CN114164175B

CN114164175B - Method for inducing PC12 cells to differentiate into neuron-like cells by eucommia ulmoides water extract

Info

Publication number: CN114164175B
Application number: CN202010950204.7A
Authority: CN
Inventors: 李辞霞; 朱晓岩; 潘佳蓉; 赵善廷; 薛玉环
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2020-09-11
Filing date: 2020-09-11
Publication date: 2023-06-09
Anticipated expiration: 2040-09-11
Also published as: CN114164175A

Abstract

The invention belongs to the technical field of cell transdifferentiation, and discloses a method for inducing PC12 cells to differentiate into neuron-like cells by using eucommia ulmoides aqueous extract. The method comprises the following steps: the eucommia bark water extract is dissolved in a basic culture medium, mixed by ultrasonic vibration, and added with 5 to 8 percent of fetal calf serum to prepare an eucommia bark induction culture medium, and PC12 cells are induced and cultured for 3 to 4 days. The PC12 cells become enlarged, the protrusions become more and elongated, and the cells differentiate into neurons under the microscope. According to the invention, NGF is not required to be added into a culture medium, and PC12 cells can be induced to differentiate into neuron-like cells and express neurofilament proteins only by virtue of the eucommia ulmoides aqueous extract with specific concentration. The invention provides powerful basis for the neurotrophic function of eucommia ulmoides, the function of promoting the synapse growth and other biological activity, and has profound practical significance for further developing and utilizing the valuable resource of eucommia ulmoides and discussing the biological activity of eucommia ulmoides.

Description

Method for inducing PC12 cells to differentiate into neuron-like cells by eucommia ulmoides water extract

Technical Field

The invention belongs to the technical field of cell transdifferentiation, and discloses a method for inducing PC12 cells to differentiate into neuron-like cells by using eucommia ulmoides aqueous extract.

Background

Eucommia ulmoides (academic name Eucommia ulmoides Oliver) is also known as bakelite, which is a eucommia family plant. Eucommia ulmoides free amino acids are very few, and a small amount of protein is similar to most foods, namely 8 amino acids which are necessary for human bodies can be detected through hydrolysis.

The medicinal eucommia bark is dry bark of eucommia bark. The water extract of eucommia ulmoides contains lignans, polysaccharides, iridoids, flavonoids, chlorogenic acid, polyphenols, amino acids, etc., and has the pharmacological effects of enhancing immunity, resisting cancer, resisting virus, resisting oxidation, resisting aging, resisting liver fibrosis, tranquilizing, etc. In recent years, research shows that chemical components in eucommia ulmoides have neuroprotective effect, and eucommia ulmoides is contained in the prescription for treating central nervous system diseases and degenerative diseases.

PC12 cells are a common nerve cell strain which is derived from pheochromocytoma of the adrenal gland of the brown rat, are in a round shape, grow semi-adherently, and are widely used for in vitro research of nervous system diseases. Under certain conditions, PC12 cells can be induced by Nerve Growth Factor (NGF) to neuron-like cells, the cell bodies change from smaller circular shapes to larger polygons, protrude from the projections, and express neurofilament protein (NF-H). PC12 cells are a cell model widely used at present for researching differentiation and apoptosis of nerve cells, and research on the pro-differentiation effect of drugs on PC12 cells is a powerful evidence for proving that drugs have nerve plasticity in neuropharmacology.

At present, the treatment effect of eucommia ulmoides water extract on nervous system diseases is well known, but whether the eucommia ulmoides water extract has functions of nourishing neurons, promoting protrusion growth and the like NGF and can induce PC12 cells to differentiate into neuron-like cells is not reported.

Disclosure of Invention

The invention discloses an eucommia ulmoides water extract with the function of inducing the differentiation of PC12 cells to neuron-like cells, and discloses a method for inducing the differentiation of PC12 cells to neuron-like cells by the eucommia ulmoides water extract.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

(1) Preparing eucommia ulmoides induction culture medium;

(2) Recovering and subculturing PC12 cells;

(3) The eucommia ulmoides inducing culture medium induces and cultures the PC12 cells to differentiate into neuron-like cells.

The operation of the step (1) is as follows: dissolving cortex Eucommiae water extract with basic culture medium at a dosage of 4mg/ml, and ultrasonic vibrating for 30min to thoroughly mix. Under the aseptic condition of an ultra-clean bench, filtering by a 0.22 mu m filter, and adding 5% -8% of fetal bovine serum to prepare the eucommia ulmoides induced culture medium.

The operation of the step (2) is as follows: taking out PC12 cells frozen by liquid nitrogen, dissolving and recovering in 37 deg.C water bath, centrifuging at 1000 rpm for 5min, discarding frozen stock solution, adding complete culture medium to resuspend cells, inoculating in culture dish, and inoculating 5% CO ₂ Culturing at 37deg.C for 1-2d. When the proliferation of the cells is attached to about 80% -90%, the attached cells are blown down by a 1000ml pipetting gun machine, and after the complete culture medium is resuspended, the culture is carried out for 1:4 subculture.

The operation of the step (3) is as follows: transferring to third generation PC12 cells, sucking off original complete culture medium, changing to Eucommiae cortex induction culture medium in step (1), and culturing for 3-4 days. The PC12 can be seen to differentiate under the microscope, the cells are changed from round shape to polygonal shape, the cell bodies are enlarged, the protrusions are gradually increased and elongated, and the cells are differentiated into neuron-like cells.

The eucommia bark water extract is eucommia bark water extract dry powder.

The basal medium was 25mM high-sugar DMEM, pH 7.4.

The complete medium was 25mM high glucose DMEM plus 5% fetal bovine serum and 5% horse serum.

The invention has the beneficial effects that: the invention provides a specific dosage, culture medium components and induction time for the eucommia ulmoides aqueous extract to induce the PC12 cells to differentiate into neuron-like cells. Compared with the existing researches, the invention can differentiate PC12 cells into neuron-like cells and express the neurofilament protein by only relying on the eucommia water extract culture medium containing specific concentration without adding NGF to the culture medium. The eucommia ulmoides induced PC12 cells are differentiated into neuron-like cells, can be widely used as a model for research of neurobiology, pharmacology and toxicology, and provides a new idea for inducing PC12 cells to differentiate by traditional Chinese medicines. The invention provides powerful basis for the neurotrophic function of eucommia ulmoides, the function of promoting the synapse growth and other biological activity, and has profound practical significance for further developing and utilizing the valuable resource of eucommia ulmoides and discussing the biological activity of eucommia ulmoides.

Drawings

FIG. 1 is a graph showing the morphology of PC12 cells before and after induction of eucommia ulmoides. A is PC12 cells cultured in a common complete medium for 4 d; b is PC12 cells cultured in eucommia induced medium for 4 d.

FIG. 2 shows NF-H immunofluorescence expression of PC12 differentiated into neuronal-like cells before and after eucommia ulmoides induction. A is PC12 cells cultured for 4d in a common complete culture medium, and NF-H is not expressed; B-D is the expression of NF-H after culturing in eucommia induced culture medium for 2-4 days.

Detailed Description

1. Materials and methods

1. Material

PC12 cells were cryopreserved by the animal medical college neurobiology laboratory of the university of North Western agriculture and forestry science and technology. Eucommia bark water extract dry powder is purchased from Shaanxi Quan Changrong biotechnology Co., ltd; 25mM high sugar DMEM, fetal bovine serum, horse serum available from Gibco, inc., USA; rabbit anti-murine NF-H antibodies were purchased from us Cell Signaling Technology company; DAPI was purchased from Sigma.

2. Preparation of complete culture medium and eucommia ulmoides induced culture medium

Preparation of complete medium: 25mM high sugar DMEM 40ml, 5% foetal calf serum and 5% horse serum were added and placed in 50ml sterile centrifuge tubes for further use.

Preparation of eucommia ulmoides induction culture medium: the eucommia ulmoides aqueous extract is dissolved by 25mM high sugar DMEM according to the dosage of 4mg/ml, and is subjected to ultrasonic vibration for 30min to be fully and uniformly mixed. Under the aseptic condition of an ultra-clean bench, filtering the mixture to a 50ml aseptic centrifuge tube by using a 0.22 mu m filter, adding 5 to 8 percent of fetal bovine serum, and marking the mixture for later use.

PC12 cell culture and neurogenesis induction

Taking out PC12 cells frozen by liquid nitrogen, dissolving and recovering in 37 deg.C water bath, centrifuging at 1000 rpm for 5min, discarding frozen stock solution, adding complete culture medium to resuspend cells, inoculating in culture dish, and inoculating 5% CO ₂ Culturing at 37deg.C for 1-2d. When the proliferation of the cells is attached to about 80% -90%, the attached cells are blown down by a 1000ml pipetting gun machine, and after the complete culture medium is resuspended, the culture is carried out for 1:4 subculture. Taking third generation cells at 10 per well ³ Individual cells were seeded in 24-well plates and grown adherent overnight. The next day, the original complete culture medium is sucked and removed, and the culture medium is replaced by eucommia ulmoides oliv induction culture medium, and the culture is carried out for 3-4 days. The morphology of PC12 was observed daily under the microscope.

NF-H immunofluorescence labeling

PC12 cell NF-H immunofluorescence labeling: fixing cells for 30min at room temperature by 4% paraformaldehyde solution, and soaking and washing with PBS for 3 times; 0.3% Triton permeant for 5min, PBS rinse 3 times (3 min each); normal goat serum is blocked, incubated for 20min at room temperature, and the goat serum is sucked and removed; dropwise adding 1:500 rabbit anti-rat neurofilament-H (NF-H) antibody, incubated overnight at 4 ℃. The next day, primary antibody was pipetted off, washed 3 times with PBS (3 min each), and 1 was added dropwise: 400 FITC-labeled goat anti-rabbit secondary antibody, incubating for 1h at room temperature, absorbing and discarding the secondary antibody, and soaking and washing with PBS for 3 times (3 min each time); 10 mug/mL DAPI counterstained nuclei for 10min, washed 3 times with PBS, and visualized under a fluorescence microscope.

2. Results

1. Eucommia ulmoides induced PC12 cell morphology change

The undifferentiated PC12 cells of 4d cultured in the normal complete medium were round in shape, small in cell mass and almost no protrusion (FIG. 1A). PC12 cells cultured by the eucommia ulmoides induction culture medium gradually start to differentiate along with the growth of the induction time, the cells are changed into polygons from circles, the cell bodies are enlarged and extend out, the gradual increase and extension of the cell protrusions can be obviously observed by the induction 4d, the cells are connected with each other to form a net, and the neuron-like cells with the morphological characteristics of nerve cells are differentiated (figure 1B).

2. Eucommia ulmoides induced positive expression condition of PC12 cell NF-H

The PC12 cell NF-H immunofluorescence mark induced by common complete culture medium and eucommia induced culture medium for 1-3 days can be found: PC12 cells cultured in normal complete medium were unable to differentiate themselves and did not express the neuron specific antibody NF-H (FIG. 2A). PC12 cells cultured by eucommia ulmoides induction medium are induced to differentiate into neuron-like cells and express NF-H on day 2; inducing part of cells on day 3 to express NF-H in a strong positive way, and inducing most of cells to express NF-H in a weak positive way; over about 90% of the cells on day 4 were induced to strongly positive express NF-H, and elongated protrusions were seen (fig. 2B, C, D).

All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope. The examples are intended to be exemplary only and not to be limiting in any way, and all ranges of reagents used are defined by the appended claims. Other modifications and equivalents of the present invention will occur to those skilled in the art without departing from the spirit and scope of the present invention, and are intended to be covered by the scope of the appended claims.

Claims

1. A method for inducing the differentiation of PC12 cells into neuron-like cells by using eucommia ulmoides aqueous extract, which is characterized by comprising the following steps:

(1) Preparing eucommia ulmoides induction culture medium;

(2) The eucommia ulmoides inducing culture medium induces and cultures PC12 cells to differentiate into neuron-like cells;

the operation of the step (1) is as follows: dissolving eucommia ulmoides water extract with basic culture medium according to the dosage of 4mg/ml, carrying out ultrasonic vibration for 30min to fully and uniformly mix, filtering with a 0.22 mu m filter under the aseptic condition of a super clean bench, and adding 5% -8% fetal bovine serum to prepare the eucommia ulmoides induction culture medium.

2. The method for inducing the differentiation of PC12 cells into neuron-like cells by using eucommia ulmoides aqueous extract as claimed in claim 1, wherein: the operation of the step (2) is as follows: transferring to third generation PC12 cells, sucking and discarding original complete culture medium, changing to Eucommiae cortex induced culture medium, culturing for 3-4 days, and culturing under microscope to obtain PC12 differentiation, wherein the cells change from round shape to polygonal shape, the cell body becomes larger, and the protrusions gradually increase and elongate, and differentiate into neuron-like cells.

3. The method for inducing the differentiation of PC12 cells into neuron-like cells by using eucommia ulmoides aqueous extract as claimed in claim 1, wherein: the eucommia bark water extract is eucommia bark water extract dry powder; the basal medium was 25mM high sugar DMEM, pH 7.4.

4. The method for inducing the differentiation of PC12 cells into neuron-like cells by using eucommia ulmoides aqueous extract as claimed in claim 2, wherein: the complete medium is a basal medium plus 5% fetal bovine serum and 5% horse serum.