CN106474144B - MiR-219a-5p relapses application in drug preparing anti-crystal methamphetamine - Google Patents
MiR-219a-5p relapses application in drug preparing anti-crystal methamphetamine Download PDFInfo
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Abstract
The present invention relates to a kind of miR-219a-5p to relapse application in drug preparing anti-crystal methamphetamine, it is characterised in that：Nucleic acid and pharmaceutically acceptable carrier or auxiliary material of the drug by effective quantity comprising SEQ NO.1 shown in sequence table form.Compared with prior art, the advantage of the invention is that confirming through a large number of experiments, in establishing crystal methamphetamine rat model, crystal methamphetamine causes microRNA to change in nucleus accumbens septi expression, after the microRNA-219a-5p nucleus accumbens septi overexpression for targeting AT1, the ignite Methamphetamine Relapse behavior of induction of Methamphetamine Relapse behavior and crystal methamphetamine to Conditioned cues induction plays inhibiting effect, illustrate that miR-219a-5p can be applied to treatment crystal methamphetamine habituation, is quit drug abuse using exploitation as gene therapy.
Description
Technical field
The present invention relates to a kind of a kind of miR-219a-5p more particularly to miR-219a-5p to prepare anti-crystal methamphetamine multiple
Inhale the application in drug.
Background technique
Crystal methamphetamine (METH), is commonly called as methamphetamine, is a kind of white amphetamine synthesis class drugs.High dose is long-term anti-
Multiple user can suffer from mental disease, including depression, mania, schizophrenia etc. altogether, and serious person will cause toxic psychosis, performance
For persecutory delusion and illusion, excessively uses and tendency of committing suiside and murder even occur.Period, methamphetamine dependent's meeting are given up in methamphetamine
There is the spiritual emotional change of insomnia, depression, mania and anxiety, and these abstinence reactions also become influence methamphetamine dependent
The major physiological factor relapsed.
The ratio that crystal methamphetamine habituation suffers from mental disease altogether has reached 20-30%, and addict usually will appear depression, coke
The symptoms such as worry, schizophrenia.Crystal methamphetamine can also generate serious neurotoxicity, and crystal methamphetamine, which is used for a long time, to be caused
Cognitive function damage.Therefore the research of crystal methamphetamine habituation mechanism and the searching of therapeutic agent, also gradually to other spirit
Extend with the nervous system disease.Periphery renin-angiotensin system (RAS) is to the function of cardiovascular system, electrolyte and body
The balance and blood pressure control of liquid play an important role.Feritin enters blood circulation through renal vein, starts chain reaction, by
Proangiotensin gradually generates angiotensin I (AngI) and II (AngII), and further generates AngIII and AngIV etc.
Mediator.In addition in peripheral-system, for maincenter there is also a relatively independent RAS system, chief component is AngII.Maincenter
AngII is the neuropeptide with pleiotropism, is present on neuron cell body, aixs cylinder and nerve endings, plays to maincenter
Multiple adjustment effect.There are two types of ATR1 and ATR2 for the major receptors of AngII.Maincenter AngII mainly by being incorporated in ATR1 in
Pivot system, which plays, adjusts endocrine, sympathetic and stress response system effect.ATR1 receptor is divided into the two kinds of Asias ATR1a and ATR1b again
Type.Research also indicates that maincenter AngII and a variety of neural and mental disease pathogenesis, and there are correlations, including Alzheimer
Disease, parkinsonism, Neurogenic inflammatory, apoplexy, two-way personality disorder, epilepsy etc..So far, there are no one to be directed to methylbenzene
The active drug of propylamine addiction therapy.Latent effect and applicant of the maincenter AngII in a variety of spirit and the nervous system disease
The previous work of study group prompts, and maincenter AngII and ATR1 may be from improving and treatment crystal methamphetamine habituation and altogether
Suffer from phrenoblabia, provides new target spot for the research of crystal methamphetamine addiction therapy drug.
Find microRNA for the first time in Caenorhabditis elegans within 1992.Later, largely research shows that miRNAs is in base
Because being of crucial importance in expression.MiRNAs is a kind of RNA of short and small non-coding, it only has 21-25 nucleotides sequence
Column.MiRNA transcribes out longer primary miRNA in nucleus first, is then processed into 60~70 by Drosha in core
The hairpin RNA of nucleotide, i.e. precursor miRNA are transported out karyon with the help of Exprotin-5 compound, in endochylema
Maturation miRNA is become by Dicer shearing, is integrated into RNA silencing complex immediately.MiRISC is by holding target non-codings with 3 '
Area targets mRNA according to basepairing rule come the translation of suppressor or the degradation of induction mRNA.Recently, increasingly
The sight of more researchers focuses adjustment effect of the miRNA in various addictive drugs, including ***e, Hai Ruoyin, phenylpropyl alcohol
Amine and alcohol.Long-term ***e can also be raised in corpus straitum using the downward for causing miR-134 and miR-135a in hippocampus
MiR-181a, miR-212 and lower terminal mucro (CPU) miR-124.It alcohol addiction and gives up and leads to miR-155 and miR-
The up-regulation of 375 expression.However, most of research is concentrated mainly on miRNA in ***e or alcohol addiction at present, methylbenzene
Propylamine habituation correlative study is less, and the microRNA adjustment mechanism in crystal methamphetamine habituation is also indefinite at present.
Summary of the invention
The technical problem to be solved by the present invention is to provide a kind of miR-219a-5p for above-mentioned state of the art to exist
It prepares anti-crystal methamphetamine and relapses application in drug.
The present invention solves technical solution used by above-mentioned technical problem：The miR-219a-5p is preparing anti-methylbenzene
Propylamine relapses the application in drug, it is characterised in that：Nucleic acid of the drug as effective quantity comprising SEQ NO.1 shown in sequence table
It is formed with pharmaceutically acceptable carrier or auxiliary material.
Further, the effective dose of the miR-219a-5p is 6~20 μ l/kg.
Further, the drug is injection medicament.
It is big establishing crystal methamphetamine compared with the prior art, the advantages of the present invention are as follows confirming through a large number of experiments
In mouse model, crystal methamphetamine cause microRNA nucleus accumbens septi expression change, target AT1 miR-219a-5p volt every
After core is overexpressed, ignite the big of induction to the Methamphetamine Relapse behavior and crystal methamphetamine of Conditioned cues induction
Mouse crystal methamphetamine Relapse behavior plays inhibiting effect, illustrate miR-219a-5p can be applied to treatment crystal methamphetamine at
Addiction is quit drug abuse using exploitation as gene therapy.
Detailed description of the invention
Fig. 1 is the foundation of crystal methamphetamine habituation model in the present invention；
Fig. 2 is the comparison figure that crystal methamphetamine causes miR-219a-5p expression quantity in nucleus accumbens septi in the present invention；
Fig. 3 is the microRNA dendrogram that crystal methamphetamine causes maincenter hypertensin system in the present invention；
Fig. 4 is the microRNA that maincenter hypertensin system is targeted in the present invention；
The expression variation that Fig. 5 is maincenter hypertensin system microRNA in the present invention；
Fig. 6 is the recession result figure that crystal methamphetamine rat subside 7 days in the present invention；
Fig. 7 is that miR-219a-5p nucleus accumbens septi is overexpressed the inhibiting effect relapsed to clue induction in the present invention；
Fig. 8 is that miR-219a-5p nucleus accumbens septi is overexpressed the ignite rat of induction of p-Methylamphetamine and relapses row in the present invention
For inhibiting effect；
Fig. 9 is that miR-219a-5p nucleus accumbens septi is overexpressed the inhibiting effect in clue induction is rebuild to AT1 in the present invention；
Figure 10 is that miR-219a-5p nucleus accumbens septi is overexpressed in drug-induced reconstruction to the inhibiting effect of AT1 in the present invention.
Specific embodiment
Below by way of accompanying drawings and embodiments are combined, the invention will be further described.
Crystal methamphetamine used in the embodiment of the present invention is standard items, is studied from Ningbo City's microcirculation and henbane class medicine
Institute, miR-219a-5p come from Shanghai JiMa pharmacy Technology Co., Ltd.
The foundation of 1 crystal methamphetamine intravenous self administration model of embodiment
Intravenous self administration (Intravenous Drug Self-Administration) is that research drug habit most passes through
One of animal model of allusion quotation, intravenous self administration model are to reflect that drug user actively looks for the classical animal mould of medicine and drug taking behavior
Type can use experimental animal and investigate administration motivation and active compulsive drug use behavior.
Classical Methamphetamine crystal methamphetamine self administration model can be very good simulation, and clinically drug abuse is suffered from
The crystal methamphetamine of person sucks behavior, and rat can produce the automedication of p-Methylamphetamine by training, while train
The behavior reaction rate and dosage of the p-Methylamphetamine constantly risen in journey can reflect drug addict and contact first from the beginning
Base amphetamine is to such a process for largely sucking crystal methamphetamine habituation.
We establish the self administration model of rat with the following method：
Firstly, SD rat carries out jugular vein intubation operation, it is inserted into one section of PE pipe in right jugular vein, and be pierced by body through back
Outside, antibacterial and restore one week or more after operation.Then, rat is trained in self administration operation cage respectively, training every time
The PE pipe of preceding connection rat back and the crystal methamphetamine injecting systems in operation cage；The two nose tentaculums in left and right are set in operation cage,
One of them is effective nose tentaculum, and rat touches nose, and once effective nose tentaculum will obtain needle crystal methamphetamine injection, is operated simultaneously
Cage lamp in cage lights (as a kind of clue with medicine), computer record；Another is invalid nose tentaculum, and rat touches nose one
Invalid nose tentaculum does not have any crystal methamphetamine injection and light down, and only computer records.Between crystal methamphetamine injection twice
There are 20 seconds refractory periods, crystal methamphetamine injection and light will not be had by during which touching the effective nose tentaculum of nose, and only computer records, often
It crystal methamphetamine injection needle number is limited to 50 needles (preventing excess injection dead).32 operation cages are using large server
Computer controls simultaneously, and self administration training software is the AniLabV652 independently write, and training program is fixed ratio
FR1, i.e. rat touch nose, and once effective nose tentaculum obtains needle crystal methamphetamine injection.Rat passes through 3 days, daily 4 hours instructions
After white silk, effectively touches rhinoreaction rate and injection volume obviously rises, tend towards stability within several days finally, so as to form stable methyl
Amphetamine addiction state.
A, method：Jugular vein intubation operation is carried out to rat, is restored 3 days later.After recovery, starts self administration, be divided into
Two groups, i.e. crystal methamphetamine group and physiological saline group, every group of 6 mouse.First three days are administration training periods, this stage allows mouse
Association.Continuous administered for fourteen days after association.
Crystal methamphetamine group：The crystal methamphetamine that solubility is 0.12mg/ml is prepared, according to 0.05mg/kg/infusion agent
Amount administration.
Physiological saline group：The group is control group, is directly administered with physiological saline substitution crystal methamphetamine.
It will be seen from figure 1 that administration number of times crystal methamphetamine group is significantly higher than physiological saline group, and maintain certain level
On, illustrate crystal methamphetamine group habituation, and self administration model has built up.
Expression of the embodiment 2 using Shanghai JiMa pharmacy Technology Co., Ltd qRCR detection kit to miR-219a-5p
It is detected
1. miR-219a-5p reverse transcription reaction system (20 μ l)：
After the above system mixes, 25 DEG C are incubated for 30 minutes, and 42 DEG C are incubated for 30 minutes, and 85 DEG C are incubated for 5 minutes, and 4 DEG C spare.
2. (PCR kit is derived from the Ma pharmacy of Shanghai Ji to microRNA real-time PCR (microRNA real-time quantitative PCR)
The primer of Technology Co., Ltd., miR-219a-5p is synthesized by Shanghai JiMa pharmacy Technology Co., Ltd)
Primer is：
F Primer:CTGATTCCCTGATTGTCCAAAC
R Primer:TATGCTTGTTCTCGTCTCTGTGTC
3. real-time quantitative PCR response procedures
What is identified by qRT-PCR method DNA amplification is purpose RNA (miR-219a-5p, nucleotides sequence
It is classified as sequence 1).
4. the results show that miR-219a-5p content relatively compares normal group mouse in crystal methamphetamine rat model nucleus accumbens septi
It decreased significantly, illustrate in crystal methamphetamine rat nucleus accumbens septi, miR-219a-5p expression lowers (result such as Fig. 2 institute
Show).
3 microRNA chip of embodiment
Since miRNA molecule fragment itself is small, type is more, and abundance is low, the global expression of overview miRNAs and mutually closes
System, under different condition differential expression, particularly with the relationship of disease and miRNA target gene and functional study etc., chip skill
Art is undoubtedly selection well.The principle of this technology is the miRNA collected in sample to be tested, the spy complementary in certain chip
Needle hybridization, the end miRNA 3' in usual sample to be tested can mark fluorophor, after hybridisation wash can scanning of fluorescent intensity, greatly
The miRNA of significant differential expression can be filtered out after amount data processing.Since chip technology uses extensive microarray technology,
Hundreds of probe can be analyzed on one chip simultaneously, substantially increases the speed and flux of screening, therefore be miRNA high
The first choice of flux research.And due to the high-throughput feature of miRNA chip, it is compared to Northern hybridization, RT-PCR, liquid phase is miscellaneous
The methods of hand over, miRNA chip technology is a kind of method for more preferably quickly and effectively detecting miRNA expression map.
Method：Microarray Experiments are carried out by LC Sciences company, and Microarray Experiments use 4-8 μ g total serum IgE sample.Make
Poly (A) tail is added at the end total serum IgE 3' with Poly (A) polymerase, then an oligonucleotide is marked and this poly
(A) tail connection (ligation) is used for subsequent fluorescent marker.Hybridization reaction utilizes micro circulation pump (Atactic
Technologies ringing) carries out overnight on μ Paraflo micro-fluid chip.On micro-fluid chip, every detection
Probe is all (complementary with target microRNA (from miRBase, http by a chemically modified nucleoside acid encoding section://www.mirbase.org/)It is complementary with other RNA (Quality Control or customization sequences)) and one be made of polyethylene glycol
The spacer spacing of coding section and matrix (expand) composition.Detection probe uses PGR (photogenerated
Reagent) chemical method carries out fabricated in situ.Hybridizing melting temperature is balanced by chemical modification detection probe.Hybridization makes
With 100 μ L 6xSSPE buffer (0.90M NaCl, 60mM the Na2HPO4,6mM EDTA, pH containing 25% formamide
6.8), hybridization temperature is 34 DEG C.After RNA hybridizes with probe, recycled on micro-fluid chip with the Cy3 dyestuff of label specific bond
Flowing is dyed.Hybridization image is acquired using laser scanner (GenePix 4000B, Molecular Device) and is used
Array-Pro image analysis software (Media Cybernetics) carries out image digitazation conversion.Data analysis is subduction first
Then background value carries out signal normalization using LOWESS filtering (Locally-Weighted Regression).
As can be seen from Figure 3 passively organize after crystal methamphetamine habituation relative to crystal methamphetamine has conspicuousness poor with physiological saline group
The microRNA of different expression has nearly 200.
4 microRNA target prediction of embodiment
According to the discrepant microRNA of the expression screened, carry out microRNA target prediction (based on TargetScan with
The databases such as KEGGpathway), filter out target microRNA.
Fig. 4 dendrogram, it is intuitive to reflect that microRNA expression status, Fig. 4 can be seen that in numerous differential expressions
In microRNA, by microRNA target prediction, have 26 it is related with maincenter hypertensin system.From Fig. 5, it can be seen that miR-
The expression multiple highest of 219a-5p, here it is targets of the invention.
The inhibiting effect that 5 miR-219a-5p of embodiment is relapsed in the overexpression of nucleus accumbens septi in Methamphetamine
Relapse be clinical treatment crystal methamphetamine habituation difficult point, crystal methamphetamine patient carries out after detoxification gives up,
It is contacted again previous drug abuse relevant environment, drugs itself or poison friend to instigate under induction, often generates and suck crystal methamphetamine
Impulsion, and eventually lead to crystal methamphetamine habituation again.The model that relapses of rat is able to reflect in crystal methamphetamine
The patient of During The Withdrawal Period is contacted again to suck behind the relevant environment of crystal methamphetamine or crystal methamphetamine sheet in the past, led to methyl
Amphetamine relapses the behavior of (habituation again).It is whether big for crystal methamphetamine habituation in order to evaluate miR-219a-5p overexpression
The Relapse behavior of mouse has an impact, we establish Conditioned cues respectively or the ignite rat of induction of crystal methamphetamine relapses mould
Type.Firstly, 48 rats of training are trained in self administration operation cage by crystal methamphetamine self administration in 14 days, subsequent institute
There is rat to subside 7 days, finally natural withdrawal 7 days in rearging cage, finally distinguishes determination condition clue or first in operation cage
Base amphetamine ignites the Methamphetamine Relapse behavior of induction.
(1) the Methamphetamine Relapse behavior measurement of Conditioned cues induction：
24 successful rats of crystal methamphetamine habituation training are chosen from all rats, subside 7 days, are subsided (such as
After Fig. 6), brain solid is done to rat, packaged slow virus is injected into rat nucleus accumbens septi (NAc), then give up within 7 days,
Crystal methamphetamine is carried out after giving up relapses measurement.It is divided into 2 groups, every group 8.①LV1CN：The slow disease of brain solid microinjection LV1CN
Poison；2. LV1-miR-219a-5p group：The slow virus of brain solid microinjection LV1-miR-219a-5p.Slow virus injection dosage can be
6-20 μ l/kg, injection volume is 14 μ l/kg herein.Wherein LV1-miR-219a-5p group microinjection contains the slow of target microRNA
Virus, microRNA of the LV1CN group microinjection containing empty plasmid.
Two groups of rats respectively correspond after giving up be placed in front of self administration training when to operation cage, utilize conditionity
Clue (sound for giving the cage lamp, syringe pump that light when crystal methamphetamine injection is trained before rat) is carried out induced rat and is generated again
The behavior of suction, program are also the FR of fixed ratio.When program starts, the cage lamp operated in cage is lighted, and the nose of rat touching at this time is effective
Nose tentaculum, syringe pump can issue sound, but inject without crystal methamphetamine, testing time 2h, thus to evaluate miR-
It is overexpressed in 219a-5p nucleus accumbens septi and whether has an impact to the Methamphetamine Relapse behavior that Conditioned cues induce.
From figure 7 it can be seen that LV1-miR-219a-5p group, relative to LV1CN group, effective nose touching is remarkably decreased (p<
0.01), it may be said that bright rat nucleus accumbens septi miR-219a-5p overexpression can significantly inhibit clue induction Relapse behavior.
(2) crystal methamphetamine ignite induction Methamphetamine Relapse behavior measurement
24 successful rats of crystal methamphetamine habituation training are chosen from all rats, subside 7 days, after recession,
Brain solid is done to rat, packaged slow virus rat nucleus accumbens septi (NAc) is injected into, then give up within 7 days, given up laggard
Row crystal methamphetamine relapses measurement.It is divided into 2 groups, every group 8.①LV1CN：The slow virus of brain solid microinjection LV1CN；②LV1-
MiR-219a-5p group：The slow virus (each 1 μ l of site) of brain solid microinjection LV1-miR-219a-5p.All rats are being tested
Preceding 15min subcutaneously injects a needle crystal methamphetamine (10mg/kg), then respectively corresponds self administration training when institute before placement
To operation cage, crystal methamphetamine ignite induced rat relapse measurement when, cage lamp and syringe pump sound no longer occur, rat
Touching the effective nose tentaculum of nose, only computer records, and similarly injects without crystal methamphetamine, testing time 2h.Thus to evaluate
Be overexpressed in miR-219a-5p nucleus accumbens septi p-Methylamphetamine itself ignite induction Methamphetamine Relapse behavior whether
Have an impact.
LV1-miR-219a-5p group is remarkably decreased (p relative to LV1CN group, effective nose touching number as can be seen from Figure 8<
0.01), it suffices to say that bright rat nucleus accumbens septi miR-219a-5p overexpression can significantly inhibit drug-induced Relapse behavior.
5 miR-219a-5p rat nucleus accumbens septi of embodiment, which is overexpressed, is relapsing inhibition to the expression of angiotensin-ii receptor 1
Influence
Nucleus accumbens septi (NAc)：It is incoming to receive prefrontal cortex, hippocampus, the Glutamatergic nerve of amygdaloid nucleus, research shows that NAc joins
With " stimulation award " related with habituation study, the nerve fibre of NAc mainly terminates at its shell region, and with project VTA
GABA serotonergic neuron formed synaptic contact, directly participate in and drug reward motivation and emotional activity.
Method：" Le rat reaches stable crystal methamphetamine habituation state by training in 14 days, and SD rat is divided into two
Group, every group 6, respectively LV1CN group and LV1-miR-219a-5p group, broken end take brain, and it is real to carry out Western Blotting
It tests, NAc brain area is removed, be divided in clean grinding pipe and be placed on ice for use.Grinding bead is added in each grinding pipe, splits
Liquid (RIPA) and protease inhibitors are solved, after various reagents accurately add well, is placed in grinder after grinding 30s to take out rapidly and set
In on ice.It is centrifuged 15 minutes under 4 DEG C of 13,000g after 5min, its supernatant is taken after centrifugation, utilize the taken egg of BCA kit measurement
White concentration, and being adjusted correspondingly is consistent protein concentration as far as possible.The egg of 5x is then added in every pipe supernatant
White buffer 35ul is simultaneously placed in boiling water bath after 10min cooling be placed on ice for use.
2. the sample after above-mentioned water-bath is carried out PAGE gel electrophoresis by electrophoresis, each brain area does 3 multiple holes.
3. transferring film, NC film, which will be pre-chilled, in transferring film liquid will balance pretreatment 10 to 15 minutes because transferring film liquid contains formaldehyde, and hand
The first albumen that bag has will cause the pollution of film, so clean gloves must be taken.Notice that sponge will be sufficiently humidified so as to, bubble will be caught up with
It walks completely, clip, which is opened, keeps black one side holding horizontal, is padding a foam-rubber cushion above, is rolled back and forth with glass rod several all over to roll away
The bubble of the inside.Three layers of filter paper are padded on Sponge cushion, fixed filter paper, rolls bubble therein with glass rod on the other hand on the other hand, stops
Horse back transferring film is wanted after running glue, otherwise albumen can be spread, and caused band to change and then influenced experimental result.In separation gel and dense
A line is drawn between contracting glue, gently push concentration glue aside, and it is removed, NC membrane cover on separation gel, and is aligned, adds
A little transferring film liquid is maintained at film in wet state, bubble cannot be had under film.
4. being immunoreacted, the above-mentioned film made is put into box, it will be such as confining liquid (generally skimmed milk power), room temperature closing
2 hours or 4 DEG C overnight.After the completion of closing, confining liquid is removed, primary antibody is added overnight (or room temperature 5h), primary antibody incubation is over
Afterwards, it recycles primary antibody and cleans 3 each 5min with the TBST of 1X, secondary antibody is added, is protected from light 2h.After being protected from light, secondary antibody is recycled, and
It is cleaned 1 time after cleaning 3 times with the TBST of 1X with 1X TBS.
5. developing the color, after the completion of above-mentioned steps, film is placed on instrument and is developed the color, and carries out observation and the ash of protein band
The measurement of angle value.
From fig. 9, it can be seen that LV1-miR-219a-5p group is relative to LV1CN group, the expression of AT1 in clue induction reconstruction
It is remarkably decreased (p<0.01), illustrate that miR-219a-5p is overexpressed the expression that can inhibit AT1.
From fig. 10 it can be seen that LV1-miR-219a-5p group is relative to LV1CN group, the table of AT1 in drug-induced reconstruction
Up to being remarkably decreased (p<0.05), illustrate that miR-219a-5p is overexpressed the expression that can inhibit AT1.
Claims (3)
1. a kind of miR-219a-5p relapses application in drug preparing anti-crystal methamphetamine, it is characterised in that：The drug by
Nucleic acid of the effective quantity comprising SEQ NO.1 shown in sequence table and pharmaceutically acceptable carrier or auxiliary material composition.
2. miR-219a-5p according to claim 1 relapses application in drug, feature preparing anti-crystal methamphetamine
The effective dose for being the miR-219a-5p is 6~20 μ l/kg.
3. miR-219a-5p according to claim 1 relapses application in drug, feature preparing anti-crystal methamphetamine
It is that the drug is injection medicament.
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| MicroRNA Expression Profile and Functional Analysis Reveal that miR-206 is a Critical Novel Gene for the Expression of BDNF Induced by Ketamine;Xiaolin Yang etal;《NeuroMolecular Medicine》;20140518;第16卷(第3期);参见第594-605页 * |
| Serum brain‐derived neurotrophic factor levels were reduced during methamphetamine early withdrawal;Pao‐Huan Chen etal;《addiction biology》;20120328;第482-485页 * |
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