CN102648977B

CN102648977B - Application of follistatin-related protein 1 in adjusting Na, K-ATPase activity

Info

Publication number: CN102648977B
Application number: CN201110046989.6A
Authority: CN
Inventors: 张旭; 鲍岚; 李开诚; 张方雄; 陆莹瑾
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Priority date: 2011-02-25
Filing date: 2011-02-25
Publication date: 2014-12-10
Anticipated expiration: 2031-02-25
Also published as: CN102648977A

Abstract

The invention relates to an application of follistatin-related protein 1 (FSTL1) in adjusting Na, K-ATPase activity, in particular to an application of FSTL1 in improving the Na, K-ATPase activity so as to treat, release or improve diseases or symptoms related to the activity, or an application of FSTL1 inhibitor in reducing the Na, K-ATPase activity so as to treat, release or improve diseases or symptoms related to the activity. The invention further provides a drug for adjusting Na, K-ATPase activity and a novel screening method of the drug. The polypeptide, nucleotide, composition and method have a great application prospect in treating, releasing or improving diseases or symptoms related to the Na, K-ATPase activity.

Description

The purposes of FSTL1 in regulating na-k-atp enzyme activity

Technical field

The invention belongs to biological technical field, more specifically, the present invention relates to FSTL1 (being FSTL1) thus na-k-atp enzyme is active to be used for the treatment of, to alleviate or improve the purposes in relevant disease or symptom (especially pain) improving.

Background technology

Current research is found: na-k-atp enzyme has very important effect in the homergy of human body, is distributed widely on the cell membrane of various histoorgans, as kidney, heart, lung and nervous system etc.The activity of sodiumpotassium pump (being na-k-atp enzyme) and some disease or symptom are closely related, and the raising of its activity or reduction produce important impact to these diseases or symptom.

For example, following disease or symptom are relevant with the hyperactivity of sodiumpotassium pump, maybe can be eased or treat by suppressing the activity of sodiumpotassium pump: research shows that sodiumpotassium pump may have certain effect in the pathogenesis of renal hypertension, the activity of sodiumpotassium pump increases promotion ion transport, thereby encourage hypertension (Wang etc., CurrOpin Nephrol Hypertens.2009Sep; 18 (5): 412-20; Ferrari etc., Cell Mol Biol (Noisy-le-grand) .2006Dec 30; 52 (8): 15-8); There is bibliographical information α subunit expression of sodiumpotassium pump in the mankind's melanoma cell obviously raise (Mathieu etc., J Cell Mol Med.2009Feb 20.[Epubahead of print]); The growth of sensory neuron projection of cultivating and the growth of tumor are also relevant with the hyperactivity of sodiumpotassium pump with generation, there is the inhibitor of bibliographical information sodiumpotassium pump as digoxin, can obviously suppress the growth of sensory neuron projection and growth and generation (Penniyainen etc., the NeurosciBehav Physiol.2009Mar of inhibition tumor that cultivate; 39 (3): 301-4.; Mijatovic etc., Expert Opin Ther Targets.2008Nov; 12 (11): 1403-17.).There is bibliographical information sodiumpotassium pump α 1 for the treatment target spot for the treatment of glioma and nonsmall-cell lung cancer, sodiumpotassium pump α 1 inhibitor be hopeful to be used to treatment (as a kind of new derivatives 2 of cardiac glycoside " oxovoruscharin (UNBS1450)) treatment glioblastoma multiforme and nonsmall-cell lung cancer (KissR etc. .Neoplasia.2008Mar; 10 (3): 198-206; Kiss R etc., J Pathol.2007Jun; 212 (2): 170-9.).

Following disease or symptom are on the low side relevant with the activity of sodiumpotassium pump, maybe can be eased or treat by improving the activity of sodiumpotassium pump: in familial hemiplegia type migraine patient, having found has more deletion mutation on sodiumpotassium pump, has confirmed that these sudden changes and clinical symptoms have close relationship; Also have some patients to there will be epilepsy symptom (Aperia, J Intern Med.2007 Jan; 261 (1): 44-52.); In the animal model of diabetes, finding the activity of sodiumpotassium pump and expressing has reduction, wherein the C polypeptide in erythrocyte also has obvious minimizing, external source is supplemented C polypeptide can obviously improve the activity of sodiumpotassium pump and the symptom of diabetes-alleviating (Vague etc., ExpDiabesity Res.2004Jan-Mar; 5 (1): 37-50.); The active obviously reduction of sodiumpotassium pump in some major depression patients on erythrocyte, gives to take a turn for the better after lithium salts treatment; The activity of the inhibition sodiumpotassium pump of moderate also can cause manic generation (Lichtstein and Rosen, Neurochem Res.2001 Sep; 26 (8-9): 971-8.); The digitalism of seeing be clinically exactly activity due to extra-inhibitory sodiumpotassium pump cause cardiac muscle be overexcited due to (Vivo etc., Am JMed Sci.2008Nov; 336 (5): 423-8.); Former studies also confirms that the activity that improves alveolar sodiumpotassium pump can obviously improve injury of lung, the symptom of pulmonary edema (Factor etc., J Clin Invest.1998 Oct 1; 102 (7): 1421-30.; Lecuona etc., J Bioenerg Biomembr.2007Dec; 39 (5-6): 391-5.; Sznajder etc., J Appl Physiol.2002 Nov; 93 (5): 1860-6).

The small neuron of dorsal root ganglion (Dorsal Root Ganglia, DRG) is responsible for transmitting thermo aesthesia and the pain sensation to spinal cord.When outer circumference end is subject to physics or chemical irritation, the centripetal fiber end of these small neurons slightly discharges excitatory transmitter glutamic acid and the neuropeptide in synaptic vesicle or large densified core bubble (LDCV) by exocytosis form at cornu dorsale medullae spinalis.Think at present, the transmission of this irritability information can be comprised the adjustings such as neuropeptide material opioid peptide, neurotransmitter γ-aminobutyric acid (GBGA), glycine (Glycine) and 5-hydroxy tryptamine (Serotonin) by the suppressive genes of the relay cell of cornu dorsale medullae spinalis and downlink release.This inhibition regulates has the significance in function for normal somesthesia and Pain Modulation.

Had been found that receptor and the passage of some selectivity high expresseds at dorsal root ganglion small neuron surface of cell membrane, this has deepened the understanding of people to sensory information pass through mechanism.These receptors and passage comprise the P2X of ATP gate ₃receptor, transient receptor potential channel V1 (TRPV1) and A1 (TRPA1), valtage-gated Na _v1.8 passages g protein coupled receptor relevant with Mas.These membrane surface molecules are relevant with the sensitization of elementary sensation transmission, sensory neuron and the facilitation process of nocuity reaction.

Although this area has had been found that the more above-mentioned molecule working in sensory information transmission at present, yet also in the urgent need to research, in sensory information transmittance process, also have those molecules in action, with the understanding that promotes sensory information to transmit, new analgesia strategy and the new analgesic of exploitation are proposed.

Summary of the invention

Thereby main purpose of the present invention is to provide FSTL1, na-k-atp enzyme is active to be used for the treatment of, to alleviate or improve the purposes in relevant disease or symptom (especially improve somesthesia, particularly pain is felt or thermo aesthesia) improving.

Another object of the present invention is to provide a kind of bioactive fragment of FSTL1, described active fragment has the somesthetic effect of good inhibition.

In a first aspect of the present invention, the purposes of FSTL1 in preparation compositions is provided, described compositions is for treating, alleviate or improvement and na-k-atp enzyme active relevant disease or symptom by improving na-k-atp enzyme activity.

In an embodiment of the invention, described disease or symptom are selected from: pain, familial hemiplegia type migraine, diabetes, depression, digitalism, injury of lung or pulmonary edema.

In yet another embodiment of the present invention, described compositions is used for the treatment of, alleviates or improves pain.In a preference, described pain is body source property pain, preferably neuropathic pain or nociceptive pain.

In yet another embodiment of the present invention, described neuropathic pain is selected from: dorsal root ganglion neuropathic pain, trigeminal ganglion neuropathic pain or sciatica.In another preference, described neuropathic pain is to be selected from the pain that the damage by maincenter or peripheral nervous system of lower group or pathological change cause: lumbago and backache, neuralgia, fibromyalgia, neuralgia that neuralgia, multiple sclerosis that diabetes are relevant are relevant, postherpetic neuralgia and with the related neurogenic neuralgia of HIV.

In yet another embodiment of the present invention, described nociceptive pain is selected from: body nociceptive pain or internal organs nociceptive pain.In a preference, described nociceptive pain is selected from: cancer pain, arthritis pain, postoperative pain or with the related nociceptive pain of HIV.

In yet another embodiment of the present invention, described FSTL1 is:

(a) protein of aminoacid sequence shown in SEQ ID NO:1 or SEQ ID NO:2;

(b) conservative variant protein matter or its active fragment of the protein defined in (a); Or

(c) by aminoacid sequence in (a), replacement, disappearance or the interpolation through 1-20 (preferably 1-10, more preferably 1-5) amino acid residue forms, and have improve na-k-atp enzyme activity by (a) derivative protein.

In yet another embodiment of the present invention, described (b) is selected from:

(1) there is the polypeptide of 156-173 aminoacid sequence in SEQ ID NO:1;

(2) there is the polypeptide of 158-175 amino acids sequence in SEQ ID NO:2;

(3) by the formed polypeptide of sequential amino acid deletion Progynon inhibin domain (being FS domain) of SEQ ID NO:1 or SEQ ID NO:2;

(4) by the formed polypeptide in sequential amino acid deletion Von Willebrand type C-structure territory (being VWC domain) of SEQ ID NO:1 or SEQ ID NO:2;

(5) by the formed polypeptide of sequential amino acid deletion EF hand-type II domain of SEQ ID NO:1 or SEQ ID NO:2; Or

(6) by the formed polypeptide in sequential amino acid deletion EF hand-type syndeton territory of SEQ ID NO:1 or SEQ ID NO:2.

In a second aspect of the present invention, a kind of polypeptide of separation is provided, described polypeptide is selected from:

(1) have the polypeptide of 156-173 amino acids sequence NGDSHLDSSEFLKFVEQN in SEQ ID NO:1, condition is that this polypeptide is not the protein shown in SEQ ID NO:1;

(2) have the polypeptide of the NGDSRLDSSEFLKFVEQN of 158-175 amino acids sequence in SEQ ID NO:2, condition is that described polypeptide is not the protein shown in SEQ ID NO:2;

In a third aspect of the present invention, a kind of polynucleotide of separation are provided, the protein shown in described polynucleotide encoding the present invention polypeptide as above or SEQ ID NO:1 or SEQ ID NO:2.

In a fourth aspect of the present invention, a kind of carrier is provided, described carrier contains the present invention's polynucleotide as above.

In a fifth aspect of the present invention, a kind of genetically engineered host cell is provided, described host cell contains the foregoing carrier of the present invention; Or in its genome, be integrated with the foregoing polynucleotide of the present invention.

In a sixth aspect of the present invention, the preparation method of the protein of the foregoing polypeptide of the present invention or SEQ ID NO:1 or SEQ ID NO:2 is provided, the method comprises:

(a) under conditions suitable for the expression, cultivate the foregoing host cell of the present invention;

(b) from culture, isolate the protein of the foregoing polypeptide of the present invention or SEQID and NO:1 or SEQID and NO:2.

In a seventh aspect of the present invention, provide a kind of and treated, alleviate or improvement and the active relevant disease of na-k-atp enzyme or the pharmaceutical composition of symptom by improving na-k-atp enzyme activity, it contains: (a) protein of the polypeptide of the present invention of safe and effective amount, SEQ ID NO:1 or SEQ ID NO:2; And (b) pharmaceutically acceptable carrier.

In an embodiment of the invention, pharmaceutical composition of the present invention is used for the treatment of, alleviates or improves disease or the symptom that is selected from lower group: familial hemiplegia type migraine, diabetes, depression, digitalism, injury of lung, pulmonary edema or pain.In a preference, described pain is body source property pain, preferably nociceptive pain or neuropathic pain.

In yet another embodiment of the present invention, described neuropathic pain is selected from: dorsal root ganglion neuropathic pain, trigeminal ganglion neuropathic pain or sciatica.In another embodiment of the present invention, described neuropathic pain is to be selected from the pain that the damage by maincenter or peripheral nervous system of lower group or pathological change cause: lumbago and backache, neuralgia, fibromyalgia, neuralgia that neuralgia, multiple sclerosis that diabetes are relevant are relevant, postherpetic neuralgia and with the related neurogenic neuralgia of HIV.

In yet another embodiment of the present invention, described nociceptive pain is selected from: body nociceptor pain and internal organs nociceptor pain.In a preference of the present invention, described nociceptive pain is selected from: cancer pain, arthritis pain, postoperative pain or with the related nociceptive pain of HIV.

In a eighth aspect of the present invention, provide the purposes of the protein shown in the protein shown in polypeptide of the present invention, SEQ ID NO:1 and/or SEQ ID NO:2 in the pharmaceutical composition of the disease for the preparation of by raising na-k-atp enzyme activity is treated, alleviated or improvement is relevant to na-k-atp enzyme activity or symptom.

In an embodiment of the invention, described pharmaceutical composition is used for the treatment of, alleviates or improves disease or the symptom that is selected from lower group: familial hemiplegia type migraine, diabetes, depression, digitalism, injury of lung, pulmonary edema or pain.In a preference, described pain is body source property pain, preferably nociceptive pain or neuropathic pain.

In yet another embodiment of the present invention, described neuropathic pain is selected from: dorsal root ganglion neuropathic pain, trigeminal ganglion neuropathic pain or sciatica.In another embodiment of the present invention, described neuropathic pain is to be selected from the pain that the damage by maincenter or peripheral nervous system of lower group or pathological change cause: lumbago and backache, neuralgia, fibromyalgia, neuralgia that neuralgia, multiple sclerosis that diabetes are relevant are relevant, postherpetic neuralgia and with the related neuropathic pain of HIV.

In a ninth aspect of the present invention, provide the purposes of polypeptide of the present invention in the pharmaceutical composition for the preparation for the treatment of rheumatoid arthritis and/or ischemic heart desease.

In a tenth aspect of the present invention, a kind of method that provides screening can pass through to improve na-k-atp enzyme active treatment, alleviation or improve the potential material of the disease relevant to this activity or symptom, described method comprises:

(1) candidate substances is contacted with the system of expressing FSTL1;

(2) detect the impact of candidate substances on FSTL1;

If described candidate substances can improve expression, activity and/or the secretion of FSTL1, show that this candidate substances is the potential material that can be used for treating, alleviating or improve by improving na-k-atp enzyme activity the disease relevant to this activity or symptom.

In a preference, described disease or symptom are selected from: familial hemiplegia type migraine, diabetes, depression, digitalism, injury of lung, pulmonary edema or pain.In another preference, described pain is neuropathic pain, preferably dorsal root ganglion neuropathic pain or trigeminal ganglion neuropathic pain.

In an embodiment of the invention, step (1) comprising: in test group, candidate substances is joined in the system of expressing FSTL1; And/or step (2) comprising: expression, activity and/or the secretion of FSTL1 in the system of detection test group, and with matched group comparison, wherein said matched group is the system of not adding the expression FSTL1 of described candidate substances; If the expression of FSTL1 in test group, activity and/or secretion statistically higher than (be preferably significantly higher than, as more than 20%, preferably high more than 50% in high; Better is high more than 80%) matched group, just show that this candidate substances is the potential material that can be used for treating, alleviating or improve by improving na-k-atp enzyme activity the disease relevant to this activity or symptom.

In another preference, described system is selected from: cell system (as small neuron cell) (or cell culture objects system), subcellular fraction system, solution system, organizational framework, organ systems or animal system.

In another preference, described method also comprises: the potential material obtaining is carried out to further cell experiment and/or animal experiment, further to select and to define the material of use from candidate substances.

In a eleventh aspect of the present invention, a kind of method of screening treatment or lenitive potential material is provided, described method comprises:

(1) candidate substances is contacted with the system of na-k-atp enzyme;

(2) detect the impact of candidate substances on na-k-atp enzyme;

If described candidate substances can improve the activity of na-k-atp enzyme, show that this candidate substances is to can be used for treatment or lenitive potential material.

In a twelveth aspect of the present invention, the inhibitor that FSTL1 is provided preparation by reduce na-k-atp enzyme activity and treat, alleviate or the compositions of disease that improvement is relevant with na-k-atp enzyme activity or symptom in purposes.

In an embodiment of the invention, described disease or symptom are selected from: renal hypertension, tumor (preferably melanoma, glioblastoma multiforme or nonsmall-cell lung cancer).

In yet another embodiment of the present invention, described inhibitor is selected from: the antibody of anti-FSTL1 or its active fragment; Antisense oligonucleotide or siRNA for FSTL1 coded sequence; Or derive from the peptide section of na-k-atp enzyme.

In yet another embodiment of the present invention, described inhibitor is selected from:

(i) there is the polypeptide of the aminoacid sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8;

(ii) conservative of the polypeptide defined in (i) variation polypeptide or its active fragment; Or

(iii) combination of polypeptide or active fragment arbitrarily (i) or (ii).

Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.

Accompanying drawing explanation

Fig. 1 .FSTL1 is high expressed in DRG small neuron

Figure 1A. by the express spectra of candidate gene in adult rat tissue and the ratio of dorsal root ganglion signal intensity and other tissue signal intensity of genechip detection.In figure, in various tissues, gene signal shows with different tones from the log2 proportion of ratio in DRG.Avil represents advillin; Calca represents CGRP α; Htr3a represents 5-hydroxytryptamine receptor 3A (5-hydroxytryptamine receptor 3A); Plekha4 representative is containing the member 4 (pleckstrin homologydomain containing, family A member 4) of the A of family of platelet homology domain; Mpz represents myelin protein 0 (myelin proteinzero); P2rx3 represents P2X3 receptor; Prph1 represents peripheral protein 1 (peripherin 1); Nef3 represents neurofilament 3 (neurofilament 3); The valtage-gated type VII α of Scn7a represent sodium ion channel (sodiumchannel, voltage-gated type VII, α); The positive contrast of dorsal root ganglion GAPDH signal intensity.

Figure 1B .RNA blotting detects the expression of Fstl1mRNA in rat Adult Nervous System, and the ribosome 18s RNA of usining contrasts as applied sample amount;

Fig. 1 C. immunoblotting detects the expression of FSTL1 albumen (～37kD) in rat Adult Nervous System, and the actin of usining contrasts as applied sample amount;

Fig. 1 D. SABC and immunoblotting show rat after period of embryo, birth and adult period dorsal root ganglion in the expression of FSTL1.The expression of FSTL1 starts embryo for 15 days, in birth, within latter 28 days, reaches top level.The actin of usining contrasts as applied sample amount;

Fig. 1 E. immunohistochemistry detects in dorsal root ganglion and cornu dorsale medullae spinalis, FSTL1 respectively with the common location situation of CGRP or IB4, FSTL1 is present in the neuron of the CGRP positive or the IB4 positive.Wherein, the green FSTL1 of expression expresses; The red CGRP of expression or IB4 express;

Fig. 1 F. immunofluorescence detects FSTL1 and Na _vthe expression of 1.8 channel proteins, FSTL1 expresses and is containing Na _vin the DRG neuron of 1.8 channel proteins.Wherein upper figure Green is expressed FSTL1 expression, the red Na that represents in middle figure _v1.8 channel proteins are expressed, and figure below is the stack (merge) of upper figure and middle figure.

The distribution situation of Fig. 1 G.FSTL1 in different DRG neuron types;

Fig. 1 H. Dorsal Root Ganglion Neurons sum situation, and the neuron situation of the Dorsal root ganglion FSTL1 positive.

Fig. 1 I. immunohistochemistry detects the expression of FSTL1 and Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 and locates altogether situation.The green FSTL1 of expression expresses, and red expression Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 is expressed, and arrow indication is the vesicle that FSTL1 expresses;

Fig. 1 J. immunohistochemistry detect FSTL1 respectively with TRPV1 or P2X ₃common location situation.Wherein, the red FSTL1 of expression expresses; Green TRPV1 or the P2X of representing ₃express;

Fig. 1 K.RNA blotting detects the expression of Fstl1 mRNA in each tissue of rat, and the ribosome 18s RNA of usining contrasts as applied sample amount.

Fig. 2. prove the experimental result that FSTL1 is secreted by little clear vesicle

Fig. 2 A. immunohistochemistry detects the vesicle distribution (shown in arrow) of FSTL1 in the peptidergic neuron of rat DRG and the positive small neuron of IB4, FSTL1 is not in the large densified core bubble (LDCV) containing CGRP, major part is not also in the positive vesicle of synaptoporin (synaptoporin, Synpr).Wherein, the green FSTL1 of expression expresses, and red expression CGRP, IB4 or Synpr express;

Fig. 2 B. immuno-gold labeling detects the location situation of FSTL1 in DRG small neuron cell space, and FSTL1 is arranged near the little clear vesicle (arrow) golgi's network outside;

Fig. 2 C. is with the different vesicle groups of the separated Rat Spinal Dorsal of method of the separated subcellular components of sucrose density gradient centrifugation, and immunoblotting detects the expression of FSTL1, Synpr and CGRP in isolated vesicle group.FSTL1 is mainly present in the vesicle group of containing Synpr, but is not present in the vesicle group containing CGRP;

The situation that contains FSTL1 vesicle in the centripetal fiber tip of the centripetal fiber of Fig. 2 D. immuno-gold labeling detection Dorsal root and cornu dorsale medullae spinalis I, II layer.FSTL1 is arranged in the little clear vesicle (shown in arrow) of the centripetal fiber tip of the centripetal fiber of Dorsal root and cornu dorsale medullae spinalis I, II layer, and the vesicle containing FSTL1 shown in arrow is positioned near presynaptic membrane.Triangle sign indication postsynaptic compact zone;

The schematic diagram that the rear root knot of Fig. 2 E. mice is pricked.Rear of ligation mice is forward transported with blocking-up vesicle, can cause centripetal fiber gathering near DRG one end FSTL1 for 24 hours;

Fig. 2 F. immunoblot experiment detects the situation of the rat drg neuron secretion FSTL1 cultivating under non-stimulated condition.The total expression of FSTL1 in cell pyrolysis liquid of usining contrasts as applied sample amount, and wherein bar diagram is the quantitative analysis to each swimming lane FSTL1 secretion situation.Can see that FSTL1 secretory volume increases in time in the culture fluid of rat drg neuron.Compare with matched group, ^*p < 0.05, ^*p < 0.01;

Fig. 2 G. immunoblot experiment detects in high potassium stimulation (55mM KCl, 1h) and has or not the situation of the rat drg neuron secretion FSTL1 cultivating under extracellular Ca2＋ condition.The total expression of FSTL1 in cell pyrolysis liquid of usining contrasts as applied sample amount, and wherein bar diagram is the quantitative analysis to each swimming lane FSTL1 secretion situation.Compare with the matched group of not increasing potassium stimulation, ^*p < 0.05 (high potassium adds calcium stimulates (being post 2) to compare with the matched group of not increasing potassium but adding calcium stimulation (being post 1)), ^#p < 0.05 (high potassium adds calcium stimulates (being post 2) to compare with the matched group of increasing potassium and do not add calcium stimulation (being post 4));

Fig. 2 H. immunoblotting detects the secretion situation that stimulates rat spinal cord brain sheet FSTL1 under (55mM KCl, 15min) condition at high potassium.High potassium stimulates significantly increases the secretory volume of FSTL1 in Spinal Cord Slices, and the expression of actin in cell pyrolysis liquid of usining contrasts as applied sample amount.In identical sample, by immunoblotting and HPLC detection method, show that CGRP and glutamic acid (Glu) level raise.Compare with the matched group of not increasing potassium stimulation, ^*p < 0.05;

Fig. 2 I. immunoblotting detects the secretion situation of FSTL1 in high potassium stimulates and has or not the synaptic glomerulus of Rat Spinal Dorsal under extracellular Ca2＋ condition.High potassium stimulates significantly increases the secretory volume of FSTL1 in the synaptic glomerulus of cornu dorsale medullae spinalis, the expression of actin in cell pyrolysis liquid of usining contrasts as applied sample amount, wherein bar diagram is the quantitative analysis to each swimming lane FSTL1 secretion situation, also represents that through immunoblotting, detecting FSTL1 is divided into and secretes with CGRP existence simultaneously.Compare with the matched group of not increasing potassium stimulation, ^*p < 0.05, ^*p < 0.01 (the high potassium stimulating group (being post 3 and post 4) that adds calcium);

Fig. 2 J. immunoblot experiment detects under KCl, capsaicin (Capsaicin, Cap) and/or capsaizepine (CPZ) exist, the FSTL1 level of the neuronal cell secretion of cultivating with young rats DRG.The total expression of FSTL1 in cell pyrolysis liquid of usining contrasts as applied sample amount, and wherein bar diagram is the quantitative analysis to each swimming lane secretion situation.Compare with the matched group that does not add stimulation, ^*p < 0.01 (only add KCl or Cap stimulate compare with the matched group that does not add any stimulation), ^##p < 0.01 (only adding Cap stimulates and add Cap and stimulate and compare with CPZ).

The inhibitory action that Fig. 3 .FSTL1 transmits for sensation

The expression plasmid of Fig. 3 A.FSTL1 and mutant M1 thereof (EF hand-type domain disappearance) and M2 (the 165th becomes A from E) builds schematic diagram, and the C end of FSTL1 and mutant thereof and Myc and His label merge;

Fig. 3 B. carries out full cell record on spinal cord of adult rats thin slice, has recorded 31 spinal cord II layer neurons at embedding voltage-70mV (V processed _h) under sEPSC.In 16 neurons, restructuring FSTL1 has reduced frequency and the amplitude of spontaneous excitatory postsynaptic current (sEPSC) simultaneously.The anti-total length FSTL1 antibody of rabbit (1: 1000) can strengthen power frequency and the amplitude of sEPSC, and preimmune serum (1: 1000) is with comparing.All quantitative analyses adopt the data that in 3min, perfusion FSTL1 collects.Compare with the matched group or the preimmune serum group that do not add FSTL1, ^*p < 0.05, ^*p < 0.01;

Fig. 3 is (intensity: 210-650 μ A) stimulate Dorsal root to induce the Glutamatergic list synapse eEPSC of C fiber, average amplitude 166 ± 23pA (37-459pA C.0.2Hz; V _h=-70mV).In 24 spinal cord II layer neurons that are recorded, single synapse that C fiber excites is brought out sexual excitation postsynaptic currents (eEPSC) by FSTL1 (60nM, n=14; 300nM, n=5) suppress, but by M1 (n=4) and M2 (n=5), do not suppressed.After being standardized as the peak current level of contrast eEPSCs, the damping time constant of repressed eEPSCs is without significant change.FSTL1 antibody (1: 1000) also strengthens the eEPSC (n=5) of C fiber.Compare with the matched group or the preimmune serum group that do not add FSTL1, ^*p < 0.05, ^*p < 0.01;

Fig. 3 D.FSTL1 (60nM) only reduces the frequency of smallness excitatory postsynaptic current (mEPSC).Compare with the matched group that does not add FSTL1, ^*p < 0.01;

Fig. 3 E. immunoblotting detects the expression of FSTL1, M1 and M2 recombiant protein;

The people of Fig. 3 F. purification recombinates Progynon inhibin to not impact of the sEPSC recording on spinal cord of adult rats thin slice.The experimental group that has added FSTL1, compares with the matched group that does not add FSTL1, ^*p < 0.01; Compare with the group that adds FSTL1, ^#p < 0.05.

Fig. 4. at DRG neuron conditional, knock out Fstl1 gene

The structure schematic diagram of Fig. 4 A.Fstl1 conditional gene knockout mice.With two loxP sequences (grey color triangle, Fstl1 ^f) exon 2 of coding FSTL1 signal peptide is deleted.Neo in intron region is also deleted by loxP.PCR detection display is containing the heterozygote (Fstl1 of single copy Fstl1 ^f/+) comprise 399bp and two bands of 505bp; Homozygote (Fstl1 ^f/F) only there is a 505bp band; And wild type (Fstl1 ^{+ /+}) there is band of 399bp.By hybridizing with SNS-Cre mice, neo and exon 2 are removed;

Fig. 4 B. immunoblotting detects Fstl1 ^{+ /+}, Fstl1 ^+/-, Fstl1 ^-/-the expression of FSTL1 in mice DRG.Show that FSTL1 is at Fstl1 ^-/-in DRG, substantially do not exist;

Fig. 4 C. is at Fstl1 ^-/-in the DRG of mice, the neuron of the FSTL1 immunoreation positive is reduced to approximately 10%, and the expression of some main molecules does not change.

The disappearance induction somesthesia of Fig. 5 .FSTL1 is super quick

The neuronic sEPSC of the full cell record spinal cord of Fig. 5 A. II layer, shows that the sEPSC frequency by 15mM KCl (1min) induction is increased in Fstl1 ^-/-in mice than at Fstl1 ^{+ /+}more remarkable in mice.Not add KCl, stimulate in contrast, ^*p < 0.01 (Fstl1 ^-/-mice);

Fig. 5 B. records the wide power neuron reaction (WDR) of mouse spinal cord dorsal horn, Fstl1 at body ^-/-the WDR neuron of the cornu dorsale medullae spinalis of mice (n=6) is more responsive (with Fstl1 for thermostimulation (left side) and mechanical stimulus (right side) ^{+ /+}mice is compared, ^*p < 0.05).In sheath, give (i.t.) FSTL1 reversible Fstl1 ^-/-the WDR neuron reaction of this enhancing of mice (n=3, ^*p < 0.05);

Fig. 5 C.Fstl1 ^{+ /+}, Fstl1 ^f/F, Fstl1 ^-/-the contract comparison on sufficient threshold value (right side) of time (left side) and mechanical stimulus is delayed in the thermal stimulus contracting foot reaction of mice.Fstl1 ^-/-for heat (left side) and the more responsive (n=12 of mechanical stimulus (right side); With Fstl1 ^{+ /+}mice and Fstl1 ^f/Fmice is compared, ^*p < 0.01);

Fig. 5 D.Fstl1 ^{+ /+}, Fstl1 ^f/F, Fstl1 ^-/-mice falls the comparative result (n=6) of the time of delaying in accelerating rotation test;

Fig. 5 E.Fstl1 ^{+ /+}, Fstl1 ^f/F, Fstl1 ^-/-mice hind paw injection of formalin induction inflammatory pain model small mouse is licked the comparison of foot time.Fstl1 ^-/-mice shows more obvious pain reaction, and (n=6, with Fstl1 ^{+ /+}mice and Fstl1 ^f/Fmice comparison, ^*p < 0.05);

Fig. 5 F.Fstl1 ^-/-after mice intrathecal injection (i.t.) variable concentrations FSTL1, the contracting foot of thermostimulation is delayed the impact of time, matched group injection carrier (0.01M PBS).FSTL1 reduces Fstl1 ^-/-the reaction of mice to thermostimulation.Fstl1 with injection carrier ^-/-mice is compared, ^*p < 0.05, ^*p < 0.01;

The antibody of the anti-FSTL1 full-length proteins of Fig. 5 G. adult rat intrathecal injection (1: 100,20 μ l) or preimmune serum, observe rat and the contracting foot of thermostimulation delayed to the variation of time.Give after antibody rat to thermostimulation more responsive (n=6, compares with the mice of serum before injecting immune, ^*p < 0.05, ^*p < 0.01).

The decline that Fig. 6 .FSTL1 expresses has contribution to neuropathic pain

Fig. 6 A. in situ hybridization shows Fstl1 mRNA level, and sciatic nerve cuts off the small neuron number that contains Fstl1 mRNA in In Rat Lumbar 4-5 sections DRG after 14 days significantly to be reduced, and the control sides that the rat of take is not cut off is contrast, ^*p < 0.01;

The variation of Dorsal root ganglion different time FSTL1 after Fig. 6 B. immunoblotting demonstration sciatic nerve sever in rats.The GAPDH of usining contrasts as applied sample amount;

The immune labeled sciatic nerve that is presented at of Fig. 6 C. cuts off the positive DRG quantity minimizing of FSTL1 after 14 days.Immunohistochemistry shows, FSTL1 disappearance in the DRG neuron [with transcriptional factors 3 (ATF3) labelling] of damage.Sciatic nerve cut off after 14 days, and in centripetal fiber in waist 5 sections (indicating with triangle arrow), FSTL1 significantly declines;

Tu6D.You sciatic nerve branch cuts off in the neurogenic pain model causing, the number that cuts off FSTL1 positive neuron in 14 days low back 5DRG significantly declines.The control sides that the rat of take is not cut off is contrast, ^*p < 0.01;

Tu6E. sciatic nerve branch cuts off in the neurogenic pain model cause, and intrathecal injection FSTL1 albumen is for the contract impact of sufficient threshold value of rat mechanical stimulus;

The combination of Fig. 7 .FSTL1 albumen and α 1 sodium-potassium ATP enzyme (NKA) subunit

Fig. 7 A. is hatched the Neurons of The Rat Dorsal Root Ganglion of cultivation with the FSTL 1-myc of restructuring, added the cross-linking agent (BS of impermeable membrane simultaneously ³), with myc antibody test to FSTL1 at the visible band in nearly 150kD place, experiment repeats 3 times;

The same sample of processing of Fig. 7 B. and Fig. 7 A detects by the monoclonal antibody of α 1NKA subunit, and visible part α 1NKA molecular weight subunit is increased to 150kD from 110kD, and α 3NKA subunit has no this phenomenon, and experiment repeats 3 times;

Fig. 7 C. immunohistochemical staining shows that α 1NKA subunit is mainly positioned in the minicell of Dorsal Root Ganglion Neurons, has common location with FSTL1;

The co-immunoprecipitation experiment of Fig. 7 D. dorsal root ganglion of rats shows, in the protein getting off, α 1NKA subunit can be detected with the co-precipitation of FSTL1 antibody mediated immunity, experiment repetition 3 times;

The α 1NKA subunit of Fig. 7 E. corotation zone flag label in COS7 cell and FSTL1-myc (FSTL1-myc (experiment repeats 3 times) in the protein under the co-precipitation of flag antibody mediated immunity, can be detected);

Fig. 7 F. Scatchard analysis shows ¹²⁵i-FSTL1 dose dependent ground is in conjunction with the COS7 cell (Kd=43nM) of coexpression α 1NKA subunit and β 1 subunit;

Fig. 7 G. is in expressing the COS7 cell of FSTL1-myc and α 1NKA subunit, and co-immunoprecipitation experiment is presented in the protein that the co-precipitation of flag antibody mediated immunity gets off and has FSTL1-myc, at coexpression FSTL1-myc and α 1NKA subunit ^e314G-flag or α 1NKA subunit ^t889N-flag or α 1NKA subunit ^{e314G, T889N}in the cell of-flag, the FSTL1-myc existing in the protein that the co-precipitation of flag antibody mediated immunity is got off significantly declines, and FSTL1-myc is not combined with α 3NKA subunit, but with α 3NKA subunit ^{g304E, N879T}-flag combination, experiment repeats 3 times.

Fig. 8 .FSTL1 activates α 1NKA subunit

Fig. 8 A. corotation α 1NKA-flag and with β 1 subunit of myc label in COS7 cell, can detect FSTL1 dose dependent and raise the activity of NKA, and corotation α can not during 3NKA-flag.The activity that FSTL1 is dose dependent and raises NKA in the Dorsal Root Ganglion Neurons of cultivating, also can be detected;

Fig. 8 B.FSTL1 is at corotation α 1NKA subunit ^e314G-flag or α 1NKA subunit ^t889N-flag or α 1NKA subunit ^{e314G, T889N}in the COS7 cell of-flag and β 1 subunit, can not increase the activity of NKA, FSTL1 ^e165Acan not improve the activity of NKA in the COS7 cell of coexpression α 1NKA subunit and β 1 subunit.Express alpha 1NKA subunit is compared with the COS7 cell of β 1 subunit together, ^*p < 0.05, ^*p < 0.01;

The full cell record of Fig. 8 C.COS7 is observed FSTL1 (30nM) cell (n=15) of corotation α 1NKA-flag and β 1-myc subunit is significantly reduced to cell membrane potential, on the not impact of cell (n=8) transmembrane potential of the cell of not transfection (n=12) or corotation α 3NKA-flag and β 1-myc subunit.Compare with the cell of not transfection, ^*p < 0.01;

Fig. 8 D. in the COS7 cell of coexpression α 1NKA subunit and β 1 subunit, preincubate M3M4 (EC ₅₀=3.6 μ M) or M7M8 (EC ₅₀=2.9 μ M) peptide section can make ¹²⁵the combination rate of I-FSTL1 significantly declines, and M1M2 peptide section can not reduce;

Fig. 8 E. is in the COS7 cell of coexpression α 1NKA subunit and β 1 subunit, and preincubate M3M4 or M7M8 peptide section can weaken the NKA increased activity effect that FSTL1 causes.Compare with not adding contrasting of FSTL1, ^*p < 0.01;

The dorsal root ganglion of adult rat small neuron of the full cell record acute isolation of Fig. 8 F., FSTL1 (30nM) reduces the frequency of action potential, and induction film hyperpolarization (record 16 cells have 7 have effect), this impact is reversed when give specific inhibitor ouabain (OB 100 μ M) time of NKA simultaneously, the effect of the film hyperpolarization that FSTL1 causes can be overturn by ouabain, also can be by the 2nd born of the same parents' outer shroud M3M4 peptide section institute antagonism of preincubate α 1NKA.

The expression of Fig. 8 G. α 1NKA in cornu dorsale medullae spinalis, SABC shows that α 1NKA and FSTL1 coexist in primary afferent termination;

The full cell record of Fig. 8 H. spinal cord brain sheet II layer neuron shows that FSTL1 (60nM) reduces the frequency of sEPSC, and this impact is reversed when give specific inhibitor ouabain (OB 100 μ M) time of NKA simultaneously.Compare with not adding contrasting of FSTL1, ^*p < 0.01 (n=5);

The full cell record demonstration of Fig. 8 I. spinal cord brain sheet II layer neuron M3M4 (20 μ M, 3min) peptide section is hatched the frequency that increases sEPSC.Compare with not adding contrasting of peptide section, ^*p < 0.05 (n=6);

The full cell record demonstration of Fig. 8 J. spinal cord brain sheet II layer neuron M3M4 (20 μ M, 3min) peptide section is hatched the eEPSC amplitude that the stimulation of C fiber causes that increases.Compare with not adding contrasting of peptide section, ^*p < 0.05 (n=5).

Each functional area of Fig. 9 .FSTL1 albumen is for the impact of intracellular Ca2+ and α 1NKA enzymatic activity

Obtain respectively removing the recombiant protein that FS, VWC, EF hand-type domain and the 165th amino acids are mutated into the eukaryotic expression of alanine, method with calcium imaging in the Primary Sensory Neuron of acute isolation detects protein to capsaicin (Capsaicin, Cap) impact that in the DRG neuronal cell causing, calcium raises, total length FSTL1 significantly reduces the intracellular Ca2+ rising that Cap causes, disappearance FS and VWC domain are on not impact of the effect of FSTL1, but the function of disappearance EF hand-type domain and the 165th the equal appreciable impact FSTL1 of sudden change.On the COS7 cell of stable transfection α 1NKA and β 1NKA subunit, detect α 1NKA enzymatic activity, FSTL1 total length can significantly activate the activity of α 1NKA enzyme, and in EF1 hand-type domain, the synthetic polypeptide (SP76) of 156-173 amino acids sequence also can significantly activate the activity of α 1NKA enzyme.

The specific embodiment

The inventor is through extensive and deep research, (Follistatin-Like 1 to have proved first FSTL1, FSTL1) or its bioactive fragment can improve the activity of na-k-atp enzyme, thereby can be used for treatment, alleviate or improve disease or the symptom relevant to this activity.For example, at sensory information transmittance process, lower FSTL1 and make the active decline of na-k-atp enzyme can cause sensitivity to pain to increase, this regulating action is that a kind of brand-new sensory information transmits regulatory mechanism.Therefore, FSTL1 itself or on it, adjustment can be active and for suppressing mammalian body sensation (as reduced the mammiferous pain sensation) by improving na-k-atp enzyme.

As used herein, " separated " refers to that material separates (if crude, primal environment is natural surroundings) from its primal environment.As the polynucleotide under the native state in active somatic cell and polypeptide do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, for separation and purification.

As used herein, " to na-k-atp enzyme active relevant disease or symptom " refers to by regulating disease or the symptom that na-k-atp enzyme activity can obtain treatment, alleviate or improve (comprise and raise or lower).Include but not limited to na-k-atp enzyme activity decreased or too low relevant disease or symptom: familial hemiplegia type migraine (Familial hemiplegic migraine), diabetes (diabetes), depression (depression), digitalism (digoxin toxicity), injury of lung (lung injury), pulmonary edema (lung edema) or mammalian body sensation (as the pain sensation or thermo aesthesia excessively obvious), these diseases or symptom can obtain medical treatment by improving na-k-atp enzyme active (in the present invention can by for example increasing FSTL1 quantity or improving that FSTL1 is active to be realized), alleviate or improve.Improve or too high relevant disease or symptom include but not limited to na-k-atp enzyme is active: renal hypertension, tumor (preferably melanoma, glioblastoma multiforme and nonsmall-cell lung cancer), these diseases or symptom can by reduce na-k-atp enzyme active (in the present invention can be by for example reducing FSTL1 quantity, reduce FSTL1 active, suppress FSTL activate na-k-atp enzyme or block FSTL be combined and realize with na-k-atp enzyme) obtain medical treatment, alleviate or improve.

As used herein, described " somesthesia " refers to that mammal is for the sensory reaction of external stimulus (as mechanical damage) or intrinsic stimuli generation, and it is transmitted by nervous system (comprising dorsal root ganglion) conventionally.Described feel to include, but is not limited to: pain is felt, thermo aesthesia.Described pain comprises neuropathic pain, as dorsal root ganglion neuropathic pain or trigeminal ganglion neuropathic pain.Described thermo aesthesia refers to body is touched to the sensory reaction after high temperature or low temperature.

With regard to pain, it can be divided into body source property (somatogenic) pain and psychogenic (psychogenic) pain.Body source property pain can be divided again nociceptive pain (nociceptive pain) and neuropathic pain (Neuropathic Pai).Neuropathic pain is to cause due to the damage of maincenter or peripheral nervous system or pathological change, include but not limited to lumbago and backache (lower back pain), neuralgia (neuralgia), fibromyalgia (Fibromyalgia), the neuralgia (diabetic neuropathic pain, DNP) that diabetes are relevant, neuralgia (pain associated with multiple sclerosis), postherpetic neuralgia (PHN) and HIV related (neurogenic) neuralgia (HIVNP) etc. that multiple sclerosis is relevant.Nociceptor pain is to cause because the nociceptor in human body is subject to machinery, heat, chemical stimulation or damage, body nociceptor pain and internal organs nociceptor pain be can be divided into, cancer pain (cancer-relatedpain), arthritis pain (arthritic pain), postoperative pain (post-operative pain) and HIV related (nociceptor) neuralgia (HIVNP) etc. included but not limited to.Wherein, neuralgia (neuralgia) includes but not limited to trident god's The pain (Trigeminal neuralgia), sciatica (sciatic neuralgia) etc.

As used herein, have comprised " containing ", " having " or " comprising " " comprising ", " mainly by ... form ", " substantially by ... form " and " by ... form "; " mainly by ... form ", " substantially by ... form " and " by ... formation " belong to the subordinate concept of " containing ", " having " or " comprising ".

FSTL1 albumen or its bioactive fragment

In finding the process of specific expressed new functional molecular on DRG neuron, the inventor finds FSTL1 selectivity high expressed on DRG small neuron.

FSTL1 is a kind of glycoprotein, is found to be the earliest a kind of transforming growth factor (TGF) β 1 induced protein.FSTL1 belongs to Progynon inhibin gene family, has Progynon inhibin spline structure territory and a pair of EF-hand-type structure, but can not with activin (Activin) combination.FSTL1 can inhibition tumor cell growth, and be the target protein of rheumatoid arthritis autoimmune antibody.Although there is Fstl1 mRNA in the nervous system of the known DRG of comprising, the physiological function of FSTL1 in nervous system was still unknown by the people in the past.

The inventor further investigation favorite outer discovery, FSTL1 can with sodium-potassium ATP enzyme (Na ⁺, K ⁺-ATPase) combination, and the key point of wherein combination is in second born of the same parents' outer shroud M3M4 of sodium-potassium ATP enzyme α 1NKA subunit and the 4th born of the same parents' outer shroud M7M8.And, after FSTL1 is combined with sodium-potassium ATP enzyme, can activate the activity of sodium-potassium ATP enzyme, thereby can be used for treatment, alleviation or improvement and na-k-atp enzyme activity decreased or too low relevant disease or symptom.

Otherwise, research has also proved and has derived from the peptide section of na-k-atp enzyme or their homologous sequence, include but not limited to: the M7M8 peptide section shown in the M3M4 peptide section shown in SEQ ID NOs:5 and 6, SEQ ID NOs:7 and 8), by suppressing FSTL1, be combined with sodium-potassium ATP enzyme, can reduce the activity of na-k-atp enzyme, thereby can treat, alleviate or improvement and the active raising of na-k-atp enzyme or too high relevant disease or symptom.

In the present invention, utilize to raise or lower FSTL1 treats, alleviates or improvement is relevant with na-k-atp enzyme activity disease or the method for symptom, exist at the same time in the tissue of FSTL1 and na-k-atp enzyme or cell (such as dorsal root ganglion, heart, lung, kidney etc.) particularly effective.

The sequence of na-k-atp enzyme α 1NKA subunit is well known in the art, and maybe can determine by this area conventional method.For example, the aminoacid sequence of rat and people's na-k-atp enzyme α 1NKA subunit is respectively as shown in SEQ ID NO:3 (1023Aa) and SEQ ID NO:4 (1023Aa), and their sequence homogeny reaches 96%.Crucial born of the same parents' outer shroud in rat and human sodium-potassium ATP enzyme α 1NKA subunit is as shown in table 1 below:

Table 1. na-k-atp enzyme α 1NKA subunit and crucial born of the same parents' outer shroud

^*it is benchmark that serial number in the 2nd row be take the aminoacid in aminoacid sequence number shown in SEQ ID NO:3

^*it is benchmark that serial number in the 3rd row be take the aminoacid in aminoacid sequence number shown in SEQ ID NO:4

Inventor is in further further investigation discovery, and FSTL1 can, from Primary Sensory Neuron and teleneuron secretion, have regulating action to sensory information transmittance process.FSTL1 is high expressed in DRG small neuron cell, by little clear vesicle, is transported to afferent nerve endings and can be jointly discharged with excitatory transmitter.Electrophysiological recording shows, FSTL1 can be by activating sodium-potassium ATP enzyme (Na ⁺, K ⁺-ATPase) make DRG small neuron cell membrane hyperpolarization.Specificity knocks out the FSTL1 in DRG neuron, and can cause importing into transportation increases, and increases mammiferous Hyperalgesia degree.Importantly, the expression of DRG neuron FSTL1 is lowered in nerve injury meeting, and spinal cord intrathecal injection FSTL1 can alleviate the pain sensation reaction that nerve injury causes.Therefore, the FSTL1 of centripetal fiber tip secretion works to maintain normal somesthesia as a kind of important regulatory factor that self suppresses.The molecule that FSTL1 can effectively alleviate the pain sensation response prompting that nerve injury causes: FSTL1 or can activate or raise FSTL1 has therapeutic value in neuropathic pain.

In the present invention, FSTL1 albumen used can be naturally occurring, such as its can be separated or purification from mammal.In addition, described FSTL1 albumen can be also artificial preparation, such as carrying out according to conventional genetic engineering recombinant technique Restruction FSTL1 albumen.Preferably, the present invention can adopt the FSTL1 albumen of restructuring.

Any applicable FSTL1 albumen all can be used for the present invention.Described FSTL1 albumen comprises the FSTL1 albumen of total length or its bioactive fragment (or be called " active fragment " or " polypeptide of the present invention ").For example, the aminoacid sequence of described FSTL1 albumen can be substantially the same with the sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

The aminoacid sequence of the FSTL1 albumen that passes through replacement, disappearance or the interpolation of one or more amino acid residues and form is also included within the present invention.FSTL1 albumen or its bioactive fragment comprise the alternative sequence of a part of conserved amino acid, and the described sequence through amino acid substitution does not affect its activity or retained the activity of its part.Suitably replacing aminoacid is technology well known in the art, and described technology can be implemented at an easy rate, and guarantees not change the biological activity of gained molecule.These technology are recognized those skilled in the art, in general, at the inessential area change single amino acids of a peptide species, substantially can not change biological activity.See Watson etc., " Molecular Biology of The Gene " (< < gene molecule > biology >, the 4th edition, 1987, Benjamin/Ku Ming publishing company, The Benjamin/Cummings Pub.Co.P224).

The bioactive fragment of any FSTL1 albumen can be applied in the present invention.Here, the implication of the bioactive fragment of FSTL1 albumen refers to as a peptide species, and it still can keep all or part of function of the FSTL1 albumen of total length.Generally, described bioactive fragment at least keeps the activity of 50% total length FSTL1 albumen.Under preferred condition, described active fragment can keep 60%, 70%, 80%, 90%, 95%, 99% or 100% activity of total length FSTL1 albumen.

As optimal way of the present invention, the bioactive fragment of described FSTL1 albumen (in the present invention also referred to as " polypeptide of the present invention ") can include, but is not limited to: (1) has the polypeptide of the base acid sequence of 158-175 position in 156-173 position in SEQ ID NO:1 or SEQ ID NO:2; (2) protein of aminoacid sequence shown in SEQ ID NO:1 or SEQID NO:2, and wherein lack Progynon inhibin (Follistatin, FS) domain; (3) protein of aminoacid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, and wherein lack Von Willebrand type C-structure territory (VWC); (4) protein of aminoacid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, and wherein lack EF hand-type II domain; Or the protein of aminoacid sequence shown in (5) SEQ ID NO:1 or SEQ IDNO:2, and wherein lack the syndeton territory between EF hand-type I domain and EF hand-type II domain.Empirical tests, the above-mentioned bioactive fragment of FSTL1 albumen has retained the biological activity of total length FSTL1 albumen, or has more excellent biological activity than total length FSTL1 albumen.

In embodiments of the present invention, in SEQ ID NO:1, the aminoacid in aminoacid sequence number shown in SEQ ID NO:1 of take is benchmark, and described Progynon inhibin (FS) domain is 28-96 position, and VWC domain is 231-285 position; EF hand-type domain is 142-226 position, and wherein: EF hand-type I domain is 142-178 position, and EF hand-type syndeton territory is 179-190 position, EF hand-type II domain is 191-226 position.

In embodiments of the present invention, in SEQ ID NO:2, the aminoacid in aminoacid sequence number shown in SEQ ID NO:2 of take is benchmark, and described Progynon inhibin (FS) domain is 30-98 position, and VWC domain is 233-287 position; EF hand-type domain is 144-228 position, and wherein: EF hand-type I domain is 144-180 position, and EF hand-type syndeton territory is 181-192 position, EF hand-type II domain is 193-228 position.

In the FSTL1 sequence of SEQ ID NO:1 and SEQ ID NO:2, the distribution of each fragment and domain is as shown in table 2 below:

The distribution of each fragment and domain in table 2.FSTL1

^*it is benchmark that serial number in the 2nd row be take the aminoacid in aminoacid sequence number shown in SEQ ID NO:1

^*it is benchmark that serial number in the 3rd row be take the aminoacid in aminoacid sequence number shown in SEQ ID NO:2

The present invention also can adopt FSTL1 albumen modified or improvement, such as, can adopt the FSTL1 albumen of being modified or improveing in order to promote the effect of its half-life, effectiveness, metabolism and/or protein.Described can be a kind of conjugate of FSTL1 albumen through the FSTL1 albumen of modifying or improve, or it can comprise substituted or artificial aminoacid.Described can be to have less common ground with naturally occurring FSTL1 albumen through the FSTL1 albumen of modifying or improve, but also can suppress mammalian body sensation, and can not bring other harmful effect or toxicity.That is to say, any bioactive version that does not affect FSTL1 albumen all can be used in the present invention.

Once the separated sequence that has obtained described protein, just can obtain this protein in large quantity with recombination method.This is normally cloned into carrier by its encoding gene, then proceeds to cell, then by conventional method separation from the host cell propagation, obtains.In addition, for the protein compared with short, also can adopt the method for synthetic (as synthesized by Peptide synthesizer) to synthesize relevant sequence, the method for synthetic can obtain needed protein easy and rapidly.

The present invention has also comprised the separated nucleic acid of the bioactive fragment of the FSTL1 albumen that coding is described, can be also its complementary strand.The DNA sequence of the bioactive fragment of coding FSTL1 albumen, can complete sequence synthetic, and also the method for available pcr amplification obtains.After the DNA sequence of bioactive fragment that has obtained the described FSTL1 albumen of encoding, be connected into suitable expression vector, then proceeded to suitable host cell.Host cell after finally transforming by cultivation, obtains desired protein by separation and purification.

The present invention has also comprised the carrier of the nucleic acid molecules of the bioactive fragment that comprises the described FSTL1 albumen of encoding.Described carrier also can comprise the expression regulation sequence being connected with the series of operations of described nucleic acid molecules, so that protein expression.Described " operability is connected " or " being operationally connected in " refer to a kind of like this situation, and some part of linear DNA sequence can regulate or control the activity of same linear DNA sequence other parts.For example, if the transcribing of promoter control sequence, it is exactly to be operationally connected in coded sequence so.

The reconstitution cell of the bioactive fragment nucleotide sequence that in addition, contains the described FSTL1 albumen of encoding is also included within the present invention." host cell " comprises prokaryotic cell and eukaryotic cell.Conventional prokaryotic host cell comprises escherichia coli, bacillus subtilis etc.; For example can be Bacillus coli cells (E.coli), as escherichia coli HMS174 (DE3) or BL21 (DE3).Conventional eukaryotic host cell comprises yeast cells, insect cell and mammalian cell.

New discovery based on the inventor, the invention provides the purposes of FSTL1 albumen or its bioactive fragment, for the preparation of the compositions that suppresses mammalian body sensation; Or for screening the material that suppresses mammalian body sensation.

The inhibitor of FSTL1 albumen or its bioactive fragment

The inhibitor of FSTL1 albumen or its bioactive fragment is also provided in the present invention, these inhibitor can be used for the treatment of, alleviate or improve the disease relevant to this activity, for example renal hypertension, tumor (preferred melanoma, glioblastoma multiforme and nonsmall-cell lung cancer) by reducing the activity of na-k-atp enzyme.

The inhibitor of FSTL1 albumen or its bioactive fragment can be selected from: the antibody of anti-FSTL1 or its active fragment; Antisense oligonucleotide, siRNA for FSTL1 coded sequence; Derive from the peptide section of na-k-atp enzyme; Or their homologous sequence, include but not limited to: the M7M8 peptide section shown in the M3M4 peptide section shown in SEQ ID NOs:5 and 6, SEQ ID NOs:7 and 8.

Those of ordinary skills, after knowing the structure of FSTL1 albumen, its bioactive fragment or its coded sequence or obtaining described albumen, fragment or coded sequence, can obtain or screen described inhibitor by the conventional method in this area.

Compositions

The invention provides a kind of compositions, it contains effective dose (as 0.000001-50wt%; 0.00001-20wt% preferably; Better, described FSTL1 albumen 0.0001-10wt%) or its bioactive fragment or can activate or raise the molecule of FSTL1, and pharmaceutically acceptable carrier.

Compositions of the present invention can be directly used in and improve na-k-atp enzyme activity, to treat, to alleviate or to improve the disease relevant to this activity or symptom, for example, regulates mammalian body sensation.In addition, also can combine use with other therapeutic agent or adjuvant simultaneously.

Conventionally, these materials can be formulated in to nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 conventionally, preferably, pH is about 6-8.

As used herein, the composition of " pharmaceutically acceptable " is applicable to people and/or mammal and without excessive bad side reaction (as toxicity, stimulation and allergy), has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various excipient and diluent.

The FSTL1 albumen that compositions of the present invention contains safe and effective amount and pharmaceutically acceptable carrier.This class carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Conventionally pharmaceutical preparation should match with administering mode, and pharmaceutical composition of the present invention can be made into injection form, for example, with normal saline or contain glucose and the aqueous solution of other adjuvant is prepared by conventional method.Described pharmaceutical composition should be manufactured under aseptic condition.The dosage of active component is treatment effective dose.Pharmaceutical preparation of the present invention also can be made into slow releasing preparation.

The effective dose of FSTL1 albumen of the present invention or its bioactive fragment can change with order of severity of the pattern of administration and disease to be treated etc.The selection of preferred effective dose can be determined (for example, by clinical trial) according to various factors by those of ordinary skills.Described factor includes but not limited to: the pharmacokinetic parameter biological example utilization rate of described FSTL1 albumen or its bioactive fragment, metabolism, half-life etc.; The order of severity of the disease that patient will treat, patient's body weight, patient's immune state, the approach of administration etc.Conventionally, the dosage when FSTL1 albumen of the present invention or its bioactive fragment every day with about 0.00001-50mg/kg the weight of animals (preferably 0.0001-10mg/kg the weight of animals) gives, and can obtain gratifying effect.For example, an urgent demand by treatment situation, can give the dosage that several times separate every day, or dosage is reduced pari passu.

In addition, also provide a kind of compositions in the present invention, it contains effective dose (as 0.000001-50wt%; 0.00001-20wt% preferably; Better, the molecule of described FSTL1 albumen 0.0001-10wt%) or the inhibitor of its bioactive fragment or its coded sequence or downward FSTL1, and pharmaceutically acceptable carrier.Compositions of the present invention can be directly used in and lower na-k-atp enzyme activity, to treat, to alleviate or to improve the disease relevant to this activity or symptom, for example, improves the sensitivity of mammalian body sensation.In addition, also can combine use with other therapeutic agent or adjuvant simultaneously.

PH in the present composition or carrier etc. can be as described above, and can be selected with general knowledge as required by those of ordinary skills.

Conventionally, the dosage when inhibitor of the present invention every day with about 0.00001-50mg/kg the weight of animals (preferably 0.0001-10mg/kg the weight of animals) gives, and can obtain gratifying effect.For example, an urgent demand by treatment situation, can give the dosage that several times separate every day, or dosage is reduced pari passu.

Improve/reduce method and the administering mode of na-k-atp enzyme activity

The invention provides a kind of na-k-atp enzyme activity that improves with treatment, alleviation or the improvement disease relevant to this activity or the method for symptom, comprise FSTL1 albumen or its bioactive fragment that gives experimenter's effective dose or the molecule that can activate or raise FSTL1.When for regulating mammalian body when sensation, preferably adopt FSTL1 albumen or its bioactive fragment of restructuring.

FSTL1 albumen of the present invention or its bioactive fragment or the administering mode that can activate or raise the molecule of FSTL1 have no particular limits, can be whole body or local.Preferably, FSTL1 albumen of the present invention or its bioactive fragment can give animal by the mode of spinal cord intrathecal injection.

After the purposes of the FSTL1 albumen described in cicada, can adopt several different methods well known in the art that described FSTL1 albumen or its encoding gene or its pharmaceutical composition are delivered medicine to mammal.Preferably, can adopt the means of gene therapy to carry out, such as can be directly by FSTL1 albumen by delivering medicine to experimenter such as methods such as injections; Or, can the ceneme (such as expression vector or virus etc.) that carry FSTL1 gene be delivered on target spot by certain approach, and make it the FSTL1 albumen of expression activity.

As one embodiment of the present invention, described FSTL1 albumen directly can be delivered medicine to mammal (such as people), or, the gene of coding FSTL1 albumen can be cloned in suitable carrier (if conventional protokaryon or carrier for expression of eukaryon or viral vector are as herpesvirus vector or adenovirus vector) by conventional method, described carrier is imported in the cell that can express described FSTL1 albumen, make described cellular expression FSTL1 albumen.Can, by appropriate described cell being incorporated into the suitable position of body of mammals, realize the expression of FSTL1 albumen.

Method well-known to those having ordinary skill in the art can be for building containing the sequence of FSTL1 protein coding gene and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technique of body etc.Described DNA sequence can be effectively connected in the suitable promoter in expression vector, to instruct mRNA synthetic.Expression vector also comprises ribosome binding site and the transcription terminator that translation initiation is used.

Comprise above-mentioned suitable gene order and the suitable carrier of promoter or control sequence, can be for transforming suitable host cell, with can marking protein.Persons skilled in the art are all known the suitable carrier of How to choose, promoter, enhancer and host cell.

Recombiant protein in the above methods can or be secreted into extracellular at cell inner expression.If needed, can utilize the albumen of and purification of Recombinant separated by various separation methods with other characteristic its physics, chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation processes, with protein precipitant, process the combination of (salt analysis method), centrifugal, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, high performance liquid chroma-tography (HPLC) and other various liquid chromatography (LC) technology and these methods.

Aspect contrary, the present invention also provides a kind of and has reduced na-k-atp enzyme activity with treatment, alleviation or improve the disease relevant to this activity or the method for symptom, comprises the molecule that gives the FSTL1 albumen of experimenter's effective dose or the inhibitor of its bioactive fragment or its coded sequence or can lower FSTL1.The administering mode of these materials has no particular limits, can be whole body or local.Preferably, FSTL1 albumen of the present invention or its bioactive fragment can give animal by the mode of spinal cord intrathecal injection.

Screening regulates the method for the potential material of low na-k-atp enzyme activity

The FSTL1 albumen described in cicada for improving after the purposes of na-k-atp enzyme activity, can screen expression or the active material that regulates FSTL1 based on this feature.

Therefore, the invention provides a kind of method that screening can be used for regulating the potential material of na-k-atp enzyme activity, described method comprises: candidate substances is contacted with the system of expressing FSTL1; With the impact of detection candidate substances on FSTL1; If described candidate substances can improve expression or the active or secretion of FSTL1, show that this candidate substances is the potential material that can be used for improving na-k-atp enzyme activity; If described candidate substances can reduce expression or the active or secretion of FSTL1, show that this candidate substances is the potential material that can be used for reducing na-k-atp enzyme activity.

In optimal way of the present invention, when screening, in order to be easier to observe expression or the active change of FSTL1, also matched group can be set, described matched group can be the system of not adding the expression FSTL1 of described candidate substances.

Described system includes, but is not limited to: solution system, subcellular fraction system, cell system, organizational framework, organ systems or animal system.In described system, can contain FSTL1, for adding therein candidate substances, observe the impact of candidate substances on FSTL1; Or, in described system, can contain the upstream and downstream material of FSTL1 and the adjusting path relevant to na-k-atp enzyme simultaneously, for adding therein candidate substances, observe candidate substances for FSTL1 simultaneously and regulate the impact of path.

As optimal way of the present invention, described method also comprises: the potential material obtaining is carried out to further cell experiment and/or animal experiment, further to select and to determine for regulating the active real useful material of na-k-atp enzyme.

On the other hand, the present invention also provides the potential material that can be used for regulating na-k-atp enzyme activity that adopts described screening technique to obtain.The material that these Preliminary screening go out can form a screening storehouse so that people finally can therefrom filter out can be for the expression and the active real useful material that regulate FSTL1.

On the other hand, the present invention also provides a kind of method of screening treatment or lenitive potential material, it is characterized in that, described method comprises: (1) contacts candidate substances with the system of na-k-atp enzyme; (2) detect the impact of candidate substances on na-k-atp enzyme; If described candidate substances can improve the activity of na-k-atp enzyme, show that this candidate substances is to can be used for treatment or lenitive potential material.

Major advantage of the present invention is:

(1) the present invention has disclosed the relation between FSTL1 and na-k-atp enzyme activity first, thereby provides a kind of by regulating na-k-atp enzyme active, thereby is used for the treatment of, alleviates or improve new method and the compositions of the disease relevant to this activity or symptom;

(2) the invention provides a kind of brand-new sensory information and transmit regulatory mechanism, having proved first FSTL1 has regulating action to sensory information transmittance process, thereby provides one to regulate somesthetic new way.

(3) screening for treatment pain medication provides good approach.Can using na-k-atp enzyme activity as drug screening target spot, the medicine of screening treatment pain.Also can FSTL1 as drug screening target spot, thereby the expression material of screening and activating FSTL1 is for the preparation of the medicine for the treatment of pain.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition as people such as Sambrook, molecular cloning: lab guide (New York, publishing house of cold spring harbor laboratory, New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and umber calculate by weight.

I. materials and methods

dRG sample preparation

Behind get respectively embryo 14 days, birth, (P) 0,7,14 and 28 day, adult SD rats are (purchased from Chinese Academy of Sciences's Shanghai Experimental Animal Center, male), after anesthesia, put to death, get waist 4 and waist 5 dorsal root ganglion, use dry ice quick-freezing, for RNA blotting and immunoblotting experiment.

immunohistochemical assay

Get respectively embryo 12 and 15 days SD rats, with 0.2% picric 4% paraformaldehyde (37 ℃, with the preparation of 0.1mol/L phosphate buffer, pH 6.9) fixative, soak 12 hours, directly carry out frozen section.Get adult SD rats, after anesthesia, by aorta, first pour into normal saline (37 ℃) 50ml, and then pour into fixative 50ml, then pour into immediately the fixative 200ml of 4 ℃ of pre-coolings, continue 5 minutes.The amount of liquid of the rat perfusion of P14 days reduces to 1/5 amount.Take out waist 4 and waist 5 spinal cords, in same fixative, after 4 ℃, fix 90 minutes, then containing in the phosphate buffer of 10% sucrose, soaking 24 hours.Cut 14 μ m frozen sections.

With primary antibodie diluent (containing the PBS of 1%BSA and 0.3%Triton X-100) dilution first antibody, at 4 ℃, place 16-24 hour.After washing with 0.01mol/LPBS, add by the fluorescently-labeled second antibody of PBS dilution (1: 100; Jackson ImmunoResearch), put into 37 ℃ of incubators and hatch 30 minutes, wash rear mounting.Section gathers pictorial data with laser confocal microscope (Leica SP2).Every rat of statistical data is got 3 sections, every group of 3 rats.

the production of FSTL1-Myc/his fusion rotein and purification

(sequence is referring to GenBank accession number: NM_024369) insert in the Xho I/Hind III restriction enzyme site of pcDNA3.1Myc/his plasmid (Invitrogen) by rat and mankind FSTL1 complete encoding sequence for the inventor, by calcium phosphate transfection method, this recombiant plasmid is proceeded in 293T cell (ATCC) again, DMEM complete medium was hatched after 12 hours, change ISCOVE culture medium (Invitrogen) into, supernatant is collected once for every 36 hours, and concentrates and purification by HisBind method of purification (Qiagen).

Rat Fstl1cDNA is subcloned in pcDNA3.1myc/His plasmid.Zones of different by pcr amplification FSTL1 builds FSTL1 ^{Δ EF}, and be cloned in pcDNA3.1myc/His plasmid, the primer sequence is as follows:

FSTL1 ^1-141: 5 '-ATGTGGAAACGCTGGCTGGC-3 ' (forward primer, SEQ ID NO:9)

5 '-ACTGCCTTTAGAGAACCAGC-3 ' (reverse primer, SEQ ID NO:10);

FSTL1 ^249-306: 5 '-TGCGCCCTGGAGGACGAAAC-3 ' (forward primer, SEQ ID NO:11)

5 '-ATCTCTTTGGTGTTCACCT-3 ' (reverse primer, SEQ ID NO:12)

FSTL1 ^e165Astructure adopted the method for two-wheeled PCR.First round PCR is with the zones of different of following primer amplification FSTL1:

5 '-ATGTGGAAACGCTGGCTGGC-3 ' (forward primer, SEQ ID NO:13)

5 '-ATTTCAGGAATGCGCTGGAGTCCAGGTGAGAGT-3 ' (reverse primer, SEQID NO:14);

5 '-GGACTCCAGCGCATTCCTGAAATTCGTGGAGCA-3 ' (forward primer, SEQID NO:15)

5 '-ATCTCTTTGGTGTTCACCT-3 ' (reverse primer, SEQ ID NO:16).

Separated PCR product carry out purification in 1% agarose gel, then mixes the template of taking turns PCR as second.The full-length cDNA of FSTL1 point mutation increases with following primer, and is cloned in pcDNA3.1myc/His plasmid.

5 '-ATGTGGAAACGCTGGCTGGC-3 ' (forward primer, SEQ ID NO:17)

5 '-ATCTCTTTGGTGTTCACCT-3 ' (reverse primer, SEQ ID NO:18).

antibody preparation

By 31 aminoacid of rat and people FSTL1C end and complete encoding sequence, (sequence is referring to GenBank accession number: NM_024369) insert pGEX-KG plasmid, then by GST, is expressed with purification and obtained corresponding protein.By after these two kinds of protein (0.5mg) and complete Freund's adjuvant mixing, be expelled to the subcutaneous of rabbit or mice, after three weeks, again inject albumen (0.25mg) and complete Freund's adjuvant mixed liquor; After fortnight, again carry out immunity.Then the serum of getting corresponding rabbit and mice is prepared into the FSTL1 antibody in rabbit source and mice source.

neuron is cultivated

Get SD rat anesthesia and put to death rear exposing spinal cord and neuroganglion, with eye scissors, fast the nerve of neuroganglion both sides is cut off, take off neuroganglion and be placed in DMEM (Gibco) culture medium that contains digestive enzyme (collagenase 1.0mg/ml, pancreatin 0.4mg/ml and DNA enzyme 0.1mg/ml), put 37 ℃ of water-bath digestion 30 minutes.For the DRG neuron of acute isolation, with the moderate Pasteur's pipe of thickness, the culture medium that contains digestive enzyme is drawn, and with extracellular fluid (ECS:150mM NaCl, 5mM KCl, 1mMMgCl ₂, 2.5mM CaCl ₂, 10mM HEPES and 10mM D-Glucose) clean after neuroganglion 2-3 time, blow and beat neuroganglion gently until individual cells is broken up out, the cell kind of breaing up is adherent on coverslip, and in extracellular fluid, cultivation can be put 8-10h room temperature or 4 ℃.Neuron to for cultivating, cleans digestive enzyme by culture medium, by the cell culture after breaing up DMEM+10%FBS (Gibco) complete medium 48 hours.

cOS-7 cell culture and transfection

The inventor by rat α 1NKA (sequence is referring to GenBank accession number: NM_012504) and α 3NKA (NM_012506) complete encoding sequence insert pIRES-EGFP2 (Clonteck), β 1NKA (NM_013113) complete encoding sequence is inserted to pcDNA3.1 (-)-A plasmid (Invitrogen).By liposome transfection method α 1NKA and β 1NKA, α 3NKA and β 1NKA recombiant plasmid respectively corotation enter in COS-7 cell (ATCC), DMEM complete medium was hatched after 36-48 hour, for NKA enzymatic activity test kit (Bioengineering Research Institute is built up in Nanjing), detected NKA enzymatic activity.

fSTL1 secretion experiment after neuron immunoblotting stimulates

The DRG neuron that (P) rat separation of 14 days obtains from being born is being cultivated 48 hours containing in the DMEM of 10% serum (FBS), the DMEM changing into afterwards containing 1% serum cultivates 4 hours again, in fresh culture, hatch after 2 hours, use respectively 55mM KCl and 1 μ M capsaicin (Sigma) to process neuron 1 hour.Obtain respectively the culture medium of processed group and matched group, with 10% trichloroacetic acid, 4 ℃ of methods of placing 10 minutes, precipitate the protein in culture medium, centrifugal 10 minutes of 15,000g, 100 μ l SDS sample buffers dissolvings for precipitation, and carry out boiling water bath 5 minutes.

The neuron lysate cracking of cultivating.Organizing also of rat and mice done same processing at 4 ℃.After sample treatment, carry out SDS-PAGE electrophoresis, then be transferred on poly difluoride membranes, 1 hour (room temperature) of TBST buffer sealing containing 5% skim milk and 0.1%Tween20 for film.Then use on demand respectively the anti-FSTL1 antibody of rabbit (Rb) (1: 4000; The gst fusion protein immune rabbit gained serum of described in " materials and methods " part, 31 aminoacid of the C-terminal of rat FSTL1 being made), the anti-actin antibody of mice is (1: 50000; Chemicon) and the anti-Myc antibody of mice (1: 4000; Chemicon), under 4 ℃ of conditions, hybridize and spend the night.With the corresponding two anti-hybridization that are connected with horseradish peroxidase, and with after TBST rinsing, by ECL developing liquid developing, exposed.

With ImageQuant 5.1, measure the ECL signal intensity of protein hybridization, the ratio of FSTL1 in the interior and full cell pyrolysis liquid of culture medium after computing, the ratio in calculating culture medium between FSTL1 and actin intensity.To test corresponding matched group at every turn, carry out standardization.

calcium record by imaging

Application fura-2 dyestuff is (to calcium sensitive fluorescent dye, Sigma) DRG cell in culture dish being carried out to lucifuge hatches 30 minutes, then take out the cell of hatching and be positioned on the object stage of fluorescence inverted microscope (OlympusIX50), carry out calcium imaging.Excite light wavelength to be respectively 340nm and 380nm, obtain respectively radiative fluorescence intensity, calculate the relative value that its ratio obtains calcium concentration.More than test on TILLvisION imaging system v3.3 (T.I.L.L. company, Germany) and complete.

spinal Cord Slices is made and record

By animal (the urethane lumbar injection of anesthesia, 1.2g/Kg) row laminectomy, expose lumbosacral enlargement place spinal cord, take out lumbosacral enlargement place spinal cord and be positioned in the frozen water mixed liquor of artificial cerebrospinal fluid, after retaining a side L5, root, before and after other, root all excises, peel off the dura mater, arachnoidea and the mantle that cover spinal cord surface, vibratome cuts the Spinal Cord Slices with root after a L5, and the thick 600-650 micron of sheet, is positioned over and is connected with 95%O ₂and 5%CO ₂artificial cerebrospinal fluid perfusion system in hatch, after 1 hour, start record.

the structure of Fstl1 conditional knock-out mice

The principle that in application P1 antibiotics, the specific Cre recombinase in position can cut the gene segment between two LoxP sites of restructuring, utilize pNeotkLoxP carrier (Nanjing University's model animal institute), build the conditionality targeting vector of Fstl1, in targeting vector, had three LoxP sites.

The exon 2 of Fstl1 (F2 includes signal peptide) is between a LoxP site, and the positive and negative sign (neo, tk) screening at embryonic stem cell for the later stage is between another LoxP site.Not I is linearizing restriction enzyme site.Subsequently linearizing targeting vector electricity is proceeded to embryonic stem cell, then the plasmid with Cre is proceeded to above-mentioned embryonic stem cell, targeting vector can be cut into three kinds of situations, select the needed conditionality targeting vector of the inventor, then the embryonic stem cell that contains this conditionality targeting vector is expelled to and is subject in stomatoblastula, be then transplanted in the female Mus body of pseudo-fetus and develop into individuality.In this Mice Body, there is the cell (cell of blastaea self and the embryonic stem cell of injection) of two class separate sources, thereby be called as chimera.Due to the totipotency of embryonic stem cell, it is expelled to after a segmentation cavity, it can bud into each organ-tissue of health together with this blastaea, comprises sexual cell.If embryonic stem cell develops into sexual cell, the genetic modification obtaining by homologous recombination can be imported in chimeric germ cell line and can pass to the next generation and will obtain the heterozygote mice that contains conditionality targeting vector, then with heterozygote mice pairing breeding, just can obtain the homozygote mice that contains conditionality targeting vector.Apply subsequently Na _v(Cre recombinase is specific expressed at Na for 1.8-Cre mice _vin the Primary Sensory Neuron of 1.8 positives, on minicell, Heidelberg, Germany university provides) breed with the mice pairing that contains conditionality targeting vector, thereby obtain specificity, knock out Na in Primary Sensory Neuron _vthe mice that on 1.8 positive cells, FSTL1 expresses.

behavioristics is observed

Coordination exercise test (Rota-rod test)

Animal is placed on the runner of Rota-rod Treadmill motion instrument uniform rotation (12 turn/min) and adapts to after 30s, make runner accelerate (acceleration is that 5min interior 3 to 30 turns), record rat from starting to accelerate to the time of falling runner, as the index of the reflection rat motor coordination ability.

Thermostimulation is measured incubation period

Animal is placed in to the transparent organic glass case that a bottom is the 20cm * 20cm * 25cm of 2mm plate glass, and be positioned over higher than on the shelf of laboratory table 30cm so that observe.Application thermostimulation instrument, select 100W halogen delineascope, regulation voltage is to 10V, regulate the distance between lamp source and glass plate, the irradiation diaphragm diameter that makes to drop on vola is 5mm, and record occurs the contracting sufficient withdrawal reflex time (s) from starting to expose to, this is the thermostimulation response latency, repeated measure 5 times, same position interval 10min, different parts interval 5min.Remove measured value for the first time, get rear 4 measured values and average, as the thermostimulation response latency and as quantitative target.As be greater than reactionless the stopping of 30s and irradiate, in order to avoid cause vola excessive tissue hot injury.

Mechanical stimulus threshold measurement

With the nylon yarn of different thicknesses and different length, make mechanical stimulus device (von-Frey) fiber of different stimulated intensity.It is on the shelf that the 30cm of wire gauze is high that the transparent organic glass case of one 20cm * 20cm * 25cm is positioned over to top, and tested animal is placed in to case.The hand-held von-Frey fiber of experimenter is through iron wire grid difference centre, vola, stimulation in rats both sides, stimulus intensity is ascending, each intensity repetitious stimulation 10 times (inferior-minor tick 3～5s), will occur that 5 above intensity of contracting foot reflex are decided to be the threshold of reaction of animal to mechanical stimulus.As there is not contracting foot reflex with 588mN von-Frey fiber stimulation in rats vola, think the painless reaction of rat, mechanical stimulus threshold value < 58.8mN is all in 58.8mN.This is a kind of widely used mechanical stimulus quantitative approach, because its most advanced and sophisticated pressure is constant after nylon fiber is obstructed bending, so can think that each measured value is constant.

statistical procedures

This is tested all data and represents with mean+SD (mean ± SEM), application SPSS software kit carries out non-ginseng (Mann-Whitney U test) check to random distribution sample, paired sample is carried out to variance analysis (ANOVA followed by posthoc Fisher ' s PLSD or Sheffe test) and t check, and p < 0.05 is considered to have remarkable statistical significance.

The checking of embodiment 1.FSTL1 selectivity high expressed in the DRG small neuron of growing up

For the protein of determining that specificity is expressed at DRG, the inventor searches for high expressed in DRG in gene bank, and in its hetero-organization, expresses relatively low gene.The inventor found 71 (candidate gene, and by self-control genechip detection they express spectras in adult rat tissue.Wherein 19 gene Selection ground high expresseds (expression in DRG is than high more than 3 times or 3 times at the average expression of its hetero-organization) in DRG, comprise that the gene of expection is as CGRP, P2X ₃receptor and Na _v1.8 passage.In several genes that specific expressed degree is the highest in the adult rat DRG detecting, FSTL1 is also one of them (Figure 1A).

Polynucleotide gene chip result shows that Fstl1 mainly expresses at the DRG of adult rat, organizes level to exceed approximately 8 times than other.And Fstl1 also has high expressed in heart, lung, kidney and the spinal cord of rat.By RNA blotting (Fig. 1 K), the Fstl1mRNA in each tissue is detected to further proof, FSTL1 expression in heart, lung, kidney is higher.

The immunoblot experiment (Fig. 1 C) of RNA blotting (Figure 1B) and use FSTL1 specific antibody detects further proof, and FSTL1 is specificity overexpression in adult rat DRG, also has expressed in spinal cord.It should be noted that in embryo development procedure, FSTL1 also has expression in Hippocampus and cortex, but the expression in these brain districts disappear (Figure 1A and 1D) after birth.On the contrary, FSTL1 appears in the time of 15 days embryo in DRG neuron, in birth, within latter 28 days, peaks and maintains its high expressed to grow up (Fig. 1 D).

In adult rat DRG, FSTL1 mainly expresses (Fig. 1 D, 1H) in small neuron.DRG small neuron comprises containing neuropeptide as the peptidergic neuron of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 and CGRP and non-peptidergic neuron IB4 (isolectin B4) neuron.Immunofluorescence double labelling shows that the positive small neuron of approximately 46% FSTL1 expresses Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2, and have an appointment, 54% locates altogether with CGRP, and 45% the FSTL1 positive cell of having an appointment is in addition simultaneously by IB4 labelling (Fig. 1 E, 1G and 1I).In addition, FSTL1 also with label TRPV1 passage, P2X3 receptor and the Na of DRG small neuron cell _vthe coexpressions such as 1.8 channel proteins (Fig. 1 F, 1G and 1J).At rat spinal cord, FSTL1 is present in I to II layer (Fig. 1 E), the region that the positive centripetal fiber of most of Toplink centripetal fibers and IB4 exists just.

As can be seen here, FSTL1 has expression in the small neuron cell space of Toplink and the IB4 positive, and can in its centripetal fiber tip, find.

Embodiment 2.FSTL1 is transported and is secreted by special clear vesicle

The inventor has detected the Subcellular Localization of FSTL1 in cell, finds that FSTL1 is stored by a kind of secretion vesicle of uniqueness.There is synaptic vesicle and large densified core bubble (LDCV) in centripetal fiber tip, when having to stimulate, causes that cellular calcium can promote these vesicles to secrete its content while raising.After exocytosis, synaptic vesicle is in the recyclable recycling of Nerve Terminals In The Human Skin, and large densified core bubble need be come to be supplemented by cell space transportation.Immunofluorescence double labelling shows, FSTL1 be associated with non-peptide class vesicle (Fig. 2 A, 1I) in DRG small neuron cell cell space.After embedding, immuno-electron microscope gold labelling shows, FSTL1 is arranged near the little clear vesicle (diameter 30-80nm) (Fig. 2 B) golgi's network outside.

The inventor has also detected the distribution of FSTL1 at centripetal fiber tip.With the method separation of the separated subcellular components of sucrose density gradient centrifugation, obtain the different vesicle groups of Rat Spinal Dorsal, discovery FSTL1 mainly appears at and contains synaptoporin (synaptoporin, Synpr) in vesicles group, rather than in containing the LDCV component of CGRP (Fig. 2 C).Synaptoporin is a member of synaptophysin (synaptophysin) superfamily, is considered to spinal cord I to the ultimate constituent of II layer afferent nerve fiber tip synaptic vesicle film.Immunofluorescence double labelling shows that most FSTL1 positive particles are all (Fig. 2 A) of synaptoporin feminine gender, and prompting FSTL1 is mainly present in vesicles subgroup another kind of and that synaptoporin is different.Electronic Speculum immuno-gold labeling shows, FSTL1 is present in the little clear vesicle (Fig. 2 D) of centripetal fiber tip of rear centripetal fiber and cornu dorsale medullae spinalis I, II layer.What is interesting is, the vesicle that contains FSTL1 also has distribution (Fig. 2 D) in the active region of presynaptic membrane.In sum, FSTL1 is synthetic at cell space, is assembled into little clear vesicle, is then transported to import end into and slightly carry out presynaptic secretion.

With immune labeled comparing stronger in DRG pericaryon, what FSTL1 will be weak in the dyeing of rat spinal cord centripetal fiber is many, and it is positive in mouse spinal cord, substantially to can't detect immunity.But, after ligation root 24 hours with blocking-up vesicle forward transportation can cause centripetal fiber near gather (Fig. 2 E) of DRG one end FSTL1, show that FSTL1 exists forward transportation.Therefore, at spinal cord centripetal fiber tip, less FSTL1 being detected may be because it is by due to rapidly secretion discharges.

The inventor uses the secretion of the rat drg neuron direct-detection FSTL1 cultivating.Immunoblot experiment shows, under non-stimulated condition, in cell culture fluid, the horizontal presentative time dependency of FSTL1 raises (Fig. 2 F, G), shows that DRG small neuron exists the Autocrine of FSTL1.In addition, the high potassium of 55mM stimulates can promote FSTL1 secretion, and in the situation that acellular outer calcium exists, the effect of high potassium is eliminated (Fig. 2 G).

With TRPV1 channel agonist capsaicin (Cap), process and can significantly increase the FSTL1 level in culture fluid, this reaction can be by the blocker capsaizepine (CPZ, Sigma) of TRPV1 passage blocking-up (Fig. 2 J).The high potassium of 55mM stimulates also can promote spinal cord of adult rats thin slice and the secretion (Fig. 2 H, I) of FSTL1 in Rat Spinal Dorsal synaptic glomerulus.It should be noted that FSTL1 can be divided into and secrete with CGRP and glutamic acid, stimulate the Rat Spinal Dorsal synaptic glomerulus FSTL1 secretion causing that (Fig. 2 I) only occurs under the condition of extracellular Ca2＋ existence.

Therefore, FSTL1 is stored in little clear vesicle transportation, and discharges at afferent nerve endings in the mode similar to other neurotransmitter.

Embodiment 3.FSTL1 suppresses the synapse of sensation nervus centripetalis and transmits

The inventor studies the synapse whether FSTL1 can regulate and control nervus centripetalis and transmits by carrying out full cell record on the Spinal Cord Slices at adult rat band Dorsal root.

At the dirty 293T of human embryo kidney (HEK293T) transit cell, dyed the plasmid that can express with the rat FSTL1 of Myc and His segment, and the FSTl1 albumen of its synthesis secretion of purification (Fig. 3 E), the structure of expressed FSTL1 is shown in Fig. 3 A.

In the spinal cord II layer neuron of about 50% (16/31 neuron) recording, perfusion restructuring FSTL1 albumen (60nM or 300nM), the minimizing that the frequency of spontaneous excitatory postsynaptic current (sEPSCs) and amplitude are dose dependent (Fig. 3 B).In addition, in about 60% (14/24) spinal cord II layer neuron of record, restructuring FSTL1 albumen can reduce excitatory postsynaptic current (eEPSCs) amplitude (Fig. 3 C) that C fiber brings out.At Fugu ocellatus toxin (TTX, while 0.5 μ M) existing, record miniature EPSCs (mEPSCs), find that FSTL1 albumen can suppress the frequency of mEPSCs, but to its amplitude without remarkable effect (Fig. 3 D), prompting FSTL1 discharges and has presynaptic inhibitory action glutamic acid.FSTL1 does not make significant difference to the decaying kinetics characteristic of sEPSCs and eEPSCs, shows that FSTL1 does not have direct effect (Fig. 3 B, C) to the character of postsynaptic membrane glutamate receptor passage.

The inventor has further checked the function (Fig. 3 E) of FSTL1 with the mutant of multiple FSTL1 albumen, FSTL1 mutant protein (structure is shown in Fig. 3 A, E) comprising calcium binding site (M2) or a pair of EF hand-type structure disappearance loss of function (M1).Inventor's discovery, the recombiant protein of these FSTL1 sudden changes has lost the inhibitory action (Fig. 3 C) to eEPSC.In addition, the people of purification recombinates Progynon inhibin (Follistatin) to centripetal fiber transmission nothing effect (Fig. 3 F).In sum, external source gives FSTL1 has special inhibitory action to the synapse transmission of spinal cord centripetal fiber.

For whether the further FSTL1 of Study on Endogenous transmits and also produces inhibitory action the synapse of centripetal fiber, the inventor adopt conventional method preparation purification the antibody of FSTL1 with the FSTL1 of sealing Spinal Cord Slices self secretion.Experiment is found, perfusion FSTL1 antibody (1: 1000) can increase the frequency of sEPSC and amplitude (Fig. 3 B of amplitude and eEPSC, C), and the not effect of the preimmune serum of contrast, the FSTL1 of prompting endogenous excretion in normal physiological situation has inhibitory action to importing synapse transmission into.

Embodiment 4.Fstl1 conditional knockout mice somesthesia sensitivity increases

The synapse transmission of cornu dorsale medullae spinalis nervus centripetalis is had after inhibitory action observing FSTL1, the inventor analyzes FSTL1 may participate in regulating normal somesthesia function.The whole body variation causing for avoiding gene entirely to knock out, inventor's application conditions formula gene knockout method, the FSTL1 in knock-out mice DRG small neuron observes its effect in elementary sensory information conduction specifically.Because express, in the DRG small neuron of FSTL1, have 96.5% and Na _v1.8 locate altogether, so take loxP as basic Fstl1 conditionality allele (Fstl1 with having ^f) mice and the Na of (Fig. 4 A) _v1.8-Cre BAC transgenic mice strain (SNS-Cre) hybridization, can obtain Fstl1 conditional knock out mice (Agarwal etc., Genesis.2004,38:122-129) Fstl1 ^f/F: SNS-Cre (Fstl1 ^-/-).

Fstl1 ^f/F: SNS-Cre (Fstl1 ^-/-) mice activeness is good, has fertility, do not occur significantly deformity, is not only the expression (Fig. 4 B, C) that can't detect FSTL1 in DRG neuron.These Fstl1 ^-/-the DRG neuronal quantity of mice and the anatomical structure of size distribution and spinal cord and wild-type mice (Fstl1 ^{+ /+}) similar.And Fstl1 ^-/-na in the spinal cord of mice and DRG neuron _v1.8 passages, P2X3 receptor, TRPV1 passage, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 and other main ion channel, receptor, neuropeptide and signal transducers are expressed significant change (Fig. 4 C) are not all occurred.But, at Spinal Cord Slices, the neuronic full cell record of the II layer of FSTL1 sensitivity is shown, with Fstl1 ^{+ /+}mice is compared, Fstl1 ^-/-the increase of the sEPSC frequency that mice 15mM KCl causes is (Fig. 5 A) significantly, and showing to stimulate the FSTL1 causing to discharge (Fig. 2 G, H) is necessary for suppressing strong synapse transmission.

The impact of body sensation being transmitted for detecting FSTL1 disappearance, the inventor carries out recording outward in somatic cell (according to Li and Chen etc. to wide power territory (WDR) neuron of mouse spinal cord relief angle, Neuroscience, 126 (2001): the method for recording in 753-762 records), these neurons and Primary Sensory Neuron have synaptic contact, and temperature and mechanical stimulus are all responded.With temperature and mechanical stimulus, stimulate the neuronic receptive field of WDR can increase the granting of these neuron action potentials.What is interesting is, the inventor finds and Fstl1 ^{+ /+}mice is compared, Fstl1 ^-/-the WDR neuron action potential granting of mouse spinal cord is stronger, and same temperature and mechanical stimulus meeting cause that electric discharge increases (Fig. 5 B).At Fstl1 ^-/-in mice, the WDR cell discharge frequency (38 ℃, 7.0 ± 1.5Hz) that non-noxious stimulation causes and compressing stimulate (15.4 ± 1.8Hz) and noxious stimulation at Fstl1 ^{+ /+}to swash (16.3 ± 3.9Hz) similar with pinching piercing for the discharge frequency of mice WDR cell induction (45 ℃, 7.9 ± 1.0Hz).In addition, at Fstl1 ^-/-mouse spinal cord intrathecal injection FSTL1 can change this reaction completely, makes Fstl1 ^-/-the neuronic discharge frequency of mice WDR fades to similar to wild-type mice (Fig. 5 B), proves Fstl1 ^-/-the increase of mice irritability is the direct result of FSTL1 disappearance, rather than due to developmental defect or other reason.These data show, it is that to maintain spinal cord sensation loop physiological equilibrium necessary that DRG small neuron is expressed FSTL1.

The inventor is further tested and is detected Fstl1 by behavioristics ^-/-the pain reaction of mice, to understand the functional meaning of FSTL1 in DRG neuron in body situation.The inventor finds, Fstl1 in heat radiation experiment ^-/-mice significantly shortens (Fig. 5 C) to the response time of thermostimulation, prompting Fstl1 ^-/-mice is improved the sensitivity of nocuity thermostimulation.In addition, mechanical stimulus device von-Frey fiber shows the mechanical irritation experiment of the mice rear foot, Fstl1 ^-/-mice also significantly reduces (Fig. 5 C) to the threshold value of mechanical stimulus.Change in these behavioristicss is not because Fstl1 ^-/-there is motor function defect in mice, because the reaction to wild-type mice similar (Fig. 5 D) of these mices in accelerating rotation test (accelerating rotarod test).In addition, to mice rear foot injection of formalin induction inflammatory pain model, Fstl1 ^-/-mice also has the pain sensation reaction of enhancing, shows that its time of licking the rear foot of II phase phase of formalin inflammatory reaction significantly increases (Fig. 5 E).In sum, FSTL1 comprises that to normal somesthesia the pain sensation is essential, and has effect in acute inflammation pain, and FSTL1 disappearance can cause Hyperalgesia.

Behavioristics tests and shows, at Normal Adult Rat intrathecal injection FSTL1, rat does not significantly change the reaction of nocuity thermostimulation.Yet, give Fstl1 ^-/-but can fully overturn its sensitivity to thermostimulation of mice intrathecal injection FSTL1 increases (Fig. 5 F).In addition, injection FSTL1 antibody (method preparation described in " materials and methods " part) also can increase rat to the reaction of heat injury sexual stimulus (Fig. 5 G).In sum, the FSTL1 of FSTL1 down-regulated expression or functional blocking-up endogenous excretion all can cause Hyperalgesia.

Embodiment 5.FSTL1 suppresses the chronic pain that nerve injury causes

Proved FSTL1 that sensory nerve ending discharges for maintain normal somesthesia essential after, the inventor thinks further to explore its may act in neuropathic pain (being mostly because nerve injury causes).The pathophysiology feature of neuropathic pain is the hyperexcitability of spinal cord sensation loop.The inventor finds that DRG neuron FSTL1 expresses minimizing after peripheral nerve injury, the small neuron number that contains Fstl1 mRNA in In Rat Lumbar 4-5 sections DRG after sciatic nerve cuts off significantly reduces (Fig. 6 A), and cause the minimizing of FSTL1 albumen in centripetal fiber in DRG small neuron cell space and cornu dorsale medullae spinalis (Fig. 6 B, Fig. 6 C).The discovery of immunofluorescence double-label experiment, its FSTL1 of cell that expresses transcriptional factors 3 (label of DRG neuron axon damage) significantly reduces, and shows that FSTL1 expresses minimizing and directly occurs in the neuron of damaged.

The neurogenic pain model being caused by sciatic nerve branch cut-out (SNI) at another kind is (according to Decosterd; I.; and Woolf; C.J. (2000); the method of recording in Spared nerve injury:an animal model ofpersistent peripheral neuropathic pain.Pain 87:149-158 is set up this model) in there is equally the downward (Fig. 6 D) of DRG neuron FSTL1, and intrathecal injection FSTL1 albumen is the elimination of dose dependent and by sciatic nerve branch, is cut off the abnormal mechanical pain (Fig. 6 E) causing.In sum, after nerve injury, the remarkable minimizing of FSTL1 can be imported by irritability the mechanism of the disinthibiting participation formation neuropathic pain of transmission into.

Embodiment 6.FSTL1 is in conjunction with α 1 sodium-potassium ATP enzyme (α 1NKA)

The inventor further carrys out to determine the receptor of FSTL1 effect.With the FSTL1-myc of restructuring, hatch the Neurons of The Rat Dorsal Root Ganglion of cultivation, added cross-linking agent (Bis (sulfosuccinimidyl) suberate, the BS of impermeable membrane simultaneously ³), with myc antibody test to FSTL1 at the nearly visible band in 150kD place (Fig. 7 A), and FSTL1-myc is positioned at 37-45kD place (Fig. 7 A) while not adding cross-linking agent, points out the protein binding of FSTL1-myc and an about 100kD.

In order to determine this albumen, to BS ³proteins after crosslinked carries out mass spectral analysis, has the peptide section sequence of FSTL1 in analyzed albumen, 3-7 peptide section of α 1 sodium-potassium ATP enzyme (α 1NKA) can be detected simultaneously, and 15% of α 1NKA aminoacid sequence has been contained in the order-checking of 3 samples.Simultaneously, at the same sample of processing, by the monoclonal antibody of α 1NKA, detect, visible part α 1NKA molecular weight is from～be increased to～145kD of 110kD (Fig. 7 B), in same sample, with the single band (Fig. 7 B) at only detect～110kD of α 3NKA antibody place, illustrate that FSTL1 is not combined with α 3NKA.

Immunohistochemical staining is presented in Dorsal Root Ganglion Neurons 51% express alpha 1NKA, and wherein 63% is small neuron, and the 36%th, large neuron (Fig. 7 C), has 66% containing FSTL1 (Fig. 7 C) in the cell of the α 1NKA positive.Co-immunoprecipitation experiment demonstration, in dorsal root ganglion of rats, endogenic FSTL1 can be in conjunction with α 1NKA (Fig. 7 D).The α 1NKA subunit of corotation zone flag label and FSTL1-myc or FSTL1 (M1)-myc in COS7 cell, in albumen under the co-precipitation of flag antibody mediated immunity, FSTL1-myc can be detected, but FSTL1 (M1)-myc (Fig. 7 E) can not be detected.

Scatchard analysis (Current Protocols in Protein Science (2000) A.3H.1-A.3H.55) shows ¹²⁵i-FSTL1 dose dependent ground is in conjunction with the COS7 cell (Kd=43nM) (Fig. 7 F) of coexpression α 1NKA subunit and β 1 subunit.Therefore, FSTL1 can be in conjunction with α 1NKA.

The inventor, in order to determine in α 1NKA subunit and the interactional critical sites of FSTL1, compares born of the same parents' outer shroud of α 1NKA subunit and α 3NKA subunit, and having found has different aminoacid (Fig. 7 G) at second and the 4th born of the same parents' outer shroud.The sequence alignment of Fig. 7 G shows that the 314th (E) in α 1NKA subunit may be relevant with the effect of FSTL1 with the 889th (T) aminoacid.In expressing the COS7 cell of FSTL1-myc and α 1NKA subunit, co-immunoprecipitation experiment is presented in the albumen that the co-precipitation of flag antibody mediated immunity gets off and has FSTL1-myc, at coexpression FSTL1-myc and α 1NKA subunit ^e314G-flag or α 1NKA subunit ^t889N-flag or α 1NKA subunit ^{e314G, T889N}in the cell of-flag, the FSTL1-myc existing in the albumen that the co-precipitation of flag antibody mediated immunity is got off significantly declines, and proves that the 314th (E) in α 1NKA subunit is really relevant with the effect of FSTL1 with the 889th (T) aminoacid.Meanwhile, FSTL1-myc is not combined with α 3NKA subunit, but with α 3NKA subunit ^{g304E, N879T}-flag, in conjunction with (Fig. 7 G), further proves the 314th (E) and the importance of the 889th (T) aminoacid in α 1NKA and FSTL1 combination in α 1NKA subunit.Therefore, in α 1NKA subunit in second born of the same parents' outer shroud M3M4 in the 314th amino acids E and the 4th born of the same parents' outer shroud M7M8 the 889th amino acids T be the pivotal role site of α 1NKA subunit and FSTL1.

Embodiment 7.FSTL1 activates α 1 sodium-potassium ATP enzyme (α 1NKA)

NKA is a kind of memebrane protein that cell membrane potential is changed by inhomogeneous transhipment sodium ion and potassium ion.The activity (Fig. 8 A) that FSTL1 is dose dependent and raises NKA in the Dorsal Root Ganglion Neurons of cultivating, also can be detected.Corotation α 1NKA-flag and with β 1 subunit of myc label in COS7 cell, can detect FSTL1 dose dependent and raise the activity (Fig. 8 A) of NKA, can not (Fig. 8 A) during corotation α 3NKA-flag.

FSTL1 is at corotation α 1NKA subunit ^e314G-flag or α 1NKA subunit ^t889N-flag or α 1NKA subunit ^{e314G, T889N}in the COS7 cell of-flag and β 1 subunit, can not increase the activity of NKA, FSTL1 ^e165Acan not improve the active alpha (Fig. 8 B) of NKA in the COS7 cell of coexpression α 1NKA subunit and β 1 subunit.COS7 is complete, and cell record is observed, in the cell of corotation α 1NKA-flag and β 1-myc subunit, FSTL1 causes film hyperpolarization (～8mV), significantly reduce cell membrane potential (Fig. 8 C), at the cell FSTL1 of corotation α 3NKA-flag and β 1-myc subunit, transmembrane potential is not affected to (Fig. 8 C).It is important that β 1 subunit and α 1NKA subunit corotation are realized to strong and stable function to FSTL1, and in the cell of transfection α 1NKA subunit separately, FSTL1 can only cause moment and low-level film hyperpolarization (～2mV).Therefore, FSTL1 can activate α 1NKA specifically, thereby causes cell hyperpolarization.These results are also illustrated in 314 (E) and 889 (T) aminoacid on α 1NKA subunit, and 165 (E) aminoacid on FSTL1 is important to the function of FSTL1 and α 1NKA.

In the COS7 cell of coexpression α 1NKA subunit and β 1 subunit, preincubate M3M4 (EC ₅₀=3.6 μ M) or M7M8 (EC ₅₀=2.9 μ M) peptide section (You Jitai company is synthetic) can make ¹²⁵the combination rate of I-FSTL1 significantly declines, and is illustrated in the calmodulin binding domain CaM that M3M4 in α 1NKA subunit and M7M8 peptide section contain FSTL1, and relatively, M1M2 peptide section can not reduce described combination rate (Fig. 8 D).Preincubate M3M4 or M7M8 peptide section can weaken the NKA increased activity effect (Fig. 8 E) that FSTL1 causes, more confirm the importance of M3M4 or M7M8 peptide section.In addition, these results represent that M3M4 or M7M8 or similar peptide section can be used as the inhibitor of FSTL1, because these peptide sections can reduce with FSTL1 combination the combination of FSTL1 and NKA.

The dorsal root ganglion of adult rat small neuron of acute isolation (diameter is 15～30 microns) is done to full cell record, FSTL1 (30nM) reduces the frequency of neuron action potential, and induction film hyperpolarization (, this impact is reversed when give specific inhibitor ouabain (OB 100 μ M) time of NKA simultaneously, the effect of the film hyperpolarization that FSTL1 causes can be overturn by ouabain (Fig. 8 F), also can be by the 2nd born of the same parents' outer shroud M3M4 peptide section institute's antagonism (Fig. 8 F) of preincubate α 1NKA.Therefore, α 1NKA is the receptor of FSTL1.

In cornu dorsale medullae spinalis, SABC shows that α 1NKA is mainly distributed in the nerve fiber of cornu dorsale medullae spinalis I-V layer (Fig. 8 G), these nerve fibers containing α 1NKA are mainly primary afferent terminations, because research in the past and current research all do not find that dorsal horn neurons has the mRNA of α 1 subunit, provides the foundation for FSTL1 presynaptic generation effect like this.The full cell record of spinal cord brain sheet with Dorsal root shows, FSTL1 reduces the frequency of sEPSC, this impact is reversed (Fig. 8 H) simultaneously when giving the specific inhibitor ouabain (OB 100 μ M) of NKA, and the 2nd born of the same parents' outer shroud M3M4 peptide section of preincubate α 1NKA can obviously strengthen the frequency (Fig. 8 I) of sEPSC.The full cell record demonstration of spinal cord brain sheet II layer neuron M3M4 peptide section is hatched the eEPSC amplitude (Fig. 8 J) that the stimulation of C fiber causes that increases.Therefore, the FSTL1 of secretion regulates and to import that to transmit be by activating α 1NKA into.

The functional area research of embodiment 8.FSTL1 albumen

The inventor further comes to determine the region that produces function in FSTL1 albumen, noxious stimulation capsaicin (Capsaicin) can cause that the capsaicin receptor VR1 on the small neuron in Primary Sensory Neuron is open, cause flow of calcium ions, by the method for calcium imaging, can detect this process.When adding capsaicin, add each mutant protein of FSTL1 (the concentration 60nM that adds capsaicin 1 μ M and mutant protein), detect the impact of flowing in the calcium that each mutant protein causes capsaicin.

Experimental result shows (Fig. 9): FSTL1 full-length molecule can significantly suppress the flow of calcium ions of capsaicin induction, disappearance FS domain, the FSTL1 albumen of VWC domain does not affect its inhibitory action to the flow of calcium ions of capsaicin induction, be illustrated in FSTL1, FS domain and VWC domain do not participate in the inhibitory action of FSTL1 to the flow of calcium ions of capsaicin induction, relatively, disappearance EFI+EF hand-type connections+EFII or the 165th amino acids be mutated into alanine FSTL1 proteins lose the inhibitory action of flow of calcium ions that capsaicin is induced, show that the 165th amino acids is important to this function of FSTL1.In fact, in this domain, the synthetic polypeptide (SP76) of 156-173 amino acids sequence just has the similar function of FSTL1 (as described below).

Further on the COS7 cell of stable transfection α 1NKA and β 1NKA subunit, detect α 1NKA enzymatic activity, FSTL1 total length can significantly activate the activity of α 1NKA enzyme, and in EF chiral structure territory I, the synthetic polypeptide (SP76) of 156-173 amino acids sequence also can significantly activate the activity of α 1NKA enzyme.

These results show, FS in FSTL 1 albumen and VWC fragment do not participate in the inhibitory action of FSTL1 to the flow of calcium ions of capsaicin induction, EFI+EF hand-type connection+EFII fragment is most important to the inhibitory action of the flow of calcium ions of capsaicin induction to FSTL1, and the 165th amino acids in EFII is most important to the inhibitory action of the flow of calcium ions of 1 pair of capsaicin induction of FSTL.In the FSTL1 of rat, 156-173 amino acids sequence may be the minimum function fragment of FSTL1.

This of rat section 156-173 amino acids sequence (SEQ ID NO:1) is equivalent to people's 158-175 amino acids sequence (SEQ ID NO:2).The 159th (H) amino acids that can find out rat from rat and people's sequence alignment is made into 161 (R) in people's sequence.This is substituting of conservative.In this area, have general Chang Shizhe all to know, these and other similar conservative substitutes the activity that (for example: Ala, Leu, Ile substitutes mutually) not too can affect albumen.So the present invention includes this function fragment that conservative substitutes.

Embodiment 9. screening of medicaments

The FSTL1 of usining expresses positive small neuron cell as the cell model for screening.

Test group: the culture of the above-mentioned cell of processing by candidate substances;

Matched group: the culture of the above-mentioned cell of processing without candidate substances.

If compared with matched group, significantly rising (for example, more than 30%) of the expression of the FSTL1 albumen in test group, activity, amount or secretion, illustrate that this candidate substances is potential na-k-atp enzyme agonist, can be used for treatment or the alleviation disease relevant to na-k-atp enzyme activity decreased by improving na-k-atp enzyme activity, such as reducing mammalian body sensation, treatment or alleviating familial hemiplegia type migraine, diabetes or major depression etc.

Otherwise, for example, if the expression of the FSTL1 albumen in test group, activity, amount or secretion significantly decline (more than 30%), illustrate that this candidate substances is potential na-k-atp enzyme inhibitor, can be used for treatment or the alleviation disease relevant to na-k-atp enzyme hyperactivity by reducing na-k-atp enzyme activity, for example, for increasing mammalian body, feel, treat or alleviate growth and the generation of melanoma or other tumor.

The inventor finds in further investigation, FSTL1 can with sodium-potassium ATP enzyme (Na ⁺, K ⁺-ATPase) combination, and the key point of wherein combination is in second born of the same parents' outer shroud M3M4 of sodium-potassium ATP enzyme α 1NKA subunit and the 4th born of the same parents' outer shroud M7M8.And, after FSTL1 is combined with sodium-potassium ATP enzyme, can activate the activity of sodium-potassium ATP enzyme, thereby prompting FSTL or its promoter can be used for treatment, alleviation or improvement and na-k-atp enzyme activity decreased or too low relevant disease or symptom.Otherwise, research has also proved by lowering the active of FSTL1 or suppressing FSTL1 and has been combined with sodium-potassium ATP enzyme, can reduce the activity of na-k-atp enzyme, thereby the inhibitor of prompting FSTL1 can be used for treatment, alleviation or improvement, improve or too high relevant disease or symptom to na-k-atp enzyme is active.

The inventor is for FSTL1 and somesthetic research discovery, and endogenous FSTL1 is somesthetic a kind of modified, and the reduction of FSTL1 contributes to the generation of the pain sensation.Those skilled in the art know, excitatory synapse transmission can be regulated by the inhibitive factor of secreting in the relay cell of cornu dorsale medullae spinalis and descending inhibitory system.It is that the inhibition that transmits of the somesthesia of a peripheral nerve endings autocrine is modified that the inventor's research discloses FSTL1.FSTL1 mainly expresses on the minicell of DRG, and by little clear vesicle, is transported to the afferent nerve endings of cornu dorsale medullae spinalis, and FSTL1 can discharge to carry out inhibitory action altogether with excitatoty neurotransmitter, to maintain normal somatosensory information transmission.Because FSTL1 selective expression is on the minicell of DRG, so the Autocrine of FSTL1 is that a kind of exceptional function of the minicell of DRG can maintain somesthetic steady-state adjustment.

FSTL1 belongs to SPARC family, has Progynon inhibin spline structure territory (FS domain) and EF hand-type domain.Existing studies show that, the Progynon inhibin domain of FSTL1 does not participate in the inhibitory action that FSTL1 induction produces, and this is also consistent with the result that Progynon inhibin does not produce direct repression.FSTL1 and Dan Baiduotang proteoglycan PG (agrin) all belong to Progynon inhibin gene subfamily, and different from the function of Progynon inhibin.Dan Baiduotang proteoglycan PG is vital for postsynaptic differentiation, and it is by suppressing Na ⁺/ K ⁺-ATP enzyme alpha 3 subunit strengthens the irritability reaction of cortical neuron.The inventor's research finds that FSTL1 high expressed is on DRG minicell, and by Na on activated membrane ⁺/ K ⁺-ATP enzyme suppresses cell excitability.

Matrix metalloproteinase (MMP) and PGE2 are considered to participate in the destruction of joint process in rheumatoid joint.Fstl1 can reduce the generation (.IntImmunol.2003Jan such as Mimori T of MMP and PGE2 in synovial fluid; 15 (1): 71-7).In the rheumatoid arthritis disease model of mice, Fstl1 and derive from the little peptide of Fstl1 155-176 amino acids can releasing arthritis disease the (.ArthritisRheum.2004Feb such as Ozaki S; 50 (2): 660-8.).Fstl1 is also considered to muscle protein or the cytokine of a secretion, and in ischemia injury, Fstl1 can improve inner skin cell function (the .J Biol Chem.2008Nov 21 such as Walsh K; 283 (47): 32802-11.), directly stimulate vascularization and suppress the death of cardiovascular cell, therefore, Fstl1 may can be used to treat ischemic heart disease, and (.Circ such as Walsh K J.2009Jan; 73 (1): 13-8.; The .Circulation.2008Jun 17 such as Walsh K; 117 (24): 3099-108).

Based on above result of study, in the minicell cell space of DRG, FSTL1 is synthesized and is sorted in little clear vesicle, the nerve ending that these vesicles that contain FSTL1 are transported to cornu dorsale medullae spinalis discharges, and its releasing pattern is similar with large dense vesicle to traditional synaptic vesicle.Under normal physiological condition, neuronic excitement causes FSTL1 to discharge to be increased, thereby makes teleneuron hyperpolarization, and then suppresses presynaptic excitatory transmitter and modified release.On the contrary, the minimizing of sensation activity causes FSTL1 to discharge minimizing, thereby makes teleneuron go levelization, and then the presynaptic excitatory transmitter of facilitation and modified release.Yet under pathological conditions, as peripheral nerve injury or arthritis, cause FSTL1 persistence to reduce, thereby cause the hypersensitivity of sensation.

Knock out the FSTL1 in neuron or give the nocuity hypersensitive phenomenon explanation that the antibody of FSTL1 causes, the hyperexcitability of the function of FSTL1 in synapse is transmitted and teleneuron and pain are quick, and relevant (removal of inhibit function that it should be noted that neurotransmission can cause the symptom of chronic pain clinically: hyperpathia and tactile bringing out bitterly.This area known in the inhibition system of spinal cord after nerve injury and upset, and shows as the loss that the minimizing of Mu opiate receptor in primary afferent fiber and GABA can spinal neurons.In the present invention, having proposed FSTL1 minimizing is a key factor of neuropathic pain, and these find that prompting FSTL1 has good therapeutical effect to neuropathic pain.

All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document, are quoted as a reference separately.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims

FSTL1 in preparation the purposes in compositions, described compositions is for by improving disease or the symptom that na-k-atp enzyme activity is treated, alleviated or improvement is relevant with na-k-atp enzyme activity; Described disease or symptom are pain.
2. purposes as claimed in claim 1, is characterized in that, described pain is familial hemiplegia type migraine.
3. purposes as claimed in claim 1, is characterized in that, described pain is body source property pain.
4. purposes as claimed in claim 3, is characterized in that, described body source property pain is selected from: neuropathic pain or nociceptive pain.
5. purposes as claimed in claim 4, is characterized in that, described neuropathic pain is selected from: dorsal root ganglion neuropathic pain, trigeminal ganglion neuropathic pain or sciatica.
6. purposes as claimed in claim 4, it is characterized in that, described neuropathic pain is to be selected from the pain that the damage by maincenter or peripheral nervous system of lower group or pathological change cause: lumbago and backache, neuralgia, fibromyalgia, neuralgia that neuralgia, multiple sclerosis that diabetes are relevant are relevant, postherpetic neuralgia and with the related neurogenic neuralgia of HIV.
7. purposes as claimed in claim 4, is characterized in that, described nociceptive pain is selected from: body nociceptive pain or internal organs nociceptive pain.
8. purposes as claimed in claim 7, is characterized in that, described nociceptive pain is selected from: cancer pain, arthritis pain, postoperative pain or with the related nociceptive pain of HIV.
9. purposes as claimed in claim 1, is characterized in that, described FSTL1 is:

(a) protein of aminoacid sequence shown in SEQ ID NO:1 or SEQ ID NO:2;

(b) conservative variant protein matter or its active fragment of the protein defined in (a);

Described (b) is selected from:

(1) polypeptide of 156-173 aminoacid sequence in SEQ ID NO:1;

(2) polypeptide of 158-175 amino acids sequence in SEQ ID NO:2;

(3) by the sequential amino acid deletion Progynon inhibin domain of SEQ ID NO:1 or SEQ ID NO:2, i.e. FS domain, formed polypeptide;

(4) by the sequential amino acid deletion Von Willebrand type C-structure territory of SEQ ID NO:1 or SEQ ID NO:2, i.e. VWC domain, formed polypeptide.
10. a separated polypeptide, described polypeptide is selected from:

(1) polypeptide of 156-173 amino acids sequence NGDSHLDSSEFLKFVEQN in SEQ ID NO:1, condition is that this polypeptide is not the protein shown in SEQ ID NO:1;

(2) polypeptide of the NGDSRLDSSEFLKFVEQN of 158-175 amino acids sequence in SEQ ID NO:2, condition is that described polypeptide is not the protein shown in SEQ ID NO:2;

(3) by the sequential amino acid deletion Progynon inhibin domain of SEQ ID NO:1 or SEQ ID NO:2, i.e. FS domain, formed polypeptide;

(4) by the sequential amino acid deletion Von Willebrand type C-structure territory of SEQ ID NO:1 or SEQ ID NO:2, i.e. VWC domain, formed polypeptide.
11. 1 kinds of separated polynucleotide, is characterized in that, described polynucleotide encoding polypeptide claimed in claim 10.
12. 1 kinds of carriers, is characterized in that, it contains the polynucleotide described in claim 11.
13. 1 kinds of genetically engineered host cells, is characterized in that, it contains the carrier described in claim 12; Or in its genome, be integrated with the polynucleotide described in claim 11.
The preparation method of 14. 1 kinds of polypeptide claimed in claim 10, is characterized in that, the method comprises:

(a) host cell described in cultivation claim 13 under conditions suitable for the expression;

(b) from culture, isolate polypeptide claimed in claim 10.
15. 1 kinds of pharmaceutical compositions for the treatment of, alleviate or improve pain by improving na-k-atp enzyme activity, it contains:

(a) protein of the polypeptide claimed in claim 10 of safe and effective amount, SEQ ID NO:1 or SEQ ID NO:2; And

(b) pharmaceutically acceptable carrier.
16. pharmaceutical compositions as claimed in claim 15, is characterized in that, described pain is familial hemiplegia type migraine.
17. pharmaceutical compositions as claimed in claim 15, is characterized in that, described pain is body source property pain.
18. pharmaceutical compositions as claimed in claim 17, is characterized in that, described body source property pain is selected from: nociceptive pain or neuropathic pain.
19. pharmaceutical compositions as claimed in claim 18, is characterized in that, described neuropathic pain is selected from: dorsal root ganglion neuropathic pain, trigeminal ganglion neuropathic pain or sciatica.
20. pharmaceutical compositions as claimed in claim 18, it is characterized in that, described neuropathic pain is to be selected from the pain that the damage by maincenter or peripheral nervous system of lower group or pathological change cause: lumbago and backache, neuralgia, fibromyalgia, neuralgia that neuralgia, multiple sclerosis that diabetes are relevant are relevant, postherpetic neuralgia or with the related neurogenic neuralgia of HIV.
21. pharmaceutical compositions as claimed in claim 18, is characterized in that, described nociceptive pain is selected from: body nociceptor pain or internal organs nociceptor pain.
22. pharmaceutical compositions as claimed in claim 21, is characterized in that, described nociceptive pain is selected from: cancer pain, arthritis pain, postoperative pain or with the related nociceptive pain of HIV.
The purposes of protein shown in protein shown in 23. polypeptide claimed in claim 10, SEQ ID NO:1 and/or SEQ ID NO:2 in the pharmaceutical composition of the disease for the preparation of by raising na-k-atp enzyme activity is treated, alleviated or improvement is relevant to na-k-atp enzyme activity or symptom; Described disease or symptom are pain.
24. 1 kinds of screenings are used for treating, alleviating or improve by improving na-k-atp enzyme activity the method for the potential material of the disease relevant to this activity or symptom, it is characterized in that, described method comprises:

(1) candidate substances is contacted with the system of expressing FSTL1;

(2) detect the impact of candidate substances on FSTL1;

If described candidate substances can improve expression, activity and/or the secretion of FSTL1, show that this candidate substances is the potential material that can be used for treating, alleviating or improve by improving na-k-atp enzyme activity the disease relevant to this activity or symptom;

Described disease or symptom are pain.