Search

CN-121975737-A - Induction method for directional differentiation of neural stem cells into dopaminergic neurons

CN121975737ACN 121975737 ACN121975737 ACN 121975737ACN-121975737-A

Abstract

The invention provides an induction method for directionally differentiating nerve stem cells into dopaminergic neurons, which comprises the following steps of (1) electroporating the nerve stem cells, enabling Fe 2 O 3 @TGA@MIL-100@PDA@Ag nano materials with the wavelength of 40-80 nm to enter the nerve stem cells, (2) placing the nerve stem cells in the step (1) in a magnetic field with the magnetic field strength unchanged for 10-20 minutes in the horizontal direction, culturing the cells, wherein the induced electromotive force generated by the induction of the rotating magnetic field is 0.1-10 mV, and the magnetic field direction is rotated for 10-20 minutes at intervals of 23-25 hours, and culturing the cells for 72-240 hours. The induction method has the advantages of high differentiation rate of the dopaminergic neurons, improvement of the synaptic function maturity, and more than 80 percent of the differentiation rate of the dopaminergic neurons, and improvement of the synaptic function maturity compared with the traditional method, can be applied to the treatment of parkinsonism and the repair of nervous system injury, and provides an efficient induction technology for the cell treatment of nervous system degenerative diseases and injuries.

Inventors

  • ZHANG XIN
  • MA RUBIAO
  • XU ZHIFENG
  • LIU GUIBO

Assignees

  • 广州沙艾生物科技有限公司

Dates

Publication Date
20260505
Application Date
20260204

Claims (10)

  1. 1. A method for inducing directed differentiation of neural stem cells into dopaminergic neurons, the method comprising the steps of: (1) After electroporation of the neural stem cells, enabling the Fe 2 O 3 @TGA@MIL-100@PDA@Ag nano material with the particle size of 40-80 nm to enter the neural stem cells; (2) And (3) placing the neural stem cells in the step (1) in a magnetic field of 0.25-0.4 Tesla, rotating the magnetic field for 10-20 minutes in the horizontal direction with the magnetic field strength unchanged, then culturing the cells, wherein the induced electromotive force generated by the induction of the rotating magnetic field is 0.1-10 mV, the magnetic field direction is rotated for 10-20 minutes at intervals of 23-25 hours, and the cells are cultured for 72-240 hours.
  2. 2. The method for inducing directional differentiation of neural stem cells into dopaminergic neurons according to claim 1, wherein the cell culture is performed in a differentiation medium and under cell culture conditions, the differentiation medium comprises, by mass, 1.5-2.5% of a B27 supplement, 0.8-1.2% of streptomycin penicillin, 0.8-1.2% of glutamine, 0.8-1.2% of FBS and the balance of a neurobasal medium.
  3. 3. The method for inducing directional differentiation of neural stem cells into dopaminergic neurons according to claim 1, wherein the method for rotating the magnetic field direction in the horizontal direction for 10-20 minutes is that a magnetic field of 0.25-0.4 tesla is generated by a bar-shaped permanent magnet or electromagnet, and the bar-shaped permanent magnet or electromagnet rotates in the horizontal direction for 10-20 minutes at a rotation speed of 400-600/min.
  4. 4. The method for inducing directional differentiation of neural stem cells into dopaminergic neurons according to claim 1, wherein the preparation method of the Fe 2 O 3 @tga@mils-100@pda@ag nanomaterial comprises the following steps: (a) Dispersing 30-50 nm Fe 2 O 3 nano particles and thioglycollic acid in ethanol, wherein the pH is 7.8-8.2, the weight ratio of the Fe 2 O 3 nano particles to the thioglycollic acid is 1:0.8-1.5, and magnetically stirring the mixture at 25-30 ℃ for 8-15 hours to obtain TGA coated Fe 2 O 3 , namely Fe 2 O 3 @TGA; (b) Dispersing Fe 2 O 3 @TGA into N, N-Dimethylformamide (DMF) in an ultrasonic manner, washing, collecting nano particles, sequentially and alternately soaking in a Fe 3+ solution and trimesic acid for 4-8 min each time, repeating 25-40 cycles, washing and centrifuging with DMF after each soaking, collecting nano particles, and vacuum-activating at 110-130 ℃ for 10-15 h to obtain a Fe 2 O 3 @TGA@MIL-100 nano material; (c) Dispersing the Fe 2 O 3 @TGA@MIL-100 nanomaterial in 1.5-2.5 mg/mL dopamine solution, magnetically stirring for 5-8 hours at 25-30 ℃, and obtaining the Fe 2 O 3 @TGA@MIL-100@PDA nanomaterial by using a buffer solution with a pH value of 8.2-8.7 as a solvent of the dopamine solution; (d) Dispersing the Fe 2 O 3 @TGA@MIL-100@PDA nanomaterial in AgNO 3 solution, and in-situ reducing to obtain the Fe 2 O 3 @TGA@MIL-100@PDA@Ag nanomaterial.
  5. 5. The method for inducing directional differentiation of neural stem cells into dopaminergic neurons according to claim 4, wherein in the step (d), the Fe 2 O 3 @ TGA @ MIL-100@ PDA nanomaterial is dispersed in AgNO 3 solution, stirred for 45-75 minutes in a dark place, naBH 4 solution is added dropwise until the color of the solution changes from colorless to pale yellow, solid particles are collected by centrifugation, washed by deionized water and dried in vacuum, and the temperature of the vacuum drying is 45-60 ℃.
  6. 6. The method for inducing directional differentiation of neural stem cells into dopaminergic neurons according to claim 4, wherein in the step (a), the preparation method of Fe 2 O 3 nano particles with a wavelength of 30-50 nm comprises the steps of adding ferric acetylacetonate, oleic Acid (OA), oleylamine (OLA) and Octadecene (ODE) into a three-neck flask, stirring until the Fe 2 O 3 nano particles are dissolved under the protection of nitrogen, heating to 280-320 ℃ at a temperature of 4.5-5.5 ℃ per minute, keeping the temperature for 45-75 minutes, forming Fe 3 O 4 nano particles, adding ethanol for precipitation, centrifuging and washing after natural cooling, and calcining for 1.5-2.5 hours at a temperature of 280-320 ℃ in air to oxidize the Fe 3 O 4 nano particles into Fe 2 O 3 nano particles.
  7. 7. The method for inducing directional differentiation of neural stem cells into dopaminergic neurons according to claim 4, wherein in the step (a), in the step (b), in the step (C), after the reaction of the step is completed, the nanoparticles are collected by centrifugation, washed with DMF, and then dried in vacuum, wherein the temperature of the vacuum drying is 45-60 ℃.
  8. 8. The method for inducing directional differentiation of neural stem cells into dopaminergic neurons according to claim 1, wherein the method in the step (1) comprises the steps of dispersing a neural stem cell suspension and a Fe 2 O 3 @TGA@MIL-100@PDA@Ag nanomaterial which is uniformly dispersed in advance in P3 PRIMARY CELL 4D-NucleofectorTM X Buffer, wherein the concentration of the Fe 2 O 3 @TGA@MIL-100@PDA@Ag nanomaterial is 5-40 mug/mL, the cell concentration is 1.5-2.5X10 6 cells/100 mug, electroporation is carried out on the dispersion, the voltage of the electroporation is 120-180V, the capacitance is 25-35 muF, the pulse time is 8-15 ms, and single pulse is carried out.
  9. 9. The method for inducing directional differentiation of neural stem cells into dopaminergic neurons according to claim 8, wherein the voltage of electroporation is 130-160V, the capacitance is 28-32 μf, the pulse time is 10-15 ms, the single pulse, the concentration of the Fe 2 O 3 @TGA@MIL-100@PDA@Ag nanomaterial is 15-35 μg/mL.
  10. 10. The method of inducing directed differentiation of neural stem cells into dopaminergic neurons according to claim 1, wherein the cell culture is performed in a CO 2 incubator.

Description

Induction method for directional differentiation of neural stem cells into dopaminergic neurons Technical Field The invention relates to the technical field of biological cells, in particular to an induction method for directional differentiation of neural stem cells into dopaminergic neurons. Background Spinal Cord Injury (SCI) is a serious disabling neurological disease that can lead to permanent and degenerative damage to the Central Nervous System (CNS), symptoms including mobility difficulties, pain and autonomic dysfunction, and presents a heavy economic burden to patients, families and society, which is the most challenging global medical problem. Rupture of the axons of neurons after SCI, death of neurons, leads to dysfunction of the neural circuits, leading to loss of sensory and motor control. Thus promoting axon regeneration, myelinating axons, and reestablishing the neural circuit is the therapeutic focus. Although researchers have developed a variety of new treatments in recent years, no effective therapies have yet been able to cure or reverse spinal cord injury. Among them, cell therapies and stem cell therapies are considered as promising candidate therapies for promoting functional recovery, and stem cell therapies rely mainly on self-renewal and differentiation capabilities, with neural stem cells standing out among many types of stem cells. The existing induction method for directionally differentiating the neural stem cells into the dopaminergic neurons is to add induction factors into the culture medium, such as hedgehog factors, cell growth factors, BMP inhibitors, ALK5 inhibitors and GSK3 Inhibitors, etc., but the rate of differentiation of the baaminergic neurons and the degree of functional maturity of synapses are to be improved. Disclosure of Invention The invention aims to overcome the defects of the prior art and provide an induction method for directionally differentiating the neural stem cells into dopaminergic neurons. In order to achieve the aim, the technical scheme adopted by the invention is that the induction method for directionally differentiating the neural stem cells into the dopaminergic neurons comprises the following steps: (1) After electroporation of the neural stem cells, enabling the Fe 2O3 @TGA@MIL-100@PDA@Ag nano material with the particle size of 40-80 nm to enter the neural stem cells; (2) And (3) placing the neural stem cells in the step (1) in a magnetic field of 0.25-0.4 Tesla, rotating the magnetic field for 10-20 minutes in the horizontal direction with the magnetic field strength unchanged, then culturing the cells, wherein the induced electromotive force generated by the induction of the rotating magnetic field is 0.1-10 mV, the magnetic field direction is rotated for 10-20 minutes at intervals of 23-25 hours, and the cells are cultured for 72-240 hours. According to the induction method for directionally differentiating the nerve stem cells into the dopaminergic neurons, after electroporation, the Fe 2O3 @TGA@MIL-100@PDA@Ag nanomaterial with the wavelength of 40-80 nm enters the nerve stem cells, electromagnetic induction is carried out by applying a magnetic field, so that the induced current generated by the Fe 2O3 @TGA@MIL-100@PDA@Ag nanomaterial stimulates the stem cells to differentiate into the neurons, the Fe 2O3 @TGA@MIL-100@PDA@Ag nanomaterial has more excellent cell compatibility, the electroporation cell death rate is lower, the Fe 2O3 @TGA@MIL-100@PDA@Ag nanomaterial stimulates under electromagnetic induction, a proper amount of active oxygen and the like are generated, and after culture, the dopaminergic neurons have high differentiation rate, the synaptic function maturity is improved, and the synaptic function maturity is improved by more than 80%, so that the induction method can be applied to the treatment of parkinsonism and the nerve system injury and provide a high-efficiency induction technology for treating the nerve system injury. Preferably, the cell culture is performed in a differentiation medium and under cell culture conditions, wherein the differentiation medium comprises, by mass, 1.5-2.5% of B27 supplement, 0.8-1.2% of streptomycin penicillin, 0.8-1.2% of glutamine, 0.8-1.2% of FBS and the balance of neurobasal medium. Preferably, the method for rotating the magnetic field direction in the horizontal direction for 10-20 minutes is that a magnetic field of 0.25-0.4 Tesla is generated by a strip-shaped permanent magnet or electromagnet, and the strip-shaped permanent magnet or electromagnet rotates in the horizontal direction for 10-20 minutes at a rotation speed of 400-600/min. Preferably, the preparation method of the Fe 2O3 @TGA@MIL-100@PDA@Ag nanomaterial comprises the following steps of: (a) Dispersing 30-50 nm Fe 2O3 nano particles and thioglycollic acid in ethanol, wherein the pH is 7.8-8.2, the weight ratio of the Fe 2O3 nano particles to the thioglycollic acid is 1:0.8-1.5, and magnetically stirring the mixture