CN110904049A - Preparation method and application of stem cell supernatant rich in VEGF and FGF - Google Patents
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Abstract
The invention discloses a preparation method and application of stem cell supernatant rich in VEGF and FGF, the technical scheme is that umbilical cord stem cells are firstly prepared, human Vascular Endothelial Growth Factor (VEGF) and human Fibroblast Growth Factor (FGF) are 'over-expressed' by a molecular cloning technology, then amplification passage is carried out, cell culture supernatant is collected, and finished product stem cell supernatant is obtained; the supernatant can be applied to the repair treatment of diabetic foot gangrene wound surface of limb ischemia, the process of the invention has reasonable design, simple operation and high feasibility, the obtained supernatant contains over-expressed VEGF and FGF, and also contains factors such as human epidermal cell growth factor, human keratinocyte growth factor, human platelet-like derived growth factor, fibronectin, immune factor and the like, can be applied to the repair treatment of diabetic foot limb ischemia, provides a new path for applying stem cells to the medical transformation industry, and has higher practicability.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method and application of stem cell supernatant rich in VEGF and FGF.
Background
According to the statistics of the international diabetes union, the number of diabetic patients in China in 2018 is up to 1.14 hundred million, the number of adult patients in the whole world is 4.15 hundred million, and the number of diabetic patients in the whole world is predicted to rise to 6.2 hundred million by 2040 years. Diabetic foot is one of chronic complications with high treatment difficulty in diabetes, and amputation is required for patients with serious conditions. At present, the common clinical treatment methods of the diabetic foot comprise blood sugar reduction, repeated cleaning of wounds, prevention of inflammation deterioration, improvement of blood supply of lower limbs, continuous negative pressure suction, hyperbaric oxygen treatment and the like, and for diabetic foot patients with severe limb ischemia, lower limb arterial lumen intervention or lower limb arterial bypass transplantation operation is generally required, but for diabetic foot patients with severe micro-vascular complications and macro-vascular complications, the curative effect of the operation on improving vascular circulation is not ideal, and the probability of amputation is still high. Therefore, it is of great significance to find a safe and effective method for treating diabetic foot.
In view of the above problems, we need to design a preparation method and application of stem cell supernatant rich in VEGF and FGF, which is used for repairing diabetic foot gangrene wounds caused by acro-ischemia, and this is one of the problems to be solved urgently.
Disclosure of Invention
The invention aims to provide a preparation method and application of stem cell supernatant rich in VEGF and FGF, 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 stem cell supernatant rich in VEGF and FGF comprises the following steps:
1) preparing mesenchymal stem cells;
2) transfecting plasmids containing VEGF and FGF 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 stem cells for stably expressing VEGF and FGF;
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, the method comprises the following steps:
1) preparing mesenchymal stem cells: the preparation of the mesenchymal stem cells in the step 1) is carried out, the mesenchymal stem cells can secrete a plurality of cytokines, such as Vascular Endothelial Growth Factor (VEGF), Keratinocyte Growth Factor (KGF), Fibroblast Growth Factor (FGF), platelet-like derived growth factor (PDGF) and Fibronectin (FN), the mesenchymal stem cells can migrate to wound tissues, secrete vascular endothelial growth factor and the like to promote the formation of new vessels, stimulate keratinocytes to participate in the re-formation of epidermis, further promote the healing of diabetic foot ulcers and improve clinical symptoms;
2) transfecting plasmids containing VEGF and FGF to the mesenchymal stem cells obtained in the step 1) to obtain over-expressed stem cells; preparing an over-expression stem cell in the step 2), and transfecting a plasmid containing VEGF and FGF into the mesenchymal stem cell to over-express the mesenchymal stem cell;
A. constructing non-viral VEGF and FGF plasmids;
B. respectively wrapping the constructed non-viral VEGF and FGF plasmids by using liposome Lipofectamine2000, and transfecting the non-viral VEGF and FGF 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 48h, detecting the transfection condition of the mesenchymal stem cells by adopting fluorescence quantitative PCR, and obtaining transfected over-expressed stem cells; performing fluorescent quantitative PCR detection in the step D of the step 2) to determine the transfection condition of the mesenchymal stem cells, detecting whether the transfection step is successful, and simultaneously comparing the expression conditions of VEGF and FGF of the transfected over-expressed stem cells with those of conventional stem cells;
3) subcloning by using a limiting dilution method to construct stem cells for stably expressing VEGF and FGF; subcloning by using a limiting dilution method in the step 3), and carrying out amplification passage on the over-expression stem cells obtained in the step 2) to obtain production stem cells for stably expressing VEGF and FGF, wherein the production stem cells are used 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 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) measuring the expression conditions of VEGF and FGF in the supernatant by adopting an ELISA detection kit;
e) 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;
and 4) continuously amplifying the production stem cells, and collecting culture supernatant to obtain a required finished product, wherein the supernatant contains over-expressed VEGF and FGF, human epidermal growth factor, human keratinocyte growth factor, human platelet-like derived growth factor, fibronectin, immune factor and other factors, and can effectively repair the diabetic foot gangrene wound.
Preferably, the stem cell in step 1) is any one of umbilical cord mesenchymal stem cell, bone marrow mesenchymal stem cell and adipose mesenchymal stem cell.
Preferably, the preparation steps of the umbilical cord 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.
Preferably, the cell concentration of the supernatant is more than or equal to 1 × 107One per ml.
Preferably, the supernatant contains over-expressed VEGF and FGF, and the supernatant also contains human epidermal growth factor, human keratinocyte growth factor, human platelet-like derived growth factor, fibronectin and immune factor.
The application of the stem cell supernatant in the medicine for repairing diabetic foot gangrene wound surface with limb ischemia according to any one technical scheme.
Compared with the prior art, the invention has the beneficial effects that:
the stem cell is an early undifferentiated cell with self-replication function and multi-differentiation potential, and is called as a universal cell in the medical field; 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 population of cells derived from a matrix, and can be obtained from most tissues of the human body. In recent years, a large number of basic and clinical studies at home and abroad prove that mesenchymal stem cells can migrate to wound tissues, secrete vascular endothelial growth factor and the like to promote neovascularization, stimulate keratinocytes to participate in epidermal reformation, further promote healing of diabetic foot ulcers and improve clinical symptoms, because the mesenchymal stem cells can secrete various 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. VEGF, also known as vascular permeability factor, is a highly specific vascular endothelial cell growth factor, and has various physiological effects of promoting vascular permeability increase, extracellular matrix degeneration, vascular endothelial cell migration, proliferation, and angiogenesis.
Meanwhile, FGF and VEGF have the effect of synergistically promoting angiogenesis. Therefore, the invention firstly leads the stem cells to 'over-express' FGF and VEGF factors by a molecular cloning technology, obviously enhances the stem cells to repair the diabetic foot limb ischemia symptom and improves the survival quality of patients.
The supernatant prepared by the invention is rich in Vascular Endothelial Growth Factor (VEGF) and Fibroblast Growth Factor (FGF); the technical scheme comprises the steps of firstly preparing and obtaining umbilical cord stem cells, performing 'over-expression' of human Vascular Endothelial Growth Factor (VEGF) and human Fibroblast Growth Factor (FGF) by a molecular cloning technology, performing amplification passage, and collecting cell culture supernatant to obtain finished stem cell supernatant; the supernatant can be applied to repair and treatment of diabetic foot gangrene wound surface of acro-ischemia, has simple and easy scheme, and is suitable for large-scale production of stem cell culture supernatants from different sources.
The invention discloses a preparation method and application of stem cell supernatant rich in VEGF and FGF, which has the advantages of reasonable process design, simple operation, high feasibility, industrialization and derivatization, and is suitable for preparing blood vessel repair supernatant of mesenchymal stem cells from different sources; meanwhile, the obtained supernatant contains over-expressed VEGF and FGF, and also contains factors such as human epidermal growth factor, human keratinocyte growth factor, human platelet-like derived growth factor, fibronectin, immune factor and the like, can be applied to the repair and treatment of diabetic foot-limb ischemia, provides a new path for applying stem cells to the medical transformation industry, and has 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 of the determination of FGF expression in supernatant by ELISA detection kit during the preparation of VEGF and FGF enriched stem cell supernatant; wherein the experimental group is culture supernatant of stem cells for over-expressing FGF and VEGF production, and the control group is culture supernatant of conventional mesenchymal stem cells;
FIG. 2 is a schematic diagram of determination of VEGF expression in supernatant by ELISA detection kit during preparation of VEGF and FGF-enriched stem cell supernatant, wherein experimental group is culture supernatant of FGF and VEGF-overexpressing production stem cells, and control group is culture supernatant of conventional mesenchymal stem cells.
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 (b):
s1: preparing mesenchymal stem cells:
s11: 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 was carried out in an ice bath.
S12: 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.
S2: transfecting plasmids containing VEGF and FGF to the obtained mesenchymal stem cells to obtain over-expressed stem cells;
s21: constructing non-viral VEGF and FGF plasmids;
s22: respectively wrapping the constructed non-viral VEGF and FGF plasmids by using liposome Lipofectamine2000, and transfecting the non-viral VEGF and FGF plasmids into the obtained mesenchymal stem cells;
s23: replacing opti-MEM containing 800. mu.g/ml G418, and continuing culturing;
s24: culturing for 48h, detecting the transfection condition of the mesenchymal stem cells by adopting fluorescence quantitative PCR, and obtaining transfected over-expressed stem cells;
s3: subcloning by using a limiting dilution method to construct stem cells for stably expressing VEGF and FGF;
s31: taking the obtained over-expression stem cells, carrying out conventional digestion by 0.25% trypsin, and collecting cell suspension;
s32: 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;
s33: culturing for 7 days, digesting with 0.25% trypsin, and amplifying to 24-well plate;
s34: 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;
s35: collecting, washing, adding stem cell freezing medium, subpackaging and marking, and freezing stem cells for production in liquid nitrogen at-196 deg.C by programmed cooling method.
S4: expanding the obtained production stem cells for twenty generations until the cell number is more than or equal to 1010(ii) a Replacing with fresh serum-free stem cell culture medium, continuously culturing the production stem cells for 3 days, collecting the production stem cell culture supernatant, and mixing; measuring the expression of VEGF and FGF in the supernatant by using an ELISA detection kit (specifically, as shown in the attached figure 1 and the attached figure 2 in the specification);
s5: 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: therapeutic effect on diabetic foot wound healing
Selecting 18g + -2 g mice, male and female half, injecting STZ55mg/kg into left lower abdominal cavity, feeding high fat and high protein containing food, selecting diabetes model with fasting blood glucose above 12.0mmol/mL after one week, lowering the temperature to 6 deg.C in the second week, and keeping the other conditions unchanged. The third week the room temperature returned to normal, and the fourth week diabetic foot models developed different degrees of acral ischemia.
10 mice with Wagner grading degree of three levels are selected, randomly divided into two groups, and injected to gangrene parts from the next day. One group was injected with 100ul of fresh medium as a control group; another group was injected with 100ul of the above-mentioned stem cell supernatant (diluted to a total protein concentration of 500ng/ml) as an experimental group.
And (4) conclusion: 1. the experimental group began to scab on the seventh day and grew new tissues on the fourteenth day, and 80% of the tissues healed in twenty-one days; the gangrene part is not healed from the control group to the end of the experiment.
2. And detecting the transfection condition of the mesenchymal stem cells by adopting fluorescent quantitative PCR in the step S24, wherein the concentration of VEGF and FGF in the transfected over-expressed stem cells is about 3 times higher than that of VEGF and FGF in the conventional stem cells.
The invention discloses a preparation method and application of stem cell supernatant rich in VEGF and FGF, which has the advantages of reasonable process design, simple operation, high feasibility, industrialization and derivatization, and is suitable for preparing blood vessel repair supernatant of mesenchymal stem cells from different sources; the prepared supernatant contains over-expressed VEGF and FGF, and also contains factors such as human epidermal growth factor, human keratinocyte growth factor, human platelet-like derived growth factor, fibronectin, immune factor and the like, can be applied to the repair and treatment of diabetic foot and extremity ischemia, provides a new path for applying stem cells to the medical transformation industry, and has 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 stem cell supernatant rich in VEGF and FGF is characterized in that: the method comprises the following steps:
1) preparing mesenchymal stem cells;
2) transfecting plasmids containing VEGF and FGF 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 stem cells for stably expressing VEGF and FGF;
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 of claim 1, wherein the method comprises the steps of: the method comprises the following steps:
1) preparing mesenchymal stem cells:
2) transfecting plasmids containing VEGF and FGF to the mesenchymal stem cells obtained in the step 1) to obtain over-expressed stem cells;
A. constructing non-viral VEGF and FGF plasmids;
B. respectively wrapping the constructed non-viral VEGF and FGF plasmids by using liposome Lipofectamine2000, and transfecting the non-viral VEGF and FGF 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 48h, detecting the transfection condition of the mesenchymal stem cells by adopting fluorescence quantitative PCR, and obtaining transfected over-expressed stem cells;
3) subcloning by using a limiting dilution method to construct stem cells for stably expressing VEGF and FGF;
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 of claim 1, wherein the method comprises the steps of: 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) measuring the expression conditions of VEGF and FGF in the supernatant by adopting an ELISA detection kit;
e) 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.
4. The method of claim 1, wherein the method comprises preparing a VEGF and FGF enriched stem cell supernatantThe method comprises the following steps: the cell concentration of the supernatant is more than or equal to 1 × 107One per ml.
5. The method of claim 1, wherein the method comprises the steps of: the supernatant contains over-expressed VEGF and FGF, 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 of claim 1, wherein the method comprises the steps of: the stem cell in the step 1) is any one of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells and adipose mesenchymal stem cells.
7. The method of claim 6, wherein the method comprises the steps of: the preparation steps of the umbilical cord 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.
8. Use of a supernatant of stem cells according to any one of claims 1 to 7 in a medicament for repairing a diabetic gangrene wound of limb ischemia.
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