阅读说明：本技术 重组人Notch1蛋白在制备神经干细胞及皮层神经元中的应用 (Application of recombinant human Notch1 protein in preparation of neural stem cells and cortical neurons ) 是由 杨潘 朱玮 王琳娜 李慧 于 2020-04-23 设计创作，主要内容包括：本发明涉及一种神经干细胞的制备方法及应用,本发明通过加入Notch信号通路受体Notch1的方法解决神经干细胞高效扩增的问题,可以获得功能稳定且活性高的神经干细胞,可进一步分化为数量大的皮层功能神经元,为皮层功能神经元的获得奠定了基础,可以满足同时筛选多种药物的目的。(The invention relates to a preparation method and application of neural stem cells, which solves the problem of efficient amplification of the neural stem cells by adding a Notch signal pathway receptor Notch1, can obtain the neural stem cells with stable functions and high activity, can be further differentiated into a large number of cortical functional neurons, lays a foundation for obtaining the cortical functional neurons, and can meet the aim of simultaneously screening various medicaments.)

1. Application of recombinant human Notch1 protein in preparing neural stem cells.

2. The use according to claim 1, wherein the neural stem cell is a neural stem cell capable of differentiating into a cortical neuron having a function of generating an action potential.

3. The use according to claim 1, in particular for the preparation of said neural stem cells, comprising the steps of:

(1) when the confluency of the human iPSCs reaches more than 100%, starting to perform induced differentiation culture on neural stem cells for 4-6 days by using a neural stem cell induction culture medium, stopping induction, then digesting, and collecting cells;

(2) carrying out digestion passage on the cells collected in the step (1) after re-suspending the cells by using a neural stem cell maintenance culture medium for continuous culture for 4-7 days; obtaining neural stem cells, wherein the components of the neural stem cell maintaining culture medium comprise recombinant human Notch1 protein.

4. The use of claim 3, wherein the neural stem cell induction medium in step (1) is Neurobasal medium +1 × N2+1 × B27+1 × GlutaMAX-I +500ng/ml human recombinant Noggin +20 μ MSB431542+4ng/ml FGF-2;

preferably, during the induced differentiation culture in the step (1), half of liquid change is carried out every 3 days;

preferably, the digestion is carried out by pancreatin in the step (1), and the cells are collected;

preferably, digestion is performed with 0.25% Trpsin-EDTA in step (1).

5. The use of claim 3, wherein the mass concentration of recombinant human Notch1 protein in the neural stem cell maintenance medium components in step (2) is 90-110 ng/ml;

preferably, the neural stem cell maintaining culture medium in the step (2) is Neurobasal culture medium +1 × N2+1 × B27+10ng/ml EGF +10ng/ml FGF-2+20ng/ml BDNF +100ng/ml recombinant human Notch1 protein;

preferably, in the process of continuing the culture in the step (2), half of liquid change is carried out every 3 days;

preferably, the culture is continued for 4 to 7 days in the step (2), and digestion passage is carried out;

preferably, the culture is continued for 4 days in the step (2), and digestion passage is carried out;

preferably, in step (2), the cells collected in step (1) are treated as 10⁵/CM²Inoculating to a neural stem cell maintenance medium;

preferably, the subculture in step (2) is performed by collecting the neurosphere culture in a centrifuge tube, centrifuging at 1000rpm for 2 min, discarding the supernatant, adding calcium-magnesium-free Phosphate Buffered Saline (PBS) to resuspend the neurospheres, centrifuging at 1000rpm for 2 min, discarding the supernatant, adding 0.25% Trpsin-EDTA into the centrifuge tube, and adding CO with a volume concentration of 5%₂Digesting in a constant temperature cell culture box at 37 ℃ until the neurospheres are single cells, stopping digestion, centrifuging at 1000rpm for 5 minutes, discarding supernatant, suspending the cells by using the neural stem cells to maintain the basic suspension of the cells according to the weight of 10⁵/CM²Inoculating into 6-well plate, and adding CO with volume concentration of 5%₂Culturing in a constant-temperature cell culture box at 37 ℃, and changing the liquid half a day every 3 days;

preferably, purified neural stem cells can be obtained by digestion passage for 2-3 times in the step (2);

preferably, the neural stem cells are obtained by digestion passage for 8-10 times;

preferably, the neural stem cells are obtained after digestion passage for 12-30 times.

6. A neural stem cell maintenance culture medium is characterized by comprising a Neurobasal culture medium +1 × N2+1 × B27+10ng/ml EGF +10ng/ml FGF-2+20ng/ml BDNF +90-110ng/ml recombinant human Notch1 protein.

7. Use of the neural stem cell prepared according to claim 1 for differentiating into cortical neurons having a function of generating action potentials.

8. The use of any one of claims 1 to 5, wherein said neural stem cells are used in screening for neuroprotective drugs.

9. The use of claim 8, wherein said neural stem cells are used for inducing differentiation to produce cortical neurons capable of generating action potentials.

10. The use of claim 9, wherein said cortical neurons are used for screening of neuroprotective drugs.

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to application of a recombinant human Notch1 protein in preparation of neural stem cells and cortical neurons.

Background

The stroke is a group of diseases causing brain tissue damage due to cerebrovascular blockage or rupture, including ischemic stroke and hemorrhagic stroke, the death rate of the stroke is the second place worldwide, and the stroke is also the leading cause of disability of adults in China. Wherein, the incidence rate of cerebral arterial thrombosis is higher and accounts for 60 to 70 percent of the total cerebral arterial thrombosis. At present, the acute treatment of ischemic stroke is to use thrombolytic drug recombinant human tissue plasminogen activator (rt-PA), but the application of the drug has strict time limit, and the application is within 4.5 hours of the attack, and the side effect of secondary intracranial hemorrhage caused by the drug also limits the application of the drug, so that the search of new neuroprotective drugs becomes urgent. In recent 20 years, scientists are continuously developing new drugs, but effective drugs in animal experiments fail clinical trials, and the experimental animal and human responses to the drugs are greatly different. With the establishment of human Induced Pluripotent Stem Cells (iPSCs), a new disease model is generated, and the model established by the iPSCs is closer to the characteristics of human beings because of being derived from normal people or patients, so that the use of cortical neurons induced by the human iPSCs for drug screening can avoid the ethical problem and greatly improve the drug conversion efficiency.

The study shows that the Epidermal Growth Factor (EGF) and fibroblast growth factor-2 (FGF-2) are important for maintaining the stable proliferation of the neural stem cells, and the addition of WNT signaling antagonist DKK1 can also enhance the derivation of the neural stem cells, at present, the commonly used neural stem cells are maintained in Neurobasal medium +1 × N2+ 1B × B27+ EGF (10/ml)) + 2(10ng/ml) + brain growth factor (BDNF, 20ng/ml) but the neural stem cells cultured in this condition for a short time are still subjected to the low neural stem cell induction strategy, which is generally indicated as a defect that the neural stem cells are not subjected to the statistics of low neural stem cell proliferation rate, and the statistics that the SCHeagth induction of the neural stem cells is low is generally indicated by the following method.

At present, human iPSCs are used for inducing neural stem cells, then the neural stem cells are amplified, and then the neural stem cells are induced into cortical neurons to screen cerebral apoplexy neuroprotection drugs. In the process, the stable derivation of the neural stem cells is the most critical, the efficiency and the activity of inducing and differentiating the neurons are determined, and the identification of the drug effect plays a crucial role, so the success or failure of drug screening is determined by the method for efficiently screening the high-activity neural stem cells.

The Chinese literature, "regulation of Notch1 signal pathway to human periodontal ligament stem cell stress differentiation process" (Zhongxiu, Qin, Cuiguanxi, etc., J.J.Op., 2017, 26(4)), which states that Notch1 is one of the most important receptors of the Notch family in human body, belongs to type I transmembrane protein, and the literature refers to the role of the Notch1 signal pathway in human periodontal ligament stem cell (PD L SCs) osteogenesis under the action of dynamic tensile stress through the agonist human recombinant Jagged1 protein and the inhibitor gamma secretase inhibitor (DAPT) up-and down-regulation of Notch1 pathway key protein (NICD), and the literature, "expression of Notch1 gene in the process of differentiation of human embryonic neural stem cell to neuron" (Wangfei, Lishi, Huangqiang, etc., journal of cell and molecular immunology, 2004: 6), in which along with the role of NSC to neuron, the expression of Notch1 is gradually reduced, the expression of Notch1 plays a role in the process of NSC neural differentiation into neuron, and the possible role of self-regulation and maintenance of the human embryonic stem cell.

Human periodontal ligament stem Cells belong to human mesenchymal stem Cells, which are derived from ectodermal mesenchyme and can be further differentiated into osteoblasts and adipocytes, while neural stem Cells are derived from ectoderm and can be further differentiated into neurons and glial Cells in the document Single-cell stem of neural stem Cells derived from human iPSCs derived from genetic differentiation promoter with neural and glial Cells (Matti L am, Tsukasa Sanosaka, Anders L undin, et al, Genes to Cells, 2019: 24), neural stem Cells derived from human iPSCs and cultured human embryonic stem Cells are subjected to sequencing, and neural stem Cells and cultured human embryonic stem Cells are differentiated into Single Cells and glia, although both have great differences in neural and glial Genes.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the application of the recombinant human Notch1 protein in the preparation of neural stem cells and cortical neurons.

Currently, the method commonly used for inducing differentiation from iPSCs to neurons is divided into two stages, the first stage is the induction differentiation of iPSCs into neural stem cells, and the second stage is the induction differentiation of neural stem cells into neurons. If a large number of neurons are needed for the next research, a large number of neural stem cells which can be stably proliferated are needed, but the current method of adding the growth factor is applied, the number of the neural stem cells is reduced along with the increase of the passage frequency in the passage process, and the proliferation is slow, and the phenomenon is considered to be caused by the reduction of the activity of the neural stem cells. The invention improves the neural stem cell maintenance culture medium in the first stage and adds the Notch signal pathway receptor Notch1 to increase the activity of the neural stem cell, shorten the proliferation cycle of the neural stem cell, stabilize the passage and obtain a large amount of functional cortical neurons in the later stage.

The invention solves the problem of high-efficiency amplification of the neural stem cells by adding the Notch signal pathway receptor Notch1, can obtain the neural stem cells with stable function and high activity, can be further differentiated into cortical functional neurons with large quantity, lays a foundation for obtaining the cortical functional neurons, and can meet the aim of simultaneously screening various medicaments.

The technical scheme of the invention is as follows:

application of recombinant human Notch1 protein in preparing neural stem cells.

The neural stem cell is a neural stem cell capable of differentiating into a cortical neuron having a function of generating an action potential.

The application of the recombinant human Notch1 protein in preparing neural stem cells, in particular to a preparation method of the neural stem cells, which comprises the following steps:

(1) when the confluency of the human iPSCs reaches more than 100%, starting to perform induced differentiation culture on neural stem cells for 4-6 days by using a neural stem cell induction culture medium, stopping induction, then digesting, and collecting cells;

(2) carrying out digestion passage on the cells collected in the step (1) after re-suspending the cells by using a neural stem cell maintenance culture medium for continuous culture for 4-7 days; obtaining neural stem cells, wherein the components of the neural stem cell maintaining culture medium comprise recombinant human Notch1 protein.

Preferably, the neural stem cell induction medium in the step (1) is Neurobasal medium +1 × N2+1 × B27+1 × GlutaMAX-I +500ng/ml human recombinant Noggin +20 mu M SB431542+4ng/ml FGF-2.

Preferably, according to the present invention, during the differentiation-inducing culture in step (1), half of the liquid change is performed every 3 days.

And (2) inducing differentiation culture for 4-6 days in the step (1) until a plurality of scattered Rosette (convex) structures can be observed under a microscope, and stopping induction.

According to a preferred embodiment of the present invention, in step (1), the cells are collected by digestion with pancreatin.

Further preferably, the digestion in step (1) is carried out with 0.25% Trpsin-EDTA.

Preferably, the mass concentration of the recombinant human Notch1 protein in the culture medium component in the step (2) is 90-110 ng/ml.

Further preferably, the neural stem cell maintaining culture medium in the step (2) is Neurobasal culture medium +1 × N2+1 × B27+10ng/ml EGF +10ng/ml FGF-2+20ng/ml BDNF +100ng/ml recombinant human Notch1 protein.

Preferably, according to the invention, during the further cultivation in step (2), half a liquid change is carried out every 3 days.

Preferably, according to the invention, in step (2), the culture is continued for 4 to 7 days and digestion passages are carried out.

And (3) continuing culturing for 4-7 days in the step (2) until the blackening phenomenon of the nerve ball center begins, and performing digestion passage.

Further preferably, in step (2), the culture is continued for 4 days, and digestion passaging is performed.

Preferably, according to the invention, the cells collected in step (1) in step (2) are treated as 10⁵/CM²Inoculating to the neural stem cell maintaining culture medium.

Preferably, according to the invention, the method for eliminating passage in step (2) isCollecting neurosphere culture in a centrifuge tube, centrifuging at 1000rpm for 2 min, removing supernatant, adding calcium-magnesium-free Phosphate Buffer Saline (PBS) to resuspend neurospheres, centrifuging at 1000rpm for 2 min, removing supernatant, adding 0.25% Trpsin-EDTA into the centrifuge tube, and adding 5% CO by volume₂Digesting in a constant temperature cell culture box at 37 ℃ until the neurospheres are single cells, stopping digestion, centrifuging at 1000rpm for 5 minutes, discarding supernatant, suspending the cells by using the neural stem cells to maintain the basic suspension of the cells according to the weight of 10⁵/CM²Inoculating into 6-well plate, and adding CO with volume concentration of 5%₂Culturing in a constant-temperature cell culture box at 37 ℃ and changing the culture solution half a day every 3 days.

According to the present invention, preferably, purified neural stem cells can be obtained by digestion passage 2-3 times in step (2).

Further preferably, the neural stem cells are obtained by digestion passage for 8-10 times.

More preferably, the neural stem cells are obtained after 12-30 digestive passages.

In the preparation method of the neural stem cell, purified neural stem cells can be obtained after 2-3 times of digestion and passage, then the neural stem cells with stable performance and large quantity can be obtained after 8-10 times of passage according to the method for digestion and passage, and then the neural stem cells can be obtained according to 10 times of passage⁷Freezing and storing the neural stem cells in a container; or further carrying out induction differentiation to cortical neurons, taking 12 th-30 th generation neural stem cells for induction, wherein the passage number has no influence on the induction differentiation to cortical neurons in the generation.

The neural stem cell prepared as described above is used for differentiation into cortical neurons having a function of generating action potentials.

A neural stem cell maintenance culture medium comprises a Neurobasal culture medium +1 × N2+1 × B27+10ng/ml EGF +10ng/ml FGF-2+20ng/ml BDNF +90-110ng/ml recombinant human Notch1 protein.

The neural stem cells prepared by the method are used for screening neuroprotective drugs.

According to the present invention, preferably, the screening of the neuroprotective drug, in particular, the neural stem cell is used for inducing differentiation to prepare cortical neurons having the function of generating action potentials.

Further preferably, the cortical neuron is used for screening for neuroprotective drugs.

The neural stem cell is used for inducing and differentiating to prepare cortical neurons with the function of generating action potentials, and the method for inducing and differentiating adopts the conventional technology.

The invention has the advantages of

The technical scheme of the invention can ensure that the neurosphere is stably proliferated, the phenomenon of neurosphere blackening is reduced, the activity of the neural stem cells is increased, the number of the neural stem cells induced into cortical neurons is increased, and the aim of screening various medicaments at one time can be realized.

Drawings

FIG. 1 is a photograph of morphological changes of humanized U1 iPSCs during nerve induction;

in the figure, scale a-i 250 μm; j-k. scale 100 μm;

the method comprises the following steps of A, cloning iPSCs, B, collecting induced iPSCs under the culture condition of an improved neural stem cell maintenance culture medium, digesting and passaging to form neural stem cells, forming a Rosette structure, collecting induced iPSCs under the culture condition of the improved neural stem cell maintenance culture medium, wherein the proliferation cycle of the neural stem cells is 4 days, collecting neurospheres generated when the induced iPSCs are passaged for the 1 st generation and the 4 th day, collecting induced iPSCs under the culture condition of the existing neural stem cell maintenance culture medium, wherein the proliferation cycle of the neural stem cells is 7 days, collecting induced neurospheres generated when the induced iPSCs are passaged for the 3 rd generation and the 4 th day, collecting induced neurospheres under the culture medium for the 12 th generation and the 4 th day, obtaining a neurospheres generated when the induced iPSCs are passaged for the 1 st generation and the 7 th generation, obtaining a neurospheres which are black and slow in proliferation, obtaining a MAP-7 th neural stem cell culture medium, wherein the MAP shows that the neurospheres are floated in the culture medium, and are obviously applied under the improved neural stem cells, the culture medium, the improved neural stem cells are obtained by the culture medium, the MAP 1, the MAP-7 culture medium, the MAP-7 neural stem cell culture medium, the invention is characterized by the MAP-7 neural stem cell culture medium, the;

FIG. 2 is a photograph of morphological changes in human DK iPSCs during neural induction;

in the figure, scale a-f. is 250 μm; scale bar 100 μm;

cloning iPSCs; rosette structure formation; collecting induced neural stem cells formed after digestion and passage of iPSCs under the culture condition of an improved neural stem cell maintenance culture medium, wherein the proliferation cycle of the neural stem cells is 4 days, the diagram C is neurospheres generated at the 4 th day of passage of the 1 st generation, and the diagram D is neurospheres generated at the 4 th day of passage of the 12 th generation; E-F, collecting induced neural stem cells formed after digestion and passage of iPSCs under the culture condition of the existing neural stem cell maintaining culture medium, wherein the proliferation cycle of the neural stem cells is 7 days, the E figure is neurospheres generated when the 1 st generation is passaged for the 7 th day, the F figure is passage for the 5 th generation for the 7 th day, visible cells float in the culture medium, and no obvious neurospheres are formed; G-H, applying the neuron differentiated from the neural stem cell cultured by the improved culture medium to perform immunofluorescence on the 10 th day, wherein the G picture is positive to a neuron marker MAP2, and the H picture is positive to a neuron marker TUJ 1;

FIG. 3 is a diagram of whole cell patch clamp recording action potentials of neurons induced to differentiate;

in the figure, A. example 1 induced differentiated neuron action potentials, neuron resting membrane potential was-72 mV, 1 action potential was recorded when the injected current reached 70pA, the action potential firing frequency increased with the increase of the injected current, and 7 action potentials were recorded when the injected current increased to 300 pA. B. Comparative example 1 induced differentiated neuronal action potential with resting membrane potential of-67 mV, no action potential was recorded when 70pA stimulation was given, no significant action potential was produced as the injected current increased, and no typical action potential waveform was recorded when the injected current increased to 300 pA.

Detailed Description

The invention is further illustrated with reference to specific examples, without the scope of protection being limited thereto.