CN104293791B - New applications of the miR 200b in the protein expression inhibitor of Rac 1 is prepared - Google Patents

Abstract

The invention belongs to biology field, and in particular to new applications of the miR 200b in the protein expression inhibitor of Rac 1 is prepared.Invention initially sets up HIBI model, carries out miRNA activators or the antagonist transfection of respective sets, is overexpressed and low expression miR 200b respectively.As a result after showing transfection miR 200b, the protein expressions of Rac 1 reduce, and after antagonism miR 200b, the protein expressions of Rac 1 further raise, and illustrate that expression of the miR 200b by adjusting the albumen of Rac 1 participates in the repair of hypoxic-ischemic brain damage.The invention provides miR 200b new opplication, can effectively adjust the expression of the albumen of Rac 1, has important clinical value.

Description

New applications of the miR-200b in Rac-1 protein expression inhibitor is prepared

Technical field

The invention belongs to biology field, and in particular to a kind of Rac-1 protein inhibitors and its application, more specifically It is related to a kind of miRNA and its application.

Background technology

MicroRNAs (miRNAs) is a kind of endogenous non-coding RNA, is primarily involved in horizontal regulation and control after genetic transcription. MiRNAs causes target mrna degradation or Translational repression by being combined with the non-translational region (UTR) of mRNA (mRNA) 3 '.It is small with other Molecule RNA is different, and miRNA does not need strict base pairing, therefore, can regulatory gene network extensively.Although miRNA life Thing function is just known, and the treatment potentiality that it is shown in disease incidence and progress has caused people greatly emerging Interest.Increasing evidence shows, the gene therapy based on miRNA, either strengthens or suppress miRNA expression and work Property, there is very big application prospect.

Rac-1 full name are related (the ras-related C3 botulinum toxin of C3 botulinum toxins substrate 1 of Ras Substrate 1), belong to Rho-GTP enzyme families.Rac-l wide expressions, exist with two kinds of conformational states, a kind of is inactivation GDP combining forms, another kind are the GTP combining forms of activation.In the presence of extracellular signal, Racl is exchanged by guanylic acid The factor (GEFs) is transformed into its activated form.And gtpase activating protein (GAPs) then makes Rac-l lose activity.Rac-l passes through energy The domain and special effector interaction for coordinating the reaction of multi-signal transduction cascade have impact on a variety of physiological actions.Rac- 1 albumen take part in a variety of vital movements of many cells, including the reconstruct of actin cytoskeleton, cell adhesion, cell fortune Dynamic, the polarity of epithelial cell, endochylema move, genetic transcription and expression and the regulation and control of Cellular cycle and apoptosis etc..It is wherein heavier Want, Rac-1 can be adjusted JNK activity, the effect such as cytoskeleton is remolded so as to play it by participating in Wnt/PCP paths. Rac-1 also can participate in Anoxia-Signaling Pathway path by the expression and activity for regulating and controlling HIF-1 α.Racl can also pass through JNK2 kinases Cause β-catenin in Serl91 and Ser605 phosphorylation, and then promote β-eatenin nuclear translocation, so as to promote Classical wnt signal transduction pathway.Rac-1 albumen take part in each stage of tumorigenesis, including tumour growth With reproduction, invasion and attack and transfer, cell cycle progress, Apoptosis and it is tumor neovasculature generation etc..Rac-1 molecular functions Diversity it is relevant with the diversity of downstream effect molecule thereon.

It is contemplated that relations of the research miR-200b in the tissue of hypoxic-ischemic brain damage with Rac-1.Invention is first HIBI model is first established, carries out miRNA activators or the antagonist transfection of respective sets, respectively mistake Expression and low expression miR-200b, take rat cerebral tissue to carry out Western blot experiments in different time points, detect Rac-1 eggs White variation tendency, so as to understand effects of the miR-200b to Rac-1.As a result after showing transfection miR-200b, Rac-1 albumen tables Up to reduction, after antagonism miR-200b, Rac-1 protein expressions further raise, and illustrate that miR-200b may be by mediating Wnt/PCP Path participates in the repair of hypoxic-ischemic brain damage.The invention provides miR-200b new opplication, can effectively adjust The expression of Rac-1 albumen is saved, this has important clinical value.

The content of the invention

It is an object of the invention to provide a kind of method of suppression Rac-1 protein expressions, including：

1) miRNA-200b analogies are prepared；

2) miRNA prepared by upper step is transfected into the cell or tissue of expression Rac-1 albumen.

The miRNA-200b mature sequences are mmu-mir-200b-3p (miRBase accession number is MIMAT0000233).

Further, the miRNA is selected from pri-miRNA, pre-miRNA, ripe miRNA, dsmiRNA and its fragment or change One or more in body.

MiRNA-200b, miR-200b, microRNA-200b belong to the different expression-forms of same substance in the present invention, Representative is equivalent in meaning.

Term-miRNA ‖ (Microrna) refer to any kind of RNA interfering, include but is not limited to, endogenous microrna RNA and Artificial microrna RNA.Interior miRNAs are the naturally occurring tiny RNA s in genome, and it is capable of the Commercial cultivation of regulating mRNA.Art The ‖ miRNA of synthesis include any kind of RNA sequence in addition to interior miRNAs to language-artificial ‖ or-, and it can be adjusted MRNA Commercial cultivation.

- Microrna flanking sequence ‖ refers to the nucleotide sequence for including miRNA machine components.MiRNA machine components are to promote Into the Min. nucleotide sequence that ripe miRNA is produced from precursor miRNA.Referred to as pri-miRNAs precursor miRNA is in cell The pre-miRNAs of about 15-70 nucleotides is processed in core, it is folded into incomplete stem-loop structure.

MiRNA analogies (miRNA mimics) have become one of instrument of research gene regulation.miRNA mimics It is a kind of RNA molecule, can be by vectors into cells, up-regulation miRNA in the cell or in vivo miRNA concentrations, from And controlling gene is expressed.The miRNA mimics of exploitation have two kinds, and a kind of is the precursor more than base of precursor molecule 60；One kind is The ripe body molecule of double-strand.Both molecules all it has proven possible to raise the abundance of miRNA in the cell well.

MiRNA inhibitor (miRNA inhibitor) be with the miRNA inhibitor that is chemically synthesized, can by with Muture miRNA molecule specifically binds and suppresses miRNA effects, can weaken gene caused by endogenous miRNA in cell and adjust Control acts on.

Nucleic acid delivery systems include desired nucleic acid, such as, but not limited to, using as-exposed ‖ nucleic acid-exposed ‖ Form, or prepare in the medium for being suitable for delivering, such as with cationic molecule or liposome formative lipid In compound, the either component as carrier or the component as pharmaceutical composition.Can be by nucleic acid delivery systems directly (such as by making it be in contact with cell) or indirectly (such as effect by any biological process) are supplied to cell.Example Such as but it is not limited to, nucleic acid delivery systems can be supplied to cell in the following manner：Encytosis, receptor target are and natural Or synthetic cell membrane-bound fragment is mutually coupled, physical means such as electroporation, make nucleic acid delivery systems and polymer support such as controlled release Film or nano particle or microparticle are combined, and using carrier, nucleic acid delivery systems are expelled into the tissue or stream around cell In body, nucleic acid delivery systems simple diffusion passes through cell membrane, or passes through cell by any actively or passively transporting mechanism Film.Furthermore it is possible to the targeting related to antibody by using such as viral vector and the technology by antibody-mediated immobilization Nucleic acid delivery systems are supplied to cell.

Further, the miRNA is incorporated into carrier.

Carrier includes but is not limited to plasmid, clay, phasmid, virus, the other media thing from virus or bacterial origin, It is by inserting or mixing the nucleotide sequence for producing Microrna and can be attached to the free core of these nucleotide sequences Acid fragment and operated.Virus and retroviral vector are preferable bearer types, and include but is not limited to come from The nucleotide sequence of lower influenza virus：Retrovirus, such as：Moloney murine leukemia virus；Murine stem cell virus, Ha Wei (Harvey) murine sarcoma virus；MuMTV；Rous sarcoma virus；Adenovirus；Adeno-associated virus；SV40- types virus； Polyomavirus；Epstein-Barr virus；Papillomavirus；Herpesviral；Vaccinia virus；Poliovirus；For example appoint with RNA virus What retrovirus.Those skilled in the art can easily use other carriers known in the art.

One or more in carrier preferred plasmid, clay, phasmid, virus and artificial chromosome.

MiRNA can synthesize acquisition, for example, by via any synthetic method known to technical staff come chemical synthesis core Acid.Then, synthesized miRNA can be purified by any method known in the art.For chemical synthesis nucleic acid Method includes but is not limited to, iii vitro chemical synthesis, and it uses phosphotriester, phosphate or phosphoramidite chemistry and solid phase technique, Or via via deoxyribonucleoside hydrogen phosphonate ester intermediate.

Further, described miRNA is the RNA through chemical modification.Described chemical modification includes glycosyl modified, phosphoric acid chain Modification, base modification, protrusion and end modified, double-spiral structure modification etc..

Modification on ribose is the most extensive, because necessary to 2 '-OH are not active siRNA.RNA 2 '-O methylate can To improve the stability of binding affinity and nuclease-resistant, and there is good compatibility in double helix, these advantages make it As one of modification being most widely used.2 '-O- methoxyethyls modification have been used to a siRNA 3 ' distal process go out and justice On chain, but antisense strand is not suitable for it.The modification of 2 '-O- pi-allyls is gone out except that can apply in 3 ' distal process, other most of sites Modification can all cause active reduction.70% 2 '-OH are that 2,4- dinitrophenyls ether can change by random replacement in siRNA double-strand It is apt to its many property, such as high-affinity, nuclease-resistant stability and potency.

Part substitutions of the 2 '-F-RNA on positive-sense strand and antisense strand can be all resistant to, some siRNA substituted entirely It is same active.In addition, 2 '-F-RNA modifications also greatly improve siRNA plasma stability, improve the combination parent of double helix And property.The spatial chemistry for changing 2 '-F-RNA fluorine atom obtains 2 '-F-ANA, and it also has resistance to well in siRNA double helixs By property, including the modification of the part of full modification to positive-sense strand and antisense strand, its introducing also bring high-affinity and nuclease-resistant Stability.

DNA is also a kind of modification in itself, does not have electronegative substituent in its 2 '-position, and this modification equally can be by SiRNA double helixs are received, in 3 '-jag, or base-paired regions.All repaiied with DNA purine and 2 '-F-RNA pyrimidines The antisense strand of decorations has silencing activity.8 base zones held to antisense strand 5 ' have been replaced silencing activity with dsDNA, and can Reduction is missed the target effect.

Sugared epoxy can also be modified, and 4 ' S-RNA have high-affinity, and the stability of nuclease is greatly improved.It There is good tolerance siRNA double helixs end.In 2 chain ends 4 ' S-RNA and 2 '-O- methyl, 2 '-O- methoxyethyl groups Close modification and show good effect and plasma stability.4 ' the S-FANA modified 2 '-and 4 '-position are inserted into 2 chains Each position also show that siRNA activity.But its relatively low compatibility only allows to insert double helix on a small quantity.

LNA modifications are applied equally well to siRNA modifications, and the rigidity characteris of its conformation significantly enhances its compatibility. Such a modification is carefully introduced on siRNA and can obtain polytype function double helix.The site for being most commonly used to modification is positive-sense strand End and antisense strand 3 '-jag.

Several changes of phosphoric acid chain can also be received by RNAi mechanism.Phosphorothioate bond (PS) modification after with it is unmodified SiRNA is compared, and potency is suitable or decreases.Whether PS modification structure dependent on chain can all be carried out.Have been observed that Cytotoxicity after a large amount of PS modifications.But PS modifies not to be appeared to significantly affect to siRNA bio distribution.

The siRNA of boranophosphate ester bond modification has active for gene silencing, can be carried compared with PS modifications and unmodified siRNA High silence efficiency.But in order to reach maximum efficiency, antisense strand center should not be modified.This modification can significantly improve siRNA's Nuclease-resistant stability.

The 2 ' of nucleotides, 5 ' connected modes (2 ', 5 '-DNA, or 2 ', 5 '-RNA) can replace 3 ', 5 ' connected modes, but Positive-sense strand is only applicable to, and amount of activated forfeiture can be caused.The amido link of nonionic have been applied in siRNA 3 '-it is prominent Go out end.

Base modification.SiRNA base modification is more confined from, can stable A-U base pairings (5-Br- uracils and 5-I- Uracil replaces uracil, and diaminopurine replaces adenine) modification can be received by siRNA, but this transformation can cause Activity decreases.The modification of 4- deracils has been applied.The pseudo- uracil that 2- deracils connect with C- can improve affine Property, and potency and specificity can be improved, on condition that the correct position in double helix introduces.The 5- of pyrimidine, which methylates, (uses T U and C is replaced with 5-Me-C) usually modification such as DNA, 2 '-F-ANA and LNA the modification combination with sugared ring.

Some off-gauge base structures are also used in siRNA.Such as difluoro-benzene base, it is identical with thymidine shape But hydrogen bond can not be formed, the uracil base of some position can be replaced in whole siRNA double-spiral structures, without significantly Degree reduces potency or changes cracking mechanism.Nonaromatic dihydrouracil can also be used, but because it is to base stacking Effect is not contributed, so the affinity of double helix can be reduced, is preferably inserted into 5 ' ends of double helix.

It is prominent and end modified.Earlier studies have shown that preferable siRNA is made up of the chain of 2 21 bases, including 2 The 3 ' of base-protrusion.These protrusions can be modified in various manners：Initially usually modified using deoxy residues, both can be with Reduce cost, it is also possible to improve its anti-3 '-exonuclease ability.Foregoing most modifications are all feasible in protrusion, can also make With flat end siRNA.Flat end siRNA is more resistant to 3 '-exonuclease.

Chain end can equally be transformed.The chemical phosphorylation that antisense strand 5 ' is held may insure high-titer.In addition, The end of siRNA double helixs can connect the end of various groups, especially positive-sense strand.These groups include upset Non-base end-blocking of upset, it helps to strengthen anti-exonuclease enzyme stability.It is generally acknowledged that 5 ' ends of antisense strand are most sensitive to modifying.

Double-spiral structure is modified.Double-spiral structure can be modified by chemical synthesis in itself.Although most siRNA are double Spiral is made up of 2 chains, but the verified (justice of 1 complete antisense strand and 2 9-13 bases being made up of 3 chains Chain) siRNA can reduce and miss the target effect and increase activity；It is referred to as small center fragmentation RNA interfering (small Internally segmented interfering RNA, sisiRNA).SiRNA also can be by chain shape in many ways Into such as hair fastener type double helix and dumbbell shape RNA or nanometer ring structure, it not only maintains RNAi activity, while also avoids completely Degraded of the exonuclease to it.There are some researches show single-stranded antisense RNA (being not folded into double helix) can enter RNAi Path, in any case, its potency is close to double helix siRNA.

The length of siRNA double helixs can change.Increase substrate of the siRNA double helixs length as Dicer, can be with Improve its potency.It is important that length is kept below 30 bases, to avoid triggering ifn response.

The miRNA can be administered as exposed RNA in salt solution, or siRNA is wrapped in into transport vehicle On.Directly applying is included with intravenous, subcutaneous, intramuscular, intranasal, intraperitoneal, Via vagina, per anum, oral, intraocular or intrathecal side The miRNA is applied directly to illing tissue by formula.Being shown in terms of carrier in terms of siRNA administrations well has chitosan, tree Shape polyamide-amide, polyethyleneimine, tree-shaped polylysine, PLGA, atelocollagen, cyclodextrin And the siRNA movement systems based on liposome or vesica and parcel siRNA lipid bilayer etc..

One of the most frequently used transfection reagent when liposome is in-vitro transfection, however, because of its toxicity, non-specific uptake and exempting from Epidemic disease is reacted so that its safety in vivo difficult to realize and efficient transfection.Most non-specific respondings and toxicity both originate from its particulate table The positive charge in face, and these positive charges be exactly with nucleic acid with reference to necessary to.Presently commercially available is several based on liposome Targeting miRNA in-vitro transfection reagent applies this principle.Researchers develop polycation lipesome-hyaluronic acid (LPH) transportation system of the nano particle as miRNA.The advantages of this induction system, is to transfect siRNA and/or miRNA Several different oncogenic pathways can be suppressed simultaneously.

Polyethyleneimine (Polyethyleni-mine, PEI)：Polyethylene is the polymerization for gene transfection most study One of thing.Under physiological environment, PEI due to amido protonate it is positively charged, so as to nucleic acid polymerization.In cell surface, PEI polymerize with the positively charged cationic polymer that nucleic acid is formed with the negatively charged polysaccharide of cell surface, passes through endocytosis Effect enters cell.The polymer is entered by so-called proton sponge effect depolymerization, a large amount of trap protons of PEI, water in vivo Cell, cause endothelia swelling, the final polymer that discharges is into cytoplasm.Using PEI as transfection reagent, successfully by DNA Each histoorgan in body animal model is transfected into siRNA.Except traditional transfection method, the gene based on PEI Transmission system also can pass through blood-brain barrier (blood-brain barrier, BBB) through further modification.From Rabies Virus Albumen (rabies virus glycoprotein, RVG) extracts small peptide, the PEI-RVG of connection PEI compositions, can combine nerve Cell surface nicotinic acetycholine specific receptor.RVG is connected by disulfide bond (RVG-SSPEI) with PEI, transfects miR- 124a, promote nerve to occur.To overcome PEI carriers because volume can not pass through greatly very much blood-brain barrier, the penetrating blood brain of mannitol can be used Barrier.

Dendritic (dendrimer) also result in larger concern in gene delivery, its unique spherical junctions Structure and higher surface-to-volume ratio, at utmost reduce cytotoxicity, improve transfection efficiency.Use polyamidoamine Dendrimer (PAMAM) adds Apoptosis after transfecting miR-21 and 5 FU 5 fluorouracil (5-FU).With only being controlled with 5-FU Treatment is compared, and the transfer ability of tumour cell declines.Compared with taxol, after transfecting anti-miR-21 with PAMAM, human brain is added The chemosensitivity of glioma cell.Although not being to transfect in vivo, this still shows that dendritic can be used for internal miRNA Transfection.

PLGA (poly (D, L-lactide-co-glycolideacid), PLGA) has combination concurrently MiRNA ability and the non-specific ability for entering cell.In past half a century, PLGAs is that one kind is widely used in biology The non-soluble polymer in field.PLGAs polymer is easily made into particulate or nano particle combination bioactive molecule.Use PLGA It can play a part of protecting medicine for nanoparticle made from material, improve bioavilability, reach the slow controlled release of long period Purpose, great potentiality are demonstrated by clinical trial.In this case it is necessary to by PLGA particulate Pegylations, surely Determine space structure, and strengthen Premeabilisation of cells ability.PLGA particulates are easily further cut by nonspecific degradation, Pegylation Weak its enters the ability of cell, so the accumulation in diseased organ is less than 5%.There is scholar to propose that RNA degradeds occurred in week In the endosome and lysosome enclosed, if this hypothesis is true, then a series of design standards on miRNA will be changed.

It is an object of the invention to provide applications of the miRNA-200b in Rac-1 protein expression inhibitor is prepared.

The miRNA-200b sequences are shown in GenBank NR_029587.1 or GI:262206115.

The miRNA is in pri-miRNA, pre-miRNA, ripe miRNA, dsmiRNA and its fragment or variant It is one or more of.

Described miRNA can the RNA through chemical modification.Described chemical modification include glycosyl modified, phosphoric acid chain modification, One or more in base modification, protrusion and end modified, double-spiral structure modification.

MiRNA is preferably used for therapeutical uses in combination with suitable pharmaceutical carrier.Such composition includes effective dose Compound and pharmaceutically acceptable carrier or excipient.Preparation of preparation is to be suitable for method of application.It is pharmaceutically acceptable Determined by particular composition to be administered and the ad hoc approach for applying said composition carrier part.Accordingly, there exist There is the appropriate formulation of the huge variety of pharmaceutical composition comprising nucleic acid.

The antisense being carried carried in the present invention is to suppress the most often applied means of microRNA functions.Krutzfeldt etc. It was found that the oligonucleotides of a kind of new chemical modification that can specifically and effectively suppress microRNA expression, is referred to as antagomirs.Antagomirs is cholesterol conjugation single strand RNA molecule (21-23nt), complementary with the target microRNA of maturation.

Brief description of the drawings

Testing result (fluorescence microscopy after miR-200b antagomir and the antagomir NC of Fig. 1 transfection Cy3 marks Mirror) A：miR-200b antagomir；B：miR-200b antagomir NC；

Fig. 2 different time points miR-200b expression quantity line charts

Fig. 3 difference group transfer rod time changes

Fig. 4 difference group escape latencies (EL) change

Fig. 5 difference group Rac-1 protein expression levels

Fig. 6 difference group Rac-1 expressing quantity line charts

Embodiment

With reference to specific embodiment, the present invention is expanded on further, is only used for explaining the present invention, and it is not intended that to this The limitation of invention.It will be understood by those skilled in the art that：Can in the case where not departing from the principle and objective of the present invention So that these embodiments are carried out with a variety of change, modification, replacement and modification, the scope of the present invention is limited by claim and its equivalent It is fixed.The experimental method of unreceipted actual conditions in the following example, generally according to normal condition or according to the bar proposed by manufacturer Part examinations.

Embodiment 1 establishes miR-200b transfection neonatal rats with ischemia-hypoxia brain injury models

First, materials and methods

Main agents and consumptive material：

microON^TM miR-200b agomir、microON^TMMiR-200b agomir NC, Cy3 are marked microOFF^TMMiR-200b antagomir, Cy3 mark microOFF^TM miR-200b antagomir NC、Bulge- LoopTM miRNA qRT-PCR Primer, (mentioned reagent is all from Guangzhou Rui Bosheng to U6 snRNA qRT-PCR Primer Thing Science and Technology Ltd.), EntransterTM-in vivo RNA transfections reagent (Beijing Ying Geen bio tech ltd), MiRNA extracts kits, the first chains of miRNA cDNA synthetic agent box, (the above-mentioned examination of miRNA fluorescent quantificationally PCR detecting kits Agent is all from CWbio.Co.Ltd)

Major experimental instrument and equipment：

Needle holder, ophthalmology dissecting forceps, ophthalmologic operation are cut, operating table (U.S. Shor-line), Ritter shadowless operating lamps (Taiwan is good to close doctor's material share for (U.S. MidMark), micro syringe, spectrophotometer, band nonabsorbable surgical suture sewing needle Co., Ltd), it is gel imaging system (BIO-RAD), fluorescence microscope (Olympus), electrophoresis apparatus (Liuyi Instruments Plant, Beijing), real When quantitative fluorescence PCR instrument 7500 (American AB I companies), superclean bench (Beijing Guan Peng cleaning equipments Co., Ltd), electric heating it is permanent Warm water bath cabinet (Beijing Xicheng District medical apparatus and instruments factory), frozen tissue section machine (German leica)

Experimental animal source：

SPF levels 3 age in days SD rats (male and female are disregarded, body weight 8-10g) of new life tie up tonneau China experimental animal technology by Beijing to be had Limit company provides [SCXK (capital) 2012-0001].

Experimental method：

(1) random packet：

Normal group (control)：Newborn 3 age in days SD rats, not specially treated.

Negative control group of hypoxia and ischemia group (HI)：Newborn 3 age in days SD rats, forever ligature left common carotid, and anoxic 2.5h, Deficiency Bilateral telocoele injects 2ul sterile PBS respectively after blood 12h.

Activator group (HI+agomir)：Hypoxia and ischemia (specific method and the same negative control group of hypoxia and ischemia group of experiment control factor) 12h Afterwards, bilateral telocoele injects 2ul miR-200b agomir and the mixture of nanometer transfection composite respectively respectively.

Activator negative control group (HI+agomir NC)：After Hypoxia and ischemia 12h, bilateral telocoele injects 2ul's respectively MiR-200b agomir NC (Negative control) and nanometer transfection composite respectively mixture.

Antagonist group (HI+antagomir)：After Hypoxia and ischemia 12h, bilateral telocoele injects 2ul miR-200b respectively Antagomir and nanometer transfection composite respectively mixture.

Antagonist negative control group (HI+antagomir NC)：After Hypoxia and ischemia 12h, bilateral telocoele injects 2ul respectively MiR-200b antagomir NC and nanometer transfection composite respectively mixture.

(2) HIBI model is established：

1st, ischemic：3 age in days SD rats, 4% chloraldurate intraperitoneal injection of anesthesia (40ul/ is only).Mouse faces upward position and is fixed on It is sterile single fixed in surgical console, after skin of anterior portion of neck sterilization, skin herein is cut off, makees throat median incision, successively separates Exposure left common carotid, 2 surgical threads are passed through below the left common carotid of separator well, are cut after ligaturing respectively from centre It is short.Sew up the incision.Postoperative 30 minutes warming, time female cage rest 3 hours completely after revival of being rested in 37 DEG C of water bath.

2nd, anoxic：Ischemic preconditioning is placed in after 3.5 hours in closed plastic box, persistently passes to nitrogen oxygen atmosphere (8% oxygen + 92% nitrogen) 2.5 hours, keep 37 DEG C of the temperature inside the box.Send female cage after the completion of anoxic back to.

(3) miRNA is transfected in body animal：

1st, the dilution of nucleic acid：Nucleic acid is diluted to 2ug/ul with pure water, takes 10ul nucleic acid, adds 10% glucose solution 10ul, make glucose final concentration of 5%, final volume 20ul, fully mix.

2nd, the dilution of transfection reagent：Take 5ul transfection reagent Entranster^TM- in vivo RNA, with the 10% of 10ul Glucose solution dilutes, and is supplied with pure water 5ul, obtains glucose final concentration of 5%, and final volume is 20ul liquid, fully Mix.

3rd, transfection composite is formed：Transfection reagent after dilution is added in the nucleic acid solution after dilution, immediately fully Mix.

4th, it is stored at room temperature 15 minutes.

5th, animal is injected：Rats with bilateral intracerebroventricular injection, per side 2ul, (let the acupuncture needle remain at a certain point 30 seconds, ensures that liquid all penetrates into brain group Knit).

6th, 1d after transfecting, randomly selects 3 rats in each group of transfection nucleic acid, and broken end takes brain, is put into 4% paraformaldehyde Solution, 8 hours are fixed in 4 degree of refrigerators, each 8 hours of 20%, 30% sucrose solution serial dehydration, row frozen section (slice thickness About 15 μm) after fluorescence microscopy Microscopic observation transfected condition.

7th, the sacrificed by decapitation after 0h, 12h, 1d, 3d, 5d, 7d after the completion of injection respectively, peel off brain tissue (remove olfactory bulb and Cerebellum), it is stored in liquid nitrogen, RNA to be extracted.

(4) real-time quantitative fluorescence PCR

The brain tissue at each time point left and taken is taken out in liquid nitrogen, row real-time quantitative fluorescence PCR detects miRNA change.Tool Body method is as follows：

1st, the total miRNA of sample is extracted：

Total serum IgE in tissue samples is extracted with miRNA extracts kits (CWbio.Co.Ltd, Cat#CW0627).

1) it will be organized in liquid nitrogen and grind, 20mg tissues add 1ml Buffer RLM, and concussion mixes.Room temperature places 5 points Clock, it is kept completely separate nucleic acid complexes.

2) ratio that 200ul chloroforms are added with every 1ml Buffer RLM adds chloroform, covers lid, acutely shakes 15s, Room temperature places 5min.

3) 4 degree of 12,000rpm are centrifuged 15 minutes, and sample is divided into three layers：Red organic phase, intermediate layer and colourless aqueous phase, will Upper strata aqueous phase is moved on in a new centrifuge tube, carries out next step operation.

4) absolute ethyl alcohol of 1.25 times of volumes is added, is mixed.

5) solution for obtaining upper step and precipitation are transferred in the adsorption column RM for having been charged into collecting pipe together, (can be several times It is transferred to).12000rpm centrifuges 30s, abandons collecting pipe waste liquid, adsorption column is placed back in collecting pipe again.

6) 700 μ l Buffer RWT ((check whether and add absolute ethyl alcohol), RT, 12000rpm are added into adsorption column RM 30S is centrifuged, abandons waste liquid.Adsorption column is placed back in collecting pipe again.

7) 500 μ l Buffer RW2 ((check whether and add absolute ethyl alcohol) RT, 12000rpm is added into adsorption column RM 30S is centrifuged, abandons waste liquid.Adsorption column is placed back in collecting pipe again.

8) repeat step 7.

9) 12000rpm centrifuges 1min, abandons collecting pipe waste liquid.Adsorption column RS is placed in room temperature several minutes, dried.

10) adsorption column RM is transferred in new 1.5ml RNase-free Collection Tube, adds 30 μ lRNase- Free Water, room temperature are placed 1 minute, 12,000rpm centrifugations 1 minute, collect RNA solution, and obtained RNA solution is stored in- 70 degree, prevent from degrading.

2nd, electrophoresis：

5ulRNA is taken to carry out electrophoresis with 1% Ago-Gel.

3rd, tailing, reverse transcription

Reverse transcription is carried out with miRNA cDNA the first chain synthetic agent box (CWbio.Co.Ltd, Cat#CW2141).

A.miRNA adds Poly (A) tail：

1. RNA amount, as follows, dilutes 10mM ATP with 1mM Tris (PH8.0) first used in.

2. the precooling without adding following reagent in RNase reaction tubes to cumulative volume 25ul into ice bath.

3. gently mixing above-mentioned reaction solution, liquid is collected in ttom of pipe by of short duration centrifugation.37 DEG C, it is incubated 15 minutes, in -20 ℃。

B. the process of the chains of miRNA cDNA first synthesis after modifying：

1. into ice bath precooling without in RNase reaction tubes add following table in reagent, to final volume 20ul：

Reagent	Volume
		Above-mentioned Poly (A) reaction solution	4ul
Ultrapure dNTPs (10Mm each)	1ul

25Um RT primer	3ul
		5XRT Buffer	4ul
SuperRT Reverse Tanscriptase	0.5ul
		RNase-Free Water	7.5ul

2. gently mixing above-mentioned reaction solution, liquid is collected in ttom of pipe after of short duration centrifugation.42 DEG C, it is incubated 50 minutes.

3.85 DEG C, it is incubated 5 minutes, terminating reaction.The cDNA reaction solutions of synthesis save backup in -20 DEG C.

4、miRNA Real-time PCR

Use miRNA Real-time PCR Assay Kit (CWbio.Co.Ltd, Cat#CW2142)

1), room temperature melts 2 × miRNA qPCR premix and Reverse primer (10uM).

2) please 2 × miRNA qPCR premix are turned upside down when, using and gently uniformly mixed, avoid bubbling, and through short Temporarily used after centrifugation.

3), reagent is placed on ice, and presses following preparation reaction system：

4), response procedures are set as follows：

2nd, statistical analysis

All data carry out statistical analysis using SPSS13.0 statistical softwares, and each group of data is represented with mean, multigroup Compare and use one-way analysis of variance (one-way ANOVA)：Homogeneity test of variance is carried out first, if response variable is between each group Homogeneity of variance, then compare between group using LSD methods；Heterogeneity of variance uses Duoett ' s methods, P<0.05 is that difference is statistically significant.

3rd, experimental result

IMAQ：

As a result as shown in figure 1, transfection after 24h, randomly select each group brain tissue row frozen section, it is seen that with Cy3 fluorescence The miRNA of mark assembles in brain tissue.

QRT-PCR results：

As a result as shown in Fig. 2 Normal group and activator group miR-200b expression, which are presented, first raises becoming of reducing afterwards Gesture, and elevated trend after first reducing is presented in negative control group of hypoxia and ischemia group, antagonist group, the negative control group of activator and antagonist.

Blank control group miR-200b expression quantity increases with incubation time and gradually increased, and reaches high when transfecting 3d Peak, it is rear slowly to decline；Compared with Normal group, negative control group of hypoxia and ischemia group miR-200b expression quantity raises (P when transfecting 0h< 0.05) it is, on a declining curve immediately, Normal group is less than after 12h is transfected, difference has statistics when transfecting 1d, 3d, 5d Meaning (P<0.05), when transfecting 5d, expression quantity is gradually increasing recovery to normal level (P>0.05).

Compared with negative control group of hypoxia and ischemia group, activator group miR-200b when transfecting 0h expression quantity is compared with negative control group of hypoxia and ischemia group indifference (P>0.05), but miR-200b expression quantity is significantly raised after 12h is transfected, and has statistics meaning in transfection 1d, 3d, 5d difference Justice (P<0.05), normal level is tended to when transfecting 5d.

Antagonist group miR-200b when transfecting 0h expression quantity is compared with negative control group of hypoxia and ischemia group also indifference (P>0.05), it is turning Each point in time after dye with negative control group of hypoxia and ischemia group difference less (P>0.05).

Activator negative control group, antagonist negative control group each time point miR-200b expression quantity compared with Hypoxia and ischemia Equal no significant difference (the P of group>0.05).

Embodiment 2 transfects influences of the miR-200b to neonatal rats with ischemia-hypoxia brain injury long term neurology prognosis

First, materials and methods

Key instrument：

(1) major experimental instrument and equipment：

1st, water maze (Anhui Zhenghua Biology Instrument Equipment Co., Ltd.)

2nd, tired instrument of transfer rod formula (Anhui Zhenghua Biology Instrument Equipment Co., Ltd.)

(2) experimental animal：Same Part I

Experimental method：

(1) model and packet are established：

It is randomly divided into 4 groups, Normal group (control), negative control group of hypoxia and ischemia group (HI), activator group (HI+agomir) is short of money Anti-agent group (HI+antagomir), each group method for establishing model and the same Part I of experiment control factor.

(2) motor coordination functional evaluation：

Turn-club test：Each group rat carried out turn-club test test, tie-in 4 days in after life 21 days.It is real before official testing Test animal and put adaptation 60s (5rpm) in transfer rod instrument, animal residence time in transfer rod instrument is then tested with 40rpm constant speed (most long timing 5min, calculated more than 5min with 5min), rest 1h carries out duplicate measurements again after every animal measures 1 time, takes 3 The average value of secondary measurement is its final result.

(3) Cognitive Function：

Morris water mazes：Test altogether 5 days, carry out within first 4 days orientation navigation experiment, carry out space exploration experiment within the 5th day.Before In the orientation navigation experiment of 4 days, each group rat receives 3 training daily, and record rat is not respectively from 3 quadrants (including platform Other 3 quadrants of place quadrant) different place of entry find time needed for platform, that is, escape the incubation period onto platform (escape latency, EL).The average value of 3 incubation period achievements is used as same day the final result to be counted.If rat exists Platform is found in 120s, then 30s is kept on platform；If not finding platform in 120s, guide its swim to platform and 30s is stopped on platform, achievement is calculated by 120s.Train after rat gives birth to 28 days, daily 8:00AM-1:00PM is carried out.5th It is carried out removing platform during space exploration experiment, and rat is put into water from any place of entry, record 120s rats'swimmings track With leap platform number.

2nd, statistical method：

Experimental data represents that the significance test between data each group uses one-way analysis of variance with mean ± standard deviation (One-Way ANOVA), compares two-by-two after with LSD methods.Comparison between two groups is examined using independent sample T.With P<0.05 is poor It is different to have significant, completed in the statistical softwares of SPSS 13.0.

3rd, experimental result

(1) turn-club test：

The each group rat transfer rod time of table 1

Note：Difference uses the multifactor analysis of variance, a between same time different grouping：Compared with control group, P<0.05；b： Compared with negative control group of hypoxia and ischemia group, P<0.05；One-way analysis of variance is used with group different time, there is the post- after significant difference Hoc is compared two-by-two using SNK, and c is relatively to have significant difference, p with the previous day<0.05.

Experiment shows that each group rat gradually extends with training number of days increase, transfer rod time, and each group was in the 3rd day transfer rod Time more previous daybreak is aobvious to extend (P<0.05).Each test point, compared with Normal group, negative control group of hypoxia and ischemia group, activator Group, the transfer rod time of antagonist group substantially shorten (P<0.05).The transfer rod time of each time point activator group is compared with Hypoxia and ischemia Group slightly shortens, but the not notable (P of difference>0.05), the transfer rod time of each time point antagonist group has compared with negative control group of hypoxia and ischemia group Extended, but the also not statistically significant (P of difference>0.05) (it is shown in Table 1, Fig. 3).

(2) water maze laboratory：

The each group rat Mirris water maze laboratories result (± s) of table 2

Note：Difference uses the multifactor analysis of variance, a between same time different grouping：Compared with control group, P<0.05；b： Compared with negative control group of hypoxia and ischemia group, P<0.05；One-way analysis of variance is used with group different time, there is the post- after significant difference Hoc is compared two-by-two using SNK, and c is relatively to have significant difference, p with the previous day<0.05.

In orientation navigation is tested and finds platform, each group experimental animal training the 3rd day, the performance of the 4th day with it is previous Its (P that is significantly increased<0.05).Train the 1st day, average escape latency (EL) is without significant difference (P between four experimental groups> 0.05).Since training the 2nd day, negative control group of hypoxia and ischemia group, activator group were obviously prolonged (P compared with Normal group EL<0.05), antagonism Agent group (P not notable compared with Normal group difference>0.05).Each time point activator group is compared with negative control group of hypoxia and ischemia group EL in training the 4th day Significantly extend (P<0.05), the remaining not statistically significant (P of time point difference>0.05), the EL of antagonist group is compared with negative control group of hypoxia and ischemia group Without significant difference (P>0.05) (2, Fig. 4 are shown in Table).

In space exploration experiment, compared with Normal group, negative control group of hypoxia and ischemia group and activator group reduce across platform number, Statistically significant (the P of difference<0.05).Compared with negative control group of hypoxia and ischemia group, activator group has been reduced across platform number, but difference Not significantly (P>0.05)；Antagonist group increased compared with negative control group of hypoxia and ischemia group across platform number, the statistically significant (P of difference< 0.05) (2 are shown in Table).

The miR-200b of embodiment 3 inquires into the repair mechanism of neonatal rats with ischemia-hypoxia brain injury

First, materials and methods

Key instrument and reagent：

Electrophoresis apparatus (Liuyi Instruments Plant, Beijing), electrophoretic blotting groove (Shanghai Tian Neng Science and Technology Ltd.s), gel imaging system (Bio-Rad), high speed low temperature centrifugal machine (U.S. BECI@IAN, AvantiJ-30I)；

Acrylamide, sub- methylene diacrylamide, SDS, Tris-base, glycine, DTT, AP, Tween-20, TEMED (above reagent is purchased from Haikang into company)；Methanol (Beijing Chemical Plant)；Efficient RIPA tissue/cell lysates (Solarbio)；Protease inhibitor cocktail(Amresco)；(Beijing Puli comes limited antibody stabilization solution Company)；BCA determination of protein concentration kit (Beijing Bo Aosen Bioisystech Co., Ltd)；Western specifically lights and detects examination Agent box (Beijing Ying Geen bio tech ltd)；Pre-dyed albumen mrker (Biomed)；Pvdf membrane (Millipore)；Degreasing Milk powder (Amresco).

Antibody：Rac1/Cdc42 Antibody, HRP-linked Antibody, Anti-mouse IgG, HRP- Linked Antibody (antibody is purchased from U.S. Cell signaling Technology).

Experimental animal：With embodiment 1

Conventional liquid dosage is standby：

1、1.0mol/L Tris·HCl

Tris(MW121.14) 30.29g

Distilled water 200ml

After dissolving, pH to 6.8 is adjusted with concentrated hydrochloric acid, finally 250ml is settled to distilled water, is protected at room temperature after high-temperature sterilization Deposit.

2、1.5mol/L Tris·HCl(pH8.8)

Tris(MW121.14) 45.43g

Ultra-pure water 200ml

After dissolving, pH to 8.8 is adjusted with concentrated hydrochloric acid, finally 250ml is settled to ultra-pure water, is protected at room temperature after high-temperature sterilization Deposit.

3rd, 10%SDS

SDS 10g

Distilled water is to 100ml

Dissolved under 50 DEG C of water-baths, room temperature preservation.Such as precipitate in long-term preserve, after water-bath is dissolved, still can be used.

4th, 10% Ammonium Persulfate 98.5 (AP)

Ammonium Persulfate 98.5 0.1g

Ultra-pure water 1.0ml

After dissolving, 4 DEG C of preservations, the holding time is 1 week.

5th, 30%Acr/Bic (37.5：1)

Acrylamide (Acr) 29g

Double acrylamide (the Bic 1g of methene

Ultra-pure water is to 100ml

After being dissolved at 37 DEG C, 4 DEG C of preservations.Recover during use to room temperature and without precipitation.

6th, electrophoresis liquid buffer solution (25mmol/L Tris, 0.25mol/L glycine, 0.1%SDS)

Room temperature preservation after dissolving, solution are reusable 3-5 times.

7th, transfering buffering liquid (48mmol/L Tris, 39mmol/L glycine, 0.037%SDS, 20% methanol)

Room temperature preservation after dissolving, secondary solution are reusable 3-5 times.

8th, TBS buffer solutions (100mmol/L TrisHCl pH7.5,150mmol/L NaCl)

1mol/LTris·HCl(pH7.5) 10ml

NaCl 8.8g

Distilled water is to 1000ml

9th, TBST buffer solutions (the TBS buffer solutions containing 0.05%Tween20)

20%Tween20 1.65ml

TBS 700ml

It can be used after mixing, best matching while using.

10th, confining liquid (the TBST buffer solutions for containing 5% skimmed milk power)

Skimmed milk power 5g

TBST 100ml

4 DEG C of preservations after dissolving.In use, recovering room temperature, dosage was to cover film surface.

11st, 5 × SDS sample-loading buffers

After mixing, it is sub-packed in 1.5ml centrifuge tubes, 4 DEG C of preservations.

12nd, 8% separation gel, 10% separation gel, 12% separation gel, 5% spacer gel

After adding TEMED, mixing immediately can encapsulating.

Experimental method：

(1) model and packet are established：

With embodiment 2.

(2), Western blot experimental procedures：

1. extract albumen：

1.1st, in precooling glass homogenizer on ice.

1.2nd, precooling RIPA histones lysate, protease inhibitors Protease inhibitor are added cocktail(1ml：10ul).

1.3rd, take appropriate brain tissue to be placed in glass homogenizer, by add protease inhibitors Tissue lysates according to 100mg：1ml ratio is added in each sample brain tissue, is homogenized.On ice be incubated 20min after, 14000rpm (4 degree) from Heart 20min, supernatant is taken, the degree of packing -80 preserves, to be measured.

2.BCA method protein quantifications：

2.1st, working solution, 25ml A liquid+0.5ml B liquid (reagent As are prepared：B=50:1), fully mix.

2.2nd, diluted protein standard items, take 15ul to be diluted to 150ul, make final concentration of 0.5mg/ml.

2.3rd, standard items are added in the standard sample wells of 96 orifice plates by 0,1,2,4,8,12,16,20ul, and diluted with PBS Liquid supplies 20ul.

2.4th, 1ul testing samples are added dropwise in sample well, 20ul (diluting 20 times) is complemented to standard dilutions.

2.5th, each hole adds 200ul BCA working solutions, 37 DEG C of placement 30min.

2.6th, room temperature is cooled to, A562nm is determined with ELIASA, canonical plotting is drawn and calculates protein concentration.

3. protein electrophoresis：

3.1 correct installation electrophoretic apparatus.

3.2 prepare separation gel.(concentration of separation gel is according to the different otherwise varied of destination protein molecular weight, Rac-1, use 12% concentration)

3.3 mix separation gel, are poured between slab-electrophoresis instrument layer glass plate, reserve needed for perfusion spacer gel Space (the tooth length of comb adds 1cm again), gently covers one layer of distilled water on glue, to prevent oxygen diffuses into gel from suppressing poly- Reaction is closed, room temperature is disposed vertically 30min.

After 3.4 glue polymerizations completely to be separated, top layer distilled water is poured out, residual liquid is exhausted with the filter paper of cleaning.Prepare 5% Spacer gel, the direct perfusion spacer gel on the separation gel having polymerize, and be immediately inserted into comb, action is soft, avoids the formation of gas Bubble, room temperature 30min is vertically arranged in by gel.

3.5 after spacer gel polymerization completely, carefully extract comb.Gel is moved into electrophoresis tank.

(Rac-1 applied sample amounts 32ug, β-actin are compareed 3.6 protein samples prepared as internal reference, and corresponding Destination protein sample-adding amount is consistent), it is placed in isometric 2 × SDS sample-loading buffers, 10min is heated in boiling water bath, is made Albuminous degeneration.

3.7 take the above-mentioned sample handled well to sequentially add in well, stay a hole to add pre-dyed albumen marker in addition, Left-right balance is kept without adding isometric 1 × SDS sample-loading buffers in the control wells of sample-adding.

3.8 are connected electrophoretic apparatus with power supply (positive pole connects lower groove), and institute's making alive is 80V on gel, when dye front enters After entering separation gel, voltage is brought up into 120V, continues electrophoresis until bromophenol blue reaches separation gel bottom (about 2.5h), closing is electric Source, gel is taken out.

4. transferring film：

4.1st, 2 clean filter paper and a pvdf membrane are cut, its size is consistent with gel.

4.2nd, pvdf membrane is impregnated with 3 minutes in methanol, is then immersed together with filter paper standby in transferring film liquid.

4.3rd, the transferring film clip of transferring film electrophoresis apparatus with transferring film liquid apply it is wet after, by filter paper, gel, pvdf membrane, filter paper by lower and On be stacked in successively on clamping plate, ensure filter paper, gel and pvdf membrane alignment, discharge bubble.

4.4th, close the lid, connection electrode, the side joint positive pole of film one, the side joint negative pole of gel one, constant current 300mA, transferring film 60 minutes.

5. closing：

After transfer terminates, take out pvdf membrane and carry out mark, be put at room temperature in confining liquid, it is warm on the shaking table gently shaken With vibration 2h, to eliminate the non-specific binding of antibody.

6. immune response：

6.1st, primary antibody is incubated：Confining liquid is discarded, pvdf membrane is put into polybag immediately, adds appropriate antibody dilution protection (primary antibody is diluted with antibody stabilization solution, and Rac-1 primary antibodies diluted concentration is 1 for liquid and primary antibody:1000, internal reference β-actin dilutions are dense Spend for 1:5000) bubble, is discharged, lies on the shaking table gently shaken after being incubated 30min and is put into 4 degree of refrigerators overnight incubations.

6.2nd, secondary antibody is incubated：Primary antibody is discarded, washes film 3 times with TBST buffer solutions, each 10min.Then pvdf membrane is put into In polybag, appropriate confining liquid and the antibody (secondary antibody) of horseradish peroxidase-labeled are added, in being incubated at room temperature 2h on shaking table.Abandon Secondary antibody is removed, washes film 3 times with TBST buffer solutions, each 10mm.

7. chemiluminescence：Take ECL reagent As liquid and each 1ml of B liquid to be mixed on preservative film, pvdf membrane albumen down and this Mixed liquor fully contacts 1min, and pvdf membrane is moved on clean preservative film, removes most raffinate, wraps, be put into ultraviolet gel imager Middle exposure.

8.Western Blot graphical analyses and processing：Western blot bars are brought into 1D-Gel image analysis softwares Row gray analysis, using destination protein band and internal reference β-actin band gray values ratio as a result,

The expression of destination protein is represented with this.

2nd, statistical method：

Experimental data represents that the significance test between data each group uses one-way analysis of variance with mean ± standard deviation (One-Way ANOVA), compares two-by-two after with LSD methods.Comparison between two groups is examined using independent sample T.With P<0.05 is poor It is different to have significant, completed in the statistical softwares of SPSS 13.0.

3rd, experimental result

As a result as illustrated in Figures 5 and 6：Rac-1 protein expressions raise after neonatal hypoxia ischemia：The Rac-1 albumen of Normal group Expression is in more stable expression at each time point.After Hypoxia and ischemia, Rac-1 protein expressions are obvious after 2d is transfected Raise (P<0.05).Negative control group of hypoxia and ischemia group Rac-1 expressing quantities are significantly raised compared with Normal group, in 2d, 3d, 4d, 5d time Point, the statistically significant (P of difference<0.05).Activator group compared with negative control group of hypoxia and ischemia group Rac-1 expressing quantities after transfection 2d, 3d, 4d, 5d reduce (P<0.05), antagonist group has raised compared with negative control group of hypoxia and ischemia group Rac-1 expressing quantities, after transfection 2d, 4d, 5d significant difference (P<0.05).

Claims (5)

1. Applications of the 1.miRNA-200b in Rac-1 protein expressions inhibitor in preparing hypoxic-ischemic brain damage.
2. 2. application according to claim 1, it is characterised in that the miRNA-200b is selected from pri-miRNA, pre- MiRNA, ripe miRNA.
3. 3. application according to claim 1, it is characterised in that described miRNA-200b is the RNA through chemical modification.
4. 4. application according to claim 3, it is characterised in that described chemical modification is repaiied including glycosyl modified, phosphoric acid chain One or more in decorations, base modification, protrusion and end modified, double-spiral structure modification.
5. 5. application according to claim 1, it is characterised in that described miRNA-200b and pharmaceutically acceptable carrier Or excipient composition prepares Rac-1 protein expression inhibitor.