may be an important prophylactic and therapeutic strategy to delay or reduce the occurrence of skin aging.
Disclosure of Invention
The invention aims to provide a method for slowing down skin aging by regulating hair follicle stem cells, so that the skin aging is effectively delayed or reduced.
To achieve the above objects, in one aspect, the present invention provides a method for slowing skin aging by modulating hair follicle stem cells by culturing hair follicle stem cells in a cell culture medium containing Taurolidine, thereby slowing the aging rate of the hair follicle stem cells and enhancing the skin repair function of the hair follicle stem cells.
Preferably, the concentration of Taurolidine in the cell culture medium is 5 μg/ml to 125 μg/ml, and the cell culture medium is DMEM medium.
Preferably, the concentration of Taurolidine in the medium is 125 μg/ml.
In another aspect, the invention provides the use of a composition comprising Taurolidine as the core active ingredient in the preparation of a formulation for reducing skin ageing.
Preferably, the composition further comprises an acceptable carrier compound, a salt-soluble or water-soluble compound.
Preferably, the composition achieves the effect of slowing skin aging by slowing the rate of aging of hair follicle stem cells and enhancing the skin repair function of hair follicle stem cells.
In another aspect, the invention provides a formulation for reducing skin aging, wherein the core active ingredient of the formulation is Taurolidine.
Preferably, the formulation further comprises an acceptable carrier compound, a salt-soluble or water-soluble compound.
Preferably, the concentration of Taurolidine in the formulation is from 5 μg/ml to 125 μg/ml.
Preferably, the concentration of Taurolidine in the formulation is 125 μg/ml.
In another aspect, the invention provides the use of Taurolidine in the preparation of a formulation for reducing the senescence of hair follicle stem cells, wherein the core active ingredient of the formulation is Taurolidine, and the concentration of Taurolidine in the formulation is 125 μg/ml.
In another aspect, the invention provides the use of Taurolidine for enhancing the skin repair function of hair follicle stem cells.
The invention has the beneficial effects that:
the inventor researches and discovers that the aging speed of the hair follicle stem cells can be slowed down by treating the hair follicle stem cells with Taurolidine. In addition, hair follicle stem cells treated with Taurolidine can promote repair of the skin.
Drawings
FIG. 1 shows the results of the detection of the effect of Taurolidine on hair follicle stem cell activity;
*p＜0.05，***p＜0.001；
FIG. 2 shows the results of the detection of the effect of Taurolidine on the expression of senescence-promoting protein P21 and senescence-inhibiting protein Cyclin-D1 in hair follicle stem cells;
FIG. 3 shows the results of the detection of the effect of Taurolidine on the senescence marker beta-galactosidase in hair follicle stem cells;
**p＜0.01，***p＜0.001；
FIG. 4 is a graph showing the effect of Taurolidine-treated hair follicle stem cells on skin repair;
***p＜0.001。
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
Taurolidine (Taurolidine) is a broad-spectrum antimicrobial agent (anti-inflammatory agent) used to prevent central venous catheter-related infections. Taurolidine has a direct and selective anti-tumor effect on brain tumor cells by inducing apoptosis (apoptosis). Meanwhile, in the research of application of the invention patent CN113491700A Taurolidine in antiviral, the Taurolidine can be used for preventing and treating lung diseases caused by influenza virus or coronavirus. However, no literature report exists on the application of Taurolidine to hair follicle stem cells.
Example 1: detection of the Activity influence of Taurolidine on hair follicle Stem cells
The experimental steps are as follows:
1) Resuscitate the frozen human hair follicle stem cells, passaging to P3 generation, preparing the cells into cell suspension, inoculating into 96-well plate, and the number of cells per well is 1×10 5 Putting the 96-well plate into a constant temperature cell incubator, setting the conditions to be 37 ℃ and 5% CO 2 ；
2) When the cell density reaches about 75%, serum-free culture medium containing Taurolidine (5 mug/ml, 25 mug/ml, 125 mug/ml, 625 mug/ml, 3125 mug/ml) with different concentrations is added, each concentration is respectively provided with 3 compound holes, a control group without Taurolidine is arranged, and 3 compound holes are also arranged;
3) After further incubation for 24h, 10 μl CCK-8 was added for 2.5h, absorbance at 450nm was detected and cell viability was calculated from the absorbance of the control group.
Experimental results:
the experimental results of example 1 are shown in FIG. 1, and from FIG. 1, we can see that when the concentration of Taurolide used is 5. Mu.g/ml, 25. Mu.g/ml, 125. Mu.g/ml, there is no effect on the viability of the cells, and when the concentrations of Taurolide used are 625. Mu.g/ml and 3125. Mu.g/ml, the viability of the hair follicle stem cells is reduced, so we select 5. Mu.g/ml, 25. Mu.g/ml, 125. Mu.g/ml of Taurolide for the subsequent study.
Example 2: detection of the effects of Taurolidine on the expression of senescence-promoting protein P21 and senescence-inhibiting protein Cyclin-D1 in hair follicle Stem cells
The experimental steps are as follows:
1. cell treatment
1) Preparing P3 generation human hair follicle stem cells into cell suspension and inoculating the cells into 10mm cell culture dishes, and adding 2ml DMEM complete medium into each dish;
2) After the cells are completely attached, changing the group a into 2ml of DMEM complete medium, changing the group b into 2ml of DMEM complete medium containing 5 mug/ml Taurolidine, and changing the group c into 2ml of DMEM complete medium containing 25 mug/ml Taurolidine; group d was replaced with 2ml DMEM complete medium containing 125 μg/ml Taurolidine;
3) When the cell density reaches 85% fusion, digesting the cells for passage, and when the cells reach P9 generation, removing the culture medium;
4) Washing 3 times with precooled PBS, removing redundant dead cells, adding 150 μl of cell lysis solution, and gently shaking on ice for 30min;
5) Scraping cells, collecting the scraped cells into a centrifuge tube, centrifuging 12000g at 4 ℃ for 15min, and collecting a supernatant;
6) The BCA method is used for measuring the protein concentration, the volume of a solution containing 20 mug of protein is calculated as the loading quantity, the loading sample is removed to a 1.5ml centrifuge tube, and 5 Xloading buffer solution is added to the final concentration of 1X;
7) The sample was boiled in boiling water for 5min to substantially denature the protein, resulting in a protein sample.
SDS-PAGE gel electrophoresis
1) Cleaning a glass plate and a sample comb by using a detergent, washing for a plurality of times by using ultrapure water, airing, and preparing 12% of separating glue and 5% of concentrating glue after the glass plate is installed;
2) Pouring the separating gel to a proper height, adding ultrapure water, and removing the ultrapure water and sucking water after the separating gel is completely solidified;
3) Adding concentrated gel, inserting into comb, completely solidifying, extracting electrophoresis comb, mounting electrophoresis frame, and adding electrophoresis buffer solution;
4) Sequentially adding a Marker and a protein sample into a sample hole, installing an electrophoresis device, running concentrated gel at a constant voltage of 90V, running separation gel at a constant voltage of 120V, and stopping electrophoresis until bromophenol blue just runs out;
3. transfer film
1) Placing the gel in 1X membrane transferring solution for standby, cutting out PVDF membrane with the same size as the gel, placing in methanol for activation for 30s, and then soaking in 1X membrane transferring buffer solution;
2) Soaking filter paper in a 1X membrane transferring buffer solution, placing a sandwich structure according to the positive-negative sequence of the membrane, ensuring no bubble, clamping, putting into a membrane transferring instrument, and adding the membrane transferring buffer solution;
3) Constant current 300mA for 2 hours, washing 3 times by using TBST on a decolorizing shaking table at room temperature after film transfer, and 10 minutes each time;
4. immunological detection
1) Soaking PVDF membrane with 5% skimmed milk powder, and sealing at room temperature on decolorizing shaking table for 1 hr;
2) Discarding skimmed milk powder, adding TBST to wash PVDF membrane for 3 times each for 10min, adding diluted primary antibodies of Cyclin-D1, P21 and GAPDH, and incubating overnight at 4deg.C;
3) Recovering the primary antibody, adding TBST to wash the PVDF membrane for 3 times, 10min each time, adding a secondary antibody solution, and incubating for 1h at room temperature;
4) Recovering the secondary antibody, adding TBST to wash the membrane for 3 times, preparing a luminescent liquid in a dark state for 10min each time, uniformly mixing, soaking the PVDF membrane in the luminescent liquid for 3min, and then placing the PVDF membrane into a luminescence imager for exposure and collecting images.
Experimental results: the experimental results of example 2 are shown in FIG. 2, and it can be seen from FIG. 2 that the expression of senescence inhibitory protein Cyclin-D1 in cells cultured using a medium containing Taurolidine increases with increasing concentration;
meanwhile, the expression of senescence-promoting protein P21 in cells is continuously decreased with the increase of the concentration;
thus, it can be derived that Taurolidine treatment can inhibit the protein expression of senescence-promoting protein P21 and can promote the protein expression of senescence-inhibiting protein Cyclin-D1.
Example 3: detection of the effect of Taurolidine on the senescence marker beta-galactosidase in hair follicle stem cells
The experimental steps are as follows:
1) Preparing P3 generation human hair follicle stem cells into cell suspension and inoculating the cells into 10mm cell culture dishes, and adding 2ml DMEM complete medium into each dish;
2) After the cells are completely attached, changing the group a and the group b into 2ml of DMEM complete medium, and changing the group c into 2ml of DMEM complete medium containing 125 mug/ml Taurolidine;
3) The cells of the group b and the group c are digested and passaged when the cell density reaches 85% fusion, and the culture medium is removed when the cells reach the P9 generation, and the group a is not passaged (P3 generation cells);
4) Removing the culture medium when the cell fusion degree of a, b and c reaches about 85%, cleaning by using PBS, and adding a cell fixing solution to fix cells for 15min at room temperature;
5) Removing the fixing solution, adding PBS to wash the cells for 2 times, adding beta-galactosidase staining solution, sealing the cell culture plate by using a sealing film, and incubating at 37 ℃ overnight;
6) After the staining solution was discarded and washed with PBS, a photograph was taken under a microscope and the positive rate of the cells was calculated.
Experimental results:
the results of example 3 are shown in FIG. 3, and it can be seen from FIG. 3 that the number of cell senescence in cells cultured from the P3 generation to the P9 generation using Taurolidine is significantly reduced as compared with cells not cultured using Taurolidine, indicating that cell senescence in cells cultured using a medium containing Taurolidine is significantly alleviated.
Example 4: detecting effect of hair follicle stem cells treated by Taurolidine on skin repair
As hair follicle stem cells in skin age, the skin repair ability is also weakened, and further skin aging is caused, so that improving the skin repair ability of hair follicle stem cells is also an effective means for slowing down skin aging.
The experimental steps are as follows:
1. cell treatment
1) Preparing P3 generation human hair follicle stem cells into cell suspension and inoculating the cells into 10mm cell culture dishes, and adding 2ml DMEM complete medium into each dish;
2) After the cells are completely attached, changing the group a into 2ml of DMEM complete medium, and changing the group b into 2ml of DMEM complete medium containing 125 mug/ml Taurolidine;
3. when the cell density reaches 85% fusion, the cells are digested for passage, when the cells are transferred to P9 generation, the cells are digested, and PBS is used for preparing the cells into 2.5X10 6 Cell suspension a and cell suspension b per ml;
2. wound repair
1) Balb/C mice at 8 weeks of age were kept under standard experimental conditions with 12 hours of light-12 hours of dark cycle, free to take water and food;
2) The mice are anesthetized by intraperitoneal injection of 10% chloral hydrate, and after the mice are completely anesthetized, the mice are fixed so that the backs of the mice face upwards;
3) Taking the spine of the back of the mouse as a middle line, shearing off hairs by using scissors, removing hairs on the back of the mouse by using 8% sodium sulfide, and wiping off the hairs by using clear water;
4) After alcohol sterilization, a circle with the diameter of 0.8cm is cut by a scalpel;
5) Randomly dividing into 2 groups of 5 cells, uniformly coating a cell suspension a in group a and uniformly coating a cell suspension b in group b;
6) Wounds were photographed and the healing rate calculated on day 0 and day 7, respectively.
Experimental results:
the results of example 4 are shown in fig. 4, and it can be seen from fig. 4 that the P9-generation hair follicle stem cells treated with Taurolidine can significantly improve the wound healing rate compared with untreated P9-generation hair follicle stem cells.
In view of the above, it can be seen that the treatment of hair follicle stem cells with Taurolidine is effective in slowing down the aging rate of hair follicle stem cells, and at the same time, taurolidine is effective in promoting skin repair, so that Taurolidine can slow down skin aging by slowing down hair follicle stem cell aging and maintaining the function of hair follicle stem cells.
Claims (3)
- The use of taurolidine for the preparation of a formulation for slowing the senescence of hair follicle stem cells and improving the skin repair function of hair follicle stem cells.
- Use of taurolidine for the preparation of a formulation for reducing skin ageing.
- 3. The use according to claim 2, wherein the Taurolidine effects a slowing of skin aging by slowing the rate of aging of hair follicle stem cells and enhancing the skin repair function of hair follicle stem cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310627929.6A CN116850193B (en) | 2023-05-29 | 2023-05-29 | Method for slowing down skin aging by regulating hair follicle stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310627929.6A CN116850193B (en) | 2023-05-29 | 2023-05-29 | Method for slowing down skin aging by regulating hair follicle stem cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116850193A CN116850193A (en) | 2023-10-10 |
| CN116850193B true CN116850193B (en) | 2024-01-30 |
Family
ID=88227554
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310627929.6A Active CN116850193B (en) | 2023-05-29 | 2023-05-29 | Method for slowing down skin aging by regulating hair follicle stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116850193B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107375903A (en) * | 2017-07-27 | 2017-11-24 | 广东科玮生物技术股份有限公司 | Skin repair spraying and its production and use |
| CN108430476A (en) * | 2015-10-07 | 2018-08-21 | 科医公司 | The Cutaneous permeation preparation of Taurolidine |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017059321A1 (en) * | 2015-10-02 | 2017-04-06 | Robert Diluccio | Composition for soft tissue augmentation providing protection from infection |
| WO2020234829A1 (en) * | 2019-05-22 | 2020-11-26 | Geistlich Pharma Ag | Methods and compositions for inhibiting gapdh |
-
2023
- 2023-05-29 CN CN202310627929.6A patent/CN116850193B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108430476A (en) * | 2015-10-07 | 2018-08-21 | 科医公司 | The Cutaneous permeation preparation of Taurolidine |
| CN107375903A (en) * | 2017-07-27 | 2017-11-24 | 广东科玮生物技术股份有限公司 | Skin repair spraying and its production and use |
Non-Patent Citations (3)
| Title |
|---|
| Studies on the Function of Taurine: Review;Yoon, Jin A等;The Korean Journal of Food And Nutrition;第28卷(第5期);第880-893页 * |
| The antibacterial substance taurolidine exhibits anti-neoplastic action based on a mixed type of programmed cell death;Stendel, Ruediger等;Autophagy;第5卷(第2期);第194-210页 * |
| 牛磺酸对小鼠抗氧化和DNA修复能力的作用;沈芳兰等;营养学报;第18卷(第03期);第258-262页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116850193A (en) | 2023-10-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105535022A (en) | Application of exosome to preparing of acute-hepatic-failure treating medicine and medicine composition | |
| EP2368974A1 (en) | Methods for isolating mesenchymal stem cells from embryos of human or animals and extracting secretion substances thereof | |
| WO2018120683A1 (en) | Multi-component injection liquid | |
| CN110269833A (en) | A kind of umbilical cord mesenchymal stem cells preparation and its preparation method and application | |
| CN109136174B (en) | Stem cell-derived exosome preparation for delaying senescence | |
| JPH02174848A (en) | Cuticle sheet;and production, storage and usage thereof | |
| CN110903348B (en) | Small peptide for promoting wound healing and application thereof | |
| Feizi et al. | Effect of amniotic fluid on the in vitro culture of human corneal endothelial cells | |
| CN111549000B (en) | Recombinant adipose-derived stem cell for over-expression of Hpgds, preparation method and application thereof | |
| Zhu et al. | Bioorthogonal DOPA-NGF activated tissue engineering microunits for recovery from traumatic brain injury by microenvironment regulation | |
| Ding et al. | Galectin-1-induced skeletal muscle cell differentiation of mesenchymal stem cells seeded on an acellular dermal matrix improves injured anal sphincter | |
| CN116850193B (en) | Method for slowing down skin aging by regulating hair follicle stem cells | |
| CN113846064A (en) | FGF18 gene modified mesenchymal stem cell and preparation method and application thereof | |
| CN111518743A (en) | Method for constructing human dermal fibroblast in-vitro thermal injury model and application thereof | |
| CN116426469B (en) | Application of LAP2 alpha in mesenchymal stem cell adipogenic differentiation | |
| KR102131569B1 (en) | Composition for hair growth containing fermented Artemisia Vulgaris extract | |
| CN110747162B (en) | Application of small molecular compound 4-aminobiphenyl in promoting stem cell proliferation and chondrogenic differentiation | |
| CN108715831A (en) | Application of the mescenchymal stem cell conditioned medium in the cellular damage for improving cardiomyocytes induced by hypoxia and reoxygenation | |
| CN111888379A (en) | Application of stem cell-derived microvesicles in preparation of scar repairing preparation | |
| Li et al. | Effective component of Salvia miltiorrhiza in promoting cardiomyogenic differentiation of human placenta‑derived mesenchymal stem cells | |
| CN116077517A (en) | Application of miR-582-5p in preparation of medicines for preventing premature skin aging | |
| KR101943203B1 (en) | Process for differentiation into dermal papilla cells from adipose-derived stem cells and uses thereof | |
| CN103468744A (en) | VEGF165 gene modified hair follicle stem cells and preparation method thereof | |
| HIRSCHBERG et al. | Stimulation of human lymphocytes by cultured allogeneic skin and endothelial cells in vitro | |
| JP6188249B2 (en) | Wound healing composition derived from porcine umbilical cord tissue, method for producing the same, and pharmaceutical product for wound healing containing the composition |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |