阅读说明：本技术 一种促进神经干细胞分化的方法 (Method for promoting differentiation of neural stem cells ) 是由 胥传来 瞿爱华 匡华 孙茂忠 徐丽广 郝昌龙 刘丽强 宋珊珊 胡拥明 高巍 于 2021-09-16 设计创作，主要内容包括：本发明提供了一种促进神经干细胞分化的方法,属于材料化学技术领域。本发明以天冬氨酸、组氨酸或者谷胱甘肽作为手性配体,镍盐、铜盐、钴盐作为原料,在弱碱性条件,制备得到具有手性的金属氢氧化物纳米簇,在近红外光作用下,可促进神经干细胞分化。本发明方法提出了一种具有手性的金属氢氧化物纳米簇在近红外光照下调控神经干细胞的分化,对于光驱动手性纳米材料与细胞的相互调节生物学行为具有重要的意义。(The invention provides a method for promoting differentiation of neural stem cells, and belongs to the technical field of material chemistry. According to the invention, aspartic acid, histidine or glutathione is used as a chiral ligand, nickel salt, copper salt and cobalt salt are used as raw materials, and the chiral metal hydroxide nanocluster is prepared under the alkalescent condition, so that the differentiation of neural stem cells can be promoted under the action of near infrared light. The method provided by the invention has the advantages that the chiral metal hydroxide nanoclusters regulate the differentiation of the neural stem cells under the near-infrared light irradiation, and the method has important significance for mutually regulating biological behaviors of the photo-driven chiral nano material and the cells.)

1. A method for promoting differentiation of neural stem cells is characterized in that chiral ligands and metal salts are used as raw materials to react under alkaline conditions to prepare chiral metal hydroxide nanoclusters, the obtained metal hydroxide nanoclusters are added into a neural stem cell differentiation culture system to be incubated with the neural stem cells, and differentiation of the neural stem cells is promoted under the action of near infrared light.

2. The method of claim 1, wherein the metal salt is selected from the group consisting of nickel chloride hexahydrate, nickel nitrate, cobalt chloride, copper chloride, cobalt nitrate, and copper nitrate.

3. The method of claim 1, wherein the chiral ligand is selected from one or more of histidine, aspartic acid and glutathione.

4. The method as claimed in claim 1, wherein the wavelength range of the near infrared light is 950-1200 nm.

5. The method of claim 1, wherein the metal hydroxide in the metal hydroxide nanoclusters is nickel hydroxide, cobalt hydroxide, or copper hydroxide.

6. The method according to claim 1, wherein the mass ratio of the chiral ligand to the metal salt is 6-8: 2-3.

7. The method of claim 1, wherein the neural hepatocyte differentiation step is:

s1: adding siRNA after adherent culture of the neural stem cells, and incubating and culturing to obtain a neural stem cell differentiation culture system;

s2: adding the aqueous solution of the metal hydroxide nanocluster into a neural stem cell differentiation culture system, performing co-incubation for 12-16 h, and then irradiating for 5-10min by adopting infrared light;

s3: the operation in step S2 is repeated 3-5 times.

8. The method of claim 7, wherein the siRNA concentration in S1 is 2-4 μ g/mL.

9. The method as recited in claim 7, wherein the concentration of the aqueous solution of the metal hydroxide nanoclusters in S2 is 400-500 μ g/mL.

10. The method as claimed in claim 7, wherein the light energy of the near infrared light in S2 is 100-250mW/cm²。

Technical Field

The invention belongs to the technical field of material chemistry, and particularly relates to a method for promoting differentiation of neural stem cells.

Background

Neurons are the most fundamental structural and functional units of the nervous system. Most nerve cells in the brain have no self-renewal ability or limited differentiation ability, and cannot regenerate after being damaged or dead. Damaged neurons can lead to the development of neurodegenerative diseases, such as alzheimer's disease, parkinson's disease, etc., which can lead to persistent memory and cognitive impairment in the brain. The neural stem cell is a stem cell with multiple differentiation potentials, and can be induced to differentiate to generate a large amount of brain cell tissues, so that damaged neural cells are supplemented. Neural stem cells are therefore considered to be the most effective means for treating neurological diseases. However, how to efficiently induce the directional differentiation of neural stem cells determines the survival rate of the neural stem cells in the brain, and is also a major bottleneck limiting the development of the technology.

A multi-level chiral nano assembly structure (application number: CN202010325740.8) is constructed in the prior art, and the chiral assembly can generate mechanical force on a cytoskeleton under the action of circularly polarized light (532nm) so as to efficiently induce the differentiation of neural stem cells, so that the problem of shallow light penetration depth exists, and the cell activity is influenced due to serious heat absorption in a stronger-power light irradiation process, so that a material with strong penetrability and weak heat absorption in the light irradiation process is urgently needed in the promotion of the differentiation of the neural stem cells.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for promoting the differentiation of neural stem cells.

A method for promoting differentiation of neural stem cells comprises the steps of taking chiral ligands and metal salts as raw materials, reacting under an alkaline condition to prepare chiral metal hydroxide nanoclusters, adding the obtained metal hydroxide nanoclusters into a neural stem cell differentiation culture system, incubating with the neural stem cells together, and promoting differentiation of the neural stem cells under the action of near infrared light.

In one embodiment of the invention the metal salt is selected from the group consisting of nickel chloride hexahydrate, nickel nitrate, cobalt chloride, copper chloride, cobalt nitrate, copper nitrate.

In one embodiment of the invention, the chiral ligand is selected from one or more of histidine, aspartic acid and glutathione.

In one embodiment of the invention, the aspartic acid is selected from aspartic acid in D or L form, the histidine is selected from histidine in D or L form, and the glutathione is selected from glutathione in D or L form.

In one embodiment of the present invention, the wavelength range of the near infrared light is 950-1200 nm.

In one embodiment of the invention, the alkaline condition is a solution pH of 8-9.

In one embodiment of the present invention, the metal hydroxide in the metal hydroxide nanocluster is nickel hydroxide, cobalt hydroxide, or copper hydroxide.

In one embodiment of the present invention, the mass ratio of the chiral ligand to the metal salt is 6-8: 2-3.

In one embodiment of the present invention, the differentiation step of the neural hepatocyte is:

s1: adding siRNA after adherent culture of the neural stem cells, and incubating and culturing to obtain a neural stem cell differentiation culture system;

s2: adding the aqueous solution of the metal hydroxide nanocluster into a neural stem cell differentiation culture system, performing co-incubation for 12-16 h, and then irradiating for 5-10min by adopting infrared light;

s3: the operation in step S2 is repeated 3-5 times.

The siRNA is used for inhibiting differentiation of the neural stem cells to the direction of the astrocytes.

In one embodiment of the present invention, the concentration of the aqueous solution of the metal hydroxide nanoclusters is 400-500. mu.g/mL.

In one embodiment of the present invention, the light energy of the near infrared light is 100-250mW/cm²。

In one embodiment of the invention, the siRNA concentration is 2-4. mu.g/mL.

Compared with the prior art, the technical scheme of the invention has the following advantages:

the invention provides a method for promoting neural stem cell differentiation of a metal hydroxide nano cluster with near infrared chirality under illumination of 950nm-1200nm, which has important significance for driving interaction of a chiral nano material and cells by near infrared light and regulating biological behaviors.

1. The absorption wavelength of the metal hydroxide nanocluster is in a near infrared region, the penetration depth of light can be improved by utilizing near infrared light irradiation, and the metal hydroxide nanocluster is hopefully applied to living bodies in the future.

2. The metal hydroxide nanoclusters are different from noble metal plasma materials, are mild in light absorption, do not generate heat under illumination, and avoid cell damage.

3. The metal hydroxide nanocluster provided by the invention is smaller in particle size, can be combined with neuron-related protein more easily when interacting with cells, and is better in differentiation efficiency.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which

FIG. 1 is a circular dichroism spectrum of an aspartic acid-modified nickel hydroxide nanocluster in example 1 of the present invention.

FIG. 2 is an absorption spectrum of the aspartic acid-modified nickel hydroxide nanoclusters of example 1 of the present invention.

FIG. 3 is a TEM image of aspartic acid-modified nickel hydroxide nanoclusters in example 1 of the present invention.

FIG. 4 is a circular dichroism spectrum of histidine-modified nickel hydroxide nanoclusters of example 2 of the present invention.

FIG. 5 is an absorption spectrum of histidine-modified nickel hydroxide nanoclusters according to example 2 of the present invention.

FIG. 6 is a circular dichroism spectrum of glutathione-modified copper hydroxide nanoclusters of example 3 of the present invention.

FIG. 7 is an absorption spectrum of glutathione-modified copper hydroxide nanoclusters of example 3 of the present invention.

FIG. 8 is a circular dichroism spectrum of aspartic acid modified cobalt hydroxide nanoclusters of example 4 of the present invention.

FIG. 9 is an absorption spectrum of aspartic acid modified cobalt hydroxide nanoclusters of example 4 of the present invention.

FIG. 10 is a statistical graph of the differentiated axon lengths of neural stem cells described in examples 1 to 4 of the present invention.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Example 1

1. Preparation of metal hydroxide nanomaterials

(1) The synthesis and purification route of the nickel hydroxide nanocluster (aspartic acid is ligand) with near-infrared chiral signals is as follows:

adding D-type aspartic acid (654mg) and L-type aspartic acid (654mg) and nickel chloride hexahydrate (238mg) into a three-neck flask containing 60mL of water respectively at room temperature, uniformly stirring for 2 minutes, adding 4.6mL of 1M sodium hydroxide, and adjusting the pH value to be alkalescent (8.4); and then continuously mixing and stirring for 12h to form the nickel hydroxide nanocluster modified by the aspartic acid. The resulting sample was washed with isopropanol and resuspended in ultrapure water for subsequent characterization.

The circular dichroism spectrogram of the synthesized nickel hydroxide nanocluster with the near-infrared chiral signal is shown in figure 1: obvious circular dichroism signals exist between 1000-1200nm, the characteristic peak is positioned at about 1100nm, and the nickel hydroxide nanoclusters modified by D-type aspartic acid and L-type aspartic acid display symmetrical chiral signals; the corresponding absorption spectrum is shown in FIG. 2, and has a certain absorption between 1000-1200 nm; the Transmission Electron Microscope (TEM) image is shown in FIG. 3, and the average particle size of the synthesized nickel hydroxide nanoclusters is about 3 nm.

2. Under the action of 980nm illumination, the method for promoting the differentiation of the neural stem cells by the nickel hydroxide nanoclusters comprises the following steps:

culturing the neural stem cells on a flat plate coated with polylysine to make the neural stem cells grow adherent to the wall; 12h later, siRNA (siSOX9, 2.6. mu.g/mL) was added, after incubation for 14h, D-type or L-type nickel hydroxide nanoclusters were added to the medium, after incubation for 12h, a 980nm laser (200 mW/cm) was used²) Irradiating for 10 minutes, repeating the operations of adding the D-type or L-type nickel hydroxide nanoclusters and irradiating by laser for five times, observing the neuron differentiation effect, and finding that the neuron axons grow, and the D-type nanocluster differentiation promoting effect is better than that of the L-type nanoclusters.

The differentiation effect of the nickel hydroxide nanoclusters on the neural stem cells under the action of 980nm light is shown in fig. 10, the nickel hydroxide nanoclusters modified by aspartic acid promote the differentiation of the neural stem cells under the action of 980nm light, and compared with a control group (the blank group is not added with the nickel hydroxide nanoclusters or siRNA, and the siRNA group is only added with siRNA), the axon length is increased; the invention also discovers that under the action of the D-type aspartic acid modified nickel hydroxide nano-cluster and 980nm illumination, the length of axon is increased to 110-140 mu m, while the L-type aspartic acid modified nickel hydroxide nano-cluster is only 70-90 mu m.

Example 2

1. The synthesis and purification route of the nickel hydroxide nanocluster (histidine is ligand) of the near-infrared chiral signal is as follows:

adding D-type or L-type histidine (754mg) and nickel nitrate (386mg) into a three-neck flask containing 60mL of water at room temperature, uniformly stirring for 2 minutes, adding 4.2mL of 1M sodium hydroxide, and adjusting the pH value to be alkalescent (8.4); and then continuously mixing and stirring for 12h to form the histidine-modified nickel hydroxide nanocluster. The resulting sample was washed with isopropanol and resuspended in ultrapure water for subsequent characterization.

The circular dichroism spectrogram of the synthesized nickel hydroxide nanocluster with the near-infrared chiral signal is shown in figure 4: the CD signal is very strong in a visible light region (430nm), the characteristic peak of a near infrared region is about 1100nm, and the nickel hydroxide nanoclusters modified by D-type histidine and L-type histidine show symmetrical chiral signals; the corresponding absorption spectrum is shown in FIG. 5, and has a certain absorption between 1000 and 1200 nm.

2. Under the action of 980nm illumination, the method for promoting the differentiation of the neural stem cells by the nickel hydroxide nanoclusters comprises the following steps:

culturing the neural stem cells on a flat plate coated with polylysine to make the neural stem cells grow adherent to the wall; 12h later, siRNA (siSOX9, 2.6. mu.g/mL) was added, after incubation for 14h, D-type or L-type nickel hydroxide nanoclusters were added to the medium, after incubation for 12h, a 980nm laser (200 mW/cm) was used²) After 10 minutes of irradiation, the above operations of adding D-type or L-type nickel hydroxide nanoclusters and laser irradiation were repeated five times to observe the neuron differentiation effect, and the experimental results are shown in fig. 10. The length of the axon of the D-type histidine-modified nickel hydroxide nano-cluster is increased to 95-120 mu m, while the length of the L-type histidine-modified nickel hydroxide nano-cluster is only 70-85 mu m.

Example 3

1. The synthesis and purification route of the copper hydroxide nanocluster (glutathione is ligand) with near-infrared chiral signals is as follows:

adding D-type or L-type glutathione (625mg) and copper chloride (265mg) into a three-neck flask containing 60mL of water at room temperature, uniformly stirring for 2 minutes, adding 4.2mL of 1M sodium hydroxide, and adjusting the pH value to be alkalescent (8.4); and continuously mixing and stirring for 12h to form the glutathione-modified copper hydroxide nanocluster. The resulting sample was washed with isopropanol and resuspended in ultrapure water for subsequent characterization.

The circular dichroism spectrum of the synthesized copper hydroxide nanocluster is shown in FIG. 6: CD signals exist at 600nm and 800nm respectively, characteristic peaks in a near infrared region are weak, and the copper hydroxide nanoclusters modified by the D-type glutathione and the L-type glutathione display symmetrical chiral signals; the corresponding absorption spectrum is shown in FIG. 7, and the absorption at 600nm is relatively strong.

2. Under the action of 980nm illumination, the method for promoting the differentiation of the neural stem cells by the copper hydroxide nanoclusters comprises the following steps:

culturing the neural stem cells on a flat plate coated with polylysine to make the neural stem cells grow adherent to the wall; after 12h, siRNA (siSOX9, 2.6. mu.g/mL) was added; after incubating for 14h, adding D-type or L-type copper hydroxide nanoclusters into the culture medium, incubating for 12h, and applying 980nm laser (200 mW/cm)²) After 10 minutes of irradiation, the above operations of adding D-type or L-type copper hydroxide nanoclusters and laser irradiation were repeated five times to observe the neuron differentiation effect, and the experimental results are shown in fig. 10. The length of the axon of the D type glutathione modified copper hydroxide nanocluster is increased to 85-100 mu m, while the length of the L type glutathione modified copper hydroxide nanocluster is only 65-75 mu m.

Example 4

1. The synthesis and purification route of the cobalt hydroxide nanocluster (aspartic acid is ligand) with near-infrared chiral signals is as follows:

adding D-type or L-type aspartic acid (758mg) and cobalt chloride (347mg) into a three-neck flask containing 60mL of water at room temperature, stirring and mixing for 2 minutes, adding 4.5mL of 1M sodium hydroxide, and adjusting the pH to be alkalescent (8.4); and continuously mixing and stirring for 12h to form the aspartic acid modified cobalt hydroxide nano cluster. The resulting sample was washed with isopropanol and resuspended in ultrapure water for subsequent characterization.

The circular dichroism spectrogram of the synthesized cobalt hydroxide nanocluster is shown in fig. 8: stronger CD signals are respectively arranged at 520nm and 1200nm, and the copper hydroxide nano-cluster modified by the D-type glutathione and the L-type glutathione shows symmetrical chiral signals; the corresponding absorption spectra are shown in FIG. 9, with stronger absorption at 520nm and 1200nm, but weaker absorption at 980 nm.

2. The method for promoting the differentiation of the neural stem cells by the cobalt hydroxide nanoclusters under the action of 980nm illumination is as follows

Culturing the neural stem cells on a flat plate coated with polylysine to make the neural stem cells grow adherent to the wall; 12h later, siRNA (siSOX9, 2.6. mu.g/mL) was added, after incubation for 14h, D-type or L-type cobalt hydroxide nanoclusters were added to the medium, and after incubation for 12h, 980nm laser (250 mW/cm) was used²) Irradiating for 10min, repeating the above steps of adding D-type or L-type cobalt hydroxide nanocluster and laser irradiationThe neuron differentiation effect is observed five times of shooting, the experimental result is shown in figure 10, and the neuron axon is found to grow. The length of the axon of the D-type aspartic acid modified cobalt hydroxide nano-cluster is increased to 85-100 μm, while the length of the axon of the L-type aspartic acid modified cobalt hydroxide nano-cluster is only 65-75 μm.

As can be seen from fig. 10, neuronal axonal growth was observed in all experimental groups compared to the control group (no nickel hydroxide nanoclusters or siRNA was added in the blank group, only siRNA was added in the siRNA group); wherein the differentiation promoting effect of the nickel hydroxide nanocluster modified by aspartic acid in example 1 under the illumination of 980nm is better than that of the nickel hydroxide modified by histidine in example 2, the copper hydroxide modified by glutathione in example 3 and the cobalt hydroxide modified by aspartic acid in example 4, and the axon length is longest, which is probably related to the absorption intensity of the nickel hydroxide nanocluster modified by aspartic acid at 980 nm. Meanwhile, the invention also finds that under the action of the D-type aspartic acid modified nickel hydroxide nano-cluster obtained in the example 1 and 980nm illumination, the length of axon is increased to 110-140 μm, while the L-type aspartic acid modified nickel hydroxide nano-cluster is only 70-90 μm.

Example 5

1. The synthesis and purification route of the nickel hydroxide nanocluster (aspartic acid is ligand) with near-infrared chiral signals is as follows:

adding D-aspartic acid (654mg) and nickel nitrate (238mg) into a three-neck flask containing 60mL of water at room temperature, stirring and uniformly mixing for 2 minutes, and adding 4.6mL of 1M sodium hydroxide to adjust the pH value to be alkalescent (8); and then continuously mixing and stirring for 18h to form the nickel hydroxide nanocluster modified by the aspartic acid. The resulting sample was washed with isopropanol and resuspended in ultrapure water for subsequent characterization.

2. Under the action of 980nm illumination, the method for promoting the differentiation of the neural stem cells by the nickel hydroxide nanoclusters comprises the following steps:

culturing the neural stem cells on a flat plate coated with polylysine to make the neural stem cells grow adherent to the wall; after 12h, siRNA (siSOX9, 4. mu.g/mL) was added; after incubation for 12h, adding D-type aspartic acid modified nickel hydroxide nanoclusters into the culture medium, and after incubation for 16h, using 980nm laser (250 mW)/cm²) Irradiating for 5 minutes, repeating the operations of adding D-type or L-type nickel hydroxide nanoclusters and laser irradiation for 3 times, observing the neuron differentiation effect, and finding the growth of neuron axons.

Example 6

1. The synthesis and purification route of the nickel hydroxide nanocluster (aspartic acid is ligand) with near-infrared chiral signals is as follows:

adding L-aspartic acid (654mg) and nickel nitrate (238mg) into a three-neck flask containing 60mL of water at room temperature, stirring and uniformly mixing for 2 minutes, and adding 4.6mL of 1M sodium hydroxide to adjust the pH value to be alkalescent (8); and then continuously mixing and stirring for 18h to form the nickel hydroxide nanocluster modified by the aspartic acid. The resulting sample was washed with isopropanol and resuspended in ultrapure water for subsequent characterization.

2. Under the action of 980nm illumination, the method for promoting the differentiation of the neural stem cells by the nickel hydroxide nanoclusters comprises the following steps:

culturing the neural stem cells on a flat plate coated with polylysine to make the neural stem cells grow adherent to the wall; 12h later, siRNA (siSOX9, 2. mu.g/mL) was added, after 16h of co-incubation, L-type aspartic acid modified nickel hydroxide nanoclusters were added to the medium, co-incubation was carried out for 16h, and a 980nm laser (250 mW/cm)²) Irradiating for 8 minutes, repeating the operations of adding D-type or L-type nickel hydroxide nanoclusters and laser irradiation for 4 times, observing the neuron differentiation effect, and finding the growth of neuron axons.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.