CN104056253B - S100 calbindin A8 prepares the new purposes of diastole airway smooth muscle medicine - Google Patents
S100 calbindin A8 prepares the new purposes of diastole airway smooth muscle medicine Download PDFInfo
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Abstract
The present invention relates to protein drug field, specifically disclose a kind of S100 calbindin A8 at the purposes of preparation diastole airway smooth muscle medicine and the pharmaceutical preparation that contains this albumen. The present invention has disclosed the new function of S100 calbindin A8 diastole airway smooth muscle, S100A8 is diastole airway smooth muscle obviously, reduce the airway resistance of Brown-Norway rats with asthma, play the effect for the treatment of asthma, can be used for the treatment of asthma disease and the preparation for the treatment of asthma medicine; Be applied to the quality of asthma disease with the form of injection or spray, have and use the advantages such as safe, effective, stable.
Description
Technical field
The present invention relates to protein drug field, specifically disclose the treating asthma medicine that a kind of S100 calbindin A8 prepares the purposes for the treatment of asthma medicine and contains this recombinant protein.
Background technology
S100A8 albumen is the important member of S100 protein family, is known as again calgranulin A (Calgranulin-A), migration inhibition factor GAP-associated protein GAP 8 (Migrationinhibitoryfactor-relatedprotein8), cystic fibrosis antigen (Cysticfibrosisantigen), leucocyte L1 light chain protein (LeukocyteL1complexlightchain), stone in urinary system albumin A (UrinarystoneproteinbandA) etc. This albumen wide expression in Cell Differentiation commitment and the granulocyte in recurrent state, monocyte, activated macrophage in marrow, also has expression at non-myeloid cell in as endothelial cell, smooth muscle cell. Studies show that the S100A8 albumen that different expressions also can be detected in extracellular fluid, show that S100A8 has in cell and extracellular function simultaneously. When S100A8 albumen is combined with Ca2+, in the HIII position of EF-2, conformation can change, thereby exposes the hydrophobic residue on HIV, and it can be combined with target protein, and then by producing different biological effects from the effect of respective target albumen. Research shows S100A8 albumen and the close phase of the pathologic process such as tumour and inflammation, S100A8 albumen is mainly activated inflammatory cell and inflammatory factor, combination and is activated membrane receptor RAGE and TLR4 by the granulocytic chemotaxis of centering, the approach such as the Inflammatory Signal Transduction in mediated cell are brought into play important regulating action in inflammation. In addition, also studies have found that the anti-inflammatory activity of S100A8 albumen, illustrated that S100A8 may bring into play more complicated biology regulatory function under different pathophysiological conditions. We study and find that S100A8 has diastole airway smooth muscle, reduce the function of airway resistance, and at present both at home and abroad still not about the report of this function, this new function may provide new method for asthma control.
Summary of the invention
The object of the invention is to overcome the defect of prior art, provide one to there is diastole airway smooth muscle function, and can treat the Protein S 100A8 (S100 calbindin A8:NP_446274) of asthma, and be applied to the preparation for the treatment of asthma medicine.
A first aspect of the present invention discloses the application of a kind of S100 calbindin A8 (S100A8) in preparation diastole airway smooth muscle medicine.
Further, described diastole airway smooth muscle medicine is treating asthma medicine.
Described S100 calbindin A8 can be both the native protein of separation and purification acquisition, can be also the recombinant protein of producing by gene technology.
The present invention tests confirmation in a large amount of experiment in vitro and body, and recombinant protein S100A8 has the function of diastole airway smooth muscle, and is applied to the treatment of asthma, has good potential applicability in clinical practice.
Second aspect present invention discloses a kind of pharmaceutical composition of diastole airway smooth muscle, comprises S100 calbindin A8.
Preferably, unique active ingredient that described S100 calbindin A8 is this pharmaceutical composition.
Further, the pharmaceutical composition of described diastole airway smooth muscle is treating asthma medicine.
Third aspect present invention discloses a kind of pharmaceutical preparation of diastole airway smooth muscle, the S100 calbindin A8 that contains safe and effective amount, and surplus is pharmaceutically acceptable carrier.
Pharmaceutically acceptable carrier is various pharmaceutically conventional auxiliary material and/or excipient, include, but is not limited to carbohydrate (as lactose, dextrose plus saccharose), starch (as cornstarch and potato starch), cellulose and derivative thereof are (as sodium carboxymethylcellulose, ethyl cellulose and methylcellulose), tragacanth gum powder, Fructus Hordei Germinatus, gelatin, talcum, kollag (as stearic acid and dolomol), calcium sulfate, vegetable oil, as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cupu oil, polyalcohol is (as propane diols, glycerine, D-sorbite, mannitol and polyethylene glycol), alginic acid, emulsifying agent is (as Tween, Emulsifier EL-60), wetting agent (as NaLS), colouring agent, flavor enhancement, tablet agent, stabilizing agent, antioxidant, anticorrisive agent, apirogen water, Deng oozing salting liquid and phosphate buffer etc., this carrier can improve stability, activity and the biological effectiveness etc. of formula as required.
Preferably, described S100 calbindin A8 accounts for 0.0004%～99% of described pharmaceutical preparation gross mass.
Pharmaceutical preparation of the present invention can be made according to the universal method in pharmacy the dosage form of any routine.
Preferably, pharmaceutical preparation of the present invention is injection or spray.
More excellent, in described injection Chinese pharmacology, acceptable carrier is selected from PBS or physiological saline.
Optimum, the pH value of described PBS is 7.2～7.4.
Further, described diastole airway smooth muscle pharmaceutical preparation is the pharmaceutical preparation for the treatment of asthma.
The present invention has disclosed the new function of S100 calbindin A8 diastole airway smooth muscle, S100A8 is diastole airway smooth muscle obviously, reduce the airway resistance of Brown-Norway rats with asthma, play the effect for the treatment of asthma, can be used for the treatment of asthma disease and the preparation for the treatment of asthma medicine; Have and use the advantages such as safe, effective, stable.
Brief description of the drawings
Fig. 1: pET-22b (+) carrier collection of illustrative plates
Fig. 2: SDS-PAGE detection display transforms the e. coli bl21 (DE3) of recombinant plasmid and expresses bacterial strain after 0.1mMIPTG16 DEG C of induction 16h, and detecting CalgranulinA (CalculatedMw:11.3KD) has overexpression.
Fig. 3: after thalline ultrasonication, supernatant soluble fraction contains CalgranulinA.
Fig. 4: through ChelatingSFF (Ni) column separation and purification, albumen mainly concentrates in D, E.
Fig. 5: S100A8 purifying protein, purity reaches 90%, Buffer:20mMTris, 20%glycerol, pH8.0.
Fig. 6: use rat S100A8 protein specific antibody (SantaCruzsc-8113,1:2000) to carry out Westernblot experimental verification, result shows that this purifying protein is rat S100A8 albumen.
Fig. 7: S100A8 albumen reduces Lung Tissues of Asthmatic Rats resistance.
Fig. 8: S100A8 albumen reduces the airway smooth muscle shrink tension of Mch induction.
Detailed description of the invention
Before further describing the specific embodiment of the invention, should be understood that protection scope of the present invention is not limited to following specific specific embodiments; It is also understood that the term using in the embodiment of the present invention is in order to describe specific specific embodiments, instead of in order to limit the scope of the invention.
In the time that embodiment provides number range, unless should be understood that the present invention is otherwise noted, between two end points of each number range and two end points, any one numerical value all can be selected. Unless otherwise defined, all technology that use in the present invention are identical with the meaning that those skilled in the art of the present technique understand conventionally with scientific terminology. The concrete grammar that uses in embodiment, equipment, material, grasp according to those skilled in the art to prior art and record of the present invention, can also realize the present invention with any method, equipment and material similar to the method described in the embodiment of the present invention, equipment, material or prior art that be equal to.
Unless otherwise indicated, in the present invention, disclosed experimental technique, detection method, preparation method all adopt the routine techniques of molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell cultivation, recombinant DNA technology and the association area of the art routine. These technology are existing perfect explanation in existing document, specifically can be referring to MOLECULARCLONING:ALABORATORYMANUAL such as Sambrook, Secondedition, ColdSpringHarborLaboratoryPress, 1989andThirdedition, 2001; Ausubel etc., CURRENTPROTOCOLSINMOLECULARBIOLOGY, john wiley & sons, NewYork, 1987andperiodicupdates; TheseriesMETHODSINENZYMOLOGY, AcademicPress, SanDiego; Wolffe, CHROMATINSTRUCTUREANDFUNCTION, Thirdedition, AcademicPress, SanDiego, 1998; METHODSINENZYMOLOGY, Vol.304, Chromatin (P.M.WassarmanandA.P.Wolffe, eds.), AcademicPress, SanDiego, 1999; And METHODSINMOLECULARBIOLOGY, Vol.119, ChromatinProtocols (P.B.Becker, ed.) HumanaPress, Totowa, 1999 etc.
Embodiment 1S100A8 Protein reconstitution expression and purification
1.1 amplification S100A8DNA
According to NCBI Tissue distribution information, select normal rat lung tissue, guanidinium isothiocyanate one-step method extracted total RNA, AMV reverse transcriptase synthesizes cDNA, as S100A8 gene PCR amplification template, carries out pcr amplification under the effect of DNAtaq enzyme, and the primer of pcr amplification is:
Pcr amplification program is: the reaction system that total amount is 20ul after 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 58 DEG C annealing 30s, 72 DEG C extend 60s, carry out altogether 35 circulations, last 72 DEG C extend 5min; Amplified production sequence is as shown in SEQIDNO:3. Mate with rat S100A8 gene 100% in ncbi database, sequence is entirely true.
1.2 express S100A8 recombinant protein
By the product after amplification after NdeI (CATATG) and XhoI (CTCGAG) enzyme are cut, T4DNA ligase is connected to the corresponding restriction enzyme site of pET-22b (+) carrier (plasmid map is shown in Fig. 1), obtains prokaryotic expression clone. Order-checking detects correct recombinant vector and is converted into BL21 (DE3) competent cell through 42 DEG C of heat shocks, and RosettapLysS expresses bacterial strain, 37 DEG C of overnight incubation. On flat board, choose that in monoclonal access 3mlLB culture medium, [BL21 (DE3) is containing 50ug/mlAmpicillin, RosettapLysS is containing 50ug/mlAmpicillin, 34 μ g/mlChloramphenicol], 37 DEG C of 250rmp shaken cultivation are to OD600=0.5-0.6, and (1) adds 0.5mMIPTG37 DEG C of induction 3h; (2) add 0.1mMIPTG16 DEG C of induction to spend the night. SDS-PAGE detection display transforms the e. coli bl21 (DE3) of recombinant plasmid and expresses bacterial strain after 0.1mMIPTG16 DEG C of induction 16h, and detecting S100A8 (CalculatedMw:11.3KD) has overexpression, as shown in Figure 2. After thalline ultrasonication, supernatant soluble fraction contains S100A8, as shown in Figure 3.
According to expression screening result, in 100ml culture medium, (containing 50ug/mlAmpicillin) access BL21 (DE3) expresses bacterial strain, cultivate 16h for 37 DEG C, be transferred to (containing 50ug/mlAmpicillin) in 1LXY1 culture medium, 37 DEG C are cultured to after OD600=2, add 0.1mMIPTG16 DEG C of induction to spend the night.
ChelatingSFF on sample (Ni) column (CV1ml), with 50mMTris, 500mMNaCl, pH8.0 buffer solution balance, with 50mMTris, 500mMNaCl, 500mMImidazole, pH8.0 buffer solution for gradient elution, 20mMTris, 20%glycerol, the dialysis of pH8.0 buffer solution, the concentrated purifying protein that obtains. The S100A8 recombinant protein of purifying is utilized to SDS-PAGE electrophoretic separation, and result shows that purity was 90% (as shown in Figure 4,5).
Carry out Westernblot experimental verification with rat S100A8 protein specific antibody (SantaCruzsc-8113,1:2000), result shows that this purifying protein is rat S100A8 albumen (as shown in Figure 6).
Embodiment 2S100A8 recombinant protein affects experimental rat model of asthma respiratory function
1. experiment material
1.1 experimental subjects
SPF level male SD rat, body weight 200～300g, is divided into 6 groups at random, 8 every group.
1.2 experiment reagent
1) lumbar injection sensitization liquid, for containing 1mgOVA and 10mgAl (OH)3Physiological saline, every rats by intraperitoneal injection 1ml.
2) exciting liquid is the normal saline solution that contains 0.5%OVA, excites by 5mg/kg dosage jugular vein.
3) other reagent all adopt the configuration of analytically pure medicine physiological saline to obtain.
4) S100A8 albumen, prepared by embodiment 1.
2. experimental technique
The preparation of 2.1 asthmatic models
Ovalbumin (OVA) lumbar injection sensitization SD rat, carries out OVA vena jugularis externa to SD rat after 14 days and excites.
The preparation of 2.2 experimental group
Ovalbumin (OVA) lumbar injection sensitization SD rat, carries out OVA vena jugularis externa to SD rat after 14 days and excites; Excite first 10 minutes, by normal control reagent (physiological saline), physiological saline group (physiological saline), positive control medicine (Terbutaline), various dose (10ng/kg, 100ng/kg, 1000ng/kg) S100A8 albumen, jugular vein enters in rat body. After OVA excites, detect rats breathing function.
Experimental group is specifically divided into following 6 groups, and 8 every group are parallel:
A. Normal group: normal rat, physiological saline sensitization, physiological saline excites.
B. physiological saline group: OVA modeling rat, 2 weeks posterior external jugular vein injecting normal salines, dosage is 0.1ml/Kg, after 10min, OVA excites.
C. Terbutaline group: OVA modeling rat, 2 weeks posterior external jugular vein injection Terbutalines, dosage is 55 μ g/kg, after 10min, OVA excites.
D.S100A8 protein 10 ng group: OVA modeling rat, 2 weeks posterior external jugular vein injection S100A8 recombinant proteins, dosage is 10ng/kg, after 10min, OVA excites.
E.S100A8 protein 10 0ng group: OVA modeling rat, 2 weeks posterior external jugular vein injection S100A8 recombinant proteins, dosage is 100ng/kg, after 10min, OVA excites.
F.S100A8 protein 10 00ng group: OVA modeling rat, 2 weeks posterior external jugular vein injection S100A8 recombinant proteins, dosage is 1000ng/kg, after 10min, OVA excites.
With yellow Jackets with 50mg/kg body weight intraperitoneal injection of anesthesia rat, insert and be connected with the tracheal catheter that the diameter of three-way cock is 2.5mm at neck center longitudinal cut, measure tidal volume (VT) and airflow rate (V) with respiratory flow analyzer and Fleisch differential pressure pickup. Esophagus is made to cross sections, and inserting head end has the water injection conduit of four side openings, and rear blood pressure transducer is for pressure in mensuration esophagus and for replacing intrathoracic pressure (IPP). In esophagus, press after SMUP-B bio signal treatment system,, after A/D conversion, automatically record VT, V and IPP curve by " respiratory flow mensuration " software, and automatically calculate airway resistance (R) with VT, V. While detecting rats breathing changes of function, after operation, record breathing state 15min, using 12-14min data mean value as initial baseline value, excite the restructuring S100A8 albumen of front 10min jugular vein control group medicine and variable concentrations in OVA, 0.5%OVA injects in rat body and excites asthma with 5mg/kg consumption, record 15min, after OVA excites, 1-10min data are as statistical value.
3. experimental result
Result shows 10ng/kg, 100ng/kg, and the S100A8 recombinant protein of 1000ng/kg dosage 3min after OVA excites all can improve the airway resistance (P is all < 0.01) of experimental rat model of asthma, is dose-dependence. 1000ng/kg dose effect best (seeing Fig. 7).
The experiment of integral animal level shows, obviously diastole airway smooth muscle of recombinant rat S100A8 albumen energy, and the airway resistance of reduction Brown-Norway rats with asthma, and present certain dosage dependence trend, S100A8 is the most obvious in the effect of 1000ng/Kg.
The in vitro tissue experiment of embodiment 3S100A8 recombinant protein on the impact of rat trachea spiral bar tension force
1. experiment material
1.1 experimental subjects
Rat trachea spiral bar preparation method (revolving the method for cutting): use yellow Jackets with 50mg/kg body weight intraperitoneal injection of anesthesia SPF level male SD rat, dissect and take out rat trachea, peel off rapidly connective tissue around, keep blade upwards by fixing blade, penetrate tracheae with a metal bar (diameter 2mm), tracheae level is pressed on blade, blade direction becomes 60 degree angles with glass rod spiral rotating axle center, keep angle constant, spiral rotating glass rod, the spiral bar by tracheae helical cut into about 3mm*20mm.
1.2 experiment reagent
The reagent that is 1.5ug/ml with the PBS cushioning liquid configuration concentration of pH7.4 by S100A8 albumen (being prepared by embodiment 1) is for subsequent use.
2. experimental technique
Tracheal strip is after balance, in bath, add respectively negative control reagent (physiological saline), positive control medicine (atropine group), BSA group or the S100 calbindin A8 reagent of various dose, detect the tension variation of tracheae spiral bar. Concrete grammar is:
The isolated rat tracheae spiral bar of preparation is fixed in line organization's thermostatic bath, upper end connects PL3508PowerLab8/35 data collecting system (ADINSTRUMENTS, the U.S.), tracheal strip tension force is converted to pulse electrical signal, by LabChart7.2 software real time record. In bath, temperature constant is at 36.5-37.5 DEG C, and (1-2 bubble/s), initial load is adjusted in 1.5g to the mist of sustainable supply 95%O2 and 5%CO2. After each group sample balance 30min, start to drip the Mch solution stimulation tracheal strip contraction of various dose, each stimulating dose records 10min, chooses the average of 6-10min data as the last tension force numerical value of this dosage.
Isolated experiment method is specifically divided into following 4 groups:
A. Normal group (n=5): add in advance physiological saline to hatch 12h;
B.S100A8 group (n=5): add in advance S100A8 purifying protein (1.5 μ g/ml) to hatch 12h;
C.BSA group (n=5): add in advance BSA (1.5 μ g/ml) to hatch 12h;
D. atropine group (n=5): after balance record end, add the Atropine of 0.1 μ M, add methacholine (Mch) to stimulate after 10min.
3. experimental result
Result shows: atropine is as the competitive inhibitor of methacholine, can with the combination of can driving in the wrong direction of M cholinergic recepter, experimental result shows that atropine blocked the combination of Mch and M cholinergic recepter completely, illustrative experiment system is reliable; BSA is as negative control albumen and relatively indifference of normal group, the effect of unrestraint Mch; 1.5 μ g/ml restructuring S100A8 albumen are hatched 12h and can obviously be reduced the tracheae spiral bar contractile response (Fig. 8 is seen in P < 0.01) that Mch induces.
The experiment of in vitro tissue level shows, obviously diastole airway smooth muscle of 1.5 μ g/ml recombinant rat S100A8 albumen energy, the rat trachea spiral bar tension force that reduction Mch stimulates.
The above; it is only preferred embodiment of the present invention; not to any formal and substantial restriction of the present invention; should be understood that; for those skilled in the art; do not departing under the prerequisite of the inventive method, also can make some improvement and supplement, these improvement and the supplementary protection scope of the present invention that also should be considered as. All those skilled in the art, without departing from the spirit and scope of the present invention, a little change of making when utilizing disclosed above technology contents, the equivalent variations of modifying and developing, be equivalent embodiment of the present invention; Meanwhile, the change of any equivalent variations that all foundations essence technology of the present invention is done above-described embodiment, modification and differentiation, all still belong in the scope of technical scheme of the present invention.
Claims (2)
- The application of 1.S100 calbindin A8 in preparation diastole airway smooth muscle medicine.
- 2. application as claimed in claim 1, is characterized in that, described diastole airway smooth muscle medicine is treating asthma medicine.
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