CN108853589A - Application of the humanization class brain organ in cerebral injury motor disorder - Google Patents
Application of the humanization class brain organ in cerebral injury motor disorder Download PDFInfo
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Abstract
The present invention relates to a kind of new applications of humanization class brain organ, i.e. the application in the graft of preparation treatment cerebral injury.Its advantage is shown：Compared with cerebral trauma group, class brain transplant can promote the nerve regneration of cerebral trauma motor cortex area and lateral ventricle region；7 days after transplanting, 14 days, 28 days, cerebral trauma motor cortex area can be detected class brain tissue cell presence, it was demonstrated that the feasibility survived after class brain transplant.Neuromuscular function test and balance beam test result show that compared with cerebral trauma group, postoperative 7 days, class brain transplant can obviously improve neuromuscular function, have preferable balance beam locomitivity.
Description
Technical field
The present invention relates to brain science technical fields, specifically, being humanization class brain organ in cerebral injury dyskinesia disease
Application in disease.
Background technique
Cerebral injury disease incidence with higher, the death rate, but it is a lack of effective treatment, it is society very serious and health
Problem.People's inductive pluripotent stem cells (iPSCs) in vivo or in vitro in the environment of, can break up generate almost all type it is thin
Born of the same parents, for simulating human developmental and disease generation, drug screening, cell replacement therapy etc..A large amount of preclinical studies show dry thin
Born of the same parents' transplantation treatment can promote the recovery of cerebral injury.But traditional monolayer cell culture may only control iPSC targeting be divided into
Specific cell type can not simulate the tissue assembling characteristic of three-dimensional (3D) cell in biosystem, cannot achieve tissue
The function replacement and repairing and treating of damage truly.
In recent years, gradually increase for the 3D culture studies of iPSCs, give its specific induced differentiation factor and culture item
Part has successfully obtained the organoids such as intestines, kidney, retina (organoids), including class brain organ (cerebral organoids,
CO).2013, class brain organ was reported on Nature magazine for the first time, opened the new sign of brain growth and cerebral disease correlative study
Journey.In this class brain nurturing research, first passage 3D culture systems obtain class brain organ, success mould in rotation generating bottle
Cortex developmental process is intended, has been cited as " the big Progress & New Products in the world ten in 2013 ".Then, the class brain of Cell magazine ran in 2016
The optimization breeding method of organ, reduces cultivation cost, and the brain area specific differentiation for realizing class brain organ is cultivated, at
Function obtains class forebrain, midbrain and hypothalamus sample organ.The structure currently, breeding method of full brain and brain area specificity class brain has succeeded
It builds, application is gradually promoted, for example, class brain is for simulating microcephaly disease caused by zika virus, probing into antenatal alcohol
Exposure is to the neurite outgrowth of class brain, the influence of nerve regneration；It is dry using the inductive pluripotent in patients with Alzheimer disease source
Cell combination presenilin-1 mutation, simulates cortex nerve degeneration microenvironment；Utilize the simulation nicotine exposure of class brain chip
Deng.
The applying date is that on May 04th, 2018 Chinese patent application 201810417891.9 is related to humanization class brain device
The cultural method of official and for constructing ischemia model, still, superiority of the class brain organ as brain organ moves in cerebral injury and hinders
Hinder after being transplanted in disease, if the dyskinesia etc. after can improving cerebral injury there is no research to report.
Summary of the invention
The object of the present invention is to probe into class brain brain motor cortex damage transplanting in feasibility, if can in body brain
Tissue connection, if the nerve regneration after promoting cerebral injury, if improve dyskinesia caused by motor cortex damages, people is provided
A kind of new application of source class brain organ.
To achieve the above object, the technical solution adopted by the present invention is that：Class brain organ is in preparation treatment cerebral injury and/or brain
Application in the graft of sequelae caused by damaging.
Further, the class brain organ is humanization class brain organ.
Further, the humanization class brain organ is the humanization class brain organ of 3D structure.
Further, the humanization class brain organ is successively undergone differentiation of germinal layers, the outer embryo of nerve by human pluripotent stem cells
Layer differentiation, neuroepithelial tissue are formed, and induction obtains the humanization class brain organ of 3D structure in rotation generating bottle.
Further, the humanization class brain organ is prepared via a method which to obtain：
(1) kind of human pluripotent stem cells are inscribed in ultralow absorption orifice plate, obtain embryoid body like cell cluster；
(2) continue induction differentiation, embryoid body like cell cluster starts differentiation of germinal layers feature occur, formed on initial nerve
Skin sample tissue, and be transferred in new ultralow absorption orifice plate and continue to cultivate；
(3) neural epithelium sample tissue continues induction differentiation, and experience Matrigel glue drips coated static culture, forms nerve
Epithelial sprout sample tissue starts the differentiation for carrying out class brain organ；
(4) the neural epithelium bud sample tissue for being coated with Matrigel glue drop is transferred in rotation generating bottle, continues to cultivate, obtains
The class brain organ that brain function subregion must be formed, express forebrain marker and choroid plexus marker.
Further, the cerebral injury is traumatic brain injury.
Further, sequelae caused by the cerebral injury is cerebral injury motor disorder.
Further, the class brain organ improves dyskinesia caused by motor cortex damages.
Further, the class brain organ promotes the nerve regneration in cerebral trauma motor cortex area and lateral ventricle region.
Further, the class brain organ improves neuromuscular function.
The invention has the advantages that：
Humanization class brain organ is used for cerebral injury transplanting for the first time by the present invention, and is verified by experiments：Compared with cerebral trauma group,
Class brain transplant can promote the nerve regneration of cerebral trauma motor cortex area and lateral ventricle region；7 days after transplanting, 14 days, 28 days, brain create
Hurting motor cortex area can be detected the presence of class brain tissue cell, it was demonstrated that the feasibility survived after class brain transplant.Neuromuscular function
It can test and balance beam test result shows that compared with cerebral trauma group, postoperative 7 days, class brain transplant can obviously improve neuromuscular
Function has preferable balance beam locomitivity.
Detailed description of the invention
Attached drawing 1：The schematic diagram (being shot under inverted microscope) of class brain organ difference cultivation stage.
Attached drawing 2：The class brain organ immunofluorescence (being shot under laser confocal microscope) of different development times.In class brain
The different phase that organ is cultivated, take culture 15 days, 30 days, 60 days class brains carry out frozen section and immunofluorescence dyeing.Pass through
Labeled neural stem cells marker SOX2 and neuron marker Tuj-1, display is with the extension for cultivating the time, stem cell ratio
It reduces, neuron ratio increases, i.e., class brain organ is gradually reached maturity.
Attached drawing 3：Class brain orga- nogenesis " forebrain and choroid plexus " brain function subregion immunofluorescence (laser confocal microscope
Lower shooting).Class brain organ culture carried out frozen section and immunofluorescence dyeing to 75 days, and forebrain and choroid plexus have been expressed in display
Marker prompts to have formed brain function subregion, i.e., successfully constructs class brain organ.
Attached drawing 4：Rat brain cortex damage zone histogenic immunity fluorescence Typical Representative figure.
Attached drawing 5：Rat brain cortex damage zone histogenic immunity fluorescence Typical Representative figure.
Attached drawing 6：Lateral ventricle of rat brain district's groups active immunity fluorescence Typical Representative figure.
Attached drawing 7：Rat brain cortex damage zone immunofluorescence BrdU/Nestin double-labeled cell number.Class brain transplant group (TBI+
CO), BrdU/Nestin double-labeled cell number is significantly more than class cerebral trauma group (TBI).* *, P<0.001；*, P<0.01；*, P<
0.05.N=12.
Attached drawing 8：Rat brain cortex damage zone immunofluorescence BrdU/DCX double-labeled cell number.Class brain transplant group (TBI+CO),
BrdU/DCX double-labeled cell number is significantly more than class cerebral trauma group (TBI).*, P<0.01；*, P<0.05.N=12.
Attached drawing 9：After class brain transplant, class brain tissue cell (human specific cytoplasm egg is can be detected in rat brain cortex damage zone
White marker STEM121).
Attached drawing 10：The scoring of rat nerve muscle function.Class brain transplant after sham-operation group (Sham), wound group (TBI), wound
Group (TBI+CO), every group of rat n=6.The results show that TBI group neuromuscular function is remarkably decreased, and TBI compared with Sham group
+ CO group is without significant difference；Compared with TBI group, TBI+CO group neuromuscular function is obviously improved.(***P<0.001,#P<
0.05)。
Attached drawing 11：The scoring of rat balance beam.Class brain transplant group (TBI after sham-operation group (Sham), wound group (TBI), wound
+ CO), every group of rat n=6.The results show that compared with Sham group, TBI group and TBI+CO group balance beam athletic performance it is significant under
Drop；Compared with TBI group, TBI+CO group balance beam athletic performance is obviously improved, and has statistical significance.(***P<0.001, * P<
0.05,#P<0.05)。
Attached drawing 12：After class brain transplant 7 days, the presence of class brain tissue cell, card can be detected in cerebral trauma motor cortex area
The feasibility survived after bright class brain transplant, class brain transplant can obviously improve neuromuscular function, have the movement of preferable balance beam
Ability.
Specific embodiment
It elaborates below with reference to embodiment to specific embodiment provided by the invention.
Embodiment 1：The culture of class brain organ
According to bibliography (Nature 2013；501:373-379 and Nat Protoc 2014；9:2329-2340), it passes through
It is a series of to grope and improve, ultralow absorption orifice plate and rotating biological generator culture systems are successively used, human pluripotent stem cells are first
By go through differentiation of germinal layers, neuroderm differentiation, neuroepithelial tissue formed, rotation generating bottle in induction obtain 3D knot
The humanization class brain organ of structure, cultivation stage are as follows.
First stage：In 96 orifice plates of ultralow absorption, according to viable cell density 9 × 104A/mL, every hole are inoculated with 150 μ L, obtain
It obtains embryoid body like cell cluster (Fig. 1, shown in 2 days).
Second stage：Continue induction differentiation, embryoid body starts differentiation of germinal layers feature occur, carries out inducing initial nerve
Epithelial tissue (Fig. 1, shown in 5 days) is transferred in 24 orifice plates of ultralow absorption and continues to cultivate.
Phase III：Neural epithelium sample tissue continues induction differentiation, and experience Matrigel glue drips coated static culture, shape
At neural epithelium bud sample tissue, that is, start the differentiation (Fig. 1, shown in 15 days) for carrying out class brain organ.
Fourth stage：The tissue that above-mentioned coating Matrigel glue drips is transferred in rotation generating bottle, with the speed of 85rpm
Degree, continues to cultivate.Class brain organ-tissue is further differentiated into ripe, and volume becomes larger (Fig. 1, shown in 30 days).With the cultivation time
Extend, neural stem cell ratio gradually decreases in class brain organ-tissue, and neuron ratio increases (Fig. 2), finally, in class brain organ
Brain function subregion is formed, forebrain marker and choroid plexus marker (Fig. 3) is expressed, successfully obtains class brain organ.
Embodiment 2：Class brain organ is transplanted for cerebral injury
Experimental method
One, rat brain trauma model and class brain transplant are constructed
Adult SD rats, weight 300g or so are purchased from Shanghai Slac Experimental Animal Co., Ltd..On the day before art,
Ciclosporin A (LC Labs C6000) 10mg/kg is given in intraperitoneal injection.Intraperitoneal injection of anesthesia rat, is then shaved using shaver
Head hair, and be fixed on mouse stereotaxic apparatus.75% alcohol wipe rat scalp disinfection, with scalpel along brain middle line
The longitudinal incision for being about 4cm is scratched, skull surface fascia is separated, sufficiently exposes skull.Half brain on the right side of rat, is bored with cranial drill
Open the rectangle skull window of long 1.5cm, width 0.6cm, exposed Rats cortex.Mouse stereotaxic apparatus is respectively on the right side of bregma
1.5mm, front 1.0mm or rear 2.0mm are the center of circle, drill through 2 connections in rat brain motor cortex using biopsy punch
The cerebral cortex of diameter 3mm, depth 2mm, cause traumatic brain injury.Cotton ball soaked in alcohol hemostasis by compression.Bone wax closes skull window,
It sews up the incision, incision smears the anti-infection of erythromycin ointment.It is postoperative to inject ciclosporin A every other day, until putting to death rat.
For cerebral trauma transplantation group, after hemostasis, class brain transplant is directly carried out in cortex wound area, number of days is cultivated in selection
45-55 days class brains are transplanted, and are then carried out bone wax and are closed skull window.For sham-operation group, only carries out operation of opening cranium, do not damage
Evil brain parenchym then carries out bone wax and closes skull window.
For needing to detect the rat of BrdU index, first post-operative day starts that BrdU (Sigma B5002) is injected intraperitoneally, agent
Measure 21mg/kg.Injection time was first post-operative day to the 7th day, was then given every other day.
Two, brain tissue is drawn materials and is fixed
Anesthetized rat, exposed heart of cutting open the belly.Infusion pump input end injection needle is inserted into left ventricle, while with scissors on the right side
Cut off notch in atrium.Infusion pump is opened, with the quick perfusion of 1 × PBS of warm, rinses blood.After blood is rinsed well, use instead
4% paraformaldehyde continues perfusion fixation.Then, rat cerebral tissue is taken out in dissection, is put into 4% paraformaldehyde, fixed, saves.
Three, brain tissue immunofluorescence assay
(1) brain tissue frozen section：Frozen section is set, after modifying tissue block, brain tissue is coated into embedding medium and carries out ice
Freeze, adjustment slice thickness is 8 μm, carries out coronal-plane slice.It is suitable according to being sliced by the tissue adsorptions cut on anticreep glass slide
Sequence number is put, -20 DEG C of storages.
(2) new using nerve regneration (BrdU/Nestin), neuron after the evaluation cerebral trauma of immunohistochemistry fluorescence chemical method
Raw and migration (BrdU/NeuN, BrdU/DCX), humanizing cells migration and existence (STEM121).This experiment detects class brain and moves
7 days after plant, 14 days, nerve regneration in 28 days and humanizing cells' Survival.
1) brain piece is placed 5 minutes, to remove vapor after -20 DEG C of taking-ups in 63 DEG C of dry insulating boxs.
2) brain piece is put into aquation 10 minutes in 0.01M PBS.
3) it is repaired using Tris-EDTA antigen retrieval buffers (pH 8.0), micro-wave oven is fiery 10 minutes low.
4) 1 × PBS is quiet washes 3 times, every time 5 minutes.(note that brain tissue piece for needing to detect BrdU index needs to carry out
Step 5,6,7；Conversely, directly carrying out step 8)
5) 1M HCL is incubated at room temperature 30 minutes.
6) 0.1M boric acid (pH 8.5) is incubated at room temperature 5 minutes.
7) 1 × PBS is quiet washes 3 times, every time 5 minutes.
8) 0.2%Triton X 100, room temperature 15 minutes, to increase permeability of cell membranes.
9) 1 × PBS is quiet washes 3 times, every time 5 minutes.
10) donkey in secondary antibody source or lowlenthal serum closing, are incubated at room temperature 2 hours.
11) primary antibody (BrdU 1:100；Nestin 1:250；DCX 1:250；STEM121 1:250) it is incubated for, 4 DEG C overnight.
12) 1 × PBS is quiet washes 3 times, every time 5 minutes.
13) secondary antibody (Alex Flour 488 or Alex Flour 594 1:500) it is incubated for, room temperature is protected from light 90 minutes.
14) 1 × PBS is protected from light quiet wash 3 times, every time 5 minutes.
15) instant DAPI, room temperature are protected from light incubation 15 minutes.
16) 1 × PBS is protected from light quiet wash 3 times, every time 5 minutes.
17) anti-fluorescence quenching, coverslip mounting, observation of taking pictures under laser confocal microscope is added dropwise.
Four, motor behavior detects
(1) neuromuscular function detects
The Testing index Score index is as shown in the table：
1 neuromuscular function of table detects Score index
(2) balance beam Function detection
All rats are preoperative to carry out one-week balance beam test, uses the balance beam width to be for 3 centimetres, length
100 centimetres, guarantee is preoperative can to pass through balance beam.Specific Score index is as shown in the table：
2 balance beam Function detection Score index of table
| Testing index | Score |
| It will not fall across balance beam | 0 |
| Across balance beam and there is the distance less than 50% shoes occur | 1.0 |
| Across balance beam and there is the distance greater than 50% shoes occur | 2.0 |
| Balance beam can be passed through but Ipsilateral hind leg cannot help to move forward | 3.0 |
| It cannot pass through balance beam but balance can kept above | 4.0 |
| Fall from balance beam | 5.0 |
Experimental result
One, the nerve regneration of at class brain transplant promotion rat cerebral trauma hindbrain cortical lesions and lateral ventricle region
The nerve regneration at rat brain damage cortex is detected after class brain transplant 7 days, 14 days, 28 days, uses BrdU/
Nestin, BrdU/DCX mark nerve regneration and newborn neuron respectively.The results show that compared with cerebral trauma group (TBI), class brain
Transplantation group (TBI+CO) nerve regneration is obvious more (Fig. 4, Fig. 5 and Fig. 6).After class brain transplant at 7 days, 14 days, 28 days, class brain is moved
Plant group (TBI+CO) BrdU/Nestin, BrdU/DCX double-labeled cell is significantly more than cerebral trauma group (TBI), but as time goes by,
Class brain transplant group (TBI+CO) BrdU/Nestin, BrdU/DCX double-labeled cell gradually decreases (Fig. 7 and Fig. 8).
Two, after class brain transplant 7 days, 14 days, 28 days, class brain cell can still survive
Human specific cytoplasmic protein marker STEM121, can express, including brain, liver and pancreas in the cell of various tissues
Gland, and highest is expressed in central nervous system cell.Therefore, this experiment uses antibody STEM121,7 days after class brain transplant,
Immunofluorescence dyeing was carried out to rat brain slice in 14 days, 28 days, the results showed that (Fig. 9), 7 days after class brain transplant, 14 days, 28 days
The presence of humanizing cells can be detected in cortex damage zone.But as time goes by, in the rat brain of class brain transplant
Humanizing cells' number gradually decreases.Result above proves, the survival feasibility of class brain transplant after rat cerebral trauma.
Three, class brain transplant can improve rat motor obstacle
Motor behavior detection display, compared with cerebral trauma group, postoperative 7 days, class brain transplant group had preferable Neuromuscular
Meat function (table 3, Figure 10), stronger balance beam locomitivity (table 4, Figure 11).
The scoring of 3 rat nerve muscle function of table
The scoring of 4 rat balance beam of table
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, under the premise of not departing from the method for the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (10)
1. the application in the graft of class brain organ sequelae caused by preparation treatment cerebral injury and/or cerebral injury.
2. application according to claim 1, which is characterized in that the class brain organ is humanization class brain organ.
3. application according to claim 2, which is characterized in that the humanization class brain organ is the humanization of 3D structure
Class brain organ.
4. application according to claim 3, which is characterized in that the humanization class brain organ is by human pluripotent stem cells elder generation
By go through differentiation of germinal layers, neuroderm differentiation, neuroepithelial tissue formed, and rotation generating bottle in induction obtain 3D structure
Humanization class brain organ.
5. application according to claim 4, which is characterized in that the humanization class brain organ is prepared via a method which
It obtains：
(1) kind of human pluripotent stem cells are inscribed in ultralow absorption orifice plate, obtain embryoid body like cell cluster；
(2) continue induction differentiation, embryoid body like cell cluster starts differentiation of germinal layers feature occur, forms initial neural epithelium sample
Tissue, and be transferred in new ultralow absorption orifice plate and continue to cultivate；
(3) neural epithelium sample tissue continues induction differentiation, and experience Matrigel glue drips coated static culture, forms neural epithelium
Bud sample tissue starts the differentiation for carrying out class brain organ；
(4) the neural epithelium bud sample tissue for being coated with Matrigel glue drop is transferred in rotation generating bottle, continues to cultivate, obtain shape
At brain function subregion, the class brain organ of expression forebrain marker and choroid plexus marker.
6. application according to claim 1, which is characterized in that the cerebral injury is traumatic brain injury.
7. application according to claim 1, which is characterized in that sequelae caused by the cerebral injury is cerebral injury movement
Disorder disease.
8. application according to claim 1, which is characterized in that caused by the class brain organ improves motor cortex damage
Dyskinesia.
9. application according to claim 1, which is characterized in that the class brain organ promote cerebral trauma motor cortex area and
The nerve regneration of lateral ventricle region.
10. application according to claim 1, which is characterized in that the class brain organ improves neuromuscular function.
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| CN113631701A (en) * | 2019-01-23 | 2021-11-09 | 英国研究与创新组织 | Choroid plexus organoids and methods of producing the same |
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| Title |
|---|
| 来源：中国科学报: "《自然》：科学家用干细胞培育人脑发育3D模型", 《科研快讯》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113631701A (en) * | 2019-01-23 | 2021-11-09 | 英国研究与创新组织 | Choroid plexus organoids and methods of producing the same |
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