CN111733133A

CN111733133A - Method for promoting differentiation and growth of epidermal stem cells

Info

Publication number: CN111733133A
Application number: CN202010708902.6A
Authority: CN
Inventors: 刘欢; 李蒙
Original assignee: Beijing Guangwei Biotechnology Co Ltd
Current assignee: Huaxiayuan (Shanghai) Life Technology Co.,Ltd.
Priority date: 2020-07-22
Filing date: 2020-07-22
Publication date: 2020-10-02
Anticipated expiration: 2040-07-22
Also published as: CN111733133B

Abstract

The invention relates to a method for promoting differentiation and growth of epidermal stem cells, which obtains neuron cells from the epidermal stem cells by inducing the epidermal stem cells by using an inducer containing a compound, and proves that the induced neuron cells have better proliferation effect and corresponding neuron characteristics by detecting the proliferation activity and the neuron electrical characteristics of the neuron cells, thereby having better application prospect.

Description

Method for promoting differentiation and growth of epidermal stem cells

Technical Field

The invention belongs to the field of stem cells, and particularly relates to a method for promoting differentiation and growth of epidermal stem cells.

Background

Cells of the central nervous system of the brain include functional nerve cells and Neural Stem Cells (NSCs) that maintain brain function. Functional nerve cells mainly include neurons, oligodendrocytes, astrocytes, and the like. The neural stem cell is an adult stem cell having self-proliferating ability and multipotentiality. Under physiological conditions, dead or apoptotic functional neural cells are replaced by functional neural cells differentiated from neural stem cells. The neural stem cells bring new eosin for the damage repair of the nervous system and the treatment of cerebral ischemia diseases due to the self-proliferation capacity and the multi-directional differentiation potential of the neural stem cells. A large number of experimental studies prove that the neural stem cells exist in the inferior ventricular duct subphragmatic region and the inferior dentate gyrus granular region on the inner side of the central nervous system of the adult mammal, and cerebral ischemia can not only trigger the local death of ischemic cells, but also start the regeneration reaction of ischemic peripheral tissues. The neural stem cells complete nerve regeneration and repair damage through proliferation, migration, differentiation and further integration into the existing neural network. Endogenous neural stem cells are not well repaired because of the limited number of stem cells and the impermissible microenvironment. In addition, studies have shown that transplanted neural stem cells are often maintained in an undifferentiated state or are mostly differentiated into glial cells after entering the body due to various factors. Therefore, promoting the differentiation rate of neural stem cells into neurons and increasing the number of functional synapses between neurons will become an important approach for the repair of neural functions.

In recent years, people have done a lot of work on the regulation factors, regulation mechanisms and applications of NSC proliferation and differentiation, and some research results have been obtained. However, the differentiation of neural stem cells into neurons is a very complex process, and is commonly regulated by internal and external factors. In addition to the intrinsic genetic program that determines their differentiation, secretion and interaction of various cytokines in vivo, cell-to-cell interactions, and environmental factors may all contribute to this. More specifically, the process, mechanism and influencing factors of neural stem cell differentiation to neurons are still the complex topic we face.

The BMSCs can be isolated from adult non-hematopoietic stem cells, bone marrow, adipose tissue, and skin. Under in vitro culture conditions, BMSCs are adherent and can differentiate into osteoblasts, chondrocytes, and adipocytes. The hMSCs can be transplanted to rat brains and can be differentiated into astrocytes, and the MSCs provide a new idea for treating diseases of a Central Nervous System (CNS). In the past few years, MSCs from different species, such as Human (Human), Rat (Rat), Mouse (Mouse), have been studied with respect to neuronal differentiation. Three methods for inducing the differentiation of MSCs into nerve cells are mainly used, namely a compound induction method, a growth factor induction method and a nerve-like cell culture method.

MSCs are a class of pluripotent stem cells belonging to the mesoderm, and have the ability to differentiate into various cells such as osteoblasts, chondrocytes, adipocytes, myocytes (tendon), bone marrow stroma, and even liver cells and nerve cells. In recent years, various scholars at home and abroad prove that MSCs which can be classified into bones, cartilages, fat, cardiac muscles, blood vessels, nerves and the like exist in bone marrow tissues. Numerous in vitro studies have shown that MSCs have the potential to be multi-differentiated. Experiments prove that a stem cell possibly exists in adipose tissues, and cells which do not express NSE originally can express NSE through induction and are represented as neurons in shape. However, the cells after induced differentiation are reported in the literature to be at most early juvenile neurons, which do not have the functions of mature neurons; there are also some theories that although it was demonstrated that the cells after induction expressed various neuron non-specific markers, it was thought that the cells were protuberant due to cytoplasmic shrinkage of the cells under external stimulation or as a result of cell attachment to the culture plane, and expression of mature neuron markers such as tubulin-2, glial neuron acidic fibril protein, etc. was not found. Zheng Yani et al confirmed that: the ADSCs of SD rats are induced and differentiated by using Trichostatin A (TSA), RG-108, 8-BrcAMP and Rolipram 4 micromolecular substances, the expression of neuron markers of the ADSCs before and after induction is detected by methods such as immunofluorescence morphology, immunoblotting, qRT-PCR and the like, and the result proves that the ADSCs can be induced and differentiated into neuron-like cells by the micromolecular compounds, and the micromolecular compounds can be used as seed cells for treating nervous system diseases and damaged stem cell transplantation.

However, in the above studies, there is no study on the differentiation of human epidermal mesenchymal stem cells into neurons, and there is no method for inducing human epidermal mesenchymal stem cells well, quickly, and efficiently.

Disclosure of Invention

In order to solve the problem that the existing epidermal stem cells are not induced to become neuron cells, the invention aims to provide the neuron cells derived from the epidermal stem cells, a preparation method and application thereof in treating central nervous system injury.

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

in the first aspect of the invention, the inducer consists of DMEM/F12 +1% penicillin +20 mu g/L bFGF +20 mu g/L compound shown as the formula (1).

The compound of formula (1) is screened to obtain the compound with the function of inducing stem cells to neuron cells when stem cell inducers are researched, and the principle of the action of the compound is preliminarily presumed to improve the metabolic activity of the cells, so that the transcription expression level of certain genes for controlling differentiation is improved. The inventor will develop research later on specific action mechanism.

Further, the structural formula of the compound of the formula (1) is shown as the formula (1).

The compound of formula (1) is prepared according to the method of CN101437785B, and the compound can also be prepared by adopting the method of compound synthesis conventional in the field.

In a second aspect of the present invention, there is provided a method for differentiating epidermal stem cells into neuronal cells using the above-mentioned inducer, comprising the steps of:

the prepared epidermal stem cells are digested by 0.25% pancreatin-EDTA and then inoculated in a polylysine coated 24-well plate, and after the cells are attached to the wall, a culture solution is replaced to be an inducer (experimental group): DMEM/F12 +1% penicillin + 20. mu.g/L bFGF + 20. mu.g/L compound of formula (1), and the solution is changed half every other day. Induction for 7 d.

In a third aspect of the invention, there is provided the use of neuronal cells derived from epidermal stem cells for the manufacture of a medicament for the treatment of central nervous system injury.

In a fourth aspect of the present invention, there is provided a pharmaceutical composition, the above-mentioned epidermal stem cell-derived neuronal cell and a pharmaceutically acceptable carrier, wherein the epidermal stem cell-derived neuronal cell is present in an amount sufficient to promote functional recovery in a patient with central nervous system injury after administration of the epidermal stem cell-derived neuronal cell to the patient.

wherein the number of the epidermal stem cell-derived neuronal cells is 1 × 10⁶～1×10⁷And (4) respectively.

Wherein the pharmaceutically acceptable carrier is normal saline.

Advantageous effects

Compared with the prior art, the invention has the following beneficial effects: the invention obtains the neuron cells from the epidermal stem cells by separating the epidermal stem cells and inducing the epidermal stem cells by the inducer containing the compound, and proves that the induced neuron cells have better proliferation effect and corresponding neuron characteristics by detecting the proliferation activity and the neuron electrical characteristics of the neuron cells, thereby having better application prospect.

Drawings

FIG. 1 Gene expression level graph

Detailed Description

The present invention is described in detail below by way of examples, it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make some insubstantial modifications and adaptations of the present invention based on the above-described disclosure.

Example 1 preparation of epidermal stem cells

And (3) taking a skin specimen, and rinsing the skin specimen with D-hank's solution containing 800u/ml gentamicin for 3 times under an aseptic condition. Cutting the skin into 1cm x 1cm pieces with an ophthalmologic scissors, placing into a culture bottle, dripping 0.25% Trypsin-0.02% EDTA with a volume ratio of 1:6, and digesting for 10h at 4 ℃. Taking out the skin piece, dripping a little fetal calf serum to stop digestion, and rinsing with D-hank's solution for 2 times. Separating epidermis and dermis, and gently peeling off epidermis with ophthalmologic forceps; add K-SFM and carefully blow the epidermis repeatedly for 15min with a pipette. Filtering with 200 mesh screen, centrifuging at 1000rpm for 5min, discarding wound, and adding K-SFM to obtain cell suspension. Inoculating the cells into a pretreated IV type collagen culture plate, and culturing in a constant temperature incubator at 37 ℃ and 5% CO2 saturated humidity; discarding culture solution and non-adhered cells after 20min, adding epidermal stem cell culture solution containing 10% fetal calf serum and 10ng/ml EGF K-SFM; culturing in a constant temperature incubator at 37 ℃ and 5% CO2 saturated humidity; and (4) observing the growth condition of the cells by an inverted phase contrast microscope, changing the liquid every 3d for 1 time, fusing about 70-80% of the cells, and then carrying out passage according to the ratio of 1: 3. After the second generation epidermal stem cells form obvious clones, the beta 1 integrin, K19 and p63 are respectively identified by immunocytochemical staining through a two-step method, and the immunocytochemical staining results are shown in Table 1.

TABLE 1 results of immunocytochemical staining of isolated epidermal stem cells

It was shown that β 1 integrin, K19, p63 were positively expressed. The in vitro separation and culture of human epidermal stem cells are successful.

EXAMPLE 2 Induction of differentiation of epidermal Stem cells into neurons by Compounds

The compound of formula (1) is prepared according to the synthesis method of CN 101437785B.

The epidermal stem cells prepared in example 1 of the 4 th generation were digested with 0.25% trypsin-EDTA and then inoculated into a polylysine-coated 24-well plate, and the culture medium was changed after the cells were attached to the wall as an induction solution (experimental group): DMEM/F12 +1% penicillin + 20. mu.g/L bFGF + 20. mu.g/L compound of formula (1), and the solution is changed half every other day. Detection was performed 7d after induction, respectively. The epidermal stem cell group prepared in example 1 of the 4 th generation to which DMEM/F12 +1% penillilin + 20. mu.g/L bFGF was added as an inducing solution was used as control 1, and the epidermal stem cell group prepared in example 1 of the 4 th generation to which no inducing solution was added was used as control 2.

After the cells induced in the 24-well plate were washed with 0.1 mol/L PBS, the cells were lysed by adding a Radio-ionization Assay (RIPA) buffer. Centrifuging at low temperature to obtain supernatant, measuring protein concentration by Bradford method, boiling in boiling water at 100 deg.C for 5min, and centrifuging for use. Separation gels were prepared, loaded, run on SDS-PAGE, the gel plates removed for membrane transfer, stained, and incubated overnight at 4 ℃ with nestin (1: 1000), β -tubulin III (1: 1000), MAP2 (1: 2000), ChAT (1: 1000) and GAPDH (1: 2000) diluted in blocking solution. The secondary antibody (1: 2000) was incubated for 2h at room temperature. Taking out the film, rinsing the film by TBST, covering a PVDF film by a preservative film, and carrying out light sensing, developing and fixing by an X film in a dark room. The results show that the expression change of the neurogenic marker protein of the epidermal stem cells after induction is detected by immunoblotting as shown in figure 1, and after 7 days of induction, the neurogenic marker proteins beta-tubulin III, MAP2, ChAT and nestin are all higher than those before induction, wherein the increase of MAP2 and ChAT is more obvious. Moreover, compared with the control group 1, the increase of the markers is quite remarkable, and the compound is a main inducer composition. Control group 2 also differed significantly from the experimental group. Microscopic examination shows that the cell shape after induction is mature, the cell body is large, and the bulges are long, thin and rich. And selecting the induced cells of the holes with the highest gene expression increase in the experimental group for the next experiment.

Example 3 cell proliferation after Induction

example 2 measurement of cell proliferation before and after Induction by MTT method neuronal cells with the highest increase in expression of marker protein were diluted to a cell concentration of about 5 × 10⁶L^-1Inoculating the cell suspension into a 96-well plate, adding 200 mu L of the cell suspension into each well, sucking out the culture solution in each well after the cells adhere to the wall, and dividing the experiment into 3 groups: the experimental group, control 1, control 2, and 6 wells in each group correspond to example 2, respectively. Blank group: freshly prepared DMEM medium containing 10% by volume calf serum was added to wells containing cells at 200. mu.L/well. Culturing for 72h, taking out 96-well plate, adding MTT25 μ L/well, incubating for 4 h in dark, aspirating out liquid, dissolving 20% sodium dodecyl sulfate in 50% dimethylformamide, adding 100 μ L of the above mixed solution into each well of 96-well plate, placing at 37 deg.C, and adding 5% CO by volume₂and (3) incubating for 24 h in a dark place, then placing the mixture into an enzyme-linked immunosorbent assay, oscillating for 10 min, measuring the absorbance value (A value) of each hole of each time phase point at 570nm, calculating the relative survival rate, wherein the relative survival rate = the A value of the sample to be measured/the A value of a normal group is × 100%, and evaluating the action degree of the cells of the sample according to the relative survival rate score standard according to the relative survival rate mean value, wherein the results are shown in table 2.

TABLE 2 Absorbance and relative Activity of cells

Group of	Absorbance at 570nm	Relative cell viability (%)
			Control 1 differentiated cellsCell	0.693±0.13	103.12^a
Cells of control 2	0.672±0.09	100
			Experimental group of differentiated cells	0.793±0.05	118.0^a

In comparison with the control 2,^aP≤0.05。

the experimental result shows that the absorbance of the cells of the control group 2 at the position of 570nm is 0.672 +/-0.09, the absorbance of the cells of the experimental group at the position of 570nm is 0.793 +/-0.05, the absorbance of the cells of the control group 1 at the position of 570nm is 0.693 +/-0.13, and the absorbance of the cells of the experimental group is obviously higher than that of the cells of the control group 1 and the control group 2, which proves that the inducer containing the compound not only can promote the induced differentiation of the cells, but also can promote the growth of the neuronal cells. The growth rate of the cell process was significantly faster than that of the control cells.

Example 4 cell functional validation

Selecting cells with typical neuron morphology and undifferentiated cells for electrophysiological detection under a patch clamp detection whole-cell patch clamp mode. Recording the cell membrane ion channel current in a voltage clamp mode; the resting potential of the cells was recorded in current clamp mode. The glass microelectrode for recording is drawn by a horizontal glass microelectrode drawing instrument in two steps, the impedance is 2-4M omega after filling electrode liquid, a high-resistance seal (1-5G omega) is formed by negative pressure after contacting cells through a micromanipulator, and a full-cell patch clamp is formed by breaking cell membranes by instant stronger negative pressure after compensating electrode capacitance. The holding voltage (HP) was set at-60 mV, the membrane current was low-pass filtered at 10kHz (-3dB), and the data was recorded on a PC computer (pClamp 10.2). Detection ofthe electrode solution used in the measurement comprises ① extracellular fluid containing NaCl 150mmol/L, KCI 5mmol/L and CaCI₂2.5 mmol/LMgC 121 mmol/L, Glucose 5mmol/L, HEPPS10 mmol/L, pH =7.4 (adjusted with 1 mol/L NaCI solution) ② electrode solution KCI 140 mmol/L, CaC1₂1mmol／L；MgC1 ₂2 mmol/L; EGTA 1 mmol/L; HEPPS10 mmol/L; pH =7.4 (adjusted with 1 mol/L KC1 solution), and the experiment was performed at room temperature (20-25 ℃). The results are shown in table 3 below.

TABLE 3 comparison of Membrane characteristics before and after cell differentiation

Cell type	Cm（n）	Rs（n）	RMP（n）
				Cells of control 2	15.31±2.17	7.44±1.28	-20.95±2.13
Experimental group of differentiated cells	39.87±5.21	22.53±2.01	-51.65±5.56

As can be seen from the results in table 3, the membrane capacitance (Cm), series resistance (Rs), and Resting Membrane Potential (RMP) of the cells before and after cell differentiation were significantly different (P < 0.01) in the experiments of the control 2 cells and the differentiated cells. (see Table 3), the committed differentiated neuronal-like cells have active electrophysiological properties. The differentiated neuron-like cells and undifferentiated cells have a significant change in membrane properties, and the differentiated cells approach the physiological properties of normal nerve cells in membrane properties.

Claims

1. An inducer for inducing epidermal stem cells into neuronal cells, consisting of DMEM/F12 +1% penicillin +20 μ g/L bFGF +20 μ g/L of a compound of formula (1);

。

2. a method of promoting differentiation and growth of epidermal stem cells, comprising the steps of:

the prepared epidermal stem cells are digested by 0.25% pancreatin-EDTA and inoculated into a polylysine coated 24-well plate, the culture solution is changed after the cells are attached to the wall as the inducer in claim 1, and the culture solution is changed half every other day for 7 days.