CN108543068A - Application of the interleukin 37 in that modulates fibrosis relevant disease - Google Patents

Application of the interleukin 37 in that modulates fibrosis relevant disease Download PDF

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CN108543068A
CN108543068A CN201810540409.0A CN201810540409A CN108543068A CN 108543068 A CN108543068 A CN 108543068A CN 201810540409 A CN201810540409 A CN 201810540409A CN 108543068 A CN108543068 A CN 108543068A
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interleukin
application
fibrosis
inhibitor
tgf
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寿娟娟
杨天舒
赵孟孟
张姗姗
曹晶晶
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Tongji University
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Tongji University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label

Abstract

The present invention is experiments prove that IL 37 significantly can aggravate pulmonary fibrosis in cell experiment and internal animal model in vitro.The present invention further confirms that IL 37 aggravates fibrosis by TGF beta ALK1 accesses by testing.Therefore, discovery through the invention, can be by regulating and controlling the horizontal to inhibit TGF signal beta accesses of IL 37, to inhibit the fibrosis of internal organ or organ.Therefore IL 37 is used as the therapy target of fibrillation related disease, such as idiopathic pulmonary fibrosis, ischemic heart disease, viral cirrhosis, acute pancreatitis.It is a discovery of the invention that IL 37 plays the role of significantly aggravating fibrosis in human desmocyte cell culture and internal animal model in vitro.Present invention discover that therapy target new as new fibrillation related disease IL 37, a direction is provided for mouse pulmonary fibrosis treatment.More directly effectively special method is provided for the treatment of fibrosis.

Description

Application of the interleukin 37 in that modulates fibrosis relevant disease
Technical field
The invention belongs to biotechnologies, more particularly, to a kind of interleukin 37 (IL-37) in that modulates fibrosis Application in relevant disease.
Background technology
Fibrotic disease includes diversified disease, and some causes of disease are it is known that some causes of disease are unknown.Influence multiple organ system And cause serious morbidity and mortality.In the fibrotic disease characterized by duration or progressive fibrosis, flesh at Fibrocyte is main effector cell.Myofibroblast also assists in proper wound healing, by promotion wound closure and carefully The synthesis of extracellular matrix (ECM) albumen.Fibroblast, which obviously disappears, in granulation tissue implies the reparation of proper wound healing Reaction terminating.The myofibroblast that this disappearance may relate to dedifferente is transformed into static progenitor cells type or apoptosis or death Myofibroblast be eliminated.In contrast, in tissue damage tissue the persistence of myofibroblast cause research and Progressivity fibrosis, such as idiopathic pulmonary fibrosis (IPF), ischemic heart disease (the alternative and interstitial after myocardial infarction Property fibrosis), viral cirrhosis, acute pancreatitis etc..
Interleukin 37 (interleukin-37, IL-37) is one of IL-1 family members, is to be closed closely recently The cell factor of note.IL-37 belongs to IL-1 families, is a kind of novel inflammation inhibiting factor, in peripheral blood mononuclear cells, dendron There is expression in shape cell, macrophage and epithelial cell.The research of IL-37 is concentrated mainly on its anti-inflammatory effect at present. IL-37 can inhibit immune system activation, there is protection to make diseases associated with inflammation and autoimmune disease such as inflammatory enteritis etc. With.Different in the extracellular mode to play a role from common cell factor, IL-37 with endonuclear Smad3 mainly by tying It closes and forms the transcription of complex adjusting gene to inhibit the release of inflammatory factor.Mechanism of action extracellular IL-37 is still unclear at present Chu.
IL-37 functional studies are concentrated mainly on anti-inflammatory effect at present.It is worth noting that IL-37 is in haze and vapour Up-regulated expression in the environment of tail gas.However IL-37 does not have research to be related to the regulating and controlling effect of fibrosis so far.
Invention content
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide 7 (IL- of interleukin 3 37) application in that modulates fibrosis relevant disease.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of application of 7 inhibitor of interleukin 3 is being used to prepare prevention or is treating the medicine of fibrillation related disease It is applied in object.
The fibrillation related disease includes silicosis, idiopathic interstitial pneumonia, pulmonary tuberculosis, ischemic heart disease, virus Property hepatic sclerosis, acute pancreatitis, vascular hypertension, spleen fibroproliferative disorders, diabetes view eye mask fibroplasia etc..
7 inhibitor of the interleukin 3 includes the substance that all make the expression of interleukin 37 reduce.
The IL-37 aggravates fibrosis by TGF-beta-ALK1 accesses.
A kind of application for the inhibitor for blocking or inhibiting TGF-beta-ALK1 accesses is being used to prepare prevention or treatment fibre It is applied in the drug of dimensionization relevant disease.
It includes TGF-β-inhibitor, such as LY21097 to block or inhibit the inhibitor of TGF-beta-ALK1 accesses.
A kind of application of interleukin 37, the application in preparing brotic cells or animal model.
The IL-37 significantly can aggravate pulmonary fibrosis in cell experiment and internal animal model in vitro.
A kind of application of interleukin 37, applications of the IL-37 as the therapy target of fibrillation related disease.
The present invention is experiments prove that IL-37 significantly can aggravate lung fibre in cell experiment and internal animal model in vitro Dimensionization.The present invention further confirms that IL-37 aggravates fibrosis by TGF-beta-ALK1 accesses by testing.Therefore, pass through this The discovery of invention, can be by regulating and controlling the horizontal to inhibit TGF-β signal path of IL-37, to inhibit the fibre of internal organ or organ Dimensionization.
Therefore IL-37 is used as the therapy target of fibrillation related disease, as idiopathic pulmonary fibrosis (IPF), Ischemic heart disease (the alternative and interstitial fibrosis after myocardial infarction), viral cirrhosis, acute pancreatitis etc..
It is a discovery of the invention that IL-37 has significant exacerbation in human desmocyte cell culture and internal animal model in vitro The effect of fibrosis.
Present invention discover that therapy target new as new fibrillation related disease IL-37, controls mouse pulmonary fibrosis Treatment provides a direction.More directly effectively special method is provided for the treatment of fibrosis.
Description of the drawings
Fig. 1：IL-37 aggravates the experimental result of the pulmonary fibrosis of bleomycin induced；
Fig. 2：IL-37 promotes the phosphorylation of Smad2/3 in TGF-β, makes COL1A1, COL1A2, COL3A1 up-regulated expressions Experimental result；
Fig. 3：IL-37 does not influence the experimental result of the inflammatory cell recruitment of bleomycin and the generation of cell factor；
Fig. 4：The air pollutions such as haze vehicle exhaust make the experimental result of IL-37 up-regulated expressions in human lung cancer cell A549；
Fig. 5：IL-37 promotes the experimental result of the expression of α-SMA in human desmocyte cell；
Fig. 6：The experimental result that IL-37 passes through activation TGF-β Signal Regulation fibroblast activation；
Fig. 7：TGF-β-inhibitor inhibition IL-37 induced fibroblasts are divided into the experiment knot of flesh fibroblast Fruit；
Fig. 8：Pulmonary fibrosis correlation TGF-β associated signal paths.
Specific implementation mode
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1
Experimental procedure includes：
(1) mouse pulmonary fibrosis model is established
The bleomycin induced fibrosis of C57BL/6 mouse snuff amounts 15mg/kg.Control group mice injects equivalent Physiological saline, IL-37 dosage are equally tracheae suckings for every 1.5 μ g/kg, in addition, be that PBS buffer solutions are added in contrast, Be every 1.5 μ g/kg simultaneously equally it is that tracheae sucking is every other day administered once from the 1st day to the 21st day.
(2) it by after four groups of mouse administration cultures, takes lung paraffin embedding within the 21st day, is contaminated using paraffin section Masson and HE Color, Western Blot, qPCR verify IL-37 and promote pulmonary fibrosis by TGF-β signal path
Collect and preserve bronchoalveolar lavage fluid：With 4% chloraldurate 0.4mg/kg anesthetized mices, by the mouse of deep anaesthesia Four limbs and head fix and make incidence layback fully to expose neck, and passivity after the skin of throat is longitudinally cut off with eye scissors Throat muscle is detached, tracheae is fully exposed.An osculum is cut on tracheae, is carried out trachea cannula, is used sterile sutures Trachea cannula is put into fix.Then, the physiological saline for 0.8m1 being drawn with lmL syringes carries out pulmonary lavage 2 times, and will Bronchoalveolar lavage fluid is recovered in 1.5mL EP pipes, is centrifuged (4 °, 3000g, 10min), and it is bronchoalveolar lavage fluid to collect supernatant, point It fills, mark and be stored in .80 DEG C of refrigerator.Precipitation is that BAL is used for doing streaming.
Mouse lung pathology detect
Dehydration and embedding：After having carried out alveolar wass, the PBS of 0.8mL in lung is injected, in tracheae close to the place hand of lung Art suture ties, and extracts intubation.Lung is completely taken out, and is put into 4% paraformaldehyde and fixes an evening.After immersion is fixed Lung tissue be sequentially placed into the alcohol of gradient concentration and be dehydrated：75% wine 20min, 75% alcohol 20min, 85% alcohol 20min, 85% alcohol 20min, 95% alcohol 20min, 95% alcohol 20min, 100% alcohol 20min, 100% alcohol 20min.Again will Dewatered lung tissue is put in transparent to replace the alcohol in tissue in dimethylbenzene：Dimethylbenzene 30min, transparent 2 times altogether.Then, Lung tissue is put in paraffin of fine quality melted in advance and is finally fixed：Paraffin 30min is fixed 2 times altogether.Finally, by lung Tissue carries out organization embedding after being trimmed.
Slice and dyeing：Embedded wax stone is fixed on slicer, it, will behind the position and direction of adjusting wax stone Lung tissue is cut into the tissue of about 3~5 μ m-thicks.Tissue is put into after being flattened in 37 DEG C of water, is gently labelled to glass slide, does Good label.Piece 2h is baked in 60 DEG C of constant temperature roasters to dry moisture and make tissue that can be fully bonded with glass slide.After the completion of roasting piece Carry out follow-up dewaxing process：Dimethylbenzene 10min, dimethylbenzene 10min, 100% alcohol 10min, 100% alcohol 10min, 95% wine Smart 10min, 95% alcohol 10min, 85% alcohol 5min, 85% alcohol 5min, 75% alcohol 5min, PBS solution 5min (3 It is secondary).
H＆E is dyed：Glass slide after dewaxing is substantially immersed in PBS solution 5min, carries out 3 times, to remove on glass slide Alcohol is in order to avoid interfere haematoxylin coloring.Glass slide is put into haematoxylin dyeing liquid and dyes about 50~60s.According to dyeing Depth is broken up using 1% hydrochloride alcohol on demand, and when differentiation asks as 3~5s.Real-time tissues observed piece bush under the microscope The case where uniformly dyeing color, and re-start dyeing or differentiation on demand, until karyon is contaminated for bluish violet and cytoplasm is colourless.Then, Glass slide is put into eosin stains liquid and is dyed about 30 seconds, tap water rinses.The case where eosin stains are observed under microscope in real time, And continues flowing water on demand and rinse or redye Yihong.
Masson is dyed：Glass slide after dewaxing is substantially immersed in PBS solution 5min, carries out 3 times, to remove on glass slide Alcohol in case interference the haematoxylin colorings of Weigert.Glass slide is put into Weigert haematoxylin dyeing liquid and is dyed about 1min.According to dye level, broken up on demand using 1% hydrochloride alcohol, divergaence time is 3~5s.Under the microscope in real time The case where tissues observed piece haematoxylin dyeing, and re-start dyeing or differentiation on demand, until karyon contaminated for bluish violet and Cytoplasm is colourless.Then, it draws enough Ponceaux acid fuchsin liquid using liquid-transfering gun and dyes 5~10min, with 0.2% glacial acetic acid Solution differentiation a moment continues to break up 5min with 1% phosphomolybdic acid, be observed in real time under mirror, differentiation effect is with muscle fibre pinkiness Preferably.Then, aniline blue is directly added dropwise and dyes about 3min, observe in real time under mirror, when collagenous fibres present vivid blue and muscle fibre is in It can be terminated with 0.2% glacial acetic acid when pink.
After H＆E dyeing or Masson dyeing, tissue is dehydrated 30 seconds with 100% alcohol, dimethylbenzene washes away on slice Alcohol is added dropwise after neutral gum and carries out mounting with coverslip, and coverslip surrounding is fixed with nail polish, after nail polish solidification just It can be under the microscope.
Quantitative PCR
Extract tissue RNA
(a) it takes appropriate tissue to be put into 1.5mL centrifuge tubes for every group, is put into 500 μ L Trizol, it is even to pour into refiner progress Slurry processing, 4 DEG C, 13000g centrifugation 15min, takes supernatant 1.5mL to be put into centrifuge tube, ice bath 10min (on ice).
(b) 0.1mL chloroforms are added, are inverted mixing, stand 10min (on ice), 4 DEG C, 13000g centrifugation 20min take Supernatant is transferred in new pipe.
(c) add isometric isopropanol, slowly mixing, -80 DEG C, 2h.12000g centrifuges 20min, abandons supernatant.
(d) 75% ethyl alcohol of 0.6mL (RNase Free ddH are added2O), RNA precipitate, 4 DEG C, 12000g centrifugations are cleaned 6min abandons supernatant.
(e) d is repeated
(f) after air-drying RNA precipitate in super-clean bench, 20 μ L RNase Free ddH are added2O re-dissolves RNA；
(g) RNA concentration is measured on Nanodrop 2000.
Reverse transcription
Reaction solution preparation carries out on ice, and to ensure the accuracy of reaction solution preparation, when carrying out every reaction, reaction is pressed The amount of stoichiometric number+1 prepares Master Mix, is then dispensed into again in each reaction tube.
Reaction condition：42 DEG C of 15min, 85 DEG C of 5s, 4 DEG C of preservations
Real-time fluorescence quantitative PCR
1. with RNase Free ddH2O dissolves primer, and all reagents is balanced at room temperature and thorough mixing.
2. carrying out the preparation of Real Time PCR reaction solutions on ice.
Reaction system：
Table 2Real Time PCR relevant primer sequences
3. using ABI 7500Real Time PCR amplification instruments, reacted by following reaction condition：
Step 1:94 DEG C of 30s pre-degenerations
Step 2:94 DEG C of 5s denaturation
Step 3:60 DEG C of 34s annealing/extensions
Step 2 and Step 3 carries out 40 cycles, extends phase acquisition fluorescence signal.
4. solubility curve：After reaction, solubility curve analysis is carried out immediately, it is synchronous to read absorbance value.
Step1：95℃ 15s
Step2：60℃ 1min
Step3：95℃ 30s
Step4：60℃ 15s
Flow cytometer detection lymphocyte, neutrophil leucocyte, macrophage.Cell factor colouring method is as follows.
1. bronchoalveolar lavage fluid (BAL) is transferred to 15ml centrifuge tubes adds 5ml erythrocyte cracked liquids after centrifugation, room temperature 5min, Add 5ml complete mediums, centrifuges.Directly outwell supernatant.
2.1ml culture mediums are resuspended, and detect total number of cells in BAL under the microscope using blood cell counting plate.
The each hole of 3.48 well culture plates spreads 500uL cells
4. fluidic cell：Add 10-50ng/ml PMA, 200-500ng/mL ionomycin and BFA (1：1000) cell is arrived 4-6h (50ng/mL PMA＆500ng/mL ionomycin) .Overnight (20ng/mL PMA and are cultivated in culture medium 200ng/mL ionomycin&BFA)。
5. receiving cell, 1500rmp, 5min.Discard supernatant
6. fixing cell, adds the fixer (4% paraformaldehyde) of 400ul, 4C to place 20min, centrifuge 500g 5min, abandon Supernatant.
7.Add 50uL FACS Buffer and antibody, the dark places 4C are incubated 30min.
8.1mL FACS Buffer are washed twice.
9.500uL FACS Buffer are resuspended.
10. being detected using BD FACSAria flow cytometers.
11. analyzing macrophage, number and the utilization of lymphocyte and neutrophil leucocyte using FlowJo 7.6.1 Prism 5 makes number and ratio chart.
FACS Buffer:PBS
1.0%Bovine Serum Albumin (BSA)
0.1%Sodium Azide
2. Hydroxyproline assay
(1) it takes and freezes lung tissue 30mg, Science and Technology Ltd.'s hydroxyl proline test specification book (sample is built up in strict accordance with Nanjing This alkali hydrolysis method).Tissue is shredded, the hydrolysis of tissue samples is carried out, adjustment PH is 6.0~6.8.
(2) hydroxyl proline standard items (100 μ g/mL of mother liquor) are dissolved and configure, diluted concentration is as schemed.Do standard curve.
(3) standard items and sample (each doing three repetitions) sequence addition reagent I, II, III to specifications, fully into Row mixing, 60 DEG C of 15min of water-bath.It is fully cooled.
(4) 3500g 20min are centrifuged.
(4) supernatant is placed at absorbance 550nm, surveys absorbance.Hydroxyproline content (μ g/mg weight in wet bases) is according to standard Curve equation is calculated.
Western blot experiments
1. prepared by protein sample
It takes lung tissue 20mg frozen or so that 200 μ L lysates are added to add 10 μ L PMSF (100mM) and be separately added into 10 μ L Inhibitors of phosphatases A liquid (100mM) and B liquid (100mM), homogenate is ground into refiner, is shaken up and is placed on ice, cracks 20min.
4 DEG C, 12000rpm centrifuges 5min.
Supernatant is dispensed into the centrifuge tube of 0.5mL, is placed on ice.
2. the measurement of protein content (BSA methods survey protein content)
Make standard curve
The dilution of standard items, it is spare after room temperature thawing from -20 DEG C of taking-up 2mg/mL BSA.27 1.5mL centrifuge tubes are taken, It 3 one group, marks respectively.
Detect sample protein content (micro-pipe assay method)：
1) the BSA titers and sample to be tested for taking Fresh in 25 μ L tables respectively, are added in 96 orifice plates.
2) 200 μ L BCA working solutions are added in every hole, and mix well.
3) it is capped, 37 DEG C are incubated 30min postcoolings and to room temperature or are placed at room temperature for 2h.
4) its absorbance is detected at 562nm with ultraviolet specrophotometer.
5) the albumen concentration in sample is calculated according to standard curve.
3.SDS-PAGE electrophoresis
Clean glass plate
After liquid detergent is added dropwise on a glass, gently clean.After positive and negative is cleaned, rinsed with tap water, then with distillation Water rinses, and is dried after cleaning.
Encapsulating and loading
Clamping in folder is put into after glass plate alignment, vertical card prepares encapsulating on the top of the shelf.
10% separation gel is prepared, being shaken up immediately after addition TEMED can encapsulating.When encapsulating, glue is drawn along glass with liquid-transfering gun Glass plate is released, when glue surface rises to greenbelt medium line height.Then, one layer of n-butanol is added on glue, is gelled after fluid-tight Faster.
When thering is fringence to occur between n-butanol and glue, illustrate that glue has solidified.After glue fully solidifies, upper layer of removing photoresist N-butanol and liquid is blotted with blotting paper.
4% concentration glue is prepared, being shaken up immediately after addition TEMED can encapsulating.When encapsulating, glue is flowed down along glass plate, with Avoid having bubble generation in glue.After remaining space is filled concentration glue, comb is inserted into concentration glue, to make comb when inserting comb Keep horizontal.After gelling to be concentrated is solid, comb is gently extracted.
It takes in sample to the EP pipes of 1.5mL, concentration is tuned into unanimously according to concentration has been surveyed, 5 × SDS sample-loading buffers are added Be diluted to 1 × final concentration, protein sample is boiled into 10min in boiling water and is allowed to be denaturalized.
Loading is begun preparing for after enough electrophoresis liquids are added in electrophoresis tank.After adherent pipette samples, pipette tips are inserted to and are added Sample is slowly added to sample in hole.
Electrophoresis：Voltage is 80V, 30min when electrophoresis time generally concentrates glue；120V, total time are used after running to separation gel About 60min or so.Electrophoresis, which has just been run to bromjophenol blue to end, can terminate electrophoresis, carry out transferring film.
4. transferring film
0.2 μm of the pvdf membrane that size is fitted in 1 opening and closing is cut, the pvdf membrane sheared is placed in methanol and activates 10s.
Clip, which is opened, keeps the one side holding of black horizontal, foam-rubber cushion is sequentially placed into above, 1 layer in the leaching of transferring film liquid The thick filter paper steeped.
Falling glass plate sled gently, is slowly rinsed in sled with flowing water.Remove glass plate after, it would be desirable to part cut Come, places it on filter paper, so that it is aligned with filter paper with hand adjustment is whole, gently roll bubble with glass rod.Pvdf membrane is covered in glue Upper and bubble removing, under membrane cover after can not move again.In 1 layer of thick filter paper moistened of film upper cover and bubble is removed, then covers another layer Foam-rubber cushion closes clip after rolling bubble removing.
Clip is placed in electric turn trough, the one side of clip black is corresponded to the black flour of slot, the one side of clip white corresponds to In the red face of slot.Meeting heat production, is put into ice bag to cool down in the side of slot when electricity turns.Electric turn trough is put into ice.Generally turned with 240V Move 60min.
5. immune response
Film is placed in the plate containing confining liquid, closes 1h at room temperature.
Primary antibody is diluted to debita spissitudo with TBST, antibody-solutions are added on preservative film；Film is taken out from confining liquid, After sucking Liquid Residue with filter paper, memebrane protein is put in down on antibody liquid level；After being incubated 1h at room temperature, at room temperature with TBST It is washed on decolorization swinging table 2 times, each 10min；1 time is washed with TBS again, 10min.
Prepare secondary antibody diluent in the same way and contacted with film, after being incubated 1-2h at room temperature, at room temperature with PBST It is washed on decolorization swinging table twice, each 10min；It is washed once with PBST again, 10min, carries out chemiluminescence reaction.
Utilize chemiluminescence imaging system acquisition image.And calculate its gray value using PS.
Whether immunofluorescence and Western Blot detection IL-37 promote the related a-SMA's of the downstreams Smad2/3 pulmonary fibrosis Expression
Western Blot：HFL-1 cells are inoculated with to 6 orifice plates, be separately added into various concentration IL-37 (0.1ng/mL, 0.3ng/mL, 1ng/mL) it stimulates, PBS does negative control.Two groups of totally 8 holes.To be stimulated 24 as a child collect cell afterwards, utilize The expression of Western blot detections a-SMA.Carry out normalization protein expression using GAPDH as internal reference.
Immunofluorescence dyeing：HFL-1 cells are inoculated with to 6 orifice plates (being put into slide in advance), are separately added into the IL- of various concentration 37 (0.3ng/mL, 1ng/mL) are stimulated, and PBS does negative control.Two groups of totally six holes, three dye a-SMA, three F-actin ginsengs According to.Also TGF-β blocking group is the same as the same 6 holes of P-Smad2/3Western Blot dispositions above.To be stimulated 24 as a child It takes out cell climbing sheet afterwards to be put into the used Tissue Culture Dish of 35mm or 60mm, PBS is washed three times.4% cold paraformaldehyde is fixed 20 minutes, PBS was washed three times.Penetrating 10 minutes of 0.2%TritonX-100, PBS is washed three times.The serum of host identical as secondary antibody seals It closes 30 minutes, PBS is washed three times.It in 4 degree of wet box of primary antibody a-SMA (green fluorescence) or F-actin (red fluorescence) overnight, also can 37 Degree 2 hours, feels that the former effect is good, PBS is washed three times.Secondary antibody room temperature 2 hours (being protected from light) or 37 degree of 1 half an hour, PBS wash three Time.Most handy DAPI contaminates core, then directly shines flourescent sheet.Distilled water washes off PBS, glycerine mounting, the surrounding of nail sheet for oil seal.
It carries out in the result such as Fig. 1 of hematoxylin eosin staining shown in A figures, scale=200 μm in figure, in Fig. 1 in A figures, Saline indicates the group of injecting normal saline, that is, indicates that control group, Bleomycin indicate the group of sucking bleomycin, that is, test Group, what PBS indicated sucking is PBS buffer solutions, and that IL-37 indicates sucking is IL-37.
Carry out the result of horse pine trichrome stain such as in Fig. 1 blue shown in B figures be collagen, scale=200 μm in figure, In Fig. 1 in B figures, saline indicates the group of injecting normal saline, that is, indicates that control group, Bleomycin indicate sucking bleomycin Group, i.e. experimental group, what PBS indicated sucking is PBS buffer solutions, and that IL-37 indicates sucking is IL-37.
Lung tissue Ashcroft score (lung tissue section's fibrosis is determined according to the severity of pulmonary fibrosis and area Grade scoring) in result such as Fig. 1 shown in C figures.Display is the result shows that IL-37 significantly increases the fiber than lung caused by bleomycin Change degree.
Hydroxyprolin levels (hydrolysate of collagen) in lung are measured according to alkali hydrolysis method, as a result such as D figures institute in Fig. 1 Show.IL-37 increases the expression of collagen.
It takes within 21 days lung to do collagen related gene expression in qPCR pulmonary fibrosis to analyze, the results are shown in Figure 2.
A：Take mouse the 21st lung tissue do real time fluorescent quantitative detect COL1A1, COL1A2 and COL3A1 mRNA tables Up to situation.GADPH is as internal reference.B：21 days after lung tissue illustrated process, Western blot detects the phosphorylation feelings of Smad2/3 Condition.GADPH is as internal reference.According to fig. 2, Western Blot show that IL-37 promotes the phosphorus of Smad2/3 in TGF-β signal path Acidification.
The 0,2,7th day and the 21st day acquisition BAL liquid after bleomycin instillation.It is assessed in BAL by flow cytometry Total cell count and neutrophil count (A in Fig. 3) and with ELISA method measure bleomycin administration after 48 hours IL-1 β The concentration (B in Fig. 3) of the factor.Data are shown as average SEM.* P ＜ 0.05.
The air pollutions such as haze vehicle exhaust make IL-37 up-regulated expressions in human lung cancer cell A549
After the processing HFL-1 of concentration shown in Fig. 4 (human lung cancer cell A549) 12 hours, Western Blot analyze IL- 37 expressions.β-tubulin are used as internal reference.
Gasoline engine exhaust particulate object (Gasoline Exhaust Particles, GEP) and Fractions of Diesel Engine Exhaust Particulates object (Diesel Exhaust Particles, DEP) is important atmosphere pollution, 4- amyl phenols (PP) and 3- methyl -4- nitre Base phenol (PNMC) is effective active composition in GEP and DEP respectively.
It is expressed it is found that the main component PP and PNMC of the pollutant in air can result in IL-37 in human fibroblasts Up-regulation.
IL-37 a concentration of 0.3 and 1ng/mg processing human desmocyte cell after immunofluorescence dyeing, F-actin be used as in Ginseng, as a result as shown in A in Fig. 5, IL-37 a concentration of 0,0.1,0.3,1ng/mg handles Western- after human desmocyte cell Blot is analyzed, and Western blot detects IL-37 expression, and GADPH is as internal reference, as a result as shown in B in Fig. 5, finds α-SMA (α- Smooth muscle actin/ α-smooth muscle actin) figure Green fluorescence is α-SMA up-regulated expressions.α-smooth muscle flesh Filamentous actin (α-SMA) is actin isomers dominant in vascular smooth muscle cells, and important work is played in fiber is formed With.Myofibroblast is metabolism and the unique fibroblast of form of express alpha-SMA, their activation is in fiberization Development in play a key role.In an active state, myofibroblast stop being proliferated and start synthesis it is a large amount of it is extracellular at Divide albumen.The expression of α-SMA is related with the activation of myofibroblast.
The results show that IL-37 can promote α-SMA (α-Smooth muscle actin/ α-smooth muscle actin) table Up to up-regulation, illustrate that IL-37 induced fibroblasts are divided into myofibroblast.
The IL-37 of the concentration shown in Fig. 6 handles HFL-1 cells, and cell is handled to indicate the time with 1ng/mL IL-37. The expression of P-SMAD2/3 and SMAD2/3 is detected with Western blot.GADPH is as internal reference, and the results are shown in Figure 6.
IL-37 can promote the phosphorylation of Smad2/3, activate TGF-β/Smad signal paths.TGF-β inhibitor is added can To inhibit the activation of Smad2/3.
Fig. 7 A:HFL-1 (human desmocyte cell) uses 1ng/ml under LY21097 (TGF-β inhibitor) or DMSO effects IL-37 processing, with the expression of western blot determination α-SMA, GADPH is as internal reference.IL-37 can promote human fibroblasts The phosphorylation of interior Smad2/3 activates TGF-β/Smad signal paths.TGF-β inhibitor, which is added, can inhibit swashing for Smad2/3 It is living.
Fig. 7 B：With immuno-fluorescence assay α-smooth muscle actin (α-SMA).It is (red to handle cell same A, F-actin Fluorescence) it is used as internal reference.Green fluorescence is α-SMA, and IL-37 makes a-SMA up-regulated expressions, illustrates IL-37 induced fibroblasts It is divided into myofibroblast.α-smooth muscle actin (α-SMA) is that dominant flesh in vascular smooth muscle cells moves egg White isomers plays an important role in fiber is formed.Myofibroblast be express alpha-SMA metabolism and form it is unique at fibre Cell is tieed up, their activation plays a key role in the development of fiberization.In an active state, myofibroblast Stop being proliferated and starting a large amount of extracellular component proteins of synthesis.The expression of α-SMA is related with the activation of myofibroblast.
Fig. 7 shows that IL-37 induced fibroblasts are divided into myofibroblast.TGF-β inhibitor inhibits IL-37 to lure It leads fibroblast and is divided into myofibroblast.
Fig. 8 is pulmonary fibrosis correlation TGF-β associated signal paths.
The therapeutic choice of fibrosis is very limited.Therefore, the identification of new therapeutic strategy is in demand.With automobile Exhaust emissions getting worse, the vehicle exhaust in air may make IL-37 up-regulated expressions in human body cell, IL-37 that can promote Fibroblast is converted into myofibroblast, TGF/Smad signal paths is activated, to activate collagen related gene Expression, makes extracellular matrix constantly accumulate, to cause the relevant diseases such as fibrosis.IL-37 combination TGF-β inhibitor for treating has Help improve the relevant diseases such as fibrosis.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention. Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general Principle is applied in other embodiment without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be the present invention's Within protection domain.

Claims (9)

1. a kind of application of 7 inhibitor of interleukin 3 is being used to prepare prevention or is treating the drug of fibrillation related disease Middle application.
2. a kind of application of 7 inhibitor of interleukin 3 according to claim 1, which is characterized in that the fibrosis Relevant disease includes silicosis, idiopathic interstitial pneumonia, pulmonary tuberculosis, ischemic heart disease, viral cirrhosis, acute pancreas Inflammation, vascular hypertension, spleen fibroproliferative disorders or diabetes view eye mask fibroplasia.
3. a kind of application of 7 inhibitor of interleukin 3 according to claim 1, which is characterized in that the leucocyte - 37 inhibitor of interleukin includes the substance that all make the expression of interleukin 37 reduce.
4. a kind of application of 7 inhibitor of interleukin 3 according to claim 1, which is characterized in that the leucocyte Interleukin -37 aggravates fibrosis by TGF-beta-ALK1 accesses.
5. a kind of application of the inhibitor of blocking or inhibition TGF-beta-ALK1 accesses is being used to prepare prevention or treatment fiber Change and is applied in the drug of relevant disease.
6. the application of the inhibitor of a kind of blocking according to claim 5 or inhibition TGF-beta-ALK1 accesses, feature It is, it includes TGF-β-inhibitor to block or inhibit the inhibitor of TGF-beta-ALK1 accesses.
7. a kind of application of interleukin 37, the application in preparing brotic cells or animal model.
8. a kind of application of interleukin 37 according to claim 7, which is characterized in that the interleukin 37 In vitro pulmonary fibrosis is significantly aggravated in cell experiment or internal animal model.
9. a kind of application of interleukin 37, therapy target of the interleukin 37 as fibrillation related disease Using.
CN201810540409.0A 2018-05-30 2018-05-30 Application of the interleukin 37 in that modulates fibrosis relevant disease Pending CN108543068A (en)

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