CN114642683B - Preparation method of dental pulp mesenchymal stem cell lysate with photoaging resistance - Google Patents
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Abstract
The invention provides a method for culturing dental pulp mesenchymal stem cells by using an optimized human platelet lysate culture system, so that dental pulp mesenchymal stem cells lysate is obtained under a low-temperature condition. The dental pulp mesenchymal stem cells obtained by the culture system have higher purity and rich growth factor content through detection. According to the invention, through in vitro experiments, the dental pulp mesenchymal stem cell can delay the aging of mouse fibroblasts subjected to ultraviolet treatment, promote the proliferation of cells, increase the content of type I collagen, and provide a key technology for clinically applying dental pulp mesenchymal stem cell lysate to resist photoaging.
Description
Technical Field
The invention belongs to the technical field of dental pulp mesenchymal stem cell treatment, and particularly relates to a preparation method of dental pulp mesenchymal stem cell lysate with light aging resistance.
Background
Aging is one of the most fundamental natural laws of the biological world, and skin aging has special significance as part of the overall aging of the body. Skin is not only the first line of defense of the human body, skin aging is not only related to many diseases, but also affects the beauty and even causes some psychological problems. With the development of stem cell technology, anti-aging treatment using 10 stem cells has also become a research hotspot.
At present, anti-aging researches are reported by using umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, fat stem cells, exosomes and lysate thereof, and the curative effects are confirmed. But its clinical transformation is limited due to seed cell sources and ethical issues. Dental pulp mesenchymal stem cells (DPSC) have the advantages of wide sources, no ethical problems and the like, and the human platelet lysate culture system has the characteristics of safety, high efficiency and the like, so that the dental pulp mesenchymal stem cell lysate can better meet clinical requirements.
Disclosure of Invention
In order to solve the problems, the invention provides a preparation method of dental pulp mesenchymal stem cell lysate with photoaging resistance.
The invention adopts the technical scheme that:
a preparation method of dental pulp mesenchymal stem cell lysate with photoaging resistance comprises the following steps:
step 1: preparing dental pulp mesenchymal stem cells;
step 2: putting dental pulp mesenchymal stem cells into an alpha-MEM culture medium added with human platelet lysate for culture;
step 3: resuspending the dental pulp mesenchymal stem cells obtained by culturing in the step 2 in ultrapure water, incubating for 10-30 min at 2-8 ℃, storing in a refrigerator at-80 ℃, taking out after 8-24 h, thawing at 2-8 ℃, and repeatedly freezing and thawing for 3-5 times;
step 4: centrifuging the cell suspension obtained in the step 3 at a low temperature of 2000-6000g at a temperature of 2-8 ℃ for 20-60 min, taking supernatant after centrifuging, and adding heparin sodium into the supernatant to obtain the dental pulp mesenchymal stem cell lysate.
Preferably, in step 1, the removed pulp tissue is cut into 1mm 3 The tissue pieces containing the double antibodies are washed 3 times by PBS leaves, then put into a 1.5ml EP tube, mixed and digested for 20-40 min by neutral enzyme and type I collagenase, placed into alpha-MEM culture medium added with human platelet lysate for culture, and after the cells are fused to about 80%, digested and centrifuged, and transferred to the fifth generation.
Preferably, the neutral enzyme is added in a mass concentration of 0.1-1%, and the type I collagenase is added in a mass concentration of 0.1-0.8%; the double antibody is added into the mixture to be 50-100U/mL penicillin and 50-150 ug/mL streptomycin.
Preferably, in step 2, after the dental pulp mesenchymal stem cells are cultured in the alpha-MEM medium added with the human platelet lysate for 36 to 72 hours, the culture is continued by changing the alpha-MEM medium added with the human platelet lysate, and the culture medium is changed every 36 to 72 hours during the subsequent culture.
Preferably, the volume concentration of the human platelet lysate in the alpha-MEM medium added with the human platelet lysate is 2-10%.
Preferably, in step 3, the concentration of cells of the resuspended dental pulp mesenchymal stem cells in ultrapure water is 5×10 6 ～1×10 8 。
Preferably, in the step 4, the concentration of the heparin sodium is 2-20 IU/ml.
The invention has the beneficial effects that:
1. the invention provides a method for culturing dental pulp mesenchymal stem cells by using an optimized human platelet lysate culture system, so that dental pulp mesenchymal stem cells lysate is obtained under a low-temperature condition. The dental pulp mesenchymal stem cells obtained by the culture system have higher purity and rich growth factor content through detection.
2. The dental pulp tissue is taken from waste or fallen wisdom teeth, no harm is caused to a donor, and the human platelet lysate culture system is efficient and safe and has no animal-derived factor. The cells obtained by the culture system can meet clinical requirements in quality and quantity, and are convenient to popularize and apply.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram showing changes in cell morphology during DPSC formation in accordance with an embodiment of the present invention, wherein A: primary DPSC 5 days after digestion, B: DPSC of the P5 generation;
FIG. 2 is a schematic diagram showing the detection of cell markers by flow cytometry in a human platelet lysate culture system and a serum culture system for DPSC according to the embodiment of the present invention;
FIG. 3 is a schematic diagram showing the detection of proliferation and migration of cells in a human platelet lysate culture system and a serum culture system for DPSC according to the present invention, wherein A: cell cloning experiments, B: cell scratch experiments;
FIG. 4 is a graph showing the measurement of the protein expression level of bFGF, KGF, VEGF, HGF in DPSC according to the embodiment of the present invention;
FIG. 5 is an electron microscope identification chart of DPSC lysate according to an embodiment of the present invention;
FIG. 6 is a schematic diagram showing the detection of aging and staining of cells of photo-aged mouse fibroblasts in vitro with DPSC lysate and deionized water added thereto, respectively, according to the present invention;
fig. 7 is a schematic diagram showing the detection of the proliferation and migration effects of DPSC lysate and deionized water on mouse fibroblasts under in vitro conditions, respectively, in the embodiment of the present invention, wherein a: cell cloning experiments, B: cell scratch experiments;
FIG. 8 is a graph showing the protein expression levels of collagenI, MMP-13 and ROCK II of photo-aged mouse fibroblasts in vitro with DPSC lysate and deionized water added thereto, respectively, in the examples of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
The embodiment provides a preparation method of dental pulp mesenchymal stem cell lysate with photoaging resistance, which comprises the following steps:
step 1: preparing dental pulp mesenchymal stem cells;
the extracted pulp tissue was cut into 1mm pieces 3 Is washed 3 times by PBS leaf containing double antibody, then put into a 1.5ml EP tube, mixed and digested for 20-40 min by neutral enzyme and type I collagenase, put into alpha-MEM culture medium added with human platelet lysate for culture, and the cells are fused to 8After about 0%, digesting and centrifuging, and transferring to a fifth generation;
step 2: putting dental pulp mesenchymal stem cells into an alpha-MEM culture medium added with human platelet lysate for culture;
step 3: resuspending the dental pulp mesenchymal stem cells obtained by culturing in the step 2 in ultrapure water, incubating for 10-30 min at 2-8 ℃, storing in a refrigerator at-80 ℃, taking out after 8-24 h, thawing at 2-8 ℃, and repeatedly freezing and thawing for 3-5 times;
step 4: centrifuging the cell suspension obtained in the step 3 at a low temperature of 2000-6000g at a temperature of 2-8 ℃ for 20-60 min, taking supernatant after centrifuging, and adding heparin sodium into the supernatant to obtain the dental pulp mesenchymal stem cell lysate. Wherein, the concentration of the added heparin sodium is 2-20 IU/mL, and the optimal concentration is 4IU/mL.
In step 2, after the dental pulp mesenchymal stem cells are cultured in the alpha-MEM medium added with the human platelet lysate for 36-72 hours, the culture is continued by replacing the alpha-MEM medium added with the human platelet lysate, and the culture medium is replaced every 48 hours during the subsequent culture. The volume concentration of the human platelet lysate in the alpha-MEM culture medium added with the human platelet lysate is 2-10%, and the optimal volume concentration is 5%.
In the step 2, the step of freezing and recovering the dental pulp mesenchymal stem cells is further included before the dental pulp mesenchymal stem cells are cultured. The freezing and storing step comprises the following steps: after digestion and centrifugation of dental pulp mesenchymal stem cells, re-suspending the dental pulp mesenchymal stem cells in serum-free frozen stock solution, putting the dental pulp mesenchymal stem cells into a gradient program frozen stock box, putting the dental pulp mesenchymal stem cells into a refrigerator at the temperature of minus 80 ℃ for 12-24 hours, and transferring the dental pulp mesenchymal stem cells into liquid nitrogen for long-term storage; the resuscitation steps include: taking out dental pulp mesenchymal stem cells from a liquid nitrogen tank, thawing at constant temperature in a constant-temperature water bath at 37 ℃, centrifuging, discarding supernatant, adding alpha-MEM culture medium of human platelet lysate to resuspend cell sediment, changing the liquid once every other day, and passaging once every 4 days.
In step 1, the mass concentration of the added neutral enzyme is 0.4%, and the mass concentration of the type I collagenase is 0.3%; the addition of the diabodies was 100U/mL penicillin and 100ug/mL streptomycin.
In step 3, the dental pulp mesenchymal stem cells are resuspended in ultrapureThe cell concentration in water was 5X 10 6 ～1×10 8 。
The method for culturing dental pulp mesenchymal stem cells by reasonably using human platelet lysate provides an optimized human platelet lysate culture system, so that dental pulp mesenchymal stem cells lysate is obtained under low temperature conditions, no animal-derived substances are required to be added in the operation process, and the method is safe and reliable, and can culture and amplify human dental pulp mesenchymal stem cells more quickly and efficiently. The dental pulp mesenchymal stem cells obtained by the culture system have higher purity and rich growth factor content through detection. The teeth are selected from the waste or fallen wisdom teeth, no harm is caused to the donor, no ethical problem exists, and the human platelet lysate culture system is efficient and safe and has no animal-derived factor. The cells obtained by the culture system can meet clinical requirements in quality and quantity, and are convenient to popularize and apply. Skin is the first line of defense of the organism, plays an important role in resisting various external invasion, maintaining homeostasis and guaranteeing physiological functions of the organism, and skin aging can cause psychological problems such as depression, spelt and the like besides physiological influence, wherein more than 80% of facial skin aging is photoaging. According to the invention, through in vitro experiments, the dental pulp mesenchymal stem cell can delay the aging of mouse fibroblasts subjected to ultraviolet treatment, promote the proliferation of cells, increase the content of type I collagen, and provide a key technology for clinically applying dental pulp mesenchymal stem cell lysate to resist photoaging.
Experimental part
1. Reagent and apparatus therefor
Dpsc (dental pulp mesenchymal stem cells): the 2-line human dental pulp mesenchymal stem cells constructed by the university of Wuhan regenerative medicine center are selected.
2. Cell culture reagent
alpha-MEM (Gbico, 12571063, gbico, inc., accession No. 12571063, the same applies hereinafter), human platelet lysate (STEMERY, RC-002-100), DPBS (Gbico, 14190144), collagenase type I (Sigma, SCR 103), neutral protease (Roche, 04942078001), heparin sodium z injection (Jiangsu Mo Bang, national standard 5H 32020612), serum-free frozen stock (Sigma, D2650), penicillin (Sigma, I9532), streptomycin (Sigma, 85886).
2. Specific induction and identification process
1. Preparation of human dental pulp mesenchymal stem cells
The experimental object: the third molar pulp tissue was isolated from 18-30 years old, and the experimental sample was from the university of martial arts people hospital, and the study of this example was passed through the university of martial arts people hospital ethics committee and was given the consent of the patient himself. It will be appreciated that in other embodiments, the dental pulp tissue or other human cell sample may also be taken from some sample repository or treatment facility, etc.
The specific process of extracting dental pulp mesenchymal stem cells by adopting a digestion method is as follows:
extracting third molar without caries and periodontal disease, sterilizing tooth surface with 75% alcohol, opening tooth body with hydraulic forceps, taking out dental pulp tissue, and cutting into 1mm 3 The tissue small block is washed 3 times by PBS containing double antibodies, dental pulp tissue is put into a 1.5mLEP tube to be digested for 20 to 40 minutes by adopting neutral enzyme and I type collagenase, and then is placed into alpha-MEM culture medium of human platelet lysate to be cultured, and after the cells are fused to about 80 percent, the cells are digested and centrifuged, and transferred to the fifth generation.
As shown in FIG. 1, pulp tissue was cut into 1mm pieces 3 After tissue, small clones appeared after about 5 days (FIG. 1A) and cell fusion reached 80% after 7-10 days after addition of the alpha-MEM medium mixed with human platelet lysate, and passaging was possible (FIG. 1B). Can be used for the next experiment. The specific detection is as follows:
1.1 flow cytometry detection of markers for mesenchymal Stem cells
The cells to be detected are digested into single cells by trypsin, and the cell concentration is adjusted to be 1 x 10 6 cells/mL; fixing cells with 4% paraformaldehyde for 30min at room temperature, washing 3 times with 0.1% BSA buffer solution, adding 01-10 mug/mL of coupling primary antibody, and incubating for 30min at room temperature in dark place; the cells were washed three times and centrifuged at 400g for five minutes and resuspendedWait for on-line in 1mL of 0.1% BSA buffer.
As shown in fig. 2, this example examined that the cells expressed markers CD44, CD73 of mesenchymal stem cells, and did not express marker CD45 of hematopoietic stem cells.
1.2 proliferation and migration Capacity analysis
(1) Cell cloning experiments: after digestion of the cells in logarithmic growth phase with pancreatin, they were resuspended into cell suspensions with human platelet lysate culture system (alpha-MEM medium+2.5% human platelet lysate) and serum culture system (alpha-mem+10% fetal bovine serum), respectively, and counted; 500 cells/well seeded in 6-well plate; culturing continuously for 14 days or until the number of cells in most single clones is greater than 50, changing liquid every 4 days in the middle, and observing the cell state; after the cloning was grown, DPBS was then washed 1 time, 1mL of 4% paraformaldehyde was added to each well for fixation for 30 minutes, and DPBS was washed 1 time; 1mL of crystal violet was added to each well and stained at room temperature for 20 minutes.
(2) Scratch experiment: after the cells in the logarithmic growth phase are digested by pancreatin, the cells are resuspended into cell suspensions by a human platelet lysate culture system (alpha-MEM culture medium+2.5% human platelet lysate) and a serum culture system (alpha-MEM+10% fetal calf serum) respectively, and inoculated into a 6-well plate, when the cells grow into a culture dish, a cell scratch is marked by a 200ul gun head compared with a ruler, and the cells are placed into a culture box for culture at intervals of 0, 12 and 24 hours, and photographed.
As shown in FIG. 3, the test in this example shows that the DPSC of the human platelet lysate culture system is significantly stronger than the DPSC of the serum culture system in proliferation capacity and migration capacity.
1.3 detection of partial protein expression level in DPSC in different culture System
DPSC cells cultured by different culture systems are inoculated in a 60mm culture dish, after the cells grow, the original culture medium is sucked, after the cells are collected, 200ul of protein lysate is added to extract protein, SDS-PAGE gel electrophoresis is performed, ECL development and gel imaging analysis are performed, a scanner is used for scanning the film, bFGF, KGF, VEGF, HGF is selected as a detection index, and GAPDH is used as an internal reference.
As shown in FIG. 4, the DPSC lysate is found to contain bFGF, KGF, VEGF, HGF and other cytokines, and the bFGF content in the human platelet lysate culture system is obviously higher than that in the serum culture system.
1.4 electron microscope detection
Preparing phosphotungstic acid into a solution with about 1-2% by using a phosphate buffer solution; dropping 10ul of sample on the sealing film by using a sample adding gun, reversely buckling the copper mesh with the film on the liquid drops, adsorbing the sample for 5 minutes, and placing the copper mesh with the film on filter paper to suck redundant liquid; dropping 10ul of phosphotungstic acid dyeing liquid on a sealing film by a sample adding gun, reversely buckling a copper mesh of a prepared sample on the phosphotungstic acid dyeing liquid drop, and sucking residual liquid by placing the copper mesh with the film on filter paper after 1-2 minutes; then a drop of pure water is dropped on the copper net, the pure water is sucked by filter paper, the process is repeated for two times and three times, the superfluous phosphotungstic acid is washed off, and then the copper net is stood and dried; and (5) observing by using an electron microscope.
As shown in FIG. 5, the test in this example found that DPSC lysate contained lipid particles showing negative staining of 150-200nm cup-shaped vesicles, 10-20 nm.
1.5 cell senescence staining
The method comprises the steps of inoculating mouse fibroblasts into a 6-hole plate, sucking an original culture medium after the cells grow fully, adding 1mLPBS, placing under a UVB ultraviolet lamp for irradiation at 0.02J/cm < 2 >, adding serum-free DMEM basic culture medium after the irradiation is finished, continuously irradiating for 3 days, adding 40ul of DPSC lysate into each hole of a treatment group, adding 40ul of ultrapure water into a control group, culturing for 2 days, and then dyeing and photographing by using a cell aging beta-galactosidase dyeing kit (specific reference kit instruction).
As shown in fig. 6, the cell senescence staining positive rate of the DPSC lysate group was found to be significantly lower than that of the control group, indicating that the DPSC lysate has the ability to resist photoaging.
1.6 proliferation and migration Capacity analysis
(1) Cell cloning experiments: the detailed steps are the same as the previous steps.
(2) Scratch experiment: the detailed steps are the same as the previous steps.
As shown in FIG. 7, the present example examined the proliferation and migration ability of the DPSC lysate group-treated mouse fibroblasts.
1.6 type I collagen and inflammatory factor detection
Inoculating mouse fibroblast cells into a 6-hole plate, sucking the original culture medium after the cells grow fully, adding 1ml of PBS, placing under a UVB ultraviolet lamp for irradiation at 0.02J/cm < 2 >, adding serum-free DMEM basic culture medium after the irradiation is finished, continuously irradiating for 3 days, adding 40ul of DPSC lysate into each hole in a treatment group, adding 40ul of ultrapure water into a control group, culturing normal group cells without UVB irradiation and serum-free DMEM basic culture medium, respectively collecting the cells after 2 days, adding 200ul of protein lysate to extract eggs, performing SDS-PAGE gel electrophoresis, performing ECL development and gel imaging analysis, scanning the films by using a scanner, and selecting Collagen I, MMP-13 and ROCK II as detection indexes and GAPDH as internal reference.
As shown in FIG. 8, the present example shows that the expression level of the Collagen I is obviously higher than that of the control group, the expression level of MMP-13 is obviously lower than that of the control group, slightly higher than that of the normal group, the expression level of ROCK II is obviously lower than that of the control group, and slightly higher Yu Zhengzu, thus indicating that the DPSC lysate has the anti-photoaging capability on the mouse fibroblasts.
The foregoing is merely illustrative of the present invention and not restrictive, and other modifications and equivalents thereof may occur to those skilled in the art without departing from the spirit and scope of the present invention.
Claims (7)
1. A preparation method of dental pulp mesenchymal stem cell lysate with photoaging resistance, which is characterized by comprising the following steps:
step 1: preparing dental pulp mesenchymal stem cells;
step 2: putting dental pulp mesenchymal stem cells into an alpha-MEM culture medium added with human platelet lysate for culture;
step 3: resuspending the dental pulp mesenchymal stem cells obtained by culturing in the step 2 in ultrapure water, incubating for 10-30 min at 2-8 ℃, storing in a refrigerator at-80 ℃, taking out after 8-24 h, thawing at 2-8 ℃, and repeatedly freezing and thawing for 3-5 times;
step 4: centrifuging the cell suspension obtained in the step 3 at a low temperature of 2000-6000g at a temperature of 2-8 ℃ for 20-60 min, taking supernatant after centrifuging, and adding heparin sodium into the supernatant to obtain the dental pulp mesenchymal stem cell lysate.
2. The method for preparing an anti-photoaging dental pulp mesenchymal stem cell lysate according to claim 1, wherein in step 1, the extracted dental pulp tissue is cut into 1mm pieces 3 The tissue pieces containing the double antibodies are washed 3 times by PBS leaves, then put into a 1.5ml EP tube, mixed and digested for 20-40 min by neutral enzyme and type I collagenase, placed into alpha-MEM culture medium added with human platelet lysate for culture, and after the cells are fused to about 80%, digested and centrifuged, and transferred to the fifth generation.
3. The method for preparing the dental pulp mesenchymal stem cell lysate with photoaging resistance according to claim 2, wherein the mass concentration of the added neutral enzyme is 0.1-1%, and the mass concentration of the type I collagenase is 0.1-0.8%; the double antibody is added into the mixture to be 50-100U/mL penicillin and 50-150 ug/mL streptomycin.
4. The method for preparing an anti-photoaging dental pulp mesenchymal stem cell lysate according to claim 1, wherein in step 2, after the dental pulp mesenchymal stem cells are cultured in an alpha-MEM medium added with human platelet lysate for 36-72 hours, the culture is continued by replacing the alpha-MEM medium added with human platelet lysate, and the culture medium is replaced every 36-72 hours during the subsequent culture.
5. A method for preparing an anti-photoaging dental pulp mesenchymal stem cell lysate according to claim 2 or 3, wherein the volume concentration of human platelet lysate in the alpha-MEM culture medium added with human platelet lysate is 2-10%.
6. The method for preparing an anti-photoaging dental pulp mesenchymal stem cell lysate according to claim 1, wherein in step 3, the cell concentration of the resuspended dental pulp mesenchymal stem cells in ultrapure water is 5×10 6 ～1×10 8 。
7. The method for preparing an anti-photoaging dental pulp mesenchymal stem cell lysate according to claim 1, wherein in step 4, the concentration of heparin sodium is 2-20 IU/ml.
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| CN110747164A (en) * | 2019-11-18 | 2020-02-04 | 北京泓信干细胞生物技术有限公司 | Preparation method of dental pulp stem cells |
| WO2021254296A1 (en) * | 2020-06-14 | 2021-12-23 | 浙江大学 | Bioactive substance composition, serum-free culture medium comprising the composition, and uses thereof |
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