CN113274412B - Application of universal calcium preparation in neural stem cell differentiation regulation - Google Patents

Application of universal calcium preparation in neural stem cell differentiation regulation Download PDF

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CN113274412B
CN113274412B CN202110467167.9A CN202110467167A CN113274412B CN 113274412 B CN113274412 B CN 113274412B CN 202110467167 A CN202110467167 A CN 202110467167A CN 113274412 B CN113274412 B CN 113274412B
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neural stem
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stem cells
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CN113274412A (en
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桑元华
郝敏
何建龙
段佳志
夏鹤
陈鑫
王书华
刘宏
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Shandong University
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/0623—Stem cells
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C12N2500/14—Calcium; Ca chelators; Calcitonin

Abstract

The invention relates to the application of general calcium preparation in the differentiation and regulation of neural stem cells, the method inoculates the neural stem cells in the neural stem cell differentiation culture medium; adding a universal calcium preparation into a culture system to culture and endocytose neural stem cells, wherein the universal calcium preparation is hydroxyapatite nanorods or calcium phosphate nanoparticles to obtain calcium preparation modified neural stem cells; the calcium preparation modified neural stem cells are transplanted for relieving and treating neurodegenerative diseases. The calcium preparation modified neural stem cells have higher differentiation speed and more obvious neuron direction differentiation tendency, can generate more mature neurons in shorter time, improve the neural tissue repair efficiency to the maximum extent, and avoid the generation of neural scars.

Description

Application of universal calcium preparation in neural stem cell differentiation regulation
Technical Field
The invention relates to application of a universal calcium preparation in neural stem cell differentiation regulation, and belongs to the technical field of biological materials and medical health.
Background
As the aging population increases, people in modern society are plagued with neurodegenerative diseases, the most common of which include Alzheimer's Disease (AD) and Parkinson's Disease (PD). According to the currently known situation, neuronal cells in specific locations in the brain (such as hippocampus or substantia nigra) are damaged, thereby creating defective neural networks in a reduced number of neuronal cells, leading to various symptoms of neurodegenerative diseases. AD is a common and incurable degenerative disease of the central nervous system, the main pathogenesis is global brain atrophy, neurofibrillary tangles, vacuolar degeneration of nerve cell granules, massive loss of neurons and the like, and the AD is characterized by low high-grade cognitive ability, continuous deterioration of brain control reasoning and unchangeable memory area function, and accounts for a large proportion of the elderly. AD is known to have a 5% incidence in people over 65 years of age and a 20% incidence in older people over 80 years of age. With the increasing aging degree of society, the population of patients is still expanding. By incomplete statistics, the population of patients worldwide has exceeded 2700 thousands, with 48% of patients in asia. It is estimated that by 2050, the number of patients will increase to 3 times the current number, and the proportion of asian patients will also increase to 59%. Also, the mortality rate of AD patients is high, and among all causes of death due to diseases worldwide, it occupies the fourth place (the first three are coronary heart disease, cancer and stroke, respectively). Treatment of AD and related research is therefore particularly pressing for worldwide reasons, particularly in asian regions. PD is also a common neurodegenerative disease, common among the elderly, and is clinically characterized by resting tremor, myotonia, hypokinesia, postural balance disorder, and the like. The incidence of PD disease in people over 65 years old in China is 1.7%, and the most important pathology is the apoptosis of mesencephalon nigral dopaminergic neurons. Therefore, the key to the alleviation and treatment of neurodegenerative diseases is to repair damaged nerve tissues, supplement neurons and construct a normal nerve signal transmission system.
Neural Stem Cells (NSCs) and Neural Progenitor Cells (NPCs), i.e., cells capable of differentiating into neural cells, are present in the adult brain. Neural stem cells are present in the subventricular zone of the lateral ventricles and the dentate gyrus of the hippocampus, and neurogenesis occurs in this region throughout the life of the animal by differentiation and proliferation of neural stem cells (Zhao et al (2008) Mechanisms and Functional Implications of additive neurogenesis. Cell 132 645-660.
Research is being conducted in various fields to find a method for treating neurodegenerative diseases, but conventional surgical and pharmaceutical treatment methods have little effect, and thus, basic treatment research for the etiology of neurodegenerative diseases is urgently required.
In recent years, neural stem cell transplantation gradually becomes the most potential treatment method, but the neural stem cell transplantation faces the problems of slow response, uncertain direction and the like of neural stem cell differentiation, and the acceleration of the differentiation process of the neural stem cell and the regulation of the differentiation direction of the neural stem cell are urgent. In the traditional method, a large amount of biological reagents are mainly used for accelerating the differentiation of the neural stem cells and regulating the differentiation direction of the neural stem cells, but the biological reagents are quick in failure, high in price, complex in components and high in quality inspection difficulty, and bring multiple uncertain and even dangerous factors while increasing the economic burden of patients. With the rapid development and the mutual cross fusion of biomedicine, material science and tissue engineering, a new solution is brought to a plurality of biomedicine problems which need to be solved urgently, and a new vitality is brought to the traditional material science research. Compared with biological reagents, the inorganic nano material has clear and adjustable and controllable components, mature synthesis technology and low price, and the novel nervous system repair scheme taking the biological materials, the seed cells and the growth factors as basic elements is expected to overcome various defects of the traditional neural stem cell transplantation and becomes a neurodegenerative disease treatment scheme with high clinical value.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the application of a universal calcium preparation in the differentiation regulation of neural stem cells. The invention combines the endocytosis of the stem cells to the general calcium preparation nanometer material with the general calcium preparation with low price, obtains the neural stem cells modified by the calcium preparation through the culture and endocytosis of the neural stem cells, and relieves and treats the neurodegenerative diseases through the stem cell transplantation.
In order to realize the purpose, the invention is realized by the following technical scheme:
the application of the universal calcium preparation in the differentiation regulation of the neural stem cells comprises the following steps:
1) Inoculating the neural stem cells into a neural stem cell differentiation culture medium;
2) Adding a universal calcium preparation into the culture system obtained in the step 1) to culture and endocytose neural stem cells, wherein the universal calcium preparation is hydroxyapatite nanorods or calcium phosphate nanoparticles, and the neural stem cells modified by the calcium preparation are obtained;
3) The calcium preparation modified neural stem cells are transplanted for relieving and treating neurodegenerative diseases.
Preferably, in step 1), the neural stem cell differentiation medium comprises a basic medium Neurobasal medium, 1-3% by volume of Gibco B-27 neural cell culture serum-free additive, and 1-2% by volume of GlutaMAX TM -I, fetal bovine serum FBS at a volume concentration of 1-2% and a diabody at a volume concentration of 1-2%.
Serum-free additive for Gibco B-27 nerve cell culture, glutaMAX TM the-I, the FBS and the double antibody are all the existing commercial products.
Preferably, in step 1), the amount of the neural stem cells to be inoculated is (1-10). Times.10 6 one/mL.
Most preferably, in step 1), the amount of the neural stem cells to be inoculated is 2X 10 6 one/mL.
Preferably, in step 2), after the universal calcium preparation is added, the concentration of the universal calcium preparation in the system is 10-400 mug/mL.
Further preferably, after the universal calcium preparation is added, the concentration of the universal calcium preparation in the system is 20-300 mu g/mL.
Further preferably, after the universal calcium preparation is added, the concentration of the universal calcium preparation in the system is 50-200 mug/mL.
Further preferably, after the universal calcium preparation is added, the concentration of the universal calcium preparation in the system is 100-200 mug/mL.
Preferably, in step 2), the culture and endocytosis of the neural stem cells are carried out by placing the culture system added with the calcium preparation in an incubator at 36-37 ℃ for 1-14 days.
Preferably, in step 2), the hydroxyapatite nanorods are prepared by the following method:
(1) Mixing octadecylamine, oleic acid and ethanol uniformly, adding NaF and Na 3 PO 4 Mixing with calcium nitrate, stirring to obtain mixed solution, naF and Na 3 PO 4 And calcium nitrate in a mass ratio of 0.1 to 0.5:1.2:1.4 calcium nitrate with octadecylamineThe ratio of the amounts of substances (1): 0.1-10, wherein the mass ratio of calcium nitrate to oleic acid is 1: 1-10, wherein the adding amount of the ethanol accounts for 1/4-2/3 of the total volume of the mixed solution;
(2) Transferring the mixed solution into a closed reactor, reacting for 8-15 hours at 120-220 ℃, and naturally cooling to room temperature;
(3) Dispersing the product in cyclohexane, centrifuging, then washing with cyclohexane and ethanol for three times, and finally dispersing in ethanol to obtain the hydroxyapatite nanorod.
Further preferably, in step 2), the specific preparation method of the hydroxyapatite nanorod is as follows:
1) Dissolving 0.6g of octadecylamine in 7mL of oleic acid, heating at 80 ℃ for 30min, and adding 20mL of ethanol under vigorous stirring;
2) 1.4mL of 0.2M NaF and 7mL of 0.168M Na were added 3 PO 4 Stirring for 5min;
3) Then adding 7mL of 0.2M Ca (NO) 3 ) 2 Stirring the solution for 1min to obtain mixed solution,
4) Heating the mixed solution at 180 ℃ for reaction for 12 hours, and naturally cooling to room temperature;
5) Dispersing the product in cyclohexane, centrifuging at 10000rpm for 10min, then washing with cyclohexane and ethanol for three times, and finally dispersing in ethanol for later use.
Preferably, in step 2), the calcium phosphate nanoparticles are prepared as follows:
a. uniformly mixing cyclohexane, trion-X100 and hexanol to obtain a mixed solution, averagely dividing the mixed solution into two parts, and adding a calcium chloride solution into one part to obtain a calcium chloride mixed solution; adding another part of the disodium hydrogen phosphate solution to obtain a disodium hydrogen phosphate mixed solution;
b. and dropwise adding the mixed solution of the disodium hydrogen phosphate into the mixed solution of the calcium chloride, reacting, and centrifuging to obtain the calcium phosphate nano-particles.
According to the invention, in the step a, the volume ratio of the cyclohexane to the Trion-X100 to the hexanol is (4-6): (1-2): (3-5).
Preferably, in step a, the volume ratio of one part of the mixed solution to the calcium chloride solution is 8:1, the volume ratio of the other mixed solution to the disodium hydrogen phosphate solution is 8:1.
preferably, in step a, the concentration of the calcium chloride solution is 35mg/ml and the concentration of the disodium hydrogen phosphate solution is 111mg/ml.
Preferably, according to the invention, in step b, the reaction time is from 0.5 to 2h.
In the methods of the invention, neural stem cells can be isolated from embryonic or adult brains according to known methods. Alternatively, the neural stem cells may be purchased from the market, or may be cultured by any conventional method known in the art. There is no particular limitation to the above. In the examples section that follows, neural stem cells isolated from cerebral cortex portions of C57 mice pregnant for 14 days were used.
The invention has the technical characteristics and advantages that:
1. in the application method, the general calcium preparation with low price is combined with the endocytosis of the stem cells to the general calcium preparation nanometer material, the neural stem cells modified by the calcium preparation are obtained through the culture and endocytosis of the neural stem cells, and the neurodegenerative diseases are relieved and treated through stem cell transplantation.
2. In the application method, the neural stem cells modified by the calcium preparation have higher differentiation speed and more obvious differentiation tendency of the neuron direction, can generate more mature neurons in shorter time, improve the neural tissue repair efficiency to the maximum extent and avoid the generation of neural scars.
3. In the application method, the related calcium preparation is simple to prepare, and compared with the traditional bioactive factor, the calcium preparation has the advantages of low cost, long time effect, low immunogenicity, low storage and transportation difficulty and wide application prospect.
Drawings
FIG. 1 is a TEM image of hydroxyapatite nanorods prepared in example 1;
FIG. 2 is a graph showing the live-dead staining of neural stem cells after 3 days of co-culture of the hydroxyapatite nanorods and the neural stem cells at different concentrations in example 1 and comparative example 1.
FIG. 3 is TEM images of neural stem cells after co-culturing the hydroxyapatite nanorods with the neural stem cells at different concentrations in example 1 and comparative example 1 for 3 days;
FIG. 4 is SEM pictures of neural stem cells after 14 days of co-culture of the hydroxyapatite nanorods with the neural stem cells at different concentrations in example 1 and comparative example 1;
FIG. 5 is a graph showing the results of electrophysiological tests on neural stem cells after 5 days of co-culture of varying concentrations of hydroxyapatite nanorods with neural stem cells in example 1 and comparative example 1.
Fig. 6 is SEM images of neural stem cells co-cultured with different concentrations of calcium phosphate nanoparticles and neural stem cells for 7 days in example 5 and comparative example 2.
Detailed Description
The following examples are intended to illustrate the features of the present invention in further detail, but the present invention is not limited to these examples.
In an embodiment, the neural stem cell extraction step is:
1.2 surgical bent discs, 1 pair of ophthalmic scissors, 4 pairs of hemostatic forceps, 3 pairs of surgical scissors, 3 pairs of straight-head forceps, 3 pairs of elbow forceps and 200-mesh filter screens are sterilized at high pressure one day before the extraction experiment, and then are placed in a 60-degree oven for drying and standby. The experimental animals were provided by the Experimental animals center of medical school of Shandong university.
2. 1C 57 mouse pregnant for 14 days is taken, injected with 1ml of 10% chloral hydrate for anesthesia, then is killed by cervical dislocation, and then is soaked in 75% alcohol for 15min for sterilization, abdominal skin and muscle are cut in a biological safety cabinet, and the embryo is immediately placed into PBS buffer solution precooled at 4 ℃. Taking off embryo head, peeling off meninges and blood vessel under 10 times of dissecting mirror, taking off cortex part of left and right brains, placing in 4 deg.C precooled PBS buffer solution, cutting into pieces with size of about 0.1cm3, repeatedly blowing, filtering with 200 mesh filter screen, adding filtrate into serum-free culture medium prepared in advance, inoculating into culture bottle, and culturing in culture box with temperature of 37 deg.C and 5% CO2. Adding 1ml of new special proliferation culture medium for the serum-free neural stem cells every day, observing the growth state of the cells by using a conventional inverted microscope, and culturing for 3-5 days and then carrying out passage. The cell morphology was observed directly in bright field or after staining the cytoskeleton and cell nucleus, it was observed with fluorescence microscope.
Example 1
Preparing a hydroxyapatite nanorod:
1) Dissolving 0.6g of octadecylamine in 7mL of oleic acid, heating at 80 ℃ for 30min, and adding 20mL of ethanol under vigorous stirring;
2) 1.4mL of 0.2M NaF and 7mL of 0.168M Na were added 3 PO 4 Stirring for 5min;
3) Then 7mL of 0.2M Ca (NO) was added 3 ) 2 Stirring the solution for 1min to obtain mixed solution,
4) Heating the mixed solution at 180 ℃ for reaction for 12h, and naturally cooling to room temperature;
5) Dispersing the product in cyclohexane, centrifuging at 10000rpm for 10min, then cleaning with cyclohexane and ethanol for three times, and finally dispersing in ethanol for later use.
The TEM image of the HAp nanorods prepared in this example is shown in FIG. 1, and it can be seen that the HAp nanorods are about 100nm long and about 20nm wide.
The hydroxyapatite nanorod is applied to the differentiation regulation of the neural stem cells as follows:
1) Inoculating the neural stem cells into a neural stem cell differentiation culture medium;
the neural stem cell differentiation medium comprises a basic medium Neurobasal medium, gibco B-27 neural cell culture serum-free additive with the volume concentration of 2 percent and GlutaMAX with the volume concentration of 1 percent TM -I, fetal bovine serum FBS at a volume concentration of 1% and a double antibody at a volume concentration of 1%. The inoculation amount of the neural stem cells is 2 multiplied by 10 6 one/mL.
2) Adding the hydroxyapatite nanorods into the culture system obtained in the step 1), placing the culture system in an incubator at the temperature of 36-37 ℃ for incubation for 1-14 days, and carrying out culture and endocytosis on neural stem cells, wherein after the hydroxyapatite nanorods are added, the concentration of the hydroxyapatite nanorods in the system is 200 mug/ml.
3) Obtaining the neural stem cells modified by the calcium preparation, and transplanting the neural stem cells modified by the calcium preparation for relieving and treating neurodegenerative diseases.
Example 2
The preparation of hydroxyapatite nanorods was the same as in example 1.
The application of the hydroxyapatite nanorod in the neural stem cell differentiation regulation is the same as that in example 1, except that:
the concentration of the hydroxyapatite nano rod in the system is 400 mug/ml.
Example 3
The preparation of hydroxyapatite nanorods was the same as in example 1.
The application of the hydroxyapatite nanorod in the neural stem cell differentiation regulation is the same as that in example 1, except that:
the concentration of the hydroxyapatite nano rod in the system is 50 mug/ml.
Example 4
The preparation of hydroxyapatite nanorods was the same as in example 1.
The application of the hydroxyapatite nanorod in the neural stem cell differentiation regulation is the same as that in example 1, except that:
the concentration of the hydroxyapatite nano rod in the system is 100 mug/ml.
Comparative example 1
The application of the hydroxyapatite nanorod in the neural stem cell differentiation regulation is the same as that in example 1, except that:
the concentration of the hydroxyapatite nano rod in the system is 0 mug/ml.
Experimental example 1:
carrying out gene expression level detection, protein expression level detection and electrophysiological signal detection on the neural stem cells modified by the calcium preparation obtained in the step 2).
1. In order to verify the biocompatibility of the hydroxyapatite nanorods and the regulation effect on neural differentiation, the neural stem cell live-dead staining was performed after the hydroxyapatite nanorods and the neural stem cells were co-cultured for 3 days in example 1 and comparative example 1 at different concentrations, and the results are shown in fig. 2.
2. TEM images of neural stem cells after 3 days of co-culture of the hydroxyapatite nanorods and the neural stem cells at different concentrations of example 1 and comparative example 1 are shown in FIG. 3.
3. SEM images of neural stem cells after the hydroxyapatite nanorods and the neural stem cells were co-cultured for 14 days at different concentrations in example 1 and comparative example 1 are shown in FIG. 4.
4. Example 1 and comparative example 1 electrophysiological test results of neural stem cells after co-culturing the hydroxyapatite nanorods with the neural stem cells at different concentrations for 5 days are shown in fig. 5.
Example 5
Preparation of calcium phosphate nanoparticles:
a. uniformly mixing cyclohexane, trion-X100 and hexanol according to a volume ratio of 5; adding another part of the disodium hydrogen phosphate solution with the concentration of 111mg/ml to obtain a disodium hydrogen phosphate mixed solution; the volume ratio of one part of mixed solution to the calcium chloride solution is 8:1, the volume ratio of the other mixed solution to the disodium hydrogen phosphate solution is 8:1.
b. and dropwise adding the mixed solution of the disodium hydrogen phosphate into the mixed solution of the calcium chloride, reacting for 1h, and centrifuging to obtain the calcium phosphate nano-particles.
The application of calcium phosphate nanoparticles in the regulation of neural stem cell differentiation is as follows:
1) Inoculating the neural stem cells into a neural stem cell differentiation culture medium;
the neural stem cell differentiation culture medium comprises a basic culture medium Neurobasal medium, 2% volume concentration Gibco B-27 neural cell culture serum-free additive and 1% volume concentration GlutaMAX TM -I, fetal bovine serum FBS at a volume concentration of 1% and a diabody at a volume concentration of 1%. The inoculation amount of the neural stem cells is 2 multiplied by 10 6 one/mL.
2) Adding the calcium phosphate nanoparticles into the culture system obtained in the step 1), placing the culture system in an incubator at 36-37 ℃ for incubation for 1-14 days, culturing and endocytosis the neural stem cells, and adding the calcium phosphate nanoparticles to obtain a calcium phosphate nanoparticle concentration of 200 mu g/ml in the system.
3) Obtaining the neural stem cells modified by the calcium preparation, and transplanting the neural stem cells modified by the calcium preparation for relieving and treating neurodegenerative diseases.
Example 6
Calcium phosphate nanoparticles were prepared as in example 5.
The calcium phosphate nanoparticles were used for the regulation of neural stem cell differentiation in the same manner as in example 5, except that:
the concentration of calcium phosphate nanoparticles in the system was 400. Mu.g/ml.
Example 6
Calcium phosphate nanoparticles were prepared as in example 5.
The calcium phosphate nanoparticles were used for the regulation of neural stem cell differentiation in the same manner as in example 5, except that:
the concentration of calcium phosphate nanoparticles in the system was 50. Mu.g/ml.
Example 6
Calcium phosphate nanoparticles were prepared as in example 5.
The application of calcium phosphate nanoparticles to the regulation of neural stem cell differentiation was the same as in example 5, except that:
the concentration of calcium phosphate nanoparticles in the system was 100. Mu.g/ml.
Comparative example 2
The application of the calcium phosphate nano-particles in the neural stem cell differentiation regulation is as follows: the difference from example 5 is that:
the concentration of calcium phosphate nanoparticles in the system was 0. Mu.g/ml.
Experimental example 2:
SEM images of neural stem cells after 7 days of coculture of the calcium phosphate nanoparticles with different concentrations of example 5 and comparative example 2 with the neural stem cells are shown in fig. 6.

Claims (3)

1. The application of the general calcium preparation for non-diagnosis and treatment purposes in the regulation of neural stem cell differentiation comprises the following steps:
1) Inoculating the neural stem cells into a neural stem cell differentiation culture medium; the inoculation amount of the neural stem cells is (1-10) multiplied by 10 6 Per mL;
2) Adding a universal calcium preparation into the culture system obtained in the step 1) to perform culture and endocytosis of the neural stem cells, wherein the universal calcium preparation is a hydroxyapatite nanorod or a calcium phosphate nanoparticle to obtain the neural stem cells modified by the calcium preparation; after the universal calcium preparation is added, the concentration of the universal calcium preparation in the system is 50-200 mug/mL; the culture and endocytosis of the neural stem cells are that a culture system added with a calcium preparation is placed in an incubator at 36-37 ℃ for incubation for 1-14 days;
the hydroxyapatite nanorod is prepared by the following method:
(1) Mixing octadecylamine, oleic acid and ethanol uniformly, adding NaF and Na 3 PO 4 Mixing with calcium nitrate, stirring to obtain mixed solution, naF and Na 3 PO 4 And calcium nitrate in a mass ratio of 0.1 to 0.5:1.2:1.4, the mass ratio of calcium nitrate to octadecylamine is 1: 0.1-10, wherein the mass ratio of calcium nitrate to oleic acid is 1: 1-10, wherein the adding amount of the ethanol accounts for 1/4-2/3 of the total volume of the mixed solution;
(2) Transferring the mixed solution into a closed reactor, reacting for 8-15 hours at 120-220 ℃, and naturally cooling to room temperature;
(3) Dispersing the product in cyclohexane, centrifuging, then sequentially cleaning with cyclohexane and ethanol for three times, and finally dispersing in ethanol to prepare a hydroxyapatite nanorod;
the calcium phosphate nano-particles are prepared by the following method:
a. uniformly mixing cyclohexane, trion-X100 and hexanol to obtain a mixed solution, averagely dividing the mixed solution into two parts, and adding a calcium chloride solution into one part to obtain a calcium chloride mixed solution; adding another part of the disodium hydrogen phosphate solution to obtain a disodium hydrogen phosphate mixed solution; the volume ratio of cyclohexane to Trion-X100 to hexanol (4-6): (1-2): (3-5); the volume ratio of one mixed solution to the calcium chloride solution is 8:1, the volume ratio of the other mixed solution to the disodium hydrogen phosphate solution is 8:1; the concentration of the calcium chloride solution is 35mg/ml, and the concentration of the disodium hydrogen phosphate solution is 111mg/ml;
b. and dropwise adding the mixed solution of the disodium hydrogen phosphate into the mixed solution of the calcium chloride, reacting for 0.5-2h, and centrifuging to obtain the calcium phosphate nano-particles.
2. The use of claim 1, wherein in step 1), the neural stem cell differentiation medium is a basal medium Neurobasal medium, gibco B-27 neural cell culture serum-free additive with a volume concentration of 1-3%, and GlutaMAX with a volume concentration of 1-2% TM -I, fetal bovine serum FBS at a volume concentration of 1-2% and a double antibody at a volume concentration of 1-2%.
3. The application of claim 1, wherein in the step 2), the specific preparation method of the hydroxyapatite nanorods is as follows:
1) Dissolving 0.6g of octadecylamine in 7mL of oleic acid, heating at 80 ℃ for 30min, and adding 20mL of ethanol under vigorous stirring;
2) 1.4mL of 0.2M NaF and 7mL of 0.168M Na were added 3 PO 4 Stirring for 5min;
3) Then 7mL of 0.2M Ca (NO) was added 3 ) 2 Stirring the solution for 1min to obtain mixed solution,
4) Heating the mixed solution at 180 ℃ for reaction for 12h, and naturally cooling to room temperature;
5) Dispersing the product in cyclohexane, centrifuging at 10000rpm for 10min, then washing with cyclohexane and ethanol for three times, and finally dispersing in ethanol for later use.
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