Method for inducing directional differentiation of neural stem cells into neuron cells by Gd:Fe3O4@RA nanoparticles

A technology of neural stem cells and neuron cells, which is applied in the field of medical biomaterials and tissue engineering, can solve the problems of low effective differentiation rate of stem cells, unfavorable stem cells, and low survival rate, and achieve the effect of easy operation and separation, simple process and environmental friendliness

Active Publication Date: 2019-03-26
JINING MEDICAL UNIV
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Problems solved by technology

However, stem cell transplantation faces several major problems: (1) the survival rate of stem cell transplantation is low, (2) the effective differentiation rate of stem cells is low, and less than 13% of surviving neural stem cells differentiate into neurons, (3) the post-evaluation method is invasive, and most traditional Tissue slices were performed after sacrificing animals to verify whether the neurological function or behavior was improved after stem cell transplantation, which is not conducive to the dynamic tracking of stem cell migration in vivo after transplantation
At present, there is no report on the method of inducing the directed differentiation of human stem cells by multifunctional gadolinium-doped ferric oxide composite nanoparticles for central nervous system injury repair and real-time MRI imaging

Method used

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  • Method for inducing directional differentiation of neural stem cells into neuron cells by Gd:Fe3O4@RA nanoparticles
  • Method for inducing directional differentiation of neural stem cells into neuron cells by Gd:Fe3O4@RA nanoparticles
  • Method for inducing directional differentiation of neural stem cells into neuron cells by Gd:Fe3O4@RA nanoparticles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Preparation of gadolinium-doped ferric oxide composite nanoparticles:

[0042] First, add 1.88g of sodium benzoate solution into 100ml of distilled water, then add 0.5g of FeCl 2 .4H 2 O and 0.46gGdCl 3 .2H 2 In an aqueous solution of O, 25 ml of acetonitrile was added to the solution; the reaction mixture was stirred at room temperature for at least 12 hours to obtain a light brown precipitate; Washing, vacuum pump drying, obtain Fe / Gd dry precursor.

[0043] Next, 0.2 g of the prepared Fe / Gd dry precursor was added to the reaction mixture containing oleylamine, oleic acid and benzyl ether in a volume ratio of 4.5 ml: 3 ml: 2.5 ml, and then heated to 110 °C under nitrogen flow to maintain 30 minutes, then slowly heated to 350°C for 30 minutes to obtain a black / brown substance; after cooling to room temperature, separate, add at least 10ml of ethanol, wash and re-separate to obtain black / brown Gd:Fe 3 o 4 Nanoparticles, finally dried under vacuum, wi...

Embodiment 2

[0044] Example 2: Preparation of gadolinium-doped ferric oxide composite nanoparticles loaded with retinoic acid:

[0045] 0.02 g of Gd:Fe 3 o 4 The nanoparticles were added to 10 mL of CHCl containing 0.1 g of the active polymer o-hydroxybenzene-PEG-carboxy-terminated compound (average molecular weight 4000) 3 Then the mixture was stirred at room temperature in the dark for 12 hours; 20ml of n-hexane was added, and centrifuged to obtain surface functionalized Gd-doped ferric oxide nanoparticles Gd:Fe 3 o 4 @DIB-PEG-COOH (o-hydroxybenzene-PEG-carboxyl terminal compound), through 10ml CHCl 3 After washing with n-hexane (1:5 v / v), disperse in 5 ml of distilled water, dialyze with distilled water for 24 hours to remove unreacted organic molecules, and store in the dark.

[0046] Secondly, the cyclodextrin loaded with retinoic acid was connected to the carboxyl group on the surface of the nanoparticles by acid-catalyzed ester bond preparation to complete the preparation of Gd:...

Embodiment 3

[0047] Example 3: Multifunctional retinoic acid-loaded gadolinium-doped ferric oxide composite nanoparticles induce directional differentiation of neural stem cells into neurons:

[0048] Put the coverslips pre-treated with poly-lysine in a 24-well culture plate, and the primary and passage neural stem cells derived from the rat embryonic brain hippocampus were mixed at 1×10 7 L -1 Inoculate into culture wells respectively, remove basic fibroblast growth factor in the complete culture medium of neural stem cells, and add 1.0ml, 1mg / ml Gd:Fe 3 o 4 @RA and 5% fetal bovine serum promote the differentiation of neural stem cells. After 6 days of culture, continue to culture in the neural stem cell medium containing 5% fetal bovine serum for 6 days, and take out the cover glass after the cells mature. piece. The coverslips loaded with conventional adherent differentiation culture and induced differentiation culture were divided into 2 groups, and the immunohistochemistry of neuro...

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Abstract

The invention relates to an experimental method for inducing directional differentiation of neural stem cells into neuron cells by Gd:Fe3O4@RA nanoparticles. The experimental method includes the following steps of putting a cover slip pre-treated with polylysine into a culture plate, and inoculating culture holes with primary and passaged neural stem cells derived from rat embryonic brain hippocampus at 1 is multiplied by 10<7> L<-1>, removing basic fibroblast growth factors from a complete culture solution of the neural stem cells, adding 1.0 ml of Gd:Fe3O4@RA complex nano ions with the concentration of 1 mg/ml and fetal bovine serum with the volume fraction of 5% to promote neural stem cell differentiation, performing culture for 6 days, continuing culture for 6 days in a neural stem cell culture medium containing the fetal bovine serum with the volume fraction of 5%, and taking out the cover slip after the cell morphology is mature.

Description

technical field [0001] The present invention relates to the technical field of medical biomaterials and tissue engineering, and more specifically relates to a nano-medical biomaterial for inducing human stem cells to differentiate into nerve cells and a material and method for repairing human central nervous system damage. Background technique [0002] The central nervous system refers to the main part of the nervous system, including the spinal cord in the spinal canal and the brain in the cranial cavity. Diseases caused by central nervous system injury seriously affect human health, and there are many patients, mainly including spinal cord injury, traumatic brain injury, cerebral apoplexy and Parkinson's disease. The incidence rate of spinal cord injury is 1 / 1000 in the middle period. There are about 1.3 million people with spinal cord injury in my country, and it is increasing at a rate of 50,000 to 70,000 per year. The number of patients is increasing year by year. Spin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0793
CPCC12N5/0619C12N2500/05
Inventor 王冠男金丽代晨谭文彬
Owner JINING MEDICAL UNIV
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