CN106367419A - Antisense oligonucleotide with neuroprotective effect, medicine composition and application thereof - Google Patents
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Abstract
The invention discloses antisense oligonucleotide with a neuroprotective effect, a medicine composition and an application thereof. The sequence of the antisense oligonucleotide is shown as SEQ ID NO:1, and the antisense oligonucleotide is modified by 3' terminal cholesterol, two 5' terminal sulfo-frameworks, four 3' terminal sulfo-frameworks and whole-chain methoxyl. The antisense oligonucleotide and acceptable pharmaceutical carriers form the medicine composition, and ischemic neuron injury related diseases, particularly ischemic stroke, are treated by intravenous, intramuscular or intraperitoneal injection. The antisense oligonucleotide is applied to global cerebral ischemia and focal cerebral ischemia, can obviously relieve cerebral injury caused by ischemia reperfusion, and has a good medicine application prospect.
Description
Technical field
The invention belongs to biomedical sector, it is related to a kind of antisense oligonucleotide, more particularly, to one kind has neuroprotective
The antisense oligonucleotide of effect, pharmaceutical composition and its application.
Background technology
Acute cerebrovascular disease, also known as apoplexy (stroke), is a kind of local caused by acute disturbance of cerebral circulation or comprehensive
Property brain function deficiency syndrome, has the characteristics that high incidence, high mortality, high disability rate.Apoplexy is divided into ischemic and goes out
Courage and uprightness, wherein acute ischemic cerebral apoplexy are modal acute cerebrovascular disease, account for the 60%～80% of whole apoplexy.Mesh
Before, clinically modal Therapeutic Method is to be recovered using Thrombolytic Drugs or improve supply of blood flow to acute ischemic cerebral apoplexy, by
In thromboembolism treatment, Reperfu- sion can cause secondary neuronal injury, and nerve protection medicine need to be coordinated to promote ischemic region tissue simultaneously
Repair and functional rehabilitation, but clinically the efficacy and saferry of nerve protection medicine is still undesirable.Therefore try to explore cerebral ischemia
The molecular mechanism of Reperfu- sion nerve injury, screening of medicaments action target spot, find more effective nerve protection medicine, have important
Medical value and social meaning.
Small rna (microrna, mirna) is the non-coding single-stranded rna molecule that a class is about 22 nucleotide.Ripe
The single-stranded silencing complex (rna-induced silencing complex, risc) being integrated into rna mediation of mirna, and foundation
Base pair complementarity principle binding purpose gene mrna, suppresses expression of target gene in post-transcriptional level.Mirna suppresses target gene table
Reach and have two ways: the first, mirna complete complementary matches and leads to genes of interest to be degraded in purpose mrna open reading frame；The
Two kinds, mirna is combined with 3 ' Duan Feibian areas of purpose mrna by non-fully complementary pairing mode, hinders it to translate into albumen
Matter.Most of mammal uses second regulatory mechanism.
Antisense technology is the biotechnology that a kind of recent two decades rise, with the table of antisense technology closing and suppression specific gene
Reaching is the important component part of gene therapy.Antisense oligonucleotide molecular weight is little, relatively easily migrates between histiocyte, and
Energy specific recognition target gene, can treat central nervous system disease as instrument.
Content of the invention
An object of the present invention is to provide a kind of antisense oligonucleotide, and it is used for combining specific nucleotide sequence, and energy
Hybridize the antisense oligonucleotide forming double-strand with specific nucleotide sequence, thus can be used for treating the nerve that ischemia-reperfusion causes
Unit is damaged.
For achieving the above object, the invention provides a kind of antisense oligonucleotide with neuroprotective, its nucleoside
Acid sequence is as shown in seq id no:1.
Described antisense oligonucleotide is prepared by chemical synthesis process, can be through different chemical modifications.
Described antisense oligonucleotide is modified through 3 ' end cholesterol, 5 ' the two thio backbone modifications in end, and 3 ' four, ends are thio
Backbone modification, full chain methoxyl group is modified.
The second object of the present invention is to provide a kind of pharmaceutical composition, can be used as nerve protection medicine and and ischemic neuronal
The medicine of relevant disease damages in unit.
The pharmaceutical composition that the present invention provides, comprises the antisense oligonucleotide of effective ingredient and pharmaceutically acceptable load
Body.Described carrier can be selected from diluent, excipient, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surface
Activating agent, absorption carrier, lubricant etc. are it may also be necessary to add flavoring agent, flavouring agent etc..
It is multiple that the pharmaceutical composition of the present invention can make tablet, powder, granule, capsule, oral liquid and injection etc.
Form.Above-mentioned various dosage form all can be prepared according to the conventional method of pharmaceutical field.
The third object of the present invention is to provide described antisense oligonucleotide in preparation treatment nervous system disease agent
Application.Wherein, described nervous system disease refers to the nervous system disease relevant with ischaemic neuronal damage, such as ischemic brain
Apoplexy.
The fourth object of the present invention is to provide application in preparing nerve protection medicine for the described antisense oligonucleotide.
The antisense oligonucleotide that the present invention provides can substantially mitigate the brain injury that ischemia-reperfusion causes, and it can be used as having
Effect composition is used for preparing nerve protection medicine, treats the disease relevant with ischaemic neuronal damage, in medical domain, especially
Neuromedicine field has a good application prospect.
Brief description
Fig. 1 shows the impact to Sector Ca 1 Hippocampus of Rats mir-7a expression for the global cerebral ischemic-reperfusion；
Fig. 2 shows the hippocampal neurons injury that the antisense oligonucleotide of the present invention causes to cerebral ischemia, reperfusion
Impact (the cresyl viollet colored graph of a: rat cerebral tissue's paraffin section；The cartogram of b:a)；
Fig. 3 shows the shadow of the brain injury that the antisense oligonucleotide of the present invention causes to Focal Ischemia-Reperfusion in Rats
Ring (a1: rat mcao cerebral tissue ttc colored graph；The cartogram of a2:a1；B: animal nerve behavioristicss longa scoring).
Specific embodiment
Experimental technique in following embodiments, if no special instructions, is conventional method.Examination used in following embodiments
Agent material etc., if no special instructions, is commercially available purchase product.
The preparation of embodiment 1 antisense oligonucleotide
The antisense oligonucleotide (aso) of the present invention is synthesized by Shanghai JiMa pharmacy Technology Co., Ltd, through 3 ' end cholesterol
Modify, 5 ' the two thio backbone modifications in end, 3 ' the four thio backbone modifications in end, full chain methoxyl group is modified, vacuum after ultraviolet quantitation
It is dried, -20 DEG C save backup.Antisense oligonucleotide through chemical modification increased its stability and targeting affinity, reduces
Cytotoxicity, can pass through vein, muscle or lumbar injection etc., can reach the purpose of Biotherapeutics ischemic brain injury.
By blast sequence alignment online with genebank, obtain Antisensedigonucleotsequence sequence as shown in seq id no:1:
5 '-acaacaaaaucacuagucuucca-3 ', it has good specificity.
The impact to Sector Ca 1 Hippocampus of Rats mir-7a expression for embodiment 2 global cerebral ischemic-reperfusion
Using four-vessel occlusion method, set up sd rat four with reference to liu y et al. (brain res, 2001,909:51-58)
Artery ligation Global Ischemia Model, in cerebral ischemia re-pouring 6h, 12h, 24h, detects Hippocampus ca1 using real time fluorescent quantitative pcr
The expression change of area's (ischemia area of liability) mir-7a.Concrete operations are as follows:
Male sd rat, with 20% chloral hydrate (300mg/kg) intraperitoneal injection of anesthesia, is fixed on electrocoagulator, and coagulation is double
Side vertebral artery, separates bilateral carotid arteries, after 24h under rat waking state, closes bilateral carotid arteries, 15min using bulldog clamp folder
Take off bulldog clamp afterwards, recover cervical blood flow supply, Reperfu- sion timing is from the beginning of the time taking off bulldog clamp.Sham operated rats give
Identical is processed, but does not press from both sides and close bilateral carotid arteries.Ischemic period keeps Rat-rectum temperature between 36.5 DEG C -37.5 DEG C.
Model construction success reference index: loss of consciousness in (1) 1min, righting reflex loss, lose mobility, extremity
Stiff with tail；(2) bilateral platycoria eyeball turns white, and after Reperfu- sion, eyeball recovers pink immediately, pupil is retracted.
Sd cerebral ischemic reperfusion in rats 6h, 12h, 24h broken end take Hippocampus ca1 district's groups to knit, and extract total rna and are used for real-time fluorescence
Quantitative pcr.
Total rna knits middle extraction, the table of mir-7a with trizol reagent (life technologies) from Hippocampus ca1 district's groups
The level that reaches, through specific primer, is detected using fast real-time pcr system (applied biosystems),
With u6snrna as internal reference.Operational approach is entered with reference to sybr premix real time pcr test kit (takara company)
OK, the calculating of expression adopts 2- δ δ ctMethod.
δ ct=ct genes of interest-ct reference gene
δ δ ct=δ ct experimental group-δ ct matched group
Relative expression quantity=2- δ δ ct
Design of primers:
Above-mentioned primer is synthesized by Shanghai Sheng Gong biotechnology company.
As shown in Figure 1: substantially to rise in global cerebral ischemic-reperfusion 6h Hippocampus ca1 area mir-7a expression, 12h mir-7a table
Reach and rise to highest, decrease compared with 12h in global cerebral ischemic-reperfusion 24h Hippocampus ca1 area mir-7a expression, but still high
In sham operated rats.Above experiment is repeated 6 times, compared with sham operated rats*P ＜ 0.05 is that difference is statistically significant.
Result shows, in cerebral ischemia, reperfusion model, the serious Hippocampus ca1 area mir-7a table of neuronal damage
Reach rising, mir-7a is relevant with ischaemic neuronal damage for prompting, and its antisense oligonucleotide is likely to be of neuroprotective.
The shadow of the hippocampal neurons injury that embodiment 3 antisense oligonucleotide (aso) causes to cerebral ischemia, reperfusion
Ring
(1) main agents
Negative control (nc) is synthesized by Shanghai JiMa pharmacy Technology Co., Ltd, its nucleotide sequence such as seq id no:8
Shown: 5 '-caguacuuuuguguaguacaa-3 '.
Isoflurane (2%) is purchased from baxter company of the U.S.；Chloral hydrate and other conventional chemical reagent are purchased from Chinese medicines group
Chemical reagent company limited；2,3,5- TTC (ttc) is purchased from ameresco company.
(2) key instrument
Toy anesthetic machine (abs-100), toy induction case (ic-r) are purchased from Shanghai Yu Yan scientific instrument company limited；
Animals stereo position finder is purchased from stoelting company of the U.S..
(3) laboratory animal
Spf level sprague-dawley (sd) health male rat, body weight is 220-300g, and by Xuzhou, medical university tests
Animal center provides, and raises in spf level Animal House, credit number: scxk (Soviet Union) 2015-0009.
(4) experiment packet
Set up sham operated rats, 5 days groups of ischemia-reperfusion, ischemia injects aso group, ischemia injects nc group, inject merely aso
Group.Wherein sham operated rats equally set up Global Ischemia Model with experimental group, but do not carry out ischemia, do not inject any medicine；Ischemia
5 days groups of Reperfu- sion equally set up Global Ischemia Model with experimental group, equally carry out ischemia 15min, but do not inject any medicine；Lack
Blood injection aso group and ischemia injection nc group are that 50min is respectively completed intracerebroventricular injection aso and nc after ischemia；Simple injection aso
Group is not ischemia but intracerebroventricular injection aso.
(5) experimental technique
Using four-vessel occlusion method, set up sd rat four with reference to liu y et al. (brain res, 2001,909:51-58)
Artery ligation Global Ischemia Model, wherein global brain ischemia 15min, complete after ischemia-reperfusion 50min intracerebroventricular injection aso or
nc.In cerebral ischemia re-pouring the 5th day, cresyl viollet dyeing observed injection medicine to Hippocampus ca1 area cone after ischemical reperfusion injury
The impact of neuronal survival.Concrete operations are as follows:
Male sd rat, with 20% chloral hydrate (0.15ml/kg) intraperitoneal injection of anesthesia, is fixed on electrocoagulator, and coagulation is double
Side vertebral artery, separates bilateral carotid arteries, after 24h under rat waking state, closes bilateral carotid arteries, 15min using bulldog clamp folder
Take off bulldog clamp afterwards, recover cervical blood flow supply, Reperfu- sion timing is from the beginning of the time taking off bulldog clamp.Sham operated rats give
Identical is processed, but does not press from both sides and close bilateral carotid arteries.Ischemic period keeps Rat-rectum temperature between 36.5 DEG C -37.5 DEG C.
Model construction success reference index: loss of consciousness in (1) 1min, righting reflex loss, lose mobility, extremity
Stiff with tail；(2) bilateral platycoria eyeball turns white, and after Reperfu- sion, eyeball recovers pink immediately, pupil is retracted.
Intracerebroventricular injection method: method (neurosci lett, the 2003,343:125- setting up by Hou Xiaoyu et al.
128).Give sd rat suction 2% Isoflurane and carry out induced anesthesia, ventilation is controlled by respirator, keep normal exhalation end o2With
co2Concentration, is fixed on after anesthesia on animals stereo position finder, cuts scalp and exposes skull so as to keep horizontal level, h2o2
Burn exposure bregma, bit bore, position is 0.8mm after bregma, 1.5mm is opened on side, and micro-injection pin inserts, and depth is from cranium
The downward 3.5mm of bone surface.Injection rate: 1 μ l/min, volume is 10 μ l, the 10min that inject that afterwards let the acupuncture needle remain at a certain point.Appropriate rnase-free is water-soluble
Solution aso or nc, is mixed using front use equal-volume transfection reagent (santa cruz, sc-29528), every lateral ventricle of rat brain injection
0.1nmol aso or nc.
Perfusion takes brain method: rats by intraperitoneal injection chloral hydrate anesthesia, opens thoracic cavity and exposes heart, by syringe needle from the left apex of the heart
Through left ventricle insert aorta, cut off right auricle, with 200ml about normal saline get express developed, to flow out normal saline
In there is no bloodstain, change ice-cold 4% paraformaldehyde perfusion, consumption is about 300ml, and perfusion within 30min terminates, and perfusion terminates big
Mus broken end takes brain, be placed in 4% paraformaldehyde 4 DEG C carry out after fixing.
Cresyl viollet colouring method: take out the brain block that fixes, rinsed with flowing water, then with 70% (3d), 80% (1d),
90% (12h), 95% (2h × 2 time), 100% (1h × 2 time) gradient alcohol dehydration；Dimethylbenzene: ethanol (1:1) (30min), two
Toluene (30min) is transparent；60 DEG C of waxdips (6h)；Take out the embedding of brain block；Embedded wax stone puts 4 DEG C of Refrigerator stores.Wax stone rewarming
Half an hour paraffin slicing machine is cut into slices, 6 μ m-thick, floats piece in 45 DEG C of warm water, and on the microscope slide that gelatin was processed, 63 DEG C are spread out paster
Piece 2h, microscope slide is stored in 4 DEG C of refrigerators and treats that cresyl viollet dyes.Rewarming microscope slide, dewaxing: dimethylbenzene (37 DEG C, 20min × 2 time),
100% ethanol (5min), 95% ethanol (5min), 80% ethanol (5min), 70% ethanol (5min), distilled water (5min),
0.1% cresyl viollet dyeing 35min, 70%, 80%, 95%, 100% graded ethanol color separation, transparent (15min × 2 of dimethylbenzene
Secondary), with micro- sem observation Neuronal Survival situation after resinenes mounting.
Result shows: normal cell is rounded, the light dye of core, cell membrane and nucleus high-visible (a and f in Fig. 2 a).Entirely
Cerebral ischemia re-pouring 5 days, the normal cell of survival is considerably less, pyramidal cell mortality, and nuclear hyperchromatism pyknosis (Fig. 2 a can be observed
Middle b and g).Compared with sham operated rats, 5 days rear side ventricles of the brain of ischemia-reperfusion are not injected medicine and are injected aso or nc Hippocampus respectively
Ca1 area neuron all there occurs obvious damage；Compared with 5 days groups of ischemia-reperfusion, intracerebroventricular aso on rear side of ischemia-reperfusion
The damage of group Hippocampus ca1 area cone neurone significantly reduces (c and h in Fig. 2 a), and intracerebroventricular nc group on rear side of ischemia-reperfusion
With 5 days group no significant differences (d and i in Fig. 2 a) of ischemia-reperfusion.Simple injection aso group no significant difference compared with sham operated rats
(e and j in Fig. 2 a).
Fig. 2 b is the cartogram of Fig. 2 a.Result shows: simple injection aso group difference compared with ischemia experimental group has statistics
Meaning, result shows, after global cerebral ischemic-reperfusion, the antisense oligonucleotide of the injection present invention can mitigate global cerebral ischemic-reperfusion and make
The neuronal damage becoming.This experiment is repeated 5 times,*P ＜ 0.05 represents that difference is statistically significant compared with sham operated rats,#P ＜
0.05 represents that difference is statistically significant compared with 5 days groups of ischemia-reperfusion.
The impact of the brain injury that embodiment 4 antisense oligonucleotide (aso) causes to focal cerebral ischemia in rats
Using line brush, set up intraluminal middle cerebral artery occlusion in rats resistance with reference to longa et al. (stroke, 1989,20 (1): 84-91)
Plug (mcao) model.Intracerebroventricular aso (every rat 1nmol) on rear side of ischemia 40min.In mcao (90min) Reperfu- sion afterwards
24h, 2,3,5- triphenyltetrazolium chlorides (2,3,5-triphenyltetrazolium chloride, ttc；2%) dyeing is seen
Examine the impact that area organized by injection medicine to ischemical reperfusion injury cerebral infarction.Concrete operations are as follows:
Set up mcao group, mcao adds aso group.Mcao adds aso group and equally sets up focal cerebral ischemia in rats with mcao group,
Intracerebroventricular injection aso is completed after ischemia 40min.
, at 25 DEG C about, water is can't help in preoperative 12h fasting for animal feeding and operation temperature control.By male sd rat with 20%
Chloral hydrate (300mg/kg) intraperitoneal injection of anesthesia, lies on the back and is fixed on operating-table, takes neck median incision, exposes right side neck and always moves
Arteries and veins, external carotid artery and internal carotid artery, ligation external carotid artery and common carotid artery proximal part, in internal carotid artery distal end for line, in neck
Total nearly crotch of tremulous pulse cuts " v " shape otch of an about 0.2mm, by a diameter of 0.26mm's processing through poly-D-lysine in advance
Nylon fishing line (the dense Science and Technology Ltd. of West Beijing, No. 2634 line bolts) through this otch along common carotid artery insertion internal carotid artery (18 ±
1) mm, feels to have stopping during light resistance, so that middle cerebral artery initial part is blocked.Tighten standby line fixing line bolt, skin suture
Line bolt is extracted about 10mm after 90min by otch, recovers Reperfu- sion.After anesthesia is clear-headed, neuroethology is carried out according to longa scoring
Check: 0 point: normal, impassivity functional impairment；1 point: right side fore paw is unable to full extension, slight neurologic impairment；2 points: OK
When walking, turn-take to the right by (paralysis side) for rat, moderate neurologic impairment；3 points, during walking, rat body (paralysis to the right
Side) topple over.Severe neurological functional impairment；It is impossible to spontaneous walking, lose consciously for 4 points.2 points or the success of 3 grouping models.
Intracerebroventricular injection method: the side setting up by Hou Xiaoyu et al. (neurosci lett, 2003,343:125-128)
Method.It is fixed on after isoflurane anesthesia rat on animals stereo brain position finder, cut scalp and expose skull so as to keep horizontal position
Put, h2o2Burn exposure bregma.Bit bore, position is 0.8mm after bregma, and 1.5mm is opened on side, and micro-injection pin inserts, depth
It is 3.5mm downward from skull surface.Injection rate: 1 μ l/min, volume is 10 μ l, the 10min that inject that afterwards let the acupuncture needle remain at a certain point.Seedless in right amount
Enzyme water dissolution aso, is mixed using front use equal-volume transfection reagent (santa cruz, sc-29528), every lateral ventricle of rat brain note
Penetrate 1nmol aso.
Rat cerebral tissue's section ttc dyeing mensure brain infarction area: focal cerebral ischemia in rats 90min, Reperfu- sion 24h,
Rapid broken end takes brain, carries out coronal section by after -20 DEG C of freezing 10min of cerebral tissue, brain piece is put into mass concentration on ice pan
In ttc phosphate buffer (ph 7.4) for 2%, 37 DEG C of lucifuge constant-temperature incubation 30min, 4% paraformaldehyde phosphate-buffered
Take pictures after the fixing 24h of liquid.Calculate brain infarction area using imaga-pro plus 6.0 image analysis software.Infarct size (%)
=(offside cerebral tissue area ischemia side normal cerebral tissue area)/offside cerebral tissue area × 100% (du w et al., j clin
Invest, 2010,120 (10): 3480-3492).
Result shows: the cerebral tissue of mcao group ischemia offside has no mistake dye region, the striatum of ischemia side and volume top skin
It is in pale asphyxia that matter loses dye；Mcao adds aso group ischemia Ce Shiran area (white portion) and is obviously reduced.
Fig. 3 a2 is the cartogram of Fig. 3 a1: it is statistically significant that mcao adds aso group difference compared with mcao group.Result carries
Show: the aso injecting the present invention after focal cerebral ischemia can substantially mitigate the brain injury that Focal Cerebral Ischemia Reperfusion causes.Experiment
It is repeated 6 times,*P ＜ 0.05 represents that difference is statistically significant compared with mcao group.
Fig. 3 b is the mcao animal pattern Neurological deficits of intracerebroventricular aso on rear side of mcao animal pattern and ischemia,
On rear side of result display focal cerebral ischemia, intracerebroventricular aso can substantially mitigate neurologic impairment.Experiment is repeated 6 times,*P ＜
0.05 represents that difference is statistically significant compared with mcao group.
In sum, on rear side of cerebral ischemia, the antisense oligonucleotide of the intracerebroventricular present invention can substantially mitigate ischemia-reperfusion
The brain injury causing.Therefore, the medicine using the antisense oligonucleotide of the present invention as effective ingredient can as nerve protection medicine,
Especially relevant with ischaemic neuronal damage such as apoplexy medicine.
Claims (10)
1. a kind of antisense oligonucleotide with neuroprotective is it is characterised in that its nucleotide sequence such as seq id no:1
Shown.
2. the antisense oligonucleotide with neuroprotective according to claim 1 is it is characterised in that described antisense is few
Nucleotide is through chemical modification.
3. the antisense oligonucleotide with neuroprotective according to claim 2 is it is characterised in that described antisense is few
Nucleotide is modified through 3 ' end cholesterol, 5 ' the two thio backbone modifications in end, 3 ' the four thio backbone modifications in end, full chain methoxyl group
Modify.
4. a kind of pharmaceutical composition is it is characterised in that the antisense described in any one of claims 1 to 3 comprising effective ingredient is few
Nucleotide and pharmaceutically acceptable carrier.
5. pharmaceutical composition according to claim 4 is it is characterised in that described pharmaceutically acceptable carrier is selected from dilution
Agent, excipient, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier, lubricant,
One or more of flavoring agent, flavouring agent.
6. the antisense oligonucleotide according to any one of claims 1 to 3 is used for treating nervous system disease agent in preparation
In application.
7. application according to claim 6 is it is characterised in that described nervous system disease is to damage with ischaemic neuronal
Hinder relevant nervous system disease.
8. application according to claim 7 is it is characterised in that described nervous system disease is cerebral infarction.
9. application in preparing nerve protection medicine for the antisense oligonucleotide according to any one of claims 1 to 3.
10. the pharmaceutical composition according to claim 4 or 5 is relevant with ischaemic neuronal damage for treatment in preparation
Application in nervous system disease agent.
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|---|---|---|---|---|
| CN107296814A (en) * | 2017-03-21 | 2017-10-27 | 复旦大学 | Purposes of the micromolecule nucleotide in the brain protection and brain repair medicine of brain damage is prepared |
| CN107296814B (en) * | 2017-03-21 | 2020-09-01 | 复旦大学 | Application of small molecular nucleotide in preparing brain protection and brain repair medicine for brain injury |
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