CN104258419A - Applications of interferon regulatory factor 1 gene in treatment of atherosclerosis - Google Patents
Applications of interferon regulatory factor 1 gene in treatment of atherosclerosis Download PDFInfo
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Abstract
The invention discloses applications of an interferon regulatory factor 1 gene in treatment of atherosclerosis, belonging to the field of the functions and applications of a gene. An atherosclerosis model is obtained by taking ApoE-/-mice and IRF1-/-ApoE-/-mice as experimental objects through induction of high-fat diet, results show that the IRF1 gene defects markedly reduce the plaque area of the aorta, enhance the stability of aortic sinus plaque, and markedly lighten the inflammatory response compared with the ApoE-/-mice. The function of the IRF1 in the atherosclerosis is mainly reflected by the fact that the IRF1 achieves the effect of promoting the formation of the aortic plaque, especially the effect of promoting the atherosclerosis. Aiming to the functions of the IRF1, the IRF1 can be used as a drug target for screening drugs preventing, alleviating and/or treating atherosclerosis, and an inhibitor of the IRF1 can be used for preparing a drug for preventing, alleviating and/or treating atherosclerosis.
Description
Technical field
The invention belongs to function and the application of gene, be specifically related to a kind of interferon regulatory factor 1(IRF1) function and application of gene in treatment atherosclerosis, specifically IRF1 gene prevents in preparation, alleviates and/or treat in atherosclerotic medicine and apply.
Background technology
Cardiovascular and cerebrovascular disease be main lethal in many developed countries because of, also raise year by year at the sickness rate of China and fatality rate.The basis of cardiovascular and cerebrovascular disease is atherosclerosis (Atheosclersisis, AS), and atherosclerosis can make ductus arteriosus wall thicken, hardening, luminal stenosis, causes a lot of cardiocerebrovasculaevents events to occur.And the coronary artery acute stenosis that causes of the breaking of atherosclerosis unstable spot, hematoblastic gathering and thrombosis and obturation are the major reasons causing acute coronary syndrome (acute coronary syndrome, ACS).
Atherosclerosis is multiple gene, a kind of chronic inflammation disease that risk factor and immunologic mechanism participate in jointly, the inflammatory and immune response of local and whole body plays an important role in atherosclerosis generation evolution, inherent immunity and adaptive immunity participate in regulating atherosclerotic lesion jointly, pathology show as large, medium-sized artery many places Mottling formation, be apt to occur in blood shunt, the regions such as the bending and arterial branch of tremulous pulse, its characteristics of lesion is that in blood, lipid deposits at endarterium, cause inner membrance stove fibrous thickening, focus deep is the medicated porridge sample material formed by slough and extracellular lipid pond.Panimmunity cell is there is in atheromatous plaque, wherein with macrophage and T cell the most common, in addition a small amount of dendritic cell (Dentritic cell is also had, DC), natural killer cell (natural killer cell, NK cell) and mastocyte (mast cell) etc., occasionally there is bone-marrow-derived lymphocyte.
The macroscopic damage of atherosclerosis earliest period is fatty streaks, forms primarily of the Macrophage derived foamy cell that intake of a large amount of cholesterol.Mononuclear cell in blood circulation is attached to reactive endothelial cells at tremulous pulse damageable zone, starts the formation of fatty streaks, under the attraction that the mononuclear cell sticked is subject to the chemistry of local generation to ingratiate with molecule subsequently moves to inner membrance, and is divided into macrophage further.A large amount of cholesterol ester, in macrophage inner accumulated, forms foam cell, and this is the early stage characteristic pathological physiological process of atherogenesis.Atherosclerotic is multifactor coefficient result, and the atherosclerosis risk sexual factor found at present is a lot, but related pins to treatment and control effects all undesirable, blood fat reducing and anti-inflammatory treatment are current topmost remedy measures.
Interferon regulatory factor (interferon regulatory factors, IRFs) family, has identified 10 family members so far, has been respectively IRF1-10, and wherein IRF1-9 only exists [1] mammal.Research prompting, IRFs participates in the signal transduction receptor-mediated with Toll sample of endochylema pattern recognition receptors mediation, regulates and controls the expression [2] of 1 type interferon.IRFs also plays important expression regulation effect [3] to the differentiation of the natural immunity and immunocyte.IRFs, by regulating Growth of Cells and apoptosis, participates in the formation and development [4] of malignant tumor.In addition, we point out in up-to-date research, and IRFs family member also plays important regulative [5] in the cardiovascular disease such as myocardial hypertrophy.IRFs also participates in lipogenesis and adipocyte lipid metabolism [6,7].IRF1 is a member of interferon regulatory factor family (IRFS), in numerous immunoreation, is a multi-functional transcription regulaton factor.Under different conditional stimuluss, work in the immune response of IRF1 optionally in different cell.At many Malignant hematologic diseases, as acute leukemia (AL), myelodysplastic syndrome (MDS) and kinds cancer, all with the unconventionality expression of IRF1 gene.In addition, the immunologic cytotoxicity effect that IRF1 gene can also make NK cell, macrophage is brought into normal play.IRF1 is as transcription factor important in body, and research IRF1 can also as the treatment of heart reconstruction [8] recently.Based on above-mentioned Research foundation, we think that IRF1 may play key player in the early stage characteristic pathological physiological process of atherogenesis, but its biological function in the early stage characteristic pathological physiological process of atherogenesis and possible mechanism of action are not yet illustrated.
Mice and genetic engineering mice resource are one of most important animal models in field such as current disease mechanisms research, gene functional research, medicine initiative, are also the necessary conditions of these field innovation researches.Utilize genetic engineering mice to study for each molecule link in Atheromatosis reason process, to the molecular mechanism illustrated in atherosclerosis generation evolution, the molecular target finding treatment of atherosclerosis has great importance.
[list of references]
1.?Savitsky?D,?Tamura?T,?Yanai?H,?and?Taniguchi?T.?Regulation?of?immunity?and?oncogenesis?bythe?IRF?transcription?factor?family.?Cancer?immunology,?immunotherapy?:?CII.2010;59(4):489-510.
2.?Honda?K,?and?Taniguchi?T.?IRFs:?master?regulators?of?signalling?by?Toll-like?receptors?andcytosolic?pattern-recognition?receptors.?Nature?reviews?Immunology.?2006;6(9):644-58.
3.?Tamura?T,?Yanai?H,?Savitsky?D,?and?Taniguchi?T.?The?IRF?family?transcription?factors?in?immunity?and?oncogenesis.?Annual?review?of?immunology.?2008;?26:535-84.
4.?Takaoka?A,?Tamura?T,?and?Taniguchi?T.?Interferon?regulatory?factor?family?of?transcriptionfactors?and?regulation?of?oncogenesis.?Cancer?science.?2008;?99(3):467-78.
5.?Lu?J,?Bian?ZY,?Zhang?R,?Zhang?Y,?Liu?C,?Yan?L,?Zhang?SM,?Jiang?DS,?Wei?X,?Zhu?XH,?Chen?M,?Wang?AB,?Chen?Y,?Yang?Q,?Liu?PP,?Li?H.?Interferon?regulatory?factor?3?is?a?negative?regulator?of?pathological?cardiac?hypertrophy.?Basic?Res?Cardiol.?2013;108(2):326
6.?Eguchi?J,?Yan?QW,?Schones?DE,?Kamal?M,?Hsu?CH,?Zhang?MQ,?Crawford?GE,?and?Rosen?ED.?Interferon?regulatory?factors?are?transcriptional?regulators?of?adipogenesis.?Cell?metabolism.2008;7(1):86-94.
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Summary of the invention
The object of the invention is to determine the expression of IRF1 gene and atherosclerotic mutual relation, provide one for preventing, alleviating and/or treat the novelty teabag of atherosclerotic target gene IRF1.
Object of the present invention is achieved through the following technical solutions:
Expression and the atherosclerotic relation of the IRF1 gene that the present invention determines are as follows:
1. IRF1 gene knockout significantly reduces atherosclerotic plaque area
The present invention dual-genely to knock out (IRF1-/-ApoE-/-) mice for experimental subject with ApoE gene knockout (ApoE-/-) mice and IRF1/ApoE, obtains atherosclerosis mouse model (AS) respectively by low fat feedstuff and high lipid food diet induced.Result show ApoE-/-and IRF1-/-ApoE-/-mice by low fat feed diet, aorta tree has a small amount of Mottling formation, after high lipid food diet, plaque area significantly increases, and the plaque area of the aorta of IRF1-/-ApoE-/-mice tree, aortic sinus and brachiocephalic trunk is all significantly less than ApoE-/-mice (Fig. 1-2).
2. IRF1 gene knockout significantly strengthens the stability of aortic sinus speckle
The present invention dual-genely to knock out (IRF1-/-ApoE-/-) mice for experimental subject with ApoE gene knockout (ApoE-/-) mice and IRF1/ApoE, obtain atherosclerosis mouse model (AS) by high lipid food diet induced, the stability of aortic sinus speckle is studied.Result shows that IRF1 gene knockout significantly reduces the content of macrophage and the area of necrotic center in aortic sinus speckle, increases content and the collagen component content of smooth muscle cell in speckle, enhances the stability (Fig. 3) of aortic sinus speckle.
3. IRF1 gene knockout significantly reduces inflammatory reaction
The present invention dual-genely to knock out (IRF1-/-ApoE-/-) mice for experimental subject with ApoE gene knockout (ApoE-/-) mice and IRF1/ApoE, obtain atherosclerosis mouse model (AS) by high lipid food diet induced, the expression of the inflammatory factor of aortic sinus speckle is studied.Result shows that IRF1 gene knockout significantly reduces the proinflammatory factors such as ICAM-1 and IL-6 of aortic sinus speckle, increases the expression (Fig. 4) that IL-10 etc. presses down the scorching factor.
During generation atherosclerosis known by above result, IRF1 genetic flaw decreases plaque area, strengthens the stability of aortic sinus speckle, significantly reduces inflammatory reaction.Therefore IRF1 has the effect promoting atherosclerosis generation development, for the research atherosclerotic novel targets of control and New Policy provide theoretical foundation and Clinical Basis.
Therefore, IRF1 gene can be used as drug target, builds In vitro cell model or the animal model of IRF1 gene overexpression, for screening prevention, alleviating and/or treating atherosclerotic medicine; IRF1 gene also can be used as the target gene in gene therapy, designs and prepares prevention, alleviates and/or treat atherosclerotic medicine and/or biological reagent, reaching prevention, alleviate and/or treat atherosclerotic object by technique for gene engineering.Be such as target gene with IRF1, design the double-strand siRNA that IRF1 can be disturbed to express, after being synthesized by chemical method, be injected into human body and make IRF1 gene silencing treat atherosclerosis by the method that RNA disturbs; Can also design and build the mutant of IRF1, after injection, entering cell, the substrate specificity of competition IRF1 original shape, thus suppressing the function of IRF1, playing therapeutic purposes; In addition, can also be shot design micromolecular compound inhibitor with IRF1, utilize In vitro cell model or the animal model of IRF1 gene overexpression, by screening, find wherein to suppress the molecule of IRF1 by specificity, thus provide new therapeutic molecules for atherosclerotic treatment.
For the above-mentioned functions of IRF1, IRF1 gene is provided to prevent as drug targets in screening, alleviate and/or treat the application in atherosclerotic medicine.
For the above-mentioned functions of IRF1, provide the inhibitor of IRF1 gene in preparation prevention, alleviate and/or treat the application in atherosclerotic medicine.
One is prevented, is alleviated and/or treat atherosclerotic medicine, comprises the inhibitor of IRF1.
The inhibitor of described IRF1 is preferably the rna interference vector of siRNA, IRF1 gene of IRF1 gene, the one in the antibody of IRF1 and other inhibitor that IRF1 can be suppressed to express.
The present invention has following advantage and effect relative to prior art:
1. the present invention finds the New function of IRF1 gene, and namely IRF1 can promote atherosclerotic effect.
2. based on IRF1 promotion atherosclerotic function, provide target for developing atherosclerotic medicine.
3. the inhibitor of IRF1 can be used for preparation prevention, alleviates and/or treat atherosclerotic medicine.
Accompanying drawing explanation
Fig. 1 is ApoE-/-and aorta tree oil red O stain and plaque area cartogram of IRF1-/-ApoE-/-mice, and result shows that IRF1 gene knockout significantly reduces the plaque area of aorta tree.
Fig. 2 be ApoE-/-and IRF1-/-ApoE-/-mouse aorta hole and brachiocephalic trunk HE dye and plaque area statistics block diagram, result shows that IRF1 gene knockout significantly reduces the plaque area (*: p < 0.05vs HFD ApoE-/-group) of aortic sinus and brachiocephalic trunk.
Fig. 3 be ApoE-/-and IRF1-/-ApoE-/-mice aortic sinus speckle in necrotic center, collagen component, macrophage and smooth muscle cell content colored graph and result statistics block diagram, result shows that IRF1 gene knockout significantly reduces the content of macrophage and the area of necrotic center in aortic sinus speckle, increases content and the collagen component content (*: p < 0.05vs HFD ApoE-/-group) of smooth muscle cell in speckle.
Fig. 4 be ApoE-/-and IRF1-/-ApoE-/-mice aortic sinus speckle in the expression immunofluorescence dyeing figure of the inflammatory factor such as ICAM-1, IL-6, IL-10 and result statistics block diagram, result shows that IRF1 gene knockout significantly reduces the expression of proinflammatory factor ICAM-1 and IL-6 of aortic sinus speckle, increases the expression (*: p < 0.05vs HFD ApoE-/-group) of anti-inflammatory factors IL-10.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Animal for research and raising
Laboratory animal kind, sex, age and source in week: ApoE gene knockout (ApoE-/-) mice and IRF1/ApoE dual-genely knock out (IRF1-/-ApoE-/-) mice, male, 8 week age, body weight 19-25g.ApoE knock out mice (ApoE-/-, purchased from Jackson Laboratory, article No. 002052); IRF1-/-ApoE-/-mice is obtained by IRF1 knock out mice (purchased from Jackson Laboratory, article No. 002762) and the hybridization of ApoE knock out mice.
Laboratory animal feed formula: high lipid food (HFD, purchased from Fukang bio tech ltd of China, Beijing, by AIN-76 A Western Diets formula, percent of calories: protein 15.8%, fat 40%, carbohydrate 44.2%); Low fat feedstuff (NC, purchased from Fukang bio tech ltd of China, Beijing, article No. D12450B): percent of calories: protein 20%, carbohydrate 70%, fat 10%.
Animal feeding and environmental condition: all experiment mices are all raised at angiocardiopathy institute of Wuhan University SPF level Animal House (credit number: SYXK(Hubei Province): 2009-0053).Alternately illumination in every 12 hours, temperature 24 ± 2 DEG C, humidity 40%-70%, mice freely drinks water feed.
Embodiment 1 rat aorta is atherosis, and model (AS) obtains
1. laboratory animal grouping: select 8 week age, body weight 19-25g, male, ApoE-/-mice and IRF1-/-ApoE-/-mice, give high lipid food (Western Diets respectively, HFD) and low fat feedstuff (Normal chow, NC) raise, ApoE-/-HFD group, ApoE-/-NC group, IRF1-/-ApoE-/-HFD group, IRF1-/-ApoE-/-NC group totally 4 groups, often organize each 20.
2. Atherosclerosis Model induces operating process by high lipid food:
Adopt ApoE-/-mice and IRF1-/-ApoE-/-mice, set up AS model, carry out phenotype correlation analysis, specify the effect that IRF1 gene pairs atheromatosis plays.Mice was from 8 week age, and the omnidistance high lipid food of HFD group is fed and put to death and collect sample for 28 weeks, and the omnidistance low fat forage feed of NC group is put to death and collects sample for 28 weeks.
Embodiment 2 AS model mice plaque area measures
1. mice last tissue sampling eventually
Mice feed high fat or low fat feedstuff until 28 weeks time, weigh, use 3% pentobarbital sodium, 90mg/kg anesthetized mice, be fixed on material drawing board with syringe needle, with the moistening mice skin of chest abdomen of gauze, cut off thoracic cavity with eye scissors, expose heart, cut off right auricle, the syringe needle of transfusion device is lunged left ventricle, slowly inject 10-15mL PBS buffer with 50mL syringe, treat that right auricle effluent is limpid, change 4% paraformaldehyde and continue to inject 10-15mL.After perfusion terminates, remove splanchnocoel internal organs, only retain heart.Under mice is placed in microscope, be separated fascia, fatty tissue around aortic arch, cut brachiocephalic trunk, put into the 5mL EP pipe that 4% paraformaldehyde is housed, heart is cut in ascending aorta initial part, cut off in the middle part of thoracic aorta, and about 3mm place cuts off under neck summation clavicle, aortic arch is put into above-mentioned EP pipe.
2. aorta tree plaque area measures
Aorta tree be placed in 4% paraformaldehyde overnight fixing → pure water rinsing 30min → 60% isopropyl alcohol process 10 min → oil red O dye liquor (company sigma, article No. 00625) 60 min → 60% isopropyl alcohol 1min × 3 time of dyeing remove remaining outer wall fat → to be laid in by tremulous pulse dye on black dissection stencil plate to clean background → anatomical lens, take pictures with digital camera after dyeing, and use Image-Pro Plus 6.0 image analysis software to carry out plaque area quantitative assay (oil red O stock solution=0.5 gram oil red O+100 milliliter 100% isopropyl alcohol, oil red O dye liquor (working solution): V(oil red O stock solution)/V(H2O)=3/2).
Aorta tree plaque area (%)=speckle gross area/aorta tree gross area * 100%.
3. pathological tissue process
3.1 paraffin specimen preparations
In 4% paraformaldehyde overnight fixing after, brachiocephalic trunk, aortic arch filter paper are carefully wrapped, in case spill from embedding frame gap.Dewaterer is put into, 30% ethanol 15min → 50% ethanol 15min → 75% ethanol 15min → 85% ethanol 15min → 95% ethanol 15min → 100% ethanol 15min → 100% ethanol 15min → dimethylbenzene 15min → dimethylbenzene 15min → paraffin 30min → paraffin 30min after running water 30min.After brachiocephalic trunk and aortic arch have dewatered, take out from dewaterer.Brachiocephalic trunk is that Y stands in paraffin, and aortic arch lies low in paraffin.
3.2 aortic tissue sections
Use microtome respectively to aortic sinus and brachiocephalic trunk paraffin specimen section (slice thickness 5 μm).
4. aortic sinus plaque area measures
Haematoxylin eosin stains (HE dyeing): get the moisture of aortic sinus paraffin white tiles 65 DEG C baking 30 minutes → dimethylbenzene 5 minutes × 3 times → 100% ethanol 1 minute → 95% ethanol 1 minute → 70% ethanol, 1 minute → pure water rinsing (slide not to hang with the globule for standard) → get rid of on most microscope slide, haematoxylin solution (Zhuhai shellfish rope, BA-4021) contaminate 5 minutes → tap water rinse (remove slide on haematoxylin loose colour) → 1% acidic alcohol 1-3 second → tap water rinse (removing acidic alcohol on slide) → Scott liquid (sodium bicarbonate 0.35g, magnesium sulfate 2g, distilled water 100mL) 1 minute → tap water embathes (removing the Scott liquid on slide) → get rid of the moisture be all on slide, Yihong solution (Zhuhai shellfish rope, BA-4024) → pure water rinsing (removing the loose colour in Yihong on slide) → 70% ethanol once → 95% ethanol once → 100% ethanol that dyes for 10 seconds 30 seconds, 3 times → dimethylbenzene 2 minutes, 3 times → take advantage of dimethylbenzene not dry time mounting (with bubble-free of cutting into slices for principle) → fume hood in dry up, microscope is taken pictures.
5. brachiocephalic trunk plaque area measures
Haematoxylin eosin stains (HE dyeing), get brachiocephalic trunk paraffin white tiles, concrete operation method is with embodiment 2.4.
HE dyeing picture statistics: directly by Image-Pro Plus 6.0 image analysis software circle aortic sinus and brachiocephalic trunk plaque area.
By aorta tree oil red O stain substantially can assess that atheromatous plaque on whole piece blood vessel formed number, distribution situation and plaque area size.Fig. 1 is oil red O stain result figure after mice AS model, and brachiocephalic trunk and aortic sinus are the most obvious positions of atheromatous plaque, ApoE-/-passing through low fat feed diet with IRF1-/-ApoE-/-mice, aorta tree just has a small amount of Mottling formation; After high fat diet, plaque burden significantly increases, and the aorta tree plaque area of IRF1-/-ApoE-/-mice is significantly less than ApoE-/-mice.Fig. 2 is mice AS model posterior sinus of Valsalva and brachiocephalic trunk HE coloration result figure, and result shows that the aortic sinus of IRF1-/-ApoE-/-mice and the plaque area of brachiocephalic trunk are significantly less than ApoE-/-mice after high fat diet.Above result shows that IRF1 gene knockout significantly reduces atherosclerotic plaque area.
The mensuration of embodiment 3 AS model mice plaque stability
1. the size of aortic sinus necrotic center measures
Haematoxylin eosin stains (HE dyeing), method is with embodiment 2.4, and the microscope of organizing chosen containing cholesterol crystal, acellular core fibre structure is taken pictures.
The area estimation of necrotic center: use Image-Pro Plus 6.0 image analysis software circle necrotic center area.
2. aortic sinus collagen component assay:
Picro-Sirius red (PSR) dyes, key step is: 55 DEG C of baking 30min → dimethylbenzene 2min, 3 times → 100% ethanol 1min → 95% ethanol 1min → 70% ethanol 1min → running water 10min → distilled water 1min → mass fraction 0.2% phosphomolybdic acid 2min → 0.1% sirius red picric acid solution drips in tissue, dye in wet box 90min → removal residual liquid → 0.01N hydrochloric acid 4s → 70% ethanol 1 time → 90% ethanol 1 time → 100% ethanol 30s, 3 times → dimethylbenzene 2min, 3 times → take advantage of dimethylbenzene not dry coverslip immediately mounting, microscope is taken pictures.
Collagen ratio measuring: use the red area of collagen of Image-Pro Plus 6.0 image analysis software circle, collagen ratio (%)=area of collagen/plaque area * 100%.
3. aortic sinus macrophage and smooth muscle cell are expressed and are measured
Immunofluorescence dyeing detects macrophage marker CD68, smooth muscle cell mark SMA(Smooth Muscle Actin) expression.Required primary antibodie information: CD68 (MCA1957; 1:100; Rat; AbD Serotec), SMA (ab5694; 1:100; Rabbit; Abcam); Required two anti-information: Alexa Flour 568 goat anti-rat IgG (A11077; Invitrogen), Alexa Fluor 488-conjugated goat anti-rabbit IgG (A11008; Invitrogen).
Key step is:
1) roasting sheet: paraffin section is placed in more than baking box 30min.
2) dewax: dimethylbenzene 5min × 3 time.
3) hydration: 100% ethanol 5min × 2 time; 95% ethanol 5min; 70% ethanol 5min; ddH
2o embathes 5min × 2 time.
4) citrate tissue antigen recovery (Pressure method): get a certain amount of pH6.0 citrate antigen retrieval working solution in reparation box, put into pressure cooker, big fire is heated to boiling, tissue slice after dewaxing hydration is placed in and repairs box, cover pot cover, buckle pressure valve, continue to be heated to jet, after starting timing 5min, take out and repair box; Room temperature places 20min, takes out section after natural cooling.
5) ddH
2o rinsing 5min × 2 time, PBS rinsing 5min × 2 time.
6) groupization stroke circle, drips 10% sheep blood serum (GTX27481, GeneTex) and closes, 37 DEG C of closed 60min in wet box.
7) abandon confining liquid, drip the primary antibodie of proper proportion dilution, 4 DEG C of overnight incubation, 37 DEG C of rewarming 30min, discard primary antibodie, PBS washes 10min × 3 time.
8) drip two to resist, in wet box, hatch 60min for 37 DEG C, discard two and resist, PBS embathes 5min × 3 time.
9) SlowFade Gold antifade reagent with DAPI(S36939, Invitrogen) mounting.
10) viewed under fluoroscopy, takes pictures.
Fluorescent quantitation is added up: use Image-Pro Plus 6.0 image analysis software to measure Positive Cell Counts, CD68/SMA(%)=positive cell number/plaque area * 100%.
Macrophage is most important cell component in speckle, it mainly contain blood circulation mononuclear cell enter interior subcutaneous after differentiate, Monocytes/Macrophages can secrete multiple adhesion, chemotactic factor as cell adhesion molecule (ICAM-1), MCAF (MCP-1) etc., promote entering of speckle inner cell, in addition macrophage also can secrete multiple matrix metalloproteinase (MMPs), reduce area of collagen in speckle, thus destroy the stability of speckle; Smooth muscle cell can secrete various kinds of cell substrate, is the cell source of atheromatous plaque endocrine collagen, and Main Function repairs destroyed cellular stromal component thus plays a protective role; Collagen component is most important extracellular matrix in speckle, is also the main component of fibrous cap, and having anti-blood flow and impact the effect preventing plaque rupture, is also the important evaluation index safeguarding plaque stability.
Fig. 3 is the analysis result figure of the speckle inclusions of ApoE-/-mice and IRF1-/-ApoE-/-mice.The dye necrotic center area of visible IRF1-/-ApoE-/-mice of aortic sinus HE is significantly less than ApoE-/-mice; PSR coloration result shows the model after high fat diet, and the collagen ratio of IRF1-/-ApoE-/-group mice is apparently higher than ApoE-/-mice; It is then remarkable in ApoE-/-mice that immunofluorescence sends out observation macrophage marker CD68 expression in IRF1-/-ApoE-/-mice speckle; Smooth muscle cell mark SMA in IRF1-/-ApoE-/-mice speckle expression apparently higher than ApoE-/-mice.Above result shows that IRF1 gene knockout significantly reduces the content of macrophage and the area of necrotic center in aortic sinus speckle, increases content and the collagen component content of smooth muscle cell in speckle; IRF1 gene knockout can strengthen the stability (Fig. 3) of aortic sinus speckle
The mensuration of inflammatory Cytokines Expression in embodiment 4 AS model mice speckle
Immunofluorescence dyeing detects the expression of the inflammatory factors such as ICAM-1, IL-6, IL-10.Required primary antibodie information: ICAM-1(AF796; 1:100; Goat; R & D systems), IL-6(AF-406-NA; 1:100; Goat; R & D systems), IL-10(AF-217-NA; 1:100; Goat; R & D systems); Required two anti-information: Alexa Flour 568 donkey anti-goat IgG (A11057; Invitrogen).
Key step is: see embodiment 3.3.
Fluorescent quantitation is added up: use Image-Pro Plus 6.0 image analysis software to carry out absorbance (IOD) to positive cell and measure.
Inflammatory reaction is one of principal element causing atheromatous plaque to break.The a large amount of cytokine of proinflammatory cytokine secretion is as interleukin-6 (IL-6), and interleukin 10 (IL-10) etc. can cause the progress of the activation of endotheliocyte, the hypertrophy apoptosis of smooth muscle cell and medicated porridge sample pathological changes.Adhesion, the chemotactic factor of impaired endotheliocyte and Monocytes/Macrophages secretion are the key factors that transmitting inflammation cell is assembled to atheromatous plaque.The expression change of the inflammatory factors such as ICAM-1, IL-6, IL-10 is observed by immunofluorescence dyeing, result shows that IRF1 gene knockout significantly reduces the expression of proinflammatory factor ICAM-1, IL-6 in aortic sinus speckle, increases the expression (Fig. 4) of IL-10 anti-inflammatory cytokine.
Above-described embodiment result shows, ApoE-/-mice and IRF1-/-ApoE-/-mice issue lively pulse atherosclerosis in the induction of high fat diet, compare with ApoE-/-mice, the dual-gene Aortic Plaque area knocking out rear mice of IRF1/ApoE significantly reduces, plaque stability also increases, and inflammatory reaction obviously alleviates.These results show, IRF1 significantly can promote the formation of Aortic Plaque and atheroscleroticly to develop.Present invention demonstrates that IRF1 has important deterioration effect in Atherosclerosis Model, the medicine that its inhibitor can be used for preparing prevention, alleviates and/or treat atheromatosis.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (4)
1.IRF1 gene prevents as drug targets in screening, alleviates and/or treat the application in atherosclerotic medicine.
The inhibitor of 2.IRF1 prevents in preparation, alleviates and/or treat the application in atherosclerotic medicine.
3. prevent, alleviate and/or treat an atherosclerotic medicine, it is characterized in that: the inhibitor comprising IRF1.
4. application according to claim 2 or medicine according to claim 3, is characterized in that: the inhibitor of described IRF1 is the one in the rna interference vector of siRNA, IRF1 gene of IRF1 gene or the antibody of IRF1 and other inhibitor that IRF1 can be suppressed to express.
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