CN109266608B - Method for regulating and controlling growth of nerve cells by using polyvinyl alcohol/silk fibroin hydrogel - Google Patents
Method for regulating and controlling growth of nerve cells by using polyvinyl alcohol/silk fibroin hydrogel Download PDFInfo
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- 239000004372 Polyvinyl alcohol Substances 0.000 title claims abstract description 75
- 229920002451 polyvinyl alcohol Polymers 0.000 title claims abstract description 75
- 239000000017 hydrogel Substances 0.000 title claims abstract description 50
- 108010022355 Fibroins Proteins 0.000 title claims abstract description 39
- 210000002569 neuron Anatomy 0.000 title claims abstract description 39
- 230000012010 growth Effects 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 20
- 230000001105 regulatory effect Effects 0.000 title claims abstract description 15
- 230000001276 controlling effect Effects 0.000 title claims abstract description 10
- 238000007710 freezing Methods 0.000 claims description 12
- 230000008014 freezing Effects 0.000 claims description 12
- 210000004027 cell Anatomy 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 210000000274 microglia Anatomy 0.000 claims description 7
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- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 6
- 208000005890 Neuroma Diseases 0.000 claims description 6
- 238000010257 thawing Methods 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 239000007850 fluorescent dye Substances 0.000 claims description 4
- 238000002791 soaking Methods 0.000 claims description 4
- 210000003855 cell nucleus Anatomy 0.000 claims description 3
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- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 2
- 206010029260 Neuroblastoma Diseases 0.000 claims description 2
- 210000001130 astrocyte Anatomy 0.000 claims description 2
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- 239000012091 fetal bovine serum Substances 0.000 claims description 2
- 239000008103 glucose Substances 0.000 claims description 2
- 210000004295 hippocampal neuron Anatomy 0.000 claims description 2
- 239000002609 medium Substances 0.000 claims description 2
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- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000005709 nerve cell growth Effects 0.000 description 2
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- 108010069514 Cyclic Peptides Proteins 0.000 description 1
- 102000001189 Cyclic Peptides Human genes 0.000 description 1
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- 108010009711 Phalloidine Proteins 0.000 description 1
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- 210000005036 nerve Anatomy 0.000 description 1
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Abstract
The invention discloses a method for regulating and controlling the growth of nerve cells by using polyvinyl alcohol/silk fibroin hydrogel. The method has strong operability, does not add any growth factor, does not adopt a complicated and complicated design route, can effectively regulate and control the growth of nerve cells only by changing the elastic modulus of the polyvinyl alcohol/silk fibroin hydrogel in a simple and easy way, and has wide application and popularization prospects in the biomedical field.
Description
Technical Field
The invention relates to the technical field of hydrogel and biomedical use, in particular to a method for regulating and controlling the growth of nerve cells by using polyvinyl alcohol/silk fibroin hydrogel.
Background
The hydrogel has a three-dimensional network structure, can absorb a large amount of water to swell but not dissolve, has a structure similar to extracellular matrix, and is one of ideal alternative materials in the field of biomedical science.
Polyvinyl alcohol (PVA) hydrogel has high mechanical property, lubricity and biocompatibility, but is not beneficial to the growth of cells. Silk fibroin (Silk) is natural polymer fibrin extracted from Silk, contains various amino acids, and is beneficial to cell adhesion, proliferation and growth. But the silk fibroin alone used as hydrogel has poor stability, low mechanical strength and poor swelling performance. Therefore, it is a better option to combine PVA with Silk to construct PVA/Silk hydrogels.
The existing literature reports about PVA/Silk hydrogel, but there is no research on the influence of the PVA/Silk hydrogel on the growth of nerve cells, such as the PVA/Silk hydrogel prepared by Zhang, etc., and the friction performance of the hydrogel is researched; the sheep spine bone powder and the nano-hydroxyapatite are doped into PVA/Silk hydrogel by Zhouqi and the like to research the bone defect repair aspect. In view of the above, the present invention provides a method for regulating the growth of nerve cells by using polyvinyl alcohol/silk fibroin hydrogel.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: aiming at the defects of the prior art, the method for regulating the growth of the nerve cells by using the polyvinyl alcohol/silk fibroin hydrogel is high in operability, does not add any growth factor, does not adopt a complicated and complicated design route, can effectively regulate the growth of the nerve cells by changing the elastic modulus of the polyvinyl alcohol/silk fibroin hydrogel only in a simple and easy mode, and has wide application and popularization prospects in the biomedical field.
The technical scheme adopted by the invention for solving the technical problems is as follows: the method for regulating the growth of nerve cells by using the polyvinyl alcohol/silk fibroin hydrogel comprises the following steps:
(1) dissolving polyvinyl alcohol in hot water, stirring until the polyvinyl alcohol is completely dissolved, cooling to room temperature to obtain a polyvinyl alcohol solution with the mass percentage concentration of 5% -15%, then dissolving silk fibroin in the polyvinyl alcohol solution, and uniformly stirring to obtain a polyvinyl alcohol/silk fibroin solution, wherein the mass ratio of the silk fibroin to the polyvinyl alcohol in the polyvinyl alcohol/silk fibroin solution is 5% -30%;
(2) putting a proper amount of polyvinyl alcohol/silk fibroin solution into a mold, and repeatedly freezing and unfreezing for many times to prepare polyvinyl alcohol/silk fibroin hydrogel for later use;
(3) and (3) soaking sterile polyvinyl alcohol/silk fibroin hydrogel in a culture solution containing nerve cells, culturing for 1 to 7 days, and fixing the nerve cells by using a glutaraldehyde solution.
Preferably, the nerve cells include primary culture cell line nerve cells and modified cell line nerve cells.
Further, the nerve cells of the primary culture cell line comprise nerve stem cells, neuron cells or astrocytes, and the modified nerve cells of the cell line comprise microglia (BV 2), mouse hippocampal neuron cells (HT 22), mouse neuroma blast cells (N2 a) or human bone marrow neuroblastoma (SH-SY 5Y).
Preferably, in the step (1), the polyvinyl alcohol is dissolved in hot water at a temperature of 70 to 99 ℃ for 1 to 5 hours.
Preferably, in the step (2), the freezing temperature is-15 ℃ to-30 ℃, the freezing time is 8h to 18h, the thawing temperature is 15 ℃ to 35 ℃, and the thawing time is 6h to 16h in each freezing-thawing cycle.
Preferably, in the step (2), the number of freeze-thaw cycles is 1 to 6, and the elastic modulus of the polyvinyl alcohol/silk fibroin hydrogel obtained by the preparation is 10 to 350 kPa.
Preferably, in the step (3), the culture solution comprises the following components in percentage by mass: 89% of high-glucose DMEM medium, 10% of fetal bovine serum and 1% of double antibody.
Preferably, in the step (3), the mass percentage concentration of the glutaraldehyde solution is 1.5-3%.
Preferably, in the step (3), the obtained nerve cells are subjected to nuclear and cytoplasmic fluorescent staining, and then laser confocal photography, so that the adhesion and proliferation rules of the nerve cells on the polyvinyl alcohol/silk fibroin hydrogel are qualitatively analyzed.
Compared with the prior art, the invention has the advantages that: the invention discloses a method for regulating and controlling the growth of nerve cells by using polyvinyl alcohol/silk fibroin hydrogel. The method has strong operability, does not add any growth factor, does not adopt a complicated and complicated design route, can effectively regulate and control the growth of nerve cells only by changing the elastic modulus of the polyvinyl alcohol/silk fibroin hydrogel in a simple and easy way, and has wide application and popularization prospects in the biomedical field.
Drawings
FIG. 1 is a graph comparing the elastic modulus of the hydrogels PS1, PS2, and PS6 of examples 1 and 2;
FIG. 2 is a fluorescent photograph of BV2 cells grown on PS1, PS2, PS6 hydrogel for 3 days in example 1;
FIG. 3 is a fluorescent photograph of N2a cells grown on PS1, PS2, PS6 hydrogel for 3 days in example 2.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
The method of polyvinyl alcohol/silk fibroin hydrogel of example 1 for modulating nerve cell growth, comprising the steps of:
(1) weighing 20g of PVA, adding 250mL of deionized water, carrying out water bath at 90 ℃, carrying out magnetic stirring for 1.5h until the PVA is completely dissolved, and then cooling to room temperature to obtain a PVA solution with the mass percentage concentration of 8%; weighing 50g of 8% PVA solution, then adding 0.93g of Silk into the PVA solution, and stirring for 2 hours to obtain PVA/Silk solution;
(2) sucking 1mL of PVA/Silk solution by using a disposable pipette, respectively dropping the PVA/Silk solution into 3 48-hole cell culture plates, respectively freezing and unfreezing the 3 48-hole cell culture plates for 1 time, 2 times and 6 times, wherein the freezing temperature of each freezing-unfreezing cycle is-20 ℃, the freezing time is 16 hours, the unfreezing temperature is 25 ℃, and the unfreezing time is 8 hours, and PVA/Silk hydrogel with 1 time, 2 times and 6 times of cycles, which are PS1, PS2 and PS6 for later use, is prepared, wherein the elastic modulus of the PVA/Silk hydrogel is compared with that of PS1, PS2 and PS6 in the figure 1, and the index represents that p is less than 0.001 in the figure 1;
(3) preparing a culture medium containing microglia (BV 2) with a microglia density of 1 × 104And (2) soaking the PS1, PS2 and PS6 hydrogels obtained in the step (2) by using the culture solution respectively, planting microglia on the PS1, PS2 and PS6 hydrogels, after culturing for 3 days, fixing the microglia by using glutaraldehyde solution with the mass percentage concentration of 2.5% for 12 hours, carrying out DAPI cell nucleus and FITC phalloidin peptide cytoplasm fluorescent staining, and taking a corresponding laser confocal microscope photo as shown in figure 2, wherein the higher the elastic modulus of the PVA/Silk hydrogel is, the more beneficial to the growth of the microglia as shown in figure 2.
The method of example 2 for modulating nerve cell growth with polyvinyl alcohol/silk fibroin hydrogel, comprising the steps of:
(1) weighing 20g of PVA, adding 250mL of deionized water, carrying out water bath at 90 ℃, mechanically stirring for 1.5h until the PVA is completely dissolved, and then cooling to room temperature to obtain a PVA solution with the mass percentage concentration of 8%; weighing 50g of 8% PVA solution, then adding 0.93g of Silk into the PVA solution, and stirring for 2 hours to obtain PVA/Silk solution;
(2) sucking 1mL of PVA/Silk solution by using a disposable suction pipe, respectively dropping the PVA/Silk solution into 3 48-hole cell culture plates, respectively freezing and unfreezing the 3 48-hole cell culture plates for 1 time, 2 times and 6 times, wherein the freezing temperature of each freezing-unfreezing cycle is-20 ℃, the freezing time is 16 hours, the unfreezing temperature is 25 ℃, and the unfreezing time is 8 hours, and PVA/Silk hydrogel with 1 time, 2 times and 6 times of cycles, which are referred to as PS1, PS2 and PS6, is respectively prepared for later use, wherein the elastic modulus of the PVA/Silk hydrogel is compared with that of PS1, PS2 and PS6 shown in figure 1;
(3) a culture medium containing mouse neuroma progenitor cells (N2 a) at a density of 1X 10 was prepared4Soaking the PS1, PS2 and PS6 hydrogels obtained in the step (2) with the culture solution, planting the mouse neuroma blast on the PS1, PS2 and PS6 hydrogels, culturing for 3 days, and then using massAfter mouse neuroma blast cells are fixed by glutaraldehyde solution with percentage concentration of 2.5% for 12h, DAPI cell nucleus and FITC phallus cyclopeptide cytoplasm fluorescent staining are carried out, a corresponding laser confocal microscope photo is shown in figure 3, and as shown in figure 3, the higher the elastic modulus of PVA/Silk hydrogel is, the more favorable the mouse neuroma blast cells grow.
Claims (8)
1. The method for regulating the growth of nerve cells by using the polyvinyl alcohol/silk fibroin hydrogel is characterized by comprising the following steps:
(1) dissolving polyvinyl alcohol in hot water, stirring until the polyvinyl alcohol is completely dissolved, cooling to room temperature to obtain a polyvinyl alcohol solution with the mass percentage concentration of 5% -15%, then dissolving silk fibroin in the polyvinyl alcohol solution, and uniformly stirring to obtain a polyvinyl alcohol/silk fibroin solution, wherein the mass ratio of the silk fibroin to the polyvinyl alcohol in the polyvinyl alcohol/silk fibroin solution is 5% -30%;
(2) putting a proper amount of polyvinyl alcohol/silk fibroin solution into a mold, and repeatedly freezing and unfreezing for 1-6 times to prepare polyvinyl alcohol/silk fibroin hydrogel with the elastic modulus of 10-350 kPa for later use;
(3) and (3) soaking sterile polyvinyl alcohol/silk fibroin hydrogel in a culture solution containing nerve cells, culturing for 1 to 7 days, and fixing the nerve cells by using a glutaraldehyde solution.
2. The method for regulating the growth of nerve cells by using polyvinyl alcohol/silk fibroin hydrogel as claimed in claim 1, wherein the nerve cells comprise primary culture cell line nerve cells and modified cell line nerve cells.
3. The method of claim 2, wherein the primary culture cell line nerve cells comprise neural stem cells, neuron cells or astrocytes, and the modified cell line nerve cells comprise microglia cells, mouse hippocampal neuron cells, mouse neuroma blasts or human bone marrow neuroblastoma.
4. The method for regulating and controlling the growth of the nerve cells by utilizing the polyvinyl alcohol/silk fibroin hydrogel as claimed in claim 1, wherein in the step (1), the dissolving temperature of the polyvinyl alcohol in hot water is 70-99 ℃, and the dissolving time is 1-5 h.
5. The method for regulating and controlling the growth of the nerve cells by utilizing the polyvinyl alcohol/silk fibroin hydrogel as claimed in claim 1, wherein in the step (2), the freezing temperature of each freezing-thawing cycle is-15 ℃ to-30 ℃, the freezing time is 8h to 18h, the thawing temperature is 15 ℃ to 35 ℃, and the thawing time is 6h to 16 h.
6. The method for regulating and controlling the growth of the nerve cells by using the polyvinyl alcohol/silk fibroin hydrogel as claimed in claim 1, wherein in the step (3), the culture solution comprises the following components in percentage by mass: 89% of high-glucose DMEM medium, 10% of fetal bovine serum and 1% of double antibody.
7. The method for regulating and controlling the growth of the nerve cells by utilizing the polyvinyl alcohol/silk fibroin hydrogel of claim 1, wherein in the step (3), the mass percentage concentration of the glutaraldehyde solution is 1.5% -3%.
8. The method for regulating and controlling the growth of the nerve cells by using the polyvinyl alcohol/silk fibroin hydrogel as claimed in claim 1, wherein in the step (3), the obtained nerve cells are subjected to cell nucleus and cytoplasm fluorescent staining, and then are subjected to confocal laser photography, so that the adhesion and proliferation rules of the nerve cells on the polyvinyl alcohol/silk fibroin hydrogel are qualitatively analyzed.
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