Efficient scheme for extracting rat neural stem cells and directionally differentiating rat neural stem cells into dopaminergic neurons

Abstract

The invention relates to the technical field of biomedicine, and discloses an efficient scheme for extracting rat neural stem cells and directionally differentiating the rat neural stem cells into dopaminergic neurons, which comprises the following steps: S1, extracting and purifying the rat neural stem cells; s2, induced differentiation of the dopaminergic progenitor cells; and S3, maturation of the dopaminergic neuron cells. According to the efficient scheme for extracting the rat neural stem cells and directionally differentiating the rat neural stem cells into the dopaminergic neurons, by optimizing culture medium components and differentiation induction factors, the efficiency of directionally differentiating the rat NSCs into the dopaminergic neurons is improved, and the number of target cells is increased, so that the requirements of experiments and treatment are met; key factors in the differentiation process are accurately regulated and controlled, so that the stability and consistency of NSCs differentiation are realized, and the experiment repeatability and reliability are ensured; by adopting an optimized differentiation and maturation scheme, the generation of non-target cells is remarkably reduced, and high-purity dopaminergic neurons are ensured to be obtained, so that the safety and curative effect of cell transplantation therapy are improved.

Description

Technical Field

[0001] The invention relates to the field of biomedical technology, in particular to a highly efficient scheme for extracting rat neural stem cells and directing their differentiation into dopaminergic neurons. Background Art

[0002] The present invention relates to the field of biomedicine, especially neurobiology and stem cell research, and aims to explore how to generate dopaminergic neurons through the directed differentiation of neural stem cells, and provide potential cell therapy for the treatment of nervous system diseases. At present, the treatment of neurodegenerative diseases such as Parkinson's disease still faces many challenges. Existing therapies such as drug therapy can only relieve symptoms, but cannot effectively prevent disease progression or repair damaged neurons.

[0003] Traditional dopaminergic neuron replacement therapy faces problems such as insufficient sources and immune rejection. Studies have shown that neural stem cells (NSCs) have good proliferation and differentiation potential and are a hot topic in regenerative medicine. Although there are some methods to differentiate neural stem cells into dopaminergic neurons, these methods often have the disadvantages of low differentiation efficiency, insufficient purity and difficulty in control. In addition, existing studies mostly focus on mouse or human cells, and lack systematic research on rats.

[0004] With the aging of the population and changes in lifestyle, the incidence of neurodegenerative diseases has increased year by year, and effective treatment methods are urgently needed. Regeneration strategies for dopaminergic neurons can not only help restore motor function, but also improve the quality of life of patients. Therefore, the development of efficient and controllable neural stem cell directed differentiation technology has important clinical application prospects. The present invention aims to provide an effective strategy to direct the differentiation of rat neural stem cells into dopaminergic neurons by optimizing culture conditions and induction factors. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the deficiencies in the prior art, the present invention provides an efficient scheme for extracting rat neural stem cells and directing their differentiation into dopaminergic neurons, which has the advantages of improving differentiation efficiency, enhancing the controllability of the differentiation process, improving cell purity, and improving cell survival rate, thereby solving the problems of low efficiency and insufficient purity in the prior art.

[0007] (II) Technical solution

[0008] In order to achieve the above-mentioned purpose of improving differentiation efficiency, enhancing the controllability of the differentiation process, improving cell purity, and improving cell survival rate, the present invention provides the following technical solution: an efficient method for extracting rat neural stem cells and directing their differentiation into dopaminergic neurons, comprising the following steps:

[0009] Extraction and purification of S1 rat neural stem cells;

[0010] Induced differentiation of S2 dopaminergic progenitor cells;

[0011] Induction of maturation of S3 dopaminergic neurons.

[0012] As a preferred technical solution of the present invention, the extraction and purification of rat neural stem cells mainly includes: microdissection and separation of hippocampal brain tissue, shearing and filtering, mechanical separation and centrifugation, cultivation and purification, and purity identification.

[0013] Under sterile conditions, the hippocampal brain tissue of fetal mice was isolated by microdissection and placed in pre-cooled D-Hank's balanced salt solution;

[0014] Use sterile ophthalmic scissors to fully chop the brain tissue in the hippocampus into erosion, filter it through a 70um filter, and gently blow it three times to mechanically separate the neural stem cells and brain tissue;

[0015] After centrifugation at 500 g for 3 min, the cells were placed in a neural stem cell-specific culture medium containing DMEM / F12 medium, 2% B27, 20 ng / mL bFGF, and 20 ng / mL EGF, and incubated at 37°C and 5% CO2;

[0016] At different time periods of 12 hours and 24 hours, the non-neural stem cells adhering to the wall were removed by aspirating the suspended cell mass, centrifuging, re-culturing, and replacing the fresh culture medium to improve the purity of the neural stem cells;

[0017] The purity of neural stem cell markers (Nestin) was identified by cell immunofluorescence.

[0018] As a preferred technical solution of the present invention, the induction differentiation of dopaminergic progenitor cells mainly includes: pre-coating of culture plates, cell inoculation and differentiation induction culture medium, culture and medium replacement, and obtaining dopaminergic progenitor cells.

[0019] After adding phosphate buffered saline (PBS) to a six-well plate, laminin 111 was added to a concentration of 1-2 μg / cm 2 After fully mixing, place in a 37°C, 5% CO2 incubator overnight for coating the day before use. When using, remove the PBS and directly add differentiation medium. Do not blow the bottom of the culture plate where the coating is located.

[0020] When the density of neural stem cells grew to 60-70% and there was no necrosis or darkening in the center of the neurospheres, the neurospheres were digested with Accutase and seeded on pre-coated culture plates at a certain density;

[0021] The medium was changed to dopaminergic progenitor cell induction differentiation medium, which contained 50% DMEM / F12, 50% Neurobasal, 1% N2, 2% B27, 1% NEAA, 1% GlutaMAX, 300 ng / mL SHH, 100 ng / mL FGF8b and Y27632. Y27632 was removed after 24 hours of culture, and the culture was continued for 10 days, with the medium changed every other day.

[0022] As a preferred technical solution of the present invention, the induction and maturation of dopaminergic neuron cells mainly includes: replacing the maturation culture medium, culturing and changing the medium and neuron identification.

[0023] The culture medium was replaced with dopaminergic neuron maturation medium preheated at 37°C, which included neuron basal medium Neurobasal, 2% B27, 1% NEAA, 1% GlutaMAX, 0.5 mM cAMP, 0.2 mM AAA, 10 μM DAPT, 1 ng / mL TGF-βIII, 20 ng / mL GDNF, 20 ng / mL BDNF, and non-adherent cells and dead cells were removed;

[0024] Culture in a 37°C, 5% CO2 incubator for 7-14 days, replacing fresh culture medium every 3 days;

[0025] The obtained dopaminergic neurons were verified by immunocytochemical fluorescence detection to detect the expression of dopaminergic neuron markers (TH).

[0026] (III) Beneficial effects

[0027] Compared with the prior art, the present invention provides an efficient scheme for extracting rat neural stem cells and directing their differentiation into dopaminergic neurons, which has the following beneficial effects:

[0028] This highly efficient rat neural stem cell extraction and directed differentiation into dopaminergic neurons protocol optimizes culture medium components and differentiation induction factors, improves the purity of rat NSCs extraction and the efficiency of directed differentiation into dopaminergic neurons, and increases the number of target cells, thereby meeting experimental and therapeutic needs.

[0029] This highly efficient protocol for extracting rat neural stem cells and directing their differentiation into dopaminergic neurons achieves stability and consistency in NSCs differentiation by precisely regulating key factors in the differentiation process (signaling pathways, induction timing, and concentration), ensuring experimental repeatability and reliability, and laying the foundation for subsequent research.

[0030] This highly efficient program for extracting rat neural stem cells and directing their differentiation into dopaminergic neurons significantly reduces the generation of non-target cells by adopting an optimized differentiation and maturation protocol, ensuring the acquisition of high-purity dopaminergic neurons, thereby improving the safety and efficacy of cell transplantation therapy.

[0031] This highly efficient program for extracting rat neural stem cells and directing their differentiation into dopaminergic neurons improves the adaptability of dopaminergic neurons in the transplantation environment and enhances their survival rate by improving the differentiation and culture process, thus providing a stable cell source for subsequent cell transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a schematic diagram of the scheme of the present invention; Figure 2 To detect the purity of neural stem cells (NSC) by cell immunofluorescence; Figure 3 For the detection of dopaminergic progenitor cells (TUJ1 / LMX1A); Figure 4 For the detection of dopaminergic neurons (TH). DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] In the description of the present invention, it should be understood that the terms "center", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship are based on the orientation or position relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0035] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a direct connection, or an indirect connection through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0036] See also Figure 1 , a highly efficient protocol for extracting rat neural stem cells and directing their differentiation into dopaminergic neurons, comprising the following steps:

[0037] Extraction and purification of S1 rat neural stem cells:

[0038] Under a microscope, the brain tissue of the fetal rat hippocampus was dissected and separated and placed in a pre-cooled D-Hank's balanced salt solution. After being fully chopped up with ophthalmic scissors, the tissue was filtered through a 70um filter. After being gently blown three times, the neural stem cells and brain tissue were mechanically separated. After centrifugation at 500g for 3 minutes, the cells were placed in a neural stem cell-specific culture medium prepared with DMEM / F12 (Gibco, 11320033) + 2% B27 (Gibco, 12587010) + 20ng / mL bFGF (R&D, 3339-FB) + 20ng / mL EGF (R&D, 2028-EG) and incubated at 37°C and 5% CO2. After 12 hours, the suspended cell mass was aspirated and centrifuged at 400g for 5 minutes before culture to remove other adherent cells except neural stem cells. After 24 hours, the suspended cell mass was gently blown to single cells and fresh culture medium was replaced for continued culture. After 2 days, the aggregated suspended neural cell mass was gently blown and the medium was replaced, and the attached cells, dead cells and their culture medium were discarded. After 3 days, the cells showed that neural stem cells aggregated into spheres. After that, the suspended neurospheres were continuously aspirated for digestion and amplification culture to improve the purity of neural stem cells. The purity of neural stem cell markers (Nestin) was identified by cell immunofluorescence. The second-generation cells after digestion and purification were all single neural stem cells and neurospheres aggregated from neural stem cells.

[0039] In this embodiment, the brain tissue of the rat hippocampus is separated by mechanical separation, and all subsequent operations need to be performed on ice or in a pre-cooled culture medium to prevent cell apoptosis. Use sterile ophthalmic scissors to fully cut into pieces to a 70um filter, and gently blow and beat 3 times to obtain a cell suspension containing neural stem cells after filtering. All operations are kept aseptically, and the blowing process should be slowly and gently blown. The number of blowing should not be too much or too fast. The isolated neural stem cells are susceptible to physical damage and cause the death of the extracted cells. After 12 hours of mixed culture, the aggregated suspended cell mass is continued to be gently blown, so as to separate the cell mass containing the neural stem cell from other attached neural cells, and prevent the attached cells from inducing the neural stem cell mass to perform non-directional differentiation. After 24 hours, the suspended cells are resuspended in a special culture medium for neural stem cells containing growth factors and suspended and cultured in a 37°C, 5% CO2 incubator to obtain a large number of neurospheres aggregated by neural stem cells. The different sources of the formulation components of the special culture medium for neural stem cells can lead to different growth rates of neural stem cells. The culture components in this operation can keep the neural stem cells growing at a high rate. Cell immunofluorescence was used to identify the purity of stem cells in neurospheres. Figure 2 shown.

[0040] It should be noted that this protocol uses a mechanical separation method to obtain brain tissue from the hippocampus. Viable neural stem cells can be obtained after gentle blowing and 70um filtration, and then a large number of neurospheres composed of neural stem cells can be obtained simply by changing the fluid.

[0041] Induction of differentiation of S2 dopaminergic progenitor cells:

[0042] Use phosphate buffered saline (PBS) to dilute laminin 111 (Biolaminin) to 10 μg / mL; place the diluted Laminin 111 solution in a six-well plate and incubate at 37°C for pre-coating, so that the coating concentration is 1-2 ug / cm 2 The coating time is overnight before use; when the neural stem cells grow to a concentration of 60-70%, the dopaminergic neurons can be induced. Accutase enzyme is used to digest the neurospheres composed of a large number of neural stem cells, and the cells are counted at 1.0×10 4 / cm 2 The density of the cells was seeded on laminin (Laminin, 1-2ug / cm 2 ) on a pre-coated six-well plate, incubate at 37°C, 5% CO2 for 2 hours, then change to dopaminergic progenitor cell induction differentiation medium and induce differentiation culture for 24 hours; the dopaminergic progenitor cell induction differentiation medium on day 0 consists of 50% DMEM / F12+50% Neurobasal+1% N2+2% B27+1% NEAA+1% GlutaMAX+300ng / mL SHH+100ng / mL FGF8b+10uMY27632, remove Y27632 after 24 hours and continue to culture, change the medium every other day, the volume of the culture medium on the 2nd, 4th, 6th, 8th and 10th days is 2ml, 2.5ml, 3ml, 3.5ml and 4ml respectively, a large number of dopaminergic progenitor cells can be obtained after the 10th day, which can further mature into dopaminergic neurons.

[0043] In this embodiment, when laminin 111 is used for pre-coating, PBS should be added first and then laminin 111. Pre-mixing in a centrifuge tube before coating will cause laminin to adhere to the tube wall, resulting in a coating concentration that is too low. In addition, laminin 111 should use the Biolaminin source in this scheme. Other sources may also lead to poor coating effects, thereby affecting the differentiation effect. The coating time is overnight coating 24 hours before use. The 2-hour rapid coating method affects the coating effect. The neural stem cells for induced differentiation are digested from transparent neurospheres without necrosis in the central area. Neural stem cells that adhere to the wall and in the central dark area are not suitable for induced differentiation. High-purity neural stem cells directly affect the induced differentiation direction and the purity of differentiated cells. The density of neural stem cell plating before induction of differentiation needs to be controlled. In a six-well plate, it should be less than 1.0×10 4 / cm 2 The differentiation speed of neural stem cells with a density lower than 100 μg / cm2 is slow, and differentiation is easily affected by the environment. The growth rate of neural stem cells with a density higher than 100 μg / cm2 is fast, and the cells have overproliferated before they are fully differentiated, which results in the inability of the cells to complete the whole differentiation process, thus affecting the differentiation efficiency. The prepared dopaminergic progenitor cell induction differentiation medium is mainly composed of sound hysteresis factor (SHH), fibroblast growth factor 8b (FGF8b), etc. The differentiation concentration of key factors is fixed. Too low a concentration leads to incomplete differentiation, and too high a concentration causes the differentiation direction to deviate and cause differentiation failure. Under the operation of this scheme, the differentiation is induced and cultured for 10 days, and the medium is changed every other day to obtain high-purity dopaminergic progenitor cells, which can further mature into dopaminergic neurons. Figure 3 For detection of dopaminergic progenitor cells (TUJ1 / LMX1A).

[0044] It should be noted that this differentiation scheme has high differentiation purity, short differentiation cycle, simple operation and low cost.

[0045] Induction and maturation of S3 dopaminergic neurons:

[0046] The dopaminergic neuron maturation medium is used instead, and non-adherent cells and dead cells are removed; the cells are cultured in a 37° C., 5% CO 2 incubator for 7-14 days, and the medium is changed every 3 days; thus, high-purity dopaminergic neurons can be obtained.

[0047] On day 10, differentiated dopaminergic progenitor cells were digested with Accutase enzyme and plated at 1.5 × 10 5 / cm 2 The cells were inoculated into six-well plates pre-coated with laminin at a density of 2-4 ug / cm. The laminin coating method and time were as described above. 2The dopaminergic maturation induction medium consists of Neurobasal + 2% B27 + 1% NEAA + 1% GlutaMAX + 0.5mMcAMP + 0.2mMAA + 10μMDAPT + 1ng / mLTGF-βIII + 20ng / mLGDNF + 20ng / mLBDNF. After culturing for 1 week, mature dopaminergic neurons can be obtained. Immunocytochemical fluorescence detection of dopaminergic neuron markers (TH) was performed to observe protein expression and maturation. Figure 4 shown.

[0048] In this embodiment, the culture medium is mainly composed of a neural cell culture medium (Neurobasal) supplemented with B27, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), transforming growth factor-β3 (TGFβ3), ascorbic acid (AA), cyclic adenosine monophosphate (cAMP) and DAPT, and high-purity dopaminergic neurons can be obtained by culturing for more than one week.

[0049] The beneficial effects of the above embodiment are:

[0050] 1. High efficiency and controllability: The present invention provides an optimized differentiation induction scheme, which can significantly improve the differentiation efficiency and purity of dopaminergic neurons, which is of great significance for large-scale cell transplantation therapy.

[0051] 2. Highly targeted: Choosing rats as the research subjects fills the gap between existing mouse and human research, provides a reliable cell source for related research on rat models, and enhances the clinical transformation potential of the experiment.

[0052] 3. Promotion of basic research: This invention not only has clinical application value, but will also provide new research ideas for the field of neurobiology and promote a deeper understanding of neural stem cells and their differentiation mechanisms.

[0053] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An efficient method for extracting rat neural stem cells and directing their differentiation into dopaminergic neurons, characterized in that: The following steps are involved: (1) Extraction and purification of rat neural stem cells; (2) Induced differentiation of dopaminergic progenitor cells; (3) Induced maturation of dopaminergic neuronal cells.

2. The method of claim 1 for efficiently extracting rat neural stem cells and directing their differentiation into dopaminergic neurons, characterized in that: The extraction and purification of rat neural stem cells mainly includes: micro-dissection and separation of hippocampal brain tissue, shearing and filtering, mechanical separation and centrifugation, cultivation and purification, and purity identification.

3. The method of claim 2 for efficiently extracting rat neural stem cells and directing their differentiation into dopaminergic neurons, characterized in that: During the extraction and purification process of the rat neural stem cells, the neurospheres growing in suspension are regularly aspirated for digestion, expansion and culture to improve the purity of the neural stem cells.

4. The method of claim 1 for efficiently extracting rat neural stem cells and directing their differentiation into dopaminergic neurons, characterized in that: The induction differentiation of dopaminergic progenitor cells mainly includes: pre-coating of culture plates, cell inoculation and differentiation induction culture medium, culture and medium replacement, and obtaining dopaminergic progenitor cells.

5. The method of claim 4 for efficiently extracting rat neural stem cells and directing their differentiation into dopaminergic neurons, characterized in that: The dopaminergic progenitor cell differentiation induction medium also contains ROCK inhibitor Y27632, which is used at the beginning of the culture and removed after 24 hours; The culture plate is pre-coated with laminin (Laminin 111) at a concentration of 1 μg / cm2, and pre-coating is performed by adding pre-heated PBS at 37°C and 5% CO2. The coating temperature is maintained before use, and the coating time is overnight.

6. The method of claim 1 for efficiently extracting rat neural stem cells and directing their differentiation into dopaminergic neurons, characterized in that: The induction and maturation of dopaminergic neuron cells mainly includes: replacing maturation culture medium, culturing and changing medium, and identifying dopaminergic neurons.

7. The method of claim 6 for efficiently extracting rat neural stem cells and directing their differentiation into dopaminergic neurons, characterized in that: The identification of the neural stem cells and dopaminergic neurons was performed by cell immunofluorescence detection.

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