WO2025007745A1 - 一种乙酰化葡甘聚糖静电纺丝膜及其制备方法与应用 - Google Patents

一种乙酰化葡甘聚糖静电纺丝膜及其制备方法与应用 Download PDF

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WO2025007745A1
WO2025007745A1 PCT/CN2024/100091 CN2024100091W WO2025007745A1 WO 2025007745 A1 WO2025007745 A1 WO 2025007745A1 CN 2024100091 W CN2024100091 W CN 2024100091W WO 2025007745 A1 WO2025007745 A1 WO 2025007745A1
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electrospinning
glucomannan
acetylated
present application
membrane
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French (fr)
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陈敏
高月
昂松
冯雁贤
赵丽娟
李文希
蔡宾
卢婉婷
孔雯婷
林佩雯
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Wuyi University Fujian
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    • D—TEXTILES; PAPER
    • D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
    • D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
    • D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
    • D04H1/70—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres
    • D04H1/72—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged
    • D04H1/728—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged by electro-spinning
    • 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
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
    • D01D5/00—Formation of filaments, threads, or the like
    • D01D5/0007—Electro-spinning
    • D01D5/0015—Electro-spinning characterised by the initial state of the material
    • D01D5/003—Electro-spinning characterised by the initial state of the material the material being a polymer solution or dispersion
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
    • D01D5/00—Formation of filaments, threads, or the like
    • D01D5/0007—Electro-spinning
    • D01D5/0061—Electro-spinning characterised by the electro-spinning apparatus
    • D01D5/0069—Electro-spinning characterised by the electro-spinning apparatus characterised by the spinning section, e.g. capillary tube, protrusion or pin
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
    • D01D5/00—Formation of filaments, threads, or the like
    • D01D5/0007—Electro-spinning
    • D01D5/0061—Electro-spinning characterised by the electro-spinning apparatus
    • D01D5/0092—Electro-spinning characterised by the electro-spinning apparatus characterised by the electrical field, e.g. combined with a magnetic fields, using biased or alternating fields
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F9/00—Artificial filaments or the like of other substances; Manufacture thereof; Apparatus specially adapted for the manufacture of carbon filaments
    • D—TEXTILES; PAPER
    • D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
    • D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
    • D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
    • D04H1/40—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres from fleeces or layers composed of fibres without existing or potential cohesive properties
    • D04H1/42—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres from fleeces or layers composed of fibres without existing or potential cohesive properties characterised by the use of certain kinds of fibres insofar as this use has no preponderant influence on the consolidation of the fleece
    • D04H1/4326—Condensation or reaction polymers
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • the present application relates to the technical field of biomaterials, and in particular to an acetylated glucomannan electrospinning membrane and a preparation method and application thereof.
  • mammalian cells mainly rely on the interaction between extracellular matrices to generate connections and signal transduction.
  • 2D cell culture is a traditional and easy-to-operate culture method, but it can only form a flat cell morphology and even change the gene expression of cells.
  • 3D cell culture has gradually become the mainstream culture method because it can better simulate the cell growth environment in vivo.
  • Cell tissues cultured in this way are often used to study cell metabolism and tissue regeneration.
  • the present application provides an acetylated glucomannan electrospinning membrane and a preparation method and application thereof.
  • the present application aims to solve at least one of the technical problems existing in the prior art.
  • the present application proposes an acetylated glucomannan electrospinning membrane and a preparation method and application thereof.
  • the preparation method of the acetylated glucomannan electrospinning membrane of the present application is simple, the prepared acetylated glucomannan molecular chain is rich in a large number of acetyl groups (-COCH 3 ), the prepared acetylated glucomannan electrospinning membrane fiber diameter is 905 ⁇ 283nm, there is no obvious beaded structure, the fibers are interlaced into a mesh, forming interconnected gaps, and the application of the membrane to cell culture can well simulate the structure of the extracellular matrix, which is conducive to promoting cell adhesion and proliferation.
  • the present application also proposes an acetylated glucomannan electrospinning membrane.
  • a method for preparing an acetylated glucomannan electrospinning membrane comprising the following steps:
  • Step S1 adding glucomannan to a mixed solution of pyridine and acid anhydride, separating the solid and the liquid after the reaction, collecting the solid phase and adding ethanol for precipitation, and then collecting the precipitate to obtain acetylated glucomannan;
  • Step S3 using the electrospinning solution as a raw material, and obtaining an acetylated glucomannan electrospinning membrane through electrospinning.
  • the reaction time is 48 h to 96 h.
  • step S1 the mass volume ratio of the solid phase to ethanol is 1 g: (2-10) mL.
  • the ethanol is analytically pure ethanol, and specifically, the ethanol is pre-cooled analytically pure ethanol.
  • the degree of acetylation of the acetylated glucomannan is 3.0.
  • step S2 the weight-to-volume ratio of the acetylated glucomannan to the mixed solution of chloroform and N,N-dimethylformamide is (150-250) mg:1 mL.
  • the volume ratio of chloroform to N,N-dimethylformamide is 1.8-2.5:1.
  • adding a certain amount of N,N-dimethylformamide helps to prevent chloroform from volatilizing too quickly.
  • the electrospinning parameters include: a sample injection rate of 0.6 mL/h to 1 mL/h,
  • the inner diameter of the needle is 0.33mm to 0.51mm, and the outer diameter of the needle is 0.64mm to 0.81mm.
  • the electrospinning parameters are: injection speed of 0.8 mL/h, needle inner diameter of 0.41 mm, and needle outer diameter of 0.64 mm to 0.81 mm.
  • the needle may be a flat needle.
  • the electrospinning solution contains an organic solvent and has a certain viscosity
  • the use of a flat needle is beneficial to avoid clogging due to corrosion.
  • the voltage at the needle tip is +10 kV to +15 kV
  • the voltage at the receiving plate is -2 kV to -4 kV
  • the distance between the needle tip and the collecting plate is 8 cm to 12 cm.
  • the voltage at the needle tip is +12 kV
  • the voltage at the receiving plate can be -3 kV.
  • the temperature is 40° C. to 45° C., and the relative humidity is 8% to 12%. Specifically, during the electrospinning process, the temperature may be 40° C., and the relative humidity may be 10%.
  • the second aspect of the present application provides an acetylated glucomannan electrospinning membrane, which is prepared by the preparation method described in the first aspect.
  • acetylated glucomannan electrospinning membrane fibers prepared by the preparation method of the present application are intertwined into a network to form interconnected gaps, which can better simulate the structure of the extracellular matrix and facilitate cell adhesion and proliferation.
  • the fiber diameter of the acetylated glucomannan electrospinning membrane is 500 nm to 1300 nm;
  • the fiber diameter of the acetylated glucomannan electrospinning membrane is 905 ⁇ 283 nm.
  • the third aspect of the present application provides a composition for cell culture, comprising the acetylated glucomannan electrospinning membrane of the second aspect and gelatin.
  • the cell culture composition of the present application includes acetylated glucomannan electrospinning membrane and gelatin, which can be used as a coating material during the cell culture process, significantly reduce cell death, promote continuous cell proliferation, and has an obvious synergistic effect.
  • the added ratio of gelatin is 0.05% to 0.2%.
  • the addition ratio of the gelatin may be 0.1%.
  • the cell culture composition is prepared by soaking the acetylated glucomannan electrospun membrane in a 0.05% to 0.2% gelatin solution.
  • the soaking time is 6 hours to 10 hours.
  • the fourth aspect of the present application provides use of the acetylated glucomannan electrospinning membrane of the second aspect or the cell culture composition of the third aspect in cell proliferation and/or cell differentiation culture.
  • acetylated glucomannan electrospinning membrane or cell culture composition of the present application can significantly reduce cell death and promote continuous cell proliferation.
  • the results of promoting the directed differentiation of NILB-hiPSCs cells show that it can significantly promote the further dispersion of NILB-hiPSCs, the continuous extension of synapses, the reduction of cell nuclei, and the formation of neuron-like cells.
  • the stem cells may be human induced pluripotent stem cells
  • the stem cells may be human induced pluripotent stem cells directed to differentiate into motor neurons.
  • the cell differentiation culture includes directed differentiation culture of human induced pluripotent stem cells
  • the directed differentiated cell type is a neuron-like cell
  • the culture medium for directed differentiation culture is Neurobasal medium supplemented with 1 ⁇ B27 additive (ThermoFish), 1 ⁇ N2 additive (ThermoFish), 0.5 ⁇ double antibody (Gibco), BDNF (10 ng/mL, Alomone Lab), GDNF (10 ng/mL, Alomone Lab) and NT3 (10 ng/mL, Alomone Lab).
  • FIG. 1 is an infrared spectrum of acetylated glucomannan and glucomannan in Example 1 of the present application.
  • FIG. 2 is a hydrogen nuclear magnetic resonance spectrum of acetylated glucomannan and glucomannan in Example 1 of the present application.
  • FIG. 3 is a diagram showing the morphology observation results of the acetylated glucomannan electrospinning membrane of the present application.
  • FIG. 4 is a fluorescence micrograph of the present invention's stem cells cultured on an acetylated glucomannan (acGM) electrospinning membrane.
  • acGM acetylated glucomannan
  • Figure 5 is a diagram showing the results of directed differentiation of stem cells at different growth stages on the acetylated glucomannan (acGM) electrospinning membrane of the present application, wherein Figure A in Figure 5 is a flow chart of induced differentiation, and Figure B in Figure 5 is a diagram showing the results of directed differentiation on the third and fifth days.
  • acGM acetylated glucomannan
  • FIG. 6 is a diagram showing the expression of TUJ1, a universal neuron marker, at day 7 of differentiation culture in the present application.
  • FIG. 7 is a diagram showing the expression of the motor neuron-specific marker HB9 at day 7 of differentiation culture of the present application.
  • FIG8 is a graph showing the expression of the neuron universal marker TUJ1 and marker MAP2 at day 14 of differentiation culture of the present application.
  • This embodiment provides a method for preparing an acetylated glucomannan (acGM) electrospinning membrane, comprising the following steps:
  • GM glucomannan
  • the electrospinning solution was transferred into a 2.5 mL plastic syringe with a flat 22G needle, fixed on an electrospinning device, and an electrospinning membrane was prepared to obtain an acetylated glucomannan electrospinning membrane, wherein the electrospinning parameters were: voltage: 15 kV, wherein the voltage at the needle was +12 kV, and the voltage at the receiving plate was -3 kV; the injection speed was 0.8 mL/h; The distance between the needle tip and the collection plate was 10 cm; the temperature was 40° C., and the relative humidity (RH) was 10%.
  • the electrospinning parameters were: voltage: 15 kV, wherein the voltage at the needle was +12 kV, and the voltage at the receiving plate was -3 kV; the injection speed was 0.8 mL/h; The distance between the needle tip and the collection plate was 10 cm; the temperature was 40° C., and the relative humidity (RH) was 10%.
  • This embodiment provides an acetylated glucomannan electrospinning membrane, which is prepared by the method of the above embodiment 1.
  • This embodiment provides a set of compositions for cell culture, comprising the acetylated glucomannan electrospinning membrane prepared in the above embodiment 1 and 1% Gelatin (purchased from STEMCELL technologies).
  • Test example Performance characterization of acetylated glucomannan electrospun membrane
  • the structure was characterized by infrared spectrometer, and the infrared spectra of glucomannan and acetylated glucomannan are shown in Figure 1. It can be seen from the figure that acetylated glucomannan has a strong absorption peak near 1747cm -1 , which is the carbonyl stretching vibration peak, and an obvious absorption peak near 1372cm -1 , which is the methyl absorption peak. A strong absorption peak appears near 1237cm -1 , which is the absorption peak of the saturated ester group, proving that there are a large number of acetyl groups (-COCH 3 ) on the acGM molecular chain.
  • acetylated glucomannan has a strong absorption peak near 1747cm -1 , which is the carbonyl stretching vibration peak, and an obvious absorption peak near 1372cm -1 , which is the methyl absorption peak.
  • a strong absorption peak appears near 1237cm
  • Example 1 The acGM electrospun membrane prepared in Example 1 was cut into 10*10 mm squares, placed in a 24-well cell culture plate, and soaked in 75% alcohol for 2 h. After discarding the 75% alcohol, the culture plate was placed in a biosafety cabinet and irradiated with ultraviolet light for 30 min for further disinfection.
  • the human induced pluripotent stem cells (NILB-hiPSCs) that had been successfully constructed in the laboratory for motor neuron-directed differentiation (this cell line conditionally expresses motor neuron-specific transcription factors Ngn2, Isl1 and Lhx3 under the induction of the drug doxycycline dox; and overexpresses the anti-apoptotic gene Bcl-xl and green fluorescent GFP) were inoculated into the culture well plate, and the stem cell culture medium mTeSR (purchased from STEMCELL technologies) containing the anti-apoptotic small molecule Y27632 (MCE, 10 ⁇ M) was added, and the cells were cultured overnight in a cell culture incubator (37°C, 5% CO2 ).
  • mTeSR purchased from STEMCELL technologies
  • Application Example 2 Application of acetylated glucomannan electrospinning membrane in promoting directional differentiation of stem cells
  • NILB-hiPSCs were seeded in the Gelatin+acGM electrospinning membrane at a seeding density of 5*10 4 cells/well (24-well plate).
  • Blocking and permeabilization Add 500 ⁇ L of tissue blocking solution to each well and block for 1 to 2 hours at room temperature.
  • NILB-hiPSCs can be 3D cultured on Gelatin+acGM electrospinning membranes and differentiated into motor neurons under drug induction. These induced neurons express a large number of motor neuron-related markers.
  • the present application provides an acetylated glucomannan electrospinning membrane and a preparation method and application thereof, wherein the preparation method comprises: firstly, acetylated glucomannan (acGM) is obtained by a mixed reaction of pyridine, anhydride and glucomannan, and alcohol precipitation is performed, and the analysis results of its organic functional groups show that there are a large number of acetyl groups (-COCH 3 ) on the obtained acGM molecular chain, and its acetylation degree reaches 3.0; further, the acetylated glucomannan prepared in the present application is mixed with chloroform and N,N-dimethylformamide for reaction, and an electrospinning solution is obtained after ultrasonic treatment, and then the electrospinning solution is used as a raw material, and an acetylated glucomannan electrospinning membrane is obtained after electrospinning treatment.
  • the preparation method is simple, does not require the addition of additional solub
  • the fibers are intertwined into a network to form interconnected gaps, which can relatively better simulate the structure of the extracellular matrix and play an important role in promoting cell adhesion and proliferation.
  • the acetylated glucomannan electrospinning membrane prepared in the present application was applied to stem cell culture, and the results showed that it could reduce cell death and promote cell proliferation and directional differentiation. Further, the acetylated glucomannan electrospinning membrane prepared in the present application was applied to directional differentiation culture of neurons, and the results showed that it could directional differentiate.

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Abstract

本申请公开了一种乙酰化葡甘聚糖静电纺丝膜及其制备方法与应用。本申请的静电纺丝膜制备方法包括：首先在吡啶和酸酐的混合溶液中加入葡甘聚糖，反应后固液分离，收集固相并加入乙醇进行沉淀，收集沉淀即得到乙酰化葡甘聚糖；然后将乙酰化葡甘聚糖溶解于氯仿和N,N-二甲基甲酰胺的混合溶液，经超声处理后得到电纺丝溶液；最后以电纺丝溶液为原料，经静电纺丝后即得到乙酰化葡甘聚糖静电纺丝膜。

Description

一种乙酰化葡甘聚糖静电纺丝膜及其制备方法与应用 技术领域
本申请涉及生物材料技术领域，尤其是涉及一种乙酰化葡甘聚糖静电纺丝膜及其制备方法与应用。
背景技术
在生物体内，哺乳细胞主要依赖细胞外基质之间的相互作用，产生连接和信号转导，为了在体外模拟细胞培养过程，2D细胞培养是一种传统的且操作简单的培养方式，但只能形成扁平的细胞形态，甚至改变细胞的基因表达。而3D细胞培养由于其能够更好地模拟体内细胞生长环境逐渐成为主流培养方式，采用该方式培养出来的细胞组织常用于研究细胞的代谢及组织再生。
在相关技术中，小鼠软骨肉瘤细胞分泌的细胞外基质(Matrigel)被广泛使用于细胞3D培养系统中，该基质主要包括胶原、粘层蛋白、明胶、纤维蛋白、透明质酸等多种成分，有利于细胞的生长和粘附。然而，这类的细胞外基质通常是动物来源的，含有疾病相关的成分，当将其作为细胞3D培养组分时，其疾病相关成分可能会对细胞培养造成不利影响，并且由于细胞外基质成分的不确定性导致其培养的重复性和可控性较差。
为解决上述问题，本申请提供了一种乙酰化葡甘聚糖静电纺丝膜及其制备方法与应用。
发明内容
本申请旨在至少解决现有技术中存在的技术问题之一。为此，本申请提出了一种乙酰化葡甘聚糖静电纺丝膜及其制备方法与应用。本申请的乙酰化葡甘聚糖静电纺丝膜制备方法简单，制得的乙酰化葡甘聚糖分子链上富含大量的乙酰基(-COCH3)，制得的乙酰化葡甘聚糖静电纺丝膜纤维直径为905±283nm，无明显的串珠结构，纤维之间相互交错成网状，形成互相通联的空隙，将其应用于细胞培养，能够很好地模拟细胞外基质的结构，有利于促进细胞黏附和增殖。
本申请还提出一种乙酰化葡甘聚糖静电纺丝膜。
本申请还提出一种细胞培养用组合物。
本申请还提出一种乙酰化葡甘聚糖静电纺丝膜或细胞培养用组合物在细胞增殖和/或细 胞分化培养中的应用。
本申请的第一方面，提供了一种乙酰化葡甘聚糖静电纺丝膜的制备方法，包括以下步骤：
步骤S1：在吡啶和酸酐的混合溶液中加入葡甘聚糖，反应后固液分离，收集固相并加入乙醇进行沉淀，然后收集沉淀即得到乙酰化葡甘聚糖；
步骤S2：将所述乙酰化葡甘聚糖溶解于氯仿和N,N-二甲基甲酰胺的混合溶液，经超声处理后得到电纺丝溶液；
步骤S3：以所述电纺丝溶液为原料，经静电纺丝后即得到乙酰化葡甘聚糖静电纺丝膜。
根据本申请实施例的制备方法，至少具有如下有益效果：
本申请乙酰化葡甘聚糖静电纺丝膜的制备方法简单，首先通过吡啶、酸酐和葡甘聚糖的混合反应，经醇沉后得到乙酰化葡甘聚糖(acGM)，其有机官能团的分析结果表明，制得的acGM分子链上存在大量的乙酰基(-COCH3)，其乙酰度达到3.0；进一步地，本申请将制得的乙酰化葡甘聚糖与氯仿和N,N-二甲基甲酰胺混合反应，经超声处理后得到电纺丝溶液，然后以电纺丝溶液为原料，经静电纺丝处理后得到乙酰化葡甘聚糖静电纺丝膜。该制备方法简单，无需额外添加助溶性物质，成本低，适用于工业化生产。
在本申请的一些实施方式中，步骤S1中，所述葡甘聚糖与吡啶和酸酐的混合溶液的重量体积比为(0.8-1.2)g：100mL。
在本申请的一些实施方式中，所述混合溶液中吡啶和酸酐的体积比为1：0.8-1.2。
在本申请的一些实施方式中，步骤S1中，所述反应的温度为45℃至60℃；
优选地，所述反应的时间为48h至96h。
在本申请的一些实施方式中，步骤S1中，所述固相与乙醇的质量体积比为1g：(2-10)mL。
在本申请的一些实施方式中，所述乙醇为分析纯乙醇，具体的，所述乙醇为预冷的分析纯乙醇。
在本申请的一些实施方式中，所述乙酰化葡甘聚糖的乙酰度为3.0。
在本申请的一些实施方式中，步骤S2中，所述乙酰化葡甘聚糖与氯仿和N,N-二甲基甲酰胺的混合溶液的重量体积比为(150-250)mg：1mL。
在本申请的一些实施方式中，所述氯仿和N,N-二甲基甲酰胺的体积比为1.8-2.5：1。
根据本申请的一些实施方式，添加一定含量的N,N-二甲基甲酰胺有助于防止氯仿过快挥发。
在本申请的一些实施方式中，所述静电纺丝的参数包括：进样速度为0.6mL/h至1mL/h， 针头内径为0.33mm至0.51mm，针头外径为0.64mm至0.81mm。
优选地，所述静电纺丝的参数为：进样速度为0.8mL/h，针头内径为0.41mm，针头外径为0.64mm至0.81mm。
在本申请的一些实施方式中，所述针头可以为扁平针头。
根据本申请的一些实施方式，由于电纺丝溶液含有机溶剂并且带一定的黏性，采用扁平针头有利于避免被腐蚀堵住。
在本申请的一些实施方式中，所述静电纺丝过程中，针头处的电压为+10kV至+15kV，接收板处的电压为-2kV至-4kV，针尖与采集板之间的距离为8cm至12cm。具体的，所述针头处的电压为+12kV，接收板处的电压可以为-3kV。
在本申请的一些实施方式中，所述静电纺丝过程中，所述温度为40℃至45℃，相对湿度为8％至12％。具体的，所述静电纺丝过程中，所述温度可以为40℃，相对湿度可以为10％。
本申请的第二方面，提供了一种乙酰化葡甘聚糖静电纺丝膜，采用第一方面所述的制备方法制得。
根据本申请实施例的乙酰化葡甘聚糖静电纺丝膜，至少具有如下有益效果：
采用本申请制备方法制得的乙酰化葡甘聚糖静电纺丝膜的纤维形貌主要为细圆柱状，其构成乙酰化葡聚糖静电纺丝膜的纤维直径为905±283nm(n＝10)，且无明显的串珠结构。
此外，本申请制备方法制得的乙酰化葡甘聚糖静电纺丝膜纤维之间相互交错成网状，形成互相通联的空隙，能够更好地模拟细胞外基质的结构，有利于细胞黏附和增殖。
在本申请的一些实施方式中，所述乙酰化葡甘聚糖静电纺丝膜的纤维直径为500nm至1300nm；
优选地，所述乙酰化葡甘聚糖静电纺丝膜的纤维直径为905±283nm。
本申请的第三方面，提供了一种细胞培养用组合物，包括第二方面的乙酰化葡甘聚糖静电纺丝膜和明胶。
根据本申请实施例的细胞培养用组合物，至少具有如下有益效果：
本申请的细胞培养用组合物包括乙酰化葡甘聚糖静电纺丝膜和明胶，其在细胞培养过程中作为包被物质，能够显著减少细胞死亡，促进细胞不断增殖，具有明显的协同增效效果。
在本申请的一些实施方式中，所述明胶的添加比例为0.05％至0.2％。
具体的，所述明胶的添加比例可以为0.1％。
在本申请的一些实施方式中，所述细胞培养用组合物是通过将乙酰化葡甘聚糖静电纺丝膜浸泡于0.05％至0.2％明胶溶液中制得。
具体的，所述浸泡的时间为6h至10h。
本申请的第四方面，提供了上述第二方面的乙酰化葡甘聚糖静电纺丝膜或上述第三方面的细胞培养用组合物在细胞增殖和/或细胞分化培养中的应用。
根据本申请实施例的应用，至少具有如下有益效果：将本申请的乙酰化葡甘聚糖静电纺丝膜或细胞培养用组合物应用于细胞增殖和/或细胞分化培养，能够显著减少细胞死亡，促进细胞不断增殖，此外，促NILB-hiPSCs细胞定向分化结果显示，其能够显著促进NILB-hiPSCs进一步散开，神经突触不断延长，细胞核变小，形成神经元样的细胞。
在本申请的一些实施方式中，所述细胞包括全能干细胞、多能干细胞或单能干细胞。
具体的，所述干细胞可以为人诱导多能干细胞；
更具体的，所述干细胞可以为运动神经元定向分化的人诱导多能干细胞。
在本申请的一些实施方式中，所述细胞分化培养包括人诱导多能干细胞的定向分化培养；
具体的，所述定向分化的细胞类型为神经元样细胞；
更具体的，所述定向分化培养的培养基是在Neurobasal medium的基础上添加1×B27添加剂(ThermoFish)、1×N2添加剂(ThermoFish)、0.5×双抗(Gibco)、BDNF(10ng/mL，Alomone Lab)、GDNF(10ng/mL，Alomone Lab)和NT3(10ng/mL，Alomone Lab)。
本申请的其它特征和优点将在随后的说明书中阐述，并且，部分地从说明书中变得显而易见，或者通过实施本申请而了解。
附图说明
下面结合附图和实施例对本申请做进一步的说明，其中：
图1为本申请实施例1乙酰化葡甘聚糖与葡甘聚糖的红外光谱图。
图2为本申请实施例1乙酰化葡甘聚糖与葡甘聚糖的核磁共振氢谱图。
图3为本申请乙酰化葡甘聚糖静电纺丝膜的形貌观察结果图。
图4为本申请干细胞在乙酰化葡甘聚糖(acGM)静电纺丝膜上培养的荧光显微图。
图5为本申请不同生长时期干细胞在乙酰化葡甘聚糖(acGM)静电纺丝膜上的定向分化结果图，其中图5中的A图为诱导分化流程图，图5中的B图为第三天和第5天的定向分化结果图。
图6为本申请分化培养至第7天的神经元通用标志物TUJ1的表达情况图。
图7为本申请分化培养至第7天的运动神经元特异性标志物HB9的表达情况图。
图8为本申请分化培养至第14天的神经元通用标志物TUJ1和标志物MAP2的表达情况图。
具体实施方式
以下将结合实施例对本申请的构思及产生的技术效果进行清楚、完整地描述，以充分地理解本申请的目的、特征和效果。显然，所描述的实施例只是本申请的一部分实施例，而不是全部实施例，基于本申请的实施例，本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例，均属于本申请保护的范围。
本申请的描述中，参考术语“一个实施例”、“一些实施例”、“示意性实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本申请的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1
本实施例提供了一种乙酰化葡甘聚糖(acGM)静电纺丝膜的制备方法，包括以下步骤：
(1)乙酰化葡甘聚糖(acGM)的制备
称取定量1g的葡甘聚糖(GM)充分溶胀在200mL水中，冷冻干燥后使用，将100mL的吡啶和酸酐的混合物(1：1，V/V)在50℃下搅拌30min后，将其缓慢倒入带有GM的圆底烧瓶中搅拌反应72h，将反应溶液过滤后，将滤渣与含有100mL的吡啶和酸酐的混合物(1：1，V/V)混合，在50℃的圆底烧瓶中搅拌72h，将反应溶液过滤后，收集滤渣并加入三倍体积的无水乙醇沉淀过夜，过滤，收集滤渣，用乙醇反复重悬，再离心5次，产物透析2天后，冷冻干燥后获得最终的反应产物乙酰化葡甘聚糖。
(2)电纺丝溶液的制备
称取200mg上述制得的乙酰化葡甘聚糖溶解于1mL混合试剂(氯仿：DMF＝2：1)中，充分搅拌，以至于完全溶解，将溶液静置超声使气泡全部析出，即可得到均一透明的电纺丝溶液。
(3)静电纺丝膜的制备
将上述电纺丝溶液转移到带有扁平22G针头的2.5mL塑料注射器中，固定在静电纺丝装置上，进行静电纺丝膜的制备，即得到乙酰化葡甘聚糖静电纺丝膜，其中静电纺丝参数为：电压：15kV，其中针头处的电压为+12kV，接收板处的电压为-3kV；进样速度为0.8mL/h； 针尖与采集板之间的距离为10cm；温度为40℃，相对湿度(R.H.)为10％。
实施例2
本实施例提供了乙酰化葡甘聚糖静电纺丝膜，该乙酰化葡甘聚糖静电纺丝膜由上述实施例1的方法制备得到。
实施例3
本实施例提供了一组细胞培养用组合物，包含上述实施例1制得的乙酰化葡甘聚糖静电纺丝膜和1％的Gelatin(购于STEMCELL technologies公司)。
检测例：乙酰化葡甘聚糖静电纺丝膜的性能表征
1、实验方法
(1)乙酰化葡甘聚糖有机官能团的鉴定：分别将实施例1中葡甘聚糖(GM)原料和乙酰化葡甘聚糖(acGM)产物与溴化钾按照1/200的质量比混合后研磨成粉压片，在红外光谱仪(FI-IR)下检测，扫描范围为4000-450cm-1，观察羰基(-C＝O)的特征峰在1735cm-1处的变化；所得产物或原料(10mg)充分溶于氘代氯仿(600μL)中，所得产物或原料(10mg)充分溶于氘代氯仿(600μL)中，观察核磁1H NMR在δ＝2.1ppm(-COCH3)处的变化。
(2)乙酰化葡甘聚糖静电纺丝膜的扫描电子显微镜(SEM)：分别取微量实施例1中葡甘聚糖(GM)原料和乙酰化葡甘聚糖(acGM)产物直接粘到导电胶上，并使用溅射镀膜仪(Quorum SC7620)对样品进行表面镀金处理，使用扫描电子显微镜(SEM,TESCAN MIRA LMS)在加速电压为3kV时拍摄样品电纺丝纤维的图像。使用ImageJ DiameterJ软件分析纤维的直径分布。
2、实验结果与分析
(1)乙酰化葡甘聚糖有机官能团的分析
通过红外光谱仪表征结构，其中葡甘聚糖和乙酰化葡甘聚糖的红外光谱图如图1所示，从图中可看出乙酰化葡甘聚糖在1747cm-1附近出现了强的吸收峰为羰基伸缩振动峰，在1372cm-1附近有明显的吸收峰，此处为甲基吸收峰。在1237cm-1附近出现强吸收峰，为饱和酯基的吸收峰，则证明了acGM分子链上存在大量的乙酰基(-COCH3)。
通过核磁共振仪检测，结果如图2所示，由图2的核磁1H NMR图可知在δ＝2.1ppm(-COCH3)范围出峰，也说明乙酰化成功。
(2)乙酰化葡甘聚糖静电纺丝膜的形貌
采用扫描电子显微镜(SEM)观察其电纺丝膜形貌，结果如图3所示，其中可观察出所得出的纤维形貌主要为细圆柱状，其构成乙酰化葡聚糖静电纺丝膜的纤维直径为905±283nm (n＝10)，均无明显的串珠结构，纤维之间相互交错成网状，形成互相通联的空隙，能够更好地模拟细胞外基质的结构，有利于促进细胞黏附和增殖。
应用例1：乙酰化葡甘聚糖(acGM)静电纺丝膜在促进干细胞生长中的应用
1、实验方法
(1)将实施例1制得的acGM静电纺丝膜剪成10*10mm正方形大小，放入24孔细胞培养板中，加入75％酒精浸泡2h；弃去75％酒精后，培养板放在生物安全柜中，紫外照射30min，进一步消毒。
(2)完成消毒的24孔细胞培养板，吸干水分后，分成3组，分别包被0.1％ Gelatin、acGM静电纺丝膜(实施例2制得)和同时包被Gelatin+acGM静电纺丝膜(实施例3制得)，其中Gelatin+acGM静电纺丝膜是用0.1％ Gelatin浸泡acGM静电纺丝膜得到的，将培养孔板放入细胞培养箱(37℃，5％ CO2)中，包被过夜。
(3)弃去包被溶液后，将实验室已构建成功的运动神经元定向分化的人诱导多能干细胞(NILB-hiPSCs，该细胞系在药物强力霉素dox诱导下，条件性表达运动神经元特异性转录因子Ngn2、Isl1和Lhx3；同时过表达抗凋亡基因Bcl-xl和绿色荧光GFP)接种在培养孔板中，加入含有抗凋亡小分子Y27632(MCE，10μM)的干细胞培养基mTeSR(购买于STEMCELL technologies公司)，在细胞培养箱(37℃，5％ CO2)中，过夜培养。
(4)次日，弃掉培养基，换成新鲜不含Y27632的干细胞培养基mTeSR，继续培养，隔天换液，显微镜下观察细胞形态。
(5)培养至第6天，荧光显微镜拍照。
2、实验结果
上述实验结果如图4所示，其中Gelatin组中的NILB-hiPSCs呈现半贴壁状态，培养过程中极易脱落和死亡，细胞数很少，并没有形成克隆；培养在acGM静电纺丝膜中的NILB-hiPSCs细胞数量比Gelatin组稍多一些，但是仍然较少，且大量变圆的细胞，提示细胞发生死亡；接种在Gelatin+acGM静电纺丝膜中的NILB-hiPSCs在培养过程中，细胞死亡较少，能不断增殖，并形成了干细胞克隆状态，提示细胞状态良好，细胞数量明显多于Gelatin组和acGM静电纺丝膜组。
上述结果表明，acGM静电纺丝膜优于Gelatin包被效果，其中以Gelatin和acGM静电纺丝膜共同包被的效果最佳，可以用于干细胞培养。
应用例2：乙酰化葡甘聚糖静电纺丝膜在促进干细胞定向分化中的应用
1、实验方法
(1)按照上述应用例1中的方法，在第0天，将NILB-hiPSCs接种在Gelatin+acGM静电纺丝膜中，接种密度为5*104个/孔(24孔板)。
(2)然后按照图5中的A实验流程图诱导分化，具体为第1天，Dox(1μg/mL，5days)加入运动神经元诱导培养基中(培养基配方为：neurobasal medium+B27添加剂+双抗+glutamax+NEAA+DAPT 5μM+SU5402 5μM)，诱导NILB-hiPSCs进行运动神经元的定向分化。
(3)诱导5天，每天半量换液。
(4)第6天开始，换成运动神经元成熟培养基(neurobasal medium+B27添加剂+N2添加剂+双抗+BDNF+GDNF+NT3)，继续培养至14天。
2、实验结果
上述细胞分化结果如图5中的B所示，结果显示Dox处理3天后，NILB-hiPSCs由克隆状散开，克隆边缘出现神经突触；Dox处理5天后，NILB-hiPSCs进一步散开，神经突触不断延长，细胞核变小，形成神经元样的细胞。
上述结果表明，与Gelatin共同包被后的acGM静电纺丝膜可以用于运动神经元的定向分化。
应用例3：乙酰化葡甘聚糖静电纺丝膜在鉴定运动神经元中的应用
1、实验方法
按照应用例2中的方法，分别在第7天和第14天采用细胞免疫荧光染色技术对Gelatin+acGM静电纺丝膜中的运动神经元进行鉴定，其中细胞免疫荧光的实验步骤包括：
(1)在含有Gelatin+acGM静电纺丝膜24孔板中，每个孔先用500μL DPBS清洗细胞3次后，每孔加300μL 4％PFA，室温固定15min至20min。
(2)吸去4％ PFA，每孔用500μL DPBS洗3次，每次洗15min。
(3)封闭通透：每孔加入500μL组织封闭液，室温封闭1h至2h。
(4)一抗孵育：弃去封闭液，加入400μL封闭液稀释好的一抗，4℃孵育过夜。
(5)次日，弃去一抗，用PBS清洗3次，每次15min。
(6)二抗孵育：洗涤完毕后，根据一抗的属性选择相应的荧光二抗，经3％ BSA稀释后，每片加入300μL稀释好的二抗，室温避光孵育1h至2h。
(7)吸去二抗，PBS清洗3遍，每次15min。
(8)滴加DAPI工作液，每孔加入400μL，室温避光孵育5min，然后用PBS洗涤5min。
(9)吸去PBS，将膜转移至载玻片，滴加50％甘油(50μL/片)，从一端甘油的位置盖 上盖玻片，防止气泡产生，挤掉多于甘油，并擦干玻片。
(10)封片完毕后，避光保存，并置于Zeiss710 NLO共聚焦显微镜下进行3D多层扫描。
2、实验结果
细胞免疫荧光实验结果如图6至8所示，结果表明NILB-hiPSCs定向分化的运动神经元分散在Gelatin+acGM静电纺丝膜中，分化至第7天的时候，大量表达神经元通用标志物TUJ1(如图6所示)，以及运动神经元特异性标志物HB9(如图7所示)。继续培养至14天，神经元进一步成熟，并表达成熟标志物MAP2(如图8所示)。
以上结果表明，基因修饰的NILB-hiPSCs可以在Gelatin+acGM静电纺丝膜进行3D培养，并在药物诱导下发生定向分化，形成运动神经元。这些诱导出来的神经元大量表达运动神经元相关标志物。
综上所述，本申请提供了一种乙酰化葡甘聚糖静电纺丝膜及其制备方法与应用，其制备方法包括：首先通过吡啶、酸酐和葡甘聚糖的混合反应，经醇沉后得到乙酰化葡甘聚糖(acGM)，其有机官能团的分析结果表明，制得的acGM分子链上存在大量的乙酰基(-COCH3)，其乙酰度达到3.0；进一步地，将本申请制得的乙酰化葡甘聚糖与氯仿和N,N-二甲基甲酰胺混合反应，经超声处理后得到电纺丝溶液，然后以电纺丝溶液为原料，经静电纺丝处理后得到乙酰化葡甘聚糖静电纺丝膜。该制备方法简单，无需额外添加助溶性物质，成本低，适用于工业化生产。
进一步的，实验表明采用本申请制备方法制得的乙酰化葡甘聚糖静电纺丝膜的纤维形貌主要为细圆柱状，其构成乙酰化葡聚糖静电纺丝膜的纤维直径为905±283nm(n＝10)，且无明显的串珠结构，纤维之间相互交错成网状，形成互相通联的空隙，能够相对更好地模拟细胞外基质的结构，对促进细胞黏附和增殖具有重要作用。
此外，将本申请制得的乙酰化葡甘聚糖静电纺丝膜应用于干细胞培养，结果表明其能够减少细胞死亡，并促进细胞增殖和定向分化。进一步地，将本申请制得的乙酰化葡甘聚糖静电纺丝膜应用于神经元的定向分化培养，结果表明其能够定向分化。
上面对本申请实施例作了详细说明，但是本申请不限于上述实施例，在所属技术领域普通技术人员所具备的知识范围内，还可以在不脱离本申请保护的前提下作出各种变化。此外，在不冲突的情况下，本申请的实施例及实施例中的特征可以相互组合。

Claims (10)

  1. 一种乙酰化葡甘聚糖静电纺丝膜的制备方法，其特征在于，包括以下步骤：
    步骤S1：在吡啶和酸酐的混合溶液中加入葡甘聚糖，反应后固液分离，收集固相并加入乙醇进行沉淀，然后收集沉淀即得到乙酰化葡甘聚糖；
    步骤S2：将所述乙酰化葡甘聚糖溶解于氯仿和N,N-二甲基甲酰胺的混合溶液，经超声处理后得到电纺丝溶液；
    步骤S3：以所述电纺丝溶液为原料，经静电纺丝后即得到乙酰化葡甘聚糖静电纺丝膜。
  2. 根据权利要求1所述的制备方法，其特征在于，步骤S1中，所述葡甘聚糖与吡啶和酸酐的混合溶液的重量体积比为(0.8-1.2)g：100mL；所述混合溶液中吡啶和酸酐的体积比为1：0.8-1.2。
  3. 根据权利要求1所述的制备方法，其特征在于，步骤S1中，所述反应的温度为45℃至60℃；
    优选地，所述反应的时间为48h至96h。
  4. 根据权利要求1所述的制备方法，其特征在于，步骤S2中，所述乙酰化葡甘聚糖与氯仿和N,N-二甲基甲酰胺的混合溶液的重量体积比为(150-250)mg：1mL。
  5. 根据权利要求4所述的制备方法，其特征在于，步骤S2中，所述氯仿和N,N-二甲基甲酰胺的体积比为1.8-2.5：1。
  6. 根据权利要求1所述的制备方法，其特征在于，步骤S3中，所述静电纺丝的参数包括：进样速度为0.6mL/h至1mL/h，针头内径为0.33mm至0.51mm，针头外径为0.64mm至0.81mm。
  7. 一种乙酰化葡甘聚糖静电纺丝膜，其特征在于，采用权利要求1至6任一项所述的制备方法制得。
  8. 一种细胞培养用组合物，其特征在于，包含权利要求7所述的乙酰化葡甘聚糖静电纺丝膜和明胶。
  9. 根据权利要求8所述的细胞培养用组合物，其特征在于，所述明胶的添加量为0.05％至0.2％。
  10. 一种如权利要求7所述的乙酰化葡甘聚糖静电纺丝膜或如权利要求8至9任一项所述的细胞培养用组合物在细胞增殖和/或细胞分化培养中的应用。
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