Culture medium for promoting neural differentiation of SH-SY5Y cells

Abstract

The invention relates to the technical field of nerve cell differentiation, and discloses a culture medium for promoting neural differentiation of SH-SY5Y cells. The culture medium disclosed by the invention is prepared from the following components: tazarotene (tazarotene) and a basic culture medium. The culture medium can promote SH-SY5Y cells to present neuron-like differentiation within 5 days, significantly improves the length of neurite and the expression level of neuron markers, and is low in cytotoxicity. The invention provides an efficient, safe and standardized new tool for neural disease in-vitro model construction, drug screening and mechanism research.

Description

Technical Field

[0001] This invention relates to the field of neural cell differentiation, and more specifically to a culture medium for promoting the neural differentiation of SH-SY5Y cells. Background Technology

[0002] The SH-SY5Y cell line is a cell line derived from human neuroblastoma and is widely used in neurobiological research. This cell line exhibits active proliferative capacity in its undifferentiated state, and under specific induction conditions, it can differentiate into neuron-like cells, exhibiting typical neuronal characteristics such as neurite growth and expression of neural markers. This makes it an ideal cell model for research scenarios such as studying neural developmental mechanisms, exploring the pathology of neurodegenerative diseases, and screening neuro-related drugs.

[0003] Although the SH-SY5Y cell line is widely used in neurobiological research, its differentiation induction technology still faces many challenges, especially in the selection and use of inducing agents. Currently commonly used SH-SY5Y cell differentiation inducers mainly include retinoic acid (RA), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF). While these inducers can induce cell differentiation to a certain extent, they are accompanied by a series of problems that urgently need to be solved.

[0004] Retinoic acid (RA), as a metabolic intermediate of vitamin A, was one of the earliest reagents used to induce SH-SY5Y cell differentiation, but its shortcomings are significant. In terms of the induction cycle, RA alone typically requires 6-8 days to induce SH-SY5Y cell differentiation, a relatively lengthy process that not only consumes considerable time but also increases the risk of cell contamination during experiments. Furthermore, its differentiation efficiency is unsatisfactory, making it difficult to achieve stable and efficient differentiation results.

[0005] Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophic factor family and plays a key role in the development, maintenance and repair of the nervous system. However, its induction period has been extended to 7-10 days, and it has relatively high costs. More importantly, BDNF has poor stability in solution and is easily inactivated by environmental factors such as temperature and pH, resulting in large fluctuations in differentiation efficiency in different batches of experiments and poor experimental reproducibility.

[0006] Nerve growth factor (NGF), as the earliest discovered neurotrophic factor, is crucial for the development, survival, and functional maintenance of the sympathetic and sensory nerves. However, it also has the problem of a long induction period, generally requiring 7-10 days to observe obvious differentiation effects.

[0007] Tazarotene is a third-generation synthetic retinoid that exerts anti-proliferative and anti-inflammatory effects by selectively activating retinoic acid receptors (RAR-β and RAR-γ) to regulate gene transcription. Currently, tazarotene is mainly used in the treatment of dermatological diseases, and there are no reports on its use in the neural differentiation of SH-SY5Y cells. Summary of the Invention

[0008] The purpose of this invention is to provide an inducer that can efficiently induce SH-SY5Y cells to differentiate into neuron-like cells, thereby solving the problems of long induction cycles and low differentiation efficiency of existing inducers.

[0009] On one hand, the present invention provides a culture medium for promoting neural differentiation of SH-SY5Y cells, the components of which include Tazarotene and basal medium.

[0010] Furthermore, the concentration of Tazarotene in the culture medium is 0.1-100 μM.

[0011] Furthermore, the culture medium also contains fetal bovine serum, penicillin-streptomycin, and L-glutamine; by volume percentage, the concentration of fetal bovine serum in the culture medium is 1%-20%, the concentration of penicillin-streptomycin is 0.5%-2%, and the concentration of L-glutamine in the culture medium is 1mM-5mM.

[0012] Furthermore, the basal culture medium includes DMEN basal culture medium, DMEN / F12 basal culture medium and / or 1640 basal culture medium.

[0013] Furthermore, the DMEM basal medium is a high-sugar DMEM basal medium.

[0014] Furthermore, the culture medium also contains BDNF (brain-derived neurotrophic factor); the concentration of BDNF in the culture medium is 10-50 ng / mL.

[0015] On the other hand, the present invention provides the use of Tazarotene or the above-mentioned culture medium in promoting the neural differentiation of SH-SY5Y cells.

[0016] On the other hand, the present invention provides the use of Tazarotene or the aforementioned culture medium in constructing in vitro models of neurodegenerative diseases.

[0017] On the other hand, the present invention provides the use of Tazarotene or the above-described culture medium in the screening of neuroprotective or neurotoxic drugs.

[0018] On the other hand, the present invention provides a method for promoting neural differentiation of SH-SY5Y cells, the method comprising: culturing SH-SY5Y cells in the above-mentioned culture medium.

[0019] Furthermore, the culture includes: placing SH-SY5Y cells in a carbon dioxide cell culture incubator for culture; the culture conditions include: a temperature of 36-38℃, a carbon dioxide concentration of 4-6%, and a humidity of 95% or higher.

[0020] The technical solution of this invention has the following advantages: Excellent induction efficiency: This invention uses tazarotene as a key inducing component, which can significantly promote the growth of neurites in SH-SY5Y cells and effectively upregulate the expression levels of neuronal markers, thereby greatly improving the quality and efficiency of cell differentiation into neuron-like cells.

[0021] Excellent therapeutic properties: In this invention, tazarotene can exert a highly efficient induction effect at the level of 2.5 μmol / L. The lower concentration significantly reduces cytotoxicity and improves the survival stability of cells during differentiation.

[0022] Shortened induction cycle: Compared with traditional induction methods such as retinoic acid and brain-derived neurotrophic factor, this invention achieves SH-SY5Y cell differentiation in just 3 days through the specific regulatory effect of tazarotene, significantly shortening the differentiation cycle of SH-SY5Y cells, reducing experimental time costs and contamination risks, and improving research efficiency.

[0023] Facilitates standardized application: The culture medium composition of this invention is clearly defined and controllable, avoiding the differentiation stability problem caused by component fluctuations in traditional induction systems. It can be stably applied to scenarios such as the construction of in vitro models of neurodegenerative diseases, screening of neuro-related drugs, and research on neural differentiation mechanisms, providing standardized tool support for related research. Attached Figure Description

[0024] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 Effects of different concentrations of Tazarotene on neurite length in SH-SY5Y cells; Figure 2 Immunofluorescence images show the expression of Tuj1 (β-III-tubulin). Detailed Implementation

[0026] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0027] Unless otherwise specified, the experimental steps or conditions in the examples were performed in accordance with conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0028] Example 1: A culture medium for promoting neural differentiation of SH-SY5Y cells This embodiment provides a culture medium for promoting neural differentiation of SH-SY5Y cells, wherein the culture medium is a DMEM high-glucose medium containing 5 μM Tazarotene, 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin.

[0029] The preparation process of the culture medium is as follows: Preparation of Tazarotene (Tazarotene, MCE, HY-15388) solution: Dissolve Tazarotene in DMSO to a final concentration <0.1% to obtain a Tazarotene solution; Preparation of culture medium: Add Tazarotene solution to DMEM high-glucose medium, then add FBS and penicillin-streptomycin, filter to sterilize, store at 4°C to obtain the culture medium.

[0030] Example 2: A culture medium for promoting neural differentiation of SH-SY5Y cells This embodiment provides a culture medium for promoting neural differentiation of SH-SY5Y cells. The culture medium is based on Example 1, but with the concentration of Tazarotene adjusted from 5 μM to 2.5 μM.

[0031] Example 3: A culture medium for promoting neural differentiation of SH-SY5Y cells This embodiment provides a culture medium for promoting neural differentiation of SH-SY5Y cells. The culture medium is based on Example 1, but with the concentration of Tazarotene adjusted from 5 μM to 10 μM.

[0032] Comparative Example 1: A culture medium for promoting neural differentiation of SH-SY5Y cells This comparative example provides a culture medium for promoting neural differentiation of SH-SY5Y cells. The culture medium is based on Example 1, but with the Tazarotene concentration adjusted from 5 μM to 0 μM.

[0033] Experimental Example 1: SH-SY5Y Cell Differentiation Experiment The experiment was divided into four groups: control group, 2.5 μM group, 5 μM group and 10 μM group. The control group was prepared by adding 10% FBS and 1% penicillin-streptomycin to DMEM high-glucose medium, filtering to remove bacteria, and storing at 4°C (i.e., comparative example 1). The culture medium for the 2.5 μM group was as follows: Tazarotene solution from Example 1 was added to DMEM high glucose medium to make the final concentration of Tazarotene in the medium 2.5 μM, then 10% FBS and 1% penicillin-streptomycin were added, filtered to remove bacteria, and stored at 4°C (i.e. Example 1). The culture medium for the 5 μM group was as follows: Tazarotene solution from Example 1 was added to DMEM high glucose medium to make the final concentration of Tazarotene in the medium 5 μM, then 10% FBS and 1% penicillin-streptomycin were added, filtered to remove bacteria, and stored at 4°C (i.e. Example 2). The culture medium for the 10 μM group was as follows: Tazarotene solution from Example 1 was added to DMEM high glucose medium to make the final concentration of Tazarotene in the medium 10 μM, then 10% FBS and 1% penicillin-streptomycin were added, filtered to remove bacteria, and stored at 4°C (i.e. Example 3). First, add 100 µL of the corresponding culture medium for each of the four groups to a 96-well plate. Then, add SH-SY5Y cells (purchased from Yuanjing Biotechnology) at a concentration of 1×10⁻⁶. 4The culture medium was seeded into 96-well plates and cultured in a CO2 cell incubator under the following conditions: temperature 37°C, CO2 concentration 5%, and humidity above 95%. The culture was carried out for 5 days, with the culture medium changed every 2-3 days.

[0034] Cell morphology was observed on days 1, 3, and 5 of culture. Results are as follows: Figure 1 As shown, compared with the control group, the neurite length of the 2.5 μM group, 5 μM group and 10 μM group were significantly increased, and the neurite length of SH-SY5Y cells in the 2.5 μM group was the most significantly increased.

[0035] Immunofluorescence staining was performed on day 5. SH-SY5Y cells were fixed with 4% PFA at room temperature for 15 min, followed by washing three times with PBS for 5 min each time. They were then permeabilized with 0.3% Triton X-100 for 10 min, and blocked with 3% BSA for 30 min. Rabbit anti-β-III-tubulin antibody (CST 5568S, diluted to 1:500 with 1% BSA / PBS) was added to the SH-SY5Y cells, and the cells were incubated overnight at 4°C in a humidified chamber. After incubation, the cells were washed three times with PBS for 5 min each time. Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, diluted to 1:1000 with 1% BSA / PBS) was then added, and the cells were incubated at room temperature in the dark for 1 h, followed by washing three times with PBS for 5 min each time. The cells were then stained with Hoechst 33342 staining solution for 5 min. Imaging was then performed to detect β-III-tubulin expression. Results are as follows: Figure 2 As shown, compared with the control group, the 10 μM group exhibited significantly increased expression of the neuronal marker protein β-III-tubulin, and the cells displayed typical neuronal morphology, indicating that SH-SY5Y cells had successfully differentiated into neuron-like cells. Therefore, the use of Tazarotene can significantly promote neuronal differentiation of SH-SY5Y cells.

[0036] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A medium for promoting neural differentiation of SH-SY5Y cells, characterized in that, The culture medium consists of Tazarotene and a basal medium.

2. The culture medium according to claim 1, characterized in that, The concentration of Tazarotene in the culture medium is 0.1-100 μM.

3. The medium of claim 1, wherein, The culture medium also contains fetal bovine serum, penicillin-streptomycin, and L-glutamine; by volume percentage, the concentration of fetal bovine serum in the culture medium is 1%-20%, the concentration of penicillin-streptomycin is 0.5%-2%, and the concentration of L-glutamine in the culture medium is 1mM-5mM.

4. The medium of claim 1, wherein, The basal culture medium includes DMEN basal culture medium, DMEN / F12 basal culture medium and / or 1640 basal culture medium; The DMEM basal medium is a high-sugar type of DMEM basal medium.

5. The medium of claim 1, wherein, The culture medium also contains BDNF; the concentration of BDNF in the culture medium is 10-50 ng / mL.

6. The use of Tazarotene or the culture medium according to any one of claims 1-5 in promoting neural differentiation of SH-SY5Y cells.

7. The use of Tazarotene or the culture medium according to any one of claims 1-5 in constructing in vitro models of neurodegenerative diseases.

8. The use of Tazarotene or the culture medium according to any one of claims 1-5 in the screening of neuroprotective or neurotoxic drugs.

9. A method of promoting neural differentiation of SH-SY5Y cells, the method comprising contacting the cells with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. The method includes culturing SH-SY5Y cells in the culture medium described in any one of claims 1-5.

10. The method of claim 9, wherein, The culture includes: placing SH-SY5Y cells in a carbon dioxide cell culture incubator; the culture conditions include: a temperature of 36-38℃, a carbon dioxide concentration of 4-6%, and a humidity of 95% or higher.

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