CN103784971B - Function of IRF (Interferon Regulatory Factor) 3 gene in atherosclerosis and application of inhibitor of IRF3 gene - Google Patents

Abstract

The invention discloses the function of an IRF3 gene in atherosclerosis and an application of an inhibitor of the IRF3 gene, belonging to the field of the function and the application of a gene. According to the invention, ApoE-/-mice and IRF3-/-ApoE-/-mice are taken as experimental subjects, an atherosclerosis model is obtained through high-fat induction, AS model mouse plaque area measurement and inclusion analysis are performed, and the results show that the aorta plaque area of the IRF3-/-ApoE-/-mice is remarkably reduced in comparison with that of the ApoE-/-mice. The invention discloses the function of the IRF3 gene in an atherosclerosis disease, which means that the IRF3 gene has an effect of promoting the formation of the aorta plaques, especially that the IRF3 gene can worsen the atherosclerosis. According to the abovementioned function, the IRF3 can be used as a drug target for screening drugs for treating the atherosclerosis disease. The inhibitor of the IRF3 can be used for preparing the drug for treating the atherosclerosis disease.

Description

The function of IRF3 gene in atherosclerosis and the application of inhibitor thereof

Technical field

The invention belongs to function and the application of gene, relate to a kind of IRF3(interferon regulatory factor 3) function of gene in atherosclerosis and the application of inhibitor thereof.

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.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.Increasing evidence display immunoreation participates in the links of progression of atherosclerosis, and the gene pairs control atherosclerosis therefore exploring regulation and control immune cell function is significant.

IRF3 is a member in interferon regulatory factor (interferon regulatory factor, IRF) family.There are some researches show that the expression of interferon gene when IRF3 and viral infection is closely related, IRF3 can express in various kinds of cell, is mainly present in cytoplasm, plays an important role in the natural immunity.

Summary of the invention

For addressing the deficiencies of the prior art and deficiency, the object of the present invention is to provide the application in the medicine preparing atheromatosis of a kind of IRF3 and inhibitor thereof.

Object of the present invention is achieved through the following technical solutions:

The present invention is with ApoE gene knockout (ApoE ^-/-) mice and IRF3/ApoE is dual-gene knocks out (IRF3 ^-/-apoE ^-/-) mice is experimental subject, obtains atherosclerosis mouse model (AS) model by high fat diet induction, carried out AS model mice plaque area and measured and inclusions analysis, result shows and ApoE ^-/-mice contrasts, IRF3 ^-/-apoE ^-/-rat aorta is atherosis, and Aortic Plaque area is starkly lower than the ApoE of diet of the same race ^-/-mice.This prompting IRF3 gene knockout can suppress atherosclerotic generation, illustrates that IRF3 gene can worsen atherosclerotic generation, for the research atherosclerotic novel targets of control and New Policy provide theoretical foundation and Clinical Basis.

The function of IRF3 gene in atherosclerosis, be mainly reflected in IRF3 gene have worsen Aortic Plaque formed effect, particularly IRF3 have worsen atherosclerotic effect.

For the above-mentioned functions of IRF3, the invention provides the application of a kind of IRF3, be mainly reflected in IRF3 as the application of drug targets in the medicine of screening suppression Aortic Plaque formation.

For the above-mentioned functions of IRF3, provide the application of a kind of IRF3, be mainly reflected in IRF3 as the application of drug targets in the medicine of screening treatment atheromatosis.

For the above-mentioned functions of IRF3, the invention provides the application of the inhibitor of a kind of IRF3, be mainly reflected in the application of inhibitor in the medicine of preparation suppression Aortic Plaque formation of IRF3.

Suppress the medicine that Aortic Plaque is formed, comprise the inhibitor of IRF3.

For the above-mentioned functions of IRF3, the invention provides the application of the inhibitor of a kind of IRF3, be mainly reflected in the application of inhibitor in the medicine of preparation treatment atheromatosis of IRF3.

Treat a medicine for atheromatosis, comprise the inhibitor of IRF3.

The inhibitor of described IRF3 is preferably the one in the rna interference vector of siRNA, IRF3 gene of IRF3 gene, the antibody of IRF3 and other inhibitor that IRF3 can be suppressed to express.

Achievement in research of the present invention shows, IRF3 ^-/-apoE ^-/-in the atherosclerosis that mice is induced in high fat diet, with ApoE ^-/-mice is compared, and Aortic Plaque area obviously reduces and collagen content increases, and illustrates that IRF3 gene has important deterioration effect in atheromatosis model.

The present invention has following advantage and effect relative to prior art:

(1) the present invention finds the New function of IRF3 gene, and namely IRF3 gene has the effect that can worsen atheromatosis.

(2) worsening the effect in atheromatosis based on IRF3, the medicine that IRF3 can be development atheromatosis provides target, and the inhibitor of IRF3 can be used for the atherosclerotic medicine of preparation treatment.

Accompanying drawing explanation

Fig. 1 is ApoE ^-/-and IRF3 ^-/-apoE ^-/-the aorta tree result figure of mice; A is mouse aorta tree oil red O stain figure, B is aorta tree plaque area statistics block diagram.

Fig. 2 is ApoE ^-/-and IRF3 ^-/-apoE ^-/-mouse aorta hole result figure; A is aortic sinus HE coloration result figure, B is aortic sinus plaque area statistics block diagram, and C is ApoE ^-/-and IRF3 ^-/-apoE ^-/-mouse Weight statistics block diagram (between NS representative group zero difference).

Fig. 3 is ApoE ^-/-and IRF3 ^-/-apoE ^-/-the analysis result figure of the speckle inclusions of mice; A is ApoE ^-/-and IRF3 ^-/-apoE ^-/-the macrophage (CD68) of mice and smooth muscle cell (SMA) mark immunofluorescence dyeing and result statistics block diagram (in figure, m represents aortic tunica media, and L represents aorta tube chamber), B is ApoE ^-/-and IRF3 ^-/-apoE ^-/-the sirius red stains of mice and result statistics block diagram.

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 in week and source: ApoE gene knockout (ApoE ^-/-) mice and IRF3/ApoE is dual-gene knocks out (IRF3 ^-/-apoE ^-/-) mice, male, 8 week age, body weight 19-25g.ApoE knock out mice (ApoE ^-/-mice, C57BL/6J background, purchased from Jackson Laboratory, article No. 002052).IRF3 ^-/-apoE ^-/-mice by IRF3 knock out mice (IRF3 knock out mice, C57BL/6J background, purchased from RIKEN BRC company, BRC number: RBRC00858) and ApoE knock out mice hybridize obtain.

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%).

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 IRF3 ^-/-apoE ^-/-mice, gives high lipid food (Western Diets, HFD) respectively and raises, ApoE ^-/-hFD group, IRF3 ^-/-apoE ^-/-hFD group is totally 2 groups.

2. Atherosclerosis Model induces operating process by high lipid food:

Adopt ApoE ^-/-mice and IRF3 ^-/-apoE ^-/-mice, sets up AS model, carries out phenotype correlation analysis, specifies IRF3 gene pairs incidence of atherosclerosis and plays a significant role.Mice fed high lipid food until 28 weeks put to death and collect sample from 8 week age.

Embodiment 2 AS model mice plaque area measures

1. mice last tissue sampling eventually

Mice feed high lipid food 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 10min → oil red O dye liquor dyeing 60min → 60% isopropyl alcohol 1min × 3 time remove remaining outer wall fat → be laid on black dissection stencil plate by tremulous pulse dye to clean background → anatomical lens, take pictures with digital camera after dyeing, and use IPP 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(H ₂o)=3/2)

Aorta tree plaque area (%)=speckle gross area/aorta tree gross area * 100%.

3. pathological tissue process

3.1 paraffin specimen

Heart preparation in 4% paraformaldehyde overnight fixing after take out, 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

Wax stone is fixed in the holder on section head, adjusts to slightly to leave and cuts into slices on the position that can switch to, and wax stone organizes tangent plane vertical parallel with the section edge of a knife, adjustment slice thickness (5 microns).The slice, thin piece cut, the curved tweezers of the right hand clamp firm paraffin section gently, section is taken off from section by the dry brush pen of left hand, puts into the glass dish containing warm water (about 30 DEG C), section is spread out on warm water face, the tangent plane of light is downward, slice fully spread out in thermostatted water and flatten after (approximately no more than 10 seconds), microscope slide vertically to be inserted in water light by section, and will cut into slices group on slide with tweezers, and adjust the position of section, uprightly mention with by slide.On the ground glass of slide one end, specimen numbering is write, air dried overnight of cutting into slices with pencil.

4, aortic sinus plaque area measures

Haematoxylin eosin stains (HE dyeing): the moisture of 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 (often take out a front cover to open, with bubble-free of cutting into slices for principle) → microscope takes pictures.Directly with IPP image analysis software circle aortic sinus plaque area (mm ²).

By aorta tree oil red O stain substantially can assess that atheromatous plaque formed number, distribution situation and plaque area size.Fig. 1 is oil red O stain result figure after mice AS model, and scalp is dry is the most obvious position of atheromatous plaque with aortic root.Fig. 2 is HE coloration result figure after mice AS model, and result shows, with ApoE ^-/-mice is compared, IRF3 ^-/-apoE ^-/-in mouse aorta tree, plaque area obviously reduces, IRF3 ^-/-apoE ^-/-mouse aorta hole plaque area obviously reduces, ApoE after 28 weeks AS models ^-/-mice and IRF3 ^-/-apoE ^-/-the body weight of mice, then without significant change (Fig. 2 C), shows that the disappearance of IRF3 gene significantly can reduce the size of atheromatous plaque.

The analysis of embodiment 3 AS model mice speckle inclusions

1. macrophage and smooth muscle cell are expressed and are measured

Immunofluorescence dyeing detects the expression of macrophage (CD68), smooth muscle actin (Smooth Muscle Actin, SMA).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, Carlsbad, CA), Alexa Fluor 488-conjugated goat anti-rabbit IgG (A11008; Invitrogen, Carlsbad, CA).

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, enough whole of the submergence sections of the necessary energy of amount of repair liquid, reparation box is put into the pressure cooker adding appropriate tap water, big fire is heated to boiling, tissue slice after dewaxing hydration is placed on high temperature resistant staining rack, again staining rack is slowly put into and repair box, cover pot cover, buckle pressure valve, continue to be heated to jet, after starting timing 5min, pressure cooker deenergization, go valve to uncap, take out and repair box; Room temperature takes out section after placing 20min 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.Preserve if need, 4 DEG C of preservations in dark wet box.

Fluorescence statistical method: SMA(%) the positive absorbance/speckle gross area * 100% of SMA in=speckle; CD68(%) the positive absorbance/speckle gross area * 100% of CD68 in=speckle.

2. Picro-Sirius red (PSR) dyeing

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.

Statistical method: the Antigen positive hybridomas gross area of collagen (%)=speckle hose lining/speckle gross area * 100%.

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; 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; 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 ApoE ^-/-and IRF3 ^-/-apoE ^-/-the analysis result figure of the speckle inclusions of mice.For the expression of macrophage in detection of plaque and smooth muscle cell, CD68(macrophage mark is carried out in aortic sinus section respectively) and SMA(smooth muscle cell mark) etc. immuning fluorescent dyeing analysis macrophage and smooth muscle cell composition.Immunofluorescence is sent out and is observed SMA, CD68 at ApoE ^-/-mice and IRF3 ^-/-apoE ^-/-all have expression in mice speckle, SMA is at IRF3 ^-/-apoE ^-/-in mice speckle, expression is apparently higher than ApoE ^-/-mice; CD68 is at IRF3 ^-/-apoE ^-/-in mice speckle, expression is then remarkable in ApoE ^-/-mice (A); PSR coloration result shows the model after high fat diet, IRF3 ^-/-apoE ^-/-the collagen ratio of group mice is apparently higher than ApoE ^-/-mice, illustrates IRF3 ^-/-apoE ^-/-the speckle more stable (B) of group mice.Result shows that IRF3 gene knockout can reduce speckle inner area in AS model, promotes the stability of speckle.

Above-described embodiment result shows, ApoE ^-/-mice and IRF3 ^-/-apoE ^-/-mice issues lively pulse atherosclerosis in the induction of high fat diet, IRF3/ApoE is dual-gene knock out after, mouse aorta plaque area comparatively ApoE ^-/-little remarkable minimizing.These results are pointed out, and IRF3 gene can promote the formation of Aortic Plaque and atheroscleroticly to develop.Present invention demonstrates that IRF3 gene has important deterioration effect in Atherosclerosis Model, it can be used as the medicine of drug targets screening treatment 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. Interferon regulatory factor-3 is as the application of drug targets in the medicine of screening suppression Aortic Plaque formation.

2. Interferon regulatory factor-3 is as the application of drug targets in the medicine of screening treatment atheromatosis.

3. the inhibitor of Interferon regulatory factor-3 suppresses the application in the medicine of Aortic Plaque formation in preparation.

4. the application of the inhibitor of Interferon regulatory factor-3 in the medicine of preparation treatment atheromatosis.