CN113755430B - Method for high expression of AFGF - Google Patents
Method for high expression of AFGF Download PDFInfo
- Publication number
- CN113755430B CN113755430B CN202111056851.4A CN202111056851A CN113755430B CN 113755430 B CN113755430 B CN 113755430B CN 202111056851 A CN202111056851 A CN 202111056851A CN 113755430 B CN113755430 B CN 113755430B
- Authority
- CN
- China
- Prior art keywords
- cells
- culture
- physiological saline
- culture medium
- afgf
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000000034 method Methods 0.000 title claims abstract description 17
- 101100120045 Bos taurus FGF1 gene Proteins 0.000 title claims abstract 6
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 title claims abstract 6
- 210000004027 cell Anatomy 0.000 claims abstract description 55
- 239000002504 physiological saline solution Substances 0.000 claims abstract description 42
- 239000001963 growth medium Substances 0.000 claims abstract description 41
- 239000006285 cell suspension Substances 0.000 claims abstract description 25
- 239000006228 supernatant Substances 0.000 claims abstract description 23
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 21
- 239000001301 oxygen Substances 0.000 claims abstract description 21
- 108010042708 Acetylmuramyl-Alanyl-Isoglutamine Proteins 0.000 claims abstract description 18
- BSOQXXWZTUDTEL-ZUYCGGNHSA-N muramyl dipeptide Chemical compound OC(=O)CC[C@H](C(N)=O)NC(=O)[C@H](C)NC(=O)[C@@H](C)O[C@H]1[C@H](O)[C@@H](CO)O[C@@H](O)[C@@H]1NC(C)=O BSOQXXWZTUDTEL-ZUYCGGNHSA-N 0.000 claims abstract description 18
- 210000002798 bone marrow cell Anatomy 0.000 claims abstract description 16
- 239000000243 solution Substances 0.000 claims abstract description 14
- 108010019160 Pancreatin Proteins 0.000 claims abstract description 13
- 230000029087 digestion Effects 0.000 claims abstract description 13
- 230000006698 induction Effects 0.000 claims abstract description 13
- 229940055695 pancreatin Drugs 0.000 claims abstract description 13
- 239000000725 suspension Substances 0.000 claims description 20
- 238000005119 centrifugation Methods 0.000 claims description 16
- 210000002966 serum Anatomy 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 8
- 238000012258 culturing Methods 0.000 abstract description 2
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 27
- 102000003971 Fibroblast Growth Factor 1 Human genes 0.000 description 26
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 12
- 230000000052 comparative effect Effects 0.000 description 12
- 238000001514 detection method Methods 0.000 description 12
- 238000008157 ELISA kit Methods 0.000 description 10
- 229910002092 carbon dioxide Inorganic materials 0.000 description 6
- 239000001569 carbon dioxide Substances 0.000 description 6
- 239000002609 medium Substances 0.000 description 4
- 210000003491 skin Anatomy 0.000 description 4
- 210000004927 skin cell Anatomy 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000019522 cellular metabolic process Effects 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 208000028990 Skin injury Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000032677 cell aging Effects 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 230000001146 hypoxic effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0669—Bone marrow stromal cells; Whole bone marrow
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/50—Fibroblast growth factor [FGF]
- C07K14/501—Fibroblast growth factor [FGF] acidic FGF [aFGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/02—Atmosphere, e.g. low oxygen conditions
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/05—Adjuvants
- C12N2501/054—Muramyle peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Rheumatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for high expression of AFGF, which comprises the following steps: passaging when the cells grow to a density of 95%; sucking out the culture solution, adding physiological saline, shaking, and discarding the physiological saline; adding pancreatin to digest and make the cells fully suspended, and adding culture medium to stop digestion; centrifuging the cell suspension in a centrifuge tube, discarding the supernatant, adding physiological saline, sucking a drop of cell count, centrifuging, discarding the physiological saline, suspending cells in a culture medium, and adding the cells in a culture bottle containing the culture medium; adding muramyl dipeptide for induction; and (5) placing the culture flask with the passage end into a low-temperature and low-oxygen incubator for culturing. The method for high expression of AFGF utilizes a low-temperature and low-oxygen environment to enable bone marrow cells to highly express AFGF, so that more AFGF is obtained; the method has the advantages of high efficiency, simplicity and good stability.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a method for high-expression of AFGF.
Background
Acidic Fibroblast Growth Factor (AFGF) is a multifunctional potent cytokine that plays an important role in promoting fibroblast metabolism and collagen formation. AFGF can promote the growth and reproduction of skin tissues, regulate the division, reproduction and growth and differentiation of skin epithelium, endothelium and stroma cells by combining with cell surface specific receptors, promote cell metabolism and enhance oxidation; can promote rapid growth and reproduction of cells related to skin injury, and regulate synthesis, secretion and decomposition of intercellular matrix; can promote regeneration of horny layer cells, accelerate repair of horny layer and matrix layer of skin, and promote growth of skin cells of human body; can enhance protein synthesis and cellular metabolism of skin cells, and has effects in delaying skin cell aging, promoting epidermal cell repair and growth, and making skin smooth and plump.
Patent CN102344930a discloses a simple industrial technology of acidic fibroblast growth factor (aFGF), an aFGF gene is amplified from the eDNA of hepG2 of human origin by an in vitro PCR amplification method, and is linked to an expression vector pET-26b by NdeI and EcoRI restriction sites, and then the plasmid is transformed into escherichia coli BL21 (DE 3) to obtain efficient soluble expression, thereby obtaining the acidic fibroblast growth factor protein of natural structure. However, this method has a drawback of complicated operation.
Disclosure of Invention
The present invention provides a method for high expression of AFGF, which overcomes the above-mentioned drawbacks of the prior art.
In order to achieve the technical purpose, the technical scheme of the invention is realized as follows:
a method of high expression of AFGF comprising the steps of:
s1, carrying out passage when bone marrow cells grow to a density of 95%;
s2, sucking out the culture solution, adding physiological saline, shaking, and discarding the physiological saline;
s3, adding pancreatin, digesting at room temperature to enable the cells to be fully suspended, adding a culture medium, and stopping digestion;
s4, sucking the cell suspension, and putting the cell suspension into a centrifugal tube for centrifugation;
s5, centrifuging, namely, discarding supernatant, adding physiological saline, sucking a drop of cell count, and centrifuging;
s6, centrifuging, discarding normal saline, and adding the culture medium suspension cells into a culture bottle containing the culture medium;
s7, adding 10-10000ng/mL Muramyl Dipeptide (MDP) into the culture flask for induction;
s8, placing the culture flask subjected to passage in the step S7 into a low-temperature and low-oxygen incubator for culturing.
Preferably, the centrifugation in S4 takes place for 5min at 1500rpm.
Preferably, the centrifugation time in S5 is 5min and the rotational speed is 1500rpm.
Preferably, the final concentration of muramyl dipeptide in the flask of S7 is 6000ng/mL.
Preferably, the low-temperature low-oxygen incubator is an incubator with oxygen of 5-40% at 20-36 ℃.
Preferably, the medium is an alpha-MEM medium.
Preferably, the medium is an alpha-MEM medium containing 10% serum replacement.
The invention has the beneficial effects that: the method for high expression of AFGF utilizes a low-temperature and low-oxygen environment to enable bone marrow cells to highly express AFGF, so that more AFGF is obtained; the method has the advantages of high efficiency, simplicity and good stability.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which are derived by a person skilled in the art based on the embodiments of the invention, fall within the scope of protection of the invention.
Example 1
S1, observing the growth of bone marrow cells to a density of about 95% by a microscope, and taking a bottle of cells for passage;
s2, sucking out the culture solution, adding 15mL of physiological saline, gently shaking, and discarding the physiological saline;
s3, adding 3mL of pancreatin into a culture flask, digesting for 5min at room temperature, observing complete suspension of cells by a microscope, adding 5mL of alpha-MEM culture medium (containing 10% of serum substitute), and stopping digestion;
s4, sucking the cell suspension, putting the cell suspension into a centrifuge tube, and centrifuging at 1500rpm for 5min;
s5, after centrifugation, removing the supernatant, adding 45mL of physiological saline, sucking one drop of cell to count, and centrifuging at 1500rpm for 5min;
s6, discarding normal saline, and adding 2mL of culture medium suspension cells into a 150 culture bottle added with the culture medium;
s7, adding 2000ng/mL MDP for induction;
s8, placing the culture flask after passage into a low-temperature and low-oxygen incubator (20 ℃/5% oxygen incubator);
s9, taking the supernatant after 24 hours, and detecting whether the AFGF factor content is improved by using an ELISA kit, wherein the detection result is shown in Table 1.
Example 2
S1, observing the growth of bone marrow cells to a density of about 95% by a microscope, and taking a bottle of cells for passage;
s2, sucking out the culture solution, adding 15mL of physiological saline, gently shaking, and discarding the physiological saline;
s3, adding 3mL of pancreatin into a culture flask, digesting for 5min at room temperature, observing complete suspension of cells by a microscope, adding 5mL of alpha-MEM culture medium (containing 10% of serum substitute), and stopping digestion;
s4, sucking the cell suspension, putting the cell suspension into a centrifuge tube, and centrifuging at 1500rpm for 5min;
s5, after centrifugation, removing the supernatant, adding 45mL of physiological saline, sucking one drop of cell to count, and centrifuging at 1500rpm for 5min;
s6, discarding normal saline, and adding 2mL of culture medium suspension cells into a 150 culture bottle added with the culture medium;
s7, adding 4000ng/mL MDP for induction;
s8, placing the culture flask after passage into a low-temperature and low-oxygen incubator (24 ℃/8% oxygen incubator);
s9, taking the supernatant after 24 hours, and detecting whether the AFGF factor content is improved by using an ELISA kit, wherein the detection result is shown in Table 1.
Example 3
S1, observing the growth of bone marrow cells to a density of about 95% by a microscope, and taking a bottle of cells for passage;
s2, sucking out the culture solution, adding 15mL of physiological saline, gently shaking, and discarding the physiological saline;
s3, adding 3mL of pancreatin into a culture flask, digesting for 5min at room temperature, observing complete suspension of cells by a microscope, adding 5mL of alpha-MEM culture medium (containing 10% of serum substitute), and stopping digestion;
s4, sucking the cell suspension, putting the cell suspension into a centrifuge tube, and centrifuging at 1500rpm for 5min;
s5, after centrifugation, removing the supernatant, adding 45mL of physiological saline, sucking one drop of cell to count, and centrifuging at 1500rpm for 5min;
s6, discarding normal saline, and adding 2mL of culture medium suspension cells into a 150 culture bottle added with the culture medium;
s7, adding 6000ng/mL MDP for induction;
s8, placing the culture flask after passage into a low-temperature and low-oxygen incubator (28 ℃/10% oxygen incubator);
s9, taking the supernatant after 24 hours, and detecting whether the AFGF factor content is improved by using an ELISA kit, wherein the detection result is shown in Table 1.
Example 4
S1, observing the growth of bone marrow cells to a density of about 95% by a microscope, and taking a bottle of cells for passage;
s2, sucking out the culture solution, adding 15mL of physiological saline, gently shaking, and discarding the physiological saline;
s3, adding 3mL of pancreatin into a culture flask, digesting for 5min at room temperature, observing complete suspension of cells by a microscope, adding 5mL of alpha-MEM culture medium (containing 10% of serum substitute), and stopping digestion;
s4, sucking the cell suspension, putting the cell suspension into a centrifuge tube, and centrifuging at 1500rpm for 5min;
s5, after centrifugation, removing the supernatant, adding 45mL of physiological saline, sucking one drop of cell to count, and centrifuging at 1500rpm for 5min;
s6, discarding normal saline, and adding 2mL of culture medium suspension cells into a 150 culture bottle added with the culture medium;
s7, 8000ng/mL MDP is added for induction;
s8, placing the culture flask after passage into a low-temperature low-oxygen incubator (32 ℃/30% oxygen incubator);
s9, taking the supernatant after 24 hours, and detecting whether the AFGF factor content is improved by using an ELISA kit, wherein the detection result is shown in Table 1.
Example 5
S1, observing the growth of bone marrow cells to a density of about 95% by a microscope, and taking a bottle of cells for passage;
s2, sucking out the culture solution, adding 15mL of physiological saline, gently shaking, and discarding the physiological saline;
s3, adding 3mL of pancreatin into a culture flask, digesting for 5min at room temperature, observing complete suspension of cells by a microscope, adding 5mL of alpha-MEM culture medium (containing 10% of serum substitute), and stopping digestion;
s4, sucking the cell suspension, putting the cell suspension into a centrifuge tube, and centrifuging at 1500rpm for 5min;
s5, after centrifugation, removing the supernatant, adding 45mL of physiological saline, sucking one drop of cell to count, and centrifuging at 1500rpm for 5min;
s6, discarding normal saline, and adding 2mL of culture medium suspension cells into a 150 culture bottle added with the culture medium;
s7, 10000ng/mL MDP is added for induction;
s8, placing the culture flask after passage into a low-temperature and low-oxygen incubator (36 ℃/40% oxygen incubator);
s9, taking the supernatant after 24 hours, and detecting whether the AFGF factor content is improved by using an ELISA kit, wherein the detection result is shown in Table 1.
TABLE 1 results of AFGF factor content detection
| Conditions (conditions) | Example 1 | Example 2 | Example 3 | Example 4 | Example 5 |
| Temperature (DEG C) | 20 | 24 | 28 | 32 | 36 |
| Oxygen% | 5 | 8 | 10 | 30 | 40 |
| MDP(ng/mL) | 2000 | 4000 | 6000 | 8000 | 10000 |
| AFGF(pg/mL) | 55.71 | 70.23 | 118.86 | 88.11 | 50.56 |
Comparative example 1
S1, observing the growth of bone marrow cells to a density of about 95% by a microscope, and taking a bottle of cells for passage;
s2, sucking out the culture solution, adding 15mL of physiological saline, gently shaking, and discarding the physiological saline;
s3, adding 3mL of pancreatin into a culture flask, digesting for 5min at room temperature, observing complete suspension of cells by a microscope, adding 5mL of alpha-MEM culture medium (containing 10% of serum substitute), and stopping digestion;
s4, sucking the cell suspension, putting the cell suspension into a centrifuge tube, and centrifuging at 1500rpm for 5min;
s5, after centrifugation, removing the supernatant, adding 45mL of physiological saline, sucking one drop of cell to count, and centrifuging at 1500rpm for 5min;
s6, physiological saline is discarded, and 2mL of culture medium is used for suspending cells and added into a 150 culture flask which is added with the same amount of culture medium as that of the example 1;
s7, adding 2000ng/mL MDP for induction;
s8, placing the culture flask after passage into a normal incubator (37 ℃/5% carbon dioxide incubator);
s9, taking the supernatant after 24 hours, and detecting whether the factor content is improved or not by using an ELISA kit, wherein the detection result is shown in Table 2.
Comparative example 2
S1, observing the growth of bone marrow cells to a density of about 95% by a microscope, and taking a bottle of cells for passage;
s2, sucking out the culture solution, adding 15mL of physiological saline, gently shaking, and discarding the physiological saline;
s3, adding 3mL of pancreatin into a culture flask, digesting for 5min at room temperature, observing complete suspension of cells by a microscope, adding 5mL of alpha-MEM culture medium (containing 10% of serum substitute), and stopping digestion;
s4, sucking the cell suspension, putting the cell suspension into a centrifuge tube, and centrifuging at 1500rpm for 5min;
s5, after centrifugation, removing the supernatant, adding 45mL of physiological saline, sucking one drop of cell to count, and centrifuging at 1500rpm for 5min;
s6, discarding normal saline, and adding 2mL of culture medium suspension cells into a 150 culture flask which is added with the same amount of culture medium as that of the example 2;
s7, adding 4000ng/mL MDP for induction;
s8, placing the culture flask after passage into a normal incubator (37 ℃/5% carbon dioxide incubator);
s9, taking the supernatant after 24 hours, and detecting whether the factor content is improved or not by using an ELISA kit, wherein the detection result is shown in Table 2.
Comparative example 3
S1, observing the growth of bone marrow cells to a density of about 95% by a microscope, and taking a bottle of cells for passage;
s2, sucking out the culture solution, adding 15mL of physiological saline, gently shaking, and discarding the physiological saline;
s3, adding 3mL of pancreatin into a culture flask, digesting for 5min at room temperature, observing complete suspension of cells by a microscope, adding 5mL of alpha-MEM culture medium (containing 10% of serum substitute), and stopping digestion;
s4, sucking the cell suspension, putting the cell suspension into a centrifuge tube, and centrifuging at 1500rpm for 5min;
s5, after centrifugation, removing the supernatant, adding 45mL of physiological saline, sucking one drop of cell to count, and centrifuging at 1500rpm for 5min;
s6, discarding normal saline, and adding 2mL of culture medium suspension cells into a 150 culture bottle added with the same amount of culture medium as that of the example 3;
s7, adding 6000ng/mL MDP for induction;
s8, placing the culture flask after passage into a normal incubator (37 ℃/5% carbon dioxide incubator);
s9, taking the supernatant after 24 hours, and detecting whether the factor content is improved or not by using an ELISA kit, wherein the detection result is shown in Table 2.
Comparative example 4
S1, observing the growth of bone marrow cells to a density of about 95% by a microscope, and taking a bottle of cells for passage;
s2, sucking out the culture solution, adding 15mL of physiological saline, gently shaking, and discarding the physiological saline;
s3, adding 3mL of pancreatin into a culture flask, digesting for 5min at room temperature, observing complete suspension of cells by a microscope, adding 5mL of alpha-MEM culture medium (containing 10% of serum substitute), and stopping digestion;
s4, sucking the cell suspension, putting the cell suspension into a centrifuge tube, and centrifuging at 1500rpm for 5min;
s5, after centrifugation, removing the supernatant, adding 45mL of physiological saline, sucking one drop of cell to count, and centrifuging at 1500rpm for 5min;
s6, discarding normal saline, and adding 2mL of culture medium suspension cells into a 150 culture bottle added with the same amount of culture medium as in the example 4;
s7, 8000ng/mL MDP is added for induction;
s8, placing the culture flask after passage into a normal incubator (37 ℃/5% carbon dioxide incubator);
s9, taking the supernatant after 24 hours, and detecting whether the factor content is improved or not by using an ELISA kit, wherein the detection result is shown in Table 2.
Comparative example 5
S1, observing the growth of bone marrow cells to a density of about 95% by a microscope, and taking a bottle of cells for passage;
s2, sucking out the culture solution, adding 15mL of physiological saline, gently shaking, and discarding the physiological saline;
s3, adding 3mL of pancreatin into a culture flask, digesting for 5min at room temperature, observing complete suspension of cells by a microscope, adding 5mL of alpha-MEM culture medium (containing 10% of serum substitute), and stopping digestion;
s4, sucking the cell suspension, putting the cell suspension into a centrifuge tube, and centrifuging at 1500rpm for 5min;
s5, after centrifugation, removing the supernatant, adding 45mL of physiological saline, sucking one drop of cell to count, and centrifuging at 1500rpm for 5min;
s6, physiological saline is discarded, and 2mL of culture medium is used for suspending cells and added into a 150 culture flask which is added with the same amount of culture medium as that of the example 5;
s7, 10000ng/mL MDP is added for induction;
s8, placing the culture flask after passage into a normal incubator (37 ℃/5% carbon dioxide incubator);
s9, taking the supernatant after 24 hours, and detecting whether the factor content is improved or not by using an ELISA kit, wherein the detection result is shown in Table 2.
TABLE 2 comparative example AFGF factor content detection results
| Conditions (conditions) | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 | Comparative example 5 |
| Temperature (DEG C) | 37 | 37 | 37 | 37 | 37 |
| Carbon dioxide% | 5 | 5 | 5 | 5 | 5 |
| MDP(ng/mL) | 2000 | 4000 | 6000 | 8000 | 10000 |
| AFGF(pg/mL) | 25.33 | 25.67 | 26.43 | 25.83 | 24.12 |
As can be seen from the data in tables 1-2, bone marrow cells were able to highly express AFGF using a low temperature hypoxic environment.
The serum substitutes described in the examples and comparative examples are of the brand thermo fisher, cat No.: 10828028.
in summary, by means of the above technical solution of the present invention, the method for high expression of AFGF of the present invention uses a low-temperature and low-oxygen environment to make bone marrow cells highly express AFGF, thereby obtaining more AFGF; the method has the advantages of high efficiency, simplicity and good stability.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (3)
1. A method for high expression of AFGF comprising the steps of:
s1, carrying out passage when bone marrow cells grow to a density of 95%;
s2, sucking out the culture solution, adding physiological saline, shaking, and discarding the physiological saline;
s3, adding pancreatin, digesting at room temperature to enable the cells to be fully suspended, adding a culture medium, and stopping digestion;
s4, sucking the cell suspension, and putting the cell suspension into a centrifugal tube for centrifugation;
s5, centrifuging, namely, discarding supernatant, adding physiological saline, sucking a drop of cell count, and centrifuging;
s6, centrifuging, discarding normal saline, and adding suspension cells of a culture medium into a culture bottle containing the culture medium, wherein the culture medium is alpha-MEM culture medium containing 10% of serum substitutes;
s7, adding muramyl dipeptide into the culture flask for induction, wherein the final concentration of the muramyl dipeptide in the culture flask is 6000ng/mL;
and S8, placing the culture flask with the passage end into a low-temperature low-oxygen incubator, wherein the low-temperature low-oxygen incubator is a 28 ℃/10% oxygen incubator.
2. The method of claim 1, wherein the centrifugation time in S4 is 5min and the rotational speed is 1500rpm.
3. The method of claim 1, wherein the centrifugation time in S5 is 5min and the rotational speed is 1500rpm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111056851.4A CN113755430B (en) | 2021-09-09 | 2021-09-09 | Method for high expression of AFGF |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111056851.4A CN113755430B (en) | 2021-09-09 | 2021-09-09 | Method for high expression of AFGF |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113755430A CN113755430A (en) | 2021-12-07 |
| CN113755430B true CN113755430B (en) | 2023-08-18 |
Family
ID=78794376
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111056851.4A Active CN113755430B (en) | 2021-09-09 | 2021-09-09 | Method for high expression of AFGF |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113755430B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1400305A (en) * | 2001-07-26 | 2003-03-05 | 广州暨南大学医药生物技术研究开发中心 | Recombinant acid fibroblast growth factor (aFGF) gene engineering bacterium and method for preparing recombinant aFGF by using said bacterium |
| CN1838962A (en) * | 2003-07-10 | 2006-09-27 | 心肌治疗有限公司 | Injection of bone marrow-derived cells and medium for angiogenesis |
| CN101709288A (en) * | 2009-09-29 | 2010-05-19 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method for separating mesenchymal stem/progenitor cells from bone marrow |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6642026B2 (en) * | 2000-08-15 | 2003-11-04 | Phage Biotechnology Corporation | Method of producing biologically active human acidic fibroblast growth factor and its use in promoting angiogenesis |
| WO2018132594A1 (en) * | 2017-01-11 | 2018-07-19 | Spinalcyte, Llc | Methods of enhancing fibroblast therapeutic activity |
-
2021
- 2021-09-09 CN CN202111056851.4A patent/CN113755430B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1400305A (en) * | 2001-07-26 | 2003-03-05 | 广州暨南大学医药生物技术研究开发中心 | Recombinant acid fibroblast growth factor (aFGF) gene engineering bacterium and method for preparing recombinant aFGF by using said bacterium |
| CN1838962A (en) * | 2003-07-10 | 2006-09-27 | 心肌治疗有限公司 | Injection of bone marrow-derived cells and medium for angiogenesis |
| CN101709288A (en) * | 2009-09-29 | 2010-05-19 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method for separating mesenchymal stem/progenitor cells from bone marrow |
Non-Patent Citations (1)
| Title |
|---|
| 孙立新.低氧条件下骨髓间充质干细胞营养因子表达的改变.《牡丹江医学院学报》.2011,第32卷(第32期),11-13. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113755430A (en) | 2021-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20190264179A1 (en) | Serum-free medium inducing differentiation of umbilical cord mesenchymal stem cell into insulin-secretion-like cell and preparation method and use thereof | |
| CN112608894A (en) | Mesenchymal stem cell culture medium | |
| CN113564111B (en) | Method for culturing umbilical cord-derived mesenchymal stem cells in low-oxygen mode | |
| CN111000868B (en) | Application of hypoxia-treated stem cell exosome in preparation of drug or scaffold material for treating spinal cord injury | |
| CN114480273A (en) | Culture medium for obtaining mesenchymal stem cells and exosomes thereof and preparation method thereof | |
| CN110257328A (en) | A kind of mesenchymal stem cell serum-free culture medium | |
| CN112641803A (en) | Stem cell exosome preparation and preparation method thereof | |
| CN113069532A (en) | Skin whitening and anti-aging medicine, health product or cosmetic containing exosome | |
| CN113755430B (en) | Method for high expression of AFGF | |
| CN113287603B (en) | Biological sample preservation solution and preparation method and application thereof | |
| CN117964694B (en) | Polypeptide capable of specifically promoting Nrf2 gene expression and application thereof | |
| EP2345715A3 (en) | Schwann cells originating in myeloid interstitial cells | |
| CN112481207B (en) | Method for promoting cord blood hematopoietic stem cell proliferation by using adipose-derived stem cells | |
| CN111617105A (en) | Preparation method of adipose-derived stem cell multi-cell active factor freeze-dried powder | |
| CN112941015B (en) | Additive and method for preparing keratinocytes based on differentiation of pluripotent stem cells | |
| CN106474157B (en) | Liver stem cell injection and preparation method thereof | |
| JP2020063192A (en) | Cosmetic composition comprising culture supernate fluid of cutis fibroblast and production method thereof | |
| CN110804584B (en) | Optimized umbilical cord mesenchymal stem cell culture solution | |
| CN113046300A (en) | Culture method for preparing keratinocytes based on differentiation of pluripotent stem cells | |
| JPS63304978A (en) | Method for breeding epithelial cell | |
| WO2018179374A1 (en) | Cosmetic composition comprising culture supernatant of dermal fibroblasts and method for manufacturing same | |
| CN115369081B (en) | Stem cell growth promoter, cell culture medium prepared from stem cell growth promoter and application of stem cell growth promoter | |
| CN116376821B (en) | Method for improving expression quantity of umbilical cord mesenchymal stem cell exosomes | |
| US10894947B1 (en) | Method for generating protein rich conditioned medium | |
| CN114350596B (en) | Hair follicle outer root sheath cell culture medium |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |