CN110452925A - A kind of method and its application of the vivo tracking cerebral nerve connection based on magnetic resonance imaging - Google Patents
A kind of method and its application of the vivo tracking cerebral nerve connection based on magnetic resonance imaging Download PDFInfo
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Abstract
The method and its application for the vivo tracking cerebral nerve connection based on magnetic resonance imaging that the invention discloses a kind of, using absorbing the adeno-associated virus variant rAAV2-retro of reverse transportation characterization with axon ends as carrier, the gene of ferritin Ferritin is loaded in its genome, it is packaged into Recombinant rAAV 2-retro-CAG-Ferritin-WPRE-pA, in the brain by the injection of above-mentioned recombinant virus, virus infection neuron simultaneously expresses Ferritin in neuron, observes the nerve that other brain areas and injection site are infected between neuron in conjunction with MRI and connects.Present invention combination magnetic resonance imaging and rAAV2-retro-CAG-Ferritin-WPRE-pA realize the vivo tracking of cerebral nerve connection, can be used for long time-histories neuromechanism loop tracer or nervous function parsing.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of vivo tracking cerebral nerve company based on magnetic resonance imaging
The method connect also relates to application of this method in the tracer of neuromechanism loop and function parsing.
Background technique
Cerebral nerve network exercises important function in body, and it is every living to participate in study, memory, cognition and mood etc.
It is dynamic, and the exception of a variety of the nervous system diseases and neural network is closely related.It is big for understanding to parse cerebral nerve structural network
Function, working mechanism, development, degeneration and the occurrence and development of neurological disease of brain have important role.In recent years, thermophilic nerve
Virus, such as rabies viruses, vesicular stomatitis virus, herpes simplex virus, Pseudorabies virus, have been widely used for neural network
Parsing, the neural circuitry tracing method based on neurotropic virus provide bulk information for neural network parsing.However, because mesh
The reporter gene that preceding neurotropic virus carries is mostly fluorescin, and the method usually requires to infect neuron one in neurotropic virus
Animal is sacrificed after the section time, in vitro tissue is sliced and carries out fluorescence imaging observation, cannot achieve neurotropic virus in the living body
Observation, be not just suitable for yet chronic, long time-histories, need the experiment of self-contrast before and after the processing.
Magnetic resonance imaging is that one kind combines Noninvasive, high-penetration, is suitable for soft tissue and higher time-space resolution
Etc. the imaging method of a variety of advantages, it has been widely used in clinical diagnosis and scientific research.In recent years, it is examined based on magnetic resonance imaging
The method for surveying cerebral nerve network structure and function is rapidly developed and is widely applied, wherein diffusion tensor imaging (DTI)
It can be used for the prediction and tracer of cerebral nerve network anatomical structure with manganese ion-enhanced MRI (MEMRI).However, DTI
Trend of the method based on mathematical model prediction fibre bundle, result cannot directly be regarded as the connection between brain area, and its
The projecting direction of unpredictable fibre bundle, therefore the upstream-downstream relationship in nerve connection can not be obtained.The neural network of MEMRI is shown
Track result is often read as forward connecting across multistage synaptic neural, but its tracer manganese ion can be expanded by cerebrospinal fluid and blood vessel
It dissipates, causes the complexity and non-specificity of traces.In addition being higher than certain density heavy metal manganese ion has relatively by force body
Toxicity, influence animal normal physiological function and even directly contribute animal dead.
Ferritin is the highly conserved existing albumen in a variety of organisms, be internal storage iron principal mode it
One.Ferritin forms a spherical housing by 24 subunits, and the cavity among shell can load iron ion and be formed paramagnetic
Iron oxide crystal, to influence MRI signal.Therefore the gene of Ferritin is considered as a kind of ideal magnetic resonance imaging report
Gene.Proof has been reported, the adenovirus for carrying Ferritin can infect neuron, and cause the T2 of injection site part brain area
The dark signal of weighted sum T2* weighting picture.However since adenovirus can only infect injection site local neuron, can not exist across cynapse
It is propagated between neuron and nerve, therefore can not show the link information between neuron.
AAV is a kind of single-stranded DNA viruses, as genophore, have immunogenicity is low, host cell species mostly and base
Because expressing the advantages such as lasting, therefore it has been widely used in neural circuitry tracer and gene therapy.The AAV of different serotypes
With not exactly the same infection characterization, 2016 Nian You study groups obtain rAAV2- by the method that artificial orientation evolves
There are efficient axon ends to absorb and pass through reverse transport to projection neuron cell space by the AAV of retro serotype, this serotype
Characteristic.Therefore the AAV carrier of rAAV2-retro serotype is displayed for the nerve from Nerve Terminals In The Human Skin to pericaryon
Project information, it can also be used to for the channel genes for the neuron for projecting specific brain area.
Summary of the invention
The method for the Vivo Tracking cerebral nerve connection based on magnetic resonance imaging that the purpose of the present invention is to provide a kind of, can
Realize the nerve connection between showing Different brain region in living body brain.
The Vivo Tracking cerebral nerve connection based on magnetic resonance imaging that it is another object of the present invention to provide a kind of
Method the tracer of neuromechanism loop and function parsing in application.
To achieve the goals above, the invention adopts the following technical scheme:
A method of the vivo tracking cerebral nerve connection based on magnetic resonance imaging, comprising the following steps:
(1) CAG promoter, Ferritin gene, WPRE transcription are sequentially inserted among the ITR of rAAV2-retro carrier
Controlling element and polyA sequence afterwards, are packaged into recombinant adeno-associated virus rAAV2-retro-CAG-Ferritin-WPRE-pA；
(2) in the brain by the injection of above-mentioned recombinant virus, virus infection neuron simultaneously expresses Ferritin in neuron,
Living body brain is observed by magnetic resonance imaging T2 weighted imaging, shows that other brain areas and injection site are infected in living body brain
Nerve connection between neuron.
Application of the above method in the tracer of neuromechanism loop or nervous function parsing, application include but is not limited to
Mouse, rat, cavy, monkey, fish, birds, rabbit or people.By Recombinant rAAV 2-retro-CAG-Ferritin-WPRE-pA
It is injected at A brain area, after a period of time, T2 weighted mri imaging display B brain area has dark signal caused by Ferritin, thus
Show that A brain area and B brain area connects in the presence of nerve, and nerve connection direction with connect series it is equal it has been determined that is, B brain area it is refreshing
There are direct axonal projections to A brain area through member.Thus resulting specific neuromechanism link information is subsequent B brain area to A brain area
Nervons projection function parsing provide structure basis.
Compared with the conventional method, the invention has the following advantages that
1. the rAAV2-retro serotype Recombinant rAAV 2-retro-CAG-Ferritin- used in the present invention
WPRE-pA can be entered neuron by Nerve Terminals In The Human Skin, and inversely enter pericaryon by axonal transport, and nerve can be explicitly indicated
Connection；
2. Recombinant rAAV 2-retro-CAG-Ferritin-WPRE-pA uses efficient starting in the present invention
CAG has ensured the high expression quantity of Ferritin albumen, and the Ferritin albumen of high abundance is the pass for generating MRI contrasting effects
Key；
3. the Ferritin egg that Recombinant rAAV 2-retro-CAG-Ferritin-WPRE-pA gives expression in the present invention
White is a kind of MRI-T2 contrast agent, this contrast agent directly affects the signal value of MRI-T2 weighted imaging, high sensitivity；
4. method proposed by the present invention can observe cerebral nerve connection in living body level, it is suitable for long time-histories and observes and handle
Front and back own control experiment etc. is of great significance to the research of cerebral nerve structural network；
5. the neural network structure connection direction that method proposed by the present invention obtains is clear, as reverse label；
6. the neural network structure connection series that method proposed by the present invention obtains is clear, as it is directly connected to, rather than across
Multistage Synaptic junction；
7. the advantage for the neurotropic virus genophore that method proposed by the present invention had both had, it may have be based on Ferritin
The MRI of contrasting effects is reported and the advantage of detection method.The method can be changed flexibly, such as replacement neurotropic virus type, more
MRI reporter gene type etc. is changed, to be applied in the neural circuitry tracer and gene transfer monitoring of different demands.
Detailed description of the invention
Fig. 1 is the recombination AAV genome schematic diagram for carrying the rAAV2-retro serotype of Ferritin gene expression frame.
Fig. 2 is MRI observation and immunohistochemistry of the rAAV2-retro-CAG-Ferritin-WPRE-pA in mouse brain
Testing result.A is MRI-T2 weighted imaging as a result, wherein brain area MC, BLA and Hipp of arrow instruction are obvious T2 dark signal brain
Area；B is Ferritin specific immunity histochemical staining as a result, wherein brain area MC, BLA and Hipp of arrow instruction are Ferritin
The high expression brain area of aggregation.
Fig. 3 is the mouse Typical AVM observation that rAAV2-retro-CAG-Ferritin-WPRE-pA injects front and back different time points
As a result.A be injecting virus before mouse brain T2 weighted mri as a result, B be injecting virus 30 days after MRI as a result, C is
MRI result after injecting virus 70 days.
Specific embodiment
Technical solution of the present invention is if not otherwise specified the ordinary skill in the art, the reagent and material,
If not otherwise specified, commercial channel is derived from.Show that the specific embodiments are only for explaining the present invention herein, rather than limit
The fixed present invention.
Embodiment 1: the constructing plan of the recombination AAV of the rAAV2-retro serotype of Ferritin gene is carried
RAAV2-retro-CAG-Ferritin-WPRE-pA genome schematic diagram (Fig. 1) is inserted into CAG among ITR and opens
Mover (shown in SEQ ID NO.1), Ferritin gene (shown in SEQ ID NO.2), WPRE posttranscriptional regulatory element (SEQ ID
Shown in NO.3) and polyA sequence (shown in SEQ ID NO.4).Pivot close brain science technology in Wuhan is transferred to have this design scheme
Limit company carries out subsequent rAAV2-retro serotype and recombinates AAV virus formulation, to obtain Recombinant rAAV 2-retro-
CAG-Ferritin-WPRE-pA。
Embodiment 2:rAAV2-retro-CAG-Ferritin-WPRE-pA is injected at the immunohistochemistry inspection after mouse brain
It surveys and is observed with MRI
By rAAV2-retro-CAG-Ferritin-WPRE-pA virus (by Wuhan Shu Mi brain science Technology Co., Ltd. structure
Build) back side corpus straitum brain area (CPu, the coordinate: AP-0.5mm of mouse are injected at by Naoliqing capsule；ML-2mm；DV-
3.3mm), titre is 5.57 × 1012Vg/ml, volume injected are 2.8 μ L.
After 40 days, living body MRI observation is carried out to animal brain using 7.0T magnetic resonance imager, wherein the T2 weighting used
The sequence of imaging is RARE rapid acquision with relaxation enhancement sequence, parameter are as follows: TR=3000ms, effTE=55ms, Rarefactor
=4, sequence total duration is 28min.FOV is 1.75 × 1.75cm, and resolution ratio is 0.136 × 0.136mm, thickness 0.5mm.
MRI result (Fig. 2A) shows that virus is successfully entered neuron, inversely enters pericaryon (outside matrix from CPu through axonal transport
The brain areas such as side amygdaloid nucleus BLA, hippocampus Hipp, motor cortex MC, in Fig. 2A shown in arrow) and give expression to and change T2 weighting enough
The ferritin of MRI signal.Wherein injection site CPu brain area shows that T2 weights highlighted signal, it may be possible to since injection site is thin
Cellular virus infection is excessive, and caused local inflammation causes.
After MRI experiment, anesthetized mice takes out mouse brain after carrying out cardiac perfusion and cooks frozen section.Then use rabbit source
Ferritin Antibody on Mouse brain sections carry out immunohistochemical staining, to show the highly expressed brain area of Ferritin.As a result
See Fig. 2 B.Wherein CPu brain area injection core region shows the fluorescence signal of opposite decrease, thus it is speculated that is caused by cellular inflammation, is tested
The result of MRI is demonstrate,proved.
Fig. 2 shows that the Ferritin that Ferritin immunohistochemistry is shown concentrates high expression region and T2 weighted mri dark signal
Region height is consistent, that is, is the brain areas such as BLA, Hipp, MC (in Tu2A &B shown in arrow), it is dark that this also demonstrates T2 weighted mri
Caused by signal is the Ferritin high expression carried as virus.
The mouse brain living body of different time points is long after embodiment 3:rAAV2-retro-CAG-Ferritin-WPRE-pA injection
Time-histories MRI observation
One of advantage proposed by the present invention for tracking neural connection method is can the long time-histories observation cerebral nerve connection of living body.
Therefore same mouse is provided in this embodiment before rAAV2-retro-CAG-Ferritin-WPRE-pA virus injection, injection
30 days and 70 days big Typical AVM results afterwards.Before injecting virus, lived using 7.0T magnetic resonance imager to animal brain
Body MRI observation, wherein the sequence for the T2 weighted imaging used is RARE rapid acquision with relaxation enhancement sequence, parameter are as follows: TR=
3000ms, effTE=55ms, Rarefactor=4, sequence total duration are 28min.FOV is 1.75 × 1.75cm, and resolution ratio is
0.136 × 0.136mm, thickness 0.5mm.Then rAAV2-retro-CAG-Ferritin-WPRE-pA virus is stood by brain
Body localization method is injected at back side corpus straitum brain area (CPu, the coordinate: AP-0.5mm of mouse；ML-2mm；DV-3.3mm), drip
Degree is 5.57 × 1012vg/ml, and volume injected is 2.8 μ L.Difference 30 days and 70 days after injection, use 7.0T magnetic resonance imaging
Instrument carries out living body MRI observation to animal brain, and MRI method is the same as the scan method before injecting virus.MRI result is shown in Fig. 3, wherein 3A
For the mouse brain before injecting virus T2 weighted mri as a result, 3B be injecting virus 30 days after MRI as a result, 3C is injection
MRI result of the virus after 70 days.The MRI of different time points the result shows that, with the growth of time, carry Ferritin in virus
Expression quantity increase, MRI contrasting effects enhancing.By this embodiment, the brain area we obtain axonal projections to CPu includes
BLA, Hipp, MC etc., this brain in the case where the information for the structure nerve connection that living body level obtains is studying physiological or pathological state
The function of neural network provides important foundation.
Sequence table
<110>Wuhan Inst. of Physics and Mathematics, Chinese Academy of Sciences
<120>a kind of method and its application of the vivo tracking cerebral nerve connection based on magnetic resonance imaging
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tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg 1020
ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg 1080
tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac 1140
ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt 1200
ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg 1260
gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc 1320
ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg 1380
cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc 1440
ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag 1500
ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg 1560
cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg 1620
accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc 1680
ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt g 1731
<210> 2
<211> 1164
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atgacctctc agattcgtca gaattattcc accgaggtgg aagctgccgt gaaccgcctg 60
gtcaacttgc acctgcgggc ctcctacacc tacctctctc tgggcttctt ttttgatcgg 120
gatgacgtgg ctctggaggg cgtaggccac ttcttccgcg aattggccga ggagaagcgc 180
gagggcgcgg agcgtctcct cgagtttcag aacgatcgcg ggggccgtgc actcttccag 240
gatgtgcaga agccatctca agatgaatgg ggtaaaaccc aggaggccat ggaagctgcc 300
ttggccatgg agaagaacct gaatcaggcc ctcttggatc tgcatgccct gggttctgcc 360
cgcgcggacc ctcatctctg tgacttcctg gaaagccact atctggataa ggaggtgaaa 420
ctcatcaaga agatgggcaa ccatctgacc aacctccgca gggtggcggg gccacaacca 480
gcgcagactg gcgcgcccca ggggtctctg ggcgagtatc tctttgagcg cctcactctc 540
aagcacgacg cgcgcggcgg cggcggcagc gattataaag atgatgatga taaaggcggc 600
ggcggcagcc gcgtgatgac caccgcgtct ccctcgcaag tgcgccagaa ctaccaccag 660
gacgcggagg ctgccatcaa ccgccagatc aacctggagt tgtatgcctc ctacgtctat 720
ctgtctatgt cttgttattt tgaccgagat gatgtggctc tgaagaactt tgccaaatac 780
tttctccacc aatctcatga ggagagggag catgccgaga aactgatgaa gctgcagaac 840
cagcgaggtg gccgaatctt cctgcaggat ataaagaaac cagaccgtga tgactgggag 900
agcgggctga atgcaatgga gtgtgcactg cacttggaaa agagtgtgaa tcagtcacta 960
ctggaactgc acaaactggc tactgacaag aatgatcccc acttatgtga cttcattgag 1020
acgtattatc tgagtgaaca ggtgaaatcc attaaagaac tgggtgacca cgtgaccaac 1080
ttacgcaaga tgggtgcccc tgaagctggc atggcagaat atctctttga caagcacacc 1140
ctgggacacg gtgatgagag ctaa 1164
<210> 3
<211> 589
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60
ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120
atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180
tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240
ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300
attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360
ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 420
gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480
aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540
cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgc 589
<210> 4
<211> 477
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca 60
gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc 120
ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca 180
acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg 240
ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg 300
ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg 360
ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct 420
tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc tgtcctt 477
Claims (3)
1. a kind of method of the vivo tracking cerebral nerve connection based on magnetic resonance imaging, which comprises the following steps:
(1) it is adjusted after being sequentially inserted into CAG promoter, Ferritin gene, WPRE transcription among the ITR of rAAV2-retro carrier
Element and polyA sequence are controlled, recombinant adeno-associated virus rAAV2-retro-CAG-Ferritin-WPRE-pA is packaged into；
(2) in the brain by the injection of above-mentioned recombinant virus, virus infection neuron simultaneously expresses Ferritin in neuron, passes through
Magnetic resonance imaging T2 weighted imaging observes living body brain, other brain areas and the infected nerve of injection site are shown in living body brain
Nerve connection between member.
2. application of the method described in claim 1 in the tracer of neuromechanism loop or nervous function parsing.
3. application according to claim 2, which is characterized in that the object be mouse, rat, cavy, monkey, fish,
Birds, rabbit or people.
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