阅读说明：本技术 miR-29c-3p抑制物在制备神经干细胞分化的促进药物中的应用 (Application of miR-29c-3p inhibitor in preparation of medicine for promoting neural stem cell differentiation ) 是由 衣昕 陆健花 缪君怡 张雅婷 王宁馨 邹嘉来 于 2021-08-11 设计创作，主要内容包括：本发明公开了miR-29c-3p抑制物在制备神经干细胞向神经元分化的促进药物中的应用,属于生物医药技术领域。本发明采用miRNA抑制物对miR-29c-3p的表达进行下调,以miR-NC作为对照,将携带有miR-29c-3p抑制物和对照miR-NC的慢病毒分别感染神经干细胞后进行筛选,得到稳定低表达和正常表达miR-29c-3p的神经干细胞系,体外分化培养,观察两种情况下神经元分化情况。结果显示,低表达miR-29c-3p能明显促进神经干细胞向神经元分化(P<0.05)。(The invention discloses application of a miR-29c-3p inhibitor in preparation of a medicine for promoting differentiation of neural stem cells to neurons, and belongs to the technical field of biological medicines. The expression of miR-29c-3p is reduced by adopting a miRNA inhibitor, the miR-NC is used as a control, the lentiviruses carrying the miR-29c-3p inhibitor and the control miR-NC are respectively infected with neural stem cells and then screened to obtain a neural stem cell line with low and stable expression and normal expression of miR-29c-3p, the neural stem cell line is subjected to in vitro differentiation culture, and the neuron differentiation condition under two conditions is observed. The result shows that the low expression miR-29c-3p can obviously promote the neural stem cells to differentiate to the neurons ( P <0.05）。)

Application of miR-29c-3p inhibitor in preparation of medicine for promoting differentiation of neural stem cells to neurons.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of a miR-29c-3p inhibitor in preparation of a medicine for promoting differentiation of neural stem cells to neurons.

Background

The main pathological feature of various degenerative changes of the central nervous system is the loss of functional neurons, and how to recover the lost neurons is a difficult point and a hot spot for the regeneration of the central nervous system. The current strategy focuses mainly on two aspects: one aspect is the use of various protection methods to delay or reduce the loss of functional neurons, and another aspect is cell replacement strategies. The former protects the loss of neurons by means of medicine or gene intervention on the basis of researching the pathogenesis of diseases, and the latter is expected to achieve the purpose of replacing the lost neurons by promoting the regeneration of the neurons in vivo or by a transplantation method. However, in the cell replacement therapy strategy, how to promote Neural Stem Cells (NSCs) to differentiate into neurons is a difficult problem to be solved, which is also a main reason for limiting the application of the therapy strategy.

Micrornas (miRNAs) are non-coding single-stranded small RNA molecules containing about 1825 nucleic acids that regulate the expression of genes following transcription. They are widely distributed, both in tissues and in all body fluids, including plasma, serum, urine, saliva, and cerebrospinal fluid. It has high stability, and is transported in blood and cerebrospinal fluid via liposome or lipoprotein, and is rarely degraded. Almost all physiological processes of the whole body including the processes of generation, learning and memory of neurons are involved, so that the neural network has close association with the degenerative disease of the central nervous system.

Therefore, if the key microRNA influencing the differentiation of the neural stem cells to the neurons can be found, the microRNA can become a new target of the neuron replacement therapy and bring breakthrough to the treatment of various degenerative diseases of the central nervous system.

Disclosure of Invention

The invention aims to provide application of a miR-29c-3p inhibitor in preparation of a medicine for promoting differentiation of neural stem cells to neurons.

DCX is a microtubule-associated protein that is widely expressed in migrating postmitotic neuronal precursors, a key protein involved in neuronal differentiation, and a major marker of neuronal precursor cells. The invention obtains miR-29c-3p (5 'UAGCACCAUUUGAAAUCGGUUA 3', SEQ ID NO. 1) by analyzing and predicting DCX mRNA upstream miRNAs through online software targetscan (http:// www.targetscan.org). Therefore, the miR-29c-3p is presumed to be involved in the process of neural stem cell differentiation to neurons.

The expression of miR-29c-3p is reduced by adopting a miRNA inhibitor, the miR-NC is used as a control, the lentiviruses carrying the miR-29c-3p inhibitor and the control miR-NC are respectively infected with neural stem cells and then screened to obtain a neural stem cell line with low and stable expression and normal expression of miR-29c-3p, the neural stem cell line is subjected to in vitro differentiation culture, and the neuron differentiation condition under two conditions is observed. The result shows that miR-29c-3p can inhibit neural stem cells from differentiating to neurons in vitro (P<0.05), the low expression of miR-29c-3p can obviously promote the neural stem cells to differentiate to the neurons (P<0.05）。

Drawings

FIG. 1 shows the DCX immunofluorescence assay results after differentiation culture. Wherein: a is a DCX immunofluorescence experiment result of neural stem cell transfection miR-NC or miR-29c-3p inhibitor differentiation culture for 72 hours, Hoechst stains nuclei, and a ruler =100 micrometers; b is the statistics of the ratio of DCX positive neuronal precursor cells (mean ± standard deviation,. vs. Control group,P<0.001；# vs.miR-NC group，P<0.001）。

Detailed Description

The invention is described in further detail below with reference to the figures and the specific examples, which should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.

Example 1

In vitro neural stem cell differentiation into neurons in this example, rat embryonic neural stem cells were used.

In the embodiment, the expression of miR-29c-3p is reduced by adopting a miRNA inhibitor, the miR-NC is used as a control, the lentiviruses carrying the miR-29c-3p inhibitor and the control miR-NC are respectively infected with neural stem cells and then screened to obtain a neural stem cell line with low expression and normal expression of miR-29c-3p, the neural stem cell line is subjected to in vitro differentiation culture, and the neuron differentiation condition under two conditions is observed.

The specific process is as follows:

1. neural stem cell culture and miRNA inhibitor transfection

1) The Neural Stem cell line was obtained from Gibco Rat Fetal Neural Stem Cells of Seimer Feishale technologies, Inc., cultured in 5% CO₂In a 37 ℃ incubator, the culture medium was DMEM (Gibco) supplemented with EGF and bFGF (both at 20 ng/mL final concentration) and 1% penicillin/streptomycin.

2) The miR-29c-3p inhibitor is from Saimer Feishale technologies, Inc., the transfection reagent of Lipofectamine RNAiMAX from Invitrogen is used for cell transfection, the transfection procedure is performed according to the instruction, and the concentration of the miRNA inhibitor is 5 nanomolar.

2. Differentiation culture and DCX immunofluorescence assay:

1) differentiation culture experiment: the neural stem cells and the neural stem cells transfected with miRNA inhibitors are divided into 1 × 10⁵Each well of a 24-well plate previously coated with a polylysine cover glass was seeded at a density of 2 mL, and 2 mL of complete medium containing 10% FBS in DMEM/F12 was added. Placing 24-well culture plate at 37 deg.C and 5% CO in saturated humidity₂Culturing in an incubator, and performing immunofluorescence detection after 3 d.

2) DCX immunofluorescence assay: after culturing for 3 days, the cells in each of the 6 wells were collected, the culture medium in the culture well was aspirated, 1 mL of 0.01 mol/L PB (pH7.2) containing 4% paraformaldehyde was added, and the mixture was fixed at room temperature for 15 min. Paraformaldehyde is aspirated, washed 3 times with 0.01 mol/L PBS (pH7.2), 200. mu.L of 0.01 mol/L PBS (pH7.2) containing 10% goat serum is added to each well, and gently shaken at room temperature for 30 min. The blocking solution was aspirated, a 1:1000 dilution of guinea pig anti-DCX 200 μ L (Abcam Co.) was added, and the mixture was placed in a refrigerator at 4 ℃ overnight with gentle shaking at room temperature for 1 hour. The next morning, the primary antibody was blotted off, washed with PBS 3 times, added with 1:800 diluted Alexa Fluor 488 goat anti-guinea pig secondary antibody (Abcam Co.), shaken gently for 2 h in the dark at room temperature, washed with PBS 3 times, added with 1:2000 diluted Hoechst (Abcam Co.) and incubated in the dark at room temperature for 30 min, after PBS was washed thoroughly, glycerol phosphate buffer seals, and DCX immunofluorescence was observed under a fluorescence microscope.

3. Statistical treatment

Data were analyzed using SPSS 21.0 data statistics software. The obtained measurement data are expressed by mean value plus or minus standard deviation, and the two-to-two comparison among the three groups adopts one-factor variance analysis. To be provided withPDifferences were considered statistically significant < 0.05.

The results are shown in FIG. 1, compared with the blank control group and the miR-NC group, after the miR-29c-3p inhibitor is transfected by the neural stem cells, the differentiation quantity of the neural stem cells to DCX positive neuron precursor cells is obviously increased, and the result of the one-factor anova analysis shows that the difference between the groups has statistical significance (theP＜0.05）。* VS.Control group，P＜0.001；# VS. miR-NC group，P＜0.001。

Sequence listing

<110> university of southeast Tong

Application of miR-29c-3p inhibitor in preparation of medicine for promoting neural stem cell differentiation

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