CN113151174B - Application of escitalopram in promoting neural stem cell to express BDNF - Google Patents

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of escitalopram in promoting expression of neural stem cells BDNF (brain derived neurotrophic factor), wherein the escitalopram with the concentration of 20ng/ml is added for intervening and inducing the neural stem cells derived from hippocampus for 7 days. The invention discloses a novel application of escitalopram in promoting the expression of BDNF (neural stem cell) by neural stem cells.

Description

Application of escitalopram in promoting neural stem cell to express BDNF

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of escitalopram in promoting neural stem cell expression BDNF.

Background

The brain-derived neurotrophic factor (BDNF) is a neurotrophic factor, is mainly synthesized in neurons at hippocampus, amygdala, midbrain limbus, striatum and substantia nigra, is transported by axon, acts on target tissues through a tyrosine kinase receptor B to play a role, and the parts are closely related to the cognitive function, long-term enhancement, learning and memory of nerves; plays an important role in the growth, development, survival and differentiation processes of neurons of the nervous system. In depression and stress-related affective disorders, the expression of hippocampal BDNF and BDNF concentration in plasma are impaired, and although the intrinsic mechanism of hippocampal injury is not yet elucidated at present, numerous studies indicate that BDNF and its conduction pathway play an important role in injury and repair of hippocampal neurons and are closely related to depressive disorders. Escitalopram (ESC) is a novel selective 5-hydroxytryptamine (5-HT) reuptake inhibitor (SSRIs) antidepressant, and unlike other SSRIs, ESC selectively binds to presynaptic membrane 5-HT transporter binding sites, and also removes interference of the dextroisomer of citalopram contained in racemic citalopram on its levorotatory enantiomer and presynaptic membrane allosteric sites, and increases the release of 5-HT, thereby exerting antidepressant and anxiolytic effects. A large number of research results prove that the SSRI antidepressant can influence multiple aspects of proliferation, differentiation and maturation of nerve cells, dendritic growth, axon formation and the like in the neurogenesis process.

It is currently believed that Neural Stem Cells (NSCs) are present in the subcentricular zone and the infragranular zone of the dentate gyrus of the hippocampus in the adult mammalian brain, and that the proliferation of these NSCs is involved in adult neurogenesis, which has an ameliorating effect on depression. NSCs have self-renewal, can differentiate into neurons and glial cells under certain conditions, and the property can make the NSCs supplement the loss of the neurons and glial cells after the central nervous system is damaged, so that a functional neural circuit is formed, and secondly, after the central nervous system is damaged, the NSCs can also secrete a plurality of trophic factors (such as BDNF, NT-3 and the like) to provide a good living environment for nerve and axon regeneration, so that the neural stem cells are seed cells for transplantation and repair after the central nervous system. According to previous researches, ESC can positively regulate survival and proliferation of rat hippocampal NSCs, and has the effects of promoting differentiation of the rat hippocampal NSCs to neurons and oligodendroglia and inhibiting differentiation to astrocytes. However, whether ESC regulates BDNF expression from rat hippocampal-derived NSCs has not been reported.

Disclosure of Invention

The invention provides an application of escitalopram in promoting neural stem cell to express BDNF in order to solve the problems in the prior art,

the invention provides an application of escitalopram in promoting neural stem cell expression BDNF, and escitalopram regulates the neural stem cell expression BDNF.

Preferably, the effect of escitalopram in increasing the expression of BDNF in hippocampal-derived neural stem cells of neonatal rats.

Preferably, the method comprises the following steps:

adding escitalopram to intervene and induce the neural stem cells derived from the hippocampus of the newborn rat.

Preferably, the concentration of escitalopram is 20ng/ml.

Preferably, the intervention time is 7 days.

The invention has the beneficial effects that: the invention discloses a pioneering method for applying escitalopram to promoting neural stem cells to express BDNF.

Drawings

FIG. 1 is an image of a third generation neural stem cell under a light microscope at a magnification of 10X with a scale bar of 100 μm;

FIG. 2 is an image of the third generation neural stem cells under a light microscope at a magnification of 20X with a scale bar of 100 μm;

FIG. 3 shows Hoechst immunofluorescent staining of cell nuclei in a control group;

FIG. 4 shows that the control group has positive BDNF staining detected by immunofluorescence staining;

FIG. 5 is an image of FIG. 3 superimposed on FIG. 4;

FIG. 6 shows that ESC intervention groups have Hoechst immunofluorescent staining of cell nuclei

FIG. 7 shows that ESC intervention groups were positive in BDNF staining by immunofluorescence staining detection;

FIG. 8 is an image of the overlay of FIGS. 6 and 7;

FIG. 9 is a bar graph of the effect of ESCs on neural stem cell protein expression;

FIG. 10 is a graph of the effect of ESCs on the relative expression of BNDF mRNA expressed by neural stem cells;

Detailed Description

The invention will be further described with reference to the following drawings and specific embodiments:

the material and the method are as follows:

1. isolation and culture of neural stem cells

Taking out hippocampus of SD rat in 24h under aseptic condition, and placing in pre-cooled DMEM/F12 culture medium; dissecting the blood vessel and meninges under a microscope, putting the separated hippocampal tissue into another precooled DMEM/F12 culture medium, and slowly and gently blowing and beating for 15-20 times to form a single cell suspension; placing in a centrifuge, centrifuging at 1000rpm/min for 5min, discarding supernatant, and adding serum-free neural stem cell culture medium for resuspension; after counting, 3 is multiplied by 10 ⁶ Order of magnitude cells seeded at 25cm ² Placing in a culture flask at 37 deg.C, with a volume fraction of 5% ₂ Culturing in an incubator. Culturing for 3d, supplementing half amount of liquid, collecting neurospheres after 5-7d, digesting with pancreatin to form single cells, counting, and subculturing. Passage 3 (P3) neural stem cells were selected for the experiments. The culture conditions are as follows: DMEM/F12 medium +2% b27+ 20. Mu.g/L basic fibroblast growth factor.

2. Intervention grouping

The experiments were divided into 2 groups: control and ESC intervention groups.

Control group: inoculating P3 neural stem cells into a 6-well plate previously coated with 0.01% polylysine to adhere to the plate, adding to an induction medium (2% by weight of Neurobasal of B27) and culturing for 7 days;

ESC intervention group: inoculating P3 neural stem cells into a 6-well plate previously coated with 0.01% polylysine to allow them to adhere, and culturing for 7 days by adding ESC intervention induction medium (2% by Neurobasal of B27+20 ng/ml ESC);

3. observation of expression of neural stem cell BDNF protein by immunofluorescence staining

After culturing the neural stem cells of the control group and the ESC intervention group for 7d, carrying out immunofluorescence staining on the two groups respectively, and washing the cells of the two groups 3 times by using 0.01mol/L PBS for 5min each time; fixing 4% paraformaldehyde at room temperature for 15min, rinsing with 0.01mol/L PBS for 3 times, 5min each time; sealing goat serum at 37 deg.C for 1h; adding rabbit anti-BDNF antibody, incubating at 4 ℃ and staying overnight; washing with 0.01mol/L PBS for 5min for 3 times; adding fluorescent secondary antibody to goat anti-rabbit TRITC (antibody working concentration 1: 100), incubating at 37 deg.C for 30min in dark, washing with 0.01mol/L PBS for 3 times, each for 5min; staining nuclei by Hoechst for 5min, washing by 0.01mol/L PBS for 3 times, and washing for 5min each time; and (4) observing by a fluorescence microscope and taking a picture. And randomly selecting 5 non-overlapping fields in each group to count the total number of the cells and the number of the BDNF positive cells respectively, and then calculating the average BDNF positive cell ratio of each field.

4. Real-time PCR observation of expression of BDNF mRNA of neural stem cells

Culturing the neural stem cells of the control group and the ESC intervention group for 7 days, and taking 5X 10 ⁶ Extracting total RNA from each cell, and strictly operating according to the instruction of a kit; reverse transcription into cDNA in 20. Mu.l reaction system containing: mu.l of 5 XmiScritHiSpec solution, 2. Mu.l of 10 XmiScrit nucleic mixture, 2. Mu.l of miScript reverse transcription mixture, 2. Mu.l of RNase-free water and 10. Mu.l of template RNA. Beta-actin is used as an internal reference, and an ABI 7500PCR instrument is used for performing Real-time PCR amplification reaction. Reaction conditions are as follows: denaturation at 95 ℃ 15min, denaturation at 94 ℃ 15s, annealing at 58 ℃ 30s, extension at 70 ℃ for 30s, repeating 32 cycles. The sequence of the beta-actin upstream primer is as follows: 5' -CCG CGA GTA CAA CCT TCT TG-3', the sequence of the downstream primer is: 5'-TGA CCC ATA CCC ACC ATC AC-3'; the BDNF upstream primer sequence is as follows: 5'-CAT AAG GAC GCG GAC TTG TAC A-3', the downstream primer sequence is: 5'-AGC AGA GGA GGC TCC AAA GG-3', the experimental results are repeated 3 times, and the results are relatively quantitatively analyzed by a 2-delta Ct method.

5. Statistical method adopts SPSS 21.0 software package to process data, and experimental results are used

Showing that the two groups are compared by adopting a t test, P<A difference of 0.05 is significant.

As a result:

1. neural stem cell culture and morphology

When the primary isolated neural stem cells are observed under an inverted microscope, the cells are in a dispersed single spherical shape and are suspended in a culture medium, and the cells have good refractive index. After 24h of culture, cell pellets of 2-4 cells were formed. Then the cells begin to aggregate to form spherical suspension growth, and the spherical or irregular-shaped small clone neurospheres with 5-7 cells are formed on the 3 rd day of culture; on days 5-7 of culture, neurospheres aggregated into clones from 15-20 cells. The stereo-spherical neural stem cells gradually increase with the increase of the culture days to form the neurospheres with diameters of 50-200 μm and different sizes. After passage, the neural stem cells still grow in a spherical suspension manner, have strong refractivity and clear internal structure, and are shown in figures 1 and 2.

2. Immune fluorescent staining detection of influence of ESC on expression of BDNF protein of neural stem cells

After the neural stem cell balls adhere to the wall for 24 hours, cells in the two groups migrate out of the neural ball under a light microscope, most of the migrated cells are round, the protrusions are not obvious, the outline is clear, the edge is neat, the stereoscopic impression is strong, and halos are arranged around the cells. When the cells are cultured for 5 days, the growth conditions of the two groups of cells are good, the cell nucleuses are uniform in size, plump and obvious in protrusion, and the cells are rarely broken. When the cells are cultured for 7d, the proportion of BDNF positive cells in the neural stem cells is detected by immunofluorescence staining as follows: control group 11.93 ± 1.15%; the ESC intervention group 27.46 +/-2.46%, and the proportion of BDNF expression of the neural stem cells in the ESC intervention group is significantly higher than that in the control group, as shown in FIGS. 3-9.

3. Effect of ESC on BDNF mRNA expression by neural Stem cells

The expression conditions of BDNF of the control group and the ESC intervention group are detected at the mRNA level by adopting Real-time fluorescence quantitative PCR (Real-time PCR), and the result shows that the BDNF expression amount of the ESC intervention group is obviously higher than that of the control group (P < 0.05), and the BDNF expression amounts are shown in the table 1 and the figure 10.

TABLE 1 Effect of ESCs on the relative expression of BDNF mRNA expressed by neural stem cells (n = 9)

In conclusion, 20ng/ml of ESC is added to intervene and induce the hippocampal-derived neural stem cells for 7 days, and the observation shows that the expression quantity of BDNF of the neural stem cells is obviously higher than that of a control group, which suggests that ESC has the function of promoting the expression of BDNF of the hippocampal-derived neural stem cells of new-born rats.

Claims (4)

1. The application of escitalopram in promoting the expression of BDNF (neural stem cell) in vitro is characterized in that the escitalopram regulates the expression of BDNF on the neural stem cell, and the concentration of the escitalopram is 20ng/ml.

2. Use of escitalopram according to claim 1 for promoting the expression of BDNF in neural stem cells in vitro, wherein the effect of escitalopram on the increase of BDNF expression in hippocampal derived neural stem cells of neonatal rats.

3. Use of escitalopram according to claim 2 for promoting the expression of BDNF in neural stem cells in vitro comprising the steps of:

adding escitalopram to intervene and induce the neural stem cells derived from the hippocampus of the newborn rat.

4. Use of escitalopram according to claim 3 for promoting the expression of BDNF in neural stem cells in vitro wherein the intervention period is 7 days.

Images