CN109536530A - A method of it generates and transmits using TRPV1 promoter and light science of heredity means specificity inhibition of pain - Google Patents
A method of it generates and transmits using TRPV1 promoter and light science of heredity means specificity inhibition of pain Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 28
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The present invention relates to a kind of using TRPV1 promoter and light science of heredity means in the method for specifically inhibiting internal pain nerve member excitement and then inhibition of pain is generated with transmitting, is the new Noninvasive of one kind, the lenitive method of specificity.With the expression of TRPV1 promoter driving Xanthophyll cycle sensitive Protein ArchT specificity in middle-size and small-size neuron, that is, nociceptor of animal DRG, when the green laser for giving 532nm stimulates, H+ in nerve cell can be pumped to extracellularly cell hyperpolarization is made and is suppressed by ArchT albumen, to achieve the effect that inhibition of pain.This is a kind of method of completely new, Noninvasive, specific inhibition of pain.The research will certainly establish good theory and practice basis for later analgesic gene therapy.
Description
Technical field
Specifically inhibiting internal pain mind using TRPV1 promoter and light science of heredity means the present invention relates to a kind of
The method with transmitting is generated through first excited and then inhibition of pain.It is widely used in biology, light science of heredity, Neuscience neck
Domain constructs a kind of recombination AAV virus more particularly to TRPV1 promoter, Xanthophyll cycle sensitive Protein ArchT and AAV5
(AAV5-TRPV1-ArchT-eGFP), it is injected directly into the DRG of animal, to destination protein ArchT a large amount and stablizes table
Up to studying its inhibiting effect reacted nervous system and ANIMAL PAIN after neuronal cell film
Background technique
Chronic ache is a kind of pathologic condition, substantially reduces people's lives quality.However so far, still do not have
The method of specific treatment chronic ache.Workable analgesic had not only been lack of pertinence while but also having had serious secondary work at present
With.Therefore a kind of Noninvasive is established, the method for specific inhibition of pain just becomes extremely important.
Recent study discovery, because the patient that pain is seen a doctor accounts for 40% or more of patient's total amount, wherein there is 20% pain again
Pain patient suffers from the torment of the chronic ache more than six months or more.Chronic ache brings great pain to patient, significantly
Reduce people's lives quality.Many patients because chronic ache torment can not ortho and work, it is more serious to lead
It causes to be changed by chronic ache partly or completely disabled.However drug, surgery are mainly passed through for the treatment of pain at present
The technological means such as operation, electro photoluminescence and physical rehabilitation treatment.Pain is treated by the method for surgical operation, in addition to bringing to patient
Outside bigger exterior trauma, wound, which also compares, to be difficult to recover, while surgery cost is expensive.The physics such as electro photoluminescence, acupuncture, massage are treated
Method also has very big drawback, such as analgesic effect is not significant, treatment cycle is long.And clinically most common method is then that drug is controlled
It treats.Opioid receptor agonist morphine, glutamate receptor antagonists NS1209 and ketamine, Na+ channel blocker mexiletine and
Lamotrigine, K+ channel opener retigabine and Flupirtine and GABA regulator are stable etc. to be made often as analgesic
With.These drugs be not only lack of pertinence while but also having had serious side effect.Such as the use of morphine can cause dose dependent
Increase, clinical manifestation is drug habit, and patient is made to bear very big pain.
Light science of heredity (optogenetics) is in neuroscience field using very extensive at present.So-called smooth science of heredity is
Refer to, optics is combined with the means of science of heredity, to activate or inactivate the knowledge and technology of specific organization and cell.It arrives
Have a large amount of light science of heredity albumen so far to be expressed and apply the nervous system in different biologies, wherein inhibition photaesthesia
Albumen has: Halorhodopsin (NpHR), Archaerhodopsin-3 and ArchT etc..Recent study show compared to
NpHR, Arch can reduce photaesthesia albumen to the refractory period of light by the inactivated state of photoinduction by quick-recovery fastly.ArchT is not only
With the same function of Arch, and photosensitivity ratio Arch it is three times higher more than.ArchT can stablize and the expression of a large amount is in nerve
On cell membrane, while it also can be very good expression on nervous process.ArchT can mediate generation maximum under external light conditions
The photoelectric current of about 900pA makes cell membrane hyperpolarization, to mediate the inhibiting effect to nervous system.
The main method of light science of heredity is that photaesthesia albumen is introduced in vitro or in body using different types of viral vectors
In animal nervous system.Then it is irradiated according to different photaesthesia albumen using the light of different wave length, activates the photaesthesia albumen
So that it is played a role and achievees the purpose that activation or the specific nerve cell of inactivation.At present since adeno-associated virus (AAV) has certainly
Right defect, safety be good, without etiology, host cell range is wide, the expression alien gene time is long in vivo the features such as, it is wide
General application and this viral vectors, carry photaesthesia albumen and enter and express in host's nerve cell.AAV has ten several serum
Type, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 etc..Research shows that directly DRG is infused
AAV5 has highest efficiency of infection in the case where penetrating.
At present for the treatment of pain without specificity, the present invention is quick with a species specific promoter driving Xanthophyll cycle
Albumen specifically expressing is felt in the nociceptor (nociceptor) of dorsal root ganglion (DRG), reaches specific inhibition
The purpose of pain.Capsaicin receptor TRPV1 is a kind of non-selective cationic channel, stablizes simultaneously a large amount expression in the wound of DRG
In evil sexuality receiver.The present invention is specifically expressed ArchT in nociceptor using the specificity of TRPV1 promoter
(nociceptor) in, to set up the town of the new Noninvasive of one kind, specificity and quick controllably inhibition of pain transmitting
Pain method.The present invention can illustrate a kind of application of the light as new light genetic strategies in terms for the treatment of pain.
Summary of the invention
Using the method that direct DRG is injected only by destination protein ArchT expression in the middle-size and small-size neuron of DRG,
The minor alteration of peripheral neverous system is exactly pertained only to, central nervous system is completely excluded.This method is controlled reaching
Peripheral neverous system is not lost as far as possible while treating pain, will not more damage central nervous system.Namely inhibiting periphery mind
Through the normal function that will not influence central nervous system while system specific cells activity.
Pass through the nociceptor of TRPV1 promoter specificity Xanthophyll cycle sensitive Protein ArchT expressed in DRG
In, these positive cells are not related to the neuron for controlling tactile and itching.It will not influence touching while inhibition of pain of the present invention
Feel and the normal of information of itching is transmitted and perceived.The present invention is not related to the processing of central nervous system, but directly inhibits from periphery
The activity of nociceptor in DRG cuts off its transmitting to central nervous system.The present invention does not have the limit of types of pain
System, therefore not only can treat different types of pain, and a plurality of types of pain can be treated simultaneously.
Detailed description of the invention
Fig. 1 is recombination TRPV1 carrier
Fig. 2 is common location of the ArchT and TRPV1 in DRG
Fig. 3 is that light science of heredity inhibits ArchT+ neuron that can alleviate the mechanical pain and heat pain of mouse, in which:
A. functionality inhibits the activity of ArchT+ neuron but still it is kept to develop the experimental principle figure of integrality；
B. the mechanical pain and hot pain Behaviors survey schematic diagram that ArchT is mediated；
C. in the case that green light according to and ArchT+ mouse (n=10) and the non-injecting virus mouse (n=9) of WT when no light machine
The tool paw withdrawal threshold of reaction；
D. in the case that green light according to and ArchT+ mouse (n=5) and the non-injecting virus mouse (n=5) of WT when no light heat
Radiate the paw withdrawal threshold of reaction.
Specific embodiment
1. the purifying of PCR reaction system and product
It introduces restriction enzyme site SnaBI and XhoI and expands TRPV1 promoter target fragment.
Upstream primer SnaBI TRPV1F:GCATACGTAGAGGACCAGAAGAAGGAGAGTC
Downstream primer XhoI TRPV1R:GTACTCGAGGTGCAGGCACACTCCAAATG
PCR reaction system:
PCR cycle system:
2. the purifying of double enzyme digestion reaction system and product
2.1 by the TRPV1 promoter target fragment of glue recovery purifying and pEGFP-N1 carrier double digestion, and 30 DEG C of water-bath digestions 1 are small
When.
Double enzyme digestion reaction system:
The purifying of 2.2 double enzyme digestion products
Double enzyme digestion product is tapped rubber after agarose gel electrophoresis, with Axygene plastic recovery kit recovery purifying.
3. utilizing TRPV1 promoter by destination gene expression in vitro DRG neuron
TRPV1 promoter is cloned into the CMV promoter for replacing the carrier itself in pEGFP-N1 carrier.The product is in the present invention
In be named as TRPV1 carrier.TRPV1 carrier is transfected into the DRG neuron of in vitro culture using the method for liposome transfection
In.
3.1 cloning TRPV1 carrier
TRPV1 promoter is the DNA fragmentation of about 2kb before TRPV1 gene transcription start site, searches for its gene order from NCBI and is
2069bp (Gene:Trpv1 ENSMUSG00000005952), and introduce two restriction enzyme site designs of SnaBI and XhoI and draw
Object, method are to obtain TRPV1 promoter sequence through PCR from C57BL/6 mouse genome.
The resulting DNA fragmentation of PCR is about 2kb, it may be determined that is TRPV1 promoter sequence.The load that TRPV1 promoter is recombinated
Body is named as TRPV1 carrier (Fig. 1).
Fig. 1 recombinates TRPV1 carrier
The middle-size and small-size neuron of DRG is responsible for the transmitting of pain sensation information caused by noxious stimulation, and the maxicell of DRG be then responsible for it is non-
The conduction of tactile data caused by noxious stimulation.Specific expressed explanation of the ArchT in DRG: when ArchT is by specific wavelength
After light irradiation activation in range, can specificity inhibition of pain, while will not influence body again to the normal of tactile data
Transmitting and perception.
3.2 ArchT and TRPV1 common location
Building recombination AAV virus is TRPV1 promoter.Recombination AAV virus injection has been done after two months with TRPV1 antibody
Mouse DRG slice dyeing, observation ArchT whether with TRPV1 in DRG common location.
The common location of Fig. 2 ArchT and TRPV1 in DRG
Immunostaining results show, ArchT with TRPV1 in DRG almost Chong Die (Fig. 2), it was demonstrated that TRPV1 promoter
High degree of specificity.Simultaneously because the height common location of ArchT and TRPV1 in DRG, ArchT is high expression in TRPV1+ mind
Through in member, then, due to the activation of ArchT albumen, it is photosensitive that ArchT plays its when the green laser for giving 532nm is irradiated
Perceptual inhibiting effect will inhibit TRPV1+ neuron.And TRPV1 is a kind of non-selective cationic channel, can be hurt
The chemical stimulation of evil property: capsaicine, proton (pH<5.9) and nocuity thermostimulation (>43 °C) are activated, so TRPV1
There is close be associated with thermostimulation.When ArchT, which plays its photosensitivity inhibiting effect, inhibits TRPV1+ neuron,
Hot pain behavior can will largely inhibit heat pain simultaneously by serious influence.
4. can be relieved mechanical pain and heat pain after Xanthophyll cycle ArchT+ neuron
Keep ArchT specific expressed after two months and successfully recombination AAV virus injection small in the nociceptor of DRG
Mouse is used to do the relevant Behavior test of pain.This test can be used to assess illumination to the inhibitory effect of ArchT positive neuron.
Fig. 3 A illustrates the functional experimental principle figure for inhibiting ArchT positive neuron.During functionality inhibits ArchT+,
Nociceptor in DRG is only inhibited by functionality, other physiological behaviors such as its growth and development can all continue it is intact into
Row.Mouse after recombination AAV virus injection is used to do mechanical pain and hot pain Behavior test, respectively by using mechanical stimulus
The hind paw (Fig. 3 B) of mouse is irradiated in 532nm green laser and no light with heat radiation stimulation.In control group WT
In mouse, the 532nm green laser (light intensity that mechanical pain behavioral experiment uses are as follows: 1.0-1.5mW/mm2, hot pain behavioral experiment makes
Light intensity are as follows: 0.15-0.2 mW/mm2) there is no the mechanical paw withdrawal thresholds of reaction (Fig. 3 C) of inducing mouse and heat radiation contracting
The sufficient threshold of reaction (Fig. 3 D) generates abnormal.However, irradiating ArchT experimental group with the 532nm green laser of same intensity of illumination
In the case of mouse, the mechanical paw withdrawal threshold of reaction (Fig. 3 C) and the heat radiation paw withdrawal threshold of reaction (Fig. 3 D) are relative to no light
There is the increase of conspicuousness.Mouse can be effectively relieved in the activity of ArchT+ neuron in the nociceptor of Xanthophyll cycle DRG
Mechanical pain and heat pain.
The mechanical pain and heat that Fig. 3 light science of heredity inhibition ArchT+ neuron can alleviate mouse are bitterly, in which:
A. functionality inhibits the activity of ArchT+ neuron but still it is kept to develop the experimental principle figure of integrality；
B. the mechanical pain and hot pain Behaviors survey schematic diagram that ArchT is mediated；
C. in the case that green light according to and ArchT+ mouse (n=10) and the non-injecting virus mouse (n=9) of WT when no light machine
The tool paw withdrawal threshold of reaction；
D. in the case that green light according to and ArchT+ mouse (n=5) and the non-injecting virus mouse (n=5) of WT when no light heat
Radiate the paw withdrawal threshold of reaction.Data represent means ± S.E.M, and analysis uses P < 0.01 Student ' s T-test. * *,
***P < 0.001.
The method of the present invention can be in Animals Adult, after all physiology and mental functioning maturation, by directly inhibiting photosensitivity
Protein expression reaches the inhibition to the cell mass function in specific cells group, after illumination, come study the cell mass be suppressed after
Which type of character mutation is experimental group can generate.Therefore when we want to study the function of certain cell type group, so that it may select
The promoter of the cell group specificity is selected, driving Xanthophyll cycle sensitive Protein ArchT special expression passes through in the cell mass
Illumination inhibits the activity of the cell mass, studies cell mass role in body.As a result, the cell mass is still complete
Good is present in body, does not damage at all, and only its cell activity is suppressed, while the cell mass and surrounding other types
Cell between substance and the interactions such as energy transmission still can be very good to carry out.This method do not develop it is compensatory,
It simultaneously because ArchT is to light stimulation quick and high efficient reaction, therefore is a kind of quickly controllable research method.Using quickly,
Controllable light genetic tool establishes the analgesia method of a kind of new Noninvasive and specific inhibition of pain transmitting, light conduct
A kind of new light genetic strategies are applied in terms for the treatment of pain.
Claims (4)
1. a kind of specifically inhibit internal pain nerve member excited and then inhibit using TRPV1 promoter and light science of heredity means
Pain generates the method with transmitting.
2. according to claim 1 a kind of internal in specifically inhibition using TRPV1 promoter and light science of heredity means
The excited inhibition of pain in turn of pain nerve member generates the method with transmitting, and photaesthesia albumen is introduced in vitro and in body animal nerve
In system, is irradiated according to different photaesthesia albumen using the light of different wave length, activate the photaesthesia albumen that it is made to play a role
The method for reaching activation or the specific nerve cell of inactivation.
3. according to claim 2 a kind of internal in specifically inhibition using TRPV1 promoter and light science of heredity means
Pain nerve member is excited and then inhibition of pain generates the method with transmitting, using TRPV1 promoter by destination gene expression from
Body and in body DRG neuron.
4. according to claim 2 a kind of internal in specifically inhibition using TRPV1 promoter and light science of heredity means
Pain nerve member is excited and then inhibition of pain generates the method with transmitting, and ArchT a large amount simultaneously expresses DRG in vivo steadily in the long term
In neuron.
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