阅读说明：本技术 高效分离人脑组织来源的神经干细胞的方法 (The method for efficiently separating the neural stem cell in human brain tissue source ) 是由 朱景敏 南炫 南都铉 洪丞彻 延济英 于 2018-02-23 设计创作，主要内容包括：本发明涉及培养和分离神经干细胞的方法,通过该方法神经干细胞可以大规模快速增殖并高效分离,以及涉及由其培养和分离的中风患者来源的人成体神经干细胞,其用于移植。(The present invention relates to cultivating and the method for separation neural stem cell, it extensive fast breeding and can be efficiently separated by this method neural stem cell, and be related to people's adult neural stem cell by its culture and isolated paralytic source, be used to transplant.)

1. a kind of method of culture of neural stem cells neural, this method comprises:

Cell is collected from brain tissue；

It is collected with clostridiopetidase A and deoxyribonuclease I or papain, cysteine and deoxyribonuclease I processing Cell to separate individual cells；

Individual cells are assigned in two or more pipes, the individual cells in each pipe are mixed with Percoll, and will each be mixed Object centrifugation is closed to recycle cell；

The cell of originally culture recycling；With

Secondary culture is carried out to primary cultured cell.

2. according to the method described in claim 1, wherein the neural stem cell is people's adult neural stem cell for transplanting, It is originated from paralytic.

3. according to the method described in claim 1, wherein carrying out the originally culture or secondary culture using adhere-wall culture method.

4. according to the method described in claim 1, wherein the secondary culture progress is three times or less secondary.

5. a kind of people's adult neural stem cell for transplanting, derived from using according to claim 1 to described in any one of 4 Method culture brain tissue.

Technical field

The present invention relates to a kind of culture of neural stem cells neural and the methods that efficiently separate neural stem cell, and thus culture and People's adult neural stem cell for transplanting of separation.

Background technique

Apoplexy is defined as tissue blood flow disorder.This may be the illness of brain privileged site, such as, it may be possible to When cardiac pumping is completely impaired, all blood flows are substantially reduced.More commonly, when the blood vessel blockage or rupture by brain When interrupting the blood flow of brain privileged site, apoplexy will occur.Angiemphraxis usually occurs in the artery to blood supply in brain, For example, being occurred by forming embolism or thrombus, this is referred to as ishemic stroke.

Apoplexy is the third-largest cause of death of developed country.In the U.S., due to apoplexy it is dead caused by medical expense and Cap loss estimation is about 40 trillion dollars.Meanwhile the unique method of effectively treatment apoplexy is application thrombolytic agent, such as Tissue plasminogen activator (t-PA), enzymatic lysis generate the protein of thrombus to eliminate thrombus.However, the application of t-PA It may cause the side effect of such as bleeding etc when too late, therefore t-PA may be applied only in 3 hours after the appearance of the first symptom With.In addition, t-PA can only be used to ishemic stroke, and when being applied to hemorrhagic stroke, such as death can usually occur The side effect of class.These curing apoplexy methods are only used for emergency treatment or postpone the progress of disease, but still not basic control Treatment measure.

It can be divided into the stem cell of internal various types tissue, i.e. neoblast, can be divided under proper condition Various histocytes, therefore regeneration that can be applied to damaged tissues etc. is treated.It is expected that stem-cell therapy agent can make to be damaged Nerve regneration, and the known various substances for improving impaired environment by secretion participate in regenerating, and directly participate in regenerating.Mesh Before, the research about mescenchymal stem cell is clinically state-of-the-art, but treats apoplexy or other nervus retrogressions for it There are contradictory viewpoints for the validity of disease.Although the neural stem cell of fetal origin is to the clinic of apoplexy and Patients of Spinal Test it is in progress, but still there are many technical problem and ethics problem.

Summary of the invention

[technical problem]

Therefore, it was found by the inventors of the present invention that the mind separated from the brain tissue extracted during the operation of burst paralytic Rapid, high volume it can be proliferated and efficiently separate and cultivating under given conditions through stem cell, so as to complete the present invention.

[technical solution]

In order to achieve the above objectives, the present invention provides a kind of methods of culture of neural stem cells neural, comprising: receives from brain tissue Collect cell；With clostridiopetidase A and deoxyribonuclease I (DNase I) or papain, cysteine and DNA The cell that enzyme I processing is collected is to separate individual cells；Individual cells are assigned in two or more pipes, it will be single in each pipe Cell is mixed with Percoll, and each mixture is centrifuged to recycle cell；The cell of originally culture recycling；And to primary training It supports cell and carries out secondary culture.

In an embodiment of the invention, brain tissue can be the brain group extracted during the operation of wind patient in a burst It knits, for example, the tissue extracted from brain or spinal cord.For example, brain tissue may include tissue, stroke surgery sample from temporal epilepsy (including temporal lobe), wound tissue's (including temporal lobe) etc..

In an embodiment of the invention, neural stem cell can be people's adult neural stem cell for transplanting, From paralytic.People's adult neural stem cell can be with autotransplantation or allograft.

In an embodiment of the invention, adhere-wall culture method can be used and carry out originally culture and/or secondary culture.

In an embodiment of the invention, secondary culture can carry out three times or less secondary.

The present invention also provides a kind of people's adult neural stem cells from brain tissue for transplanting, by according to this The method culture of the culture of neural stem cells neural of invention.

[beneficial effect]

The present invention can not only be solved by separating from the brain tissue of paralytic itself with culture of neural stem cells neural Ethics problem, but also transplantable people's adult neural stem cell can be easily provided.

According to the present invention, neural stem cell rapid, high volume can be proliferated under specific separation and condition of culture.Particularly, Separated from brain tissue it is unicellular can assign in two pipes and mix with Percoll to recycle cell, so that nerve cord is thin About 2 times of the yield increase of born of the same parents.Further, it is possible to use adhere-wall culture method carries out originally culture or secondary culture, thus with existing Spherical method compare, can steadily improve culture efficiency and improve purity.In addition, sufficient amount of patient's body to be transplanted to Cell can be by carrying out three times or less secondary culture is cultivated, so as to fast breeding culture, especially will be thin In born of the same parents' Rapid transplant to burst paralytic's body.

Brief Description Of Drawings

Fig. 1 shows the process of embodiment culture of neural stem cells neural according to the present invention.

Fig. 2 shows the cell numbers of an embodiment according to the present invention handled according to Percoll.

Fig. 3 shows the method for the differentiation neural stem cell of an embodiment according to the present invention.

Fig. 4 is a display embodiment according to the present invention, is confirmed neural stem cell differentiating at nerve according to differentiation condition One group of image of the result of member and the ability of astroglia.

Specific embodiment

The present invention provides a kind of methods of culture of neural stem cells neural, comprising: cell is collected from brain tissue；Use clostridiopetidase A And deoxyribonuclease I or papain, cysteine and deoxyribonuclease I processing cell are to separate individually carefully Born of the same parents；Individual cells are assigned in two or more pipes, the individual cells in each pipe are mixed with Percoll, and will each be mixed Object centrifugation is closed to recycle cell；The cell of originally culture recycling；And secondary culture is carried out to primary cultured cell.

Neural stem cell of the invention is separated from the brain tissue extracted during apoplexy patients surgery, can be certainly Body is transplanted in paralytic's body or allograft.

In general, using Percoll to the unicellular process for being removed impurity separated from biological tissue.Of the invention In method, it can assign to unicellular in two pipes and mixed with Percoll, be then centrifuged for recycle cell, thus and in tradition Single pipe in it is unicellular with Percoll mixing the case where compared with, about 2 times of cell yield increase (see Fig. 2).

Culture medium for originally culture or secondary culture of the invention can be commonly used in any of culture stem cell Culture medium.It is, for example, possible to use the culture mediums for containing serum (such as fetal calf serum, horse serum and human serum).It can be used for this hair Bright culture medium can be, for example, RPMI is serial, such as RPMI 1640 (Moore et al., J.Amer.Med.Assoc.199: 519 (1967)), the MEM (Eagle minimum essential medium, Eagle, H.Science 130:432 (1959)) of Eagles, α- MEM (Stanner, C.P. et al., Nat.New Biol.230:52 (1971)), Iscove's MEM (Iscove, N. et al., J.Exp.Med.147:923 (1978)), 199 culture mediums (199medium) (Morgan et al., Proc.Soc.Exp.Bio.Med., (1950) 73:1), CMRL 1066, F12 (Ham, Proc.Natl.Acad.Sci.USA 53:288 (1965)), (Dublecco improves Eagle training by F10 (Ham, R.G.Exp.Cell Res.29:515 (1963)), DMEM Support base, Dulbecco, R. et al., Virology8:396 (1959)), the mixture of DMEM and F12 (Barnes, D. et al., Anal.Biochem.102:255 (1980)), MB752/1 (Waymouth, the C.J.Natl.Cancer of Way mouth Inst.22:1003 (1959)), the 5A of McCoy (McCoy, T.A., et al., Proc.Soc.Exp.Biol.Med.100:115 (1959)) and MCDB is serial (Ham, R.G. et al., In Vitro 14:11 (1978)), and but the invention is not restricted to this.Culture Base may include other components, such as antibiotic or antifungal agent (such as penicillin and streptomysin), glutamine etc..

The secondary culture of stem cell usually carries out seven times or nine times or more times.However, in the method for the invention, due to Mix and use adhere-wall culture method in two Guan Zhongyu Percoll, can carry out three times or less secondary culture process, from And the neural stem cell of the sufficient amount for transplanting can be obtained.

With reference to the embodiment being described below in detail, advantages and features of the invention and the method for realizing them will become aobvious And it is clear to.Hereinafter, the present invention will be described in further detail with reference to following embodiment.However, provide these embodiments be for The specific explanations present invention, the range that is not intended to be limiting of the invention.