CN106959373B - Purposes of the interferon-γ as cough drug target - Google Patents

Purposes of the interferon-γ as cough drug target Download PDF

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CN106959373B
CN106959373B CN201710186335.0A CN201710186335A CN106959373B CN 106959373 B CN106959373 B CN 106959373B CN 201710186335 A CN201710186335 A CN 201710186335A CN 106959373 B CN106959373 B CN 106959373B
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CN106959373A (en
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赖克方
邓政
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First Affiliated Hospital of Guangzhou Medical University
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/6866—Interferon
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Abstract

Purposes the invention discloses interferon gamma (Interferon γ, IFN γ) as cough drug target.Cough after Viral Infection caused by virus infection and the core cell factor of cough Gao Min syndrome patients lung release are IFN γ.The present invention is analyzed by whole guinea pig cough's signal, it is found that IFN γ intratracheal instillations increase guinea pig cough's sensibility；By calcium imaging technique, it is found that IFN γ cause the extracellular fluid flow of calcium ions of rat vagus nerve member by JAK PKA AMPA signal paths；Using patch clamp technique, it is found that IFN γ not only increase rat vagus nerve metaaction potential frequency under electro photoluminescence, also directly cause action potential granting (its mechanism is the same).Vagus nerve metaaction current potential can be passed to maincenter upwards, cause cough reflex.These also directly cause to cough the result shows that IFN γ can not only increase Cough reflex sensitivity by JAK PKA AMPA signal paths.The present invention prompts IFN γ or its gene that can be applied to as potential drug target in the drug of screening or preparation treatment cough.

Description

Purposes of the interferon-γ as cough drug target
Technical field
The present invention relates to the function of albumen and application fields, in particular to interferon-γ as cough drug target Purposes.
Background technology
Chronic cough be with cough be only symptom or cardinal symptom, time be more than 8 weeks, chest X-ray is without obviously different Chang Zhe.The country causes the common known reason of chronic cough to include cough variant asthma, epithelium healing cough syndrome, acidophilus grain Cellularity bronchitis, allergia cough are coughed with Gastroesophageal reflux1.It is viscous that the receptor of cough reflex is predominantly located at respiratory tract Film.Include mainly unmyelinated fiber C and medullated A δ fibers, wherein fiber C master with the relevant afferent nerve of cough reflex It is sensitive to chemical stimulation；There are marrow A δ fibers mainly sensitive to mechanical stimulus.Then afferent impulse is prolonged by Vagus-pressor responses Marrow triggers cough reflex.The mechanism of chronic cough may be cough receptor by chemistry, physical stimulation increase, or It is that Cough reflex sensitivity increases.
Cough reflex sensitivity refers to body when receiving environmental stimuli (including chemistry, machinery, warm), the cough showed Complexity.The high quick syndrome (cough hypersensitivity syndrome, CHS) of cough refers to Cough reflex sensitivity liter Height checks some the idiopathic chronic coughs (also known as chronic idiopathic cough) that cannot be still attributed to the known cause of disease comprehensively.CHS It is a kind of clinical common type (account for about chronic cough total number of persons 8%~40%) of chronic cough, often with upper respiratory tract sense Contaminate the startup factor (about 50%) as morbidity2.The startup factor (such as virus infection, toxic gas exposure) of CHS is often It has been disappeared that, but dry cough symptoms last3.Cough reflex sensitivity, which increases, is mainly shown as that cough threshold value declines, and minimal irritation will draw Play cough symptom (such as speak, laugh, contacting cold air, smog, the cough of part patient CHS even can be constant).By It is unknown in mechanism, the specific medicament for CHS is not yet found at present.
Delayed allergy (delayed-type hypersensitivity, DTH) is sensitized T lymphocyte and phase After answering antigenic action, cause anti-as the inflammation of main feature using lymphocyte, monocytes/macrophages infiltration and tissue cell insult It answers, belongs to cellular immunity type.Virus is the important antigen for causing DTH with intracellular bacterial parasite, and main effects cell is differentiation group 8+(cluster of differentiation 8+,CD8+) cytotoxic T cell (cytotoxic T cell, CTL) and CD4+It is auxiliary Helping property T cell 1 (T helper type1cell, Th1 cell).CD8+CTL cells can directly kill the target of pathogenic infection Cell；CD4+After t cell activation formed Th1 cells, by secrete cytokines [such as interferon-γ (interferon- γ, IFN-γ), tumor necrosis factor-alpha (tumor necrosis factor- α, TNF-α), interleukin 2 (interleukin-2, IL-2) etc.] attract and assembles in reactive site with activated macrophage, form the chronic inflammation of tissue4-6； Certainly, CD8+CTL cells and CD4+The division of labor of T cell is not absolute, CD4 when pathogenic infection+T cell may be shown simultaneously Dissolved cell activity, and CD8+CTL cells also can the cell factors such as secretion of gamma-IFN7,8.In these cell factors, IFN-γ is Τ h1 cells and CD8+The most important cell factor of CTL cells secretion, and play core in the inflammatory signals access of downstream and make With8-10.Some diseases with cough symptom such as Cough after Viral Infection, pertussis and pulmonary tuberculosis etc. are often with lung's IFN-γ Level increases11-13.In the research of one, the U.S., researcher has collected the adult cold patients with fever with cough symptom, with Bacterial origin is compared, and the IFN-γ in virus flu patients serum is horizontal significantly raised11.It (is deposited with IFN-γ using I type IFN Acting synergistically) antiviral therapy can cause some patients cough symptom occur14,15.Certainly, respiratory virus infection is in addition to leading It causes outside cell immune response, moreover it is possible to stimulate CD4 to a certain degree+Th2 cell differentiations are formed, and humoral immune reaction is caused16。
There is the clinical test that researcher carries out IFN-γ treatment mycosis fungoides in Japan, it is found that most common side effect is stream Feel sample symptom, has an example dead after IFN-γ is treated 50 days in 15 patients, also an example is coughed due to drug induccd and exits examination It tests (cough disappears after drug withdrawal)17.In addition, Neulized inhalation IFN-γ treats caused most common secondary work when idiopathic pulmonary fibrosis With being exactly to cough18.In fact, studies have found that CHS has with the disorder of organ autoimmune function, lung's lymphocytosis It closes19.More studies have found that the T lymphocytosises of patient CHS lung20, T lymphocytosises are one of performances of DTH.Thus, It is concluded that the DTH characterized by lung's IFN-γ level increases may be also one of the pathogenesis of CHS.Inventor is studying The middle not only possible increase Cough reflex sensitivity of IFN-γ for finding DTH releases, it is also possible to be drawn by Jak-PKA-AMPA signal paths It is excited to play vagus nerve member, so as to cause causing to cough.The result of study prompt IFN-γ of inventor and Jak- downstream PKA-AMPA signal paths are Cough after Viral Infection and the potential target spot of CHS treatments.
【Bibliography】
1.Lai K,Chen R,Lin J,Huang K,Shen H,Kong L,Zhou X,Luo Z,Yang L,Wen F, Zhong N.A prospective,multicenter survey on causes of chronic cough in China.Chest 2012；143(3):613-20.
2.Haque RA,Usmani OS,Barnes PJ.Chronic idiopathic cough:a discrete clinical entityChest 2005；127(5):1710-3.
3.Chung KF.Chronic'cough hypersensitivity syndrome':a more precise label for chronic cough.Pulm Pharmacol Ther 2011；24(3):267-71.
4.Brusselle GG,Joos GF,Bracke KR.New insights into the immunology of chronic obstructive pulmonary disease.Lancet 2011；378(9795):1015-26.
5.Koltsova EK,Garcia Z,Chodaczek G,Landau M,McArdle S,Scott SR,von Vietinghoff S,Galkina E,Miller YI,Acton ST,Ley K.Dynamic T cell-APC interactions sustain chronic inflammation in atherosclerosis.J Clin Invest 2012；122(9):3114-26.
6.Sow FB,Gallup JM,Olivier A,Krishnan S,Patera AC,Suzich J,Ackermann MR.Respiratory syncytial virus is associated with an inflammatory response in lungs and architectural remodeling of lung-draining lymph nodes of newborn lambs.Am J Physiol Lung Cell Mol Physiol 2011；300(1):L12-24.
7.Kilduff CE,Counter MJ,Thomas GA,Harrison NK,Hope-Gill BD.Effect of acid suppression therapy on gastroesophageal reflux and cough in idiopathic pulmonary fibrosis:an intervention study.Cough2014；10:4.
8.McElroy AK,Akondy RS,Davis CW,Ellebedy AH,Mehta AK,Kraft CS,Lyon GM,Ribner BS,Varkey J,Sidney J,Sette A,Campbell S,Stroher U,Damon I,Nichol ST,Spiropoulou CF,Ahmed R.Human Ebola virus infection results in substantial immune activation.Proc Natl Acad Sci U S A2015；112(15):4719-24.
9.Kidd P.Th1/Th2balance:the hypothesis,its limitations,and implications for health and disease.Altern Med Rev 2003；8(3):223-46.
10.Kreutzfeldt M,Bergthaler A,Fernandez M,Bruck W,Steinbach K,Vorm M, Coras R,Blumcke I,Bonilla WV,Fleige A,Forman R,Muller W,Becher B,Misgeld T, Kerschensteiner M,Pinschewer DD,Merkler D.Neuroprotective intervention by interferon-gamma blockade prevents CD8+ T cell-mediated dendrite and synapse loss.J Exp Med 2013；210(10):2087-103.
11.Haran JP,Buglione-Corbett R,Lu S.Cytokine markers as predictors of type of respiratory infection in patients during the influenza season.Am J Emerg Med 2013；31(5):816-21.
12.Dirix V,Verscheure V,Vermeulen F,De Schutter I,Goetghebuer T,Locht C,Mascart F.Both CD4(+)and CD8(+)lymphocytes participate in the IFN-gamma response to filamentous hemagglutinin from Bordetella pertussis in infants, children,and adults.Clin Dev Immunol 2012；2012:795958.
13.de Souza-Galvao ML,Latorre I,Altet-Gomez N,Jimenez-Fuentes MA,Mila C,Solsona J,Seminario MA,Cantos A,Ruiz-Manzano J,Dominguez J.Correlation between tuberculin skin test and IGRAs with risk factors for the spread of infection in close contacts with sputum smear positive in pulmonary tuberculosis.BMC Infect Dis 2014；14:258.
14.Carbonelli C,Montepietra S,Caruso A,Cavazza A,Feo C,Menzella F, Motti L,Zucchi L.Sarcoidosis and multiple sclerosis:systemic toxicity associated with the use of interferon-beta therapy.Monaldi Arch Chest Dis 2012；77(1):29-31.
15.Botnaru V,Munteanu O,Rusu D.Interstitial pneumonitis after treatment for hepatitis C virus infection.Pneumologia 2015；64(1):46-50.
16.Khatri M,Dwivedi V,Krakowka S,Manickam C,Ali A,Wang L,Qin Z, Renukaradhya GJ,Lee CW.Swine influenza H1N1virus induces acute inflammatory immune responses in pig lungs:a potential animal model for human H1N1influenza virus.J Virol 2010；84(21):11210-8.
17.Sugaya M,Tokura Y,Hamada T,Tsuboi R,Moroi Y,Nakahara T,Amano M, Ishida S,Watanabe D,Tani M,Ihn H,Aoi J,Iwatsuki K.Phase II study of i.v.interferon-gamma in Japanese patients with mycosis fungoides.J Dermatol 2014；41(1):50-6.
18.Diaz KT,Skaria S,Harris K,Solomita M,Lau S,Bauer K,Smaldone GC, Condos R.Delivery and safety of inhaled interferon-gamma in idiopathic pulmonary fibrosis.J Aerosol Med Pulm Drug Deliv 2012；25(2):79-87.
19.Birring SS,Brightling CE,Symon FA,Barlow SG,Wardlaw AJ,Pavord ID.Idiopathic chronic cough:association with organ specific autoimmune disease and bronchoalveolar lymphocytosis.Thorax2003；58(12):1066-70.
20.Mund E,Christensson B,Gronneberg R,Larsson K.Noneosinophilic CD4 lymphocytic airway inflammation in menopausal women with chronic dry cough.Chest 2005；127(5):1714-21.
Invention content
Purposes the purpose of the present invention is to provide IFN-γ as cough drug target.The result of study of the present invention is aobvious Show, IFN-γ and its gene and internal Cough reflex sensitivity are closely related, and for the later stage, the medicament research and development of cough provides one Completely new drug target and new treatment means and thinking.
In order to realize the above-mentioned purpose of the present invention, we use following technical scheme：
In a first aspect, being analyzed by whole guinea pig cough's signal, it is found that IFN-γ intratracheal instillation can increase cavy cough It coughs sensibility, shows that internal IFN-γ can improve Cough reflex sensitivity in integral level.
Second aspect passes through calcium imaging technique, it has been found that IFN-γ can pass through Janus kinases-protein kinase A-α- Amino-(3- hydroxy-5-methyl base -4- isoxazoles propionic acid), i.e. JAK-PKA-AMPA signal paths cause rat vagus nerve member Extracellular fluid flow of calcium ions improves calcium ion concentration in vagus nerve member, and then improves Cough reflex sensitivity.
Finally utilize patch clamp technique, it has been found that it is dynamic that IFN-γ can not only increase cavy vagus nerve member under electro photoluminescence Make potential frequency, moreover it is possible to directly cause action potential granting (its mechanism may relate to JAK-PKA-AMPA signal paths).Fan walks Neuron action potential can be passed to maincenter upwards, cause cough reflex.
The studies above result illustrates that IFN-γ can not only increase Cough reflex sensitivity, moreover it is possible to pass through JAK-PKA-AMPA signals Access directly causes to cough.
These results of study prompt interferon-γ or its gene that can be used as drug target in cough drug is treated in screening Application.The drug includes the expression for 1. inhibiting the interferon-γ, active drug；2. blocking the interferon- γ is combined to inhibit vagus nerve cell Ca with IFN-γ R2+The drug of interior stream；3. the inhibitor of targeted interferon-γ Preparing the application in treating cough drug；4. the cough includes Cough after Viral Infection or the high quick syndrome of cough.
Compared with prior art, beneficial effects of the present invention are：
There are no research report IFN-γ to have the effect for improving Cough reflex sensitivity or causing cough reflex in the world at present. Inventor, which is put forward for the first time and demonstrates IFN-γ, can not only increase Cough reflex sensitivity, moreover it is possible to logical by JAK-PKA-AMPA signals Road directly causes to cough.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technology description to be briefly described.
Fig. 1 is " IFN-γ pretreatment increases guinea pig cough the influence of number " result figure (figure of the embodiment of the present invention 1 In：*, the P compared with bone peptide group is indicated<0.1；* indicates the P compared with bone peptide group<0.05；N=5~7)；
Fig. 2 is that " indirect influence of the IFN-γ on rat vagus ganglion intracellular calcium concentration " is tied in the embodiment of the present invention 2 (ordinate indicates the ratio of F340/F380 to fruit figure in curve graph；Abscissa indicates the record time, records 3.6 × 10 in total3S, That is 1h).A schemes, and the peak value of the lower F340/F380 ratios of reference substance Cap stimulations before and after the processing does not have significant change；B schemes, at IFN-γ The peak value of the lower F340/F380 ratios of Cap stimulations obviously increases after reason.
Fig. 3 is " statistical chart of F340/F380 ratios variation " (wherein, *, expression and ground state in the embodiment of the present invention 2 (Baseline) compare P<0.05；# indicates the P compared with (After Vehicle) after reference substance processing<0.05；N=6).
Fig. 4 is " direct effect of the IFN-γ to rat vagus ganglion intracellular calcium concentration " in the embodiment of the present invention 2 Result figure (in figure, first with control buffer solution processing, has no apparent F340/F380 ratios variation；IFN-γ is used after about 15 minutes (1ng/mL) handles 5min, it is seen that apparent F340/F380 ratios increase).
Fig. 5 is " IFN-γ of various concentration handles the influence to F340/F380 ratios " figure (its in the embodiment of the present invention 2 In, * indicates the P compared with ground state (Baseline)<0.05；N=6).
Fig. 6 is that " extracellular fluid flow of calcium ions is the main way that IFN-γ increases intracellular calcium concentration in the embodiment of the present invention 2 The result figure of diameter " (in figure, is handled first using IFN-γ in no calcium buffer solution, has no apparent F340/F380 ratios variation； Then calcic buffer solution is changed into, it is seen that the lower F340/F380 ratios of IFN-γ stimulation obviously increase).
Fig. 7 is " statistical chart of influence of the IFN-γ to extracellular fluid flow of calcium ions " (its in the embodiment of the present invention 2 In, * indicates the P compared with base state (Baseline)<0.05, n=6).
Fig. 8 is " Jak inhibitor AG490, PKA inhibitor H89 and ampa receptor inhibitor C NQX in the embodiment of the present invention 2 Pre-process to the antagonism of IFN-γ " result figure (effect of figure A, control buffer solution IFN-γ before and after the processing is not apparent Variation；Scheme B, the liter calcium effect of IFN-γ is apparent after (50 μM) pretreatments of AG490 weakens；Then high potassium stimulation is still obviously increased Calcium concentration excludes cell activity problem；Scheme C, the liter calcium effect of IFN-γ is apparent after (10 μM) pretreatments of H89 weakens；Scheme D, The liter calcium effect of IFN-γ is apparent after (10 μM) pretreatments of CNQX weakens).
Fig. 9 is " Jak inhibitor AG490, PKA inhibitor H89 and the ampa receptor inhibitor in the embodiment of the present invention 2 CNQX pre-processes the statistical chart to the antagonism of IFN-γ " (wherein, * indicates the P compared with ground state (Baseline)< 0.05；# indicates the P compared with (After Vehicle) after comparison liquid processing<0.05；N=6).
Figure 10 is " IFN-γ pretreatment increases neuron action potential frequency under electro photoluminescence " figure in the embodiment of the present invention 3 (in figure, under appropriate electro photoluminescence (20pA × 280ms), when base state, scheme the action potential (action of A neuron grantings Potential, AP) negligible amounts；After comparison liquid perfusion, the AP quantity of figure B neurons is not substantially change；IFN-γ(1ng/ ML) after perfusion, the action potential frequency showed increased of the same intensity electro photoluminescence initiations of figure C).
Figure 11 be the embodiment of the present invention 3 " after IFN-γ pretreatment under electro photoluminescence neuron action potential frequency diagram system (wherein, * indicates the P compared with (After Vehicle) after comparison liquid processing to meter figure "<0.05, n=4).
Figure 12 be the embodiment of the present invention 3 " IFN-γ perfusion causes the granting of neuron action potential " result figure (in figure, A is basic status representative figure；B is representative figure after comparison liquid (CS) perfusion；C is representative figure after 0.25ng/mL IFN-γ perfusions；D To represent figure after 1ng/mL IFN-γ perfusions；E is representative figure after 4ng/mL IFN-γ perfusions；F fills for 16ng/mL IFN-γ Figure is represented after stream；G is representative figure after 40ng/mL IFN-γ perfusions；H is representative figure after 160ng/mL IFN-γ perfusions).
Figure 13 be the embodiment of the present invention 3 " IFN-γ perfusion causes the granting of neuron action potential to act electricity in 12s (wherein, * indicates the P compared with after comparison liquid (CS) perfusion to bit quantity statistical chart "<0.05, n=4~5).
Figure 14 is " Jak inhibitor AG490, PKA inhibitor H89 and the ampa receptor inhibitor C NQX of the embodiment of the present invention 3 Pre-processing the antagonism to IFN-γ " ((in figure, A is basic status representative figure to result figure；B is IFN-γ after comparison liquid perfusion The influence of (16ng/mL) to neuron action potential represents figure；C is IFN-γ (16ng/mL) after AG490 (50 μM) perfusion to god Influence through metaaction current potential represents figure；D is IFN-γ (16ng/mL) after H89 (10 μM) perfusion to neuron action potential It influences to represent figure；E is influence representative figure of the IFN-γ (16ng/mL) to neuron action potential after CNQX (10 μM) perfusion；F is Statistical chart：*, the P compared with base state is indicated<0.05；# indicates to compare with (IFN After Vehicle) after comparison liquid processing Compared with P<0.05；N=4).
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment.The following example is merely to illustrate this Invention, and be not construed as limiting the scope of the invention.
First, to arrived used in experimental animal, main agents and experimentation used in following embodiments solution, And an explanation is done in the processing of cell climbing sheet：
1. experimental animal and raising：
Animal FMMU cavys 40, male, weight 300-350g are provided, animal by Guangdong Medical Lab Animal Center Quality certification number：44002100004818.SD rats, male, weight 200-250g are carried by Guangdong Medical Lab Animal Center For animal quality certification number：44007200036817；It is placed in room temperature (22 ± 2) DEG C, humidity (55 ± 5) % is alternately illuminated for 12 hours Lower life, free water diet.
2. main agents and instrument：
Physiological saline (Jiangxi moisten medicine company)；Bone peptide (Heilongjiang ZBD Pharmaceutical Co., Ltd.)；People source interferon- Gamma (IFN-γ) (Shanghai KaiMao biological medicine Co., Ltd tests for guinea pig animal)；Rat source IFN-γ (Peprotech is used for rat primary cell experiment)；Citric acid (Guangzhou Chemical Reagent Factory)；Ether (Guangzhou Chemical Reagent Factory)； DMEM/F12 culture mediums (Gibco)；Fetal calf serum (Gibco)；Dual anti-(Gibco)；F12 culture mediums (Gibco)；Neure growth Additive (Sciencecell)；35mm culture dishes (Nest)；25mm cell climbing sheets (Nest)；Laminin (Invitrogen)；Poly-D-lysine (the green skies)；Type Ⅳ collagenase (Gibco)；II type dispase (Sigma)；Phosphate is slow Fliud flushing/PBS (doctor's moral)；D-Hanks liquid (lucky promise biology)；HVG high vacuum seals fat (dow corning)；Capsaicine (Tokyo Chemical Industry)；Yellow Jackets (Merck)；Fura-2AM(Thermo Fisher Scientific)；Small animal laryngoscope (Penn-century)；The noninvasive Pulmonary function system (Buxco) of toy；Fluorescence is inverted Microscope (Leica)；MCM syringe filters (Agela companies)；Sutter glass electrodes (Sutter companies)；12mm cells are climbed Piece (Fisher companies)；35mm Tissue Culture Dish (Corning companies)；Inverted microscope (Zeiss)；Patch clamp amplifier (HEKA_EPC10 series)；Glass electrode draws instrument (Sutter companies).
3. the preparation of solution：
Cell inoculation liquid：+ 10% fetal calf serum+1% of DMEM/F12 culture mediums is dual anti-.
Cell culture fluid：+ 1% dual anti-+ 1% neure growth additive of+3% fetal calf serum of F12 culture mediums.
The digestion of Collagenase Type containing IV (1.5mg/mL) and II types dispase (1.5mg/mL) are prepared using D-Hanks liquid Liquid, filtration sterilization, -20 DEG C of preservations after packing.
Poly-D-lysine (0.1mg/mL), filtration sterilization, -20 DEG C of preservations after packing are prepared using aqua sterilisa.
Laminin (10mg/mL), filtration sterilization, -20 DEG C of preservations after packing are prepared using DMEM/F12 culture mediums.
There is the outer buffer solution (ECS) of calcium cell：3.8025g containing NaCl, KCl 0.175g in per 500mL deionized waters, CaCl20.1g, MgCl2·6H2O 0.1017g, NaH2PO4·2H2O 0.025g, C6H12O6·H2O 0.594g, HEPES 1.19g；Using the NaOH tune pH value of 1M to 7.4.
Without buffer solution outside calcium cell：3.8025g containing NaCl, KCl 0.175g, MgCl in per 500mL deionized waters2· 6H2O 0.203g, NaH2PO4·2H2O 0.025g, C6H12O6·H2O 0.594g, HEPES 1.19g；Use the NaOH tune of 1M PH value is to 7.4.
Liquid in electrode：Contain CaCl in per 100mL deionized waters211.1mg, EGTA 380.35mg, HEPES 238.3mg, MgCl2·6H2O 10.17mg, KCl 298mg, K-IAO 2155.1mg, NaCl 46.8mg；Use the KOH tune pH value of 1M To 7.1, adjusts infiltration using mannitol and be depressed into 285mosM.
Electrode external solution：3.978g containing NaCl, KCl 0.2011g, CaCl in per 500mL deionized waters20.1g, MgCl2· 6H2O 0.1017g,NaH2PO4·2H2O 0.025g,C6H12O6·H2O 0.495g, HEPES 1.19g；Use the NaOH tune of 1M PH value adjusts infiltration to 7.4, using mannitol and is depressed into 295mosM.
4. the processing of cell climbing sheet：
It by cell climbing sheet, is rinsed 15 times, then with pure water rinsing 10 times, high pressure sterilization after drying, then dried standby with tap water With；Cell climbing sheet is put into 35mm culture dishes bottom in super-clean bench, 1mL poly-D-lysines (PDL, 0.1mg/mL) are added dropwise, make Be completely covered cell climbing sheet, ultraviolet irradiation 30min is moved into 37 DEG C of incubators overnight；PDL is abandoned in suction, and two are rinsed with aqua sterilisa Time, it is added 1mL laminins and 1mL cell inoculation liquid, mixing moves into spare in incubator, faces the used time with cell inoculation liquid It washes one time.(this step needs bubble removing, ensures that cell climbing sheet sinks).
Embodiment 1
IFN-γ pre-processes the influence to whole guinea pig cough's sensibility
1. experimental animal is grouped and medication：
Cavy 40 is only randomized into 5 groups, is pre-processed respectively to cavy with different solution：Saline control Group, bone peptide control group (concentration is similar with high dose IFN-γ group), low dosage, middle dosage and high dose IFN-γ group (8 × 103、 4×104、2×105IU, user source IFN-γ).
Utilize cavy basis cough number (0.3M citric acids atomization excitation under Buxco system non-invasive measurement waking states 1min observes 8min).
Using etherization cavy, physiological saline of the intratracheal instillation containing above-mentioned modeling agent under laryngoscope light source-guide 0.2mL；Twice a week, continue one week, use Buxco system non-invasive measurement waking states within 5 hours or so after last time instils Lower guinea pig cough's number (method is the same).
2. experimental result：
The cough number that every cavy last time measures is subtracted into basis cough number, resulting value is to increase number If (the cough number that last time measures is less than basic value, then it represents that this guinea pig cough's number does not increase, and is denoted as 0). The results are shown in Figure 1, and compared with bone peptide control group, the IFN-γ pretreatment of low dosage has the tendency that increasing guinea pig cough's number (P <0.1)；Middle and high dosage IFN-γ pretreatment significantly increases guinea pig cough's number (P<0.05), thus illustrate that IFN-γ is located in advance Reason increases guinea pig cough's sensibility.
Embodiment 2
Calcium imaging experiment
1. experimental method：
A. primary vagus nerve member culture：
Anesthesia, sacrificed by exsanguination rat, operation open cranium from throat and take bilateral vagus ganglion；It carefully removes, indulge under microscope To tearing, shred；It is collected in the 5mL centrifuge tubes containing cold PBS.400g × 5min is centrifuged, and is removed supernatant, is added 0.5mL digestive juices, close Be honored as a queen 37 DEG C of water-bath digestion 40min.Add 2mL cell inoculation liquid, it is close with the carefully piping and druming 40~60 times (avoiding bubble) of 1mL pipette tips Envelope.400g × 5min is centrifuged, and removes supernatant；Add 2mL cell inoculation liquid, carefully 30~40 times (avoiding bubble) blown and beaten with 1mL pipette tips, Sealing.400g × 5min is centrifuged, and removes supernatant；Add 2mL cell inoculation liquid, with the carefully piping and druming 20 times (avoiding bubble) of 1mL pipette tips, connects Kind moves into incubator in the culture dish of the 35mm containing cell climbing sheet (processed).It is carefully inhaled after about 2 hours and abandons stoste, use PBS It washes 1 time, changes 2mL cell culture fluids, stayed overnight in incubator.It can be used to test within second day.
B. calcium record by imaging：
This research uses the primary vagus nerve member for being inoculated in 25mm circular glass creep plates.Calcium is added in culture solution first It is imaged indicator Fura-2AM (final concentration of 5 μM), 30min is loaded in 37 DEG C of incubators.Then cell climbing sheet is taken out, is used ECS buffer solutions wash 1 time；Cell climbing sheet is pasted on cell bath using high vacuum seal fat, the continuous perfusion of ECS buffer solutions (flow velocity 1mL/min).Cell bath is positioned over to the fluorescence inverted microscope (Leica) for being connected with xenon lamp and CCD camera Under, it is to be measured.Acquiring Two Colour Fluorescence intensity, (excitation wavelength is respectively 340nm and 380nm, and launch wavelength is 510nm；It is acquired per 12s 1 time), pass through Ca in the ratio calculation neuron plasma of F340/F3802+Concentration.Every time by the nerve in the visual field at the end of experiment Member is irised out, and the calcium transient curve graph of single neuron is obtained.
3. experimental result：
As a result as shown in Figures 2 and 3, the ratio of F340/F380 can reflect Ca in neuron plasma2+Concentration.As a result it carries Show Cap (5 × 10-8M) perfusion 10s moment can cause Ca2+Concentration increases；Same dosage Cap perfusions draw before and after comparison liquid perfusion The calcium concentration increase degree risen does not have a significant change, after IFN-γ (1ng/mL) perfusion 5min caused by same dosage Cap perfusions Calcium concentration increase degree obviously increases.
As shown in Figure 4 and Figure 5, control buffer solution perfusion 5min does not cause apparent intracellular calcium concentration to change；IFN-γ (1ng/mL) perfusion 5min obviously increases neuron intracellular calcium concentration.The IFN-γ perfusion of 1~200ng/mL can be with dose-dependant Increase intracellular calcium concentration (the P of property<0.05).
As shown in Figure 6 and Figure 7, in no calcium buffer solution, IFN-γ (1ng/mL) perfusion 5min can hardly change nerve First intracellular calcium concentration；Change calcic buffer solution into, same dose of IFN-γ perfusion 5min obviously increases neuron intracellular calcium concentration (P<0.05).It is the main of calcium concentration in IFN-γ increase neuron to illustrate that extracellular fluid calcium ion flows into endochylema by cell membrane Approach.
As shown in Figure 8 and Figure 9, compared with compareing buffer solution, Jak inhibitor AG490, PKA inhibitor H89 and ampa receptor The liter calcium effect of same dose of IFN-γ (100ng/mL) perfusion 5min is apparent after inhibitor C NQX pretreatments 10min weakens (P <0.05).Illustrate that IFN-γ is to cause extracellular fluid flow of calcium ions by Jak-PKA-AMPA signal paths.
Embodiment 3
Patch clamp experiments
1. experimental method：
This research is first (cultural method is with embodiment 2) using the primary vagus nerve for being inoculated in 12mm circular glass creep plates. Cell climbing sheet is taken out first, is washed 3 times with electrode external solution；Cell climbing sheet is pasted on cell bath using high vacuum seal fat On, the continuous perfusion (flow velocity 1mL/min) of electrode external solution.Cell bath is positioned over be connected with CCD camera inversion it is micro- It is to be measured under mirror (Zeiss).
It is about 4~8M Ω to draw instrument glass electrode is pulled to tip resistance using electrode (in pipe in charging extremely when liquid).Quilt The silver wire electrode of chlorination need to immerse in electrode in liquid.Under micro-manipulator control, glass electrode tip and neuronal cell film Sealing-in is carried out, resistance must reach G Ω or more；Then negative pressure rupture of membranes realizes that (setting Clamping voltages are -60mV to whole-cell recording technique；String The connection resnstance transformer time is 10s, and compensation depth is 80% or so).The neuron of experimental record also needs to meet the following conditions：It is adherent Good oval and no aixs cylinder is grown；Cell size is smaller (diameter is less than 35m, and membrane capacitance is between 15pF and 30pF)； There is reaction to Cap stimulations；Resting membrane electric potential is less than -40mV.It then switches under current clamp logging mode and detects neuron to electricity The reaction (recording frequency 20KHz) of stimulation or chemical stimulation.Electro photoluminescence uses between 0~90pA with pattern incremental 10pA, Single electro photoluminescence continues 280ms.
2. experimental result：
As shown in Figure 10 and Figure 11, under appropriate electro photoluminescence (20pA × 280ms), when base state neuron granting it is dynamic Make current potential (AP) negligible amounts (excitability is relatively low)；The excitability of neuron is not substantially change after comparison liquid perfusion；IFN-γ Perfusion obviously increases the excitability (P of neuron<0.05).
As shown in Figure 12 and Figure 13, compared with comparison liquid, the IFN-γ perfusion of 0.25~160ng/mL leads to vagus nerve The AP quantity showed increaseds that member generates, and there is a certain amount to imitate relationship.Illustrate IFN-γ be the excited stimulation of vagus nerve member because Element.
As shown in figure 14, compared with compareing buffer solution, Jak inhibitor AG490, PKA inhibitor H89 and ampa receptor inhibit AP quantity significantly reduces (P in same dose of IFN-γ (16ng/mL) perfusion causes after agent CNQX pretreatments 12 seconds< 0.05).Illustrate that IFN-γ is to cause vagus nerve member excited by Jak-PKA-AMPA signal paths.
In conclusion being analyzed by whole guinea pig cough's signal, it is found that IFN-γ intratracheal instillation can increase cavy cough It coughs sensibility, shows that internal IFN-γ can improve Cough reflex sensitivity in integral level.By calcium imaging technique, IFN-γ is found It can cause extracellular fluid flow of calcium ions by JAK-PKA-AMPA signal paths, improve calcium ion in rat vagus nerve member Concentration, and then improve Cough reflex sensitivity.Using patch clamp technique, it is found that IFN-γ can not only increase cavy fan under electro photoluminescence and walk Neuron action potential frequency, moreover it is possible to which directly causing action potential granting, (it is logical that its mechanism may relate to JAK-PKA-AMPA signals Road).Vagus nerve metaaction current potential can be passed to maincenter upwards, cause cough reflex.In short, IFN-γ can not only increase cough It coughs sensibility, moreover it is possible to directly cause to cough by JAK-PKA-AMPA signal paths.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from the present invention's Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims Including belonging to all such changes and modifications in the scope of the invention.

Claims (6)

1. application of the interferon-γ as drug target in the drug that the high quick syndrome of cough is treated in screening.
2. application according to claim 1, which is characterized in that the drug is the expression water for inhibiting the interferon-γ Flat drug.
3. application according to claim 1, which is characterized in that the drug is the active of the inhibition interferon-γ Drug.
4. application according to claim 1, which is characterized in that the drug is to block the interferon-γ and interferon- γ receptors are in conjunction with inhibiting vagus nerve cell Ca2+The drug of interior stream.
5. application of the gene of interferon-γ as drug target in the drug that the high quick syndrome of cough is treated in screening.
6. application of the inhibitor of targeted interferon-γ in preparing the drug for treating the high quick syndrome of cough.
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