CN110878287A - Preparation method and application of stem cell supernatant over-expressing TAT-KGF - Google Patents
Preparation method and application of stem cell supernatant over-expressing TAT-KGF Download PDFInfo
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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Abstract
The invention discloses a preparation method and application of stem cell supernatant liquid for over-expressing TAT-KGF, which combines cell penetration rate of KGF by using cell penetrating peptide (TAT), clones TAT-KGF fusion gene into production stem cells by using a molecular cloning technology, obtains a stem cell strain for stably expressing TAT-KGF factors after screening and subcloning, and can autocrine FGF2 by the cell strain, thereby maintaining the characteristics of the stem cells of the cells, avoiding the transverse or longitudinal differentiation of the stem cells, enabling the stem cells to become engineering cell strains with long-term passage and stable cell factor expression capacity, ensuring the stability and sustainability of the production stem cells, and further achieving the stability of product quality. The invention discloses a preparation method and application of stem cell supernatant liquid over-expressing TAT-KGF, which are high in feasibility, can be industrialized and derivatized, are suitable for preparing mesenchymal stem cells from different sources, can treat alopecia from different causes, and are excellent in alopecia regeneration effect and high in practicability.
Description
Technical Field
The invention relates to the technical field of biological stem cells, in particular to a preparation method and application of stem cell supernatant for over-expressing TAT-KGF.
Background
Alopecia is a ubiquitous modern disease at present, and although the alopecia does not belong to a serious disease, the alopecia can negatively affect the appearance and the mind of people and further affect the life quality of patients; the types of hair loss are many, including androgenetic alopecia, alopecia areata, and post-traumatic hair loss such as burns.
In recent years, the irregular living habits and the overload of learning workload of people lead to the increase and the trend towards the youthfulness of alopecia patients year by year, at present, finasteride and minoxidil are commonly used for treating alopecia, although the two medicines have certain curative effects, the alopecia is easy to relapse after the medicine is stopped, and certain side effects such as skin itch, dry hair, dizziness, cold sweat, anorexia, myocardial infarction and the like can be caused after long-term use; the traditional Chinese medicine treatment effect is slow, and the long-term administration can also cause the worry of the patients about the side effects of the traditional Chinese medicine, such as liver and kidney toxicity. Therefore, there is a great practical interest and need to develop safe and effective methods for treating alopecia.
In view of the above problems, there is a need to design a method for preparing a stem cell supernatant over-expressing TAT-KGF and an application thereof, which can effectively treat alopecia, and is one of the problems to be solved.
Disclosure of Invention
The invention aims to provide a preparation method and application of stem cell supernatant over-expressing TAT-KGF, so as to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for preparing a stem cell supernatant over-expressing TAT-KGF, comprising the following steps:
1) preparing mesenchymal stem cells;
2) transfecting a plasmid containing TAT-KGF to the mesenchymal stem cells obtained in the step 1) to obtain over-expressed stem cells;
3) subcloning by using a limiting dilution method to construct a stem cell for stably expressing TAT-KGF;
4) carrying out amplification passage of stem cells for production, collecting culture supernatant, aseptically packaging into 1ml per cell, and storing to obtain the final product.
Stem cells are early undifferentiated cells with self-replicating function and multi-differentiation potential, and are called "universal cells" in the medical community. Stem cells have a wide variety of biological properties, most notably self-renewal and multipotentiality and the ability to proliferate indefinitely. Mesenchymal Stem Cells (MSCs) are a heterogeneous group of cells derived from a matrix, and can be obtained from most tissues of the human body. In the past few years, mesenchymal stem cells have been mainly used for repairing wound tissues, promoting the formation of new blood vessels, stimulating keratinocytes to participate in the regeneration of epidermis, promoting the healing of diabetic foot ulcers and the like, and are now also commonly used for the treatment of alopecia. Mesenchymal stem cells secrete a variety of cytokines, such as Vascular Endothelial Growth Factor (VEGF), Keratinocyte Growth Factor (KGF), Fibroblast Growth Factor (FGF), platelet-like derived growth factor (PDGF), Fibronectin (FN), and the like. Among them, Fibroblast Growth Factors (FGF) are a class of cell growth factors secreted from cells derived from mesoderm and neuroectoderm and having important biological activities, and are also an important class of mitogenic factors and inducers of cell differentiation.
Research shows that FGF can promote tissue and nerve repair, regeneration, wound healing, angiogenesis and other physiological functions. FGF is one of the most effective angiogenesis factors found in vivo at present, and has obvious promotion effect in the processes of capillary basement membrane degradation, endothelial cell migration and proliferation, collagen synthesis and other angiogenesis. Keratinocyte Growth Factor (KGF) belongs to one of the members of Fibroblast Growth Factor (FGFs), also called FGF-7, and is mainly produced by mesenchymal cells, and KGF is specifically combined with receptor KGFR on epithelial cells to participate in the regulation of signal transmission cascade reaction, and participate in various biological processes such as development of tissue organs and proliferation of cells, improve skin microcirculation, promote vasodilatation, promote wound healing, and play an important role in promoting hair regeneration.
However, factors secreted by stem cells belong to macromolecular proteins, and the ratio of absorption of the factors by skin and hair follicle precursor cells is low, so that the improvement of the skin transmittance of the factors secreted by the stem cells is of great significance, and related researches show that TAT can efficiently mediate macromolecular substances into cells and even cell nuclei, and is widely applied to the development of medicaments such as carried medicaments, DNA (deoxyribonucleic acid) and the like.
The invention combines cell penetration rate of cell-penetrating peptide (TAT) to improve KGF, utilizes molecular cloning technology to clone TAT-KGF fusion gene into production stem cell, after screening subcloning, obtains stem cell strain which stably expresses TAT-KGF factor, and the cell strain can autocrine FGF2, thereby maintaining stem cell characteristic of cell, avoiding transverse or longitudinal differentiation, making it become engineering cell strain with long-term passage and ability of stably expressing cell factor, ensuring stability and sustainability of production stem cell, and achieving stability of product quality.
Preferably, the method comprises the following steps:
1) preparing mesenchymal stem cells: in the step 1), the preparation of the mesenchymal stem cells can be carried out firstly, and during actual operation, the human umbilical cord mesenchymal stem cells can be prepared by adopting an improved tissue block adherence method;
2) transfecting a plasmid containing TAT-KGF to the mesenchymal stem cells obtained in the step 1) to obtain over-expressed stem cells;
A. constructing TAT-pET28a-KGF eukaryotic expression plasmid;
B. respectively wrapping constructed TAT-pET28a-KGF eukaryotic expression plasmids by using liposome Lipofectamine2000, and transfecting the plasmids into the mesenchymal stem cells obtained in the step 1);
C. replacing opti-MEM containing 800. mu.g/ml G418, and continuing culturing;
D. culturing for 72h, detecting the transfection condition of the mesenchymal stem cells by adopting an RT-PCR method, and obtaining transfected over-expressed stem cells; constructing TAT-pET28a-KGF eukaryotic expression plasmids in the step 2), and then transfecting the genes into mesenchymal stem cells to enable TAT-KGF genes to be over-expressed in the mesenchymal stem cells; meanwhile, an RT-PCR method is adopted for detection, which is used for determining the transfection condition of the mesenchymal stem cells, detecting whether the transfection step is successful or not, and simultaneously comparing the TAT-KGF expression condition of the transfected over-expressed stem cells with that of the conventional stem cells;
3) subcloning by using a limiting dilution method to construct a stem cell for stably expressing TAT-KGF; subcloning by using a limiting dilution method in the step 3), and carrying out amplification passage on the over-expressed stem cells obtained in the step 2) to obtain production stem cells stably expressing TAT-KGF for batch production;
a) taking the over-expression stem cells obtained in the step 2), carrying out conventional digestion by 0.25% trypsin, and collecting cell suspension;
b) counting with a hemocytometer and diluting the cells to 10 using limiting dilution5Each well is inoculated with 100 mul of cell culture plate with 96 wells, and selection culture is carried out by using selection culture medium containing 1600 mug/ml G418, wherein the volume of the selection culture medium is 100 mul/well;
c) culturing for 7 days, digesting with 0.25% trypsin, and amplifying to 24-well plate;
d) carrying out amplification passage, gradually reducing the dosage of G418 to 10 mug/ml, and continuously culturing until the amplification amount reaches the required quantity to obtain the stem cells for production;
e) conventionally collecting, washing, adding stem cell freezing solution, subpackaging and marking, and freezing stem cells for production in liquid nitrogen at-196 ℃ by adopting a programmed cooling method;
4) carrying out amplification passage of stem cells for production, collecting culture supernatant, aseptically packaging into 1ml per cell, and storing to obtain the final product.
Preferably, in the step B, the mesenchymal stem cells are arranged at 5X 10 per hole4The cells were inoculated in a 24-well plate, cultured for 24 hours, the supernatant was removed, the medium was replaced with a serum-free medium and cultured for 4 hours, and then opti-MEM medium containing Lipofectamine2000 liposome encapsulated with TAT-pET28a-KGF eukaryotic expression plasmid was added for transfection.
Preferably, in the step 4), the specific operation steps are as follows:
a) expanding and subculturing twenty generations of the stem cells for production obtained in the step 3) until the number of the cells is more than or equal to 1010；
b) Changing into fresh serum-free stem cell culture medium, and continuously culturing the stem cells for production for 3 days;
c) collecting the culture supernatant of the stem cells for production, and mixing uniformly;
d) dialyzing the collected supernatant, concentrating until the total protein concentration is 20 μ g/ml, aseptically packaging into 1 ml/piece, and storing at-20 deg.C to obtain the final product.
Preferably, the supernatant contains over-expressed TAT-KGF, and the supernatant also contains human epidermal growth factor, human keratinocyte growth factor, human platelet-like derived growth factor, fibronectin and immune factor.
Preferably, the mesenchymal stem cell is prepared by the following steps:
a) taking 20cm of umbilical cord near fetal end under aseptic condition, cutting off 2cm at both ends, rinsing umbilical cord with physiological saline containing 200U streptomycin for 4-5 times until no blood stain exists, rinsing with physiological saline without double antibody, placing into culture dish containing little DMEM/F12 culture medium, cutting into 2-3cm segments, cutting the segments longitudinally, removing artery and vein blood vessels in umbilical cord, tearing tissue block into thin strip with tissue forceps, placing into culture dish containing little DMEM/F12 culture medium, cutting into 1-2mm with ophthalmic scissors3The whole process is carried out in ice bath;
b) dipping a little serum on the cut tissue blocks, transferring the tissue blocks into breathable T25 culture bottles, adding 20 cells in each bottle, adding 1.5ml of DMEM/F12 culture medium containing 20% fetal calf serum, culturing for 48h, changing the culture medium, culturing for about 8-12 days after the cells adhere to the wall, and removing the tissue blocks; culturing for about 20 days, allowing the cells to grow until 80% of the cells are fused, and collecting the cells by trypsinization to obtain the mesenchymal stem cells.
Preferably, the cell concentration of the supernatant is more than or equal to 1 × 107One per ml.
Use of a supernatant of stem cells according to any one of the preceding claims in a medicament for the treatment of alopecia.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a preparation method of stem cells for treating alopecia rapidly and regenerating hair, wherein the stem cells can improve skin microcirculation and promote new hair follicle regeneration. The TAT-KGF fusion protein is 'over-expressed' by using stem cells obtained by conventional separation through a molecular cloning technology, and the supernatant of the stem cells is rich in TAT-KGF fusion protein and stem cell self-autocrine cytokines, including human epidermal growth factors, human vascular endothelial growth factors, human fibroblast growth factors, human platelet-like derived growth factors, fibronectin and the like, and can be applied to promoting the hair regeneration of patients suffering from alopecia.
The invention relates to a preparation of a stem cell supernatant for over-expressing TAT-KGF fusion protein and application of the stem cell supernatant to promoting hair regeneration, wherein the stem cell is obtained by introducing a cell-penetrating peptide (TAT) and Keratinocyte Growth Factor (KGF) fusion gene into the stem cell through a molecular cloning technology, and the stem cell for over-expressing TAT-KGF fusion protein is applied to hair regeneration; the method is simple and easy to implement, is suitable for large-scale production of stem cell culture from different sources, can be used for promoting the regeneration of alopecia from different causes, and provides a new path for applying the stem cells to the medical transformation industry.
The invention discloses a preparation method and application of stem cell supernatant liquid over-expressing TAT-KGF, which has the advantages of reasonable process design, high feasibility, industrialization and derivatization, is suitable for preparing mesenchymal stem cells from different sources, can treat alopecia from different causes, and has excellent alopecia regeneration effect and higher practicability.
Drawings
In order that the present invention may be more readily and clearly understood, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments that are illustrated in the appended drawings.
FIG. 1 is a schematic diagram showing the detection results of RT-PCR methods of groups ① and ② in experiment 1 of a method for preparing a TAT-KGF-overexpressing stem cell supernatant of the present invention.
Detailed Description
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, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
s1: preparing mesenchymal stem cells:
taking 20cm of umbilical cord near fetal end under aseptic condition, cutting off 2cm at both ends, rinsing umbilical cord with 200U streptomycin-containing normal saline for 4 times until no blood stain exists, rinsing with nonreactive normal saline, cutting into 2cm segments in a culture dish containing a small amount of DMEM/F12 culture medium, cutting the segments longitudinally, removing artery and vein blood vessels in umbilical cord, tearing tissue block into strips with tissue forceps, placing into a culture dish containing a small amount of DMEM/F12 culture medium, and cutting into 1-2mm segments with ophthalmic scissors3The whole process is carried out in ice bath;
dipping a little serum on the cut tissue blocks, transferring the tissue blocks into breathable T25 culture bottles, adding 20 cells in each bottle, adding 1.5ml of DMEM/F12 culture medium containing 20% fetal calf serum, changing the culture medium after culturing for 48h, changing the culture medium after the cells adhere to the wall, culturing for about 8 days, and removing the tissue blocks; culturing for about 20 days, allowing the cells to grow until 80% of the cells are fused, and collecting the cells by trypsinization to obtain the mesenchymal stem cells.
S2: transfecting a plasmid containing TAT-KGF to the obtained mesenchymal stem cells to obtain over-expressed stem cells;
constructing TAT-pET28a-KGF eukaryotic expression plasmid; then respectively wrapping constructed TAT-pET28a-KGF eukaryotic expression plasmids by liposome Lipofectamine2000, and transfecting the plasmids into the obtained mesenchymal stem cells;
mesenchymal stem cells were plated at 5X 10 per well4Inoculating the cells into a 24-well plate, culturing for 24h, removing supernatant, replacing the culture medium with a serum-free medium for culturing for 4h, and adding liposome Lipofectam containing TAT-pET28a-KGF eukaryotic expression plasmidine2000 in opti-MEM medium, for transfection; culturing for 72h, detecting the transfection condition of the mesenchymal stem cells by adopting an RT-PCR method, and obtaining transfected over-expressed stem cells;
s3: subcloning by using a limiting dilution method to construct a stem cell for stably expressing TAT-KGF;
taking the over-expression stem cells obtained from S2, conventionally digesting with 0.25% trypsin, and collecting cell suspension; counting with a hemocytometer and diluting the cells to 10 using limiting dilution5Each well is inoculated with 100 mul of cell culture plate with 96 wells, and selection culture is carried out by using selection culture medium containing 1600 mug/ml G418, wherein the volume of the selection culture medium is 100 mul/well; culturing for 7 days, digesting with 0.25% trypsin, and amplifying to 24-well plate;
carrying out amplification passage, gradually reducing the dosage of G418 to 10 mug/ml, and continuously culturing until the amplification amount reaches the required amount to obtain the production stem cells; conventionally collecting, washing, adding stem cell freezing solution, subpackaging and marking, and freezing stem cells for production in liquid nitrogen at-196 ℃ by adopting a programmed cooling method;
s4: expanding and passaging the production stem cells for twenty generations until the number of the cells is more than or equal to 1010(ii) a Replacing with fresh serum-free stem cell culture medium, and continuously culturing the stem cells for production for 3 days; collecting the culture supernatant of the stem cells for production, and mixing uniformly; dialyzing the collected supernatant, concentrating until the total protein concentration is 20 μ g/ml, aseptically packaging into 1 ml/piece, and storing at-20 deg.C to obtain the final product.
Experiment 1: RT-PCR method for detecting expression level of TAT-KGF
① group, S2 in example 1, incubation for 72h after transfection, extraction of total RNA using TRIzon total RNA extraction reagent, reverse transcription of total RNA using PrimeScript mRNA qPCR starter kit (37 ℃, 2h), 10-fold dilution of cDNA after completion of reverse transcription, and detection of RNA expression using SybrGreen II 2 XPCR Mix (Perfect Real-time).
② group is tested with conventional mesenchymal stem cells, ① and ② group form control, and the test results are shown in figure 1 (① group is rightmost data, ② group is leftmost data)
And (4) conclusion: because TAT-KGF contains an RNA sequence of KGF, the expression level of the KGF can indirectly react with the expression level of TAT-KGF compared with a control group; the detection result shows that the TAT-pET28a-KGF loaded liposome
2000 after transfection, the KGF expression of MSCs was significantly increased, thereby reflecting that the MSCs overexpress TAT-KGF.
Experiment 2: evaluation of therapeutic Effect on alopecia patients
Mr. 45 years old. Severe alopecia, extensive hair loss at the top of the head, excessive androgenic hormone secretion alopecia and poor common treatment effect, and then the stem cell supernatant is used as a volunteer.
When in use, the injection is injected into the edge of the alopecia area by a water light instrument, the injection dose is 5mL, and the concentration is 1 multiplied by 107One per mL, one injection per month, and new small villi are grown at hair loss after three months.
And (4) conclusion: the invention discloses a preparation method and application of stem cell supernatant liquid over-expressing TAT-KGF, which has the advantages of reasonable process design, high feasibility, industrialization and derivatization, capability of treating alopecia caused by different reasons, excellent alopecia regeneration effect and higher practicability.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (8)
1. A method for preparing a stem cell supernatant over-expressing TAT-KGF, which is characterized by comprising the following steps: the method comprises the following steps:
1) preparing mesenchymal stem cells;
2) transfecting a plasmid containing TAT-KGF to the mesenchymal stem cells obtained in the step 1) to obtain over-expressed stem cells;
3) subcloning by using a limiting dilution method to construct a stem cell for stably expressing TAT-KGF;
4) carrying out amplification passage of stem cells for production, collecting culture supernatant, aseptically packaging into 1ml per cell, and storing to obtain the final product.
2. The method for preparing a supernatant of a stem cell overexpressing TAT-KGF as defined in claim 1, wherein: the method comprises the following steps:
1) preparing mesenchymal stem cells:
2) transfecting a plasmid containing TAT-KGF to the mesenchymal stem cells obtained in the step 1) to obtain over-expressed stem cells;
A. constructing TAT-pET28a-KGF eukaryotic expression plasmid;
B. respectively wrapping constructed TAT-pET28a-KGF eukaryotic expression plasmids by using liposome Lipofectamine2000, and transfecting the plasmids into the mesenchymal stem cells obtained in the step 1);
C. replacing opti-MEM containing 800. mu.g/ml G418, and continuing culturing;
D. culturing for 72h, detecting the transfection condition of the mesenchymal stem cells by adopting an RT-PCR method, and obtaining transfected over-expressed stem cells;
3) subcloning by using a limiting dilution method to construct a stem cell for stably expressing TAT-KGF;
a) taking the over-expression stem cells obtained in the step 2), carrying out conventional digestion by 0.25% trypsin, and collecting cell suspension;
b) counting with a hemocytometer and diluting the cells to 10 using limiting dilution5Each well is inoculated with 100 mul of cell culture plate with 96 wells, and selection culture is carried out by using selection culture medium containing 1600 mug/ml G418, wherein the volume of the selection culture medium is 100 mul/well;
c) culturing for 7 days, digesting with 0.25% trypsin, and amplifying to 24-well plate;
d) carrying out amplification passage, gradually reducing the dosage of G418 to 10 mug/ml, and continuously culturing until the amplification amount reaches the required quantity to obtain the stem cells for production;
e) conventionally collecting, washing, adding stem cell freezing solution, subpackaging and marking, and freezing stem cells for production in liquid nitrogen at-196 ℃ by adopting a programmed cooling method;
4) carrying out amplification passage of stem cells for production, collecting culture supernatant, aseptically packaging into 1ml per cell, and storing to obtain the final product.
3. The method for preparing a supernatant of a stem cell overexpressing TAT-KGF as defined in claim 2, wherein: in the operation of step B, the mesenchymal stem cells are arranged at 5X 10 per well4The cells were inoculated in a 24-well plate, cultured for 24 hours, the supernatant was removed, the medium was replaced with a serum-free medium and cultured for 4 hours, and then opti-MEM medium containing Lipofectamine2000 liposome encapsulated with TAT-pET28a-KGF eukaryotic expression plasmid was added for transfection.
4. The method for preparing a supernatant of a stem cell overexpressing TAT-KGF as defined in claim 2, wherein: in the step 4), the specific operation steps are as follows:
a) expanding and subculturing twenty generations of the stem cells for production obtained in the step 3) until the number of the cells is more than or equal to 1010；
b) Changing into fresh serum-free stem cell culture medium, and continuously culturing the stem cells for production for 3 days;
c) collecting the culture supernatant of the stem cells for production, and mixing uniformly;
d) dialyzing the collected supernatant, concentrating until the total protein concentration is 20 μ g/ml, aseptically packaging into 1 ml/piece, and storing at-20 deg.C to obtain the final product.
5. The method for preparing a supernatant of a stem cell overexpressing TAT-KGF as defined in claim 1, wherein: the supernatant contains over-expressed TAT-KGF, and the supernatant also contains human epidermal growth factor, human keratinocyte growth factor, human platelet-like derived growth factor, fibronectin and immune factor.
6. The method for preparing a supernatant of a stem cell overexpressing TAT-KGF as defined in claim 1, wherein: the preparation steps of the mesenchymal stem cells are as follows:
a) taking 20cm of umbilical cord near fetal end under aseptic condition, cutting off 2cm at both ends, rinsing umbilical cord with physiological saline containing 200U streptomycin for 4-5 times until no blood stain exists, rinsing with physiological saline without double antibody, placing into culture dish containing little DMEM/F12 culture medium, cutting into 2-3cm segments, cutting the segments longitudinally, removing artery and vein blood vessels in umbilical cord, tearing tissue block into thin strip with tissue forceps, placing into culture dish containing little DMEM/F12 culture medium, cutting into 1-2mm with ophthalmic scissors3The whole process is carried out in ice bath;
b) dipping a little serum on the cut tissue blocks, transferring the tissue blocks into breathable T25 culture bottles, adding 20 cells in each bottle, adding 1.5ml of DMEM/F12 culture medium containing 20% fetal calf serum, culturing for 48h, changing the culture medium, culturing for about 8-12 days after the cells adhere to the wall, and removing the tissue blocks; culturing for about 20 days, allowing the cells to grow until 80% of the cells are fused, and collecting the cells by trypsinization to obtain the mesenchymal stem cells.
7. The method for preparing a supernatant of a stem cell overexpressing TAT-KGF as defined in claim 1, wherein: the cell concentration of the supernatant is more than or equal to 1 × 107One per ml.
8. Use of a supernatant of stem cells according to any one of claims 1 to 7 in a medicament for the treatment of alopecia.
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| CN112029802A (en) * | 2020-09-03 | 2020-12-04 | 复旦大学附属中山医院 | Preparation method and application of exosome rich in human keratinocyte growth factor-2 |
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