Sodium ion channel mutant gene induced epilepsy brain tissue and application thereof

Abstract

The invention discloses an epilepsy brain tissue induced by a sodion channel mutant gene and application of the epilepsy brain tissue. Specifically, the epilepsy brain tissue is prepared by using a sodion channel mutant gene carrying SCN1A gene c.5768Agt; the epilepsy brain-like tissue is obtained through induction culture of the embryonic stem cells with G mutation, and the epilepsy brain-like tissue obtained through culture is used for pathogenesis, treatment and drug research and development of epilepsy diseases. Meanwhile, the invention also provides a construction method of the gene carrying the SCN1A gene c.5768Agt; the invention relates to a method for obtaining brain-like tissues through induction culture of G-mutated embryonic stem cells.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to the cultivation of epilepsy-like brain tissue induced by sodium ion channel mutant genes and the application of the cultivated brain-like tissue. Background Art

[0002] Sodium channel α-subunit type 1 (SCN1A) is the most clinically relevant epilepsy gene. Its mutation leads to a wide spectrum of epilepsy phenotypes, ranging from mild generalized epilepsy with febrile seizures plus (GEFS), moderate partial epilepsy with antecedent febrile seizures plus (PEFS) to severe Dravet syndrome. SCN1A plays an important role in maintaining sodium channel function and neuronal excitability. SCN1A knockout mice show spontaneous epileptic seizures.

[0003] At present, there is no research on using embryonic stem cells carrying the SCN1A gene c.5768A>G mutation to induce the cultivation of brain-like tissue and use this type of brain tissue as a model of epilepsy brain tissue to study epilepsy treatment and drug development experiments. Summary of the invention

[0004] The present invention provides a method for inducing and cultivating brain-like tissues by using embryonic stem cells carrying the SCN1A gene c.5768A>G mutation, and using the induced epilepsy-like brain tissues for the pathogenesis, treatment and drug development of epilepsy;

[0005] In order to achieve the above technical objectives, the present invention is implemented by the following technical solutions:

[0006] Application of SCN1A gene c.5768A>G mutation in inducing and cultivating epilepsy-like brain tissue;

[0007] Preferably, the epilepsy-like brain tissue is used for the pathogenesis, treatment and drug development research of epilepsy;

[0008] Another object of the present invention is to provide a method for inducing and cultivating epilepsy-like brain tissue using embryonic stem cells carrying the SCN1A gene c.5768A>G mutation:

[0009] S1: Cultivate embryonic stem cells carrying the SCN1A gene c.5768A>G mutation to obtain subcultured cells;

[0010] S2: The subcultured cells obtained in S1 were digested with disapase, suspended in stem cell culture medium NEM supplemented with 2 μM SB431542 and 2 μM DMH1 for 4 days, and then subjected to neural induction differentiation, i.e., from the 5th day after digestion, the stem cell culture medium NEM was replaced with neural induction medium NIM, and the suspension culture was continued until the 7th day, wherein the neural induction medium NIM was supplemented with 2 μM SB431542 and 2 μM CHIR factor, to obtain differentiated neuronal precursor cells, which were embryoid body-like neurospheres;

[0011] S3: The embryoid body-like neurospheres obtained in S2 were cultured in a neural induction medium NIM supplemented with 2 μM SB431542 and 2 μM CHIR factor until the 16th day to form neural rosette neural tube-like structure neural progenitor cells, which were then blown up and cultured in suspension for 7 days to obtain neural rosette neural tube-like structure neurospheres;

[0012] S4: The neural rosette neural tube-like structure neurospheres obtained in S3 were embedded in matrigel gel, and cultured in neural induction medium NIM supplemented with insulin, N2 and B27 until the 30th day to obtain matrigel-coated beads; the matrigel-coated beads were then cultured on a shaker until the 70th day; and then cultured in neural differentiation medium NDM supplemented with BDNF, GDNF, non-essential amino acids and cAMP until the 120th day to obtain human brain organoids containing VZ / SVZ / CP / MZ layer neurons.

[0013] S5: Sox2, Ctip2, Tbr1, SATB2, and Reelin staining were used to mark the neurons in the VZ / SVZ / CP / MZ layers of the human brain organoids to observe the polarity, proliferation, differentiation, and migration of the cells. Those expressing the above markers were identified as mature.

[0014] The beneficial effects of the present invention are:

[0015] The present invention uses the provided induction and cultivation method to induce and cultivate epileptic brain tissue from embryonic stem cells carrying the SCN1A gene c.5768A>G mutation, thereby providing a model that meets clinical standards for the pathogenesis, treatment and drug development of epilepsy. DETAILED DESCRIPTION

[0016] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0017] Example 1

[0018] Application of SCN1A gene c.5768A>G mutation in inducing and cultivating epilepsy-like brain tissue;

[0019] Preferably, the epilepsy-like brain tissue is used for the pathogenesis, treatment and drug development research of epilepsy;

[0020] Another object of the present invention is to provide a method for inducing and cultivating epilepsy-like brain tissue using embryonic stem cells carrying the SCN1A gene c.5768A>G mutation:

[0021] S1: Cultivate embryonic stem cells carrying the SCN1A gene c.5768A>G mutation to obtain subcultured cells;

[0022] S2: The subcultured cells obtained in S1 were digested with disapase, suspended in stem cell culture medium NEM supplemented with 2 μM SB431542 and 2 μM DMH1 for 4 days, and then subjected to neural induction differentiation, i.e., from the 5th day after digestion, the stem cell culture medium NEM was replaced with neural induction medium NIM, and the suspension culture was continued until the 7th day, wherein the neural induction medium NIM was supplemented with 2 μM SB431542 and 2 μM CHIR factor, to obtain differentiated neuronal precursor cells, which were embryoid body-like neurospheres;

[0023] S3: The embryoid body-like neurospheres obtained in S2 were cultured in a neural induction medium NIM supplemented with 2 μM SB431542 and 2 μM CHIR factor until the 16th day to form neural rosette neural tube-like structure neural progenitor cells, which were then blown up and cultured in suspension for 7 days to obtain neural rosette neural tube-like structure neurospheres;

[0024] S4: The neural rosette neural tube-like structure neurospheres obtained in S3 were embedded in matrigel gel, and cultured in neural induction medium NIM supplemented with insulin, N2 and B27 until the 30th day to obtain matrigel-coated beads; the matrigel-coated beads were then cultured on a shaker until the 70th day; and then cultured in neural differentiation medium NDM supplemented with BDNF, GDNF, non-essential amino acids and cAMP until the 120th day to obtain human brain organoids containing VZ / SVZ / CP / MZ layer neurons.

[0025] S5: Sox2, Ctip2, Tbr1, SATB2, and Reelin staining were used to mark the neurons in the VZ / SVZ / CP / MZ layers of the human brain organoids to observe the polarity, proliferation, differentiation, and migration of the cells. Those expressing the above markers were identified as mature.

[0026] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0027] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. Application of SCN1A gene c.5768A>G mutation in inducing and cultivating epilepsy-like brain tissue.

2. The use of the SCN1A gene c.5768A>G mutation in inducing and cultivating epilepsy-like brain tissue according to claim 1, characterized in that: The epilepsy-like brain tissue is used for the research on the pathogenesis, treatment and drug development of epilepsy.

3. A method for inducing and cultivating epilepsy-like brain tissue using embryonic stem cells carrying the SCN1A gene c.5768A>G mutation, characterized in that: The following methods are included: S1: Cultivate embryonic stem cells carrying the SCN1A gene c.5768A>G mutation to obtain subcultured cells; S2: The subcultured cells obtained in S1 were digested with disapase, suspended in stem cell culture medium NEM supplemented with 2 μM SB431542 and 2 μM DMH1 for 4 days, and then subjected to neural induction differentiation, i.e., from the 5th day after digestion, the stem cell culture medium NEM was replaced with neural induction medium NIM, and the suspension culture was continued until the 7th day, wherein the neural induction medium NIM was supplemented with 2 μM SB431542 and 2 μM CHIR factor, to obtain differentiated neuronal precursor cells, which were embryoid body-like neurospheres; S3: The embryoid body-like neurospheres obtained in S2 were cultured in a neural induction medium NIM supplemented with 2 μM SB431542 and 2 μM CHIR factor until the 16th day to form neural rosette neural tube-like structure neural progenitor cells, which were then blown up and cultured in suspension for 7 days to obtain neural rosette neural tube-like structure neurospheres; S4: The neural rosette neural tube-like structure neurospheres obtained in S3 were embedded in matrigel gel, and cultured in the neural induction medium NIM supplemented with insulin, N2 and B27 until the 30th day to obtain matrigel-coated beads; the matrigel-coated beads were then cultured on a shaker until the 70th day; and then cultured in the neural differentiation medium NDM supplemented with BDNF, GDNF, non-essential amino acids and cAMP until the 120th day to obtain human brain organoids, which contained VZ / SVZ / CP / MZ layer neurons; S5: Sox2, Ctip2, Tbr1, SATB2, and Reelin staining were used to mark the neurons in the VZ / SVZ / CP / MZ layers of the human brain organoids to observe the polarity, proliferation, differentiation, and migration of the cells. Those expressing the above markers were identified as mature.