CN110938598B - Culture method and application of Schwann cells - Google Patents
Culture method and application of Schwann cells Download PDFInfo
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- CN110938598B CN110938598B CN201911418347.7A CN201911418347A CN110938598B CN 110938598 B CN110938598 B CN 110938598B CN 201911418347 A CN201911418347 A CN 201911418347A CN 110938598 B CN110938598 B CN 110938598B
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Abstract
The invention discloses a culture method of schwann cells, which comprises the following steps: cycloastragenol and isoflurane are used in the culture process. The culture steps are as follows: (1) separating and purifying schwann cells and performing primary culture; (2) subculturing schwann cells; (3) expression of nerve growth factor in schwann cells: when 3 rd generation cells grow to fill the bottom of the cell bottle, subculturing the cells in a 25mL culture bottle for 5d by shaking culture, wherein the culture solution is as follows: separately introducing a culture solution of cycloastragenol with a mass fraction of 0.5-0.8% into mixed air containing 3.0% of isoflurane for 3 hours for 2 times.
Description
Technical Field
The invention relates to a culture method of schwann cells and application thereof, belonging to the technical field of cell culture.
Background
Schwann cells (Schwann cells), discovered by Theoder Schwann, form an insulating coating of axons extending from neurons of the peripheral nervous system, are characterized by phagocytic capacity and clearance of cell debris. Schwann cells (Schwann's cells) appear in the peripheral nervous system, form myelin sheaths to insulate and cover axons extending from neurons of the peripheral nervous system, do not cover the entire axons, leave gaps between Schwann cells, called as Langerhans' knots, and accelerate the transmission speed of nerve signals in a jumping type transmission manner. Schwann cells (Schwann's cells) have phagocytic capacity, can remove cellular debris, and provide space for neuronal regeneration.
Nerve Growth Factor (NGF) is one of the most important bioactive factors in the nervous system, and has the effects of promoting nerve cell differentiation and determining the growth direction of axons in peripheral nerve injury. Under certain conditions, Schwann cells cultured by in vitro proliferation can synthesize and secrete a certain amount of nerve growth factors, but the traditional culture method has slow cell proliferation and low nerve growth factor content, and greatly limits the clinical application of the nerve growth factors in the repair of peripheral nerve injury.
The invention aims to provide a culture method of Schwann cells and application thereof, and particularly provides the capacity of the Schwann cells for secreting Nerve Growth Factor (NGF) greatly, and the Schwann cells have great application prospects in preparation of medicines for treating and preventing nerve injury repair.
Disclosure of Invention
The invention aims to provide a culture method of Schwann cells and application thereof, in particular to the capability of the Schwann cells for secreting Nerve Growth Factor (NGF) which has great application prospect in preparing medicaments for treating and preventing nerve injury repair.
Cycloastragenol (CAG) is a main saponin component extracted from traditional Chinese medicine radix astragali, belongs to triterpenoid saponin compounds, and is aglycone of astragaloside. CAG has immunomodulatory, anti-inflammatory, antioxidant, anti-ischemic brain injury, anti-aging effects, such as: CAG inhibits inflammation by inhibiting NLRP3 inflammatory corpuscle, and prevents mouse myocardial cell fibrosis; or as an adjunct to inflammation-induced prostate cancer chemotherapy by inhibiting NF- κ B activity, and the like. Meanwhile, CAG is the only telomerase activator found in the traditional Chinese medicine extracted compound, can effectively activate the telomerase activity of neuron cells, and can be used for the adjuvant therapy of depression; or delaying human embryonic lung fibroblast aging by affecting telomerase activity and klotho gene expression, thereby being considered to have an anti-aging effect. In addition, studies have shown that high concentrations (5, 10, 20 μmol/L) of CAG inhibit CON a-induced T cell activation and proliferation; the low concentration (1 mu mol/L) of CAG can moderately delay the shortening of telomeres of human peripheral blood-derived CD8+ T lymphocytes infected with HIV virus and improve the potential of T cell expansion.
It is generally considered that after the isoflurane inhalation anesthesia treatment, the proliferation of the neural stem cells is remarkably inhibited, namely, obvious neurotoxicity which has certain toxicity to schwann cells is provided, and specific researches are not reported at present.
The inventor finds that the cycloastragenol has weak function of promoting schwann cells to secrete Nerve Growth Factor (NGF); the introduction of isoflurane inhibits the ability of schwann cells to secrete nerve growth factor. However, the addition of trace amounts of isoflurane in combination with cycloastragenol significantly increases the ability of schwann cells to secrete Nerve Growth Factor (NGF).
The research and development team firstly researches the physiological activity of the cycloastragenol and the isoflurane on the Schwann cells and finds that the combined application can be used for stimulating the proliferation of the neural stem cells.
The technical problem to be solved by the invention can be realized by the following technical scheme.
A method of culturing schwann cells, comprising:
cycloastragenol and isoflurane are used in the culture process.
Preferably, the culturing steps are as follows:
(1) separation and purification of schwann cells and primary culture:
killing 20 SD rats born 3-5d, soaking in 75% ethanol for 5min, taking out bilateral sciatic nerves under aseptic condition, removing adventitia and adhesion tissue under microscope as much as possible, washing with PBS for 2 times, shearing into 0.5mm3 size under ophthalmic scissors, adding 0.25% trypsin and 0.06% collagenase, mixing and digesting in 37 deg.C water bath for 30min, shaking, precipitating for 5min, sucking the cell suspension of the upper list, transferring into DMEM culture solution containing 10% fetal calf serum to stop digestion, gently blowing with a suction tube to disperse tissue blocks, centrifuging (1000r/min, 5min), adding appropriate amount of DMEM culture solution containing 10% fetal calf serum, blowing to obtain suspension, adjusting cell concentration to 1 × 108L-1, planted in a 25mL flask pre-coated with polylysine, placed in a 5% volume CO2 incubator at 37 ℃ for 30min, and then partially depleted of fibroblasts. After the cells are cultured for 24 hours, the liquid is changed in a full amount, the non-adherent hybrid cells are removed, cytarabine (the final mass concentration is 2mg/L) is added to inhibit the fibroblasts, the cells are placed in a CO2 incubator with the temperature of 37 ℃ and the volume fraction of 5 percent, the liquid is changed for the first time after 48 hours, the liquid is changed for 1 time every 3 days, and the growth condition of the cells is observed under an inverted microscope every day;
(2) subculturing of Schwann cells
After the cells are 90% full of the bottom of the bottle, washing the cells with PBS for 2 times, repeatedly beating with 0.125% trypsin for 2.0-3.0min, removing pancreatin, adding culture solution, repeatedly beating, counting, and counting at 1 × 107The cell concentration of L-1 is subcultured in a 25mL culture flask;
(3) expression of nerve growth factor in schwann cells:
when the 3 rd generation cells grow to 90% of the cell bottle bottom, 1 × 107The cell concentration of L-1 is subcultured in a 25mL culture flask for 5d by shaking, and the culture solution is: DMEM culture solution containing 10% fetal calf serum and 0.5-0.8% cycloastragenol by volume fraction, and introducing 3 hr of mixed air containing 3.0% isoflurane at the 1 st and 5 th days of culture.
The invention uses ELISA to detect the level of nerve growth factor secreted by Schwann cells, after culturing for 5 days, each group of Schwann cells in a logarithmic growth phase is taken and digested by trypsin-EDTA mixed solution to prepare cell suspension, and 5 × 10 is used8Inoculating L-1 cells into 24-well culture plate coated with polylysine, wherein each group has 6 wells, the culture solution is DMEM complete culture medium containing 10% fetal calf serum by volume fraction, each well has 1mL, placing at 37 deg.C, and has 5% CO by volume fraction2Culturing in incubator for 48h, centrifuging (1000 r/min) for 5min, collecting supernatant, transferring the supernatant into sterile EP tube, centrifuging again for 5min (1000 r/min), collecting supernatant, and refrigerating at-20 deg.C for testing. After the samples are completely collected, detection is carried out according to the specification of an ELISA test box of the nerve growth factor, the absorbance value of each hole is measured after the blank control hole is adjusted to zero at 450nm, and the obtained absorbance value is in direct proportion to the level of the nerve growth factor in the supernatant of each group of cells.
The invention has the advantages that:
the inventor finds that the cycloastragenol has weak function of promoting schwann cells to secrete Nerve Growth Factor (NGF); the introduction of isoflurane inhibits the ability of schwann cells to secrete nerve growth factor. However, the addition of trace amounts of isoflurane in combination with cycloastragenol significantly increases the ability of schwann cells to secrete Nerve Growth Factor (NGF). The research and development team researches the physiological activity of the cycloastragenol and the isoflurane to the Schwann cells for the first time, and finds that the combined application can be used for stimulating the proliferation of the neural stem cells and has great application potential in neural restoration.
Detailed Description
The experiment of the invention is completed in a laboratory of a subsidiary hospital of Qingdao university.
The following examples of the present invention are described in detail, and are only for the purpose of illustrating the present invention and are not to be construed as limiting the present invention.
Specific examples of the present invention are described below.
Example 1
Experimental group 1:
(1) separation and purification of schwann cells and primary culture:
killing 20 SD rats born 3-5d, soaking in 75% ethanol for 5min, taking out bilateral sciatic nerves under aseptic condition, removing adventitia and adhesion tissue under microscope as much as possible, washing with PBS for 2 times, shearing into 0.5mm3 size under ophthalmic scissors, adding 0.25% trypsin and 0.06% collagenase, mixing and digesting in 37 deg.C water bath for 30min, shaking, precipitating for 5min, sucking the cell suspension of the upper list, transferring into DMEM culture solution containing 10% fetal calf serum to stop digestion, gently blowing with a suction tube to disperse tissue blocks, centrifuging (1000r/min, 5min), adding appropriate amount of DMEM culture solution containing 10% fetal calf serum, blowing to obtain suspension, adjusting cell concentration to 1 × 108L-1 is planted in a 25mL culture bottle pre-coated with polylysine, placed in a CO2 incubator with the volume fraction of 5% at 37 ℃ for 30min, and then part of fibroblasts are attached to the wall at a differential speed of two 30 min. After the cells are cultured for 24 hours, the liquid is changed in a full amount, the non-adherent hybrid cells are removed, cytarabine (the final mass concentration is 2mg/L) is added to inhibit the fibroblasts, the cells are placed in a CO2 incubator with the temperature of 37 ℃ and the volume fraction of 5 percent, the liquid is changed for the first time after 48 hours, the liquid is changed for 1 time every 3 days, and the growth condition of the cells is observed under an inverted microscope every day;
(2) subculturing Schwann cells until the cells are 90% full of the bottom of the flask, washing the cells with PBS for 2 times, repeatedly beating with 0.125% trypsin for 2.0min, removing pancreatin, adding culture medium, repeatedly beating, counting, and counting at 1 × 107The cell concentration of L-1 is subcultured in a 25mL culture flask;
(3) expression of nerve growth factor in schwann cells:
when the 3 rd generation cells grow to 90% of the cell bottle bottom, 1 × 107Cell concentration of L-1 was subcultured toShaking and culturing the mixture in a 25mL culture bottle for 5d, wherein the culture solution is as follows: DMEM culture solution containing 10% fetal calf serum and 0.5% cycloastragenol by volume fraction, and introducing mixed air containing 3.0% isoflurane for 3 hours at the 1 st and 5 th days of culture.
Experimental group 2:
(1) separation and purification of schwann cells and primary culture:
killing 20 SD rats born 3-5d, soaking in 75% ethanol for 5min, taking out bilateral sciatic nerves under aseptic condition, removing adventitia and adhesion tissue under microscope as much as possible, washing with PBS for 2 times, shearing into 0.5mm3 size under ophthalmic scissors, adding 0.25% trypsin and 0.06% collagenase, mixing and digesting in 37 deg.C water bath for 30min, shaking, precipitating for 5min, sucking the cell suspension of the upper list, transferring into DMEM culture solution containing 10% fetal calf serum to stop digestion, gently blowing with a suction tube to disperse tissue blocks, centrifuging (1000r/min, 5min), adding appropriate amount of DMEM culture solution containing 10% fetal calf serum, blowing to obtain suspension, adjusting cell concentration to 1 × 108L-1 is planted in a 25mL culture bottle pre-coated with polylysine, placed in a CO2 incubator with the volume fraction of 5% at 37 ℃ for 30min, and then part of fibroblasts are attached to the wall at a differential speed of two 30 min. After the cells are cultured for 24 hours, the liquid is changed in a full amount, the non-adherent hybrid cells are removed, cytarabine (the final mass concentration is 2mg/L) is added to inhibit the fibroblasts, the cells are placed in a CO2 incubator with the temperature of 37 ℃ and the volume fraction of 5 percent, the liquid is changed for the first time after 48 hours, the liquid is changed for 1 time every 3 days, and the growth condition of the cells is observed under an inverted microscope every day;
(2) subculturing Schwann cells until the cells are 90% full of the bottom of the flask, washing the cells with PBS for 2 times, repeatedly beating with 0.125% trypsin for 3.0min, removing pancreatin, adding culture medium, repeatedly beating, counting, and counting at 1 × 107The cell concentration of L-1 is subcultured in a 25mL culture flask;
(3) expression of nerve growth factor in schwann cells:
when the 3 rd generation cells grow to 90% of the cell bottle bottom, 1 × 107Cell concentration of L-1Inoculating the culture solution into a 25mL culture bottle for shaking culture for 5d, wherein the culture solution comprises: DMEM culture solution containing 10% fetal calf serum and 0.8% cycloastragenol by volume fraction is introduced into the culture medium at 1d and 5d for 3 hours to form mixed air containing 3.0% isoflurane.
Blank control group:
(1) separation and purification of schwann cells and primary culture: same as experimental group 1;
(2) subculturing schwann cells: same as experimental group 1;
(3) expression of nerve growth factor in schwann cells: the other experiment is the same as the experiment group 1, but 10% of fetal calf serum does not contain the DMEM culture solution of the cycloastragenol, and 3.0% of mixed air of the isoflurane is not introduced into the 5 th day.
Comparative example 1:
(1) separation and purification of schwann cells and primary culture: same as experimental group 1;
(2) subculturing schwann cells: same as experimental group 1;
(3) expression of nerve growth factor in schwann cells: the other experiment was the same as the experiment 1, but 10% fetal bovine serum was used alone, and the culture solution of DMEM without cycloastragenol was used.
Comparative example 2:
(1) separation and purification of schwann cells and primary culture: same as experimental group 1;
(2) subculturing schwann cells: same as experimental group 1;
(3) expression of nerve growth factor in schwann cells: the other experiment was the same as the experiment 1 except that the 5 th day mixed air containing 3.0% of isoflurane was not introduced.
Example 2
The invention uses ELISA to detect the level of nerve growth factor secreted by Schwann cells, after culturing for 5 days, each group of Schwann cells in a logarithmic growth phase is taken and digested by trypsin-EDTA mixed solution to prepare cell suspension, and 5 × 10 is used8Inoculating L-1 cells into 24-well culture plate coated with polylysine, wherein each group has 6 wells, the culture solution is DMEM complete culture medium containing 10% fetal calf serum by volume fraction, each well has 1mL, placing at 37 deg.C, and has 5% CO by volume fraction2Culturing in an incubator for 48h, centrifuging1000 r/min) for 5min, transferring the supernatant into a sterile EP tube, centrifuging for 5min (1000 r/min) again, taking the supernatant, and refrigerating at-20 ℃ for testing. After the sample is completely collected, detection is performed according to the nerve growth factor ELISA test kit instructions. The results are shown in table 1 below.
Statistical treatment: statistical analysis was performed using SPSS16.0 and the results of the data were expressed as mean ± standard deviation using the t test between groups.
TABLE 1 nerve growth factor Mass concentration
| Mass concentration of nerve growth factor (ng/L) | |
| Experimental group 1 | 112.75±12.64* |
| Experimental group 2 | 121.34±9.75* |
| Blank control group | 50.80±9.37 |
| Comparative example 1 | 49.11±8.43 |
| Comparative example 2 | 59.77±7.54 |
Note: t-test, x: p <0.05 (compare with blank group)
It can be seen that cycloastragenol has weak function of promoting schwann cells to secrete Nerve Growth Factor (NGF); the introduction of isoflurane inhibits the ability of schwann cells to secrete nerve growth factor. However, the addition of trace amounts of isoflurane in combination with cycloastragenol significantly increases the ability of schwann cells to secrete Nerve Growth Factor (NGF). The research and development team researches the physiological activity of the cycloastragenol and the isoflurane to the Schwann cells for the first time, and finds that the combined application can be used for stimulating the proliferation of the neural stem cells and has great application potential in neural restoration.
It is to be understood that the foregoing is only a preferred embodiment of the invention and that modifications, variations and changes may be made in the invention without departing from the spirit or scope of the invention as defined in the appended claims.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (2)
1. A method of culturing schwann cells, comprising:
cycloastragenol and isoflurane are applied in the culture process;
the culture steps are as follows:
(1) separating and purifying schwann cells and performing primary culture;
(2) subculturing schwann cells;
(3) expression of nerve growth factor in schwann cells:
when the cells were passaged to passage 3 and grown to 90% of the cell bottom, the cells were cultured at 1 × 107The cell concentration of L-1 is subcultured in a 25mL culture flask for 5d by shaking, and the culture solution is: DMEM culture solution containing 10% fetal calf serum and 0.5-0.8% cycloastragenol by volume fraction, and introducing 3 hr of mixed air containing 3.0% isoflurane at the 1 st and 5 th days of culture.
2. The culture method according to claim 1, wherein:
the mixed air is 3.0% of isoflurane, 5% of carbon dioxide and 92% of oxygen.
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