CN101792765A - Small RNA of human-mouse homologous Caspase-3 and application thereof - Google Patents

Abstract

The invention belongs to the field of biological genes. Chronic pain is one of expression forms of the changes of neuron plasticity and has the physiological characteristics that the reactivity of pain sense is enhanced, the treatment is difficult, and the mechanism is unknown; and opiate receptor antagonists which have more side effects are mainly used in treatment. The research shows that the increase of Caspase-3 is related to the occurrence of the chronic pain, thus the invention provides a small RNA of human-mouse homologous Caspase-3, and the sequence of the small RNA is shown in SEQ ID NO: 1. The invention also provides application of the small RNA in medicines for treating the chronic pain. The small RNA of the invention can be directly injected, can inhibit expression of the Caspase-3 of human bodies and rats in vivo and in vitro, achieves the effect of treating the chronic pain, has a convenient administration route, does not have reactions such as habituation, tolerance and the like, and can be used for a long time.

Description

A kind of little RNA and application thereof of human-mouse homologous Caspase-3

Technical field

The invention belongs to field of biological genes, relate to little RNA and disturb and use, be specifically related to suppress little RNA that the Caspase-3 of people and rat expresses and in the application of preparation treatment chronic pain medicine.

Background technology

Chronic pain is one of manifestation of neuron plasticity variation, and its physiologic characteristic is that the reactivity of the pain sensation increases, and mainly shows as hyperpathia (hyperalgesia) and allergy (allodynia).This type of pain therapy difficulty mainly is because its mechanism is not clear.In treatment, mainly be to be the opioid receptor retarding agent of representative with the morphine at present, but have reactions such as numerous side effects and habituation, tolerance, thereby limited its life-time service.

Joachim Scholz, Hassanzadeh P had the report that on neurogenic pain and the inflammatory pain model there is the discovery Neuron Apoptosis respectively at 2005,2006, infer that Neuron Apoptosis may be one of reason that causes neurogenic pain.Apoptosis (apoptosis) is a procedural biological process of cell oneself's destructive, finally all work by proteolytic enzyme Caspases mediation protein cleavage, a Caspases part is the startup person (initiators) that preceding apoptotic signal is reacted, mainly comprise caspase-2,8,9,10, another part is as the effect person (effectors) of apoptosis, mainly comprise caspase-3,6,7, and in neural system, caspase-3 plays a crucial role.

RNA disturbs that (RNA interference RNAi) is meant when importing with endogenous mRNA coding region homologous double-stranded RNA in the cell, and this mRNA degraded takes place and causes the phenomenon of gene silencing, is a kind of phenomenon that suppresses the specific gene expression in the organism.The double-stranded RNA that external source imports or introduced by variety of ways such as transgenosis, virus infectiones will by one in the RNase III family can the specific recognition double-stranded RNA enzyme Dicer, the mode that relies on ATP progressively be cut into 19～23nt the small molecules interference RNA fragment (smallinterfering RNAs, siRNA).SiRNA two strands two strands under the RISC effect is untied, and homologous target RNA combination under the effect of endonuclease, is cut off target mRNA, thereby blocked genetic expression.It is at present efficient gene " silence " technology that RNA disturbs, and theoretically, the gene that excitability increases in the chronic pain path all may become RNA and disturb target position, thereby reduces its excitability, reaches therapeutic purpose.But, the jamming effectiveness height, the acquisition of the little RNA of high specificity is difficult, and not homotactic difference can cause the forfeiture of validity, thereby influences its pharmacological value.

Summary of the invention

The object of the present invention is to provide a kind of little RNA of human-mouse homologous Caspase-3, and its application in preparation treatment chronic pain medicine

We are through screening, obtain people and the big mouse homologous little RNA of caspase-3, can be simultaneously the caspase-3mRNA of people and rat be degraded, thereby suppress the proteic expression of Caspase-3 of people and rat, ease the pain by suppressing apoptosis, can become a kind of new medicine of treatment chronic pain.

Concrete technical scheme is: rat caspase-3 gene order is obtained by Gene bank, and sequence number is NM 004346, and total length is 2484bp.People's caspase-3 gene order is obtained by Gene bank, and sequence number is NM 004346, and total length is 2689bp.Again the open reading frame in its identical sequence is partly imported the RNAi design software that http://i.cs.hku.hk/～the sirna/software/sirna.php website is provided and carried out online screening, then select GC to compare between 40%-60%, be positioned at 5 different sequences of open reading frame, as follows:

AGCCGAAACTCTTCATCAT

GATACCAGTGGAGGCCGAC

TACCAGTGGAGGCCGACTT

GTCGATGGACTCTGGAATA

ACCTCCGTGGATTCAAAAT

We therefrom select three sequence construct expression vector pShuttleH1-siCas3, make up the pEGFPC1 reporter plasmid that contains the caspase-3 gene simultaneously, with liposome method with pShuttleH1-siCas3 and pEGFPC1-Cas3 cotransfection 293 cells, carry out the detection of RNA jamming effectiveness, and then filtering out the highest sequence of RNA jamming effectiveness: GATACCAGTGGAGGCCGAC, corresponding RNA sequence is: GAUACCAGUGGAGGCCGAC.

The invention provides a kind of little RNA of human-mouse homologous Caspase-3, its sequence is GAUACCAGUGGAGGCCGAC (shown in SEQ ID NO:1).

The present invention detects carrying out the RNA jamming effectiveness by pEGFPC 1 reporter plasmid that contains the caspase-3 gene, obtains the RNA of higher primary school of jamming effectiveness; Act on the human nerve cell HN of endotaxin induction again by little RNA, find that little RNA has good inhibition humanized Neuron Apoptosis; By the little RNA subarachnoid injection of caspase3, find inhibited again to the hyperpathia of rat models of neuropathic pain.Principle of work of the present invention: caspase-3 is apoptotic a kind of proteolytic enzyme, and apoptosis is relevant with chronic pain, therefore reduces spinal cord caspase-3 and expresses the effect with inhibition chronic pain.Thereby the expression of RNA perturbation technique degradable mRNA arrestin, so, the expression of adopting this technology to suppress caspase-3 just can reach the purpose of treatment pain.

Therefore the present invention also provides the application of little RNA in preparation treatment chronic pain medicine of above-mentioned human-mouse homologous caspase-3.

Application of the present invention can be adopted the mode of little RNA through the direct injection of subarachnoid space, can reach the purpose of treatment chronic pain, and route of administration is convenient, respond well.Simultaneously,, there are not reactions such as habituation, tolerance because the present invention is not traditional opioid receptor retarding agent, but life-time service.

Description of drawings

Fig. 1 is that flow cytometry is measured the RNA jamming effectiveness

Fig. 2 is that RT-PCR detects apoptosis neurons caspase-3mRNA

Wherein: 1. the RNA of mispairing organizes (MM group); 2. siRNA group (siRNA group);

3. physiological saline group (NS group); 4.Marker

Fig. 3 is that the neuronal cell vigor of different time endotaxin induction apoptosis compares

Fig. 4 is the variation of the CCI rat machinery threshold of pain and the hot threshold of pain

Wherein: A is the variation of the mechanical threshold of pain; B is the variation of the hot threshold of pain

Fig. 5 is a CCI rat spinal cord Caspase-3 mRNA change of Expression

Embodiment

Describe the present invention below in conjunction with drawings and Examples, but enforcement of the present invention is not limited only to this.

Embodiment 1: the screening process of the little RNA sequence of human-mouse homologous caspase-3

One. make up 3 carrier pShuttleH1-siCas3 that express the little RNA of caspase-3

Rat caspase-3 gene order is obtained by Gene bank, and sequence number is NM 004346, and total length is 2484bp, and people's caspase-3 gene order is obtained by Gene bank, and sequence number is NM 004346, and total length is 2689bp.We have selected 3 different interference target genes according to RNA interferential principle, and the purpose of design gene is as follows in view of the above:

Forward 5 '-gatcccAGCCGAAACTCTTCATCATttcaagagaATGATGAAGAGTTTCGGCTttt ttt-3 '

(shown in SEQ ID NO:2)

The reverse 5 '-agctaaaaaaAGCCGAAACTCTTCATCATtctcttgaaATGATGAAGAGTTTCGGC Tgg-3 ' of I

(shown in SEQ ID NO:3)

Forward 5 '-gatcccGATACCAGTGGAGGCCGACttcaagagaGTCGGCCTCCACTGGTATCttt ttt-3 '

(shown in SEQ ID NO:4)

The reverse 5 '-agctaaaaaaGATACCAGTGGAGGCCGACtctcttgaaGTCGGCCTCCACTGGTAT Cgg-3 ' of II

(shown in SEQ ID NO:5)

Forward 5 '-gatcccACCTCCGTGGATTCAAAATttcaagagaATTTTGAATCCACGGAGGTttt ttt-3 '

(shown in SEO IDNO:6)

The reverse 5 '-agctaaaaaaATTTTGAATCCACGGAGGTtctcttgaaACCTCCGTGGATTCAAAA Tgg-3 ' of III

(shown in SEQ IDNO:7)

The aim sequence two ends contain BamH I and HindIII restriction enzyme site respectively.Plasmid pShuttleH1 (available from American I nvitrogen company) utilizes the Bgl II cohesive end identical with BamH I with Bgl II and HindIII double digestion, uses T ₄16 ℃ of dna ligases are connected with goal gene after the annealing and spend the night called after: pShuttleH1-siCas3.Transformed E .coli.DH5 α competent cell (available from Canadian MicrobixBiosystems company) is chosen clone's extracting plasmid DNA, identifies with HindIII and the correct laggard pacing preface of Nde I double digestion.

1, pShuttleH1 endonuclease reaction system

pShuttleH1 30μl

Bgl?II 2μl

HindIII 2μl

Buffer 25μl

ddH ₂O 11μl

Total 50μl

Behind the mixing, of short duration centrifugal, 37 ℃ of incubations 6 hours.

2,1% agarose gel electrophoresis, with the Gel Extraction test kit recovery of QIAgen company, product is dissolved among the 30 μ l ddH2O, and-20 ℃ of preservations are standby.

3, ligation system

pShuttleH1 3μl

dsDNA 4μl

T ₄Dna ligase 1 μ l

Buffer 1μl

ddH ₂O 1μl

Total 10μl

Behind the mixing, 16 ℃ are incubated overnight.

4, recombinant plasmid transformed DH5 α bacterium

Get among the intestinal bacteria E.coli.DH5 α competent cell 50 μ l that connect the preparation of product 5 μ l adding Calcium Chloride Method, ice bath 42 ℃ of heat-shockeds 90 seconds after 1 hour, ice bath adds nonresistant LB nutrient solution 200 μ l after 4 minutes again, gentle jolting is 1 hour in 37 ℃ of shaking tables, the kalamycin resistance (Kan of shop preheating ⁺) the LB agar plate.Be inverted in and cultivate after 12 hours the mono-clonal bacterial strain on the picking flat board in 37 ℃ of biochemical incubators to Kan ⁺Among the LB, 37 ℃ of violent joltings logarithmic phase in the bacterium is chosen clone's extracting plasmid DNA.

The clone is called pShuttleH1-siCas3.

5, HindIII and Nde I double digestion are identified

pShuttleH?1-siCas3 3μl

HindIII 0.5μl

Nde?I 0.5μl

Buffer 1μl

BSA 1μl

ddH ₂O 4μl

Total 10μl

Behind the mixing, of short duration centrifugal, 37 ℃ of incubations 3 hours, 1% agarose gel electrophoresis.The clone that electrophoretic band fulfills the expectation serves the sea and gives birth to the order-checking evaluation of worker bio-engineering corporation.

Two, contain the structure and the evaluation of the pEGFPC1 reporter plasmid of rat caspase-3 gene

According to the caspase-3 sequence, the design primer:

5 '-CGGAGCTCGACAACAACGAAACCTCCGTG-3 ' (shown in SEQ ID NO:8);

5 '-CGCGTCGACGGCCACCTTCCGGTTAACAC-3 ' (shown in SEQ ID NO:9).

The primer two ends contain Sac I and Sal I restriction enzyme site respectively.Get normal SD rats nephridial tissue extracted total RNA, row RT-PCR obtains the partial sequence that contains in the caspase-3 expression cassette that disturbs target sequence, and length is 741bp.This fragment and pEGFPC1 plasmid (available from American I nvitrogen company) are cut with Sac I and Sal I enzyme respectively, then use T ₄Dna ligase spends the night for 16 ℃ and connects into pEGFPC1-Cas3.Transformed E .coli.DH5 α competent cell is chosen clone's extracting plasmid DNA, carries out pcr amplification and goes out the laggard pacing preface evaluation of correct band.

1, PCR identification system:

cDNA 2.5μl

Upstream primer 1 μ l

Downstream primer 1 μ l

dNTPs(2.5mmol/L) 2μl

MgCl ₂(25mmol/L) 2μl

Taq archaeal dna polymerase (5u/ μ l) 0.5 μ l

10 * Taq damping fluid, 2.5 μ l

ddH ₂O 16μl

Total 25μl

94 ℃ of 5min unwind; 94 ℃ of 50s, 55 ℃ of 50s, 72 ℃ of 90s totally 30 circulations; 72 ℃ of 10min extend.Get 5 μ l, 1% agarose gel electrophoresis is identified the PCR product.PCR with QIAgen company reclaims the test kit recovery, carries out next step Sac I enzyme and cuts.

2, PCR product S ac I enzyme is cut

PCR product 22.5 μ l

Sac?I 1.5μl

Buffer1 3μl

BSA 3μl

ddH ₂O 0μl

Total 30μl

Behind the mixing, of short duration centrifugal, 37 ℃ of incubations 6 hours.1% agarose gel electrophoresis, with the Gel Extraction test kit recovery of QIAgen company, product is dissolved in 30 μ l ddH ₂Among the O, carry out next step Sal I enzyme and cut.

3, PCR product S al I enzyme is cut

(Sac I enzyme is cut 22.5 μ l to the PCR product

The back)

Sal?I 1.5μl

Buffer3 3μl

BSA 3μl

ddH ₂O 0μl

Total 30μl

Behind the mixing, of short duration centrifugal, 37 ℃ of incubations 6 hours.1% agarose gel electrophoresis, with the Gel Extraction test kit recovery of QIAgen company, product is dissolved in 30 μ l ddH ₂Among the O ,-20 ℃ of preservations are standby.

4, pEGFPC1 plasmid Sac I enzyme is cut

pEGFPC1 30μl

Sac?I 2.5μl

Buffer1 5μl

BSA 5μl

ddH ₂O 7.5μl

Total 50μl

Behind the mixing, of short duration centrifugal, 37 ℃ of incubations 6 hours.

5,1% agarose gel electrophoresis, with the Gel Extraction test kit recovery of QIAgen company, product is dissolved in 30 μ l ddH ₂Among the O, carry out next step Sal I enzyme and cut.

6, reclaiming product S al I enzyme cuts

Sac I enzyme is cut product 22.5 μ l

Sal?I 1.5μl

Buffer3 3μl

BSA 3μl

ddH ₂O 0μl

Total 30μl

Behind the mixing, 37 ℃ of incubations 6 hours, 1% agarose gel electrophoresis reclaims with the GelExtraction test kit of QIAgen company, and product is dissolved in 30 μ l ddH ₂Among the O, be used for next step and be connected with the RT-PCR product.

7, ligation system

PEGFPC1 (enzyme is cut the back) 3 μ l

RT-PCR product (enzyme is cut the back) 4 μ l

T ₄Dna ligase 1 μ l

Buffer 1μl

ddH ₂O 1μl

Total 10μl

Behind the mixing, 16 ℃ are incubated overnight.

8, recombinant plasmid transformed DH5 α bacterium

Get among the intestinal bacteria E.coli.DH5 α competent cell 50 μ l that connect the preparation of product 5 μ l adding Calcium Chloride Method, behind the ice bath 1 hour, 42 ℃ of heat-shockeds 90 seconds, ice bath adds nonresistant LB nutrient solution 200 μ l after 4 minutes again, gentle jolting is 1 hour in 37 ℃ of shaking tables, the kalamycin resistance (Kan of shop preheating ⁺) the LB agar plate.Be inverted in and cultivate after 12 hours the mono-clonal bacterial strain on the picking flat board in 37 ℃ of biochemical incubators to Kan ⁺Among the LB, 37 ℃ of violent joltings logarithmic phase in the bacterium is chosen clone's extracting plasmid DNA.The clone is called pEGFPC1-Cas3.

9, HindIII and Nde I double digestion are identified

pEGFPC1-Cas3 3μl

Xbal?I 0.5μl

Xho?I 0.5μl

Buffer2 1μl

BSA 1μl

ddH ₂O 4μl

Total 10μl

37 ℃ of incubations 3 hours, 1% agarose gel electrophoresis.Clone's performing PCR that electrophoretic band fulfills the expectation is identified.

10, PCR identification system:

pEGFPC1-Cas3 2.5μl

Upstream primer 1 μ l

Downstream primer 1 μ l

dNTPs(2.5mmol/L) 2μl

MgCl ₂(25mmol/L) 2μl

Taq archaeal dna polymerase 0.5 μ l

(5u/μl)

10 * Taq damping fluid, 2.5 μ l

ddH ₂O 16μl

Total 25μl

94 ℃ of 5min unwind; 94 ℃ of 50s, 55 ℃ of 50s, 72 ℃ of 90s totally 30 circulations; 72 ℃ of 10min extend.Get 5 μ l, 1% agarose gel electrophoresis is identified the PCR product.The clone that electrophoretic band fulfills the expectation serves the sea and gives birth to the order-checking evaluation of worker's biotechnology company.

Three, the detection of RNA jamming effectiveness

With conventional 293 cells (available from Shanghai Inst. of Cytobiology, Chinese Academy of Sciences) of cultivating of six orifice plates, (dye DNA transfection .J. Sha nurse Brooker work of mediation referring to: fat with liposome method, Huang Peitang translates. the molecular cloning experiment guide. and the 3rd edition. Science Press publishes .2002,1276-1288.) with pShuttleH1-siCas3 and each 2 μ g cotransfections, 293 cell of pEGFPC1-Cas3, and establish negative contrast of blank group and the positive contrast of pEGFPC1-Cas32 μ g transfection group.Observe the intensity of green fluorescence next day, trypsin digestion cell, flow cytometer is further analyzed.

1, pShuttleH1-siCas3 and pEGFPC 1-Cas3 cotransfection HEK-293 cell strain

(1) 24h before the transfection, the cell that will be in logarithmic phase with 0.25% trysinization after, be inoculated in 6 orifice plates by certain density, continue cultivation 18-24h, treat that cell covers with floorage and carried out transfection at about 80% o'clock.

(2) the DMEM nutrient solution with the serum-free antibiotic-free cleans twice with cell, and the DMEM nutrient solution that adds an amount of serum-free antibiotic-free is cultivated standby.

Each hole in (3) 6 orifice plates, the consumption of pShuttleH1-siCas3 (expressing 3 little RNA of difference) and pEGFPC1-Cas3 respectively is 2 μ g, positive control only adds 2 μ g pEGFPC1-Cas3 and does not add pShuttleH1-siCas3, and the negative contrast of blank group neither adds.PShuttleH1-siCas3 and pEGFPC1-Cas3 are mixed with 200 μ l Opti-MEM I jointly, be made into A liquid, room temperature is placed 5min.

(4) 5 μ l Lipofectamin, 2000 reagent are mixed at another Guan Zhongyu 200 μ l Opti-MEM I, be made into

B liquid, incubation 5min under the room temperature.

(5) 200 μ l B mixed solutions are joined in the 200 μ l A mixed solutions, abundant mixing, room temperature is placed 20min.

(6) abandon serum-free medium in six orifice plates.

(7) the every hole 1ml of AB mixed solution is added in six orifice plates, culturing cell, soft front and back push away shakes culture plate with mixing liquid, makes it evenly to cover on the cellular layer.

2, fluorescence differs the inverted microscope observation

Transfection 24,48 is being observed the expression of EGFP in the HEK-293 cell strain behind the 72h under the fluorescence inverted microscope, be placed on multiple at object lens and eyepiece simultaneously to be respectively * 100 o'clock, takes imaging with Digital Video (LeicaTCS-TN, the U.S.).

3, flow cytometer detects the GFP fluorescence intensity

Collecting cell behind the conventional digestion of the 0.25% trypsinase HEK293 cell behind the 24h, the PBS washed twice, detect the average fluorescent strength (MFI) and the EGFP express cell positive rate of every porocyte with flow cytometer, each siRNA of quantitative analysis is to the influence of EGFP fluorescent protein expression.

Flow cytometry measurement result such as Fig. 1 compare with positive control, and pShuttleH1-siCas3 intervention group fluorescence obviously weakens, and weakens the most obvious with pShuttleH1-siCas3II.Illustrate pShuttleH1-siCas3 can better disturb pEGFPC1-Cas3 in the Cas3 fragment, thereby the EGFP that transcribes simultaneously with it of influence causes the strength reduction of green fluorescence, thereby determine the little RNA that No. 2 little RNA are high interference efficient of the present invention.

Embodiment 2: little RNA of the present invention is to the restraining effect of human nerve cell HN apoptosis

Adopt conventional cultivator neurocyte HN (available from Shanghai Inst. of Cytobiology, Chinese Academy of Sciences), be divided into three groups: the RNA group (MM group) of mispairing; Caspase-3 siRNA group (siRNA group) and physiological saline group (NS group), the employing liposome method (referring to: fat dyes DNA transfection .J. Sha nurse Brooker work of mediation, Huang Peitang translates. the molecular cloning experiment guide. and the 3rd edition. Science Press publishes .2002,1276-1288.) with RNA and each 5 μ g (20 μ l) transfection human nerve cell HN of Caspase-3 siRNA of mispairing, the physiological saline group adds equivalent physiological saline.Back 12h after the transfection, adding intracellular toxin to final concentration in the nutrient solution is 5 μ g/ml, 0,6,12,24, go RT-PCR and tetrazolium salts colorimetric experiment (MTT) behind the 36h respectively.

1, tetrazolium salts colorimetric test (MTT) is a kind of by measuring the detection method of cellular energy metaboilic level in order to indirect reflection cell proliferation situation.Ultimate principle and method be referring to document: Wu Junzheng, Si Tuzhenqiang, the discussion of relevant condition in the tetrazolium salts colorimetric test. The Fourth Military Medical University's journal, 1991,12 (4): 304-306.

2, RT-PCR identification system:

cDNA 2.5μl

Upstream primer 1 μ l

Downstream primer 1 μ l

dNTPs(2.5mmol/L) 2μl

MgCl ₂(25mmol/L) 2μl

Taq archaeal dna polymerase 0.5 μ l

(5u/μl)

10 * Taq damping fluid, 2.5 μ l

ddH ₂O 16μl

Total 25μl

The upstream and downstream primer is the same, and 94 ℃ of 5min unwind; 94 ℃ of 50s, 55 ℃ of 50s, 72 ℃ of 90s totally 30 circulations; 72 ℃ of 10min extend.Get 5 μ l, 1% agarose gel electrophoresis is identified the PCR product.The clone that electrophoretic band fulfills the expectation serves the order-checking of sea base Kanggong department and identifies.

Found that, apoptotic body occurs in the cell behind the neurocyte endotaxin induction, do not dye and add endotoxic cell indigo plant, cell walls is complete.Apoptosis-induced RT-PCR the results are shown in Figure 2, and the little RNA group of Caspase-3 (siRNA group) neurocyte HN caspase-3 mRNA obviously is weaker than control group.

Tetrazolium salts colorimetric experiment (MTT) the results are shown in Figure 3, and the human nerve cell vigor of siRNA group endotaxin induction apoptosis obviously is better than the little RNA group of mispairing (MM group), physiological saline group (NS group).Illustrate: the little RNA of Caspase-3 can better suppress the expression of caspase-3 mRNA; the human nerve cell HN apoptosis that suppresses endotaxin induction; have good neuroprotective, for suppressing nerve cell apoptosis in the intravital clinical experiment of people from now on, the treatment chronic pain lays the first stone.

Embodiment 3: the little RNA subarachnoid injection of caspase3 of the present invention is to the influence of the hot threshold of pain of this rat, the mechanical threshold of pain

Adopt the ligation of rat right sciatic nerves to make up the neurogenic pain model, observe of the influence of the little RNA subarachnoid injection of caspase3 this hot threshold of pain of rat.Rat adopts the PE-10 conduit to put pipe through subarachnoid space, and rat is divided into sham operated rats (Sham group), sciatic nerve ligation group (CCI group), little RNA group and MM group, and little RNA 1d before art begins intrathecal injection, 20 μ g/d, 7d continuously.MM organizes to the little RNA of mispairing.Not ligation of sham operated rats sciatic nerve.

1, the structure of CCI model

Rat begins model surgery in the Animal House recuperation after 3 days.Adopt Sodital (40ml/kg) intraperitoneal injection of anesthesia, increase about 40mg when needing.Four limbs bundle slightly and open on operating table behind the right lateral thigh postmedian butt, after the sterilization of iodine fluorine, cut thigh postmedian skin, the passivity separating muscle, expose stage casing on the sciatic nerve, free neural to the nervus peronaeus crotch, tie up 4 knots successively lightly with the catgut of 4-0, the about 1mm of spacing of knot, the dynamics of knotting for leg muscle occurs and twitch or kick one's legs reflect till.Sham operated rats is not tied up sciatic nerve, and other step is all identical.Suture muscles and skin successively, iodine fluorine sterilization behind the involutory skin, abdominal injection 0.3ml penbritin (concentration 100mg/ml).Anesthesia places plastics cage separately before waking up, the wood fragments bits of cage middle berth 3-6cm, and water and food should be able to obtain easily.

2, the hot threshold of pain is measured

Observe rat and latent period of pawl reflection under the radiant heat irradiation, occurs contracting as the hot threshold of pain.Rat is positioned in the transparent synthetic glass cage, cage does not have diapire, cage is placed on (little wooden cupboard on the thick sheet glass of 3mm, the upper strata is a sheet glass, the determinator pendulum is in the middle of cabinet), the direct contact glass plate in the vola of animal is close to the following mensuration of sheet glass and measure to pop one's head in like this, reduces systematic error.Irradiating source is 8v, the bulb of 50w, in have concavees lens to focus on, the outer cover metal shell, there is the hole of a diameter 5mm upper end.Bulb is 40mm from front end like this.Probe connects accurate timing register, and timing phase and irradiating source power supply are synchronous, and start-of-record shines the time interval that the pawl that occurs contracting reacts.Latent period of pawl reflection occurs contracting as the hot threshold of pain under radiant heat irradiation with rat, measured value is accurate to 0.1S, the impetus of inboard the 1st toes of all animals irradiations, and each time point is measured 3 times, each 5min at interval, averaging is the hot threshold of pain.The single fraction irradiation time is no more than 20S, in order to avoid the damage irradiated site.All mensuration are finished under identical time point and same experimental conditions by a unwitting experimenter.

3, the rat subarachnoid space is put pipe and little RNA injection

Behind the rat anesthesia, the PE-10 conduit prolongs puncturing hole and inserts, and visible animal shouts or whipping or kick one's legs during insertion, insert about 1cm, but visible cerebrospinal fluid flows out or inject the backflow of physiological saline water breakthrough, can seal the mouth of pipe.Little RNA 1d before art begins intrathecal injection, 20 μ g/d, 7d continuously.The NS group is only given the physiological saline of equivalent.Found that before the sciatic nerve ligation inject the little RNA of caspase3 of the present invention (give birth to worker Bioisystech Co., Ltd by Shanghai and synthesize, add liposome 1 μ g/ μ l), change (P＞0.05) is not seen in the hot threshold of pain of rat and the mechanical threshold of pain; The hot threshold of pain of physiological saline group and the mechanical threshold of pain (see figure 4) that obviously descends after the ligation is compared with sham operated rats and to be had significant difference (P＜0.05).Little RNA group the 1st, 3, in the 7d time point, obviously increase the hot threshold of pain of siRNA group, compares significant difference (P＜0.05) with the physiological saline group.And 10d compares no significant difference (P＜0.05) with the physiological saline group, illustrates that the little RNA of caspase3 has the thermal hyperalgesia effect that suppresses the sciatic nerve ligation.

In order to detect the expression whether RNA disturbs influences spinal cord caspase-3, we adopt the method for real-time quantitative PCR, level to spinal cord L4-6 caspase-3 mRNA is measured, the result shows, the level of caspase-3mRNA is starkly lower than MM group (see figure 5) on little RNA injection back 1d, 3d, 7d, 10d time point, illustrates that the little RNA of caspase-3 can obviously suppress the expression of caspase-3 mRNA.

SEQUENCE?LISTING

＜110〉Second Military Medical University, PLA

＜120〉a kind of little RNA and application thereof of human-mouse homologous Caspase-3

＜130〉specification sheets, claims

<160>9

<170>PatentIn?version?3.1

<210>1

<211>19

<212>RNA

＜213〉artificial sequence

<400>1

gauaccagug?gaggccgac 19

<210>2

<211>59

<212>DNA

＜213〉artificial sequence

<400>2

gatcccagcc?gaaactcttc?atcatttcaa?gagaatgatg?aagagtttcg?gcttttttt 59

<210>3

<211>59

<212>DNA

＜213〉artificial sequence

<400>3

agctaaaaaa?agccgaaact?cttcatcatt?ctcttgaaat?gatgaagagt?ttcggctgg 59

<210>4

<211>59

<212>DNA

＜213〉artificial sequence

<400>4

gatcccgata?ccagtggagg?ccgacttcaa?gagagtcggc?ctccactggt?atctttttt 59

<210>5

<211>59

<212>DNA

＜213〉artificial sequence

<400>5

agctaaaaaa?gataccagtg?gaggccgact?ctcttgaagt?cggcctccac?tggtatcgg 59

<210>6

<211>59

<212>DNA

＜213〉artificial sequence

<400>6

gatcccacct?ccgtggattc?aaaatttcaa?gagaattttg?aatccacgga?ggttttttt 59

<210>7

<211>59

<212>DNA

＜213〉artificial sequence

<400>7

agctaaaaaa?attttgaatc?cacggaggtt?ctcttgaaac?ctccgtggat?tcaaaatgg 59

<210>8

<211>29

<212>DNA

＜213〉artificial sequence

<400>8

cggagctcga?caacaacgaa?acctccgtg 29

<210>9

<211>29

<212>DNA

＜213〉artificial sequence

<400>9

cgcgtcgacg?gccaccttcc ggttaacac 29

Claims (3)

1. the little RNA of a human-mouse homologous Caspase-3, the sequence that it is characterized in that this little RNA is shown in SEQID NO:1.

2. the application of the little RNA of a human-mouse homologous Caspase-3 as claimed in claim 1 in preparation treatment chronic pain medicine.

3. the application of the little RNA of human-mouse homologous Caspase-3 according to claim 2 in preparation treatment chronic pain medicine, it is characterized in that treating the chronic pain medicine is injection.

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