CN103467570B - Transmembrane polypeptide with analgesic effect - Google Patents

Abstract

The invention discloses a transmembrane polypeptide with an analgesic effect. The transmembrane polypeptide is any polypeptide shown in (a) to (c), wherein the amino acid sequence of the polypeptide shown in (a) is shown by amino acid residues 10 to 25 of a sequence I in a sequence table; the amino acid sequence of the polypeptide shown by (b) is shown in the sequence I in a sequence table; and the polypeptide shown by c is a derived polypeptide, which is obtained by mutating the polypeptide shown by (a) or (c) in the following manner and has the at least one effect of (1) to (3), the mutation manner comprises the steps of: replacing, deleting and/or adding one to ten of the amino acid residues 10 to 25 of the sequence I in the sequence table except for the amino acid residue 12 of the sequence 1, the effect (1) is the prevention and/or treatment of pain, the effect (2) is the disturbance of phosphorylation of actin-depolymerzing protein/cofilin, and the effect (3) is reduction of the function of transient receptor potential vanillic acid subtype 1. The transmembrane polypeptide with the analgesic effect can be used for preventing and/or treating pain.

Description

A kind of there is analgesic activity can transmembrane polypeptide

Technical field

The present invention relates to a kind of there is analgesic activity can transmembrane polypeptide.

Background technology

Pain under physiological condition can, as a kind of early warning system of body for physical impairment, cause the defensive protection of body to react.But chronic pathological pain can be produced under some pathological conditions, a kind of insufferable torment is but formed for body.Thus, pain, as a kind of disease, has become and has had a strong impact on one of people's routine work life and life quality and endanger greatly.With the quickening pace of modern life with the arrival of social senilization, the sickness rate of various pain disease especially chronic inflammatory pain about comes higher.But due to chronic pathological cause of disease complexity bitterly, molecular mechanism and pathophysiological process are not completely understood, so still lack effective and special treatment means.

About the pathogenesis of pain, think at present mainly caused by periphery sensitization and central sensitization.Several factors take part in generation and the maintenance of pain sensation sensitization, as many neurotrophic, protein kinase and ionic channel etc., finally causes cynapse to transmit the enhancing of usefulness, the generation of sensitization.Pain sensation sensitization can betide two levels, i.e. the modification of post-transcriptional level and the change of transcriptional level, has mediated early stage and long-term pain sensation sensitization respectively.But up to now, the Forming Mechanism of pain sensation sensitization still can be explained completely without any a factor, but by interacting, mutually restricting between each messenger molecule intracellular, form complicated signal transmission network, the common generation participating in pain sensation sensitization.In the network of this complexity, no matter be the modification of post-transcriptional level or the change of transcriptional level, protein phosphorylation all plays central role.Have been found that multiple protein kinases at present, as PKA (protein kinase A), PKC (protein kinase C), CaMK (calcium/calmodulin dependent protein kinase), PI3K (phosphatidylinositol3kinase), ERK (extracelluar signal regulated kinase) etc., both can have been participated in the regulation and control of early stage pain sensation sensitization by phosphorylation ionic channel, acceptor etc., in also can being conducted by the regulation and control pain sensation, key molecule transcribes the regulation and control participating in long-term pain sensation sensitization.Therefore, if determine that some protein kinase participates in pain transduction and Pain Modulation, and then find the target protein played a role, and by determining the structural domain of its phosphorylation site or key, the comparatively strong and analgesic that side effect is less of some specificitys can be developed accordingly.

Summary of the invention

The object of this invention is to provide a kind of polypeptide and the application thereof that prevent and/or treat pain.

The polypeptide preventing and/or treating pain provided by the present invention, name is called TAT-S3, is a)-c) in arbitrary shown in polypeptide:

A) polypeptide of aminoacid sequence as shown in sequence in sequence table 1 10-25 amino acids residue;

B) polypeptide of aminoacid sequence as shown in sequence in sequence table 1;

That c) carries out that following sudden change obtains to the polypeptide shown in a) or b) has 1)-3) in the derivative polypeptide of at least one effect: by the amino acid residue sequence in the 10-25 amino acids residue of sequence in sequence table 1 except the 12nd of sequence 1 through the replacement of one to ten amino-acid residue, disappearance and/or interpolation;

Described 1) be-3):

1) pain is prevented and/or treated;

2) phosphorylation of actin depolymerizing factor/cofilin (ADF/cofilin) is disturbed;

3) function of transient receptor potential vanillic acid hypotype 1 is reduced.

Wherein, the sequence 1 of sequence table is made up of 25 amino-acid residues, and the 1-9 amino acids residue composition of sequence 1 has the region of penetration cell membrane interaction, and this region can be replaced by other sequence with phase same-action; The 10-25 amino acids residue of sequence 1 is formed can LIMK(LIM motif-containing protein kinase in competitive inhibition cell, LIMK) to the region of the 3rd Serine (Ser-3) phosphorylation of actin depolymerizing factor/cofilin cofilin, wherein, the 12nd Serine of sequence 1 is the phosphorylation site (i.e. the site of antagonize endogenous Ser-3 phosphorylation) of LIMK.

Wherein, b) shown in polypeptide be the polypeptide shown in a) is carried out that following sudden change obtains have described 1)-3) and in the polypeptide of at least one effect: the aminoterminal of the polypeptide shown in a) adds 9 amino-acid residues of the 1-9 position of sequence 1.In actual applications, also can carry out following sudden change to the polypeptide shown in a) and obtain having described 1)-3) in the polypeptide of at least one effect: ensure that the 12nd amino acids residue of sequence 1 in sequence table is constant, the 10-11 amino acids residue of sequence 1 is carried out more than one disappearance, the 13-25 amino acids residue of sequence 1 is carried out more than one interpolation; Or in guarantee sequence table, the 12nd amino acids residue of sequence 1 is constant, and the 10-11 amino acids residue of sequence 1 is carried out more than one interpolation, and the 13-25 amino acids residue of sequence 1 is carried out more than one disappearance.

The nucleic acid molecule (DNA or RNA) of above-mentioned arbitrary described polypeptide of encoding also belongs to protection scope of the present invention.Described nucleic acid molecule specifically can be the encoding gene of above-mentioned arbitrary described polypeptide.Contain the expression cassette of the nucleic acid molecule of the above-mentioned arbitrary described polypeptide of coding, recombinant vectors, transgenic cell line (non-animals and plants reproductive material) or recombinant bacterium and also belong to protection scope of the present invention.

The nucleic acid molecule of above-mentioned arbitrary described polypeptide and/or coding said polypeptide is being prepared the application that prevents and/or treats in the product of pain and is also being belonged to protection scope of the present invention with the product preventing and/or treating pain that the nucleic acid molecule of above-mentioned arbitrary described polypeptide and/or coding said polypeptide is activeconstituents.

The product of the product of the phosphorylation of the interference actin depolymerizing factor/Cofilin (cofilin) made for activeconstituents with the nucleic acid molecule of above-mentioned arbitrary described polypeptide and/or coding said polypeptide and/or the function that reduces transient receptor potential vanillic acid hypotype 1 also belongs to protection scope of the present invention.

Above, described pain can be inflammatory pain.

The described pain that prevents and/or treats can be presented as the sensitization of the alleviation pain sensation.The sensitization of the described alleviation pain sensation specifically can be alleviates the sensitization of the inflammatory pain sensation, as alleviated the sensitization of inflammatory thermalgesia.

The said products specifically can be medicine.

When needing, can also add one or more pharmaceutically acceptable auxiliary materials in said medicine, described auxiliary material comprises the thinner of pharmaceutical field routine, vehicle, weighting agent, tackiness agent, wetting agent, absorption enhancer, tensio-active agent, lubricant and stablizer etc.

Medicine of the present invention can make the various ways such as injection liquid, dry powder injection, tablet or granula.The medicine of above-mentioned various formulation all can be prepared according to the ordinary method of pharmaceutical field.

TAT-S3 can disturb the phosphorylation of actin depolymerizing factor/Cofilin (cofilin), reduces the function of transient receptor potential vanillic acid hypotype 1, can alleviate the sensitization of inflammatory thermalgesia, can be used for preventing and/or treating pain.

Accompanying drawing explanation

Fig. 1 is vitro kinase experiment and western blot result

Fig. 2 is the sequence of TAT-S3 and control peptide TAT-S3A.

Fig. 3 is the impact of subarachnoid injection TAT-S3peptide on the phosphorylation of cofilin in rat body.

Fig. 4 is the impact of subarachnoid injection TAT-S3 on the function of TRPV1

Fig. 5 is the impact of subarachnoid injection TAT-S3 on the rat thermalgesia sensitization that CFA induces.

Fig. 6 is the thermalgesia sensitization that subarachnoid injection TAT-S3 does not affect rat inflammation offside.

Fig. 7 is that TAT-S3A does not ease pain or promotes the effect of pain sensation sensitization.

Embodiment

Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.

The data processing adopted in following embodiment and statistical test as follows:

Experimental result adopts GraphPad Prism5.01 version software analysis, represents with mean value ± standard error (Mean ± SEM).In histogram shown in Fig. 1,3,5, two groups are compared use non-paired t test (unpaired t Test).Behaviors survey and calcium imaging experiment result adopt two-way analysis of variance (two-way ANOVA), and test with Bonferroni posttests.Using p<0.05 as the standard having significant difference statistically.Western blot result adopts Quantity One(Bio-Rad) image analysis software carries out photo densitometry.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

Rat in following embodiment is healthy male SD rat, original body mass 160-180g.

TAT-S3 solution in following embodiment, TAT-S3A solution are all prepared as solvent by stroke-physiological saline solution.

The functional experiment of embodiment 1, TAT-S3

1, the design of TAT-S3peptide

LIMK (LIM motif-containing protein kinase, LIMK) protein kinase family is a kind of serineprotein kinase, only comprises LIMK1 and LIMK2 two members.The foundation structure feature of two members is identical, all comprises two continuous print LIM structural domains and a PDZ structural domain of N end, and the protein kinase domain of C end, in the middle of PDZ structural domain and protein kinase domain, also have one section of Serine/proline-rich region.Since LIMK1 and LIMK2 finds, people focus mostly in the regulation and control to neuronal development to its functional study in neural system, find that LIMK can regulate and control the form of growing tip and the extension of motion and nervous process by phosphorylation cofilin, participate in the various neurone guiding growth stimulating induction.There are many evidence implicates LIMK may participate in the regulation and control of pain sensation sensitization: the mRNA of LIMK1 has the expression of high level in neural system, two key position DRG(dorsal root ganglion particularly in pain sensation intracellular signaling, DRG) and spinal cord, the mRNA of LIMK2 also has distribution in neural system; The mouse of LIMK1 gene knockout shows the exception of dendritic spine form and the impaired of synaptic function; the dual-gene mouse knocked out of LIMK1 and LIMK2 then shows as even more serious impaired of synaptic function; suppress the phosphorylation of dentate gyrus cofilin also can intervene long term potentia￣tion in late period (late phase long term potentiation, L-LTP); The activation of LIMK1 in thrombocyte can be caused, and thrombin receptor PAR(protease-activated receptor, PAR by stimulated by thrombin) regulation and control of pain sensation sensitization then can be participated in by number of mechanisms; Stimulate PC12 cell can cause the slow rising of the quick active of LIMK1 and LIMK2 activity with NGF, and a kind of pro-inflammatory cytokine that NGF plays a significant role in inflammatory pain exactly; Cytoskeleton can participate in the regulation and control of chronic pain by the protein kinase be anchored on above them.

LIMK only has a kind of substrate specificity of classics, i.e. actin depolymerizing factor/Cofilin cofilin.LIMK can by the polymerization state of phosphorylation cofilin the 3rd Serine modulate actin skeleton, thus wide participation is in the processes such as the motion of cell, form generation, division, differentiation, apoptosis, neurite-outgrowth and tumour generation.

The research of this seminar shows, in the inflammatory pain that complete Freund's adjuvant (CFA) is induced, LIMK can pass through its classical substrate cofilin of phosphorylation, the polymerization state of modulate actin skeleton, thus regulate TRPV1---experience the function of the important molecule integrator of noxious stimulation, then participate in generation and even the maintenance of the sensitization of inflammatory thermalgesia.On this basis, the present invention has designed and synthesized one section LIMK can be disturbed the film the worn fusogenic peptide TAT-S3 of the Ser-3 phosphorylation of cofilin.

Wherein, following experiment confirm LIMK be by phosphorylation cofilin participate in the sensitization of inflammatory thermalgesia regulation and control.

Local inflammation is that modal reason occurs pain.The solitary adjuvant arthritic rats model that chronic inflammatory diseases pain animal model conventional in the world at present has Butler to create and the rat inflammation pain model caused by subplantar injection CFA, they are all using freund's adjuvant as proinflammatory agent, the detection of the latter's pain index is relatively simple, stable, thus more conventional compared with the former.

Chronic pain can produce allodynia, and main manifestations is two kinds of symptoms: spontaneous pain and bring out pain.Bring out pain and can be divided into two kinds: thermalgesia sensitization and mechanical bitterly quick or title Mechanical allodynia.The application selects the rat inflammation pain model caused by subplantar injection CFA, and after injection, red and swollen heat pain phenomenon appears in In The Rat Sole local, and other state is all good.Get the administration homonymy L4-L6DRG of the rat of 0.5h, 1h, 2h, 6h and 1d after normal rat (Naive) and subplantar injection CFA respectively, carry out vitro kinase activity experiment, sds polyacrylamide gel electrophoresis and western blot after extracting total protein and test.

Result such as Fig. 1 shows, in inflammation homonymy DRG LIMK1 kinase activity standard state under relatively low, increase sharply after CFA injection, peak at 1h, and after this falling after rise, but still maintain a relatively high level from 2h to 1d.The result of Western blot shows, in whole process, the protein expression level of LIMK1 be substantially constant (in Fig. 1 a).

Namely LIMK2 then reaches active peak at the rear 0.5h of CFA injection, and in after this falling after rise rapidly.In whole inflammatory process, the protein expression level of LIMK2 be also substantially remain unchanged (in Fig. 1 b).

As mentioned before, there occurs activation in the inflammatory pain process that LIMK induces at CFA, therefore we infer that corresponding change may occur the phosphorylation degree of the 3rd Serine of the most classical substrate cofilin of LIMK.The result display of Western blot, namely phosphorylation level 0.5h after CFA injection of cofilin increases sharply, and peak in 1h, arrive 1d ever since and still remain on higher level (in Fig. 1 c), its Changing Pattern changes with the kinase activity of LIMK1 and LIMK2 and is consistent, and this prompting LIMK regulates and controls inflammatory thermalgesia sensitizing by its classical substrate cofilin of phosphorylation.

In order to study the impact of phosphorylation for the effect of LIMK in inflammatory thermalgesia sensitizing of cofilin the 3rd Serine further at body, design for Ser-3 site and construct the film worn fusogenic peptide---the TAT-S3(TAT-S3peptide that can disturb its phosphorylation), by the Ser-3 of TAT-S3 replace by L-Ala, obtain control peptide TAT-S3A(TAT-S3A peptide)).

2, the preparation of TAT-S3

The sequence of TAT-S3 is the sequence 1 in sequence table, the sequence of control peptide TAT-S3A be sequence 2(in sequence table as shown in Figure 2).

TAT-S3 and TAT-S3A all adopts solid phase polypeptide synthesis (Fmoc method) by carboxyl terminal to aminoterminal direction composition.The polypeptide of synthesis adopts high performance liquid chromatography to carry out purifying and obtains TAT-S3 and TAT-S3A that purity is >=98%.TAT-S3 and TAT-S3A of purifying is through Mass Spectrometric Identification, and the aminoacid sequence of TAT-S3 is as shown in sequence in sequence table 1; The aminoacid sequence of TAT-S3A is as shown in sequence in sequence table 2.

3, the function of TAT-S3

3.1TAT-S3 disturbs the phosphorylation of cofilin

Give TAT-S3 or TAT-S3A half an hour in 5d sheath after spinal subarachnoid space in rats intubate after, L4, L5DRG total protein is extracted after the subplantar injection 100 μ l25%CFA1h of left side, carry out Western Blot and test the phosphorylation level detecting actin depolymerizing factor/Cofilin (cofilin), result such as Fig. 3 shows, can be good at reducing phosphorylation level (the * * p=0.0048 in the inflammatory pain that cofilin induces at CFA after giving TAT-S3 in sheath, n=6(often organizes 6 SD rats)), and the total protein concentration of cofilin is not affected.As can be seen here, in vivo, the film the worn fusogenic peptide TAT-S3 of structure can be good at the phosphorylation disturbing cofilin.In Fig. 3, the total protein concentration of p-cofilin to be the cofilin of phosphorylation, cofilin be cofilin.

Wherein, the experimental technique used is as follows:

A, organize the extraction of total protein

1) animal broken end, is separated both sides L4, L5DRG rapidly ,-80 DEG C of preservations;

2) one-sided DRG adds the above-mentioned Tissue lysates of 200 μ l, tissue homogenizer (Pellet motor, Rontes) grinding, add 200 μ l lysates respectively, 4 DEG C of suspendible 1h after grinding fully;

3) 4 DEG C of 12,000g × 5min are centrifugal, and chorista fragment is got supernatant and is total protein extract.

B, vitro kinase activity experiment (Kinase Assay)

1) DRG total protein is extracted.

2) pearl (protein A-Sepharose CL-4, Amersham Pharmacia, CAT#17-5280) often pipe 30 μ l, wash 3 times with PBS, jointly hatch with LIMK1 or LIMK2 antibody, antibody final concentration is 1:50, hatches more than 3h for 4 DEG C.

3) by BCA method quantitatively (Pierce Biotechnology, Rockford, IL, USA), 400-500 μ g albumen got by each sample, and the mixed solution equal-volume of pearl and antibody joins in each sample, final volume lysate is mended to 400 μ l, 4 DEG C of overnight incubation.

4) pearl is washed 4 times, each 1ml with 0.1%Triton X-100/1 × TBS.Pearl is washed 2 times, each 1ml with kinase reaction damping fluid (the substrate protein bletilla isotropic substance without DTT, purifying).

5) pearl is collected, with 25-30 μ l kinase reaction damping fluid (Tris-HCl (pH7.6) 20mM, MgCl ₂20mM, DTT0.1mM, [ ^γ-32p] ATP10 μ Ci/50 μ l, the substrate protein of purifying) resuspended pearl, 30 DEG C of water-bath 30min.

6) add 6 × sample-loading buffer and boil sample 5min, centrifugal loading after ice bath 5min.

7) SDS-PAGE (10%) electrophoresis.

8) electrophoresis after stain glue, decolouring, dry glue spends the night.

9) darkroom compressing tablet ,-80 DEG C of refrigerators place the suitable time, radioautograph.

C protein matter immunoblotting assay (Western Blot)

1) first partition is from sol solution (10% or 12%) 10ml, adds between two sheet glass, washes down unpolymerized polyacrylamide with distilled water.Concentrated glue (5%) 4ml of preparation, injects separation gel upper end.

2) after gelling, take out comb, rinse loading wells, gel is put into electrophoresis chamber, adds electrophoretic buffer, the dipped gel of upper tank liquor.

3) loading in order.

4) constant voltage electrophoresis (80V), when sample arrives separation gel and concentrated glue line of delimitation, changes voltage into 120V, arrives bottom gel to dyestuff.

5) gel is put into transferring film liquid, room temperature washes glue 20min.

6) cut one and gel equal-sized NC (nitrocellulose, NC) film (Pall Gelman Laboratory, USA) and six 3MM filter paper (Whatman, UK), 4 DEG C are soaked in transferring film damping fluid stand-by.4 DEG C of precoolings put by transferring film damping fluid.

7) three filter paper, NC film, gel and other three filter paper are overlayed on transfer table to negative pole successively by positive pole, drive away bubble.In room temperature constant current transferring film (150mA) 1.5-2h.

8) close: NC film is put into confining liquid, be placed in 1h under shaking table room temperature.

9) primary antibodie (confining liquid dilution) (cofilin antibody (1:100, sc-33779 is added; Santa Cruz Biotechnology) or p-cofilin antibody (1:2000, sc-12912-R/sc-21867-R; Santa Cruz Biotechnology) or GAPDH antibody (1:2000, CW0100; CoWin Biotech Co., Ltd.), 4 DEG C of overnight incubation.Wherein, the expression amount of GAPDH albumen is internal reference.

10) TBST rinsing NC film 3 times are used under room temperature, each 5min.

11) two anti-(Zhong Shan company, import packing) of HRP mark are added, incubated at room 1h.

12) TBST rinsing NC film 4 times are used under room temperature, front twice 5min, rear twice 10min.

Chemical luminescence for liquid A, B liquid balanced mix (Santa Cruz, Biotechnology, CA, USA.sc-2048) hatches 1-2min, and darkroom exposes.

3.2 subarachnoid injection TAT-S3 can weaken the function of TRPV1 on DRG neurone

LIMK is by its classical substrate cofilin of phosphorylation, the polymerization state of modulate actin skeleton, thus regulates the function of TRPV1, then participates in generation and even the maintenance of the sensitization of inflammatory thermalgesia.So, can the phosphorylation of interference cofilin affect the function of TRPV1? we give 30 g TAT-S3 or TAT-S3A after spinal subarachnoid space in rats intubate in 5d sheath, 25%CFA is given at the bottom of 0.5h metapedes, acute isolation L4, L5DRG neurone when giving CFA1h, after adherent 1-1.5h, Fura-2AM hatches 40min-1h, carries out calcium image checking after recovery.Result, as Fig. 4, obviously reduces through the reactivity of TAT-S3 process to capsaicine (Cap) relative to control peptide TAT-S3A, DRG neurone.This illustrates that TAT-S3 disturbs the phosphorylation of cofilin obviously can reduce the function of TRPV1.* * p<0.0001, n=44-48(often organize 44-48 neurone), two-way analysis of variance (two-way ANOVA), and test with Bonferroni posttests.

Wherein, in this experiment, experimental technique is as follows:

The configuration of A main agents and solution

DMEM nutrient solution: DMEM solid medium (Gibco-BRL, USA) 13.48g is dissolved in 800ml ddH ₂in O, use 3.7g NaHCO ₃adjust pH, to 7.2-7.4, is settled to 1L.0.22 μm of filtering with microporous membrane is degerming, 4 DEG C of preservations.

0.25% pancreatin: take 0.625g pancreatin (Sigma) and be dissolved in 200ml DMEM nutrient solution, be settled to 250ml, 0.22 μm of little frit of micropore is degerming.

3mg/ml collagenase: take 0.75g collagenase (Sigma) and be dissolved in 200ml DMEM nutrient solution, be settled to 250ml, 0.22 μm of little frit of micropore is degerming.

Extracellular fluid: weigh 7.605g NaCl, 0.3725g KCl, 0.272g KH ₂pO ₄, 0.094gMgCl ₂, 0.2775g CaCl ₂, 1.8g glucose and 2.38g HEPES is dissolved in 800ml ddH ₂in O, with NaOH adjust pH to 7.2, Sucrose adjusts osmotic pressure to 300mOsm, is settled to 1L.0.22 μm of filtering with microporous membrane is degerming, 4 DEG C of preservations.

Poly-lysine (poly-D-lysine, PDL): 0.5mg/ml (Sigma-Aldrich).

Fura-2AM: store concentration 1mM (Invitrogen).

Capsaicine (Capsaicin): be dissolved in DMSO, stores concentration 100mM (Enzo Life Sciences).

PBS:NaCl8g, Na ₂hPO ₄12H ₂o2.08g, KCl0.2g, KH ₂pO ₄0.2g is dissolved in 800ml ddH ₂in O, adjust pH, to 7.2-7.4, is settled to 1L.0.22 μm of filtering with microporous membrane is degerming, 4 DEG C of preservations.

B acute isolation DRG neurone

Before experiment, 12h is at calcium imaging special culture dish middle berth poly-lysine, and 37 DEG C of incubators spend the night.Apparatus needed for high pressure.Siphon away poly-lysine (recyclable use again) after 12h, with the high distilled water wash pressed through three times, insert super clean bench and dry up.

The SD rat of sheath interpolation pipe administration gives anaesthetic by required time point and deeply anaesthetizes execution after CFA modeling, get L4-L6DRG cell space and be placed in 1.5ml collagenase, 37 DEG C, 100rmp digests 50min, sucking-off collagenase, add 1ml pancreatin, 37 DEG C, 100rmp digests 10min, digestion is stopped with 500 μ l FBS, glass dropper piping and druming 10-30 time, move into 15ml centrifuge tube, the centrifugal 4min of 500rmp, carefully suck supernatant, add suitable inoculation liquid (DMEM containing 10%FBS), cell is inoculated in and is covered with in the culture dish of poly-lysine by mixing, be placed in 37 DEG C of adherent 1-1.5h of incubator, carry out calcium imaging experiment.

The imaging of C calcium

Loading buffer: with extracellular fluid dilution Fura-2AM to 5mM.

1) twice, extracellular fluid fine laundering cell is contained with 37 preheatings;

2) add the loading buffer of appropriate 37 DEG C of preheatings, room temperature 50min is hatched;

3) loading buffer is discarded, with the extracellular fluid fine laundering cell 2 times of 37 DEG C of preheatings;

4) add extracellular fluid, room temperature recovers 1h;

5) detect under laser confocal microscope;

6) observe time-histories comprise: 10*10s baseline, give to observe 30*10s after 5 μMs of capsaicines stimulate, observe 30*10s after giving 5 μ l10%KCl solution and detect cytoactive.

3.3 subarachnoid injection TAT-S3 can alleviate the inflammatory thermalgesia sensitization of rat

The existing research of this study group shows, LIMK is the polymerization state by its classical substrate cofilin modulate actin skeleton of phosphorylation, thus the function strengthening TRPV1 participates in generation and even the maintenance of the inflammatory pain of CFA induction.Can so intervene LIMK affect the inflammatory thermalgesia sensitization of LIMK mediation generating process on the phosphorylation of cofilin? next, we select the phosphorylation by intervening cofilin Ser-3 and then interference LIMK to be studied this problem the method for the regulation and control of TRPV1 in the inflammatory pain rat model of CFA induction.The method of subarachnoid injection TAT-S3 is specifically adopted to observe its impact on the behavior of rat pain.

The TAT-S3 solution and the TAT-S3A solution that concentration are 1mg/ml inject rat with following dosage respectively: 10 μ g, 17 μ g or 30 μ g TAT-S3 or TAT-S3A are injected into the subarachnoid space of SD rat, afterwards during 30min, to rats with left subplantar injection 100 μ l25%CFA incite inflammation, and there is 1h in inflammation, when 2h, 6h, 1d, radiant heat stimulation instrument is utilized to measure thermal stimulus contracting foot latent period (paw withdrawal latency, PWL) of rat.Wherein, the rat of subarachnoid injection TAT-S3 is TAT-S3 treatment group, and the rat of subarachnoid injection TAT-S3A is TAT-S3A treatment group (control group).

Result is as Fig. 5, when there is 1h and 2h in inflammation, the thermal stimulus contracting foot of the rat (being expressed as in Figure 5 " TAT-S3 ") of the TAT-S3 treatment group of 30 μ g and 17 μ g (is expressed as " TAT-S3A " in Figure 5 apparently higher than the rat of TAT-S3A treatment group latent period, illustrate that TAT-S3 can be alleviated thermalgesia sensitization that CFA causes and present dose-dependently and (give the rat of 30 μ g TAT-S3 or TAT-S3A, group difference ###p=0.0001, n=7(often organize 7 SD rats) (in Fig. 5 A); Give the rat of 17 μ g TAT-S3 or TAT-S3A, group difference #p=0.0350, n=10-11(often organize 10-11 SD rat) (in Fig. 5 B); Give the rat of 10 μ g TAT-S3 or TAT-S3A, group difference p=0.6684, n=8-9(often organize 8-9 SD rat) (in Fig. 5 C)).When 1h and 2h occurs inflammation, the thermal stimulus contracting sufficient latent period of 30 μ g TAT-S3 pre-treatment energy significant prolongation rats, (PWL of the pretreated rat of inflammation generation 1h:TAT-S3 was 10.77 ± 2.416s; The PWL of the pretreated rat of TAT-S3A is 4.935 ± 0.5547s; * p<0.01.The PWL that the pretreated rat of 2h:TAT-S3 occurs inflammation is 8.404 ± 1.645s; The PWL of the pretreated rat of TAT-S3A is 3.956 ± 0.2916s; * p<0.05).In Fig. 5, the area under curve (AUC) of A, B, C also shows the thermalgesia sensitization (in Fig. 5 D) that TAT-S3 obviously can be alleviated CFA and causes.And all have no significant effect (as Fig. 6 to the offside inflammation Thermal allodynia giving CFA, A is the rat giving 30 μ g TAT-S3 or TAT-S3A, B is the rat giving 17 μ g TAT-S3 or TAT-S3A, C is the rat giving 10 μ g TAT-S3 or TAT-S3A), illustrate that subarachnoid injection TAT-S3 does not affect the basal heat susceptibility (basal heat sensitivity, namely under base state to the reactivity of nocuity thermal stimulus) of rat.In Fig. 5 and Fig. 6, baseline represents the basic Thermal allodynia of rat before administration, and baseline does not have Discrepancy Description two groups of rats not have difference.Above-mentioned statistics all adopts two-way analysis of variance method to calculate the overall difference between two groups, and adopts Bonferroni posttests to test to the group difference on same time point.

Then, be again the effect that contrast determines that TAT-S3A do not ease pain or promote pain sensation sensitization with stroke-physiological saline solution: before SD rats with left subplantar injection 100 μ l25%CFA incite inflammation during 30min to the TAT-S3A solution that subarachnoid injection 30 μ g TAT-S3A(30 μ l concentration is 1mg/ml) or the stroke-physiological saline solution of same volume, and there is 1h in inflammation, the PWL of rat is measured when 2h, 6h and 1d.When result shows that 1h, 2h, 6h and 1d occur inflammation, two kinds of process rats show thermalgesia sensitization (there was no significant difference between as Fig. 7, PWL group, the p=0.5966 of same degree after CFA injection.Wherein, the rat 7 of injection TAT-S3A, the rat 6 of injection stroke-physiological saline solution), illustrate that control peptide (TAT-S3A) itself does not have the effect of easing pain or promoting pain sensation sensitization.Above-mentioned statistics all adopts two-way analysis of variance method to calculate the overall difference between two groups, and adopts Bonferroni posttests to test to the group difference on same time point.In Fig. 7,30 μ l normal saline represent the process to subarachnoid injection 30 μ l stroke-physiological saline solution, and 30 μ gtat-S3A peptide represent the process to subarachnoid injection 30 μ g TAT-S3A.

Wherein, in this experiment, experimental technique is as follows:

Healthy male SD rat, original body mass 170-190g, is provided by Laboratory Animal Science portion of Department Of Medicine, Peking University.Rat is all placed in the plastics casing of place mat sawdust, and every cage 4-5 only raises.Give natural illumination, freely drink water and diet.

The foundation of A inflammatory pain model

Adopt rat left rear side vola subcutaneous injection CFA as chronic inflammatory diseases pain animal model.Rat etherization, right arm reclining, with 75% alcohol by the bottom of the metapedes of left side and surrounding skin sterilization, then inject 25%CFA0.1ml, respectively at the 30th minute (min), inflammation after injection, the mensuration that 1,2,6 hour (h) and 1 day (d) carries out behavioral indexes occurs.

B heat brings out the mensuration of pain contracting foot latent period (paw withdrawal latency, PWL)

Contracting foot latent period (PWL) is stimulated to reflect the inflammation thermalgesia sensitization of rat by radiant heat.Carry out rat being placed on sheet glass before test, cover with transparent plexiglass tent (18cm × 8cm × 8cm), rat is conformed 15min, after the combing of rat and inquiry activity disappear substantially, start test.During test, the focus of radiant heat lamp is aimed at rat sole centre, open radiant heat lamp and start timing, occur that contracting foot phenomenon is positive reaction rapidly, closes radiant heat lamp immediately and stops timing when positive reaction appears in rat depending on rat.Adjusting photothermal intensity makes the PWL of rat base state at 10-15 second (sec, s), if rat stimulates 30s to occur positive reaction not yet, closes radiant heat lamp, stops timing to avoid scalding.Every rat replicate measurement 5 times, each test interval 15min, removes a maximum and a Schwellenwert, and the mean values remaining three values be its radiation heat-shrinkable sufficient latent period.

C subarachnoid space intubation procedure

SD rat is anaesthetized through 10% Chloral Hydrate (300mg/kg, i.p.), back cropping, sterilization.Touch T12, T13 spinal crest of Rauber boundary (Lumbar Enlargement, waist marrow L3-L4 level) and ilium sour jujube (lumbar vertebra L4 level), draw horizontal line respectively and make marks, measure line-to-line distance, be inlet pipe length (for 250g rat, about 3.5-4.0cm).Longitudinally skin is scratched in the horizontal median line of ilium sour jujube, the stainless steel tube of 5cm is about as outer tube (external diameter 0.9mm with one, front end is polished into inclined-plane, PE-10 pipe can be held pass through), vertically in L4 with L5 vertebrae gap thrust canalis spinalis (now the tail of animal or hind leg there will be slight twitch), change stainless steel tube direction and point to head end, PE-10 pipe (is used 75% alcohol disinfecting in advance, stroke-physiological saline solution is rinsed) send into subarachnoid space to waist marrow L3-L4 level by outer tube, pull out steel pipe, mattress suture, the fixing PE-10 pipe in local.After neck, skin cuts osculum, and PE-10 pipe is passed thus through subcutaneous, and mattress suture is fixed, and about exposes about 5cm, skin suture.Postoperative i.p. injects penicillin 10,000 unit with preventing infection.Rat recovers 4d and gets final product administration.

Claims (7)

1. Polypeptide 1.a) or b):

   A) polypeptide of aminoacid sequence as shown in sequence in sequence table 1 10-25 amino acids residue;

   B) polypeptide of aminoacid sequence as shown in sequence in sequence table 1.
2. 2. the nucleic acid molecule of polypeptide described in coding claim 1.
3. 3. the expression cassette containing nucleic acid molecule described in claim 2, recombinant vectors, transgenic cell line or recombinant bacterium.
4. 4. polypeptide according to claim 1 and/or nucleic acid molecule according to claim 2 alleviate the application in the product of inflammatory pain sensation sensitization in preparation.
5. 5. alleviate a product for inflammatory pain sensation sensitization, its activeconstituents is polypeptide according to claim 1 and/or nucleic acid molecule according to claim 2.
6. Application 6.e) or f):

   E) polypeptide according to claim 1 and/or nucleic acid molecule according to claim 2 application in the product of the phosphorylation of preparation interference actin depolymerizing factor/Cofilin;

   F) polypeptide according to claim 1 and/or nucleic acid molecule according to claim 2 reduce the application in the product of the function of transient receptor potential vanillic acid hypotype 1 in preparation.
7. Product 7.g) or h):

   The product of the phosphorylation of the interference actin depolymerizing factor/Cofilin g) made for activeconstituents with polypeptide according to claim 1 and/or nucleic acid molecule according to claim 2;

   The product of the function of the reduction transient receptor potential vanillic acid hypotype 1 h) made for activeconstituents with polypeptide according to claim 1 and/or nucleic acid molecule according to claim 2.