CN110982788A

CN110982788A - Method for inducing transdifferentiation of satellite glial cells into neurons

Info

Publication number: CN110982788A
Application number: CN201911354423.2A
Authority: CN
Inventors: 李力燕; 马微; 郭建辉; 杨金伟; 王先斌; 刘矿嫔; 刘洁; 刘伟; 臧成昊; 吴朕; 梁宇; 李春艳
Original assignee: Kunming Medical University
Current assignee: Kunming Medical University
Priority date: 2019-12-25
Filing date: 2019-12-25
Publication date: 2020-04-10
Anticipated expiration: 2039-12-25
Also published as: CN110982788B

Abstract

The invention discloses a method for inducing transdifferentiation of satellite glial cells into neurons, which comprises the following steps: and (3) carrying out induction culture on the satellite glial cells after subculture in an induction culture solution containing Anti-probBDNF for 3-7 days to obtain the neurons. The invention can inhibit endogenous proBDNF to induce the differentiation of satellite glial cells to neuron phenotype under physiological conditions (without exogenous gene transfection). The invention provides a new theoretical support for the generation of peripheral nervous system, and finds a new mode of peripheral neurogenesis, namely the direct transdifferentiation of glial cells into neurons. Provides a new theoretical basis for the research of peripheral nerve development and injury repair treatment, and the method of the invention has simpler operation and higher transdifferentiation rate.

Description

Method for inducing transdifferentiation of satellite glial cells into neurons

Technical Field

The invention relates to the field of cell transdifferentiation, in particular to a method for inducing satellite glial cells to transdifferentiate into neurons.

Background

The Nervous System (NS), including the Central Nervous System (CNS) and the Peripheral Nervous System (PNS), is the system in the body that is the most important system for the body to play a leading role in the regulation of physiological functional activities. The nervous system is complex in structure and function, and still has many unbolved puzzles. Therefore, development, damage and repair of the nervous system have been one of the hot spots in the field of medical research.

In the central nervous system, damage to nerve cells and repair after apoptosis are mainly induced and differentiated into nerve cells by Neural Stem Cells (NSCs) to replace nerve cells at the damaged site. The neurogenesis phenomenon exists in the central nervous system of adult mammals, the result and importance of which is a well-recognized medical research view, and plays a key role in the cognitive functions such as brain plasticity, learning and memory and the like and in injury repair. Then, is there a neurogenesis phenomenon similar to that of the central nervous system in the peripheral nervous system? Studies have found that neurogenesis or increased neuronal numbers are also present in the peripheral nervous system, but are less well recognized.

The Dorsal Root Ganglion (DRG) is located in the foramen between two vertebrae, belongs to the peripheral nervous system, is a transfer site for peripheral afferent and efferent nerves, is a place where primary sensory neurons gather, and is a primary afferent neuron for somatosensory sensation. Peripheral nerve diseases or injuries can cause the reduction of dorsal root ganglion neurons, the disturbance of the connection between neurons, the formation of traumatic neuroma, etc., cause abnormal sensory conduction, and cause neuropathic pain. Neuropathic pain is a difficult-to-cure chronic pain state caused by damage to the nervous system. After the injury occurs, not only neuropathic pain is caused, but also a nerve repair effect and a nerve regeneration process exist, and a certain relation exists between the neuropathic pain and the nerve repair regeneration, so that the influence on the nerve repair and the nerve regeneration must be considered while a new treatment method for the nerve injury is explored.

Satellite Glial Cells (SGCs) are a group of Cells surrounding sensory neurons in the dorsal root ganglia, have the same role as astrocytes in the central nervous system, are important Glial Cells in the peripheral nervous system, and are widely distributed in the Dorsal Root Ganglia (DRG) and Trigeminal Ganglia (TG) to support, protect, and nourish neurons. It was originally thought that the satellite glial cells provided structural support, trophic support, and thus promoted the growth of neurons.

proBDNF (precusor for broad-derived neurotrophic factor) is a precursor form of mature brain-derived neurotrophic factor, which cleaves to form mature BDNF. The precursor of the brain-derived neurotrophic factor not only can be used as the precursor form of the brain-derived neurotrophic factor, but also can be synaptically secreted to the extracellular region by nerve cells, and has different functions with the brain-derived neurotrophic factor. proBDNF is involved in neuronal death after nerve injury and nervous system diseases such as neurodegeneration, and the action mode and conduction pathway of proBDNF are different from those of mature BDNF.

At present, there is no literature report that the satellite glial cells can transdifferentiate into neurons. In the invention, after a stable, efficient and high-purity DRG-SGCs in-vitro primary culture model is successfully established in the early stage, the transdifferentiation process of the satellite glue quality cells is researched, and the neurons are successfully obtained.

Disclosure of Invention

The invention provides a method for inducing the transdifferentiation of satellite glial cells into neurons, which adopts Anti-proBDNF (proBDNF antiserum or proBDNF antibody) to induce the transformation of Satellite Glial Cells (SGCs) from Dorsal Root Ganglia (DRG) to the neurons, provides a new theoretical support for the generation of peripheral nervous system, and discovers a new mode of peripheral neurogenesis: directly transdifferentiated into neurons by glial cells. Provides a new theoretical basis for the research of the development of peripheral nerves and the repair and treatment of injuries.

In order to solve the technical problems, the technical scheme of the invention is as follows:

a method of inducing transdifferentiation of satellite glial cells into neurons, the method operating as follows: and (3) carrying out induction culture on the satellite glial cells after subculture in an induction culture solution containing Anti-probBDNF for 3-7 days to obtain the neurons.

Preferably, the concentration of Anti-probBDNF in the induction culture solution is 3-5 ug/mL.

Preferably, the induction culture solution is prepared by adding 46-48 mL of DMEM/F12, 0.4-1.2 mL of B27 cell culture additive, 0.4-1.2 mL of penicillin-streptomycin mixed solution, 1.8-2.2 mmol/L of L-glutamine, 15-20 mg/mL of BSA, 8-12 μ g/mL of NRG1- β, 22-27 μ g/mL of dexamethasone, 3-7mg/mL of Insulin, 8-12 μ g/mL of T3, 396-404 μ g/mL of T4, 3-5 μ g/mL of Anti-proBDNF and the like in 50mL of DRG-SGCs culture solution, and the like in a cell culture system, wherein the cell culture additive and penicillin-streptomycin mixed solution are prepared by adding 50 × 50 and 100 × required dilution of the reagent, and the reagent is prepared by adding 0.6-100 × 7 mL of penicillin-streptomycin culture solution, and the cell culture solution is prepared by adding the other reagent in culture solution according to the cell culture system, or by adding 0.8-6 × 1.8-6 × 1-100 × 1 mL of the cell culture additive in culture system.

Furthermore, the satellite glial cells are derived from dorsal root ganglia of newborn rats (suckling mice within 24 hours of birth), and are subcultured for 3-5 generations.

Further, the induction culture solution is changed every 1-2 days when the satellite glial cells are transdifferentiated into the neurons.

Further, the induction culture conditions were as follows: in the presence of 4.5-5.5% CO₂The culture is carried out in an incubator at the temperature of 36.5-37.5 ℃ and the humidity of 70-80%.

Preferably, the satellite glial cells after subculture are subjected to induced culture in an induction culture solution containing Anti-probdNF for 3-7 days to obtain neuron-like cells, and the neuron-like cells are continuously cultured in a neuron culture solution for 2-4 days to obtain mature neurons. Since fetal bovine serum-containing media for glial cell culture may not be optimal for neuronal growth and maturation, changing the media to neuronal media was found to promote maturation of neuronal-like cells.

Preferably, the formula of the neuron culture solution is as follows: 0.8-2.2mL of penicillin-streptomycin mixed solution, 0.8-2.2mL of L-glutamine, 0.8-2.2mL of B27 cell culture additive and 93.4-97.6 mL of Neurobasal medium A. The reagent of penicillin-streptomycin mixed solution, L-glutamine and B27 cell culture additive in the neuron culture solution is usually 50X and 100X, 50X and 100X represent the dilution times of the reagent, and the specification of 50X or 100X can be adopted by the person skilled in the art. For example, 1.8-2.2 mL of B27 cell culture additive 50X may be added to 100mL of neuron culture solution, or 0.8-1.2 mL of B27 cell culture additive 100X may be selected according to the purchased specifications, and the penicillin-streptomycin mixture and L-glutamine may be added in this manner.

The invention also provides the neuron obtained by the method, the change of cell morphology is identified by morphological observation and immunofluorescence staining, the electrophysiological properties of each group of cells after induced differentiation are detected by a patch clamp technology, and the induced differentiation cells are further proved to be the neurons.

The invention is characterized in that under the guidance of no existing literature, the application firstly researches the transdifferentiation process of the satellite glial cells from the dorsal root ganglion and successfully transdifferentiates the satellite glial cells to obtain the neurons. Different from the prior transdifferentiation means such as gene editing, the invention inhibits the expression of endogenous paracrine or autocrine proBDNF by adding Anti-proBDNF (Anti-proBDNF serum) into the culture solution, regulates the expression of proBDNF in a cell microenvironment, and induces the transdifferentiation of a satellite glial cell phenotype to a neuron phenotype. The present invention provides a first example of transdifferentiation from SGC into neurons regulated by the autocrine/paracrine regulatory mechanism of physiological factors, which is capable of inducing glial cell differentiation into neurons under physiological conditions (without exogenous gene transfection).

Compared with the prior art, the invention has the following beneficial effects: the invention successfully induces the glial cells to differentiate into neurons under physiological conditions (without exogenous gene transfection), has simpler and more convenient operation, is economic and has higher transdifferentiation rate.

Drawings

FIG. 1 is a graph of the morphology of satellite glial cells at 3, 7, 14 and 21 days of primary culture of dorsal root ganglia;

FIG. 2 is a cell morphology after satellite glial cell subculture;

FIG. 3 shows the positive rates of three markers in satellite glial cells;

FIG. 4 is a graph of the morphological changes of DRG-SGC after treatment with Anti-probBDNF serum at 4 μ g/mL;

FIG. 5 is a graph of immunofluorescence identification of satellite glial cells three days after Anti-probBDNF treatment;

FIG. 6 shows the dual-marker identification of marker-specific markers on the third and seventh days after Anti-probBDNF stem prediction;

FIG. 7 is an electrophysiological characterization of neuronal cells transformed from DRG-SGC;

FIG. 8 is a graph of action potential peak, threshold and duration of cells at various time points in the same group;

FIG. 9 is an Elisa kit for detecting autocrine and expression levels of proBDNF and BDNF in the culture media of satellite glial cells from

groups

1d and 3 d;

fig. 10 is a cell map identifying SGC-specific marker GS and neuron-specific markers Tuj1 and NeuN.

Detailed Description

The technical solutions of the present invention will be described in further detail with reference to the drawings and specific examples, but the present invention is not limited to the following technical solutions.

The following examples used the following reagents DMEM/F12 (1: 1) medium, B-27 additive (50X), diabody (penicillin and streptomycin solution) (50X), BSA (15mg/mL), dexamethasone (25ug/mL), bovine Insulin (5mg/mL) from Biochar corporation, T3(10ug/mL), T4(400ug/mL), NRG- β 1 from abcam, fetal bovine serum, 0.25% pancreatic enzymes from Life Technology, L-lysine, PBS from Genview, Anti-ProBDNF from the presentation of the Proc. professor laboratories, university of south Australia, L-Glutamine (L-Glutamine, 100X), Neurobasal A nerve-based media from Gibco reagent, Inc.

Example 1

Carrying out primary culture on dorsal root ganglion of a newborn SD rat (according to the operation of 201611170545.2 a method for carrying out primary culture on dorsal root ganglion satellite glial cells), and carrying out subculture purification after the satellite glial cells migrate out.

DRG-SGC (dorsal root ganglion-satellite glial cell) Primary, subculture, purification and grouping

After SGCs occupied 70-80% of the dish bottom (approximately 10 days DRG incubation), DRG was transferred to a six-well plate coated with poly D-lysine hydrobromide (PDL) (P7405-5MG, Sigma-Aldrich, MO, USA). SGCs were then digested with 0.25% trypsin-EDTA (25200-056, Gibco life technologies, Invitrogen, CA, USA) for a typical digestion time of 3min depending on the time of DRG culture and the trypsin titer, and when the shedding of the satellite glial cells from the culture plate began, an equal volume of 10% FB was usedS (12657029, Gibco life technologies, Invitrogen, CA, USA) termination of digestion. Repeatedly pumping, collecting suspension, centrifuging for 6 min at 1000prm/min, discarding supernatant, and determining cell concentration as 6 × 10⁵Adding DRG-SGC culture medium, blowing and mixing the cells uniformly, and enabling the cell suspension to be plated into a 24-hole plate for immunocytochemical staining. Place the satellite glial cells plates in 25cm flasks, and place the plates and flasks at 5% CO₂In an incubator (37 ℃). The culture medium was changed every other day, and the morphology and growth state of the cells were observed under an inverted microscope, as shown in FIGS. 1 and 2.

In fig. 1, the migration rate of satellite glial cells is higher as the culture time is prolonged, and the morphology of satellite glial cells is observed when the back root segments are cultured for 3d, 7d, 14d and 21 d. Scale bar 500 μm. In FIG. 2, A: morphology of satellite glial cells emigration from dorsal root ganglion, B: the morphological observation after the passage of the satellite glial cells shows that the cell bodies are full and elliptical, the two poles of the cell bodies are generated with bulges, and the bulges between the cells are mutually connected.

Satellite glial cell after immunofluorescence identification passage and purity thereof

(1) Removing the cultured satellite glial cells from the 24-well plate; the medium in the plate was aspirated off and rinsed 2 times for 3min each with 0.01 mol/LPBS;

(2) soaking and fixing the glass slide in 4% paraformaldehyde solution for 20min to prevent the cells on the glass slide from falling off;

(3) rinsing with 0.01mol/L PBS for 3 times, 10min each time;

(4) sealing 5% sheep serum at room temperature for 1 h;

(5) rinsing with 0.01mol/L PBS for 3 times, 10min each time;

(6) the prepared primary antibody was added and incubated overnight at 4 ℃ in a refrigerator. Selecting proper dilution ratio according to the antibody specification and experimental experience, and adding 5% sheep serum into a negative control group;

(7) the antibody incubation box was taken out and left to stand for 30min to return to room temperature. PBST rinsing for 3 times, 10min each time;

(8) adding fluorescent secondary antibody to incubate for 2h at room temperature, wherein the fluorescent secondary antibody is goat anti-mouse Cy3 and is diluted at a ratio of 1: 1000. Attention is paid to light protection;

(9) PBST rinsing for 3 times, 10min each time;

(10) mounting the chip by using fluorescent mounting agent containing DAPI;

(11) the images are collected under a Nikon 90i fluorescence microscope, and at least 5 pictures with different fields are collected for each tissue under the same multiple.

(12) From each group, 5 pictures at 200-fold magnification in different fields were randomly drawn and the number of positive cells was counted by the technical tool of the PS tool.

After 3-5 passages, the cells were cultured for 3 days and then identified by labeling the three SGC-specific markers GS, GFAP and S100 β. all cultured cells showed GS, GFAP and S100 β immunoreactivity as examined by fluorescence microscopy as described previously. DAPI showed blue. therefore, the cultured cells were confirmed to be SGC expressing the three SGC-specific markers GS with SGAP and S100 β having SGC positivity rates of 97.10%, 67.69% and 91.66%, respectively, as shown in FIG. 3.

Identified Satellite Glial Cells (SGCs) are divided into two major groups: control group (SGCs group), Anti-probBDNF dried group, SGC group using the cell amount in one flask as one group without any intervention, an Anti-probdNF induction culture solution with a concentration of 4ug/mL is used for interfering satellite glial cells for 12 hours to form an Anti-probdNF0.5d group, an Anti-probdNF induction culture solution with a concentration of 4ug/mL is used for interfering satellite glial cells for 24 hours to form an Anti-probdNF1d group, an Anti-probdNF induction culture solution with a concentration of 4ug/mL is used for interfering satellite glial cells for 3 days to form an Anti-probdNF3d group, and an Anti-probdNF induction culture solution with a concentration of 4ug/mL is used for interfering satellite glial cells for 7 days to form an Anti-probdNF7d group, after the Anti-probdNF induction culture solution with the concentration of 4ug/mL intervenes satellite glial cells for 7 days, the cells are changed into neuron culture solution to be cultured for 3 days to form an Anti-probdNF7d + NC3d group. The total experimental grouping is therefore shown in table 1.

TABLE 1 Experimental grouping

Six control groups without Anti-probDNF intervention were cultured using DRG-SGC medium alone, the formulation of which is shown in Table 2. The Anti-probDNF dry pre-culture group is cultured in an induction culture solution, and Anti-probDNF is added into the induction culture solution on the basis of the DRG-SGC culture solution in the table 2, and the concentration is 4 ug/mL. The control group and the intervention group were changed every other day, and the culture conditions were 37 deg.C and 5% CO₂Culturing in an incubator.

After each group reached the required intervention time, cells were taken at each time point for the corresponding experiment. The change of the cell morphology is identified by morphological observation and immunofluorescence staining. The patch clamp technology detects the electrophysiological properties of each group of cells after induced differentiation.

TABLE 2 DRG-SGC culture fluid formulation

Intervention and identification of Anti-probBDNF on satellite glial cells

Morphological observation of Anti-probdNF after intervention on satellite glial cells

Phase difference images are observed under an inverted microscope at all time points, and the result shows that the satellite glial cells before induction are round and bright, and the process is short. After Anti-proBDNF was added to the culture for 3 days, the size and morphology of some cells changed significantly, the cells became compact, the cell bodies increased, the cell bodies became round and bright, the cells showed polygonal shapes, the protrusions showed multi-polar behavior, and the cell protrusions were connected to each other to form a network structure similar to neurons, as shown in fig. 4. FIG. 4A shows the morphological characteristics of DRG-SGC when cultured in normal medium for 7 d; in fig. 4, B-D represent morphological features of DRG-SGC after Anti-proBDNF treatment for 3D, 5D, and 7D days, respectively, and the scale bar is 200 μm in fig. 4.

Immunofluorescence identification of Anti-probDNNF after intervention on satellite glial cells

After 3 days of Anti-proBDNF treatment, it was identified by immunofluorescence as shown in fig. 5. SGC was labeled with Nestin, a neural stem cell-specific marker, and the results showed that cells cultured with Anti-probBDNF treatment all showed Nestin immunoreactivity and DAPI was shown to be blue.

In fig. 5 (a): the cell mass collected by migration from the dorsal root ganglion was labeled GS (green). (B) The method comprises the following steps DAPI marks the nuclei, showing blue. (C) The method comprises the following steps Labeled as neural stem cell specific marker (Nestin) (red) (D): and combining ABC with images (merge). Scale bar 100 μm. Abbreviations: DAPI, 4 ', 6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole). The figure 5 results suggest: SGCs are multipotent progenitor cells whose differentiation is influenced by environmental signals and can differentiate into mature neural cells or glial cells where appropriate.

Detection by labelling SGC-specific markers S100 β and P75 on days three and seven after Anti-probBDNF Stem prognosis^NTRThe Anti-proBDNF intervention on the third day in the experiment, the detection of the satellite glial cell, a positive rate of 8.7%, and the Anti-proBDNF intervention on the seventh day, the CGRP positive rate was significantly increased and 42%, as shown in table 3 and fig. 6.

TABLE 3 Positive rate of CGRP markers

The electrophysiological operation of patch clamp technique is as follows:

(1) preparation of glass microelectrode

1) The glass microelectrode is prepared by a vertical two-step drawing method (the first drawing temperature is 58.1 ℃, and the second drawing temperature is 48.0 ℃), and the drawn electrode is thermally polished by a polisher to eliminate residual burrs so as to be beneficial to forming giant barrier sealing. The water inlet impedance of the polished electrode is about 8-14M.

(2) Patch clamp recording procedure

1) Taking cells: the slide with the attached cells was removed from the medium, placed in a new dish with extracellular fluid, and the dish was placed on the microscope stage. Extracellular fluid (mM) used in this experiment: 144 NaCl, 2.5 KCl, 2 CaCl₂，0.5 MgCl₂5 HEPES, 10 glucose, adjusted to pH 7.4 with NaOH.

2) The Patch process: and selecting the DRG-SGC with better state under the mirror. The glass microelectrode filled with the electrode solution is fixed on an electrode holder, and at the moment, the circuit is in an open circuit state and has high resistance. And (3) applying positive pressure to the electrode by using an injector, putting the electrode into water by using an electric micromanipulator, switching on a circuit at the moment, displaying the impedance of the electrode by using software, observing the impedance of the electrode to be 8-14M, and clicking an Auto key in the liquid connection potential compensation to perform liquid connection potential compensation. Finding the electrode tip under a microscope, moving the electrode to the vicinity of a target cell by using a micromanipulator, adjusting the position of the electrode, enabling the electrode to be just pressed right above the cell and slightly deflected to the direction of an electrode holder, observing the contact of the cell surface and the electrode under a microscope, then increasing the resistance of the electrode, removing the positive pressure and applying slight negative pressure until the resistance is increased to more than 1G to form high-resistance sealing. Clicking the "Auto" key in "C-Fast" automatically performs Fast capacitance compensation. And applying negative pressure into the microelectrode again to break the cell membrane below the tip of the electrode, clicking the Auto key in the C-Slow to perform Slow capacitance compensation. Setting the cell clamping potential between-70 mV to-90 mV, adopting an 'I-CLAMP' mode, wherein the signal sampling frequency is 5kHz, the low-pass filtering frequency is 1kHz, and carrying out whole-cell recording according to the preset protocol.

(3) Recording process

1) Current clamp mode: the recording mode is set as current clamp, the cell injection current is 0pA, the Resting Membrane Potential (RMP) of the satellite glial cells is recorded at the moment, and then the conditions of the satellite glial cell potential of the control group and the intervention group are observed.

Using neurons from the cerebral cortex of SD ratsThe action potential threshold value, the duration and the amplitude of the SD rat cerebral cortex isolated culture neuron are respectively positive control group

And

in the absence of Anti-proBDNF induction, no action potentials were detected in DRG-SGC. The magnitude of the action potential was detected at day 7 of Anti-proBDNF induction treatment and SGCs cultured with neuronal media groups were similar to the neuronal groups, see figure 7.

In FIG. 7A: electrophysiological properties of DRG-SGC cultured for 7 days. B, C and D: Anti-proBDNF intervenes in the electrophysiological properties of cells induced to differentiate after 1, 3 and 7 days. E: after the cells are cultured in SGC culture medium added with Anti-probBDNF for 7 days, the cells are replaced by neuron culture solution to culture the cells for 3 days to induce differentiation. F: electrophysiological measurements of neurons from the cerebral cortex of SD rats served as a control group. In the same group, the peak, threshold and duration of action potential of the cells at each time point are shown in graphs G, H and I of fig. 8, respectively,. P < 0.05 relative to the previous time point (e.g. action potential threshold for 7d versus 3 d); the delta P for the SGC, 3d, 7d and SGC groups cultured in neuronal medium and neuronal groups was < 0.05.

In the Anti-probBDNF induction 1, 3 and 7 day groups, the action potential threshold was

And

the action potential amplitude is respectively

And

the action potential time is respectively

And

after the induced DRB-SGC was cultured in the neuron medium for 3 days, the dynamic potential threshold, amplitude and amplitude were respectively shown as

And

it was found that neuronal action potentials were detected similar after 3 days of Anti-proBDNF treatment. Electrophysiological experiments show that the neuron-like cells induced by proBDNF to SGC transdifferentiate can generate action potentials similar to neurons, and have no significant difference with positive controls, and further prove that Anti-proBDNF induces SGC to differentiate into neurons.

Elisa detection of protein expression in cell culture solution

The presence and expression level of proBDNF and BDNF in the culture medium of the 1d and 3d groups of cells were determined by Elisa kit. As shown by the A result in FIG. 9, the expression of the probDNNF protein in the culture medium was significantly reduced in both the Anti-probDNNF 1d group and the Anti-probDNNF 3d group (P < 0.05) compared to the control group. As shown by the B result in FIG. 9, the expression of BDNF protein in the Anti-probBDNF 1d group in the culture medium was significantly reduced compared with the control group. While the expression of BDNF protein in Anti-proBDNF3d group in the culture medium is obviously up-regulated, the expression of BDNF protein in 3d normal group is obviously reduced in the culture medium compared with 1 d. (P < 0.05). It was shown that the expression level of BDNF/proBDNF in the extracellular environment had an effect on the transdifferentiation of DRG-SGC. Satellite glial cells respond to endogenous proBDNF and rely on proBDNF to maintain the satellite glial cell phenotype. Anti-proBDNF stem prognosis, mature BDNF signaling may play a dominant role and promote the transformation from a satellite glial phenotype to a neuronal phenotype.

EXAMPLE 2 characterization of DRG-SGCs after replacement of SGC Medium with neuronal Medium

After intervention of DRG-SGCs 7d with Anti-probNFanti, the SGC medium was replaced with neuronal medium, the formulation of which is shown in the table below.

TABLE 4 preparation of neuronal Medium

After 3 days of SGC culture in neuronal medium, the cell morphology was similar to neurons. Cells were identified by labeling the SGC-specific marker GS and the neuron-specific markers Tuj1 and NeuN by inverted fluorescence microscopy, as shown in fig. 10. All cultured cells showed satellite glial cell specific marker GS positive (green in fig. 10A, B), neuronal specific marker NeuN positive (red in fig. 10C) and Tuji positive (red in fig. 10D). Co-expression of GS and Tuj1 (yellow in fig. 10H), as well as GS and NeuN (yellow in fig. 10G) was observed in the cells. E, F represent nuclear 4 ', 6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole, DAPI) staining.

Example 3 selection of optimal intervention dose of Anti-proBDNF

After grouping the satellite glial cells, interfering the satellite glial cells with induction culture solutions with different Anti-probdNF concentrations, and selecting the optimal Anti-probdNF interference concentration through cell morphology and immune cell fluorescence chemical detection. The Anti-probdNF was used at 0ug/mL, 2ug/mL, 4ug/mL, 6ug/mL, 8ug/mL and 10ug/mL as gradient concentrations, and the cells were observed under an inverted microscope at different time points after the satellite glial cells were dried by Anti-probdNF at different concentrations, and the status of the cells was identified by immunofluorescence cytochemical assay.

During the anti-proBDNF serum concentration gradient treatment: 6 mu g/mL, 8 mu g/mL and 10 mu g/mL of the anti-proBDNF serogroup, all cells die on the 3 rd day of addition; 2. mu.g/mL of anti-proBDNF serogroup did not have a positive Tuj1 result for a long period of time, while 4. mu.g/mL of anti-proBDNF serogroup could detect a positive expression of Tuj1 on the third day of addition, so 4. mu.g/mL was chosen as the preferred concentration for anti-proBDNF serum treatment of cells.

Claims

1. A method of inducing transdifferentiation of a satellite glial cell to a neuron, the method operated as follows: and (3) carrying out induction culture on the satellite glial cells after subculture in an induction culture solution containing Anti-probBDNF for 3-7 days to obtain the neurons.

2. The method for inducing transdifferentiation of glial cells into neurons according to claim 1, wherein the concentration of Anti-proBDNF in the induction medium is 3-5 μ g/mL.

3. The method for inducing transdifferentiation of glial cells into neurons according to claim 1, wherein the formulation of the induction culture medium is that DMEM/F12 is 46-48 mL, B27 cell culture additive is 0.4-1.2 mL, penicillin-streptomycin mixture is 0.4-1.2 mL, L-glutamine is 1.8-2.2 mmol/L, BSA is 15-20 mg/mL, NRG1- β 1 is 8-12 μ g/mL, dexamethasone is 22-27 μ g/mL, Insulin is 3-7mg/mL, T3 is 8-12 μ g/mL, T4 is 396-404 μ g/mL, and Anpro BDNF is 3-5 μ g/mL per 50mL of DRG-SGCs culture medium.

4. The method for inducing transdifferentiation of satellite glial cells into neurons according to claim 1, wherein the satellite glial cells are derived from dorsal root ganglia of newborn rats and subcultured for 3-5 generations.

5. The method for inducing the transdifferentiation of satellite glial cells into neurons according to claim 1, wherein the induction culture medium is changed every 1-2 days when the satellite glial cells transdifferentiate into neurons.

6. The method for inducing transdifferentiation of glial cells from satellites into neurons according to claim 1, wherein the inducing culture conditions are as follows: in the presence of 4.5-5.5% CO₂The temperature of the medium is 36.5 to37.5 ℃ and the humidity is 70-80 percent.

7. The method for inducing the transdifferentiation of satellite glial cells into neurons according to claim 1, wherein the satellite glial cells after subculture are induced and cultured in an inducing culture solution containing Anti-proBDNF for 3-7 days to induce and differentiate into neuron-like cells, and the mature neurons are obtained after the further culture in the neuron culture solution for 2-4 days.

8. The method for inducing transdifferentiation of glial cells from satellites into neurons according to claim 7, wherein the neuron culture fluid is formulated as follows: 0.8-2.2mL of penicillin-streptomycin mixed solution, 0.8-2.2mL of L-glutamine, 0.8-2.2mL of B27 cell culture additive and 93.4-97.6 mL of Neurobasal medium A.

9. A neuron obtainable by a method according to any one of claims 1 to 8.