CN114642683A - 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 an optimized human platelet lysate culture system, so that the dental pulp mesenchymal stem cell lysate can be obtained under the low-temperature condition. The dental pulp mesenchymal stem cells obtained by the culture system have high purity and rich growth factor content through detection. In-vitro experiments show that the dental pulp mesenchymal stem cell not only can delay the aging of mouse fibroblast subjected to ultraviolet treatment and promote the proliferation of cells, but also can increase the content of I type collagen, and provides a key technology for clinically applying dental pulp mesenchymal stem cell lysate to light aging resistance.
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 a dental pulp mesenchymal stem cell lysate with photoaging resistance.
Background
Aging is one of the most basic natural laws in 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, but also 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, the application of 10 stem cells for anti-aging treatment has also become a research hotspot.
At present, the research on anti-aging is carried out by umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose-derived stem cells and exosomes and lysis solutions thereof, and the curative effect of the anti-aging is proved. However, the transformation of the seed cells in clinic is limited due to the problems of seed cell source, ethics and the like. The dental pulp mesenchymal stem cells (DPSC) have the advantages of wide source, no ethical problem and the like, and a 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 the clinical requirement.
Disclosure of Invention
In order to solve the problems, the invention provides a preparation method of a dental pulp mesenchymal stem cell lysate with photoaging resistance.
The technical scheme adopted by the invention is as follows:
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: placing dental pulp mesenchymal stem cells into an alpha-MEM culture medium added with human platelet lysate for culturing;
and step 3: resuspending the dental pulp mesenchymal stem cells cultured 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;
and 4, step 4: and (4) centrifuging the cell suspension obtained in the step (3) at the low temperature of 2-8 ℃ and the rotating speed of 2000-6000g for 20-60 min, taking the supernatant after centrifugation, and adding heparin sodium into the supernatant to obtain the dental pulp mesenchymal stem cell lysate.
It is preferable thatIn step 1, the extracted dental pulp tissue is cut into 1mm pieces3Washing the small tissue blocks with PBS (phosphate buffer solution) leaves containing double antibodies for 3 times, then placing the small tissue blocks into a 1.5ml EP (ethylene propylene glycol) tube, performing mixed digestion for 20-40 min by using neutral enzyme and collagenase I, placing the small tissue blocks into an alpha-MEM (membrane-organic membrane) culture medium added with human platelet lysate for culture, after cells are fused to about 80%, performing digestion and centrifugation, and transferring the cells to the fifth generation.
Preferably, the mass concentration of the added neutral enzyme is 0.1-1%, and the mass concentration of the collagenase type I is 0.1-0.8%; adding 50-100U/mL penicillin and 50-150 ug/mL streptomycin as double antibody.
Preferably, in step 2, after the dental pulp mesenchymal stem cells are cultured in the alpha-MEM culture medium added with the human platelet lysate for 36-72 hours, the alpha-MEM culture medium added with the human platelet lysate is replaced to continue culturing, and the culture medium is replaced every 36-72 hours in the subsequent culturing process.
Preferably, the volume concentration of the human platelet lysate in the alpha-MEM culture medium added with the human platelet lysate is 2-10%.
Preferably, in step 3, the cell concentration of the resuspended dental pulp mesenchymal stem cells in ultrapure water is 5 × 106～1×108。
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 an optimized human platelet lysate culture system, so that the dental pulp mesenchymal stem cell lysate is obtained under the low-temperature condition. The dental pulp mesenchymal stem cells obtained by the culture system have high purity and rich growth factor content through detection.
2. The dental pulp tissue is taken from the discarded or fallen wisdom teeth, has no harm to the donor, and the human platelet lysate culture system is efficient and safe and has no animal derived factors. The cells obtained by the culture system can well meet clinical requirements in both quality and quantity, and are convenient to popularize and apply.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
Fig. 1 is a schematic diagram of cell morphology change in the DPSC formation process in the example of the present invention, wherein a: primary DPSCs 5 days after digestion, B: DPSCs of P5 generation;
FIG. 2 is a schematic diagram of the detection of cell markers by flow cytometry in a human platelet lysate culture system and a serum culture system of the DPSC according to the embodiment of the invention;
fig. 3 is a schematic diagram of detecting the proliferation and migration of cells in a human platelet lysate culture system and a serum culture system by DPSC according to an embodiment of the present invention, wherein a: cell cloning experiments, B: performing cell scratching experiments;
FIG. 4 is a graph showing the protein expression levels of bFGF, KGF, VEGF, HGF in DPSC according to an embodiment of the present invention;
FIG. 5 is an electron microscope identification chart of the DPSC lysate according to the embodiment of the present invention;
FIG. 6 is a schematic diagram of the cell senescence staining detection of photoaged mouse fibroblasts in vitro, to which the DPSC lysate and deionized water were added respectively, in an example of the present invention;
fig. 7 is a schematic diagram of detecting the effect of DPSC lysate and deionized water on the proliferation and migration of mouse fibroblasts under in vitro conditions, where a: cell cloning experiments, B: cell scratching experiments;
FIG. 8 is a graph showing the detection of the protein expression levels of Collagen I, MMP-13, and ROCK II in mouse fibroblasts aged in vitro and added with DPSC lysate and deionized water, respectively, in the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.
The embodiment provides a preparation method of a dental pulp mesenchymal stem cell lysate with photoaging resistance, which comprises the following steps:
step 1: preparing dental pulp mesenchymal stem cells;
the pulp tissue taken out was cut into 1mm3Washing the small tissue blocks by PBS (phosphate buffer solution) leaves containing double antibodies for 3 times, then putting the small tissue blocks into a 1.5ml EP (EP) tube, performing mixed digestion for 20-40 min by using neutral enzyme and collagenase I, putting the small tissue blocks into an alpha-MEM (membrane-organic membrane) culture medium added with human platelet lysate for culture, and digesting and centrifuging the small tissue blocks until the cells are fused to about 80 percent, and transferring the cells to the fifth generation;
step 2: placing dental pulp mesenchymal stem cells into an alpha-MEM culture medium added with human platelet lysate for culturing;
and step 3: resuspending the dental pulp mesenchymal stem cells cultured 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;
and 4, step 4: and (3) centrifuging the cell suspension obtained in the step (3) at the temperature of 2-8 ℃ at the rotating speed of 2000-6000g for 20-60 min at low temperature, taking the supernatant after centrifugation, 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 4 IU/mL.
In the step 2, after the dental pulp mesenchymal stem cells are cultured in the alpha-MEM culture medium added with the human platelet lysate for 36-72 hours, the alpha-MEM culture medium added with the human platelet lysate is replaced for continuous culture, and the culture medium is replaced once every 48 hours in the subsequent culture process. 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 step 2, before the culture of the dental pulp mesenchymal stem cells, the method further comprises a step of performing cryopreservation and resuscitation on the dental pulp mesenchymal stem cells. Wherein, the step of cryopreserving includes: digesting and centrifuging dental pulp mesenchymal stem cells, then suspending the dental pulp mesenchymal stem cells in serum-free freezing medium, putting the dental pulp mesenchymal stem cells into a gradient program freezing box, storing the dental pulp mesenchymal stem cells in a refrigerator at the temperature of-80 ℃ for 12-24 hours, and then transferring the dental pulp mesenchymal stem cells into liquid nitrogen for long-term storage; the resuscitation step comprises: taking out dental pulp mesenchymal stem cells from a liquid nitrogen tank, immediately thawing in a constant-temperature water bath tank at 37 ℃, centrifuging, discarding supernatant, adding alpha-MEM culture medium of human platelet lysate for resuspending cell sediment, changing the culture solution once every other day, and carrying out passage once every 4 days.
In the step 1, the mass concentration of the added neutral enzyme is 0.4 percent, and the mass concentration of the collagenase I is 0.3 percent; the double antibody was added at 100U/mL penicillin and 100ug/mL streptomycin.
In step 3, the cell concentration of the resuspended dental pulp mesenchymal stem cells in ultrapure water is 5 × 106～1×108。
The method for obtaining the dental pulp mesenchymal stem cell lysate under the low-temperature condition by reasonably using the human platelet lysate to provide an optimized human platelet lysate culture system to culture the dental pulp mesenchymal stem cells. The dental pulp mesenchymal stem cells obtained by the culture system have high purity and rich growth factor content through detection. The teeth are selected from waste or fallen wisdom teeth, no harm is caused to donors, no ethical problem exists, and a human platelet lysate culture system is efficient and safe and has no animal derived factors. The cells obtained by the culture system can well meet clinical requirements in both quality and quantity, and are convenient to popularize and apply. Skin is the first line of defense of the body, playing an important role in resisting various external aggressions, maintaining homeostasis and guaranteeing physiological functions of the body, and skin aging may cause psychological problems such as depression and self-inferior in addition to physiological influences, wherein over 80% of facial skin aging is photoaging. In-vitro experiments show that the dental pulp mesenchymal stem cell not only can delay the aging of mouse fibroblast subjected to ultraviolet treatment and promote the proliferation of the cell, but also can increase the content of type I collagen, and provides a key technology for resisting light aging by clinically applying dental pulp mesenchymal stem cell lysate.
Experimental part
Reagent and equipment thereof
DPSC (dental pulp mesenchymal stem cells): 2 lines of human dental pulp mesenchymal stem cells constructed by the regenerative medicine center of Wuhan university are selected.
2. Cell culture reagent
alpha-MEM (Gboco, 12571063, Gboco, 12571063, the same applies below), human platelet lysate (STEMERY, RC-002-100), DPBS (Gboco, 14190144), collagenase type I (Sigma, SCR103), neutral protease (Roche, 04942078001), heparin sodium z injection (Jiangsu Wanbang, Chinese pharmacopoeia 5H32020612), serum-free stock solution (Sigma, D2650), penicillin (Sigma, I9532), streptomycin (Sigma, 85886).
Second, specific induction and identification process
1. Preparation of human dental pulp mesenchymal stem cells
Subjects: the third molar pulp tissue of 18-30 years old was isolated, and the experimental sample was obtained from Wuhan university Hospital, and the study of this example was conducted by discussion of the ethical Committee of Wuhan university Hospital and the patient's own consent was obtained. It is understood that in other embodiments, the dental pulp tissue or other human cell samples may be obtained from some frozen stock of samples or from a treatment facility, etc.
The specific process for extracting the dental pulp mesenchymal stem cells by adopting a digestion method comprises the following steps:
extracting third molar without dental caries and periodontal disease, disinfecting tooth surface with 75% alcohol, opening tooth body with hydraulic forceps, taking out dental pulp tissue, and cutting into 1mm3Washing the small tissue blocks with PBS containing double antibodies for 3 times, placing the dental pulp tissues into a 1.5mLEP tube, performing mixed digestion for 20-40 minutes by using neutral enzyme and I-type collagenase, placing the dental pulp tissues into an alpha-MEM culture medium of human platelet lysate for culture, and culturing the dental pulp tissues until the cells are fused to 80 DEG CAfter% left and right, the mixture was digested and centrifuged to reach the fifth passage.
As shown in FIG. 1, the pulp tissue was cut to 1mm3After the tissue is organized, the alpha-MEM culture medium mixed and added with human platelet lysate shows that a small clone appears after about 5 days (figure 1A), and after 7-10 days, the cell fusion reaches 80%, and can be subjected to passage expansion (figure 1B). Can be used for the next experiment. The specific detection is as follows:
1.1 flow cytometry detection of markers of mesenchymal Stem cells
Digesting the cells to be detected into single cells by trypsin, and adjusting the cell concentration to 1 x 106cell/mL; fixing the cells with 4% paraformaldehyde for 30 minutes at room temperature, washing with 0.1% BSA buffer solution for 3 times, adding 01-10 μ g/mL of coupled primary antibody, and incubating for 30 minutes at room temperature in a dark place; the cells were washed three times, centrifuged at 400g for five minutes, resuspended in 1mL of 0.1% BSA buffer, and left to sit on the machine.
As shown in fig. 2, this example detected that the cells expressed markers CD44 and CD73 of mesenchymal stem cells, but did not express marker CD45 of hematopoietic stem cells.
1.2 analysis of proliferation and migration Capacity
(1) Cell cloning experiments: after the cells in the logarithmic growth phase are digested by pancreatin, a human platelet lysate culture system (alpha-MEM culture medium + 2.5% human platelet lysate) and a serum culture system (alpha-MEM + 10% fetal bovine serum) are respectively resuspended into cell suspensions, and the cell suspensions are counted; inoculating 500 cells/well in a 6-well plate culture plate; continuously culturing for 14 days or until the number of cells in most single clones is more than 50, changing the culture medium every 4 days in the middle and observing the cell state; after the clone grows, then washing with DPBS for 1 time, adding 1mL of 4% paraformaldehyde into each hole, fixing for 30 minutes, and washing with DPBS for 1 time; 1mL of crystal violet was added per well and stained for 20 minutes at room temperature.
(2) Scratch test: after the cells in logarithmic growth phase are digested by pancreatin, the cells are respectively 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 bovine serum), the cell suspensions are inoculated into a 6-hole plate, when the cells grow over a culture dish, a 200ul gun head is used for drawing a cell scratch along a ruler, the cell scratch is placed into an incubator for culture at intervals of 0, 12 and 24 hours, and pictures are taken.
As shown in FIG. 3, the present example found that the proliferation ability and migration ability of DPSC of human platelet lysate culture system are significantly stronger than those of DPSC of serum culture system.
1.3 detection of partial protein expression in DPSC under different culture systems
Inoculating DPSC cells cultured by different culture systems into a 60mm culture dish, sucking the original culture medium after the cells grow full, collecting the cells, adding 200ul of protein lysate to extract protein, performing SDS-PAGE gel electrophoresis, performing ECL development and gel imaging analysis, scanning the film by using a scanner, and selecting bFGF, KGF, VEGF and HGF as detection indexes and GAPDH as an internal reference control.
As shown in FIG. 4, the detection in this example shows that the DPPC lysate contains cytokines such as bFGF, KGF, VEGF, HGF, etc., and the bFGF content in the human platelet lysate culture system is significantly higher than that in the serum culture system.
1.4 Electron microscopy
Phosphotungstic acid is prepared into a solution with the concentration of 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 net with the film on the drop, adsorbing the sample for 5 minutes, and placing the copper net with the film on filter paper to absorb redundant liquid; dropping 10ul of phosphotungstic acid staining solution on a sealing film by using a sample adding gun, reversely buckling the copper mesh of the prepared sample on the phosphotungstic acid staining solution drop, and after 1-2 minutes, placing the copper mesh with the film on filter paper to suck out the multi-residual solution; dropping a drop of pure water on a copper net, sucking off the pure water by using filter paper, repeating the process for two times and three times, washing off redundant phosphotungstic acid, and then standing and drying; observing by using an electron microscope.
As shown in FIG. 5, the DPSC lysate contains lipid particles with negative stain of 150-200nm cup-shaped membrane bubble and 10-20 nm.
1.5 cell senescence staining
Inoculating mouse fibroblast into a 6-hole plate, absorbing an original culture medium after the cell grows full, adding 1ml PBS, placing under a UVB ultraviolet lamp to irradiate with 0.02J/cm2, adding a serum-free DMEM basic culture medium after the irradiation is finished, continuously irradiating for 3 days, adding 40ul DPSC lysate into each hole of a treatment group, adding 40ul ultrapure water into a control group, culturing for 2 days, and dyeing and photographing by using a cell aging beta-galactosidase staining kit (specifically referring to the kit specification).
As shown in FIG. 6, the present example found that the positive rate of cell senescence staining in the DPSC lysate group is significantly lower than that in the control group, indicating that the DPSC lysate has the anti-photoaging ability.
1.6 analysis of proliferation and migration Capacity
(1) Cell cloning experiments: the detailed steps are the same as the previous steps.
(2) Scratch test: the detailed steps are the same as the previous steps.
As shown in FIG. 7, it was found that the proliferation and migration of the mouse fibroblasts treated with the DPSC lysate group were enhanced.
1.6 type I collagen and inflammatory factor assay
Inoculating mouse fibroblasts into a 6-hole plate, sucking out original culture medium after the cells grow full, adding 1ml PBS, placing under a UVB ultraviolet lamp to irradiate with 0.02J/cm2, adding serum-free DMEM basic culture medium after irradiation, continuously irradiating for 3 days, adding 40ul DPSC lysate into each hole of a treatment group, adding 40ul ultrapure water into a control group, culturing cells of a normal group without UVB irradiation and serum-free DMEM basic culture medium, respectively collecting cells after culturing for 2 days, adding 200ul 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 GAROC II as detection indexes and GAPDH as an internal reference control.
As shown in FIG. 8, it was found that the expression level of Collagen I was significantly higher than that of the control group, MMP-13 was significantly lower than that of the control group, and ROCK II was significantly lower than that of the control group, and slightly higher than that of the positive group, indicating that the DPSC lysate has anti-photoaging ability for mouse fibroblasts.
The above description is only for the purpose of illustrating the technical solutions of the present invention and not for the purpose of limiting the same, and other modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention should be covered within the scope of the claims of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (7)
1. The preparation method of the dental pulp mesenchymal stem cell lysate with photoaging resistance is characterized by comprising the following steps of:
step 1: preparing dental pulp mesenchymal stem cells;
step 2: placing dental pulp mesenchymal stem cells into an alpha-MEM culture medium added with human platelet lysate for culturing;
and step 3: resuspending the dental pulp mesenchymal stem cells cultured 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;
and 4, step 4: and (3) centrifuging the cell suspension obtained in the step (3) at the temperature of 2-8 ℃ at the rotating speed of 2000-6000g for 20-60 min at low temperature, taking the supernatant after centrifugation, and adding heparin sodium into the supernatant to obtain the dental pulp mesenchymal stem cell lysate.
2. The method for preparing the lysate of dental pulp mesenchymal stem cells with photoaging resistance according to claim 1, wherein the extracted dental pulp tissue is cut into 1mm in step 13Washing the small tissue blocks by PBS (phosphate buffer solution) leaves containing double antibodies for 3 times, then putting the small tissue blocks into a 1.5ml EP (EP) tube, performing mixed digestion for 20-40 min by using neutral enzyme and collagenase I, putting the small tissue blocks into an alpha-MEM (membrane-organic membrane) culture medium added with human platelet lysate for culture, and digesting and centrifuging the small tissue blocks until the cells are fused to about 80 percent, and transferring the cells to the fifth generation.
3. The method for preparing a lysate of dental pulp mesenchymal stem cells with photoaging resistance according to claim 2, wherein the added neutral enzyme has a mass concentration of 0.1% to 1%, and the collagenase type i has a mass concentration of 0.1% to 0.8%; adding 50-100U/mL penicillin and 50-150 ug/mL streptomycin as double antibody.
4. The method for preparing a dental pulp mesenchymal stem cell lysate with photoaging resistance according to claim 1, wherein in step 2, after dental pulp mesenchymal stem cells are cultured in the alpha-MEM culture medium added with human platelet lysate for 36-72 hours, the alpha-MEM culture medium added with human platelet lysate is replaced for continuous culture, and the culture medium is replaced every 36-72 hours in the subsequent culture process.
5. The method for preparing a dental pulp mesenchymal stem cell lysate with photoaging resistance according to claim 2 or 3, wherein the volume concentration of the human platelet lysate in the alpha-MEM medium added with the human platelet lysate is 2-10%.
6. The method for preparing the lysis solution of dental pulp mesenchymal stem cells with photoaging resistance of claim 1, wherein in step 3, the cell concentration of the resuspended dental pulp mesenchymal stem cells in ultrapure water is 5 x 106～1×108。
7. The method for preparing a lysate of dental pulp mesenchymal stem cells with photoaging resistance according to claim 1, wherein in step 4, the concentration of heparin sodium is 2-20 IU/ml.
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| CN105708860A (en) * | 2016-01-26 | 2016-06-29 | 深圳爱生再生医学科技有限公司 | Stem cell preparation for repairing skin ulcer |
| CN106361770A (en) * | 2016-09-30 | 2017-02-01 | 广州赛莱拉干细胞科技股份有限公司 | Application of dental pulp mesenchymal stem cell supernate to preparing dental ulcer treating medicine, gargle and preparation method of gargle |
| 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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